Q1 lavasanifar_micelles

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Advanced Drug Delivery Reviews 54 (2002) 169190www.elsevier.com/ locate/ drugdeliv

Poly(ethylene oxide)-block-poly(L-amino acid) micelles fordrug delivery

a a b ,*Afsaneh Lavasanifar , John Samuel , Glen S. Kwon

aFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada

bSchool of Pharmacy, University of WisconsinMadison, 777 Highland Ave., Madison, WI 53705-2222, USA

Abstract

Block copolymer micelles encapsulate water insoluble drugs by chemical and physical means, and they may target

therapeutics to their site of action in a passive or active way. In this review, we focus on micelles self-assembled from

poly(ethylene oxide)-block-poly(L-amino acid) (PEO-b-PLAA). A common theme in these studies is the chemical

modification of the core-forming PLAA block used to adjust and optimize the properties of PEO-b-PLAA micelles for drug

delivery. Micelle-forming block copolymerdrug conjugates, micellar nanocontainers and polyion complex micelles have

been obtained that mimic functional aspects of biological carriers, namely, lipoproteins and viruses. PEO-b-PLAA micelles

may be advantageous in terms of safety, stability, and scale-up. 2002 Elsevier Science B.V. All rights reserved.

Keywords:Block copolymer micelles; Drug delivery; Poly(ethylene oxide); Poly( L-amino acid); Conjugates; Encapsulation; Water insoluble

drugs; Passive drug targeting

Contents

1. Introduction ............................................................................................................................................................................ 170

2. Functional properties of polymeric micelles for passive drug targeting .................. .................... .................... ................... ........... 170

2.1. Water solubility .................... .................... .................... ................... .................... .................... ................... .................... . 170

2.2. Biocompatibility ................... .................... .................... ................... .................... .................... ................... .................... . 171

2.3. Micellar stability ................... .................... .................... ................... .................... .................... .................... ................... . 171

2.4. Biological half-life ................... .................... .................... .................... ................... .................... .................... ................. 171

2.5. Morphology.................... ................... .................... .................... .................... ................... .................... .................... ....... 172

2.6. Drug loading .................. ................... .................... .................... .................... ................... .................... .................... ....... 173

2.7. Release characteristics. .................... .................... .................... ................... .................... .................... ................... ........... 173

3. Polymeric micelles versus surfactant micelles for drug delivery .................. .................... .................... ................... .................... . 174

4. Block copolymer micelles used for drug delivery.................... ................... .................... .................... .................... ................... . 175

5. Micelle-forming PEO-b-PLAAs for drug delivery .................. ................... .................... .................... ................... .................... . 178

5.1. Micelle-forming block copolymerdrug conjugates ................... ................... .................... .................... ................... ........... 178

5.2. Micellar nanocontainers...................... .................... .................... .................... ................... .................... .................... ....... 181

5.3. Polyion complex micelles... .................... ................... .................... .................... ................... .................... .................... .... 184

6. Conclusions ............................................................................................................................................................................ 185

References ................... .................... .................... .................... ................... .................... .................... ................... .................... . 186

*Corresponding author. Tel.: 11-608-265-5183; fax: 11-608-262-3397.

E-mail address: [email protected] (G.S. Kwon).

0169-409X/ 02/ $ see front matter 2002 Elsevier Science B.V. All rights reserved.

P I I : S 0 1 6 9 - 4 09 X ( 0 2 ) 0 0 0 1 5 - 7


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170 A. Lavasanifar et al. / Advanced Drug Delivery Reviews 54 (2002) 169190

1. Introduction enhance properties of PEO-b-PLAA micelles for

drug delivery [33,34].

The concept of selective delivery of drugs to their

site of action was first introduced by Paul Erhlich

early in the 20th century. He proposed a Magic 2. Functional properties of polymeric micellesBullet, i.e. carriers with specific affinity for certain for passive drug targeting

organs, tissues or cells for drug targeting [1]. Since

then delivery systems such as liposomes, micro- Carriers should meet several requirements for

spheres and nanoparticles have been developed for effective drug delivery, including water solubility,

this purpose. In many cases they have been able to non-toxicity, non-immunogenicity, lack of long-term

widen the gap between the efficacy and toxicity, accumulation in host, in vivo stability and selective

i.e. therapeutic index of drugs. There is still a long delivery to the target site. Besides a capacity for the

way towards a real Magic Bullet, however. encapsulation of poorly water-soluble drugs, the

Among different drug carriers used for controlled carrier is required to prevent drug release before

drug delivery, there has been a rising interest in reaching the site of action to achieve targeted drug

self-assembled block copolymers over the past de- delivery. The multiple properties of the core / shellcade [214]. This is owed to the similarity of structure in polymeric micelles (Fig. 1) allow the

polymeric micelles to natural carriers, e.g. viruses simultaneous fulfilment of these somewhat opposing

and serum lipoproteins. Polymeric micelles mimic requirements.

aspects of biological transport systems in terms of

structure and function. A hydrophilic shell helps 2.1. Water solubility

them to stay unrecognized during blood circulation

[15,16]. A viral-like size ( , 100 nm) prevents their Polymeric carriers often tend to precipitate in

uptake by the reticuloendothelial system and facili- water due to a localized hydrophobicity caused by

tates their extravasation at leaky sites of capillaries, the drug and the hydrophobic portion of the poly-

leading to passive accumulation in certain tissues meric chain. The problem is more significant for

[1720]. The small size may also ease further drugpolymer conjugates where functional water-

penetration of the micellar carrier to cells. The soluble groups of the drug (e.g. amino and carboxylincorporation of recognizable moieties on micellar groups) are converted to more hydrophobic groups

surfaces [2124] or the development of thermo or

pH sensitive block copolymers [25,26] has been

pursued to enhance cellular interaction in specific

sites for active targeting. Finally, polymeric micelles

have been used for gene delivery and have shown a

great potency in directing therapeutics to sub-cellular

targets [11,27].

The multifunctional nature of polymeric micelles

appears to fulfil several tasks required for an ideal

carrier capable of selective drug delivery at differentlevels. Emphasis has been placed on micelles made

of poly(ethylene oxide)-b-poly(L-amino acid) (PEO-b-PLAA) as synthetic analogs of natural carriers with

a unique ability for chemical modification. Free

functional groups on a PLAA block provide sites for

the attachment of drugs [2830], drug compatible

moieties [31,32] or charged therapeutics such as

DNA [27]. In either case, it may be possible to fine

tune the structure of the core-forming block and Fig. 1. Sketch of a polymeric micelle loaded with drug in core.


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A. Lavasanifar et al. / Advanced Drug Delivery Reviews 54 (2002) 169190 171

(e.g. amide) by the conjugation process. Typically, sociation rate may exist for polymeric micelles

with a core/ shell structure, the polymeric carrier below the CMC, and polymeric micelles may not

may stay water-soluble if the number of monomers necessarily exist in equilibrium with polymeric un-

in the shell-forming block is more than that of the imers [8,9]. Kinetic stability may be high for poly-

core-forming block. meric micelles with stiff or bulky core-formingblocks due to hindrance of rotation. The strength of

2.2. Biocompatibility cohesive forces may be characterized by glass transi-

tion temperature (T) [45], degree of crystallinity andgToxicity studies have been mostly carried out on cross-linkage in the micellar cores.

Pluronics , block copolymers composed of PEO and Micelles are subject to extreme dilution upon

poly(propylene oxide) ( PPO) (PEO-b-PPO-b-PEO). intravenous injection into humans. If kinetically

They are fairly safe, particularly those with a high stable, slow dissociation allows polymeric micelles

content of PEO [35,36]. PEO is widely used in the to retain their integrity and perhaps drug content in

design of non-immunogenic carriers. Block copoly- blood circulation above or even below CMC for

mers with biodegradable core-forming blocks such as some time. This may give them a chance to reach the

polyesters and poly(L

-amino acids) (PLAAs) are of target site before decaying to single chain unimers.increasing interest because they may undergo hy- In this situation, interactions of polymeric micelles

drolysis and/ or enzymatic degradation, producing with components of blood (serum proteins) and cells

biocompatible monomers. Polyesters such as poly- must be weighed in terms of micellar stability and

(D,L-lactic acid) (PDLLA) have been used safely in drug release.

humans for a long time. Studies on the biocom-

patibility of PLAAs are few. Nevertheless, results 2.4. Biological half-life

indicate a dependence of enzymatic degradability

and immunogenicity on the chemical structure and/ Long circulation times are prerequisite to achieve

or physicochemical properties of PLAA chains [37 depot properties. Carriers with insufficient stabilities

41]. tend to break up and be removed rapidly from blood

Lastly, block copolymers have molecular weights by kidneys. The molecular weight of polymeric

21 6

21less than 50,000 g mol and can undergo renal micelles ( . 10 g mol ) prevents renal elimination

clearance [5]. unless the micelle structure dissociates to unimers

[42,46]. Supramolecular structures with sufficient

2.3. Micellar stability stability often end up accumulating in the liver and

spleen due to a large size or protein adsorption, both

The stability of micellar structures should be triggering a rapid uptake by the reticuloendothelial

assessed in two different aspects: thermodynamic system (RES). For this reason, drug delivery to

stability and kinetic stability. organs other than liver and spleen is limited for such

Large molecular dimensions of the core-forming carriers.

segment in block copolymers induce a strong ten- Delivery systems that are smaller than 200 nm

dency for aggregation, in other words high thermo- have low uptake by RES and may circulate in blood

dynamic stability. This pushes the CMC to very for prolonged periods [9,16,17,19,4749]. Polymericsmall levels [4,42]. A reverse relationship between micelles usually range in size between 10 and 50 nm

the hydrophobicity of the core-forming block and [5,9]. This range is much smaller than the size of

CMC has been shown in many studies [4244]. The other self-assembled delivery systems and similar to

entropy driven self-assembly of block copolymers the size of serum lipoproteins and viral particles.

may be followed by a hydrophobic or electrostatic Based on the results obtained for other colloidal

interaction in the core, depending on the structure of delivery systems, the nanoscopic size is expected to

the core-forming blocks. Strong cohesive forces facilitate the extravasation of polymeric micelles at

resulting from these interactions make the micellar leaky sites of capillaries, e.g. tumours and sites of

system kinetically stable. As a result, a slow dis- inflammation [19,47,48] (Fig. 2). They may even


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Fig. 2. Proposed model for the in vivo fate of polymeric micelles.

enter cells by different mechanisms [5053]. Other on the surface of those carriers was found to affect

advantages associated with nanoscopic dimensions of the pharmacokinetics and biological fate of the

polymeric micelles include the ease of sterilization delivery system, leading to a long circulation and

via filtration and safety of administration [5,7]. accumulation in sites with leaky capillaries [16,54].

The presence of a hydrophilic polymeric brush on

the surface of polymeric micelles (e.g. PEO shell) 2.5. Morphology

induces steric repulsive forces and stabilizes themicellar interface. This prevents the adsorption of Most of the polymeric micelles designed and used

proteins to the delivery system. As a result, poly- for drug delivery are reported to be spherical,

meric micelles may escape the uptake of RES evidenced by atomic force microscopy (AFM), dy-

efficiently. The extent of steric stabilization is depen- namic light scattering (DLS), regular and cryo-TEM

dent on the length of the hydrophilic block and its [30,34,43,44,5559]. A transfer to ellipsoid, rod and

density on colloidal particles [15]. In fact, block lamellar micelles may occur as the proportion of core

copolymers were originally used as stabilizers for to shell-forming block, copolymer concentration,

colloidal dispersions such as emulsions, liposomes or type and concentration of electrolytes in the medium,

nanoparticles. The adsorption of block copolymers temperature, organic solvent and the method of


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micellar preparation are altered. The effect of such ture is a prerequisite for control over the rate of drug

factors on the shape of polymeric micelles has been release. For drugs physically encapsulated in stable

reviewed [9,60]. The effect of micellar morphology structures of polymeric micelles, release is controlled

on loading, release and efficacy of drugs remains to by the rate of drug diffusion in the micellar core or

be explored. break up of the micelles (Fig. 3). The diffusion ratemay be quite low if a favourable interaction exists

between the solubilizate and the core-forming block2.6. Drug loadingin a rigid core [32,69,70]. An inordinate slow release

rate of indomethacin in an unionized form compat-Micellar cores serve as a nanoreservoir for loadingible with a nonpolar core of a micellar carrier hasand release of hydrophobic molecules that are conju-been shown [64].gated or complexed with the polymeric backbone or

The physical state of the micelle core and en-physically encapsulated in the core [8,9,27]. Thecapsulated drug plays an important role. The sameextent of drug incorporation in polymeric micelles byfactors may contribute to enhanced kinetic stabilityphysical means is dependent on several factors,for the micelle structure as described earlier. Theincluding the molecular volume of the solubilizate,

design of block copolymer micelles with glassy coresits interfacial tension against water, length of the under the physiological condition (37 8C) wouldcore and shell-forming blocks in the copolymer, andfavour the release in a sustained manner. Glassythe polymer and solubilizate concentration [6163].cores of poly(styrene) and poly(tert-butyl acrylate)The partition coefficient of the hydrophobic moleculehave been proposed to slowly release pyrene from abetween the micellar core and surrounding aqueous

218 216micellar carrier (diffusion constant of 10 to 10medium describes the extent of drug entrapment in

2cm / s). In contrast, pyrene was released too rapidlypolymeric micelles [61]. The greatest degree ofto be assessed from swollen cores of poly(2-vin-solubilization occurs when high compatibility existsylpyridine), which are liquid-like under the ex-between the micellar core and the solubilizate,perimental conditions [71]. Drug release may beassessed by the FloryHuggins interaction parametersustained following an increase in the loading con-(x ):sptent, owing to the crystallization of solubilizate in the

2 polymeric carrier, evidenced by differential scanningx 5 (d2d )V /RT (1)sp s p scalorimetery (DSC) in a few cases [43,44,55]. The

where d and d are the ScatchardHildebrand localization of the solute in the core/ shell structure,s psolubility parameter of the solubilizate and the core- micellar size and molecular volume of the drug areforming polymer, respectively, V is the molar vol- among other factors influencing the rate of drugsume of the solubilizate, R is the gas constant and T diffusion in the polymeric carrier.the Kelvin temperature. The highest compatibility is In case of drug conjugates, the covalent bondachieved when d5d . between the therapeutic molecule and the polymers p

The chemical conjugation of drugs or complex has to be cleaved for drug release. Water penetrationformation between block copolymers and charged and hydrolysis of the liable bonds in the micellartherapeutics has been used as an alternative approach core (bulk erosion), followed by drug diffusion may

in drug delivery by polymeric micelles [27]. In either occur in relatively hydrophilic liquid-like core struc-case, existence and accessibility of functional groups tures (Fig. 3). Water diffusion into hydrophobic andon the polymeric backbone is a requirement. rigid cores may be restricted. Therefore, in this case

release may be dependent on the rate of micellar2.7. Release characteristics dissociation. The slow dissociation of the micellar

structure to single polymeric chains and further

Evidence points to sustained release characteristics hydrolysis of the liable bonds may result in a

for many solubilizates encapsulated in polymeric sustained drug release [34]. If the micellar structure

micelles by chemical or physical means is sufficiently stable, drug release might even be

[30,34,55,6468]. The stability of the micellar struc- delayed until carrier reaches target cells. It might be


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Fig. 3. Mechanisms of drug release for polymeric micelles.

possible to tailor the chemical structure of polymeric dynamic stability is characterized by low CMC

micelles and adopt either of these mechanisms to values, which are usually in the mmolar range for

fulfil specific requirements of drug release. polymeric micelles. This contrasts with typical mil-

limolar CMC levels of low molecular weight surfac-

tants [42,57]. Following self-association, the con-

3. Polymeric micelles versus surfactant micelles centration of free amphiphile remains at the CMC

for drug delivery level. As a result, one can assume a higher number

of micelles to be formed in a given concentration for

Similar to block copolymers, low molecular block copolymers in comparison to surfactants.weight amphiphiles are well known to form micelles Schechter et al. suggest the involvement of two

that solubilize hydrophobic drugs. For the purpose of different mechanisms for solubilization in Pluronic

drug delivery clear advantages may exist for poly- micelles versus surfactant micelles [72]. Their results

meric micelles, mainly due to the polymeric nature also indicate the advantage of some polymeric

of these systems (Fig. 4). micelles to surfactant micelles in solubilization

The tendency for micellization is overall much capacity, which might be attributed to the higher

higher in block copolymers in comparison to surfac- number of micelles formed from self-assembly of

tants since the exposure of a long hydrophobic block block copolymers and / or larger cores.

to water is unfavourable to a greater extent. Thermo- Upon dilution in blood, the polymeric micelles


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Fig. 4. Break up of polymeric micelles versus low molecular weight surfactant micelles.

may remain kinetically stable, while the surfactant drug depot and influence the pharmacokinetics of

micelles are diluted to values below CMC and drugs in a favourable manner. In this sense, poly-

rapidly dissociate [4]. The concentration of the low meric micelles mimic serum lipoproteins for drug

molecular weight surfactant cannot be raised to delivery.compensate for the situation, since high concen-

trations of low molecular weight surfactants are

usually toxic and not suitable for administration [73]. 4. Block copolymer micelles used for drug

The slow dissociation rates (in hours and days) have delivery

been reported for polymeric micelles under sink

conditions even below their CMC [2]. The kinetic Micelle-forming block copolymers have been the

stability of polymeric micelles with rigid cores is in focus of several studies over the past few years

sharp contrast to surfactant micelles, which tend to [3,4,9,50,63,7479]. Efforts have led to the prepara-

break up in milliseconds upon dilution and are in tion of micellar carriers that can be safely adminis-

continuous exchange with their unimers in solution tered to humans and adequately solubilize drugs. The1

[2]. H NMR and fluorescent probe studies [30,42] hydrophilic block in these systems is usually PEOprovide evidence for the existence of rigid cores in with a molecular weight ranging from 1000 to

21polymeric micelles. 20,000 g mol . PEO has been used safely in

Unlike low molecular weight surfactant micelles humans and is approved by regulatory agencies for

that typically have mobile cores, sustained and administration. The use of other hydrophilic poly-

controlled drug release may be achieved with polymers as shell-forming blocks has been reported for

meric micelles. As a result, the rapid loss of drug bioadhesive [80] or thermoresponsive [56,77,81]

with attendant risk of intravascular precipitation of properties. Unlike the shell-forming block, the choice

water-insoluble drug poses less risk. Further, poly- for a core-forming block is relatively diverse. The

meric micelles may potentially act as a nanoscopic length of the core-forming block is usually equal or


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shorter than the PEO block to maintain water testosterone in 63.9 and 0.74% w/ w drug to poly-

solubility and form spherical core/ shell micelle mer, respectively [57]. The greater extent of solubili-

structures. zation for sudan black B was attributed to its higher

Most of the studies on block copolymers have hydrophobicity. A family of PEO-b-poly(lactic acid)

been conducted on Pluronics . Like low molecular (PEOPLA) block copolymers with different lengthsweight surfactants, Pluronics demonstrate solubiliz- of the PLA block were prepared recently and used to

ing effects for parenteral drug administration encapsulate a water-soluble drug [90]. In this case an

[35,36,82]. Pluronics have been used to solubilize increase in the length of the PLA block caused an

haloperidol [21,83], indomethacin [84], doxorubicin increase in the size of nanoparticles but did not affect

(DOX) [78], epirubicin [78] and amphotericin B their encapsulation properties.

(AmB) [85]. Overall, many Pluronics used for drug Micelles of PEO and PDLLA, poly(D,L-lactic acid-

solubilization have high ratios of PEO to PPO and co-caprolactone) (PDLLACL) or poly(glycolic acid-

are non-toxic relative to many low molecular weight co-caprolactone) (PGACL) have been used to en-

surfactants, e.g. Tween 80, especially in terms of cell capsulate taxol [91]. The greatest physical stability

membrane lysis, e.g. haemolysis. was observed for the taxol formulation in PEO-b-

Relatively hydrophobic Pluronics , on the other PDLLA micelles with no sign of precipitation in 24hand, have been used to induce immune responses, h. This was attributed to higher hydrophobicity and

i.e. act as an adjuvant [86,87]. Pluronics have T of the PDLLA block. The loading weight propor-gshown other important biological effects, inhibiting tion of taxol in PEO-b-PDLLA micelles with higher

P-glycoprotein, which is believed to be at least partly PDLLA contents reached 25% and its solubility

responsible for multi-drug resistance in cancer cells increased 5000-fold [92], which contrasts with the

[50,78]. Lastly, Pluronics have been used to in- loading of 0.5% for the same drug in Pluronic

crease the transport of drugs across membrane micelles.

barriers [21,51]. Taxol in PEO-b-PDLLA micelles shows a similar

To avoid long-term toxicities, biodegradable block in vitro cytotoxicity, a fivefold increase in maximum

copolymers with polyester core-forming structures tolerable dose and an increased efficacy after intra-

such as poly(lactic acid), poly(glycolic acid), poly- peritoneal injection in murine P388 leukemia model

(caprolactone) and their copolymers have been de- in comparison to its standard formulation in Cre-veloped and used for drug delivery. Polyesters have mophor [93]. Slow drug release from the micellar

a history of safe use in humans as biodegradable carrier was anticipated based on a high core viscosity

surgical sutures, bone fracture fixture devices and and an interaction between taxol and PDLLA seg-1

controlled drug delivery systems. PEO-b-polyesters ment evidenced by H NMR and solubility data,

were introduced back in the 1950s [88]. In 1994, respectively [92]. A similar distribution in protein

Gref et al. prepared block copolymers of PEO-b- components of the human plasma for free and PEO-

poly(lactic-co-glycolic acid) (PEO-b-PLGA) and b-PDLLA incorporated taxol [94], rapid dissociation

PEO-b-poly(caprolactone) (PEO-b-PCL). Following of tritium-labelled taxol after i.v. injection to rat

self-assembly by an O / W emulsion process, nanos- models and the elimination of micellar carrier within

pheres with an average diameter of 140 nm were 15 h, however, did not support a sustained drug

formed from PEO-b-PLGA, showing an enhanced release behaviour for taxol in PEO-b-PDLLA mi-blood circulation particularly at a high PEO content. celles [91,95]. This formulation of taxol is in clinical

The carrier was used successfully to encapsulate trials in Canada.

lidocaine and prednisolone (45% w/w drug to PEO-b-PCL self assembles into micelles that can

polymer) [55]. encapsulate indomethacin [70,96], dihydrotestos-

In a separate study long circulating nanospheres of terone [69], taxol [90] and a number of neurotrophic

PEO-b-PDLLA were developed [89]. The same agents with hydrophobic properties [76,97]. There

block copolymer has been shown to form a micellar are reports on the use of PGA [98], PLGA [99] and

fraction (,50 nm in size), which is able to solubilize PLA [100] as core-forming blocks for the encapsula-

model hydrophobic drugs, such as sudan black B and tion of indomethacin and DOX. The size of resulting


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particles was found to be above 100 nm in those micellar peak in SEC chromatograms. Recently, the

cases, however. authors have reported on the conjugation of peptidyl

Kataoka et al. have reported on the preparation of ligands to the aldehyde group of the micellar surface

functional PEO-b-PDLLA micelles with aldehyde through Schiff base formation and further reduction

groups on their surface by a method illustrated in [23]. Chemical engineering of the core-formingFig. 5 [22,59]. More than 80% of the acetal group on blocks in such carriers is underway [101].

the micelle surface was converted to aldehyde within In a separate approach, PEO blocks having site-

4 h of reaction under acidic conditions [59]. No specific sugar groups at their chain ends have been

change in micellar size and shape was evidenced achieved through ring opening polymerization using

after this conversion. The reaction of aldehyde group D,L-lactide. The block copolymer forms micelles with

with benzoic hydrazide as a model compound was glucose or galactose moieties on their surfaces. The

evidenced by an increase in the UV absorption of the galactose residues bind sugar binding sites of an

Fig. 5. Preparation of PEO-b-PDLLA micelles with functional groups on their surface. (Reprinted with permission from Ref. [22].

Copyright 1995 American Chemical Society.)


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RCA1-lecetine molecule [102]. Such strategies may systems based on PEO-b-PLAA block copolymers

be used to achieve intelligent polymeric micelles for have been investigated (Table 1).

active targeting by receptor-mediated endocytosis.

1. Micelle-forming block copolymerdrug conju-

gates.2. Micellar nano-containers.

5. Micelle-forming PEO-b-PLAAs for drug 3. Polyion complex micelles.

delivery

5.1. Micelle-forming block copolymerdrug

Micelles based on PEO-b-PLAA block copoly- conjugates

mers are unique among drug carriers owing to a

tailor-made non-polar core of PLAA, which can take In the 1980s, Ringsdorf et al. were the first to

up and protect water-insoluble drugs. A primary prepare a micelle-forming drugcopolymer conju-

advantage of PEO-b-PLAA over other micelle-form- gate. They attached a cytotoxic agent, a cyclophos-

ing block copolymers is a potential for attachment of phamide-sulfido derivative, to the poly(L-lysine) of a

drugs, drug compatible moieties, genes or intelligent PEO-b

-poly(L

-lysine) block copolymer and usedvectors in the micellar core through free functional hydrophobic moieties (palmitic acid) to induce the

groups (e.g. amine or carboxylic acid) of the amino required amphiphilicity for micelle-formation ( Fig.

acid chain. A systemic alteration in the structure of 6) [104]. Micelle formation was evidenced by

the core-forming block may lead to better control solubilization of a hydrophobic dye [53]. The libera-

over the extent of drug loading, release or activation. tion of the active metabolite, 4-hydro-cyclophospha-

Preparation of PEO-b-PLAA micelles with func- mide, could be varied within a time scale of minutes

tional groups on the surface for the attachment of to hours, depending on the structure of the conjugate.

recognizable moieties has recently been reported Pendant palmitic acid residues had a marked effect

[103]. Lastly, there is evidence that PEO-b-PLAA on drug release. In vitro studies indicated that the

micelles may easily be sterilized by filtration, freeze- cyclophosphamide-containing block copolymer acts

dried, reconstituted and administered safely [5]. as an intracellular depot for the active metabolite. In

To date, three different types of drug delivery vivo studies indicated enhanced antitumor effects of

Table 1

Micelle-forming block copolymers based on PEO-b-PLAA used for drug delivery

Polymeric micelle Core-forming block Encapsulated Reference

delivery system molecule

Drug conjugates Poly(L-lysine) Cyclophosphamide [104]

P(Asp) Doxorubicin [28,105]

Cisplatin [29,115]

Poly(hydroxyalkyl-L-aspartamide) Methotrexate [30,34]

Nano-containers P(Asp)-(DOX) Doxorubicin [114]

PBLA Pyrene [119]

Doxorubicin [66,67,125]Indomethacin [64]

Amphotericin B [127,128]

PHSA Amphotericin B [129]

P(BLA, C-16) KLN-205 [31,126]

PBLG Norfloxacin [44]

Clonazepam [43]

Polyion complex micelles Poly(L-lysine) Poly(L-aspartic acid) [133,134]

Plasmid DNA [135]

P(Asp) Lysozyme [136,137]

Poly(L-lysine/L-glycine) DNA [138]


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cores filled with self-aggregated drug. A 4050% ofDOX substitution on P(Asp) and a decrease in

proportion of P(Asp)-DOX to PEO segment was

necessary to achieve stable micelles [33,108]. Long

PEO chains were favorable avoiding the formation ofsecondary aggregates, and micelle interaction by

biological components [2,28,109]. The physicochem-

ical properties of this system have been reviewed

elsewhere [110].

Optimized structures of PEO-b-P(Asp)DOX

formed stable micelles that could dissociate into

unimers at a slow rate over a period of days in

phosphate buffer saline [108]. In the presence of

10% rabbit serum, the dissociation rate of PEO-b-

P(Asp)DOX micelles doubled [33,108]. Sub-

sequently, radioiodinated PEO-b-P(Asp)DOX mi-

celles were found to circulate for prolonged periods

in blood in healthy ddy mice in comparison to free

DOX. The volume of distribution was 2000 ml for

DOX and 3.6 ml for PEO-b-P(Asp) DOX. The

accumulation of DOX in major organs was reduced,

especially at the heart, where DOX expresses dose-

limiting cardiotoxicity [109,111].Fig. 6. First models of micelle-forming block copolymerdrug

The biodistribution of long-circulating PEO-b-conjugates and first example with cyclophosphamide. (ReprintedP(Asp)DOX conjugates was assessed in tumor-with permission from Ref. [104]. Copyright 1985 Plenum Press.)bearing mice (female CDF1 mice, transplanted with

C26 tumor cells). For PEO-b-P(Asp)DOX micelles

21cyclophosphamide-containing block copolymer com- with a 12,000 and a 2100 g mol of PEO and

pared to free drug (L1210 model) [104]. P(Asp) blocks, respectively, the delivery of DOX to

The hydrophobicity of a therapeutic molecule solid tumors was enhanced in comparison to drug

itself has been utilized to achieve the amphiphilicity alone, and the tumor to heart selectivity of DOX was

required for micellization of a conjugate prepared improved from 0.9 to 12 at 24 h [112].

from PEO-b-poly(L-aspartic acid) and doxorubicin In a murine carcinoma model (P338), DOX was

(PEO-b-P(Asp)DOX) (Fig. 7). Yokoyama et al. active at 15 mg/ kg after intraperitoneal injection

attached DOX onto the P(Asp) backbone through an [106]. DOX caused a remarkable weight loss in

amide bond between the carboxylic group of aspartic animals at that dose. A similar level of activity for

acid and the amino group of the glycosidyl residue PEO-b-P(Asp)DOX was obtained at a much higher

on DOX [28,105]. The level of conjugated DOX was dose (200 mg/kg of DOX), accompanied by a

varied by changes in reaction conditions, e.g. level of temporary weight loss. A similar trend was observeddrug. Depending on the length of the PEO chain, for several tumor models after intravenous injection

substitution level of DOX on the polymeric back- of PEO-b-P(Asp)DOX [111]. Judging from these

bone had to be under a certain level to avoid results, the superiority of the PEO-b-P(Asp)DOX

precipitation. DLS and SEC studies were able to conjugates over free drug was mainly due to lowered

reveal the presence of micelles with an average toxicity (20 times decrease in DOX maximum

diameter of 15 to 60 nm [2,28,106,107]. The quench- tolerable dose) as opposed to improved efficacy1

ing of DOX fluorescence and lack of H NMR peaks [106,107,111]. This allowed for the administration of

for the P(Asp) block for the conjugate in water higher doses of DOX. Adjusting the composition of

(D O) signaled the presence of micelles with rigid the block copolymer and the dose of DOX led to2


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Fig. 7. Synthesis of micelle-forming poly(ethylene oxide)-b-poly(aspartic acid)doxorubicin conjugates. (Reprinted with permission from

Ref. [28]. Copyright 1992 American Chemical Society.)

improved efficacy, evidenced by a complete dis- existence of micelles with an average diameter of 20

appearance of C26 tumors in animal subjects [113]. nm at a critical substitution molar ratio of cisplatin to

It was later found that PEO-b-P(Asp)DOX conju- Asp units of 0.5. The cisplatin complexed micelle

gates are not the active species causing tumor was stable in distilled water at room temperature. In

disappearance in C26 transplanted mice [32,114]. 0.15 M NaCl, however, an exchange between theThe antitumor activity was in fact caused by non- chloride ion and cisplatin led to a sustained release

conjugated DOX encapsulated in the micellar struc- of drug from its polymeric complex over 50 h [115].

ture. As it turns out, the amide linkage was too stable Accordingly, a time-dependent cytotoxicity was ob-

for drug release. This finding led to the use of served for cisplatin complexes of PEO-b-P(Asp),

PEO-b-P(Asp)DOX conjugates as nanocontainers with an overall fivefold increase in the cytotoxic

for physically encapsulated DOX [114]. concentration against B16 melanoma cells [29].

Cisplatin has been complexed with carboxyl Cisplatin complexes of PEO-b-P(Asp) demonstrated

groups on PEO-b-P(Asp) to form a metal complex plasma AUC and tumor accumulation 5.2 and 14

micelle (Fig. 8). DLS and SEC [29] showed the times greater than cisplatin alone, respectively, and


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Fig. 8. Complex formation of cisplatin and PEO-b-P(Asp). (Reprinted with permission from Ref. [29]. Copyright 1996 Elsevier Science.)

caused less nephrotoxicity [116]. A cisplatin com-

plex of PEO-b-poly(a-glutamic acid) (PEO-b-

P(Glu)) has shown greater stability, a prolonged

circulation in blood stream and an improved accumu-

lation in tumor site in comparison to the PEO-b-

P(Asp) complex of cisplatin [117].

To overcome the excessive stability of an amide

linkage between a drug and core-forming block, an

ester bond has been utilized to attach another cyto-

toxic agent, methotrexate (MTX), to create a hydro-lysable micelle-forming conjugate (Fig. 9). The slow

release of steroids attached to poly(hydroxyalkyl-L-

glutamate) through an ester bond for long periods of

time after a subcutaneous injection of conjugate

microparticles had been reported by Feijen et al.

[118]. MTX esters of PEO-b-poly(2-hydroxyethyl-L-

aspartamide) were prepared and shown to self assem-

ble to micellar structures with an average diameter of

14 nm as determined by TEM. MTX conjugate

micelles gradually released the drug, owing to ester Fig. 9. Synthesis of MTX conjugates of PEO-b-poly(2-hydroxy-

ethyl-L-aspartamide). (Reprinted with permission from Ref. [34].hydrolysis in PBS, pH57.4 at room temperature. Copyright 2000 Plenum Press.)Notably, MTX conjugate micelles were quite stable

and could elute entirely as micelles during SEC

HPLC. Adjusting the level of attached MTX was the block copolymer, thereby influencing micelle

used to control the stability of the polymeric micelles stability and controlling drug release.

as well as influence the rate of drug release. It was

concluded that MTX esters of PEO-b-poly(2-hy- 5.2. Micellar nanocontainers

droxyethyl-L-aspartamide) could be structurally

modified by varying the degree of drug substitution, The physical encapsulation of drugs within poly-

which in turn changed the overall hydrophobicity of meric micelles is generally a more attractive ap-


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proach than micelle-forming polymerdrug conju- solubilized haloperidol was enhanced by Pluronic

gates since many polymers as well as drug molecules micelles, presumably owing to increased uptake into

do not bear reactive functional groups, e.g. carboxyl, the brain. The penetration of the micellar carrier to

hydroxyl or amino groups, for chemical conjugation, brain was enhanced when a monoclonal antibody

or the free functional site may be required for the against specific antigen of brain glial cells, i.e. anti-pharmacological effectiveness of the drug. In addi- a -GP Ab, was partially inserted on the Pluronic2tion, conjugates of drugs may exhibit markedly micelles (P85). As a result, mortality was drastically

dissimilar biological properties relative to parent decreased in animals receiving haloperidol in anti-

drugs, leading to inherent difficulties in characteriza- a -GP Pluronic micelles in comparison to subjects2

tion and regulatory approval even for already ap- treated with haloperidol in standard Pluronic mi-

proved drugs. celles. The delivery of digoxin to brain has been

Physical encapsulation of drugs in the polymeric revealed to be enhanced by Pluronic P85 by the

micelles is usually carried out through dialysis or same group [120].

O/ W emulsion methods [31,32,64,66,67]. In the Physically encapsulated DOX in Pluronic mi-

dialysis method polymer and drug are both dissolved celles (L61 and F127) significantly increased the

in an organic solvent. The solution is then dialyzed antitumor effects of the drug in vivo, owing toagainst distilled water to remove the free drug and enhanced delivery to solid tumors, increase in the

organic solvent. In the O / W emulsion method, drug influx of DOX, a decrease in the efflux of DOX

is dissolved in a volatile solvent, which is also (inhibition effects on P-glycoprotein), and changes in

immiscible with water, such as chloroform, and intracellular trafficking of DOX (reviewed elsewhere

added to an aqueous solution of polymeric micelles. [121]). A parenteral formulation of DOX in

The mixture is homogenized by sonication and Pluronic micelles has entered phase I clinical trials

chloroform is evaporated in an air open system. Free in Canada.

drug is removed by ultra-filtration. The choice of The primary advantage of PEO-b-PLAA micelles

organic solvent and loading process seem to be as tailor-made nanocontainers for encapsulation and

important factors affecting micellar stability, size and release of compatible drugs is best illustrated for

extent of encapsulation [31,64,66]. DOX and PEO-b-P(Asp)DOX conjugate micelles

Similar to micelle-forming polymerdrug conju- [32]. A strong interaction between the conjugatedgates, micellar nanocontainers are expected to resist and physically encapsulated DOX is believed to be

uptake by the RES and dissociation in blood com- the basis for the improved micellar stability and

partment, which may lead to preferential accumula- sustained release properties. A careful control of the

tion of the carrier at target sites. The carrier may pH during the encapsulation was necessary to keep

then act as a depot, releasing its drug content without DOX in its unionized form, which favours a non-

going through an extra step of drug cleavage. The polar environment. The same factor affected drug

drawbacks for micellar nanocontainers are the possi- release from polymeric micelles. DOX encapsulated

bility of low encapsulation capacity or the rapid in PEO-b-P(Asp)DOX conjugate micelles were

release of encapsulated drugs, i.e. dose dumping. The active against P388DI mouse leukemia cells in vitro

encapsulation and release characteristic of polymeric and against C26 tumours in vivo. The presence of

micelles might be modified for each particular drug physically encapsulated DOX was important foror class of drugs through the attachment of drug antitumour activity in both cases [32,122]. Physical

compatible moieties to the core-forming block, encapsulation resulted in an increase in the maxi-

which is easily attainable for PEO-b-PLAA micelles mum tolerable dose of DOX from 20 to 40 mg / kg/

[5,32,57,119]. day. In a C26 murine model, tumours completely

In the late 1980s, Kabanov et al. reported on the disappeared at a dose of 10 mg/kg/ day for all

physical encapsulation of drugs in polymeric mi- animals treated with the micelle formulation. At the

celles as nanocontainers for drug delivery. They same dose, DOX alone caused tumour disappearance

encapsulated haloperidol [21,83] and DOX [78] in in just two out of five animals.

Pluronic micelles. The neuroleptic activity of In pharmacokinetic studies, free DOX disappeared


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in 15 min from the blood of tumour-bearing mice probably due to ionization of the 39 NH group at214

[122]. For C labelled DOX encapsulated in PEO-b- that pH, resulting in a reduced hydrophobic inter-

P(Asp)DOX conjugate micelles, on the other hand, action between drug and micellar core.

24.6% of the injected dose remained in blood Overall, the results of biological studies with this

circulation after 24 h and 9.6% of the dose was system were not as impressive as DOX encapsulateddetected per g of the tumour at this time. The latter in PEO-P(Asp)DOX micelles. Twenty-four hours

level was 1.3% for free drug. The highest con- after intravenous injection into healthy BDF1 mice,14

centration of free drug in tumour site was observed 1 only 5% of the C labelled DOX encapsulated in

h after its administration. In case of PEO-b-P(Asp ) PEO-b-PBLA micelles was in blood. The tolerable

DOX conjugate micelles, DOX levels at tumour site dose of DOX increased from 10 mg / kg to 23 mg/ kg

increased between 15 min and 24 h. Based on the for DOX loaded in PEO-b-PBLA micelles after

findings, the superior antitumour activity of DOX in administration to C26 tumour transplanted CDF1polymeric micelles was attributed to the accumula- mice. In comparison to free DOX-treated animals,

tion of PEO-b-P(Asp)DOX conjugate micelles at the tumour volume after 24 days decreased sig-

tumour sites. This system has been characterized in nificantly for DOX encapsulated in PEO-b-PBLA

detail [122,123] and is going to move into clinical micelles administered in its maximum tolerable dose.trials in Japan [27]. After 60 days tumours disappeared in three out of

The intermediate polymer in the synthesis of PEO- five animal subjects. For free DOX complete re-

b-P(Asp)DOX conjugates (Fig. 7), poly(ethylene covery took place in one animal subject [125].

oxide)-b-poly(b-benzyl-L-aspartate) (PEO-b-PBLA), PEO-b-PBLA micelles were also used to solubil-

was also used by Kataoka et al. to encapsulate ize indomethacin in a separate study [64]. SEC

hydrophobic model molecules [42,119], anticancer confirmed encapsulation and DLS provided data,

[66,67] and anti-inflammatory drugs [64]. The exist- illustrating an increase in the diameter of PEO-b-

ence of aromatic groups was the common feature in PBLA micelles as a result of drug loading. Similar to

the chemical structures of all encapsulated mole- DOX, the rate of indomethacin release was sustained

cules. A pp interaction between the benzyl core of from PEO-b-PBLA micelles in a pH-dependent

the micelles and aromatic ring of the drug provided manner. The maximum control was achieved in

means for formation of a stable system even in the acidic pH, where indomethacin was unionized andpresence of serum proteins [32]. PEO-b-PBLA was favoured the nonpolar environment of PBLA core in

shown to form spherical micelles around 20 nm in polymeric micelles. A sharp rise in the rate of drug

size with rigid cores at very low concentrations [42]. release was observed between pH 4 to 5, which is

A spherical shape and narrow size distribution of a close to the pK of indomethacin (4.5).apyrene conjugate of this block copolymer micelle has The synthesis of another class of PEO-b-PLAA-

recently been illustrated by AFM [124]. SEC showed based block copolymer with aromatic structure in the

prohibition of protein adsorption to PEO-b-PLAA core has been reported by Kim et al. Di and tri block

micelles where micelles were incubated with serum copolymers of PEO-b-poly(g-benzyl-L-glutamate)

albumin in PBS, pH 7.0 [67]. (PEO-b-PBLG) were prepared and self-assembled

The first attempt for drug loading for PEO-b- [43,44]. The dimensions of the colloidal system were

PBLA micelles was carried out with DOX [66,67]. in the range of nanoparticles rather than micellesEvidence for the encapsulation of DOX by PEO-b- (200300 nm) possibly due to the formation of

PBLA micelles was provided by SECHPLC and secondary aggregates. This system has been utilized

fluorescence techniques. PEO-b-PBLA micelles to solubilize clonazepam and norfloxacin.

protected DOX from chemical degradation in an Yokoyama et al. engineered the chemical structure

aqueous environment. The release of DOX from of the core-forming block in PEO-b-PBLA through

PEO-b-PBLA micelles was slow over several days partial replacement of its benzyl group by an ali-

and pH-dependent [66]. Fifty percent of encapsulated phatic chain, cetyl ester residue, PEO-b-P(BLA,C16)

DOX was released in 72 h in pH 7.4 at 37 8C. The to encapsulate a cytotoxic agent, KRN 5500, which

release was accelerated by decreasing the pH to 5.0 has an aliphatic moiety (Fig. 10) [31]. The average


16/22


Fig. 10. Synthesis of PEO-b-P(BLA,C16) and chemical structure of KRN5500. (Reprinted with permission from Ref. [31]. Copyright 1998

Elsevier Science.)

diameter of the colloidal system was found to be acid ester core was enhanced 13 times in comparison

above 100 nm, which was reduced to 70 nm by to the benzyl core of PEO-b-PBLA micelles. The

sonication without any loss in the drug content. haemolytic activity of AmB was reduced drastically

Similar to free drug, cytotoxic effects of KRN 5500 as a result of its encapsulation in poly(ethylene

encapsulated in PEO-b-P(BLA,C16) micelles was oxide)-b-poly(N-hexyl stearate-L-aspartamide) (PEO-

not strongly time dependent in in vitro and in vivo b-PHSA) micelles. This is attributed to a reduced

assessments. This contrasts with previous findings rate of AmB release from the micellar carrier

for formulation of DOX in PEO-b-P(Asp)DOX (unpublished data).

micelles, implying the possibility of a rapid drugrelease or a direct interaction of the micellar system 5.3. Polyion complex micelles

with tumor cells [126].

The physical encapsulation of amphotericin B Depending on the type of amino acid, PEO-b-

(AmB) in PEO-b-PBLA at an alkaline pH has been PLAA block copolymers may bear positive or nega-

reported [127,128]. We have developed a PEO-b- tive charge at their side chains. Therefore, oppositely

PLAA-based micellar system with AmB compatible charged macromolecules such as DNA or peptides

moieties, i.e. saturated fatty acid esters, in the core can form poly ion complexes with the PLAA seg-

that can encapsulate AmB effectively at a neutral pH ment of the block copolymer, neutralize the charge

(Fig. 11) [129,130]. AmB encapsulation in a stearic and induce required amphiphilicity for micellization


17/22


Fig. 11. Chemical structure of PEO-b-PHSA and AmB.

of the complex. The incorporation of DNA and of polymeric micelles over other colloidal systems

peptides in polymeric micelles may lead to stabiliza- for site-specific drug delivery. While small size maytion against digestive enzymes such as nuclease and lead to an improved penetration of the micellar

facilitate their penetration in cells. The polyion carrier to extravascular environment and cell mem-

complex micelles are salt-sensitive. They will fall branes, it may also restrict the space for drug

apart and release their content as the salt concen- encapsulation and limit the ability of polymeric

tration increases above a certain value [131]. There micelles for a sustained drug release, due to a large

has been a rising interest in this novel application of total surface area. The problem might be overcome if

PEO-b-PLAA micelles over the past few years the drug is stably encapsulated in the core of

(Table 1) with promising results for the use of polymeric micelles via chemical or physical means.

polyion complex micelles in the areas of diagnosis, In this context polymeric micelles of PEO-b-PLAA

biotechnology and gene therapy. These delivery are of interest since the attachment of drugs, drug

systems have been reviewed elsewhere compatible moieties or charged molecules through[11,12,27,132]. the functional side chain of the PLAA block is easily

attainable in those structures. This concept has been

used to design self-assembling block copolymers for

6. Conclusions drug and gene delivery as summarized above. The

effect of variations in the chemical structure of

Polymeric micelles have a great potential for micelle-forming PEO-b-PLAA block copolymers on

selective drug delivery in a passive or active manner functional properties of polymeric micelles for site-

[11,2127]. The stability of micellar structure as specific drug delivery requires further attention. Such

well as nanoscopic size are the two main advantages assessments may lead to the development of finely


18/22


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