P EGylated nanoparticles for biological and pharmaceutical applications Hidenori ... ·...

Advanced Drug Delivery Reviews 55 (2003) 403–419www.elsevier.com/ locate/addr

P EGylated nanoparticles for biological and pharmaceuticalapplications

a ,1 b c ,*Hidenori Otsuka , Yukio Nagasaki , Kazunori KataokaaBiomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan

bDepartment of Materials Science, Tokyo University of Science, Noda, Chiba 278-8510, JapancDepartment of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, Hongo 7-3-1,

Tokyo 113-8656, Japan

Received 22 April 2002; accepted 16 September 2002

Abstract

The utility of polymeric micelles formed through the multimolecular assembly of block copolymer was comprehensivelydescribed as novel core–shell typed colloidal carriers for drug and gene targeting. Particularly, novel approaches for theformation of functionalized poly(ethylene glycol) (PEG) layers as hydrophilic outer shell were focused to attain receptor-mediated drug and gene delivery through PEG-conjugated ligands with a minimal non-specific interaction with otherproteins. Surface organization of block copolymer micelles with cross-linking core was also described from a standpoint ofthe preparation of a new functional surface-coating with a unique macromolecular architecture. The micelle-attached surfaceand the thin hydrogel layer made by layered micelles exhibited nonfouling properties and worked as the reservoir forhydrophobic reagents. Furthermore, the potential utility of multimolecular assembly derived from heterobifunctional PEGsand block copolymers were explored to systematically modify the properties of metal and semiconductor nanostructures bycontrolling their structure and their surface properties, making them extremely attractive for use in biological and biomedicalapplications. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Poly(ethylene glycol); Block copolymers; Biological and biomedical applications

Contents

1 . Introduction ............................................................................................................................................................................ 4042 . Polymeric micelles for drug delivery ........................................................................................................................................ 4053 . Surface modification with polymeric micelles for the design of functional biointerface................................................................. 407

3 .1. Adsorption of protein on micelle-coated surface ................................................................................................................. 4073 .2. Release of pyrene from micelle-coated surfaces.................................................................................................................. 408

4 . Metal and semiconductor nanoparticles as biological labels ........................................................................................................ 409

*Corresponding author. Tel.:1 81-3-5841-7138; fax:181-3-5841-7139.E-mail address: [email protected](K. Kataoka).1Present address: Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.

0169-409X/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.doi:10.1016/S0169-409X(02)00226-0

mailto:[email protected]

404 H. Otsuka et al. / Advanced Drug Delivery Reviews 55 (2003) 403–419

4 .1. Metal nanoparticles .......................................................................................................................................................... 4094 .1.1. Metal nanoparticles synthesized in amphiphilic block coplymer micelle ..................................................................... 4094 .1.2. Gold nanoparicles synthesized by heterobifunctional PEG......................................................................................... 410

4 .1.2.1. Dispersion stability in physiological condition ............................................................................................. 4114 .1.2.2. Quantitative and reversible lectin-induced association of gold nanoparticles................................................... 412

4 .1.3. Sensitivity enhancement for surface plasmon resonance (SPR) .................................................................................. 4134 .2. Luminescent quantum dots for biological detection............................................................................................................. 414

5 . Conclusion.............................................................................................................................................................................. 415Acknowledgements...................................................................................................................................................................... 415References .................................................................................................................................................................................. 415

1 . Introduction [17–24]. Furthermore, surface organization of reac-tive micelles with cross-linking cores was described,

Block copolymers with amphiphilic character, allowing the surface to have extremely high non-having a large solubility difference between hydro- fouling character and working as a reservoir forphilic and hydrophobic segments, have a tendency hydrophobic agents, as summarized in Fig. 1. Coreto self-assemble into micelles in a selective solvent segregation from aqueous milieu is the direct driv-[1–4]. In an aqueous solution, micelles with core– ing force for micellization and proceeds through ashell structure are formed through the segregation of combination of intermolecular forces, including hy-insoluble hydrophobic blocks into the core, which is drophobic interaction [12,15,25–30], electrostaticsurrounded by a shell composed of hydrophilic interaction [31–34], metal complexation [35,36],blocks. This self-assembling property of amphiphilic and hydrogen bonding [37] of constituent blockblock copolymers provides their high utility in the copopymers. A variety of drugs including genes andbiomedical field as drug carriers, surface modifiers, proteins, metals, and semiconductors with diverseand colloidal dispersants [5–8]. This review de- characteristics can be incorporated into the core-scribes the recent progress in the field of block forming segment of the block copolymer so that onecopolymer assembly in the solution and on the can expect a sufficiently strong interaction withsurface, focusing on the biological and biomedical core-incoorporated molecules. In order to prepareapplication of poly(ethylene glycol) (PEG) based the drug delivery system for site specificity, theblock copolymers. PEG chains as a hydrophilic outer shell of the polymeric micelle was buit inpolymers with a flexible nature can be selected as such a way that it was covered with fuctionalshell-forming segments, which assemble into dense groups, which react readily with potential pilotpalisades of tethered chains to achieve unique prop- molecules or target-specific antibodies. Theseeries; The biocompatibility was guaranteed by the strategy to construct functionalized PEG layers wasdense PEG shell, which endows the micelle with a further applied to metal and semiconductorstealth character in the blood compartment, achiev- nanoparticles, which have recently attracted muching a long circulation [9]. PEG chains attached to a interest in biological assay system due to theirsurface or forming the corona of a nanosphere unique photochemical and photophysical propertiesexhibit rapid chain motion in an aqueous medium [38–42]. These photonic properties depend on theand have a large excluded volume. The steric particle size and composition, which can be variedrepulsion resulting from a loss of conformational with the method of preparation, including the use ofentropy of the bound PEG chains upon the approach Langmuir-Blodgett films [43,44], reverse micellesof a foreign substance and the low interfacial free [45,46], vesicles [47], and various polymer net-energy of PEG in water contribute to the extra- works [48–50], yet the resulting nanoparticles wereordinary physiological properties of nanospheres not effective in preventing non-specific aggregationcovered with PEG [10–16]. PEG grafted to surfaces in aqueous medium unless their surface was modi-of biomedical devices also proved to increase their fied with hydrophilic coatings including PEGylation.biocompatibility and to reduce thrombogenicity Accordingly, the surface organization of PEG on

H. Otsuka et al. / Advanced Drug Delivery Reviews 55 (2003) 403–419 405

Fig. 1. Schematic representation of the application for the multimolecular assembly using heterobifunctional PEG and their blockcopolymer.

these nanoparticles may open the new opportunities raised in the application of block copolymer mi-to their use in the biological fields. celles as a novel carrier systems for anticancer

agents because of the high drug-loading capacity ofthe inner core as well as of the unique disposition

2 . Polymeric micelles for drug delivery characteristics in the body [9–12]. Compared tosurfactant micelles, polymeric micelles are generally

Drug targeting for efficient accumulation in the more stable, with a remarkably lowered criricalbody is often hampered by the rapid recognition of micelle concentration (CMC), and have a slowercarrier system by the reticuloendothelial system rate of dissociation, allowing retention of loaded(RES) and by the subsequent kidney and/or hepatic drugs for a longer period of time and, eventually,elimination. Moreover, for modulated drug delivery achieving higher accumulation of a drug at theto solid tumors, which locate outside the blood target site [9]. Polymeric micelles have a size ofcompartment, the carrier is required to exhibit not 30–50 nm in diameter, ranging closely to that ofjust a sufficient half-life in the blood compartment, viruses, and apparently, this size range is favorablebut also the capability of extravasation at the tumor for extravasation to achieve so-called enhancedsite. Recent developments led to the design of drug permeation retention effect (EPR effect) [51].carriers with prolonged circulation in the vascular Special focus is focused here to polymeric mi-system [9]. Cancer chemotherapy may cause severe celles formed from heterobifunctional block copoly-side effects, leaving patients under extreme distress. mers. A challenge in the development of novelTo overcome this problem, an interest has been micellar carrier systems is to design targetable


polymeric micelles in which pilot molecules are employed to prepare the polymeric micelle, i.e. afterinstalled on their surface to achieve a specific-bind- the block copolymer was dissolved in a good sol-ing property to target cells. Of particular importance vent for both segment, such as dimethylacetamidein this regard is the establishment of a novel and (DMAc), the solution was dialyzed against watereffective synthetic route for end-functionalized am- [57,58]. The size and shape of the polymeric mi-phiphilic block copolymers with appreciable celles were estimated by dynamic light scatteringbiocompatibility and biodegradability, allowing (DLS/SLS), revealing the micelle to be| 30 nmconjugation of the pilot molecules at the tethered with a very low polydispersity. The conversion ofend of the hydrophilic segment. the acetal end group into the aldehyde group was

As reported previously, we have developed a conducted after the micelle formation by adjustingfacile and quantitative synthetic method for the pH of the medium to pH 2 with an addition of a

1heterobifunctional PEG, which denotes PEG having small quantity of hydrochloric acid. From the H-different functional groups at thea- and v-ends NMR analysis, the conversion of acetal to aldehyde[52–56]. This polymerization procedure is further was estimated as up to 90%.applicable to the preparation of an end-function- The availability of the aldehyde groups positionedalized block copolymer by extending a sec-end at the micelle’s periphery for further modificationpolymer segment from thev-end of the thus-pre- was then confirmed through the reaction of thepared hetero-PEG, keeping the other functional aldehyde groups with ligand molecules such asgroup at the PEG chain end (a-end) to be available tyrosyl-glutamic acid (Tyr-Glu) as a model peptidylfor further reaction. An introduction of an aldehyde ligand as well as saccharide moieties includinggroup at the PEG end was achieved by the use of glucose, galactose, and lactose [58,59].potassium 3,3-diethoxypropanolate (PDP) as an The concept of polymeric micelle stabilizationinitiator for the polymerization of ethylene oxide through the formation of a hydrophilic palisade(EO). The acetal group at thea-end of resultant surrounding a water-incompatible core can be ex-PEG can be easily converted into an aldehyde group tended to include the case of macromolecular as-by acid treatment. In this way,a-acetal-poly- sociation through electrostatic interaction. It was(ethylene glycol)-block-poly(D,L-lactide) (a-acetal- shown that a pair of block copolymers with anPEG-PLA) was synthesized by a one-pot anionic oppositely charged polyelectrolyte segment, poly-ring-opening polymerization of EO followed byD,L- (ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL)lactide (LA) initiated with PDP as an initiator at and poly(ethylene glycol)-block-poly(a,b-asparticroom temperature under argon (Scheme 1) [21– acid) (PEG-b-P(Asp)), spontaneously associates to24,57]. Because the PEG-PLA block copolymer has form micelles with a core composed of polyionan amphiphilic character, it forms polymeric mi- complex of poly(L-lysine) and poly(a,b-asparticcelles in an aqueous milieu. A dialysis method was acid) segments. The polyion complex (PIC) mi-

celles opened the way to incorporate chargedmacromolecules of synthetic and biological originsincluding proteins and nucleic acids into the mi-celles and have been developed as non-viral DNAdelivery systems [32,60–62]. The strategy to coatthe polyion complex core of a polycation and DNAwith a hydrophilic segment of the cationic blockcopolymer was applied to prepare a variety of nano-associates. These includes PEG-b-PLL [31,61,62],poly(ethylene glycol)-block-poly(ethyleneimine)(PEG-b-PEI) [63], and poly(ethylene glycol)-block-poly(dimethylaminoethyl methacrylate) (PEG-b-Scheme 1. Synthetic procedure of end-functionalized block co-PAMA) [64], which were polymerized from corre-polymer [a-acetal-poly(ethylene glycol)-poly(D,L-lactide) block

copolymer]. sponding monomers using PEG with the terminal


functional group as a macroinitiator. Synthesis of fouling property. Further, by incorporating hydro-these cationic block copolymers as well as physico- phobic drugs into the core of surface-immobilizedchemical and biological properties of their micelles micelles, one can create surfaces releasing drugs inwith DNA are reviewed in detail elsewhere [5,65]. a controlled manner.

3 .1. Adsorption of protein on micelle-coated3 . Surface modification with polymeric micelles surfacefor the design of functional biointerface

The aminated glass slides and polypropyreneProviding polymeric micelles are able to be im- (PP) films were coated with monolayer and multi-

mobilized on the surface with maintaining their layers of micelles mediated by polyallylaminecore–shell architecture, the aforementioned advan- (PAlAm). The substrate was immersed in the mi-tages of micelles in the solution, particularly non- celle solution in HEPES (pH 6.7) containingfouling property, should be also expected on the NaCNBH for 2 h. After the light rinsing with3

surface. However, physical coagulation forces may Milli-Q water, the micelle-coated substrate wasnot be stable enough to maintain the micelle struc- immersed into 0.6% (w/v) PAlAm in HEPES (pHture in the process of surface fixation. A disruption 6.7) containing NaCNBH for 2 h. The above3

of the micelle upon attachment to the surface is procedure was repeated until the desired number ofreported both experimentally and theoretically [66– coatings was obtained.68]. Johner and Joanny [68] used scaling arguments Surface covered with core-polymerized reactiveto show the disruption of the micelles during their micelle was evaluated by the protein adsorptionadsorption process. The disruption results in the measurements. The quantitative analysis of adsorbedformation of loops and trains on the surface, leading proteins on the surface was carried out by the microto the formation of a loosely packed layer structure. BCA method that determines small amount of pro-

1This discrepancy may be solved by the preparation tein in aqueous solution by the absorption of Cuof reactive and structurally stabilized micelles. chelated with bicinchoninic acid (BCA) [23,75].Heterobifunctional block copolymers of PEG-PLA Fig. 2 shows the equilibrated amount of BSA on thethat possess reactive group at the PEG-end and PP films after the exposure to 45 mg/ml of BSApolymerizable group at the PLA-end were success- solution for 60 min. The micelle coating exhibitedfully prepared. The polymerization of PLA-end afterthe micellization resulted in the formation of core-polymerized micelles with high stability in harshenvironment [69].

Then, the core-stabilized reactive micelles werecovalently linked to the surface, forming a singlelayer of micelle as well as multilayered highly-organized micelle hydrogel [70–72]. Although thestar polymers of PEG with micelle-like structurehave been utilized for the surface modification toresist protein adsorption and to apply for the scaf-fold in tissue engineering by Merrill et al. andGriffith et al. [73,74], the polymeric micelle isexpected to present more extended application forits easy preparation and unique structure. SincePEG density in the shell of the micelle is appreciab-

Fig. 2. Adsorption of BSA to aminated polypropylene surfacesly high, the surfaces treated with PEG-PLA micelles modified with grafted PEG-aldehyde and reactive micelles. Theare eventually to have a dense layer of tethered numbers denote the numbers of layers of micelle and poly-PEG chain, which may provide an effective non- allylamine (PAlAm).


significantly reduced amount of BSA compared with Kwon et al. [76] The pyrene-incorporated micellesbare and plasma-treated PP plates (the surfaces were coated on an aminated glass slide in theaminated by plasma treatment necessarily for mi- aforementioned manner. The sample was then ex-celle coating through the reaction between the posed to an excess amount of Mili-Q water. Byamine and aldehyde groups on the micelle). The measuring the fluorescence atl 5 336 nm, theex

non-fouling property against BSA was more signifi- release of pyrene from the micelle-coated surfacecant than the surface grafted with PEG-aldehyde was monitored. It is interesting to note that theunder the same condition as the micelle-coating. It initial amount of pyrene as well as the rate ofshould be noted that the protein adsorption reduced release were dependent on the number of micellewith increasing the number of micelle coating. coatings; as the number of coatings increased, the

initial fluorescence was higher, and release-rate was3 .2. Release of pyrene from micelle-coated slower. Repetitive loading and release of pyrenesurfaces were also successfully accomplished with the mi-

celle-gel layer as shown in Fig. 3. After the hexa-The micellar gel has another unique property that poid coatings of pyrene-free micelle, the samples

is not typical of conventional hydrogels. The gel were exposed to the micelle solution containingconsists of micelles that have hydrophobic cores of pyrene for 12 h to transfer pyrene from solution to|10 nm in size. The micelles on a surface are the gel phase. The loading and release of pyreneexpected to hold drugs in the same manner as the can be repeated reproducibly. The third cycle of theones in the solution, and to release them in a pyrene release carried out at 48C showed slowercontrolled manner. As a model drug, pyrene was decline of fluorescence presumably due to theincorporated into the micelle by mixing pyrene with change in the mobility of the PLA segment withthe micellar solution, following the procedure by temperature, suggesting a variation in the release

Fig. 3. Loading and releasing of pyrene from the aminated glass coated with hexapoid layer of micelle.


rate obtained by the use of block copolymers with a 4 .1. Metal nanoparticleshydrophobic segment that is different from PLA.These results indicate that the loading capacity and The use of gold colloid in biological applicationsthe release-rate of a drug can be controlled by the began in 1971, when Faulf and Taylor invented thenumber of coatings. immunogold staining procedure. Since that time, the

labeling of targeting molecules, especially proteins,with gold nanoparticles has revolutionized the vis-ualization of cellular or tissue components by elec-

4 . Metal and semiconductor nanoparticles as tron microscopy [99]. The optical and electronbiological labels beam contrast qualities of gold colloid have pro-

vided excellent detection qualities for imaging tech-Metal and semiconductor nanoparticles are of nologies in electron microscopy. Although metal

considerable current interest because of their unique and inorganic nanoparticles can be prepared fromsize-dependent properties [77–80]. By controlling various materials by several methods [100–104], thethe structure precisely at nanoscale dimensions, one coupling and functionalization with biological com-can control and tailor properties of nanoparticles. In ponents has only been carried out with a limitedaddition, one can modify the nanostructures to number of chemical methods.better suit for biological systems; for example, To apply gold colloids in newly developed bio-modifying their surface layer for enhanced aqueous medical assay systems, a simple and facile means ofsolubility, biocompatibility, and biorecognition [81]. anchoring different ligand biomolecules onto par-These ability to systematically modify the properties ticle surfaces are strongly required as well as theof nanoparticles by controlling their structure and stability in the physiological condition should betheir surface properties at a nanoscale level makes improved. Particularly, color changes induced bythem extremely attractive candidates for use in the association of nanometer-sized gold particlesbiotechnological systems, from fundamental scien- provide a basis of a simple yet highly selectivetific studies to commercially viable technologies method for detecting specific biological reactions[82]. The use of exquisite recognition properties of between acnchored ligand molecules and receptorbiomolecules in organizing non-biological inorganic molecules in the milieu. Mirkin and co-workersobjects into functional materials becomes an im- have shown that gold colloidal particles modifiedportant frontier in materials science. Recent research with oligonucleotides form large assemblies throughby several groups has linked colloidal nanoparticles the hybridization with complementary oligonucleo-to biomolecules such as sugars [83–86], peptides tide strands, providing a new method for colorimet-[87], proteins [88–90], and DNA [91–93]. These ric detection of targeted DNA sequences [91–nanoparticle bioconjugates are being used for as- 94,96,98]. With decreasing gold colloidal particlesembling new materials [94,95], for developing size, however, colloidal stability decreases signifi-homo- and heterogeneous bioassays [96–98], par- cantly due to increased particle surface energy.ticularly, multicolor fluorescent labels for ultrasensi- Such gold nanoparticles aggregate in high ionictive and high-throughput detection and imaging strength milieu as well as adsorb biomolecules such[88–92]. Many advantages are expected by replac- as proteins and DNA nonspecifically, resulting ining conventional molecular tags, such as fluorescent reduced sensitivity and selectivity when used aschromophores, with nanoparticles. These include colloidal sensor systems in biological fluids.higher quantum efficiencies, greater scattering orabsorbance cross sections, optical activity over more 4 .1.1. Metal nanoparticles synthesized inbiocompatible wavelengths, and significantly in- amphiphilic block coplymer micellecreased chemical or photochemical stability. The Several methods have attempted to synthesize andavailability of these new nanoparticles will greatly stabilize nano-scale gold particles in aqueousfacilitate in situ probes and sensor methods. milieu. Most of them have utilized surfactant and/


or polymer stabilizers, yet were not effective in block copolymer is adopted, where both blocks arepreventing aggregation of nanoparticles particularly soluble in water, but only one block is able tounder physiological conditions (concentrated salt coordinate with metal ions. The example of such anmedium) [105]. application was reported for the interaction of PEG-

Block copolymer micelles could successfully be block-PEI with AuCl , PdCl , H PtCl , Na PtCl ,3 2 2 6 2 6

used as nanoreactors for noble metal colloid forma- K PtCl , and Na PdCl [108]. Addition of the gold2 6 2 4

tion; in such micelles, chemical and physical re- salt to a PEG-b-PEI solution resulted in the forma-actions can be confined to the fluid micellar cores, tion of polydisperse micelles, and PEG-b-PEI in-the size of which are confirmed as a nanometer duced reduction of the gold salt to form goldscale. For the amphiphilic block copolymer in the nanoparticles. Neither PEG nor PEI itself showednon-polar selective solvent, the unpolar blocks form this behavior of auto-reduction. Analytical ultracen-the corona, which provides solubilization and trifugation confirmed that 75–80% of the gold wasstabilization, while the polar or hydrophilic and formed inside the micelle, suggesting particle for-functionalized blocks form the core, which is able mation arround the PEI chains [108,109]. Micelleto dissolve metal compounds due to coordination, formation upon salt addition to a PEG-b-PEI solu-followed by the nucleation and growth of metal tion was also observed for PdCl and K PtCl with2 2 4

particles upon reduction. The important step in- polydisperse, large, and unstable properties, but novolves the solubilization of inorganic compounds self-reduction occurred in these cases. It was furtherinto the micellar core. As a guideline for optimum found for PEG-b-PEI that branched copolymersprecursor materials and micellar core blocks, one with muliple PEG blocks attached to PEI are bettercan use Pearsons hard/soft acid /base (HSAB) con- stabilizers for metal nanoparticles as compared tocept [106], which has been generalized to include the diblock system. Light scattering and transmis-metals and semiconductors [107]. The general sion electron microscopy (TEM) revealed the exist-strategy is to start from weakly coordinated metals, ence of large micellar aggregates for the diblocke.g. Pd(OAc) or Pd(ClO ) which are complexes system. If stable micelles were formed with the2 4 2

of a soft acid (the transition metal ions) and a hard metal salts (H PtCl , Na PtCl , Na PdCl ), effi-2 6 2 6 2 4

base (acetates, perchlorates, etc.). The formation of cient control of the nanoparticle growth and stabili-a more stable complex of a soft acid with a softer zation was possible although the equilibration of thebase, e.g. polyvinylpyridine, to assemble the micel- micelle architecture could be a slow process takinglar core, is the driving force for solubilizatikon. The up to weeks [110]. Besides the identified parameter,polymer complex should not be too stable since such as polymer/metal ratio and type of reducingover-stabilization could prevent the formation of the agent on the metal nanoparticle size and shape,desired colloid in the subsequent chemical reaction. complex ion geometry and also the pH of theChemical reactions typically involve reduction pro- solution were found to be of importance.cess to obtain noble metal colloids or the prepara-tion of sulfides or oxides. Using polystyrene-poly- 4 .1.2. Gold nanoparicles synthesized by4vinylpyridine (PS-b-P4VP) as the constituting am- heterobifunctional PEGphiphilic block copolymer, Sidorov et al. prepared Recently, Wuelfing et al. reported that surfacemono- and bimetallic colloids with size controlled PEGylation of gold particles by CH O–PEG–SH3

by varying such parameters as species of metal salt, significantly improved their dispersion stability intype of reducing agent, and block copolymer com- aqueous milieu due to steric repulsion effects ofposition. However, the most study using am- tethered PEG strands [111]. Yet, the CH O–3

phiphilic block copolymers were successfully em- PEGylated nanoparticles possess no reactive groupsployed only in nonpolar organic solvents, because to further immobilize ligand molecules. Thus, aappropriate polymers to show both the ability to system possessing both sufficient colloidal stabilityform micelles in water and to bind metals in the and biofunctionality is strongly desired [112,113] tocore are not available. Use of water as reaction construct quantitative bioassays using goldmedium becomes possible when double-hydrophilic nanoparticles. Recently, we have reported a facile


route to functionalize PEGylated gold nanoparticles, properties. As a model ligand, lactose was success-having a potential utility for colloidal sensor sys- fully introduced in the distal end of PEG chain totems [114], by the use of newly-designed induce lectin-mediated quantitative and reversibleheterobifunctional PEG derivatives. association of gold nanoparticles under physiologi-

Our strategy to synthesize various types of cally relevant conditions producing a concomitantheterobifunctional PEGs is based on the ring-open- color change (red→ purple→ red) as described ining polymerization of ethylene oxide using a metal Scheme 3.alkoxide initiator with a protected functional group[52–56]. Synthesis of heterobifunctional PEG con- 4 .1.2.1. Dispersion stability in physiological con-taining both mercapto and acetal terminal groups dition(a-acetal-v-mercapto-PEG, acetal-PEG-SH) was Gold nanoparticles were prepared by reduction ofpreviously reported as shown in Scheme 2a. The metal salt (HAuCl ) with NaBH in the presence of4 4

acetal moiety can readily be transformed into a acetal-PEG-SH. The formation of gold nanoparti-reactive aldehyde group by simple treatment with cles, an average diameter of 8.9 nm as measured bydilute acid. An aldehyde-functionalized PEGylation TEM, was confirmed by the UV–visible spectrumof gold nanoparticles using this acetal-PEG-SH of an absorption band nearl5520 nm, assigned to(M 5 3090) was successfully achieved, obtaining a gold nanoparticle plasmon band.n

gold nanoparticles with high dispersion stability, Acetal-PEG-SH coated gold nanoparticles wereparticularly in physiological milieu, and appreciable appreciably stable in buffers of elevated salt con-aldehyde reactivity to immobilize ligand molecules centrations (d, m) and also in serum-containingon the PEG coronas which additionally impart medium (s), while gold nanoparticles physicallysensitivity and selectivity due to their non-fouling stabilized through the adsorption of acetal-PEG-OH

Scheme 2. Synthetic procedure ofa-acetal-v-mercapto-poly(ethylene glycol) (a) and cationic block copolymer [poly(ethylene glycol)-block-poly(dimethylaminoethyl methacrylate)] (b).


Scheme 3. Schematic representation of the reversible aggregation-dispersion behavior of Lac-PEGylated gold nanoparticles by sequentialaddition of RCA lectin and galactose with actual concomitant change in color from pinkish red→ purple→ pinkish red.120

(M 5 3000) (j, .) aggregated immediately evenn

in 0.03 M PBS (Fig. 4). Highly enhanced stabiliza-tion as well as reduced nonspecific interactions withbiological components including cells and proteinsof gold nanoparticles bearing acetal-PEG-SH allowsthis system to be developed for diagnostics in bloodserum-containing samples.

4 .1.2.2. Quantitative and reversible lectin-inducedassociation of gold nanoparticles

The PEG acetal terminal group was converted toaldehyde by gentle acid treatment, followed by thereaction with sugar derivatives havingp-amino-phenyl moieties at the C-1 position (p-amino-phenyl-b-D-lactopyranoside: Lac) in the presence of(CH ) NHBH . Reaction of these Lac-derivatized3 2 3

gold nanoparticles with bivalent galactose-bindinglectin (recinus communis agglutinin, RCA )120

[115,116] was followed as a function of timeFig. 4. Dispersion stability of gold nanoparticles with time inthrough optical changes in the surface plasmon bandvarious environments. Acetal-PEG-SH protected gold nanoparti-in the UV–Vis spectrum. These solutions werecles in 0.15 M PBS (d), 0.3 M PBS (m), and 0.15 M PBS with

initially a pinkish-red color due to the well-dis- 2%-serum (s); acetal-PEG–OH adsorbed gold nanoparticles inpersed nature of the particles (Scheme 3). After 0.03 M PBS (j), 0.15 M PBS (.), 0.03 M PBS with 2%-serumintroduction of the RCA lectin in 0.15 M PBS, (h), and 0.15 M PBS with 2%-serum (,).120


the color gradually changes from red to purple ing the system to be utilized to quantitate lectin(Scheme 3). In line with this directly observable concentration with nearly the same sensitivity aschange in appearance, significant differences in the enzyme-linked immunosorbent assay (ELISA) ((1optical spectra over time were observed, specifically mg/ml (1 ppm)). This simple yet highly effectivea broadening and red shift in the particle surface derivatization of gold nanoparticles withplasmon resonance from 523 nm to longer wave- heterobifunctional PEG provides a convenient meth-length [117] (Fig. 5). This is attributed to distance- od to construct practical colloidal sensor systemsdependent changes in the optical properties of three- currently applied in bioassays and biorecognition.dimensionally aggregated gold nanoparticles cross- As an another interesting method to prepare goldlinked by RCA lectin which recognizes lactose nanoparticles with narrow size distribution, we have120

residues on the PEGylated gold surface. The lectin- developed to use the PEG based cationic blockparticle system were well-ordered and three-dimen- copolymer, PEG-b-PAMA as a stabilyzer for goldsionally linked, as deduced from TEM and SAXS nanoparticle preparation. Recent advances in ouranalysis. Images of two-dimensional, single layer study showed that the metal–ligand coordinationaggregates revealed close-packed assemblies of the bond is able to be utilized as a driving force tocolloids with uniform particle separations of about form the core of the block copolymer micelles20 nm, which corresponds to the length of the [35,36]. PEG-b-P(Asp) and cisplatin (cis-dich-combination of lectin and PEG linker. The aggrega- lorodiamineplatinum (II):CDDP) form micelles withtion by the RCA lectin was reversible, recovering the core of the polymer–metal complex through120

the original dispersed phase and color by addition ligand exchange from chloride to carboxyl group ofof excess galactose. Notably, the degree of aggrega- the poly(aspartic acid) segment. The similar concepttion was proportional to lectin concentration, allow- of these micelle stabilization can be adopted to

prepare and stabilize the gold nanoparticles par-ticularly in physiological condition. The copoly-merization of PEG-b-PAMA was achieved by thesequential anionic polymerization of ethyleneoxideand dimethylaminoethyl methacrylate (AMA). PDPwas also employed as an initiator to introduce anacetal group at the end of the PEG segment of theblock copolymer (Scheme 2b). Addition of the goldsalt to a PEO-b-PAMA solution induced reductionof the gold salt to form extremely monodispersegold nanoparticles without reductive agents. Furtherfunctionalization with biotin molecules at the PEGchain-end demonstrated extended aggregation ofbiotinylated gold nanoparticles through biotin–avidin interaction.

4 .1.3. Sensitivity enhancement for surface plasmonresonance (SPR)

Gold nanoparticles can also be applied to enhancedetection limits in SPR-based real-time biospecificinteraction analysis [118,119]. The dynamic en-hancement of SPR biosensing with colloidal gold

Fig. 5. Change in the surface plasmon band intensity (DA(5 A 20 was initially observed in a sandwich immunoassayA) /A ) with RCA concentration ([RCA ]) for nanoparticle0 120 120 in which gold nanoparticles were coupled to aAu-Lac(0.5) (m, d, .) and Au-Lac(0.2) (j) at different times

secondary antibody. This system was used to detectafter onset of the aggregation reaction.A is the surface plasmon0

band intensity in the absence of RCA . a primary antibody bound through specific immuno-120


sorption to the antigen immobilized on the gold real-time monitoring or tracking of intracellularsensor surface. Our recent study demonstrated that prosesses over long periods of time. Another advan-an appreciable enhancement in sensitivity was ob- tage is the ability to use multicolor nanoparticles totained to detect the avidin immobilized on the SPR simultaneously image multiple targets inside livingsensor surface through biotin-avidin interaction cells or on the cell surface.using a-biotynylated-PEG functionalized gold A typical QDs in these applications, CdS, havenanoparticles. The immunosorptive binding of the been synthesized via arrested precipitation fromcolloidal gold to the sensor surface led to a large simple inorganic ions using polyphosphate and lowshift in plasmon angle, a broadened plasmon reso- molecular weight thiols as stabilizers [122,123],nance, and an increase in minimum reflectance, from dimethylcadmium in trioctyl phosphine usingthereby allowing picomolar detection of antigen trioctyl phosphine oxide as a stbilizer, and from[118]. Similarly, an improvement in sensitivity by cadmium 2-ethylhexanoate in dimethyl sulfoxideabout 1000-fold was obtained in nucleic acid hy- (DMSO) using ethylhexanoate as a stabilizer. How-bridization analysis, when a colloidal gold /oligo- ever, these QDs are practically synthesized at highnucleotide conjugate was used as a probe [119]. temperature (| 3008C) and the following bioconju-These results suggest that the detection limit of SPR gation was applied using several method; use of a[120] now begins to approach that of traditional bifunctional ligand such as mercaptoacetic acid forfluorescence-based DNA hybridization detection linking QDs to biomolecules [89], trioctylphosphinesystem. oxide (TOPO)-capped QDs bound to a modified

acrylic acid polymer by hydrophobic forces, and4 .2. Luminescent quantum dots for biological QD solubilization and bioconjugation using a mer-detection captosilane compound [88]. All of these require

high temperture and many processes for both syn-A unique property of semiconductor nanoparti- thesis and bioconjugation, further resulting QDs

cles, known as quantum dots (QD) and metal seem to be lack of stability and selectivity innanoshell, is that they absorb and scatter light of the physiological condition. In contrast, as well as thenear infrared, a spectrum region where tissue is case for synthesis of gold nanoparticles, the PEG-b-essentially transparent. These nanoparticles are often PAMA solution containing the Cd (II) salt andcomposed of atoms from II–VI or III–V elements in Na S, respectively, were simultaneously introduced2

the periodic table. QDs are highly light-absorbable to form CdS nanoparticles stabilized by PEG-b-and luminescent with absorbance onset and emis- PAMA at room temperature in aqueous solution.sion maximum shift to higher energy with decreas- These nanoparticles showed not only the stability ining particle size due to quantum confinement effects concentrated salt solution but also biorecognition[88]. These nanoparticles are in the size range of properties, demonstrating the high utility in the2–8 nm in diameter. Unlike molecular fluorophores, biomedical applications, particularly, high-through-which typically have very narrow exitation spectra, put detection and imaging.semiconductor QDs absorb light over a very broad Interestingly, CdS/dendrimer nanocomposite wasspectral range. This makes it possible to optically synthesized by the arrested precipitation of CdS inexcite a broad spectrum of QD colors using a single the presence of Starburst (PAMAM:poly-excitation laser wavelength, which enables one to (aminoamine)) dendrimers as stabilizers [124]. Re-simultaneously probe several markers. Thus, cently, amino-derivatized polysaccharides (amino-bioconjugated QDs have been used in DNA hybridi- dextran or Amdex) were employed as a stabilizer forzation [91–94], immunoassay [89], receptor-medi- the preparation of stable aqueous dispersions consist-ated endocytosis [89], and time-gated fluorescence ing of CdS nanoparticles, and the resultant Amdex-imaging of tissue sections [121]. QDs are also CdS nanoparticle complexes could be activated andemerging as a new class of fluorescent labels for in conjugated with an antibody [125]. Due to the factvivo cellular imaging. An important advantage is that the CdS/polymer nanocomposite underwentthat the extremely high photostability of QDs allows further aggregation in solution to produce mi-


crometer-scale composite flocs, individual CdS nan- ticolor fluorescent labels for high-throughput detec-particles cannot be isolated in these CdS/polymer tion and imaging.nanocomposites, resulting in the loss of photocmemi-cal activity.

A cknowledgements

5 . ConclusionThe authors gratefully acknowledge Professors Y.

Sakurai and T. Okano, Tokyo Women’s MedicalRecently, the tremendous progress has been at-

University, and Professor M. Kato, Science Universi-tained in the characterization and application of

ty of Tokyo for their help and stimulating discus-nanostructured materials using block copolymers.

sions. The authors would like to acknowledge Dr K.Nanostructure fabrication from block copolymers

Emoto and Mr M. Iijima for carrying out a part ofinvolves polymer design, synthesis, self-assembly,

this study. This study was partly supported by bothand derivatization. Block copolymers self-assembled

CREST and The Research Fund for the Paten-into micelle afford a powerful means of manipulating

tization(RFP) of JST (Japan Science and Technologythe characteristics of surfaces and interfaces, and

Corporation).therefore, are expected to have novel applications. Inthis review, nanoparticle fabrication usingheterobifunctinal poly(ethylene glycol) (PEG) andtheir block copolymer is explored to construct func- R eferencestionalized PEG layers on surfaces, achieving thebio-specific adsorption of a target protein through an [1] M. Moffitt, K. Khougaz, A. Eisenberg, Micellization of ionic

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