1.1. Nanotherapy Nanoparticles

5/25/2018 1.1. Nanotherapy Nanoparticles

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1.-NANOTHERAPY: TARGETED DRUG DELIVERY

Despite the significant progress in the development of anticancer technology, there

is still no common cure for patients with malignant diseases. In addition, the long-

standing problem of chemotherapy is the lack of tumor-specific treatments.

Traditional chemotherapy relies on the premise that rapidly proliferating cancer

cells are more likely to be killed by a cytotoxic agent. In reality, however, cytotoxic

agents have very little or no specificity, which leads to systemic toxicity, causing

undesirable severe side effects such as hair loss, damages to liver, kidney, and

bone marrow.

Nowadays, drug-delivery systems with nanoparticles show a clear potential for

cancer treatments in view of advantages such as, i) the ability of targeting specific

locations in the body, ii) the ability of reducing the quantity of drug that needs to

be delivered to attain a particular concentration level in the vicinity of the target,

and iii) the ability of decreasing the concentration of the drug at non-target sites.1

As a consequence, controlled drug delivery is one of the fastest-growing segments

of the pharmaceutical market, and in the United States alone the demand is

expected to grow nearly 9 % annually to reach more than US$ 82 billion by 2007.2

1.1.-Preparation of nanoparticles for drug delivery.

The idea of directing a therapeutic agent to the proximity of a damaged tissue was

postulated by the Nobel Prize winner P. Ehrlich already in 1906 with the concept ofmagic bullets which were compounds that would have a specific attraction to

disease-causing microorganisms. He envisioned that these magic bullets would

seek out these organisms and destroy them, avoiding other organisms and having

no side effects on healthy tissue.3

In general, there are two possibilities of localizing a drug in the proximity of a

target (i.e., tumor): passive and active. The former implies using characteristic

properties of the tumor to locate in its proximity an encapsulated, bonded or

adsorbed drug on nanoparticles. That is by using the characteristic enhanced

permeation and retention of the vasculature of the tumor (EPR-effect) is possible to

concentrate drug-loaded nanoparticles within the tumor tissue. This is because

1J. Ritter, A.Ebner; K. Daniel, K. Stewart. Application of high gradient magnetic separation principles tomagnetic drug targeting.J. Magn. Magn. Mater. 2004, 280, 184.

2S. K. Sahoo, V. Labhasetwar. Nanotech approaches to delivery and imaging drug. Drug Discov. Today.

2003,8,1112.3T. M. Fahmy, P. M. Fong, A. Goyal and W. M. Saltzman, Targeted for drug delivery, Mat. Today. 2005,8, 18.


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tumors develop a leaky vasculature as well as a poor lymphatic drainage.4 The

majority of solid tumors exhibit a vascular pore cut-off size between 380 and 780

nm.5 The healthy capillary vessels are permeable showing a pore size cut-off

depending on the location and kind of blood capillary. The tight-junction capillary

(blood-brain barrier), including the central nervous system has exhibited a vascularpore cut-off size < 0.1 nm; continuous capillaries, including most tissues, such as

muscle, lung, and skin (cut-off < 6 nm); fenestrated capillaries, including kidney,

intestine, and some endocrine and exocrine glands (cut-off < 50-60 nm); and

sinusoid capillaries, including liver, spleen, and bone marrow (cut-off 100-1000

nm).6 Therefore it is possible to synthesize drug-loaded nanoparticles with a

tailored size and with a shell that delays or avoids the action of the reticulo-

endothelial system (RES) expecting to be naturally located within a specific organ.

Other examples of passive targeting imply using specific affinity or physicochemical

properties of the tumor tissues. For instance, Kukowska-Latallo et al.7used the high

affinity folate receptor for the vitamin folic acid as a target for the delivery of

folate-conjugated drugs to cancer tissue. Another example of passive targeting with

nanoparticles uses pH-dependent drug release due to the tumor has a pH slightly

lower than healthy tissues. Most solid tumors have pH values of less than 7.2 8

contrary to the normal blood pH of 7.4 0.05.9 When these pH sensitive

nanocarriers (i.e., polymers, liposomes) encounter acidic environments such as

tumor tissues, they break apart and release the molecules they contain.10,11

Another physical property of tumor tissue is its higher temperature (hyperthermia)

compared to healthy tissues12 and it is considered another utilization of passive

4 M. J. Vicent and R. Duncan, Polymer conjugates: nanosized medicines for treating cancer, TrendsBiotech., 2006, 24, 39.5S. K. Hobbs, W.L. Monsky, F. Yuan, W.G. Roberts, L. Griffith and V.P. Torchilin, R. K. Jain, Regulationof transport pathways in tumor vessels: role of tumor type and microenvironment, Proc. Natl. Acad. Sci.1998, 95, 4607.6Y. Okuhata, Delivery of diagnostic agents for magnetic resonance imaging, Adv. Drug Delivery Rev.,1999, 37, 121.7J. F. Kukowska-Latallo, K. A. Candido, Z. Y. Cao ZY, S. S. Nigavekar, I. J. Majoros, T. P. Thomas, L. P.Balogh, M. K. Khan, J. R. Baker, Nanoparticle targeting of anticancer drug improves therapeuticresponse in animal model of human epithelial cancer, Cancer Res., 2005, 65, 5317.8 O. M. Koo, I. Rubinstein and H. Onyuksel, Role of nanotechnology in targeted drug delivery andimaging: a concise review, Nanomed.: Nanotech. Biology Med.2005, 1, 193.9 G. Gaucher, M. H. Dufresne, V. P. Sant, N. Kang, D. Maysinger and J. C. Leroux, Block copolymermicelles: preparation, characterization and application in drug delivery,J. Controll. Release.2005, 109,169.10T. Ishida, M. J. Kirchmeier, E. H. Moase, S. Zalipsky and T. M. Allen, Targeted delivery and triggeredrelease of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells, Biochim.biophys. acta-biomem.2001, 1515, 144.11D. E. L. de Menezes, L. M. Pilarski, A. R. Belch and T. M. Allen, Selective targeting of immunoliposomal

doxorubicin against human multiple myeloma in vitro and ex vivo, Biochim. biophys. acta-biomem.2000, 1466, 205.12V. P. Torchilin, Drug targeting, Europ. J. Pharmac. Sci.,2000, 11, S81.


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targeting by using temperature-sensitive release nanoparticles.13The PathFinder

technology is based on the identification of naturally occurring mechanisms for the

localization of material (i.e., proteins) indifferent parts of the body and uses these

principles for the controlled production of site-specific nanoparticulate carriers.14

On the other hand, active targeting is based on inducing external properties to the

nanoparticles to target them to specific tumor tissues and involves the use of light,

magnetism, and the specific recognition mechanisms (i.e., interaction antigen-

antibody, peptide-based targeting, DNA or RNA-based ligands, etc.) Magnetic drug

delivery is not a new therapeutic tool; in 1978 Senyei et al.15 used an in vitro

analog of the human circulatory system to test the magnetic retention of magnetic

microspheres which consisted of adriamycin hydrochloride and ultrafine magnetite

encapsulated in an albumin matrix. From then to now, several scientific papers

have been published describing different drug-loaded magnetic nanoparticles

encapsulated in polymeric or inorganic matrices.16,17,18 Currently, magnetic

nanoparticles are commercially used to treat tumors by hyperthermia and

thermoablation (Magforce Nanotechnologies AG, Germany) and by using the

magnetic properties of the core to activate an encapsulated prodrug (Nanoboiotix

, France, Alnis BioScience, USA). The TNTSystem (Triton Biosystems) consist of

polymer-coated iron oxide nanoparticles tagged with an antibody, and an external

magnetic field is used to kill diseased cells. Pre-clinical animal models test not show

any side effects in healthy tissue.

On the other hand, active targeting using the specific interaction antigen-antibody

is widely studied19,20 and currently exist commercially a drug conjugated with an

antibody (Mylotarg) used to treat a form of bone marrow cancer (CD33 positive

acute myeloid leukemia). Several papers have been published using dendrimers,

dendritic polymers and inorganic matrices with a loaded drug and with an antibody

functionalized surface.

13A. Chilkoti, M. R. Dreher, D. E. Meyer and D. Raucher, Targeted drug delivery by thermally responsivepolymers,Adv. Drug Delivery Rev.2002, 54, 613.14R. DAquino, T. Harper and C. Roman-Vas, Nanobiotechnology. Fulfilling the promise ofnanomedicine, Chem. Eng. Prog.2006, 102, 35.15R. DAquino, T. Harper and C. Roman-Vas, Nanobiotechnology. Fulfilling the promise ofnanomedicine, Chem. Eng. Prog.2006, 102, 35.16[16] X. H. Gao, Y. Y. Cui, R. M. Levenson, L. W. K. Chung and S. M. Nie, Magnetic nanoparticle designfor medical diagnosis and therapy,J. Mater. Chem.2004, 14, 2161.17A. K. Gupta and M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles forbiomedical applications, Biomater.2005, 26, 3995.18Z. P. Xu, Q. H. Zeng, G. Q. Lu and A. B. Yu, Inorganic nanoparticles as carriers for efficient cellulardelivery, Chem. Eng. Sci.2006, 61, 1027.19

E. R. Gillies and J. M. J. Frechet, Dendrimers and dendritic polymers in drug delivery, Drug Discov.Today,2005, 10, 35.20X. Wu and I. Ojima, Tumor specific novel taxoid-monoclonal antibody conjugates, Current Medicnl.Chem.2004, 11, 429.


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We can separate the drug-loaded nanoparticles in two groups according to their

structure, core-shell and matrix-like nanoparticles. Nanoparticles are defined

as solid, submicron-sized drug carriers that may or may not be biodegradable. The

term nanoparticle is a collective name for both nanospheres and nanocapsules.

Nanospheres have a matrix type of structure. Drugs may be absorbed at the spheresurface or encapsulated within the particle. Nanocapsules are vesicular systems in

which the drug is confined to a cavity consisting of an inner liquid core surrounded

by a inorganic or polymeric shell. In this case the active substances are usually

dissolved in the inner core but may also be adsorbed to the capsule surface.

Nanoparticles are receiving considerable attention for the delivery of therapeutic

drugs.

1.1.1.-Core-shell based nanoparticles

Core-shell nanoparticles have recently attracted a huge scientific effort due to the

possibility of combining different properties in individual particles, based on

different compositions of the core and the shell. In addition, many interesting

technological applications can be foreseen for this kind of materials, including

analytical chemistry (chromatography), separation technology (ion exchange),

catalysis, biochemistry and medicine, etc.21These types of particles can be defined

by their different core and shell composition. The core often shows a useful physicalproperty, e.g. semiconductors, metals, magnetic oxides, encapsulated molecules,

while the shell can be useful to stabilize the core and make compatible the core and

the environment. It is also possible to change the charge, functionality or reactivity

of the surface. This is especially important for medical purposes, e.g. for drug

delivery applications.

We will focus in core-shell nanoparticles composed of a magnetic core

encapsulated in an organic (i.e., polymeric) or inorganic shell. Those nanoparticles

are based on the use of a magnet to locate them in the proximity of a tumor (activetargeting). At this location the nanoparticles can release the conjugated drug which

is associated to the magnetic core or an alternating magnetic field is used to induce

heat in the magnetic nanoparticles and produce ablation of the tumoral tissue

(hyperthermia). In all these applications the coating of the particles is a very

important issue. It helps to make the particles biocompatible, preventing

aggregation and the degradation of the metallic core, and reducing the extent of

21G. Kickelbick, L.M. Liz-Marzn, Core-shell nanoparticles, in Encyclopedia of Nanoscience andNanotechnology, H.S. Nalwa (Ed.), American Scientific Publishers, 2004, Vol. 2, pp. 199-220.


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clearance by the reticuloendothelial system. Moreover, the outer coating surface of

the particles can be functionalized to allow the binding of drugs or biomolecules to

the system to cause a therapeutical action or to get local targeting. The magnetic

core is composed of 3d metals (iron, cobalt, iron/cobalt alloys, iron/platinum alloys,

iron/nickel alloys, and their oxides). The shell is composed of polymeric materials(i.e., dextrane, albumin, starch, etc.) or inorganic materials (ie., silica, graphite and

gold). Inorganic, amorphous silica is biocompatible, non-toxic, and posses hydroxyl

surface groups (4.6 OH/nm2, though this value strongly depends on the synthesis

temperature) which provide intrinsic hydrophilicity and allow surface attachment by

covalent linkages of specific drugs or biomolecules. Also, amorphous silica is a

heat-resisting material, with a low specific gravity, high surface area and good

mechanical strength. In addition, the isoelectric point of magnetite ferrofluids is

reached at pH close to 7. Aqueous ferrofluids therefore flocculate in the pH range of5 to 9 and are stable only under highly acidic or basic conditions. On the other

hand, the isolectric point of silica is reached at pH 2-3 and therefore silica

nanoparticles are negatively charged at the pH of the blood. As consequence,

dispersions of silica particles in biological media are stable and prevent magnetite

agglomeration. However, the magnetic nanoparticles used for MRI are based on a

magnetic core encapsulated in an organic shell (generally dextrane), for example

superparamagnetic or ferromagnetic iron oxide nanoparticles are widely used as

negative contrast agents in MRI for oral and parental administration (i.e., Feridex,Endorem or Resovist, GastroMARK or Lumirem, Sinerem, etc). Currently,

magnetic nanoparticles are commercially used to treat tumors by hyperthermia and

thermoablation (Magforce Nanotechnologies AG, Germany) and by using the

magnetic properties of the core to activate an encapsulated prodrug (Nanoboiotix

, France, Alnis BioScience, USA). The TNTSystem (Triton Biosystems) consists

of polymer-coated iron oxide nanoparticles tagged with an antibody, and an

external magnetic field is used to kill diseased cells. Pre-clinical animal models test

not shown any side effects in healthy tissue when using silica or carbonencapsulated iron and iron oxide nanoparticles.

-Silica coated nanoparticles

A versatile method for the preparation of core-shell nanomaterials is the

growth of the shell material on pre-existing cores by chemical methods. This has

been used often for the deposition of silica shells on various nanoparticles, including

metallic,22 semiconductor,23 or magnetic24,25 materials. A schematic view of the

22L.M. Liz-Marzn, P. Mulvaney, The assembly of coated nanocrystals,J. Phys. Chem. B2003, 107,7312-7326.


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synthetic steps followed for silica encapsulation of magnetic nanoparticles in

solution is shown in the figure below, comprising coprecipitation of Fe(II) and

Fe(III) hydroxides, slow deposition of sodium silicate for thin silica shells, and shell

growth in ethanol.

For example, it has been recently demonstrated26that semiconductor quantum dots

can be assembled on the surface of silica-coated magnetic nanoparticles, so that

single particles containing magnetic and luminescent functionalities can be obtained

through relatively simple chemical processes.

23M.A. Correa-Duarte, M. Giersig, L.M. Liz-Marzn, Stabilization of CdS Semiconductor NanoparticlesAgainst Photodegradation by a Silica Coating Procedure, Chem. Phys. Lett.1998,286, 497-501.24M.A. Correa-Duarte, M. Giersig, N.A. Kotov, L.M. Liz-Marzn, Control of Packing Order of Self-Assembled Monolayers of Magnetite Nanoparticles with and without SiO2 Coating by MicrowaveIrradiation, Langmuir 1998, 14, 6430-6435.25Y. H. Deng, C. C. Wang, J. H. Hu, W. L. Yang and S. K. Fu, Investigation of formation of silica-coated

magnetite nanoparticles via solgel approach, Colloids Surf. A: Physicochem. Eng. Aspects, 262 (2005)(1-3), pp. 87-93.26V. Salgueirio-Maceira, M.A. Correa-Duarte, M. Spasova, L.M. Liz-Marzn, M. Farle, Composite SilicaSpheres with Magnetic and Luminescent Functionalities,Adv. Funct. Mater.2006, 16, 509-514.

EFTEMGreen SiO2

Red Fe

EFTEM (708 eV)Fe L3Edge

EFTEM (22 eV)SiO2Plasmon

HRTEM images and EFTEM colormap showing the Iron (red)

atomic distribution (in this case

forming the Fe3O4nanoparticles),

and the silicon (green)

distribution, as part of the shells.

The silica coating was provided by

the sol-gel method. (M. Arruebo,

R. Fernndez-Pacheco et al.

publishing)


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The assembly of nanoparticles on the surface of silica particles can be

achieved trough a technique based on the alternate deposition of oppositely

charged species,27,28 using polyelectrolytes as molecular cement, and can be

implemented for the incorporation of drugs or other biomolecules within the

nanoparticles. Additionally, the nature of the polyelectrolytes can be tailored, so

that they are sensitive to external stimuli, such as pH or temperature, to trigger

drug encapsulation and release. Combination of these techniques can be useful to

fabricate multifunctional nanoparticles including a magnetic core (for external

manipulation), a luminescent, intermediate shell (for labelling) and an external

polyelectrolyte shell (for drug encapsulation). The various steps can be

schematically represented as shown in the figure below.

A B CA B C

-Carbon coated iron nanoparticles

Carbon coated iron and iron oxide nanoparticles are suitable for biomedicalapplications.29 The inert carbon encapsulation, provide a way to make

biocompatible, functionalize and also gives the possibility to adsorb and desorb

therapeutical agent as doxorrubicine.30 These nanoparticles are obtained by two

procedures: by the discharge arc method designed by Krtschmer-Huffman in

27F. Caruso, R. A. Caruso, H. Mhwald, Nanoengineering of inorganic and hybrid hollow spheres bycolloidal templating, Science1998, 282, 1111-1114.28V. Salgueirio-Maceira, F. Caruso, L.M. Liz-Marzn, Coated Colloids with Tailored Optical Properties,

J. Phys. Chem. B2003, 107, 10990-10994.29Kuznetsov, A. et al , J. Mag. Mag. Mat. 194, 22 (1999).30De Teresa J.M., Marquina C., Algarabel P.A., Morelln L,.Fernandez-Pacheco R. Ibarra M.R.Interantional Journal of Nanotechnology (2005)

Schematic view of the synthesis of multifunctional nanoparticles. First, magnetic

cores are encapsulated in a silica shell, onto which luminescent quantum dots are

assembled. Subsequently, a second silica shell is deposited and

polyelectrolyte/biomolecule multilayers are deposited.


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1990,31 or by high energy ball mill grinding.32 The Krtschmer method uses a

cylindrical chamber, in which there are two graphite electrodes: a stationary anode

containing 10 microns starting iron powders, and a moveable graphite cathode. An

arc is produced between the graphite electrodes in a helium atmosphere. The

graphite electrode is sublimed and builds up a deposit on the inner surface of thechamber. In the material collected from this deposit we found: carbon

nanostructures, amorphous carbon and iron and iron oxide nanoparticles

encapsulated in graphitic layers. High energy grinding is performed in a ball mill. A

suspension of iron micrometric powders and graphite powders in ethanol is

grounded for several hours to obtain a viscous solution that is dried up to obtain

the final product as fine iron carbon powders.

-Gold coated iron nanoparticles

In many cases gold could be the ideal coating material because of its well-

known optical properties,33 easy chemical functionalization with thiolated organic

molecules34 and high stability of the gold-coated nanoparticles in solutions of

physiological pH. Recently, gold-coated nanoparticles of about 60 nm in size with

31Kratschmer W. et al., Nature 347, 354 (1990)32Tapolsky, G. et al, Eur. Cells and Materials Vol. 3 Suppl. 2, 12 (2002).33T.A. Taton et al. Science 289, 1757 (2000)34M. Brongesma, Nat. Mater. 2, 296 (2003)

HRTEM images and EFTEM

color map showing the iron

(green) atomic distribution (in

this case forming the Fe3O4

nanoparticles), and the

carbon (red) distribution, as

part of the shells. (R.

Fernandez- Pacheco et al.)


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an iron oxide core of 9 nm have been synthesized by a wet-chemical method in two

steps.35However, metallic iron is expected to be a better core magnetic material

than iron oxides for drug delivery and biosensor applications because it is

magnetically softer and its saturation magnetization is about a factor two larger

than that of iron oxides.

Unfortunately, successful preparation of Fe/Au core-shell nanostructures has

been a lasting scientific challenge. Several works have reported the synthesis of

non-well structural characterized and/or non-long term stabilized nanoparticles,

until a novel method combining wet chemistry for synthesis of the Fe cores and

laser irradiation of Fe nanoparticles and Au powder in liquid medium has been very

recently proposed36.

In this CONSOLIDER project we aim to study the synthesis of gold-coated

metallic and oxidized iron nanoparticles and their feasibility for drug-delivery

applications. Au/(-Fe2O3, Fe3O4) core-shell nanoparticles will be prepared by

reduction of Au3+onto the Fe oxide surfaces of previously synthesized nanoparticles

(co-precipitation and further oxidation) using a modification of iterative

hydroxylamine seeding procedure37. We intend to get a good control of the Au shell

thickness, since this layer is responsible of the resulting optical properties, total size

and colloidal stability of the core-shell nanoparticles.

Gold-coated metallic iron nanoparticles will be synthesized following a twosteps method. First, Fe cores will be obtained by thermal decomposition of iron

pentacarbonyl in the presence of oleic acid38which acts as a surfactant, and further

particle precipitation by addition of ethanol. In the second step, we will explore two

techniques to achieve gold coating of the iron cores.

In the first method, Fe and Au nanoparticles will be dispersed together in a

water-based solution, which will be irradiated with a pulsed laser beam in order to

promote the selective fusion of the gold nanoparticles and ulterior condensation

onto the Fe cores, as described in reference36.

The second method is based on sonochemistry, which is an alternative

technique that can be used for the production of coated particles39. Power

ultrasound effects provoke chemical changes due to cavitation phenomena

involving the formation, growth and implosive collapse of bubbles in liquids.

35J.L. Lyon et al., Nano Letters 4, 719 (2004)36

J. Zhang et al., J. Phys. Chem. B 110, 7122 (2006)37K. J. Brown et al., J. Chem. Mater. 12, 306 (2000)38D. Farrell et al., J. Phys. Chem. B 107, 11022 (2003)39V.G. Pol, A. Gedanken and J. Caldern-Moreno, Chem. Mater. 15, 1111 (2003)


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Sonication of a solution containing a gold precursor in the presence of

(metallic/oxidized) iron cores provides an alternative means of trapping gold

nanometer seeds, which produce active supported heterogeneous catalysis giving

rise to the formation of core-shell nanoparticles.

A careful structural characterization of the obtained core-shell

nanostructures will be carried out to determine the quality and continuity of the

resulting gold coatings which ensure the long-term stability of the iron core.

Work Plan 1.1.1

-Optimization of core-shell nanoparticles with high magnetic response

Until now, silica and carbon coated magnetic nanomaterials have been mostlyrestricted to iron oxides, since these present a better chemical compatibility with

the silica shell. The magnetic response of such core-shell nanoparticles can be

improved by using other magnetic materials, such as metals (Co, Ni) or alloys

(CoPt3, FePt) in the case of silica. The formulation of such novel core-shell materials

is expected within the duration of the project.

-Development of novel multifunctional core-shell nanoparticles

The process outlined above for the incorporation of multiple functionalities in a

single particle can be extended to other materials. For instance, magnetic cores can

be combined with metallic shells, which can be continuous or assemblies of smaller

nanoparticles. In either case, the morphology can be tailored for high absorption

coefficients in the visible or NIR.

-Drug encapsulation in multifunctional nanoparticles

For the described multifunctional nanoparticles, drug encapsulation is envisaged via

wrapping with polyelectrolyte multilayers. As mentioned above, such multilayers

can be designed so that they are sensitive toward pH or temperature, and this canbe used to trigger encapsulation and release.

1.1.2.-Matrix-like nanoparticles

-Nanoparticles based on polymeric matrices

Natural occurring polymers and synthetic polymeric (dendrimers, dendritic

polymers and micelles) are used to deliver an adsorbed or encapsulated drug. Their

advantages are their biodegradability in many cases and their well-known

chemistry. These advantages have made that different research groups and


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pharmaceutical companies have invested all their efforts in developing drug-

conjugated nanoparticles for drug delivery. Hence, interdisciplinary research at the

interface of polymer chemistry and the biomedical sciences has produced the first

polymer-based nanomedicines for the diagnosis and treatment of cancer. These

water-soluble hybrid constructs, designed for intravenous administration, fall intotwo main categories: polymerprotein conjugates or polymerdrug conjugates.

Polymer conjugation to proteins reduces immunogenicity, prolongs plasma half-life

and enhances protein stability. Polymerdrug conjugation promotes tumor targeting

through the enhanced permeability and retention (EPR) effect and, at the cellular

level following endocytic capture, allows lysosomotropic drug delivery. The

successful clinical application of polymerprotein conjugates (PEGylated enzymes

and cytokines) and promising results arising from clinical trials with polymer-bound

chemotherapy (i.e., doxorubicin, paclitaxel, camptothecins) has provided a firmfoundation for more sophisticated second-generation constructs that deliver the

newly emerging target-directed anticancer agents (e.g. modulators of the cell cycle,

signal transduction inhibitors and antiangiogenic drugs) in addition to polymerdrug

combinations (i.e., endocrine- and chemo-therapy). First-generation technologies

include antibodydrug conjugates, for example Mylotarg

(http://www.pharmacist.com/pdf/mylotarg.pdf), and also several polymer

conjugates carrying either low-molecular-weight drugs or proteins.

Besides these drug-polymer conjugates, biodegradable polymers such as polyesters

and polyanhydrides and their copolymers with hydrophilic segments (e.g. PEO,

PPO) have been efficiently used for the nanoencapsulation and delivery of drugs.

These polymers have the specific advantage of being degraded by a hydrolitic

mechanism which leads to the erosion of the nanomatrix and subsequent delivery

of the associated drug. Moreover, these polymers have a long safety record as

shown by the fact that they are major components of several marketed

formulations (Lupon depot, Decapeptil, Gliadel..among others). Recently, the

attention has been directed to the use of copolymers of poly(lactic acid/glycolicacid) with PEG. This is due to the ability of these copolymers to organize forming

PEG-coated PLGA nanoparticles. This PEG coating is critical for drug targeting

purposes since it provides the nanoparticle with long-circulating properties in the

blood stream as well as targeting capabilities (PEG can be linked to peptide or

antibody molecules).


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-Polipeptide matrix nanoparticles

Polypeptide nanoparticles will be exploited to entrap drugs and obtain a controlled

release. Moreover, the entrapped drug will be protected from degradation.

Two kind of polypeptide nanoparticles will be explored:polypeptide dendrimers andSAP-basednanoparticles. In the first group, the covalent assembly of the multiple

peptide chains will provide a matrix nanoparticle, with the ability to entrap the

drug, either covalently or non-covalently. In the case of SAP-based nanoparticles,

the assembly of different copies will be achieved due to the non-covalent self-

assembly properties of sweet-arrow peptide (SAP).

i ) P o ly p e p t i d e d e n d r i m e r s

Dendrimers are nanoparticles that are creating great interest. Dendrimers

are synthetic, highly branched, monodispersed macromolecules. As their molecular

size increases, they adopt a spherical shape that has a vacant inner core that can

encapsulate drug molecules, and a highly functionalized surface that can be

derivatized with a ligand. Biocompatible dendrimers are obvious candidates for drug

delivery applications.

A particularly interesting class of biocompatible dendrimers is obtained when

peptides are the base of the dendrimeric structrure. Polypeptide dendrimers havebeen previously described, 39and the pioneering work of the group of J. Tam has

demonstrated the application of lysine dendrimers as immunogens.40In the present

project we plan to focus our attention onpolyproline dendrimersa particular class

of polypeptide dendrimers. Proline is singular among the 20 genetically coded

amino acids in that it contains both a cyclic backbone as well as a secondary, as

opposed to primary, -amino group. These structural features impart unique

stereochemical properties to proline. Polyproline oligomers exist in two distinct

conformations. In organic solvents, they adopt a conformation known as polyprolineI, a right-handed helix in which all peptide bonds are cis-oriented ( = 0 ).41 In

aqueous solvents, they adopt the conformation known as polyproline II, a left-

handed helix in which all peptide bonds are trans-oriented ( = 180 ).42 The

transition from polyproline I to polyproline II implies a considerable increase in the

long dimension of the helix that changes from 1.9 to 3.1 per residue.

39 L. Crespo, G. Sanclimens, M. Pons, E. Giralt, M. Royo, F. Albericio. Chem. Rev., 2005, 105, 1663-

1681.40Nardelli, B.; Lu, Y. A.; Shiu, D. R.; Profy, A. T.; Tam, J. P. Immunology1992, 148, 914-920.41Traub, W.; Shmueli, U. Nature1963, 198, 1165-1166.42Cowan, P. M.; McGavin, S. Nature1955, 176, 501-503


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This unique conformational plasticity of polyproline chains allows to modulate the

dendrimer properties by changing the length of the branches in response to the

environment. In this way, drugs could be trapped in organic solvents, where

polyproline chains adopt a polyproline I conformation, and released under

physiological conditions in which polyproline spacers form more extended

polyproline II helices. A more classical alternative to this non-covalent drug

entrapment could be attachment by covalent bond formation with the building

blocks of the dendrimer.

One of the characteristics of dendrimers is the facility to modulate its properties by

modification of the dendrimer surface.43 This surface modification would be used

either for covalent attachment of the drug or, even more interesting, to

functionalize the dendrimer surface with vector peptide sequences able to target

the nanovector to a given tissue, and especially to a tumoural tissue (see point

1.2.). The biocompatibility of peptide structures and the possibility of cellular

43Lee, J. W.; Ko, Y. H.; Park, S. H.; Yamaguchi, K.; Kim, K. Angew. Chem., Int. Ed.2001, 40, 746-749

polyproline I polyproline III


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internalization of polyproline structures44,45 are additional potential benefits of this

approach.

i i ) S A P - b a s ed n a n o p a r t i c l e s

Sweet Arrow Peptide (SAP), VRLPPPVRLPPVRLPP, is an amphipathic Pro-rich

cell-penetrating peptide, i.e., it has the special ability to cross the cell membrane.

Because of its amhiphathic character, SAP was expected to self-assemble. In

aqueous media SAP adopts polyproline II structure, with hydrophobic residues

pointing to one face of the helix and hydrophilic ones facing to the opposite (see

figure). The self-assembling properties of SAP were proven by circular dichroism

(CD) and transmission electron microscopy (TEM). TEM micrographs of the replicas

showed fibrils of 16 (3) nm width and variable length.

In the present project, nanoparticulate molecular materials of SAP will be obtained

by top down methods. In order to covalently attach the drug, a percentage of the

arginines of the SAP sequence, (VRLPPP)3, will be replaced by lysine or serine in

order to bind carboxylic containing-drugs to the SAP-based nanoparticles through

amide or ester bonds, respectively.

44L. Crespo, G. Sanclimens, B. Montaner, R. Prez-Toms, M. Royo, M. Pons and F. Albericio, E. Giralt.Peptide Dendrimers Based on Polyproline Helices.J. Am. Chem. Soc., 2002, 124, 8876-8883.


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Apart from preventing the drug degradation in the biological media and to

offer a controlled release, SAP-based nanoparticles will have the unique ability to

internalize the entrapped drug inside the cell.46,47,48

Workplan 1.1.2

a) Synthesis of polyproline dendrimers with reactive functional groups at the

surface.

b) Covalent attachment of antitumoral drugs to polyproline dendrimers. We will

focus on doxorubicin (as a proof of concept) and on antiangiogenic drugs.

c) Non-covalent entrapment of antitumoral drugs in polyproline dendrimers. We will

exploit the conformacional plasticity of the polyproline branches in response to

changes in solvents polarity. The effect of the size of the drug will be also studied.

Again, we will focus on antiangiogenic drugs.

d)Synthesis of SAP-based nanoparticles. Monomeric SAP will be prepared using well

established solid-phase peptide synthesis methodologies. SAP-based nanoparticles

will be obtained by top down methods (use of critical superfluids) from the

nanofibrilar structures formed in highly concentrated SAP aqueous solutions.

e)Synthesis of functionalized SAP-based nanoparticles. Lysine- and serine-

containing SAP monomers will be prepared by solid-phase synthesis. Attachment ofantiangiogenic drugs will be done in solution by reaction with the amino- and the

hydroxy- groups of lysine and serine side-chains, respectively. Then, drug-loaded

nanoparticles will be prepared as described above.

In an alternative procedure that will also be explored, the drug will be attached in

solution to preformed lysine- or serine-containing SAP-based nanoparticles.

45Farrera-Sinfreu, Josep; Giralt, Ernest; Castel, Susanna; Albericio, Fernando; Royo, Miriam. Cell-Penetrating cis-g-Amino-L-Proline-Derived Peptides.J. Am. Chem. Soc., 2005, 127, 9459-9468.46S. Pujals, J. Fernndez-Carneado, C. Lpez-Iglesias, M. J. Kogan and E. Giralt, BBA-Biomembranes,

2006, in press.47[9 J. Fernandez-Carneado, M. J. Kogan, S. Pujals, and E. Giralt, Biopolymers 2004, 76 196-203.4810 J. Fernandez-Carneado, M. J. Kogan, S. Castel, and E. Giralt,Angewandte Chemie, Int. Ed. 2 0 0 4 ,43,1811-1814.

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