Block Copolymer Micelles for Drug Delivery Design Characterization and Biological Significance 2001...

7/21/2019 Block Copolymer Micelles for Drug Delivery Design Characterization and Biological Significance 2001 Advanced Dru…

http://slidepdf.com/reader/full/block-copolymer-micelles-for-drug-delivery-design-characterization-and-biological 1/19

Advanced Drug Delivery Reviews 47 (2001) 113–131www.elsevier.com/ locate/ drugdeliv

Block copolymer micelles for drug delivery: design,characterization and biological significance

a , a b*Kazunori Kataoka , Atsushi Harada , Yukio Nagasaki

a Department of Materials Science, Graduate School of Engineering, The University of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo

113 -8656, Japanb Department of Materials Science, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278 -8510, Japan

Abstract

Recently, colloidal carrier systems have been receiving much attention in the field of drug targeting because of their high

loading capacity for drugs as well as their unique disposition characteristics in the body. This paper highlights the utility of

polymeric micelles formed through the multimolecular assembly of block copolymers as novel core–shell typed colloidal

carriers for drug and gene targeting. The process of micellization in aqueous milieu is described in detail based on

differences in the driving force of core segregation, including hydrophobic interaction, electrostatic interaction, metal

complexation, and hydrogen bonding of constituent block copolymers. The segregated core embedded in the hydrophilic

palisade is shown to function as a reservoir for genes, enzymes, and a variety of drugs with diverse characteristics.

Functionalization of the outer surface of the polymeric micelle to modify its physicochemical and biological properties is

reviewed from the standpoint of designing micellar carrier systems for receptor-mediated drug delivery. Further, the

distribution of polymeric micelles is described to demonstrate their long-circulating characteristics and significant tumoraccumulation, emphasizing their promising utility in tumor-targeting therapy. As an important perspective on carrier systems

based on polymeric micelles, their feasibility as non-viral gene vectors is also summarized in this review article. © 2001

Elsevier Science B.V. All rights reserved.

Keywords: Polymeric micelle; Drug targeting; Gene vector; Block copolymer; Polyion complex; Tumor targeting; Poly(ethylene glycol);

Poly(amino acid); Polylactide; Poly(ethyleneimine); Poly(dimethylaminoethylmethacrylate)

Contents

1. Introduction ............................................................................................................................................................................ 114

2. Polymeric micelles incorporating cytotoxic agents in the core......................... ................... .................... .................... ................. 1153. Synthesis of amphiphilic block copolymers possessing a reactive group at the PEG chain end ..................................... ................. 117

4. Preparation of reactive polymeric micelles and their characteristics .................... .................... ................... .................... .............. 119

5. Formation of polyion complex micelles (PIC micelles) from charged block copolymers ................... .................... .................... .... 120

6. Novel polyion complex micelles entrapping enzyme molecules in the core.. .................... .................... ................... .................... . 122

7. Design and functionality of DNA-loaded PIC micelles............... .................... ................... .................... .................... ................. 123

8. Synthesis of PEG–polycation block copolymers possessing a reactive PEG end group and their micellization with DNA ............... 125

*Corresponding author. Tel.: 181-3-5841-7138; fax: 181-3-5841-7139.

E -mail address: [email protected] (K. Kataoka).

0169-409X/ 01/ $ – see front matter © 2001 Elsevier Science B.V. All rights reserved.

P I I : S 0 1 6 9 - 4 09 X ( 0 0 ) 0 0 1 2 4 - 1



K . Kataoka et al. / Advanced Drug Delivery Reviews 47 (2001) 113 –131 115

Fig. 1. Features of polymeric micelles that are relevant for drug delivery.

2. Polymeric micelles incorporating cytotoxic [12,32,33]. PEG was selected as the shell-forming

agents in the core segment because of its physicochemical characteris-

tics, including high water solubility and significant

Effective targeting of cytotoxic agents to solid chain mobility as well as its low toxicity. DOX was

tumors by polymeric micelles has been achieved by covalently introduced into the side chain of the PAsp

our group with a system based on doxorubicin- segment by condensation between the flanking car-

conjugated poly(ethylene glycol)– poly(a,b-aspartic boxylic groups of the PAsp segment and the

acid) block copolymer [PEG–PAsp(DOX)] (Fig. 2) glycosidic primary-amino-group of the DOX mole-

Fig. 2. Structure of doxorubicin-conjugated poly(ethylene glycol)– poly(a,b-aspartic acid) block copolymer.



116 K . Kataoka et al. / Advanced Drug Delivery Reviews 47 (2001) 113 –131

cule using carbodiimide compounds. In this way, bound and physically entrapped DOX), the PEG–

approximately 50% of the carboxylic moieties in the PBLA micelles seem to become more stable due to

PAsp segment can be conjugated with DOX, making the incorporation of DOX even in the presence of

the PAsp segment sufficiently hydrophobic to form serum proteins [42]. The entrapped drug, in this case

micelles in an aqueous milieu [34]. Further, the DOX, may act as a filler molecule and even enhanceappreciable self-associating property of DOX moi- the stability of the micelle itself, preventing the

eties through p –p interaction contributes substan- micelle from dissociating upon dilution. Thus, in

tially to increasing the cohesive force in the core, order to achieve successful drug loading into the

which causes the additional entrapment of DOX polymeric micelle system, structure matching of the

molecules simply through physical interaction [35]. block copolymer with the candidate drug should be

It is worth mentioning that the micelle structure was taken into account. A remarkable improvement in the

further stabilized by increasing the amount of phys- blood circulation of DOX was recently demonstrated

ically entrapped DOX in the core, reducing the using PEG–PBLA micelles as a carrier, resulting in

systemic leakage of DOX and achieving enhanced micelle-entrapped DOX achieving a considerably

DOX accumulation into a solid tumor with less toxic higher antitumor activity, compared to free DOX,

side effects caused by non-specific organ distribution against a subcutaneously inoculated mouse C26[36]. Eventually, PEG–PAsp(DOX) micelles, with tumor by i.v. injection [43].

both chemically bound and physically entrapped In addition to hydrophobic interaction, the metal-

DOX in the core, achieved prolonged circulation in complex formation of ionic block copolymers should

the blood compartment due to reduced uptake into be of great interest as a driving force for block

the RES and accumulated notably in the solid tumor copolymer micellization. cis-Diaminedichlorop-

through the EPR effect, leading to complete tumor latinum(II) (cisplatin, CDDP) is a well-known metal

regression mainly by the sustained release of phys- complex that exhibits high antitumor activity [44,45].

ically entrapped DOX from tumor-localized micelles However, its clinical use is limited due to its low

[37–39]. This system is now in the final stage of water solubility and significant toxic side effects, in

animal experimentation and is expected to move into particular, acute as well as chronic nephrotoxicity

a phase I clinical trial in the very near future. [46]. Furthermore, the high glomerular clearance of

From the standpoint of carrier design with wide CDDP leads to an extremely short circulation periodapplicability to a variety of hydrophobic drugs, it is in the blood compartment [47]. These problems may

attractive to obtain a simple block copolymer that be overcome by incorporating CDDP into a long-

can form stable polymeric micelles with high effica- circulating carrier with high accumulating efficacy

cy to physically entrap hydrophobic drugs in the toward solid tumors. Chloride ligands in the leaving

core. Encouraged by the success of the PEG–PAs- groups of the platinum(II) [Pt(II)] atom in CDDP

p(DOX) micelles, we extended our research to can be substituted by a variety of reacting groups

develop a simpler system composed of poly(ethylene depending on the concentration of chloride ions in

glycol)–poly(b-benzyl-L-aspartate) block copolymer the surroundings [48]. Carboxylates are of interest in

(PEG–PBLA) to entrap DOX only in a physical this regard because the chloride ligands in CDDP

manner [40]. DOX loading into the PEG–PBLA may be substituted with carboxylate ligands in a

micelles was established using either dialysis or an chloride-free medium, yet the newly formed car-oil / water (o / w) emulsion method with a substantial boxylate ligands are still able to undergo an ex-

loading level (5–18 w /w%) [40,41]. The benzyl change reaction with chloride ion to regenerate

moiety located in the side chain of PEG–PBLA may CDDP at physiological salt concentrations due to

contribute to stabilize the core through p– p inter- their fairly low nucleophilicity [48]. This property of

action with entrapped DOX molecules. DOX mole- carboxylate can be utilized for designing a tumor-

cules in the micelle became less susceptible to directed micellar carrier system of a cytotoxic

chemical degradation than that in aqueous solution platinum complex with carboxylate-containing block

[41]. Further, as is the case with the PEG–PAs- copolymers.

p(DOX) system (micelles with both chemically Several studies have been reported on carboxylate-




containing polymeric carriers of CDDP [49–53]. segments in the block copolymer. It is worth noting

However, a solubility problem is often encountered that the micelle starts to dissociate after approximate-

in these conjugate systems due to the increased ly a 10-h induction period, when the molar ratio of

cohesive force as well as interpolymer cross-link- CDDP to Asp residues (CDDP/Asp) in the micelle

ings. For example, the introduction of CDDP into the decreases to the critical value of 0.5. This inductiveside chains of poly(L-glutamic acid) through ligand decay profile of the micelle structure in physiological

substitution caused precipitation when the molar saline is of great interest from the viewpoint of

ratio of CDDP to L-glutamic acid residues in the tumor targeting, because the induction period re-

polymer exceeded 0.2 [49]. On the other hand, this quired for micellar dissociation is in the same range

apparent problem of precipitation turns into an as that required for macromolecular drugs to ac-

advantage for designing a stable block copolymer cumulate in solid tumors through the intravenous

micelle with a CDDP-loaded core surrounded by a route [5,54]. Indeed, micelle-incorporated CDDP

shell of hydrophilic tethered chains, such as poly- was confirmed recently to have a 5.2-times higher

(ethylene glycol). Indeed, simply mixing CDDP with plasma AUC value than that of free CDDP, achiev-

PEG–PAsp in distilled water led to the spontaneous ing impressive levels in tumours (14 times higher

formation of narrowly distributed polymer–metal than that of free CDDP based on AUC) with lesscomplexed micelles with diameters of approximately nephrotoxicity, as seen in Fig. 3 [54].

20 nm [28,29]. The critical substitution molar ratio

of CDDP to Asp residues in PEG–P(Asp) (CDDP/

Asp) to form a stable micelle structure was de- 3. Synthesis of amphiphilic block copolymers

termined to be 0.5. possessing a reactive group at the PEG chain

In physiological saline (0.15 M NaCl solution) at end

378C, sustained release of CDDP from the micelle

occurs for over 50 h. The release rate of CDDP was A further challenge in the development of novel

inversely correlated with the chain length of P(Asp) micellar carrier systems is in the design of polymeric

micelles for cellular-specific targeting in which pilot-

molecules are installed on their surface to achieve a

specific-binding property to target cells. Of particularimportance in this regard is the establishment of a

novel and effective synthetic route for end-function-

alized amphiphilic block copolymers with appreci-

able biocompatibiity and biodegradability, allowing

conjugation of the pilot-molecules at the tethered end

of the hydrophilic segment.

As reported previously, a series of potassium

alkoxides possessing a protected functional group

was found to initiate the polymerization of ethylene

oxide (EO) without any side reaction to form

heterobifunctional PEG (heteroPEG), which denotesPEG possessing different functional groups at the a-

and v-ends [55–60]. This polymerization procedure

is further applicable to the preparation of an end-

functionalized block copolymer by extending a sec-

ond polymer segment from the v-end of the thus-

prepared heteroPEG. In this way, a one-pot synthesisFig. 3. Blood clearance of micelle-incorporated cis-diaminedich-

of a-acetal–poly(ethylene glycol)-block–poly(D,L-loroplatinum(II) (CDDP) and free CDDP. Male C57BL/6N mice

lactide) (a-acetal–PEG–PLA) was accomplished, aswere injected (i.v.) with free CDDP (1 mg/ml) or micelle-

incorporated CDDP at equivalent doses of free CDDP (n 54). shown in Scheme 1, through the block copolymeriza-




of a-acetal PEG, were in good accordance with the

initial monomer/initiator ratio, indicating that poly-

merization proceeds almost quantitatively.

After the block copolymerization of LA, the1

segment length of the PLA was determined by HNMR. Using PLA and acetal-ended PEG as refer-

ence compounds, assignment of the spectrum was

carried out and the results are depicted in Fig. 4. As

the weight-averaged molecular weight (M ) of PEGv

is known from the Gel Permeation Chromatography

(GPC) is correct, the M of the PLA unit can beScheme 1. One-pot synthesis of end-functionalized block co- v

calculated from this spectrum by comparing the peak polymer [a-acetal–poly(ethylene glycol)– poly(D,L-lactide) block

copolymer]. intensity of methine in the LA unit, at 5.2 ppm, to

that of methylene in the EO unit, at 3.6 ppm.1

tion of ethylene oxide (EO) and D,L-lactide (LA) The H NMR spectrum of the a-acetal–PEG–

using potassium 3,3-diethoxypropanolate (PDP) as PLA provides other critical information regarding thean initiator [61,62]. The acetal moiety located at the end group of the copolymer. As can be seen in Fig.

PEG chain-end of the block copolymer can easily be 4, a triad signal appearing at 4.6 ppm is attributable

converted into a reactive aldehyde group by gentle to the methine proton of the acetal moiety located at

treatment with a weak acid solution, as described the a-end of the PEG segment. The peak intensity

later. ratio of this acetal proton to the methylene protons in

The molecular mass of each segment can be the PEG segment and methine protons in the PLA

controlled by the initial monomer/ initiator ratio. The segment agreed well with the assumed structure,

M and the molecular weight distribution (MWD), where each block copolymer chain quantitativelyn

determined by size exclusion chromatography (SEC) possesses an acetal end group.

1Fig. 4. H NMR spectrum of a-acetal–poly(ethylene glycol)– poly(D,L-lactide) block copolymer (a-acetal–PEG–PLA). (Reprinted with

permission from Ref. [62]. Copyright 1998 American Chemical Society.)




PEG chain-end of the micelle can be converted into

peptidyl groups. Zeta potential values of Tyr–Glu

derivatized micelles correlated well with the amounts

of conjugated ligands, as shown in Fig. 7, which was

controllable over the range of 0 to 2 9 mV. Thesemicelles with peptidyl ligands may have utility for

exploring the effects of surface charge on the

pharmacokinetic behavior of colloidal carrier sys-

tems as well as for modulated drug delivery where

cellular peptidyl receptors play a substantial role. In

a similar way, sugar moieties can be introduced on

the micelle surface in a regio-selective manner [64].

5. Formation of polyion complex micelles (PIC

micelles) from charged block copolymers

Fig. 6. Effect of the weight ratio of poly( D,L-lactide) (PLA) to The concept of polymeric micelle stabilizationpoly(ethylene glycol) (PEG) segments in poly(ethylene glycol)–

through the formation of a hydrophilic palisadepoly(D,L-lactide) block copolymer (PEG–PLA) on the critical

surrounding a water-incompatible core can be ex-association concentration (CAC) of polymeric micelles. (Reprint-tended to include the case of macromolecular as-ed with permission from Ref. [19]. Copyright 1999 Elsevier

Science B.V.) sociation through electrostatic interaction. Unlike

polyion complexes formed from an oppositely

charged pair of simple homopolymers or statistical

copolymers, polyion complex micelles (PIC mi-

celles) from charged block copolymers are totally

water-soluble and are narrowly distributed. In this

regard, this is a totally new entity of polyioncomplex and is of great interest from the basic

standpoint of the supramolecular assembly of macro-

molecules. The formation of PIC micelles was first

evidenced by our group for the pair of poly(ethylene

glycol)-poly(L-lysine) and poly(ethylene glycol)-

poly(a,b-aspartic acid) block copolymer [20]. The

distribution of PIC micelles thus formed was ex-

tremely narrow, with a polydispersity index of less

than 0.1. A detailed static light scattering study

revealed that the association number of the micelle

was closely correlated with the length of the chargedsegments and, consequently, the core size of the

micelle was precisely regulated by changing the

degree of polymerization of the lysine and aspartic

acid units in the charged segments [69]. On the other

hand, the shell thickness remained constant as longFig. 7. Zeta-potential of poly(ethylene glycol)– poly(D,L-lactide) as the molecular weight of the PEG segment re-block copolymer (PEG–PLA) micelles with varying degrees of

mained constant, suggesting that the single PIC coresubstitution of the PEG chain-end with a tyrosyl–glutamic acid

is surrounded by a palisade of tethered PEG chains(Tyr–Glu) group. (Reprinted with permission from Ref. [68].

Copyright 1999 Elsevier Science B.V.) with an appreciably stretched conformation [69].




This simple, yet well-defined, size regulation of the regular alignment of the molecular junction between

PIC micelle from a pair of oppositely charged block PEG and the charged segment at the interface of the

copolymers can be explained clearly from a thermo- two domains, and even a strict recognition based on

dynamic viewpoint, as shown schematically in Fig. the length of the charged segments occurs in the

8. mixture of block copolymers with different lengthsThe requirement for decreasing excess free energy of charged segments: only matched pairs form

at the core–shell interface apparently drives the multimolecular micelles, with the remaining block

block copolymer micelles to increase in association copolymers of unmatched length being left in an

number, allowing the relative surface area of the isolated form [23].

interface to be lowered. However, in turn, this Narrowly distributed PIC micelles can also be

increase in the association number leads to an obtained when one of a pair is changed from a block

increased core radius, resulting in the stretching of ionomer to polyelectrolytes of synthetic [21,25–27]

the core-forming segments whose junction with or natural origin (DNA and enzymes) [22,24,70–83].

shell-forming segments should align at the interface In contrast to PIC micelles from a block copolymer

to avoid thermodynamically unfavorable mixing of pair, chain-length matching is not necessary for these

the two phases. Furthermore, the increased density of micelles, lending them broader availability as car-the shell with block copolymer association causes the riers for charged compounds, including proteins and

shell-forming segment to assume a more stretched nucleic acids. The PIC core of the micelle can serve

conformation. Stretching of both the core- and shell- as a microreservoir for these compounds, allowing

forming segments, with an increased association, modulation of their inherent properties, such as

apparently decreases the conformational entropy, stability, solubility, and reactivity. Furthermore, the

which should compensate for the decreased interfa- core of the PIC micelles may provide a unique field

cial free energy upon micellization. It is this balance for biochemical reactions because it forms a sepa-

between interfacial energy and conformational en- rated phase from the outer aqueous phase. For

tropy of the polymer strands that uniquely deter- example, the enzyme in the core might be active

mines the thermodynamically stable size of the PIC even under conditions where the enzyme is usually

micelles. Segregation of the PIC core from the PEG inactive, e.g., high temperature and organic media,

shell seems to be unprecedentedly sharp, with a due to its segregation from the outer phase. In this

Fig. 8. Determining factors for the size of polymeric micelles from block copolymers.




sense, the core of the micelles is regarded as a micelle with varying PEG–PAsp/ lysozyme ratios in

nano-compartmentalized reactor. the region with excess lysozyme agreed well with

calculated values, assuming a cooperative association

mechanism [79]. The diffusion coefficients of lyso-

6. Novel polyion complex micelles entrapping zyme / PEG–PAsp micelles prepared at a stoichio-enzyme molecules in the core metric mixing ratio showed neither an angular nor a

concentration dependence, indicating no secondary

Lysozyme was selected as a model protein to be aggregate formation. The association numbers of

incorporated into the micelle because it has a high lysozyme and PEG–PAsp (12–15) in the stoichio-

isoelectric point (pI 5 11), it is positively charged metric micelle were calculated from the apparent

over a wide pH range, and has practical usage in molar mass and were determined to be 56 and 62 for

drug delivery applications as a lytic enzyme. De- lysozyme and PEG–PAsp (12–15), respectively.

tailed physicochemical characteristics are also avail- Such PIC micelles entrapping enzymes in the core

able for this protein, which is the additional advan- are expected to be useful as functional materials

tage from the standpoint of gaining insight into including carrier systems in drug delivery applica-

complexation mechanisms. Dynamic light scattering tions and as a nanometric-scale reactor for enzymesmeasurements were determined for solutions of [80].

lysozyme/ PEG–PAsp (12–15; abbreviation for the The enzymatic activity of lysozyme in the micelle

block copolymer with a M of PEG of 12,000 g/ mol was then evaluated using p-nitrophenyl-penta-NAc-w

and a degree of polymerization (DP) of the Asp unit b-chitopentaoside as the substrate [82]. Interestingly,

of 15), prepared at various mixing ratios of r , which a remarkable increase in the V value was observedmax

is the ratio of the number of aspartic acid residues in for micellized lysozyme, indicating that the apparent

PEG–PAsp to the total number of lysine and arginine activity of lysozyme was enhanced through micelli-

residues in lysozyme (r 5 [Asp in PEG–PAsp]/ [Lys zation. Condensation of substrates into the micelle

and Arg in lysozyme]) [79,80]. The concentration of was believed to be the reason for this unique

lysozyme in the solution was held constant, changing acceleration of the enzymatic reaction. From the

the PEG–PAsp concentration to modulate the value standpoint of nano-reactor design, this result is of

of r . It should be noted that these solutions showed worth because a regulated enzymatic reaction isno precipitate formation and were optically clear achievable by modulating the microenvironment of

even after 1-month of storage at room temperature, the micelle core.

which is in sharp contrast to the obvious and prompt Salt concentration is a key parameter for the

precipitation observed in the mixture of lysozyme dissociation of the PIC micelle because coulombic

with PAsp homopolymer solutions. This apparent interactions between charged segments are screened

transparency of the lysozyme / PEG–PAsp system is by the added salt. Apparently, this salt sensitivity of

due to the formation of water-soluble polyion com- PIC micelles can be utilized to construct a nanomet-

plex micelles that are detectable through dynamic ric-scaled enzymatic reactor whose activity is con-

light scattering. trolled by the salt concentration in the milieu [81].

Cumulant analysis of DLS data revealed that Our recent study demonstrated that lysozyme en-

lysozyme/ PEG–PAsp micelles had an extremely trapped in the core of PIC micelles showed no2¯narrow distribution (m / G , 0.04) with an average enzymatic activity against Micrococcus luteus cells,2

diameter of 50 nm. In line with the result of the because the PEG corona effectively inhibits the cells

cumulant analysis, the z -weighted size distribution of from interacting with lysozyme in the core. How-

the micelles prepared under stoichiometric conditions ever, increasing the ionic strength resulted in the

(r 5 1.0) were monomodal in nature. Steric stabiliza- dissociation of PIC micelles, allowing lysozyme to

tion by the PEG corona was suggested for this be exposed to the milieu and, thus, to exhibit its

system because of the very low absolute value of the native lytic activity against Micrococcus luteus cells.

zeta-potential. It is worth noting that the association of lysozyme

A change in the apparent molar mass of the and PEG–PAsp by a decrease in ionic strength is




totally reversible even in the presence of substrate averaged scale, of the micelles from PEG–PLys and

cells, and that the enzymatic activity of lysozyme plasmid DNA (pDNA) decreased with an increase in

was inhibited completely through the reformation of the unit molar ratio (r ) of the lysine residue of

the PIC micelles. This synchronized switching of the PEG–PLys to the phosphate residue of DNA, and

enzymatic activity with the micellization is a good finally, it leveled off at around r 5 2. At this point,example of an intelligent bioreactor, which might be the size of the micelle was as small as 80 nm, with

useful in the areas of diagnosis and biotechnology. moderate polydispersity. The small size of the PIC

micelles compared to the dimensions of free DNA

strongly suggests the compaction of complexed DNA

7. Design and functionality of DNA-loaded PIC to form a collapsed core in the micelles. DNA

micelles compaction may be facilitated in the complex with

PEG–PLys because of a decrease in the local

Water-soluble complexes were obtained by direct- dielectric constant due to the dense PEG corona

ly mixing a solution of block copolymers (PEG– surrounding the complexed DNA [72]. Further, the

PLys) with DNA solution under stoichiometric con- sterically repulsive nature of the PEG corona pre-

ditions in 10 mM Tris–HCl buffer, pH7.4, over a vents the micelle particles from secondary aggrega-wide range of DNA concentrations ( # 150 mg / ml tion, retaining the high solubility of the micelles in

DNA) [72]. On the other hand, no water-soluble aqueous medium.

complex was obtained by mixing solutions of DNA Because of the transparent nature of the PEG–

and the PLys homopolymer under stoichiometric PLys/ DNA complex solution, the kinetics of the

conditions due to precipitation. The formation of inter-exchange reaction of complexed DNA with the

micelles with well-defined particle size was revealed polyanion can be evaluated directly from spectros-

for the PEG–PLys/DNA system by dynamic light copy [73]. Toluidine blue (TB) dye is known to

scattering (DLS) measurement. As summarized in change its absorption spectrum following interaction

Fig. 9, the cumulant diameter, representing a z- with anionic polysulfates such as poly(vinylsulfate)

(PVS) and dextran sulfate (Dex-sulf). This phenom-

enon is called ‘metachromasy’ and was used to

follow the exchange kinetics of complexed DNAwith polysufate. Metachromasy of TB is not ob-

served for polysulfate complexed with PEG–PLys

because the interaction site of polysulfate with TB is

blocked by PEG–PLys. Further, TB shows no

metachromasy with DNA, although DNA has

anionic character. These characteristics provide a

basis for evaluating DNA release from the complex

through the exchange reaction with polysulfate,

because only free polysulfate in the system induces

the metachromasy of TB in a concentration-depen-

dent manner.A metachromasy assay revealed that all of the

DNA in the complexes was quantitatively released

into the medium by the addition of an equi-unitmolarFig. 9. Change in the cumulant diameter of poly(ethylene gly-

col)–poly(L-lysine) block copolymer (PEG–PLys)/plasmid DNA ratio of polysulfate to the PEG–PLys / DNA stoichio-(pDNA) micelles with the mixing charge ratio. Dynamic light metric complexes. The half-lives for this exchangescattering (DLS) measurement was carried out at 25.010.28C for reaction of complexed DNA with polysulfate are30 mg/ml of pDNA in 10 mM Tris–HCl buffer using a DLS-700

summarized in Fig. 10 as a function of the degree of instrument (Olsuka Electronics Co., Ltd., Japan). Vertically polar-

polymerization of the PLys segment of the block ized light of 488 nm wavelength from an Ar ion laser (15 mW)

was used as the incident beam. copolymer. Obviously, the stability of PEG–PLys /




DNA complexes against the exchange reaction with

polysulfate increased progressively with an increase

in the molecular weight of the PLys segment. Note

that the structure and molecular weight of polysulfate

also substantially affect the rate of exchange re-action.

The nuclease resistance of complexed DNA in

serum-supplemented physiological saline was then

examined using a pGL3 plasmid DNA (pDNA). It

should be noted that increased nuclease resistance is

one of the characteristics required for gene vector

systems. Free pDNA and complexed pDNA with

PEG–PLys block copolymers were incubated for

various time periods at 378C in DMEM medium

containing 8 vol% fetal bovine serum (FBS) without

heat-inactivating treatment. pDNA was extractedfrom the sample solution by the sodium iodide

method (DNA extractor kit) and then subjected to an

agarose gel electrophoresis assay. The ratio of intactFig. 10. Release of complexed DNA from poly(ethylene glycol)–

supercoiled pDNA to open, circular pDNA thatpoly(L-lysine) block copolymer (PEG–PLys)/DNA micelles with

suffered from nuclease attack was quantitated den-varying degrees of polymerization of PLys segments through the

sitometrically. Fig. 11 shows the time-dependentexchange reaction with polysulfates of different structure and

molecular weight. change in the percentage of remaining supercoiled

form (sc-DNA) for free pDNA and the complexed

pDNA with block copolymers having varying

lengths of PLys segments. The abbreviation X–Y

was used to express the composition of the block 23copolymers, where X stands for PEG Mw 3 10

and Y for the degree of polymerization (DP) of the

PLys segment. Obviously, the stability of PEG–

PLys/ DNA complexes vs. the exchange reaction

with PVS increased progressively with an increase in

the molecular weight of the PLys segment, and the

rate of degradation dramatically decreased following

complexation with the block copolymers. The degra-

dation rate of pDNA decreased with an increase in

the degree of polymerization of the PLys segment.

Consequently, nuclease resistance increased in the

order of: free DNA<12–7/pDNA , 12–19/ pDNA , 12–28/pDNA 5 12–42/ pDNA. Compared

to free pDNA, an extension in the half-life of more

than 27 times was achieved for the 12–42/pDNA

system.

Correlation between the transfection efficiency of Fig. 11. Improved stability of supercoiled DNA (sc-DNA) against the PEG–PLys/pDNA system and the rate of DNAnuclease attack by complexation with poly(ethylene glycol)–poly-

exchange as well as nuclease resistance was then(L-lysine) block copolymer (PEG–PLys) of varying composition.

evaluated and the results are summarized in Fig. 12.(Reprinted with permission from Ref. [73]. Copyright 1998

American Chemical Society.) A luciferase assay against 293 cells was used in this




forming segment is of particular importance to

achieve an effective uptake into target cells through a

receptor–ligand interaction. For this purpose, a-acet-

al–PEG-block–poly(2-( N , N -dimethylamino)ethyl m-

ethacrylate) (acetal–PEG–PAMA) was recently syn-thesized by our group, based on the previously

established procedure for hetero-bifunctional PEG

synthesis as well as of anionic polymerization of

( N , N -dimethylamino)ethyl methacrylate (AMA)

initiated by a metal alkoxide [83]. It should be noted

that, as stated above, the acetal end-group of the

block copolymer is readily transformed into a reac-

tive aldehyde group by gentle acid treatment in

aqueous medium.

Acetal–PEG–PAMA was demonstrated to form

micelles through complexation with pDNA in aque-ous milieu. The micelle system maintained its appar-

ent transparency even after long-term storage for

more than 1 month. Cumulant analysis of the DLSFig. 12. Transfection efficiency of lipofectin and poly(ethylene

glycol)–poly(L-lysine) block copolymer (PEG–PLys)/ plasmid data revealed the presence of PIC micelles having aDNA (pDNA) system for 293 cells evaluated by a luciferase assay diameter of 149.0 nm, with a polydispersity indexat 24 h of cultivation. Micelles formed at the mixing charge ratio 2

(m / G ) of 0.19 [83]. Also, monomodal distribution2of 2.0 were used in this study. The transfection medium (DMEM)

of the PIC micelles was confirmed by histogramcontained 100 mM chloroquine.

analysis.

The DNA-entrapped PIC micelles were then im-

evaluation. Obviously, the gene transfection ef- mersed in an acidic environment (pH 2.5) to trans-

ficiency was progressively improved by complexing form the acetal groups located on the surface of the

pDNA with PEG–PLys containing longer PLys PIC micelles into aldehyde groups. Acid treatmentsegments, which is in line with the trend in the induced no change in the micelle distribution, and no

stability of PEG–PLys / pDNA micelles. Excess scission or denaturation of the supercoiled form of

PEG–PLys in the micelle was required to achieve pDNA took place in this process, as confirmed by

high efficiency, probably due to increased cellular gel electrophoresis. The presence of an aldehyde

uptake via adsorptive endocytosis, although the zeta- group was further confirmed using 1,2-diamino-4,5-

potential of the micelles represented a very small dimethoxybenzene dihydrochloride (DDB), which

positive value (|5 mV), even under conditions of generates a strong fluorescence at 402 nm through

excess PEG–PLys. This is consistent with the as- the reaction with an aldehyde group under acidic

sumed structure in which the PEG shell surrounds conditions. A variety of ligands, including sugars and

the pDNA complexed with the PLys segment of the peptides, can easily be installed on this micelle

block copolymer. surface using the aldehyde functionality of each PEGsegment.

The alternative approach for the preparation of a8. Synthesis of PEG–polycation block PEG– polycation block copolymer possessing a func-

copolymers possessing a reactive PEG end tional group at the PEG-end is based on cationic

group and their micellization with DNA polymerization utilizing heterotelechelic PEG as a

macroinitiator. The polycation segment that we

From the standpoint of utilizing PIC micelles in focused on was poly(ethyleneimine) (PEI), because

the field of gene delivery, the addition of ligand PEI-complexed DNA showed an impressive transfec-

molecules on the tethered chain end of the shell- tion efficiency [84]. This is believed to be due to the




buffering effect of PEI, maintaining the microen-

vironmental pH neutral in the endosome where the

PEI/ plasmid complex should be located after cellular

internalization and thus preventing activation of the

endosomal nuclease to attack the plasmid DNA[85,86]. It should be noted that comb-type graft

copolymers of PEI with PEG were synthesized to

show the improved solubility of their complex with

DNA [87,88].

Synthesis of the acetal–PEG–PEI block copoly-

mer was carried out via a two-step reaction, i.e.,Scheme 2. Synthetic procedure for end-functionalized block cationic polymerization of oxazoline initiated with ancopolymer of poly(ethylene glycol) and poly(ethyleneimine)

acetal–PEG macroinitiator, followed by the alkaline(PEG–PEI).

hydrolysis of pendent acyl groups in the poly(ox-

azoline) segment, as shown in Scheme 2 [89]. In

order to utilize acetal–PEG–OH as a macroinitiatorfor oxazoline polymerization, the hydroxyl group

was converted to the methanesulfonyl group (acetal–

PEG–SO CH ), which can initiate the polymeri-2 3

zation of oxazoline, while retaining the acetal group

at the other end intact.

Using acetal–PEG–SO CH as a macroinitiator,2 3

the cationic polymerization of 2-methyl-2-oxazoline

(Oz) was carried out successfully, obtaining an a-

acetal–PEG–POz block copolymer with a sufficient-

ly narrow molecular-weight distribution (M / M 5w n

1.41). The preparation of acetal–PEG–PEI block

copolymer was then accomplished by the alkalihydrolysis of the acetyl group of each repeating unit

in the block copolymer, using NaOH in an ethylene1

glycol/ ethanol (1:1) cosolvent. Fig. 13 shows the H

NMR spectra of the block copolymer before (Fig.

13a) and after (Fig. 13b) the deacylation reaction.

The acetal signals (1.1 and 1.8 ppm) are clearly

observable even after treatment with strong base,

because the acetal groups are known to be stable in

an alkali environment.

9. Polyion complex micelles as an

environmentally sensitive vehicle for antisense

DNA

PIC micelles entrapping pDNA have been shown1

Fig. 13. H NMR spectra of a-acetal–poly(ethylene glycol)– to be stable even in the presence of serum proteins.poly(oxazoline) block copolymer (acetal–PEG–POz) (a) and a-

On the other hand, the stability of PIC micellesacetal–poly(ethylene glycol)– poly(ethyleneimine) block copoly-

becomes a critical issue from the standpoint of theirmer (acetal–PEG–PEI) (b). (Reprinted with permission from Ref.

[89]. Copyright 2000 American Chemical Society.) utility as vehicles in the targeted delivery of DNA




with considerably lower molecular weight, i.e., anti- palisades of tethered chains to achieve effective

sense oligo-DNA. There are several reports on the steric stabilization propensities. Core segregation

stabilization of the polymeric micelle by cross-link- from the aqueous milieu is the direct driving force

ing of the core or the shell [90,91]. In these cases, for micellization and proceeds through a combination

the cross-linkage fixed the structure of the micelle of intermolecular forces including hydrophobic inter-and permanently suppressed the dissociation. For action, electrostatic interaction, metal complexation,

application in drug delivery systems, however, the and hydrogen bonding of the constituent block

micelle must dissociate to release the entrapped copolymers. The segregated core embedded in the

drugs at the targeted site. To this end, cross-linking hydrophilic palisade serves as a reservoir for a

by reversible bonds is a promising method if the variety of drugs with diverse characteristics. Further,

bond is cleaved in response to physical or chemical pilot molecules can be installed on the periphery of

stimuli given in the environment at the site of drug the micelles, allowing an increase in the uptake of

action. Recently, we prepared PIC micelles with micelles into the particular cells expressing targeted

cores cross-linked by disulfide bonds [92]. The core receptors. Even the targeting of polymeric micelles

of the PIC micelle composed of PEG–PLys and guided by physical stimuli, for example, local heat-

oligo-DNA was cross-linked by the oxidation of ing, is feasible using polymer strands with ther-thiols introduced in the side chains of the lysine units mosensitive properties, such as poly( N -iso-

of PEG–PLys. The stability, at a high salt con- propylacrylamide) [9]. Thus, the widespread use of

centration, and the dissociation behavior, after the polymeric micelles is expected in the field of drug

addition of a reducing reagent, such as dithiothreitol delivery, particularly for the modulated delivery of

and glutathione, were determined by light scattering genes and cytotoxic agents.

measurements. The advantage of the PIC micelles

with a disulfide cross-linkage in the field of drug

delivery is that the cleavage of the disulfide bondAcknowledgements

would occur within the cell because the intracellular

compartment has a stronger reducing environmentOur work cited in this review paper was supported

than the extracellular fluid. Glutathione, the most

in part by The Special Coordination Funds forabundant reducing agent in most cells, has an Promoting Science and Technology from The Sci-intracellular concentration of approximately 3 mM,

ence and Technology Agency, Japan, and Grant-in-while the concentration in blood is 1/300 of that,

Aid for Scientific Research, Ministry of Education,being in the range of 10 mM [93,94]. This significant

Science, Sports, and Culture, Japan.difference in glutathione concentration between the

extra- and intracellular environments provides a

rationale for the intracellular delivery of antisense

DNA using disulfide-stabilized PIC micelles with a Referencestailored property to promptly dissociate under the

physiological salt conditions found inside cells. [1] M. Moffitt, K. Khougaz, A. Eisenberg, Micellization of ionic

block copolymers, Acc. Chem. Res. 29 (1996) 95–102.

[2] Z. Tuzar, P. Kratochvil, Block and graft copolymer micellesin solution, Adv. Colloid Interface Sci. 6 (1976) 201–232.10. Conclusions

[3] P. Munk, K. Prochazka, Z. Tuzar, S.E. Webber, Exploiting

polymer micelle technology, CHEMTECH 28 (1998) 20–28.Block copolymer micelles hold promise for the [4] M.R. Talingting, P. Munk, S.E. Webber, Z. Tuzar, Onion-

type micelles from polystyrene-block–poly( 2-vinylpyridine)delivery of drugs and genes. The most relevantand poly(2-vinylpyridine)-block–poly(ethylene oxide),feature of block copolymer micelles for this purposeMacromolecules 32 (1999) 1593–1601.is their formation of distinguished core–shell ar-

[5] G.S. Kwon, K. Kataoka, Block copolymer micelles as long-chitecture. A variety of hydrophilic polymers with a

circulating drug vehicles, Adv. Drug Deliv. Rev. 16 (1995)flexible nature, including PEG, can be selected as the 295–309.

shell-forming segments, which assemble into dense [6] J. Kreuter, Nanoparticles, in: J. Kreuter (Ed.), Colloidal Drug




Delivery Systems, Marcel Dekker, New York, 1994, pp. micelles in aqueous milieu from a pair of oppositely-charged

219–342. block copolymers with poly(ethylene glycol) segments,

Macromolecules 28 (1995) 5294–5299.[7] P.D. Scholes, A.G.A. Coombes, M.C. Davis, L. Illum, S.S.

[21] A. Harada, K. Kataoka, Formation of stable and monodisper-Davis, Particle engineering of biodegradable colloids for

sive polyion complex micelles in aqueous medium fromsite-specific drug delivery, in: K. Park (Ed.), Controlled Drug

poly(L-lysine) and poly(ethylene glycol)–poly(aspartic acid)Delivery. Challenges and Strategies, American Chemicalblock copolymer, J. Macromol. Sci., Pure Appl. Chem. A34Society, Washington, DC, 1997, pp. 73–106.

(1996) 2119–2133.[8] S. Cammas, K. Kataoka, Site specific drug-carriers: Poly-

[22] K. Kataoka, H. Togawa, A. Harada, K. Yasugi, T. Mat-meric micelles as high potential vehicles for biologically

sumoto, S. Katayose, Spontaneous formation of polyionactive molecules, in: S.E. Webber, P. Munk, Z. Tuzar (Eds.),

complex micelles with narrow distribution from antisenseSolvents and Self-Organization of Polymers, NATO ASI

oligonucleotide and cationic block copolymer in physiologi-Series E: Applied Sciences, Vol. 327, Kluwer, Dordrecht,

cal saline, Macromolecules 29 (1996) 8556–8557.1996, pp. 83–113.

[23] A. Harada, K. Kataoka, Chain length recognition: core–shell[9] G.S. Kwon, T. Okano, Polymeric micelles as new drug

supramolecular assembly from oppositely charged block carriers, Adv. Drug Deliv. Rev. 21 (1996) 107–116.

copolymers, Science 283 (1999) 65–67.[10] K. Kataoka, G.S. Kwon, M. Yokoyama, T. Okano, Y.

Sakurai, Block copolymer micelles as vehicles for drug [24] A.V. Kabanov, S.V. Vinogradov, Y.G. Suzdaltseva, V.Y.

delivery, J. Controlled Release 24 (1993) 119–132. Alakhov, Water-soluble block polycations as carriers for

oligonucleotide delivery, Bioconjug. Chem. 6 (1995) 639–[11] H. Bader, H. Ringsdorf, B. Schmidt, Water soluble polymers

643.in medicine, Ang. Makromol. Chem. 123/124 (1984) 457–

485. [25] A.V. Kabanov, T.K. Bronich, V.A. Kabanov, K. Yu, A.

Eisenberg, Soluble stoichiometric complexes from poly( N -[12] M. Yokoyama, M. Miyauchi, N. Yamada, T. Okano, Y.

ethyl-4-vinylpyridinium) cations and poly(ethylene oxide)-Sakurai, K. Kataoka, S. Lnoue, Polymer micelles as novel

block–polymethacrylate anions, Macromolecules 29 (1996)drug carrier: adriamycin-conjugated poly(ethylene glycol)–

6797–6802.poly(aspartic acid) block copolymer, J. Controlled Release

11 (1990) 269–278. [26] T.K. Bronich, A.V. Kabanov, V.A. Kabanov, K. Yu, A.

Eisenberg, Soluble complexes from poly(ethylene oxide)-[13] A.V. Kabanov, V.P. Chekhonin, V. Yu, V. Alakhov, E.V.

block–polymethacrylate anions and N -alkylpyridinium cat-Batrakova, A.S. Lebedev, N.S. Melik-Nubarov, S.A. Ar-

ions, Macromolcules 30 (1997) 3519–3525.zhakov, A.V. Levashov, G.V. Morzov, E.S. Severn, V.A.

Kabanov, The neuroleptic activity of haloperidol increases [27] E.A. Lysenko, T.K. Bronich, A. Eisenberg, V.A. Kabanov,

after its solubilization in surfactant micelles, FEBS Lett. 258 A.V. Kabanov, Block ionomer complexes from polystyrene-

(1989) 343–345. block–polyacrylate anions and N -cetylpyridinium cations,

Macromolecules 31 (1998) 4511–4515.[14] G.S. Kwon, M. Naito, M. Yokoyama, T. Okano, Y. Sakural,K. Kataoka, Micelles based on AB block copolymers of [28] M. Yokoyama, T. Okano, Y. Sakurai, S. Suwa, K. Kataoka,

poly(ethylene oxide) and poly(b-benzyl L-aspartate), Lang- Introduction of cisplatin into polymeric micelle, J. Controlled

muir 9 (1993) 945–949. Release 39 (1996) 351–356.

[15] D. Bazile, C. Prud’homme, M.-T. Bassoullet, M. Marland, G. [29] N. Nishiyama, M. Yokoyama, T. Aoyagi, T. Okano, Y.

Spenlehauer, M. Veillard, Me. Stealth, PEG–PLA nanoparti- Sakurai, K. Kataoka, Preparation and characterization of cles avoid uptake by the mononuclear phagocytes system, J. self-assembled polymer–metal complex micelle from cis-

Pharm. Sci. 84 (1995) 493–498. dichlorodiamine platinum (II) and poly(ethylene glycol)–

[16] S.A. Hagan, A.G.A. Coombes, M.C. Garnett, S.E. Dunn, poly(a,b-aspartic acid) block copolymer in an aqueous

M.C. Davis, L. Illum, S.S. Davis, S.E. Harding, S. Purkiss, medium, Langmuir 15 (1999) 377–383.

P.R. Gellert, Polylactide– poly(ethylene glycol) copolymers [30] K. Kataoka, A. Ishihara, A. Harada, H. Miyazaki, Effect of as drug delivery systems. 1. Characterization of water secondary structure of poly(L-lysine) segments on the micel-dispersible micelle-forming systems, Langmuir 12 (1996) lization of poly(ethylene glycol)– poly(L-lysine) block co-2153–2161. polymer partially substituted with hydrocinnamoyl-group at

´[17] R. Gref, Y. Minaniitake, M.T. Peracchia, V. Trubetskoy, V. the N -position in aqueous milieu, Macromolecules 31

Torchilin, R. Langer, Biodegradable long-circulating poly- (1998) 6071–6076.

meric nanosphere, Science 263 (1994) 1600–1603. [31] Y. Matsumura, H. Maeda, A new concept for macromolecu-

[18] X. Zhang, J.K. Jackson, H.M. Burt, Development of am- lar therapeutics in cancer chemotherapy: mechanism of

phiphilic diblock copolymers as micellar carriers of taxol, tumoritropic accumulation of proteins and the antitumor

Int. J. Pharm. 132 (1996) 195–206. agent smancs, Cancer Res. 46 (1986) 6387–6392.

[19] K. Yasugi, Y. Nagasaki, M. Kato, K. Kalsoka, Preparation [32] M. Yokoyama, S. Lnoue, K. Kataoka, N. Yui, Y. Sakurai,

and characterization of polymer micelles from poly(ethylene Preparation of adriamycin-conjugated poly(ethylene glycol)–

glycol)–poly(d ,l-lactide) block copolymer as potential drug poly(aspartic acid) block copolymer: A new type of poly-

carrier, J. Controlled Release 62 (1999) 89–100. meric anticancer agent, Makromol. Chem., Rapid Commun.

8 (1987) 431–435.[20] A. Harada, K. Kataoka, Formation of polyion complex




[33] M. Yokoyama, M. Miyauchi, N. Yamada, T. Okano, Y. [45] P.M. Takahara, A.C. Rosenzweig, C.A. Frederick, S.J.

Sakurai, K. Kataoka, S. Inoue, Characterization and anti- Lippard, Crystal-structure of double-stranded DNA contain-

cancer activity of micelle-forming polymeric anticancer ing the major adduct of the anticancer drug cisplatin, Nature

drug, adriamycin-conjugated poly(ethylene glycol)–poly(as- 377 (1995) 649–652.

partic acid) block copolymer, Cancer Res. 50 (1990) 1693– [46] V. Pinzani, F. Bressolle, L.J. Hang, M. Galtier, J.P. Blayac, P.

1700. Balmes, Cisplatin-induced renal toxicity and toxicity-modu-[34] M. Yokoyama, T. Okano, Y. Sakural, K. Kataoka, Improved lating strategies—a review, Cancer Chemother. Pharmacol.

synthesis of adriamycin-conjugated poly(ethylene oxide)– 35 (1994) 1–9.

poly(aspartic acid) block copolymer and formation of unim- [47] Z.H. Siddik, D.R. Newell, F.E. Boxall, K.R. Harrap, The

odal micellar structure with controlled amount of physically comparative pharmacokinetics of carboplatin and cisplatin in

entrapped adriamycin, J. Controlled Release 32 (1994) 269– mice and rats, Biochem. Pharmacol. 36 (1987) 1925–1932.

277. [48] M.E. Howe-Grant, S.J. Lippard, Aqueous platinum(II)

[35] M. Yokoyama, S. Fukushima, R. Uehara, K. Okamoto, K. chemistry: binding to biological molecules, in: Metal Ions in

Kataoka, Y. Sakurai, T. Okano, Characterization of physical Biological Systems, Vol. 11, Marcel Dekker, New York,

entrapment and chemical conjugation of adriamycin in 1980, pp. 63–125.

polymeric micelles and their design for in vivo delivery to a [49] B. Schechter, A. Neumann, M. Wilchek, R. Arnon, Soluble

solid tumor, J. Controlled Release 50 (1998) 79–92. polymers as carriers of cis-platinum, J. Controlled Release

[36] M. Yokoyama, T. Okano, Y. Sakurai, S. Fukushinia, K. 10 (1989) 75–87.

Okamoto, K. Kataoka, Selective delivery of adriamycin to a [50] Y. Ohya, T. Masurnaga, T. Baba, T. Ouchi, Synthesis and

solid tumor using a polymeric micelle carrier system, J. Drug cytotoxic activity of dextran carrying cis-dichloro(cyclohex-Target. 7 (1999) 171 –186. ane-trans-l-1,2-diamine) platinum(II) complex, J. Biomater.

[37] G.S. Kwon, S. Suwa, M. Yokoyama, T. Okano, Y. Sakurai, Sci. Polym. Ed. 7 (1996) 1085–1096.

K. Kataoka, Enhanced tumor accumulation and prolonged [51] A. Bogdanov Jr., S.C.Wright, E.M. Marecos, A. Bogdanova,

circulation times of micelle-forming poly(ethylene oxide– C. Martin, P. Petherick, R. Weissleder, A long-circulating

aspartate) block copolymer–adriamycin conjugate, J. Con- copolymer in ‘passive targeting’ to solid tumors, J. Drug

trolled Release 29 (1994) 17–23. Target. 4 (1997) 321–330.

[38] M. Yokoyama, T. Okano, Y. Sakurai, H. Ekimoto, C. [52] M.J. Han, T.J. Cho, S.J. Park, Synthesis, characterization,

Shibazaki, K. Kataoka, Toxicity and antitumor activity and biological activity of polyanion-cis-diamineplatinum(II)

against solid tumors of micelle-forming polymeric anticancer complexes as antitumor agents, J. Bioact. Compat. Polym. 7

drug and its extremely long circulation in blood, Cancer Res. (1992) 358–369.

51 (1991) 3229–3236. [53] R. Perez-Soler, I. Han, S. Al-Baker, A.R. Khokhar, Lipo-

[39] M. Yokoyaxna, G.S. Kwon, T. Okano, Y. Sakurai, H. philic platinum complexes entrapped in liposomes: Improved

Ekimoto, K. Okamoto, H. Mashiba, T. Seto, K. Kataoka, stability and preserved antitumor activity with complexes

Composition-dependent in vivo antitumor activity of ad- containing linear alkyl carboxylato leaving groups, Cancer

riamycin-conjugated polymeric micelle against murine colon Chemother. Pharmcol. 33 (1994) 378–384.

adenocarcinoma 26, Drug Deliv. 1 (1993) 11–19. [54] Y. Mizumura, Y. Matsumura, T. Hamaguchi, N. Nishiyama,

[40] G.S. Kwon, M. Naito, M. Yokoyama, T. Okano, Y. Sakurai, K. Kataoka, T. Kawaguchi, M. Kawaguchi, T. Saito, T.

K. Kataoka, Physical entrapment of adriamycin in AB block Kakizoe, Cisplatin-incorporated polymeric micelles reducing

copolymer micelles, Pharm. Res. 12 (1995) 200–203. nephrotoxicity, while maintaining antitumor activity. Jap. J.

[41] G.S. Kwon, M. Naito, M. Yokoyama, T. Okano, Y. Sakurai, of Cancer Res. Submitted for publication.

K. Kataoka, Block copolymer micelles for drug delivery: [55] M. Yokoyama, T. Okano, Y. Sakurai, A. Kikuchi, N. Ohsako,

loading and release of doxorubicin, J. Controlled Release 48 Y. Nagasaki, K. Kataoka, Synthesis of poly(ethylene oxide)

(1997) 195–201. with heterobifunctional reactive groups at its terminals by an

[42] S. Cammas, T. Matsumoto, T. Okano, Y. Sakurai, K. anionic initiator, Bioconjug. Chem. 3 (1992) 275–276.

Kataoka, Design of functional polymeric micelles as site- [56] Y.J. Kim, Y. Nagasaki, K. Kataoka, M. Kato, M. Yokoyama,

specific drug vehicles based on poly(a-hydroxy ethylene T. Okano, Y. Sakurai, Heterobifunctional poly(ethylene

oxid-co-b-benzyl L-aspartate) block copolymers, Materials oxide)—One pot synthesis of poly(ethylene oxide) with aScience & Engineering C: Biomimetic Materials, Sensors primary amino group at one end and a hydroxyl group at the

and Systems 4 (1997) 241–247. other end, Polym. Bull. 33 (1994) 1–6.

[43] K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano, Y. [57] S. Cammas, Y. Nagasaki, K. Kataoka, Heterobifunctional

Sakurai, S. Fukushima, K. Okamoto, G.S. Kwon, Doxorubi- poly(ethylene oxide)(PEO): Synthesis of a-methoxy-v-

cin-loaded poly(ethylene glycol)– poly(b-benzyl-L-aspartate) amino and a-hydroxy-v-amino PEOs with the same molecu-

copolymer micelles: their pharmaceutical characteristics and lar weights, Bioconjug. Chem. 6 (1995) 226–230.

biological significance, J. Controlled Release 64 (2000) 143– [58] Y. Nagasaki, T. Kutsuna, M. Lijima, M. Kato, K. Kataoka, S.

153. Kitano, Y. Kadoma, Formyl-ended heterobifunctional poly-

[44] B. Rosenberg, L. VanCanp, J.E. Trosko, V.H. Mansour, (ethylene oxide)—A synthesis of poly(ethylene oxide) with a

Platinum compounds: A new class of potent antitumor formyl group at one end and a hydroxyl group at the other

agents, Nature 222 (1969) 385 –386. end, Bioconjug. Chem. 6 (1995) 231 –233.




[59] Y. Nagasaki, M. Lijima, M. Kato, K. Kataoka, Primary Nazarova, L.W. Seymour, Characterization of vectors for

amino-terminal heterobifunctional poly(ethylene oxide)— gene therapy formed by self-assembly of DNA with syn-

Facile synthesis of poly(ethylene oxide) with a primary thetic block co-polymers, Hum. Gene Ther. 7 (1996) 2123–

amino group at one end and a hydroxyl group at the other 2133.

end, Bioconjug. Chem. 6 (1995) 702–704. [75] P.R. Dash, V. Toncheva, E.H. Schacht, L.W. Seymour,

[60] T. Nakamura, Y. Nagasaki, K. Kataoka, Synthesis of Synthetic polymers for vectorial delivery of DNA: charac-heterobifunctional PEG with a reducing monosaccharide terisation of polymer–DNA complexes by photon correlation

residue at one end, Bioconjug. Chem. 9 (1998) 300–303. spectroscopy and stability to nuclease degradation and

disruption by polyanions in vitro, J. Controlled Release 48[61] C. Scholz, M. Lijima, Y. Nagasaki, K. Kataoka, A novel

(1997) 269–276.reactive polymeric micelles. Polymeric micelle with alde-

hyde groups on its surface, Macromolecules 28 (1995) [76] V. Toncheva, M.A. Wolfert, P.R. Dash, D. Oupicky, K.

7295–7297. Ulbrich, L.W. Seymour, E.H. Schacht, Novel vectors for gene

delivery formed by self-assembly of DNA with poly(L-[62] Y. Nagasaki, T. Okada, C. Scholz, M. lijima, M. Kato, K.

lysine) grafted with hydrophilic polymers, Biochem. Bio-Kataoka, The reactive polymeric micelle based on an

phys. Acta 1380 (1998) 354–368.aldehyde-ended poly(ethylene glycol) / poly(lactide) block

copolymer, Macromolecules 31 (1998) 1473–1479. [77] D. Oupicky, C. Konak, P.R. Dash, L.W. Seymour, K. Ulbrich,

[63] Y. Nagasaki, K. Kataoka, A reactive polymeric micelle as Effect of albumin and polyanion on the structure of DNA

drug vehicle for active targeting, Polymer Preprints 40 complexes with polycation containing hydrophilic nonionic

(1999) 286–287. block, Bioconjug. Chem. 10 (1999) 764–772.

[64] K. Yasugi, T. Nakamura, Y. Nagasaki, M. Kato, K. Kataoka, [78] L.W. Seymour, K. Kataoka, A.V. Kabanov, Cationic block

Sugar-installed polymer micelles: Synthesis and micelliza- copolymers as self-assembling vectors for gene delivery, in:

tion of poly(ethylene glycol)–poly(D,L-lactide) block copoly- A.V. Kabanov, P.L. Felgner, L.W. Seymour (Eds.), Self-

mers having sugar groups at PEG chain end, Macromole- assembling Complexes for Gene Delivery, Wiley, Chichester,

cules 32 (1999) 8024–8032. 1998, pp. 219–239.

[65] K.C. Dowling, J.K. Thomas, A novel micellar synthesis and [79] A. Harada, K. Kataoka, Novel polyion complex micelles

photophysical characterization of water-soluble acrylamide– entrapping enzyme molecules in the core: Preparation of

styrene block copolymers, Macromolecules 23 (1990) 1059– narrowly-distributed micelles from lysozyme and poly-

1064. (ethylene glycol)– poly(aspartic acid) block copolymer in

aqueous medium, Macromolecules 31 (1998) 288–294.[66] C. Zhao, M.A. Winnik, G. Riess, M.D. Croucher, Fluores-

cence probe techniques used to study micelle formation in [80] A. Harada, K. Kataoka, Novel polyion complex micelles

water-soluble block copolymers, Langmuir 6 (1990) 514– entrapping enzyme molecules in the core. [II]: Characteriza-

516. tion of the micelles prepared at non-stoichiometric mixing

ratios, Langmuir 15 (1999) 4208–4212.[67] K. Yasugi, Y. Nagasaki, M. Kato, K. Kataoka, Preparationand characterization of polymer micelles from poly(ethylene [81] A. Harada, K. Kataoka, On–off control of enzymatic activity

glycol)–poly(d ,l-lactide) block copolymer as potential drug synchronizing with reversible formation of supramolecular

carrier, J. Controlled Release 62 (1999) 89–100. assembly from enzyme and charged block copolymers, J.

Am. Chem. Soc. 121 (1999) 9241–9242.[68] Y. Yamamoto, Y. Nagasaki, M. Kato, K. Kataoka, Surface

charge modulation of poly(ethylene glycol)–poly(D,L-lactide) [82] A. Harada, K. Kataoka, Pronounced activity of enzymes

block copolymer micelles: conjugation of charged peptides, through the incorporation into the core of polyion complex

Colloids and Surfaces B: Biointerfaces 16 (1999) 135–146. micelles made from charged block copolymers. J. Controlled

Release (submitted).[69] A. Harada, K. Kataoka, Manuscript in preparation.

[83] K. Kataoka, A. Harada, D. Wakebayashi, Y. Nagasaki,[70] A.V. Kabanov, V.A. Kabanov, Interpolyelectrolyte and block

Polyion complex micelles with reactive aldehyde groups onionomer complexes for gene delivery: physicochemical

their surface from plasmid DNA and end-functionalizedaspects, Adv. Drug Deliv. Rev. 30 (1998) 49–60.

charged block copolymers, Macromolecules 32 (1999)[71] A. Harada, H. Togawa, K. Kataoka, Physicochemical prop-

6892–6894.erties and nuclease resistance of antisense-oligodeoxynucleo-

tides entrapped in the core of polyion complex micelles [84] O. Boussif, M.A. Zanta, J.P. Behr, Optimized galenics

composed of poly(ethylene glycol)–poly(L-lysine) block improve in vitro gene transfer with cationic molecules up to

copolymers. Eur. J. Pharm. Sci., in press. 1000-fold, Gene Ther. 3 ( 1996 ) 1074 –1080.

[72] D. Katayose, K. Kataoka, Water-soluble polyion complex [85] O. Boussif, F. Lezoualc’h, M.A. Zanta, M.D. Mergny, D.

associates of DNA and poly(ethylene glycol)–poly(L-lysine) Scherman, B. Demeneix, J.P. Behr, A versatile vector for

block copolymer, Bioconjug. Chem. 8 (1997) 702–707. gene and oligonucleotide transfer into cells in culture and

in-vivo-polyethyleneimine, Proc. Natl. Acad. Sci. USA 92[73] S. Katayose, K. Kataoka, Remarkable increase in nuclease

(1995) 7297–7301.resistance of plasmid DNA through supramolecular assembly

with poly(ethylene glycol)– poly(L-lysine) block copolymer, [86] J.P. Behr, The proton sponge, a means to enter cells viruses

J. Pharm. Sci. 87 (1998) 160–163. never thought of, M/ S-Med. Sci. 12 (1996) 56–58.

[74] M.A. Wolfert, E.H. Schacht, V. Toncheva, K. Ulbrich, O. [87] S.V. Vinogradov, T.K. Bronich, A.V. Kabanov, Self-assembly




of polyamine–poly(ethylene glycol) copolymers with phos- [91] K.B. Thurmond, T. Kowalewski, K.L. Wooley, Water-soluble

phorothioate oligonucleotides, Bioconjug. Chem. 9 (1998) knedel-like structures: the preparation of shell-cross-linked

805–812. small particles, J. Am. Chem. Soc. 118 (1996) 7239–7240.

[88] S. Vinogradov, E. Batrakova, S. Li, A. Kabanov, Polyion [92] Y. Kakizawa, A. Harada, K. Kataoka, Environment-sensitive

complex micelles with protein-modified corona for receptor- stabilization of core–shell structured polyion complex mi-

mediated delivery of oligonucleotides into cells, Bioconjug. celle by reversible cross-linking of the core through disulfideChem. 10 (1999) 851–860. bond, J. Am. Chem. Soc. 121 (1999) 11247–11248.

[89] Y. Akiyama, A. Harada, Y. Nagasaki, K. Kataoka, Synthesis [93] A. Meister, O.W. Griffith, S.S. Tate, Glutathione deficiency

of poly(ethylene glycol)-block–poly(ethyleneimine) posses- produced by inhibition of its synthesis, and its reversal;

sing an acetal group at the PEG end, Macromolecules 33 applications in research and therapy, Ciba Found. Symp. 72

(2000) 5841–5845. (1979) 135–161.

[90] A. Guo, G. Liu, J. Tao, Star polymers and nanospheres from [94] A. Meister, Glutathione deficiency produced by inhibition of

cross-linkable diblock copolymers, Macromolecules 29 its synthesis, and its reversal; applications in research and

(1996) 2487–2493. therapy, Pharmacol. Ther. 51 (1991) 155–194.

Block Copolymer Micelles for Drug Delivery Design Characterization and Biological Significance 2001...

Documents

Transcript of Block Copolymer Micelles for Drug Delivery Design Characterization and Biological Significance 2001...