SURFACE MODIFICATION OF POSITIVELY AND NEGATIVELY...

SURFACE MODIFICATION OF POSITIVELY AND

NEGATIVELY CHARGED POROUS SILICON

NANOPARTICLES AND THEIR BIOLOGICAL

APPLICATIONS

Mezbah Uddin

Masterrsquos Thesis

Department of Chemistry

Medicinal Chemistry

4062012

ii

UNIVERSITY OF EASTERN FINLAND School of Pharmacy

Master Degree Program for Research Chemists

MEZBAH UDDIN

Master Thesis 40 p

Supervisors

Senior Scientist Docent Ale Naumlrvaumlnen and Researcher MSc Jussi Rytkoumlnen

September 2012

KEY WORDS Nanotechnology PEGylation Opsonization Zeta potential Fluorescence intensity

Biofunctionalization

Porous silicon nanoparticles (PSiNPs) have drawn significant attention in recent years in the

study of oncology due to their biocompatibility favorable biodistribution and efficient

clearance or biodegradability The clinical use of PSiNPs as nanocarriers in drug delivery

depends on biologically nonspecific adsorption and immunological response Minimization

of the opsonization in the blood increases the half life of the particles and the capability to

target tumors In this project the opsonization of PEGylated porous silicon nanoparticles was

investigated as it is expected that PEGylation of TCPSi-NH2NPs with Me-PEG-COOH can

contribute to minimization of the opsonization PEGylated PSiNPs were also prepared using

single step reaction between terminal amines in thermally carbonized porous silicon

nanoparticles (TCPSi-NH2NPs) and carboxylic acid groups in mPEG-COOH by refluxing

PEGylated particles were characterized by measuring their size and zeta potential and the

opsonized particles were analyzed by SDS-PAGE The surface of TCPSi-NH2NPs was also

conjugated with fluorescent molecule such as fluorescein-5-isothiocyanate (FITC) and by

tailoring of the acid treated thermally hydrocarbonized porous silicon (UnTHCPSi) NPs by

associating with fluoresceinamine isomer-1 (FluorA) activated with DICNHS activator

FITC labeling TCPSi-NH2NPs were further modified by conjugating stearylated NickFect 51

(NF51) cell penetrating peptides (CPPs) and radioactive iodine labeled stearylated NickFect

cell penetrating peptides Fluorescent labeled NPs were characterized with zeta sizer and

fluorescence spectroscopy The surface modified PSiNPs can be loaded with targeted

therapeutic compounds and studied their biodistribution both in vivo and in vitro

iii

ACKNOWLEDGEMENT

I am heartily thankful to my supervisor Ale Naumlrvaumlnen and Jussi Rytkoumlnen whose

encouragement guidance and support from the initial to the final level enabled me to develop

an understanding of the subject

Finally I would like to offer my regards and blessings to all of those who supported me in

any respect during the completion of the project

Mezbah Uddin

iv

Contents Abbreviations vi

1 Introduction 1

11 Nanotechnology 1

12 Porous Silicon (PSi) 2

121 Properties of Porous Silicon 2

122 Structural Properties of Porous Silicon and Their Effect 2

123 Chemical Properties of the Porous Silicon 2

1 3 Surface Functionalization of Porous Silicon 3

14 Advantages of Porous Silicon 3

15 Porous Silicon Nanoparticles 3

151 Amino-derivatized Thermally Carbonized Porous Silicon Nanoparticles (TCPSi-

NH2 NPs) 4

152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous Silicon

Nanoparticles (UnTHCPSiNPs) 5

16 Polyethylene Glycol (PEG) Molecule 5

17 Fluorescent Molecules and Their Applications 6

171 Fluorescence Detection 7

18 Cell-penetrating Peptides (CPPs) 7

19 Opsonization 8

110 Role of Zeta Potential in Characterization of Nanoparticles 10

111 Surface Modification 11

112 PEGylation 12

1121 Affecting Factors on PEGylation 13

1122 Different Sized NP 13

1123 Length amp Conformation of PEG Chain 14

1124 Terminal Portion 15

113 Biofunctionalization 16

114 General Conjugation Reactions 17

2 Experimental 20

21 Materials 20

22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation) 21

221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and NHS as an Activator

Reagent 21

v

222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and DIPEA as an

Activator Reagent 21

223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of Amino-modified TCPSi-

NH2NPs and MeO-PEG-COOH 21

224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different Size (5768kDa

11kDa and 25kDa) 22

225 Opsonization 22

23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent

Molecules 23

231 Standard Calibration Curve with FITC and FluorA 23

232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC 23

2321 Absorption of Stearylated NickFect 51 (NF51) Cell Penetrating Peptides (CPPs)

onto the Surface of FITC Labeled TCPSi-NH2NPs 23

2322 Conjugation of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51) Cell

Penetrating Peptides (125I-CPPs) to the Surface of FITC Labeled TCPSi-NH2NPs 24

233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1 (FluorA) 24

24 Characterization Methods 25

241 Dynamic Light Scattering (DLS) 25

242 Gel Electrophoresis 25

243 Fluorescence Intensity Measurement 25

3 Results and discussion 26

31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) 26

32 Opsonization of the PEGylated TCPSi-NH2 NPs 28

33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH

30

34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2)

Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC) 31

341 Characterization of Stearylated Stearylated NickFect 51 (NF51) CPPs Adsorped

FITC Labeled TCPSi-NH2NPs 34

342 Characterization of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51)

Cell Penetrating Peptides Conjugated FITC Labeled TCPSi-NH2 NPs 35

35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA 35

4 Conclusion 38

5 References 40

vi

Abbreviations AEASP minus3-(2-Aminoethylamino)propyldimethoxymethylsilane

AFM minus Atomic force microscopy

CPPsminusCell-penetrating peptides

CTminus Cytotoxic ions

DICminusN N-Diisopropylcarbodiimide

DIPEAminusN N-Diisopropylethylamine

DLSminusDynamic Light Scattering

DLVOminusDerjaguin-Landau-Verwey-Overbeek

DMFminusDimethylformamide

DNAminusDeoxyribonuclic Acid

EDACminus1-ethyl-3-(3 dimethylaminopropyl)carbodiimide

EDTAminus Ethylenediaminetetraacetic acid

EPRminusenhanced permeability and retention

EtOHminusEthanol

FminusFlory radius

Fc minus Fragment crystallizable

FDAminusfood and drug administration

FEminus Field Emission

FESEMminus Field Emission scanning electron microscopy

FITCminusfluorescein isothiocyanate

FluorAminus fluoresceinaminisomer-1

FLminusFluoroskan Ascent

HBTUminus O-Benzotriazole-N N N N-tetramethyl-uronium-hexafluoro-phosphate

HEPESminus4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HIV-1minusHuman Immunodeficiency Virus 1

HPminus Human plasma

MRIminusMagnetic resonance imaging

NHSminusN-hydroxysuccinimide

NIRminus Near infra red

NPs minus Nanoparticles

PBSminus Phosphate Buffer Saline

PEGminuspolyethylene glycol

PSminus Porous silicon

RESminusReticuloendothelial system

SDS-PAGEminusSodium dodecyl sulphate polyacryl amide gel electrophoresis

SEFminus Surface-enhanced fluorescence

SFMminusScanning force microscopy

SOIminusSilicon-on-insulator

SOSminusSilicon on sapphire technology

TCPSi-NH2 NPsminusAmino-treated thermally carbonized porous silicon nanoparticles

TatminusTrans-activating transcriptional activator

TEMminusTransmission electron microscopy

UnTHCPSiNPsminus Undecylenic acid-treated thermally hydrocarbonized porous silicon

nanoparticles

vdWminus Van der Waals

X-ray CTminus X-ray computed tomography

ZSminus Zetasizer

1

1 Introduction 11 Nanotechnology

Biomedical process has been remarkably impacted over the preceding decades due to

evolution of micro and nanotechnology Quantum dots controlled released nanoparticles

targeted delivery and cancer nanotechnology are ideal examples Practical employment of

many applications is still difficult but high rate of evolution is raising hope for innovative

biomedical process in near future [1]

Nanotechnology is an interdisciplinary study area comprising physics chemistry biology

engineering and precise diagnosis Treatment of different diseases especially cancer can

adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which

fits well with the size of biological molecules such as antibodies Nanoparticles can also have

very good interactions both on surface and interior cells of biomolecules which is very

effective for syndrome diagnosis and treatment used in nanomedicine

Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo

purposes [4 5] Biocompatibility in vivo kinetics capability to break out the

reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost

effectiveness are main challenges in preclinical animal models and clinical translation of

nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where

the absorbent tumor vasculature can let for superior tissue permeation than normal organs

Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening

molecular interactions and to identify molecular changes in a susceptible approach

Nanotechnology made a great progress in biological imaging and drug delivery They are

now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and

diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and

many more are used in nanomedicine We are interested to monitor biological characteristics

of porous silicon nanoparticles

2

12 Porous Silicon (PSi)

Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -

m2cm

3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the

VS Porous silicon has many applications in drug delivery biological imaging photonics

chemical sensing and so on

121 Properties of Porous Silicon

122 Structural Properties of Porous Silicon and Their Effect

Basic structural parameters are pore size porosity and porous thickness Field emission

scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic

force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study

structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable

depending on porosity [11] In in-vitro study porous silicon samples were exposed to

simulate body fluid containing similar ion concentration as in human blood They were

investigated for longer time High porosity mesoporous layers were completely removed

within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite

growth

123 Chemical Properties of the Porous Silicon

Porous silicon has high surface area with high density of Si dangling bonds and adulterations

like hydrogen and fluorine Adulterations are usually formed during PSi formation from

electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades

during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally

whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in

luminescence spectra is observed after oxidation [12] and electrical conductivity and optical

properties are also influenced Level of impurity of fluorine depends on type of electrolyte

and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is

3

replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride

concentration decreases with time

1 3 Surface Functionalization of Porous Silicon

PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx

termination Meta-stable hydro-silicon can oxidize and degrades surface structure So

passivation of PSi surface is important to have good electrical contacts on PSi

14 Advantages of Porous Silicon

Porous silicon micro-particles are very useful for controlled drug delivery because they are

less toxic They are good for chronic use as they disintegrate in body whereas carbon

nanotubes degrade Secondly desired surface area free volume and pore size (few

nanometers to several hundreds of nanometers) can be achieved by electrochemical

fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or

biological molecules (drugs peptides antibodies proteins etc)

Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence

deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic

purposes Additionally porous silicon is simple enough to replace more complicated clinical

devices [15-17]

15 Porous Silicon Nanoparticles

Porous silicon nanoparticle is commonly used as nanomedicine They have excellent

properties like biocompatibility favorable bio-distribution biodegradability for controlled

drug delivery Nanoparticles have large pay loads stability avidity and enhancement They

can be used in multiple purposes because of their unique size and on elevated ratio of surface

to volume tunable optical electric magnetic and biological features They can have various

4

sizes and shapes chemical constituents surface chemical features and hollow or solid

structures So they are suitable for drug delivery vehicles distinction agents and diagnostic

devices NPs are smaller than cells and can effect in vivo applications For example in cancer

tissue NPs can remain in the area even after extravasating from leaky tumor and shows

enhanced permeability and retention effect [18] for signaling or therapy [19]

Moreover SiNPs are commercially available and easily separable from aqueous suspension

Large specific surface of most SiNPs can be easily functionalized and delivered into living

cell

151 Amino-derivatized Thermally Carbonized Porous Silicon

Nanoparticles (TCPSi-NH2 NPs)

The most important characteristics of TCPSi-NH2-NPs are summarized in following table

Table 11

Table 11 Features of the plain TCPSi-NH2

NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)

Pore Size (nm) Particle Size

Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170

Figure 11 Chemical structure of the TCPSi-NH2

5

TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is

more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when

primary amine forms amide bond with succinimidyl ester terminated surface layer is formed

during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of

TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group

to primary amine

152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous

Silicon Nanoparticles (UnTHCPSiNPs)

Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size

distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in

presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface

This terminated surface further reacts with primary amine to form amide bond

Figure 12 Chemical structure of the UnTHCPSi

16 Polyethylene Glycol (PEG) Molecule

PEG has repeating ethylene ether units and very attractive for conjugation It is most

frequently used in biomedical applications due to high solubility in water PEG is very

flexible and can be cleared from body

6

17 Fluorescent Molecules and Their Applications

Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein

isothiocyanate (FITC) are often utilized in many biological purposes These derivatives

contain a reactive substituent on the phenyl ring for coupling reactions

Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)

FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-

N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure

This derivative can easily react with nucleophiles including amine and sulfhydryl groups

FluorA is the amine derivative of the original fluorescein molecule which reacts with

carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak

wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency

to photobleaching This feature is challenging so it has to be overcome for the biological

applications where greater photostability higher fluorescence intensity or different

attachment groups are needed

Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for

labeling These probes are used in vivo purposes for example to image target tissue in live

animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used

for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized

with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes

fluorescent micro-particles and nanoparticles are used in many imaging techniques

7

171 Fluorescence Detection

Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is

required to identify low concentrated objective molecule [26-29] There are many methods

for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]

When a fluorophore is confined near to surface of metallic nanoparticles [33 34]

fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic

particles causes SEF When fluorophore is near to metal core then fluorescence quenched

completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]

Small particles quench fluorescence due to absorption and big particles increases

fluorescence due to dispersion [35]

18 Cell-penetrating Peptides (CPPs)

CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian

cells Cargoes are from small chemical molecules to nanosize particles and large fragments of

DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or

through non-covalent interactions

CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid

composition of high relative abundance of positively charged amino acids or non polar

hydrophobic amino acids Initially it was found that trans-activating transcriptional activator

(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media

by numerous cell types in culture [36] Many CPPs have been identified and small synthetic

analogues with more effective protein transduction properties have been established [37]

8

Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_

for_CPPjpg 26 April 2009)

Extracellular materials are gene or nanoscale synthetic delivery system They can not

transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can

overcome this problem through receptor independent pathway CPP mediated NPs can be

internalized by a cell and have high potential for delivering imaging and therapeutic agents

into the cells

19 Opsonization

A process in which pathogens are layered and neutralized by immune system is called

opsonization An opsonized pathogen is killed either by uptaking or direct neutralization

RES is an immune system where circulating and macrophages liver kupffer cells and spleen

and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin

protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]

9

Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)

opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T

Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash

728)

Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation

(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For

example Kupffer cells serve as major filter for many types of NPs with major interference

(long t12) [41]

Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver

(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy

Nanomedioine 2011 6 (4) 715ndash728)

PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It

protects recognition by monocytes and macrophages and thus NPs circulates [39 40]

Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially

NPs aggregate because attraction between particles is stronger than solvent [40 42]

According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to

their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls

attraction potential is important factor for aggregation of spherical NPs PEG reduces surface

energy of NPs and Vander Waals attraction [45-47]

10

110 Role of Zeta Potential in Characterization of Nanoparticles

Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-

characterizationtechnologyzeta-potential)

Zeta potential is the electric potential at the slip plane between the bound layer of diluents

molecules surrounding the particle and bulk solution It depends on particles surface charge

and diluents solution Higher zeta potential means greater electrostatic repulsion between

particles and less aggregation

Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution

can be measured with dynamic light scattering Zeta potential determines stability of colloidal

system and particle ndashparticle interactions within suspension

Minimum value of zeta potential to prevent aggregation depends on particular sample but

usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery

and pharmaceutical applications higher zeta potential is required For removing too small

particles in water treatment applications low zeta potential is necessary

According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability

depends on attractive Vander Waals and repulsive electrostatic forces If repulsive

electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of

aggregation So nanoparticles with adequate density of surface charge will not aggregate By

screening charges with counter ions can favor vdw forces which are crucial for surface

11

functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle

clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)

Biofunctionalization can cause surface charge neutralization which decreases dispersion

Zeta potential can identify interaction between active substance and carrier It ensures

whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be

protected from enzymatic degradation or released fast after degradation

111 Surface Modification

NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial

system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone

marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES

accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment

in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is

necessary to overcome these problems and it can be done with PEGylation

Fluorescence molecular probes are limited for real time imaging studies due to several

limitations such as short stokes shift poor photochemical stability sensitivity to buffer

composition photo-bleaching and decomposition non-targeted accumulation and faster rate

of photo-bleaching still they are used due to low cost commercial availability and ease of

use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa

dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical

and optical properties Usually organic fluorophores are used as luminescent bio-pointer but

they have some limitations Reliable determination of cancer biopointers needs extremely

sensitive and photo-stable probes with sophisticated fluorescent imaging detection system

Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray

CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of

optical signal by body tissue and fluid in UV and visible range weak tissue penetration

depth light dispersion by tissues weak spatial image resolution

Optimized particle sized of nanoscales materials is necessary because they are easily

consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real

time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based

12

and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than

organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and

quenching in aqueous environment Under intense excitation of light they exhibit irreversible

photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use

fluorescence molecule with porous silicon nanoparticles to overcome those limitations

Covalent and non-covalent bonding processes are two usual chemical approaches for

conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon

nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently

bound PEG chains are more effective because they have longer blood circulation half lives

Covalently bound fluorescent molecule can reduce dye leakage

112 PEGylation

Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol

(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and

pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases

circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular

interactions and bio-barrier transport may prove superior with targeting ligands like

antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)

PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG

chains are constantly available PEG chains can attach to NP surface or other candidate

molecule and reduce some challenges (fig 17)

Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG

chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy


13

PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated

NPs [40] It is helpful in drug delivery and imaging purposes

Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also

reduced Charged based contact of proteins and small molecule are diminished Solubility

increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to

PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver

than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]

1121 Affecting Factors on PEGylation

Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus

group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and

therapy Nanomedioine 2011 6 (4) 715ndash728)

Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)

interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as

polyethylene oxide The behavior of PEG-NP construct is affected with increased t12

1122 Different Sized NP

t12 depends on type of NP to be PEGylated like their size charge and composition For liver

accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are

14

size independent Positively charged NPs with size more than 100 nm can be rapidly cleared

from circulation [64 65] NP composition is also very important for PEGylation

1123 Length amp Conformation of PEG Chain

Grafted PEG layer on NP surface and polymer conformation are related

F= αn35

Here F represents Flory radius Polymer conformation can be described on basis of Flory

radius n represents the number of monomer per polymer chain and length of one monomer in

Angstrom is α [66 67]

Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS

Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)

PEG chains can have two main conformations on basis of surface coverage

Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains

brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging

and therapy Nanomedioine 2011 6 (4) 715ndash728)

15

PEG chains with lower surface density are called mushroom conformation where distance

between attachment points of polymer is larger than F (Figure 110A) In mushroom

conformation polymer chain have roughly sphere with Flory radius Polymer with bigger

graft density DltF) is called brush room configuration In brush configuration long thin

bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units

necessary to transform from mushroom to brush arrangement depends on types of PEG and

NP When distance of PEG molecules on surface close to F then mushroom conformation

coverts to brush conformation NPs containing brush PEG have denser coating and they

shield NPs from RES better Usually brush PEG conformation has longer circulation time

Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys

and maintains high concentration for longer time by increasing recirculation So oligos and

small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius

and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG

(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular

weight but liver uptake increases For example liposome circulation with PEG 5000 is very

long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on

QD [70-72] In mushroom configuration If PEG is larger then less copy number can be

loaded on a NP

1124 Terminal Portion

In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups

diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very

ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively

charged and causes fewer phagocytic events Simple hydroxyl group can diminish non

specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of

secondary targeting ligand [76] but it is challenging due to steric hindrance

16

113 Biofunctionalization

NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body

exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies

aptamers streptavidin or peptides are common address tags They are covalently coupled and

should be in controlled number per nanoparticles

For successful biofunctionalization reactions [77] few requirements are very important

Activity of biomolecules must be removed Signal of nanoparticles should not be slowed

down Linkage sites of NP surface should be covered and stability of biomolecule-

nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as

tiny as nanoparticles

Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption

(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct

chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia

amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences

Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA

Weinheim ISBN 3-527-31381-8

17

There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules

to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules

change and it is difficult to control amount of adsorbed biomolecules per nanoparticle

Physisorption or noncovalent coupling of biomolecules to other biomolecules is another

technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive

groups of biomolecules can also conjugate with cross-linker molecules by physisorption or

chemisorptions (figure 111C) Chemical conjugation is possible for coupling of

oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control

number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done

by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin

or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding

proteins avidin or streptavidin is used as linker molecules

114 General Conjugation Reactions

Nanoparticles are conjugated with covalent coupling One popular coupling process is

reaction between primary amine and carboxylic acid group

Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide

(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester

reacts with primary amine to form a stable amide bond

18

Another approach is reaction between thiol and maleimide groups At certain pH a good

yield of stable thioester bond is formed It directly connects maleimide functional ligands to

thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with

one thiol end group or by reducing disulfide bonds within the target protein

Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is

usually labile in biological fluids

Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation

of carbohydrate forms reactive aldehyde group

Chemical reaction between two primary amines is applied for bioconjugation

Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]

19

In this study one of our goal was to ensure efficient surface modification of the positively

charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)

which has primary amine on the surface through PEGylation so that resulting particles can

minimize the opsonization which involved in mononuclear phagocytic uptake of

nanoparticles for optical data storage and other technical applications in related fields Our

another goal was surface functionalization of negatively charged acid treated thermally

hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group

on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with

Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can

optimize photostability sensitivity and photbleaching and eventually they can act as an

effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was

introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled

stearylated NickFect CPPs to check the CPPs conjugation

20

2 Experimental

21 Materials

Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)

nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and

Astronomy University of Turku Amino-modified thermally carbonized porous silicon

(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied

Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid

poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-

Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide

(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate

(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-

isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell

penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS

PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma

Ethanol (EtOH)

21

22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)

221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and

NHS as an Activator Reagent

To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of

5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625

125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725

23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to

prepare an active succinimidyl ester-terminated intermediate After activation 02 mg

(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction

system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end

mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the

reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation

(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF

and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS

222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and

DIPEA as an Activator Reagent

In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of

PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10

mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064

758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA

(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified

TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles

were washed as described above

223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of

Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH

In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-

COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring

with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-

22

COOH was removed by washing three times with 1mL of DMF and then three times with

1mL of water as described above After each centrifugation an ultrasonic bath was used to re-

suspend the PEGylated-TCPSiNPs in solution

224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different

Size (5768kDa 11kDa and 25kDa)

780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken

into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of

NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs

(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer

overnight at room temperature to complete the reaction Centrifugation was performed to

remove the supernatant and particles were washed three times with 1mL of EtOH and finally

washed three times with 1mL of water as described above After washing 50 microg of sample

was taken from each tube and diluted with water to 3 mL for size and zeta potential

measurement

225 Opsonization

150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution

containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for

15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times

with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted

to 3 mL with water for the size and zeta potential measurement as mentioned earlier

Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel

electrophoresis (SDS-PAGE)

23

23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules

231 Standard Calibration Curve with FITC and FluorA

Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and

standard calibration curve was plotted with fluorescence intensity versus concentration of

FITC and FluorA

232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC

FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into

02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf

tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer

overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed

by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and

three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath

(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-

NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50

mM HEPES (pH 72) and stored at +40C

2321 Absorption of Stearylated NickFect 51 (NF51) Cell

Penetrating Peptides (CPPs) onto the Surface of FITC Labeled

TCPSi-NH2NPs

Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at

13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed

two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend

the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs

were added into sample After adding CPPs the solution was sonicated for 3 hours followed

by centrifugation and discarding of supernatant The particles were washed thrice with

HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a

similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample

24

2322 Conjugation of Radioactive Iodine Labeled Stearylated

NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the

Surface of FITC Labeled TCPSi-NH2NPs

Prior to 125

I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were

centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-

NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was

used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then

13microL (57 mgmL) of 125

I-CPPs (9KBq) were added into sample After adding 125

I-CPPs the

solution was mixed with end-over-end mixture overnight at room temperature Then the

mixture was centrifuged and the supernatant was discarded The particles were washed thrice

with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into

500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube

The radioactivity of 125

I-CPPs conjugated particles was measured with Gammacounter (LKB-

Wallac Clingamma 1272 Turku Finland)

233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1

(FluorA)

The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by

adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-

hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol

was added on NPs and the mixture was protected from light by wrapping with tinfoil By

using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at

37oC in Termaks incubator followed by centrifuging for 20 min and discarding the

supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of

centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with

1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove

unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in

solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM

HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned

earlier

25

24 Characterization Methods

241 Dynamic Light Scattering (DLS)

Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS

by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was

diluted to 3mL with water Before measuring the particles were sonicated for a while

242 Gel Electrophoresis

SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine

samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size

markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples

were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5

glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block

heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer

containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel

with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes

Every lane showed separation of the plasma proteins from the original mixture After running

the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were

washed with water and molecular sizes of the stained proteins were compared to the

molecular weight standards

243 Fluorescence Intensity Measurement

20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)

and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the

particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-

TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was

measured using excitation wave length of 488 nm and emission wave length of 520 with

Fluoroskan Ascent FL (Thermo Lab systems)

26

3 Results and discussion

To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-

COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-

NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic

acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)

31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was

Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH

groups in MeO-PEG-COOH

27

achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group

(reaction scheme shown with DICNHS Figure 31)

Prior to any modification of the surface amino groups are responsible for the positive zeta

potential of the core particle The size of the original nanoparticles was 170 nm and the zeta

potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower

values (mostly negative zeta potential) Although the zeta-potential and the size changed

during the conjugation there was no direct correlation between size zeta potential and the

concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is

suggested to be aggregated In conclusion the changes in size and zeta potential of the

particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs

were successful

Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as

a surface modifier with different concentration of DIC and NHS as an activator

Sample Name Size (nm) Zeta potential (mV)

Original (plain) NPs 170 +70

NP+5mM of DIC and NHS 196 -287

NP+10mM of DIC and NHS 183 +163







The coupling efficiency was further tested using HBTU as a coupling reagent The size and

the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had

insignificant changes (table 32) suggesting insufficient conjugation of PEG

Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and

size of refluxed nanoparticles showed significant change indicating successful conjugation

(Table 32 last row)

28

Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a

surface modifier and different concentration of HBTU and DIPEA as an activator except last row

Sample Size (nm) Zeta potential (mV)

NP+ 10mM of HBTU and DIPEA 188 +52


NP+80mM of HBTU and DIPEA 1701 +494




NP+ MeO-PEG-COOH (reflux) 240 -300

32 Opsonization of the PEGylated TCPSi-NH2 NPs

Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric

hindrance for preventing protein binding Comparing the obtained size and zeta potential

after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein

adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)

Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS

Sample Size(nm) Zeta-potential(mV)

Plain NPs 357 -313









The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive

zeta potential was changed to the negative values after opsonization Nanoparticles

29

conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta

potential after opsonization suggesting the optimal shielding properties

Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=

only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC

and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and

NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS

+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and

NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma

The molecular weight and relative adsorption rates of the human plasma proteins were

analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60

mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2

well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we

cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we

can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly

better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high

concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was

achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From

the aforementioned discussion we can conclude that the concentration of NHS and DIC

influenced the activation and the optimum result was found with 40 and 60 mM of DIC and

NHS From this point of view 40 mM concentration of DIC and NHS were used for the

further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs

during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin

(HSA)

30


Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was

investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with

different size of PEG molecules were prepared several times but surprisingly the consistent

results (size and zeta potential) were not found We got different results every time Two of

test series are shown in the table 34(a) and 34(b)

Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs

Sample Size(nm) Zeta potential (mV)

PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78

Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs


PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201

Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct

correlation was also observed between the molecular weight of the PEG molecule and the

zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-

Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish

of the positive charges introduced and thus causing a negative zeta potential which could be

partially interpreted by the charge-shielding effect of PEG backbones A significant

correlation was observed between the molecular weight of the PEG molecule and the size

values Size was increased with increasing the length of the PEG molecule

31

34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)

FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine

labeling reagent forming a thiourea bond upon reaction The particles are conjugated with

FITC by a covalent attachment of the dye with an appropriate functional group Using FITC

the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the

dye and the primary amino group of the TCPSi-NH2 (Figure 33)

Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine

Mechanism of the reaction

32

The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group

resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen

of primary amine Finally negatively charged nitrogen abstracts a proton from the positively

charged nitrogen and consequently thiourea bond is formed

The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS

solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum

emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-

labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in

basic conditions

In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent

adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed

fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently

conjugated SiNPs was considered free of adsorbed labels because they did not fade during

washing





basic conditions

The determination of a number of FITC conjugated inside the NPs is challenging due to the

potential effect of self-quenching of dye molecules in close proximity Calibration curves

with the pure fluorophore were performed in order to elucidate the number of FITC

33

molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on

FITC concentration was observed with regression coefficient 0995

Figure 34 Fluorescence intensity vs FITC concentration

Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration

Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also

observed (Figure 35) with regression coefficient 09943

y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC

Concentration of FITC(micro-mol)

y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty

Concentration of FITC labeled NPs (microg)

34

The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In

comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-

modified NPs The obtained fluorescence intensity of the particles reveals that the surface

conjugation of FITC to the TCPSi-NH2 NPs were successful

After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES

the fluorescence of the supernatant was measured (data not showed) but surprisingly

fluorescence of the supernatant was higher than the precipitate NPs interacted with

fluorescence material and lowered their intensity and this may be a reason for lower

fluorescence intensity in suspension compared to supernatant as in supernatant there are no

NPs and for that such interaction is not expected there

Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the

FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and

monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant

change in particle size was not shown with a mean diameter around (176 nm) in deionized

water at ambient temperature after fluorescence labeling The zeta potential of the plain

TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with

FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain

NPs have some silanol groups which were remained after FITC labeling and higher negative

charged was observed on account of silanol groups that were dissociated or deprotonated in

de-ionized water or oxidation of silanol groups

341 Characterization of Stearylated Stearylated NickFect 51

(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs

Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by

measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)

of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of

the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were

flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs

(125

I-CPPs) were used for the conjugation onto the surface

35

342 Characterization of Radioactive Iodine Labeled Stearylated

NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC

Labeled TCPSi-NH2 NPs

The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-

counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to

NPs surface

35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA

FluorA is an organic dye containing primary amine group which is used to carboxyl acid

labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-

1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic

acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH

through reaction route in Figure 36

Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester

intermediates

36

As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)

with a primary amine first proceed through an unstable intermediate an O-acylisourea ester

(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this

intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to

the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which

is a much more stable that remains reactive with amines Upon the final addition of an amine

(6) the preferred coupling product an amide (7) is formed by displacement of the NHS

Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine

group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA

to the UnTHCPSiNPs was investigated by measuring fluorescence intensity

The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the

fluorescence signal of the NPs solution with a calibration curve of the pure dye under the

same conditions Though it is not possible to determine the accurate number of dye molecules

because of the significant effect of self-quenching (proved) of dye molecules in close

proximity the determination of the average fluorescence of the NPs is possible Figure 37

and 38 shows a correlation between fluorescence intensity and concentration of the pure dye

and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA

labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and

09901

Figure 37 Fluorescence intensity vs concentration of FluorA

y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty

Concentration of fluoresceinamine (microM)

37

Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs

The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In

comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the

acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals

that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful


fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform

size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs

was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter

around (148 nm) in deionized water at ambient temperature after fluorescence labeling The

zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid

groups Following modification with fluorA the potential changes to -36 mV

y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion

Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was

carried out by acid termination into succinymidyl (activated) ester by using DICNHS action

Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-

COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal

activation The activated PEG polymers were conjugated to the amine groups of the amino

derivatized thermally carbonized porous silicon nanoparticles through the covalent amide

bond Aminated thermally carbonized porous silicon nanoparticles were successfully

functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA

reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which

was also successful The degree of the conjugation was approximated by size change and zeta

potential measurements Potential aggregation was observed in PBS for coated nanoparticles

after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the

PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human

plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High

degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the

nanoparticles The DICNHS activated particles showed lowest opsonization degree

according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar

concentration was not observed after opsonization suggesting that plasma proteins or other

plasma components interrupt the aggregation

Desirable surface modification of the acid derivated thermally hydrocarbonized porous

silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)

through the amide bond formation and the surface of the aminated thermally carbonized

porous silicon nanoparticles were functionalized with FITC through the thiourea bond

formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were

screened lower fluorescence intensity because of the self quenching The stearylated

NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-

NH2NPs which was screened higher fluorescence intensity and to check the CPPs

conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled

stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2

39

NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated

NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can

be used as an effective bioimaging probe

Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a

number of limitations and challenges Size and specifically zeta potential was not consistent

for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs

was surprising The minimization of the opsonization was monitored but total minimization

still to be observed The fluorescence intensity was higher in the supernatant for fluorescent

labeled NPs which can be overcome by performing further study To summarize our

experiment turned out successful in minimization of opsonization and obtaining desired

surface modification of PSiNPs and now next turn is to check out effectiveness these

modified PSiNPs and continue further research to make those achieved results better

40

5 References

1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653

2 Cai W Chen X Small 2007 3 1840

3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17

4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20

5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307

6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W

Science 2003 300(5624) 1434ndash1436

7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482

8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS

Nano 2009 3(1) 197 -206

9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)

H18ndashH40

10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash

1555

11 Canham L T Advanced Materials 1995 7(12) 1033

12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202

(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242

13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)

1149-1155

14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31

15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res

2000 182 505ndash513

16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260

17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333

18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash

284

19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586

20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599

21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38

22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309

23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139

24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York

1994 4

(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999

25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds

Birkhauser Verlag Berlin Germany 1997

41

(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds


26 (a) Ulman A Chem Rev 1996 96 1533

(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305

(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J

Am Chem Soc 2000 122 9071

(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215

(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820

(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320

27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957

(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723

(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784

(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918

28 (a) Ho W J Phys Chem B 2002 117 11033

(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802

(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463

(d) Lee S J amp Kim K Chem Commun 2003 2 212

29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd

Ed Kluwer AcademicPlenum Publishers

New York 1999

30 (a) Gosling J P A Cell 1990 36 1408

(b) Walker N J Science 2002 296 557

31 (a) Kronick M N J Immunol Methods 1986 92 1

(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology

Applications Kluwer Academic Publishers New York 1998

(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in

Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press

New York 1994 4 183-222

(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke

K Van Dyke R Eds CRC Press New York 1990 234-250

32 (a) Lakowicz J R Anal Biochem 2005 337 171

(b) Lakowicz J R Anal Biochem 2001 298 1

(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106

2191

(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160

(e) Gersten J I amp Nitzan A Surf Sci 1985 158165

33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031

(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898

(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958

(d) Kamat P V J Phys Chem B 2002 106 7729

42

(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp

Artemyev M Nano Lett 2002 2 1449

34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A

2003 107 3443

(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236

(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003

107 8829

35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and

Heidelberg Germany 1995

(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082

(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605

36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)

37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C

Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159

38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052

39 Owens D 3rd

amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102

40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414

41 Decker K Eur J Biochem 1990 192(2) 245ndash261

42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427

43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693

44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901

45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985

46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217

47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371

48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)

6288ndash6308

49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1

50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113

51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008

20 2677

52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005

114 15

53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface

Sci 2008 320 132

54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524

55 Addison C J amp Brolo A G Langmuir 2006 22 8696

56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263

57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J

PhysChem B 2006 110 11399

43

58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235

59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12

60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009

23(6) 1007ndash1013

61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20

2294ndash2295

62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403

63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603

64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515

65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56

66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209

67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075

68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281

69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194

70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044

71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359

72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004

15(1) 79ndash86

73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)

5605ndash5620

74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167

75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034

76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410

77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464

78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123

79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031

80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474

81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M

A Larabell C A amp Alivisatos A P Nanotechnology 2003 14

82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P

Avidin J Am Chem Soc 2002 124 6378ndash6382

83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534

84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85

1114ndash1119

85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato

R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971

ii

UNIVERSITY OF EASTERN FINLAND School of Pharmacy

Master Degree Program for Research Chemists

MEZBAH UDDIN

Master Thesis 40 p

Supervisors

Senior Scientist Docent Ale Naumlrvaumlnen and Researcher MSc Jussi Rytkoumlnen

September 2012

KEY WORDS Nanotechnology PEGylation Opsonization Zeta potential Fluorescence intensity

Biofunctionalization

Porous silicon nanoparticles (PSiNPs) have drawn significant attention in recent years in the

study of oncology due to their biocompatibility favorable biodistribution and efficient

clearance or biodegradability The clinical use of PSiNPs as nanocarriers in drug delivery

depends on biologically nonspecific adsorption and immunological response Minimization

of the opsonization in the blood increases the half life of the particles and the capability to

target tumors In this project the opsonization of PEGylated porous silicon nanoparticles was

investigated as it is expected that PEGylation of TCPSi-NH2NPs with Me-PEG-COOH can

contribute to minimization of the opsonization PEGylated PSiNPs were also prepared using

single step reaction between terminal amines in thermally carbonized porous silicon

nanoparticles (TCPSi-NH2NPs) and carboxylic acid groups in mPEG-COOH by refluxing

PEGylated particles were characterized by measuring their size and zeta potential and the

opsonized particles were analyzed by SDS-PAGE The surface of TCPSi-NH2NPs was also

conjugated with fluorescent molecule such as fluorescein-5-isothiocyanate (FITC) and by

tailoring of the acid treated thermally hydrocarbonized porous silicon (UnTHCPSi) NPs by

associating with fluoresceinamine isomer-1 (FluorA) activated with DICNHS activator

FITC labeling TCPSi-NH2NPs were further modified by conjugating stearylated NickFect 51

(NF51) cell penetrating peptides (CPPs) and radioactive iodine labeled stearylated NickFect

cell penetrating peptides Fluorescent labeled NPs were characterized with zeta sizer and

fluorescence spectroscopy The surface modified PSiNPs can be loaded with targeted

therapeutic compounds and studied their biodistribution both in vivo and in vitro

iii

ACKNOWLEDGEMENT






Mezbah Uddin

iv


1 Introduction 1

11 Nanotechnology 1









NH2 NPs) 4







19 Opsonization 8



112 PEGylation 12







2 Experimental 20

21 Materials 20



Reagent 21

v






11kDa and 25kDa) 22

225 Opsonization 22


Molecules 23
















30








4 Conclusion 38

5 References 40

vi














EtOHminusEthanol

FminusFlory radius





























nanoparticles



ZSminus Zetasizer

1


























2



m2cm














growth










3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



iii

ACKNOWLEDGEMENT






Mezbah Uddin

iv


1 Introduction 1

11 Nanotechnology 1









NH2 NPs) 4







19 Opsonization 8



112 PEGylation 12







2 Experimental 20

21 Materials 20



Reagent 21

v






11kDa and 25kDa) 22

225 Opsonization 22


Molecules 23
















30








4 Conclusion 38

5 References 40

vi














EtOHminusEthanol

FminusFlory radius





























nanoparticles



ZSminus Zetasizer

1


























2



m2cm














growth










3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



iv


1 Introduction 1

11 Nanotechnology 1









NH2 NPs) 4







19 Opsonization 8



112 PEGylation 12







2 Experimental 20

21 Materials 20



Reagent 21

v






11kDa and 25kDa) 22

225 Opsonization 22


Molecules 23
















30








4 Conclusion 38

5 References 40

vi














EtOHminusEthanol

FminusFlory radius





























nanoparticles



ZSminus Zetasizer

1


























2



m2cm














growth










3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



v






11kDa and 25kDa) 22

225 Opsonization 22


Molecules 23
















30








4 Conclusion 38

5 References 40

vi














EtOHminusEthanol

FminusFlory radius





























nanoparticles



ZSminus Zetasizer

1


























2



m2cm














growth










3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



vi














EtOHminusEthanol

FminusFlory radius





























nanoparticles



ZSminus Zetasizer

1


























2



m2cm














growth










3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



1


























2



m2cm














growth










3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



2



m2cm














growth










3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



3

















devices [15-17]







4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



4








cell




Table 11


NPs

Sample Surface Area

(m2g)

Pore Volume

(cm3g)


Distributions

(nm)

TCPSi-

NH2

970372 0508017 926 170


5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



5






to primary amine












6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



6





















7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



7






















8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



8







into the cells

19 Opsonization






9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



9




728)




(long t12) [41]












10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



10




















11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



11






























12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



12











112 PEGylation













13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



13


















14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



14





F= αn35










15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



15


















loaded on a NP








16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



16

















17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



17



















18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



18











19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



19














20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



20

2 Experimental

21 Materials














Ethanol (EtOH)

21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



21





























22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



22














measurement

225 Opsonization








23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



23





FITC and FluorA













TCPSi-NH2NPs









24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



24




Prior to 125







I-CPPs the









(FluorA)













earlier

25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



25


























26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



26









27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



27











were successful


















(Table 32 last row)

28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



28


















Plain NPs 357 -313











29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



29























(HSA)

30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



30









PEG 5768kDa 210 -163

PEG 11kDa 243 -134

PEG 25kDa 245 -78



PEG 5768kDa 1662 -343

PEG 11kDa 1761 -140

PEG 25kDa 187 -201









31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



31









32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



32









basic conditions





washing





basic conditions




33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



33







y = 34477x Rsup2 = 09959

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty o

f FI

TC


y = 15057x Rsup2 = 09943

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

Flu

ore

sce

nce

inte

nsi

ty


34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



34




























(125


35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



35






NPs surface








intermediates

36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



36



















09901


y = 34959x Rsup2 = 09933

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

Flu

ore

sce

nce

inte

nsi

ty


37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



37













y = 0305x Rsup2 = 09901

0

05

1

15

2

25

3

35

0 2 4 6 8 10 12

Flo

ure

sce

ne

inte

nsi

ty

microg of NPs well

38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



38

4 Conclusion






























39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



39













40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



40

5 References







Science 2003 300(5624) 1434ndash1436



Nano 2009 3(1) 197 -206


H18ndashH40


1555





1149-1155



2000 182 505ndash513




284







1994 4




41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



41



26 (a) Ulman A Chem Rev 1996 96 1533



Am Chem Soc 2000 122 9071








28 (a) Ho W J Phys Chem B 2002 117 11033






New York 1999








New York 1994 4 183-222






2191







42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



42




2003 107 3443



107 8829









39 Owens D 3rd











6288ndash6308




20 2677


114 15


Sci 2008 320 132





PhysChem B 2006 110 11399

43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



43




23(6) 1007ndash1013


2294ndash2295












15(1) 79ndash86


5605ndash5620














1114ndash1119



SURFACE MODIFICATION OF POSITIVELY AND NEGATIVELY...

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