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Block copolymer micelles as delivery vehicles of hydrophobic drugs


Journal of Drug Targeting, July 2006; 14(6): 343–355

Block copolymer micelles as delivery vehicles of hydrophobic drugs: Micelle – cell interactions
? 1, ADI EISENBERG2, & DUSICA MAYSINGER1 RADOSLAV SAVIC
Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada, and 2Department of Chemistry, McGill University, Montreal, Quebec, Canada
(Received 1 October 2005; revised 26 January 2006; accepted 1 February 2006) Dedicated to Professor H. Ringsdorf
1

Abstract One-third of drugs in development are water insoluble and one-half fail in trials because of poor pharmacokinetics. Block copolymer micelles are nanosized particles that can solubilize hydrophobic drugs and alter their kinetics in vitro and in vivo. However, block copolymer micelles are not solely passive drug containers that simply solubilize hydrophobic drugs; cells internalize micelles. To facilitate the development of advanced, controlled, micellar drug delivery vehicles, we have to understand the fate of micelles and micelle-incorporated drugs in cells and in vivo. With micelle-based drug formulations recently reaching clinical trials, the impetus for answers is ever so strong and detailed studies of interactions of micelles and cells are starting to emerge. Most notably, the question arises: Is the internalization of block copolymer micelles carrying small molecular weight drugs an undesired side effect or a useful means of improving the effectiveness of the incorporated drugs?

Keywords: Micelles, drug delivery, cells, block copolymer

Introduction: Block copolymer micelles from nanoscience to nanomedicine Nanomedicine (Freitas 1999) is concerned with applications of nanoscience and nanotechnology (Lavan et al. 2002; Li et al. 2005) in diagnosing, treating and monitoring of the diseases (BoguniaKubik and Sugisaka 2002; Twaites et al. 2005). Feynmann (1960) is credited with introducing the concept of nanoscience and an atom-by-atom building of molecules. The introduction of nanotechnology as a synonym for molecular technology and concepts of working nanosized devices constructed with atomic precision is credited to Drexler Eric K. (Drexler 1981, 2004; Freitas 1998). As opposed to nanotechnological devices, nanostructured bulk materials such as block copolymers fall into the area of nanoscience. Professor Helmut Ringsdorf, a 2006 recipient of an Annual Lifetime Achievement Award for an outstanding contribution

to the ?eld of drug targeting (Ringsdorf 2006), is credited with introducing drug-carrying micelles as drug delivery systems in the 1980s (Hirano et al. 1979; Ringsdorf 1980; Gros et al. 1981; Pratten et al. 1985). The groups of Kazunori Kataoka (Kataoka et al. 2001; Kakizawa and Kataoka 2002) and Alexander V. Kabanov (Kabanov et al. 2002a; Kabanov and Okano 2003) contributed greatly to the ?elds of nanoscience and nanomedicine in the area of block copolymer micelle-based drug delivery vehicles. Steadfast efforts of all the contributors to the ?eld led to several independent clinical trials conducted in the 2000s (Danson et al. 2004; Kim et al. 2004; Matsumura et al. 2004). Present milestones and the increasing importance of polymer therapeutics notwithstanding (Duncan 2003), continued collaborations between pharmacologists, biologists and polymer chemists are necessary to explore the full potential of micelles and similar particulate delivery systems as future nanomedicines.

Correspondence: D. Maysinger, Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada. E-mail: dusica.maysinger@mcgill.ca
ISSN 1061-186X print/ISSN 1029-2330 online q 2006 Informa UK Ltd. DOI: 10.1080/10611860600874538

? et al. 344 R. Savic In particular, more information is needed on interactions of micelles (release of drugs and elimination of delivery vehicles in vivo) and cells (attainment of desired pharmacological effects in cells of targeted organs and tissues) and their respective fates following such interactions. Hence, the focus of this review is critical evaluation of newer literature available on micelles and their interactions with cells, the data from completed clinical trials and future directions and approaches for development of advanced block copolymer micelles-based drug delivery vehicles. Micelles from low and high molar mass amphiphiles Micelles (mica, Latin—grain) are assemblies of amphiphilic molecules in which water hating (hydrophobic) ends of amphiphilic molecules make the center of the micelles (core) and water-loving (hydrophilic) ends make the corona (shell). Micelles form when a suf?cient concentration of free molecules is reached and each micelle contains a de?ned number of amphiphiles. For example, bile micelles of taurodeoxycholic acid are formed at the concentration of 1023 M with an average of 20 molecules per micelle (Small 1971; Steele et al. 1978). Commonly used membrane detergents sodium dodecyl sulfate and Tween 20 require 1023 and 1025 M concentrations of respective molecules to form micelles (Menger et al. 1978; Menger 1979). Drugs can form micelles as well (Mukerjee 1974) with as little as 3 molecules per micelle and drug concentrations of 1021 M (Schreier et al. 2000). The concentration of amphiphilic molecules at which the micelles are formed is called a critical micelle concentration (CMC). The higher the CMC, the less stable the micelles are towards dilution, which leads to their disassembly. The above examples of CMCs are high and micelles with low CMCs (nM to low mM) are made from synthetic block copolymers (Kumar et al. 2001; Rosler et al. 2001; Torchilin 2001). In general, micelles are in a dynamic equilibrium with free amphiphilic molecules, however, exchange of amphiphilic block copolymers between block copolymer micelles is negligible to nondetectable (van Stam et al. 2000). The latter is achieved by designing the characteristics of the coreforming block such that, for example, below 408C, there is no observable exchange of the unimers (Yamamoto et al. 2002). This in turn contributes to the stability of micelles towards disassembly (Van Stam et al. 2000) as micelles with frozen cores are very stable against dilution. The drug release is slowed down as the molecular motions of the chains in the cores are “frozen” (Allen et al. 1999a).

Block copolymer micelles for delivery of hydrophobic drugs Polymeric micelles are supramolecular (Lehn 1993) self-assemblies (Whitesides and Grzybowski 2002) of synthetic macromolecules (Ringsdorf et al. 1988; Mulhaupt 2004) where individual block copolymers (unimers) are held together by non-covalent interactions. Block copolymer micelles possess a typical coreshell structure (Scheme 1). The cores of the micelles solubilize drugs (Table I) while the corona allows for their suspension in aqueous media (Kwon 2003). Polyion complex micelles (Harada et al. 2001) made of ionic block copolymers (Kakizawa and Kataoka 2002) are used in delivery of genetic material. Non-ionic Pluronicw micelles have also been investigated (Kabanov and Alakhov 2002) in this regard, for example, for oral delivery of DNA (Chang et al. 2004). The use of block copolymer micelles in gene delivery, however, is not the topic of this article and the reader is referred to excellent reviews available on the subject (Kabanov and Alakhov 2002; Kakizawa and Kataoka 2002; Funhoff et al. 2005;Gaucher et al. 2005). As well, an essential reading about the development of the concepts of macromolecules and

Scheme 1. Core-shell structure of block copolymer micelles.

Block copolymer micelles

345

Table I. Examples of increased amount of drugs that can be administered per unit volume of water/media when the drugs are incorporated in micelles. Solubility in water (A) 0.40 mg ml21 (Cavallaro et al. 2004) Solubility in micelle suspension (B) 0.41 mg ml21 (Cavallaro et al. 2004) Fold increase (B/A) 1000

Drug Tamoxifen

Structure, formula, molecular weight

Cyclosporin A

0.23 mg ml21 (Miyake et al. 1999)

1.28 mg ml21 (Aliabadi et al. 2005)

5565

Dipyridamole

7.6 mg ml21 (Tang et al. 2003)

0.1 mg ml21 (Tang et al. 2003)

13

17 Beta-hydroxy-5 alpha-androstan- 3-one

, 0.5 mg ml21 (Sigma Aldrich Inc. 2003)

12.7 mg ml21 (Allen et al. 2000b)

25

FK506

0.97 mg ml21 (Arima et al. 2001)

8.84 mg ml21 (Allen et al. 2000a)

9

? et al. 346 R. Savic tailor-made polymeric materials has recently been written by Dr. Ringsdorf (Ringsdorf 2004). The weight of both the core- and corona-forming blocks can be controlled by controlling their respective lengths, leading to different morphologies of the formed block copolymer aggregates (Zhang and Eisenberg 1995; Zhang et al. 1996). To obtain spherical micelles with hydrophobic cores, the molecular weight of the corona-forming blocks should not exceed that of the core-forming parts of the block copolymer (Allen et al. 1999a). The de?ning properties of micelles include CMC, aggregation number, size and shape (Riess 2003). Typical block copolymer micelles will have low CMC values, . 100 individual polymer chains, hydrodynamic radius of 25 – 50 nm and largely spherical shape (Kwon 2003). The core forming blocks can be classi?ed into amino acids, polyesters and poloxamers (Adams et al. 2003). Poly(beta-benzyl-L -aspartate) – b – poly(ethylene oxide) micelles are an example of amino acidbased micelle cores and examples of CMCs for these systems include 5 –18 mg l21 (Kwon et al. 1993; La et al. 1996). Poly(propylene oxide) –poly(ethylene oxide)– poly(propylene oxide) micelles are an example of poloxamer (also known as Pluronicw) based micelle cores with CMCs in a range of 10 – 1000 mg l21 (Alakhov and Kabanov 1998). Poly(caprolactone) – b –poly(ethylene oxide) (PCL – b– PEO) micelles are an example of micelles with polyester-based cores with CMCs in a range of 1.2 –25 mg l21 (Allen 1999; Luo et al. 2002; Park et al. 2002; Letchford et al. 2004). The low CMC is takes as an indication that dilution after intravenous administration does not lead to break down of micelles, allowing them to circulate in blood for at least several hours and accumulate in tumor sites (Kataoka et al. 2001; Kabanov and Alakhov 2002; Adams et al. 2003) by enhanced permeability and retention (EPR) effect (Kwon et al. 1994 a,b; Maeda et al. 2000). However, the dynamics of the micelles breakdown after in vivo administration remain to be investigated. A potential breakthrough in this regard is highlighted later in this article under the section Importance of the micelles’ integrity assessment in sera, in cells and in vivo in animals. Enhanced permeability and retention effect Endothelium of blood vessels presents a physical barrier for extravasation of intravenously administered drug delivery systems to the surrounding tissues. However, in disease states such as in?ammation or tumors, the permeability of blood vessels increases through the loss of junction integrity between endothelial cells and loss of cell –cell and cell – matrix interactions (Amerongen and van Hinsbergh 2002). Under such conditions, distribution of molecules with molecular weights . 40 kDa is several folds greater in tumors than in normal tissues, while small molecular weight compounds are rapidly cleared from plasma and excreted in urine (, 4.5 kDa) (Noguchi et al. 1998). Micelles (hydrodynamic radii , 50 nm, MW . 80 kDa) traverse the pores (several hundred nm) between the capillary endothelial cells that were caused by in?ammation or abnormal vasculogenesis in tumors and aided by impaired lymphatic drainage they then accumulate in the interstitial ?uid (Maeda et al. 2000) (Scheme 2). Hence, by incorporating the small molecular weight drugs inside the micelles, the drugs are accumulated in tumors as well (Maeda et al. 2000). Usually, micelles are considered to eventually disassemble into smaller aggregates amenable to elimination by kidney ?ltration. However, considering the EPR dictated accumulation of micelles/polymer, local polymer concentrations may become suf?ciently high to allow the cellular uptake of micelles, which could lead to polymer retention.

Rationale for following the fate of block copolymer micelles To facilitate the development of advanced, controlled, micellar drug delivery vehicles, we have to understand the fate of micelles and micelle-incorporated drugs under physiologically relevant conditions. Experiments addressing the effects of charge and size on modifying the cellular uptake of water-soluble polymers (Duncan et al. 1981; Pratten et al. 1982) and pinocytotic uptake of the block copolymers by macrophages (Pratten et al 1985) were available in the early 1980s. One of the early evidence of the cellular internalization of ?uorescent-labeled micelles with covalently attached ligands capable of receptormediated endocytosis came in 1992 (Kabanov et al. 1992; micelles with ligands attached to their coronas are called actively targeted micelles—the ligand has to mediate their internalisation without destabilizing the micelles (or preventing their formation) hence its amount is usually kept low but suf?cient (Torchilin

Scheme 2. Illustration of parameters contributing to the accumulation of micellar delivery systems by EPR effect.

Block copolymer micelles et al. 2003; Nasongkla et al. 2004). These Pluronicw (P85) block copolymer micelles (Kabanov et al. 1992) had covalently attached staphylococcus aureus entertoxin B (SEB) to drive their internalization through receptor mediated endocytosis. By order of magnitude, block copolymer used to make the micelles was in mM range, ?uorescent dye used to tag the polymer was in mM range and SEB ligand was in nM range (Kabanov et al. 1992). The results showed increased uptake of SEB decorated micelles in both Jurkat and MDCK cells (Kabanov et al. 1992). Importantly, control treatments with passively targeted micelles (no SEB ligand attached) also showed detectable signals in Jurkat cells (Kabanov et al. 1992), suggesting that block copolymer micelles without the attached ligands could also be taken up by the cells. This has, since, been con?rmed with non-Pluronicw micelles as well (Liaw et al. 1999; Yoo and Park 2001; Luo et al. 2002; Rapoport et al. 2002; Shuai et al. 2004a,b; Liu et al. 2005). Considering the protective role of PEO in decreasing the interactions of PEO-coated materials with cells, it did not prevent the internalization of micelles. It is clear that block copolymer micelles are not merely inert drug containers that simply solubilize and release hydrophobic drugs—cells internalize micelles. This demands investigations into the internalization and subcellular fate of micelles with relevance not only to the mechanisms of micelle internalization but, importantly, to the effectiveness of incorporated drugs, retention of the polymer and the ultimate fate of the affected cells. In this regard, tracking the block copolymer micelles becomes imperative. Tracking the block copolymer micelles Fluorescent labeling of the block copolymers, combined with the use of organelle selective dyes (Table II) can provide effective means of following the micelles in ? et al. 2003). Red ?uorescent polymer live cells (Savic ? et al. 2003), green tags (Luo et al. 2002; Savic ?uorescent dyes (Rapoport et al. 2002) and drugs (Yoo and Park 2004) have all been successfully used to label the polymers. Alternatively, block copolymers can be labeled with heavy atoms (Sakai and Alexandridis 2004; Sidorov et al. 2004; Kang and Taton 2005) and individual delivery vehicles detected in cells by electron microscopy. No studies have yet tried that approach for micelles, although electron dense osmium tetroxide was used to investigate the fate of nanoparticles (Panyam et al. 2003). This study (Panyam et al. 2003) successfully demonstrated clearly distinguishable individual nanoparticles within cells. Quantum dots (semiconductor nanoparticles) may represent an interesting novel label allowing both confocal and electron microscopy analyses of the cellular fate of micelles. Dubertret et al. (2002) recently used quantum dots to simultaneously
Mitochondria and endoplasmic reticulum, 3 mM, 30 min Golgi apparatus and endoplasmic reticulum, 3 mM, 30 min Mitochondria, 10 nM, 30 min

347

Plasma membrane, 1 mM, 5 min

Nuclei, 2 –10 mM, 10–30 min

Green

Green

Acidic organelles, 50 nM, 30 min

Suggested use

Green

Table II. Fluorescent, cell-permeable, organelle-selective dyes for live cell imaging.

Emission (nm)

518

511

Green 510 C30H37BF2N2O5 B-7447, 25 mg 554.44 503

Blue

461

Excitation (nm)

495

504

501

C27H37Cl3N6O4

Cl8H26BClF2N4O

C32H35NO6

398.69

572.53

529.63

H-1399, 100 mg

D-109, 100 mg

Cat, no., unit size

615.99

L-7526, 20 ? 50 mL D-273, l00 mg

3,30 -Dihexyl oxacarbocyanine iodide (DiOC6(3)) Brefeldin A, BODIPYw FL conjugate

5-Dodecanoyl amino?uorescein

Lysotracker green DND 26

Mito trackerw green FM

Hoechst 33342

Dye name

Molecular Probes Molecular probes Molecular probes Molecular probes Molecular probes Molecular probes

Source

M-7514, 20 ? 50 mg

671.88

MW

C34H28Cl5N3O

C29H37IN2O2

Formula

490

350

484

516

Green

Color

? et al. 348 R. Savic visualize both the phospholipid micelles and the incorporated quantum dots. Lastly, polymers can be labeled with radioactive probes to allow the quantitative analyses of the polymer distribution in vivo in animals (Araujo et al. 1999) and in humans (Batrakova et al. 2004). Regardless of the approach used, the goal is to understand the fate of both the delivery vehicle and of the incorporated drug. Following the drug is critical in comparing the distribution and effectiveness pro?les of free and delivered drugs. The fate of both the micelles and the drugs can be discerned (Scheme 3) if labeled micelles are combined with differently labeled micelle-incorporated drugs. Relying on labeled drug alone (Scheme 3A) carries with it the risk of mistaking the localization of the released drug for that of the micelles. In a similar manner, cellular adhesion could be mistaken for cellular internalization of the delivery vehicle if only the amount of the drugs within cells is measured (De et al. 2005). For example, as demonstrated by De at al. (2005) particles of increasing size loaded with paclitaxel or etoposide both exhibited increased amount of drug delivered to cells when larger particles were used. However, the adhesion of larger particles, rather than their cell uptake, contributed to the observed greater amounts of the drug in the cells (De et al. 2005). Hence, the delivery vehicle must be labeled and followed separately (Scheme 3B). Quantitative analyses of ?uorescence interactions between the two ?uorophores by FRET and FLIM (Emptage 2001; Wallrabe and Periasamy 2005) may provide additional, presently unexploited, opportunities to investigate where and when is the drug released. Determinants of the internalization of block copolymer micelles Present research suggests a signi?cant involvement of endocytosis in the internalization of micelles but the mechanistic details remain to be elucidated. There is no doubt that the internalization of micelles is a time, energy, temperature and concentration dependent process (Kabanov et al. 1992; Liaw et al. 1999; Allen et al. 1999b), with involvement of clathrin mediated endocytosis (Luo et al. 2002; Mahmud and Lavasanifar 2005). However, the unimers, types of block copolymers used and cell lines in which the studies are carried out all play a role in the internalization of micelles and their interaction with cells. Pluronicw micelles have been the most studied block copolymer micelle delivery vehicles to date (Kabanov and Alakhov 2002; Kabanov et al. 2002b). The known interactions of Pluronicw block copolymers with cells include inhibition of P-gp drug ef?ux transporters (Venne et al. 1996) and preferred energy depletion in MDR expressing cells (Batrakova et al. 2001), by, at least in part, interfering with the electron transport chain in the mitochondria (Rapoport et al. 2000) and by membrane ?uidization (Melik-Nubarov et al. 1999; Batrakova et al. 2001; Krylova and Pohl 2004; Demina et al. 2005). These interactions contribute to the sensitisation of tumor cells to anticancer drugs and are observed below CMC, i.e. they are attributed to the action of Pluronicw unimers (Kabanov et al. 2002b). Conversely, a series of chemically different (PCL – b– PEO) diblock copolymers increased the accumulation of rhodamine-123 (P-gp substrate) above the CMC (Zastre et al. 2002). The exact

Scheme 3. Fluorescence-based approach to study the subcellular fate of micelles and incorporated agents.

Block copolymer micelles attribution of this effect, however, to micelles, unimers or combinations thereof is not clear. In the follow up study, Zastre et al. (2004) suggest that the internalization of rhodamine-123 above the polymer CMC was not altered by inhibitors of endocytosis, however, a 16 and 19% decrease in the cell accumulation of rhodamine-123 in presence of sucrose was observed (polymer concentrations (0.5 and 1% w/v) were above the CMC (0.01%) (Zastre et al. 2002)). A 14% decrease in presence of brefeldin A (Zastre et al. 2004) was observed with 1% w/v as well—40% of rhodamine was incorporated in micelles under these conditions (Zastre et al. 2002). Therefore, more detailed inhibition studies are needed to exclude a contribution of micelle-incorporated rhodamine-123 to the cell accumulation of rhodamine-123. When comparing the results between chemically similar polymers with different chain lengths, the ratios of hydrophilic and hydrophobic parts of the block copolymer should not be neglected as they may play a role in the mechanism of the internalization of micelles (Mahmud and Lavasanifar 2005). In addition to above-discussed contributors to internalization, size plays a role in the mechanism of internalization of particles as well. Mechanistic studies of the internalization of nanosized, latex particles, including the ones similar in size to micelles, revealed that inhibition of the clathrin-mediated endocytosis decreased the uptake of 50 nm particles (Rejman et al. 2004). This is corroborated by results obtained with ?uorescent microspheres (20 – 1010 nm), the uptake of which varied between six different cell lines in accordance with their size, cell type, cell density and cell growth rate (Zauner et al. 2001). All six cell lines internalized the small 20 nm particles under both growing and con?uent conditions (Zauner et al. 2001). Three cell lines (KLN 205 cells, Hepa 1 –6, HepG2) internalized very few or none of the 93, 220, 560 and 1010 nm particles, while the remaining cells (HNX 14C, ECV 304 and HUVEC) internalized particles of all sizes with signi?cant decrease in the amounts of larger particles (Zauner et al. 2001). These data suggest that low nm range particles were internalized irrespective of the cell type or their growth state, possibly by constitutive endocytosis (Mellman 1996). A direct consequence of these observations on the effectiveness of the incorporated drugs was observed using poly(D,L -lactide-co-glycolide) nanoparticles loaded with plasmid DNA (Prabha et al. 2002). The transfection ef?ciency of particles with 200 nm average diameter was over 20 times lower than that of those with average 70 nm diameter. These observations are relevant to block copolymer micelles since micelles can form secondary aggregates the size of which can exceed several hundred nm (Lele and Leroux 2002; Dimitrov et al. 2004; Zeng et al. 2004). Together, these data suggest that in order to discern

349

the internalization of micelles, we have to consider all of the presently demonstrated contributors to the internalization of similarly sized particles and known physical chemical properties of micelles. Pinning down the mechanisms of the micelle internalization will require the assessment of contribution of each potential population of particles, or their building blocks, to the internalization and effectiveness of micelles and incorporated drugs (Scheme 4). However, these studies should be balanced with investigation and improvement of the integrity of micelles under physiological conditions, as the recent clinical trials suggest this may be a culprit to their effectiveness. Effectiveness of micellar drugs in clinical trials Recently, several clinical trials using micellar formulations of anticancer drugs have been completed (Danson et al. 2004; Kim et al. 2004; Matsumura et al. 2004). The results from these studies suggest that improvements in micelle design are necessary to realize their potential as successful nanoparticulate drug delivery vehicles. For example, in a doxorubicin trial (Matsumura et al. 2004), the drug was incorporated in doxorubicin –poly(aspartic acid)– b– poly(ethylene glycol) micelles and administered onceevery-three weeks to patients with metastatic or recurrent solid tumors. The recommended dosage of micellar formulation was similar to that of free doxorubicin (50 mg m22). Pharmacokinetic parameters of micellar doxorubicin at this dose, compared to free doxorubicin, showed a moderate change in area under the plasma-drug-concentration over-time curve (AUC) (2-fold increase), elimination (t1/2beta) (3.6fold increase), clearance (CLtot) (2-fold decrease) and volume of distribution (Vss) (1.5-fold decrease) (Matsumura et al. 2004). On the other hand, liposomal formulation of doxorubicin at the same dose had 556, 57, 720 and 300-fold differences in AUC, t1/2beta, CLtot and Vss (Gabizon et al. 1994; Matsumura et al. 2004), which is in better accordance with the behavior of particulate delivery systems and their effects on the pharmacokinetics of incorporated drugs (Allen and Cullis 2004). As the authors acknowledge (Matsumura et al. 2004), these data suggest a poor stability of micelles carrying the doxorubicin. Furthermore, a micellar formulation of doxorubicin using Pluronicw L61 and Pluronicw F127 block copolymers (Danson et al. 2004) with non-covalently incorporated doxorubicin showed similar AUC to free doxorubicin with delayed terminal clearance of a drug. While initially a partial response was seen in 3 out of 21 patients (14%) it did not persist—the response was not observed 4 months after the last treatment (once every three weeks, for up to 6 cycles, MTD 70 mg m22) (Danson et al. 2004). As well, in the clinical trial with methoxy polyethylene

? et al. 350 R. Savic

Scheme 4. Possible contributors to the internalization of micelles and micelle-incorporated drugs.

glycol – poly(D,L -lactide) incorporated paclitaxel (Kim et al. 2004), the t1/2 and AUC were shorter and lower than those for free paclitaxel (two patients (9.5%) excluded from the analyses due to low paclitaxel concentrations). In terms of response to therapy a partial response was observed in 3 out of 21 patients (14%) and it continued for 3 –6 months, however, the response rate of free paclitaxel is estimated at 21% with 40% one year survival (Bonomi 1998). Taken together (Danson et al. 2004; Kim et al. 2004; Matsumura et al. 2004) these data suggest that approaches to enhance the stability of micelles and control their disassembly may be needed. For example, stereocomplexation (Kang et al. 2005) and cross-linking of the core/shell (Xu et al. 2004; Shuai et al. 2004b) can enhance the stability of micelles and modify the kinetics of drug release in vitro, while use of building blocks responsive to changes in phosphorylation (Katayama et al. 2001), pH or temperature (Gil and Hudson 2004; Gillies et al. 2004) allows environmentally responsive disassembly of micelles (Gil and Hudson 2004; Gillies and Frechet 2004). However, conditions (such as solvents, duration, and initiator) under which the chemical cross-linking of the core is performed have to be optimized to obtain micelles with desired drug incorporation, release kinetics and micelle size (Shuai et al. 2004b).

Pluronicw polymers have been successfully crosslinked using a UV light induced free radical polymerization of cross-linker monomer (Petrov et al. 2005). The resulting micelles showed a diameter of 32 – 50 nm and stability against 1 h long sonication which normally disrupts the micelles within a few minutes (Petrov et al. 2005). In addition, there are interesting approaches to control the stability of micelles by engineering the block copolymers to respond to a particular change in the environment. In this regard, a control over the stability of micelles was attempted with a polymer – peptide conjugate (Katayama et al. 2001). Thermoresponsive polymer poly(N-isopropylacrylamide) was coupled with protein kinase A substrate—GLRRASLG peptide. Upon the addition of activated protein kinase A (proof of concept in test tubes), phosphorylation of GLRRASLG increased the extent of hydration of the polymer, which led to gradual disintegration of the micelles (Katayama et al. 2001). The approach remains to be tested in vitro in cells. The modi?cations of the polymers continue by using pH responsive polymers which in a similar manner undergo a change in hydration with the decreasing pH, leading to disintegration of micelles and drug release in response to the environment (Taillefer et al. 2001; Yoo et al. 2002; Gillies and Frechet 2004; Gillies and Frechet 2005; Vamvakaki

Block copolymer micelles et al. 2005). In addition, attachment of ligands to the outer corona of block copolymer micelles is used to increase their internalization in target cells. Attachment of vitamins (Dalhaimer et al. 2004; Yoo and Park 2004), antibodies (Torchilin et al. 2003) and peptides (Nasongkla et al. 2004) may confer targeted accumulation of drug delivery vehicles in tumor cells by targeting folate receptor (Dalhaimer et al. 2004), nucleosomes (Torchilin et al. 2003) or endothelial cells over expressing integrin (Nasongkla et al. 2004). Notably, introducing the above-discussed modi?cations, namely varying the molecular weights and cross-linking block-copolymers to stabilize the micelles, should not adversely affect the elimination of micelle building blocks out of the body (e.g. smaller building blocks are amenable to kidney ?ltration).

351

Importance of micelles’ integrity assessment in sera, in cells, and in vivo in animals Drug delivery vehicles should not fail to deliver drugs, and one of the critical parameters in achieving this goal is the vehicles’ integrity. In that regard, the properties of incorporated drugs and vehicles’ ability to incorporate and deliver such drugs must be kept in mind. For example, hydrophobic drugs are commonly potent, sparingly water-soluble molecules with adverse and unwanted side effects (Scheme 5A, free drug) that can be solubilized in delivery systems such as micelles to provide more localized delivery (Scheme 5A). Micelles can solubilize hydrophobic drugs at concentrations several hundred times greater than those attainable in water (Table I). Therefore, should the drug be released from the micelles suddenly and fully in an uncontrolled fashion, it

Scheme 5. The importance of the integrity of delivery vehicles such as micelles and a ?uorogenic-based approach of integrity assessment under physiologically relevant conditions.

? et al. 352 R. Savic would immediately precipitate (Scheme 5B). The ability of both actively and passively targeted micelles to resist premature disassembly and drug loss is critical to their success as drug carriers (Scheme 5B). Challenges to their integrities arise through contacts with biological ?uids, macromolecules and cells encountered in vivo and only suf?ciently stable carriers will succeed in improving the poor drug solubility and pharmacokinetics of selected drugs. However, this fundamental parameter of micelle integrity remained under investigated in vitro and most notably no data was available in vivo (Kabanov and Alakhov 2002). During the preparation of this review, an important step towards monitoring the integrity of micelles in ? et al. 2006a). To vitro and in vivo has been made (Savic address the integrity of micelles under complex biological conditions we have used a ?uorogenic? based approach (Scheme 5C, modi?ed from Savic et al. 2006b). The integrity was assessed in a time and concentration dependent manner in sera and in cells, and a proof of principle was obtained in vivo in mice. Upon disruption of micelle integrity the activation (cleavage of ester groups of ?uorescein derivative) of the inherently non-?uorescent dye attached to the polymer yielded a ?uorescence signal allowing the integrity of micelles in media, sera, cells and in vivo to ? et al. 2006a; Scheme 5C). A be assessed (Savic detailed discussion of the results and relevance of the ? et al. integrity assessment of micelles is available (Savic 2006a) and will not be repeated here. Markedly, with a suspected need for improvement of the integrity of block copolymer micelles to enhance their performance in clinical trials, examination of their integrity in vivo using ?uorogenic-based or similar approaches will be an essential step towards development of improved self-assembled drug delivery vehicles with drug release pro?les and elimination kinetics tailored for achieving desired pharmacological effects. Correspondingly, looking at the pre-clinical investigations of mechanisms of the internalization of micelles and cellular fate of the incorporated drugs, the investigations of the micelle integrity may facilitate the dissection of the exact contributions of factors involved in the internalization of micelles and incorporated drugs (Scheme 4). Conclusions and summary It is dif?cult to answer the question posed in the abstract of this review crisply and clearly with critical data on micelle– cell interactions still largely emerging. Logic would dictate that if one aims to kill the cells, as in cancer, any possible cytocidal side effects due to polymer would be complementary as long as they did not occur in healthy, non-target cells. However, since we cannot exclude the polymer accumulation in nontarget cells and tissues (Jewell et al. 1997; Batrakova et al. 2004), or if we consider cell-rescue, rather than cell demise, the issue of toxicity becomes critical and more detailed studies are needed for these effects to be accounted for. Novel reports investigating polymerrelated changes in cells’ gene expression pro?les (Kabanov et al. 2005; Minko et al. 2005) are encouraging steps in this regard. The modest improvements in pharmacokinetic parameters of micelle-incorporated drugs in clinical trials have to be addressed as well, most notably with attention to improved stability of the micelles to preserve their integrity and payload after administration. The drug must be released to act, but it should not be lost en route to target in an uncontrolled, premature manner. To insure the effectiveness of micelle-incorporated drugs, we must understand their integrity under physiologically relevant conditions and be able to follow the micelles and drugs from the site of administration to eventually out of the body. Efforts to sort out the mechanisms of internalization of micelles and improve their stability in vivo should be balanced by parallel studies of the polymer retention and toxicity. Further studies are needed to understand exactly where the delivery system goes, when and where is the drug released and how can we further utilize the advances in vehicle design and targeting to overcome the present challenges and realize the full potential of polymeric micelles. Acknowledgement The authors thank Canadian Institutes for Health Research, Canada and Juvenile Diabetes Research Foundation. References
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