pH-Responsive Copolymer Assemblies for Controlled Release of

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pH-Responsive Copolymer Assemblies for Controlled Release of Doxorubicin Elizabeth R. Gillies and Jean M. J. Fre´chet* Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, California 94720-1460. Received June 26, 2004; Revised Manuscript Received December 13, 2004

pH-Responsive drug carriers have the potential to provide selective drug release at therapeutic targets including tumors and in acidic intracellular vesicles such as endosomes and lysosomes. We have developed a new approach to the design of acid-sensitive micelles by incorporating hydrophobic acetal groups on the core block of a micelle-forming block copolymer. Hydrolysis of the acetals at mildly acidic pH is designed to reveal polar groups on the core-forming block, thus changing its solubility and disrupting the micelle, triggering drug release. The anticancer drug doxorubicin (DOX) was encapsulated in these pH-sensitive micelles, and the acetal hydrolysis rates and DOX release rates were determined in the pH range of 4.0 to 7.4 and were compared to those of control systems. The micelle disruption was investigated by dynamic light scattering. The in vitro toxicities of the empty and DOX-loaded micelles were determined, and the intracellular fate of the encapsulated DOX was compared to free DOX using fluorescence confocal microscopy.

INTRODUCTION

In recent years there has been increased interest in the development of drug delivery systems to improve the properties, increase the effectiveness, and reduce the harmful side effects of therapeutic molecules. Micelles formed by the assembly of amphiphilic block copolymers in aqueous solution have been investigated as potential drug carriers and offer many attractive characteristics (1-3). For example, these nanocontainers are capable of encapsulating hydrophobic drugs in their core, thus improving the drug’s water solubility. In addition, the size of copolymer micelles, typically between 20 and 100 nm, is effective in avoiding rapid renal exclusion, but is also small enough to avoid undesirable uptake by the reticuloendothelial system (4). Therefore, the circulation of the micellar carrier and encapsulated drug is prolonged, but since the micelles are composed of polymer chains that are small enough to be eliminated by renal filtration, the eventual disintegration of the micelle will allow the polymer to be excreted. This is important since the long-term build-up of polymer in the body could lead to toxicity. The size and prolonged circulation of copolymer micelles also facilitates their passive accumulation at pathological sites such as tumors where the vasculature has increased permeability (5). This selective targeting of the drug carrier is ideal in terms of increasing the effectiveness and lowering the systemic toxicity of the drug payload. An important issue determining the effectiveness of a micellar drug carrier is the ability to control the location and time over which drug release occurs. This challenge has motivated the development of micelle systems that are designed to release their drug load in a controlled manner, upon arrival at the target site. For example, temperature has been used to modulate drug release from thermoresponsive micelles (6, 7), while ultrasound has * To whom correspondence should be addressed. E-mail: [email protected].

been reported to trigger drug release from Pluronic micelles (8, 9). Both these approaches require the presence of external stimuli, but it is also possible to exploit naturally occurring changes in the physiological environment. Change in acidity is a particularly useful stimulus to consider in the development of drug carriers because of the numerous pH gradients that exist in both normal and pathophysiological states. For example, it is well documented that the extracellular pH of tumors is slightly more acidic than the blood and normal tissue (10-12). In addition, it is proposed that micelles are taken up by cells via an endocytosis process (13, 14). Although the endocytic pathway begins near the physiological pH of 7.4, it drops to a lower pH of 5.5-6.0 in endosomes and approaches pH 4.5-5.0 in lysosomes (15). Therefore, polymeric micelles that are responsive to pH can be designed to selectively release their payload in tumor tissue or within tumor cells. Several different approaches have been taken to the development of pH-sensitive micelles (16). One approach is to incorporate “titratable” groups such as amines or carboxylic acids into the block copolymers such that the micelle formation is altered by the protonation of these groups. However, only a few of these systems have been demonstrated to undergo transitions in the physiologically relevant pH range of 5.0-7.4 and have the proven capacity to encapsulate drugs (17-21). Another approach that remains less explored is to incorporate acid degradable linkages into the copolymer for the direct attachment of drugs to the copolymer backbone or such that degradation of the linkage provides sufficient structural change in the polymer to trigger drug release. The groups of Park (22) and Kataoka (23) have demonstrated the first approach by attaching the anticancer drug doxorubicin (DOX) to the micelle-forming copolymer by an acidsensitive hydrazone linkage. Heller and co-workers have developed micelles based on poly(ethylene oxide)(PEO)polyortho ester block copolymers where the core-forming polyortho ester block degrades under acidic conditions (24, 25).

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We have recently introduced a new approach to acidsensitive micelles using PEO-dendrimer hybrids as backbones (26, 27). The approach involves the attachment of hydrophobic groups to the periphery of the coreforming dendrimer block by an acid-sensitive acetal linkage. The system is designed such that upon hydrolysis of the linkage and loss of the hydrophobic groups, the core-forming block becomes hydrophilic, thus destabilizing the micelle and enabling escape of the drug from its encapsulating micellar compartment. The stepwise synthesis and controlled multivalency of the PEO-dendrimer backbone afford a high degree of control over the polymer structures. This has allowed us to tune properties such as the rate of micelle disruption, the critical micelle concentration (CMC), and the size of the micelles by adjusting the length of the PEO block, the generation and chemical structure of the dendrimer block, and the method for attaching the hydrophobic acetals to the polymer (27). Here we describe the application of this new approach for the development of a pH-responsive delivery system for DOX. DOX is an ideal candidate for incorporation into this micelle system because it has previously been shown to benefit from the increased circulation time, tumor targeting, and decreased systemic toxicity provided by drug delivery systems such as liposomes (28-30), polymeric micelles (31, 32), and polymer-drug conjugates (33, 34). The loading and pH-dependent release of DOX from micelles based on a PEO-dendritic polyester hybrid are described here. Investigations of the micelle degradation by dynamic light scattering (DLS) and UV-visible spectroscopy are also described, and preliminary in vitro studies of the DOX-loaded micelles are reported. MATERIALS AND METHODS

Preparation of DOX-Loaded Micelles. Copolymers 1 and 4 were synthesized as previously reported (26, 35). DOX was loaded into the copolymer micelles using a modification of the previously reported oil/water emulsion method (31, 36). With sonication, DOX (0.75 mg) was dissolved in 0.15 mL of chloroform with 3.0 equiv of NEt3. This solution was added dropwise to a stirred solution containing 2.5 mg of copolymer in 5 mL of water. The resulting emulsion was stirred overnight in the dark at room temperature, open to the atmosphere allowing the chloroform to evaporate. Insoluble, free DOX was removed by centrifugation at 2000g for 5 min. Soluble, free DOX was removed from the micelles by ultrafiltration (triplicate) using a membrane with a molecular weight cutoff of 30 000 g/mol (Amicon YM30). DOX loading was quantified by absorbance using a UV-visible spectrometer (CARY 50 Conc) (37). Fluorescence spectra of DOX and DOX-loaded micelles were obtained at a concentration of 10 µg/mL DOX equiv using an ISA/SPEX Fluorolog 3.22 equipped with a 450 W Xe lamp, double excitation and double emission monochromators, and a digital photon-counting photomultiplier. An excitation wavelength of 490 nm was used, and the emission spectra were recorded from 510 to 700 nm. Determination of Acetal Hydrolysis Rates. A solution of DOX-loaded micelles was adjusted to a buffer concentration of 0.1 M at the desired pH (pH 4.0, 5.0: acetate; pH 6.0: citrate; pH 7.4: phosphate). A micelle concentration of approximately 0.5 mg/mL was used. The samples were stored at 37 °C, and formation of the 2,4,6-trimethoxybenzaldehyde hydrolysis product was detected over time by measuring the absorbance of the sample at 290 nm in a quartz cuvette having a path length of 1 mm. To determine the absorbance at 100%

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hydrolysis, a drop of concentrated HCl was added to the sample to effect complete hydrolysis. Determination of DOX Release Rates. A solution of DOX or DOX-loaded micelles (approximately 20 µg/ mL DOX equiv) was added to a Slid-A-Lyzer dialysis cassette (Pierce) with a molecular weight cutoff of 10 000. The solution was dialyzed at 37 °C against 0.1 M buffer of the required pH (pH 4.0, 5.0: acetate; pH 6.0: citrate; pH 7.4: phosphate). The solution was removed periodically from the dialysis cassette and the absorbance at 490 nm was measured, then the solution was returned to the dialysis cassette. The amount of released DOX was determined by the decrease in absorbance of the solution in the dialysis cassette. Dynamic Light Scattering. Micelle sizes were determined by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments) equipped with a 4 mW He-Ne laser at 633 nm. Copolymer concentrations were 1 mg/mL in the appropriate 0.1 M buffer (pH 5.0: acetate; pH 7.4: phosphate). The samples were incubated at 37 °C in the temperature-controlled instrument and size measurements were taken automatically every 20-30 min. over a period of 72 h. Cell Cytotoxicity Assay. MDA-MB-231 breast cancer cells were cultured by the UC Berkeley Cell Culture Facility in a medium consisting of Dulbecco’s Modified Eagle medium (DMEM), containing 10% FBS. The cytotoxicity of the empty micelles, DOX-loaded micelles, and free DOX were determined using the MTT assay (38). Cells were seeded onto a 96-well plate at a density of 1.6 × 104 cells per well in 100 µL of medium and incubated overnight (37 °C, 5% CO2). The medium of each well was then replaced by 100 µL of new medium (DMEM, 10% FBS, 1% penicillin-streptomycin) containing various concentrations of micelles, DOX-loaded micelles, or free DOX. The tests were conducted in replicates of four for each concentration. The cells were incubated for 72 h, the medium was aspirated, and 100 µL of fresh medium was added, followed by 20 µL of MTT solution (5 mg/mL). The cells were incubated for 4 h, and then the medium was carefully aspirated. To the resulting purple crystals was added 200 µL of DMSO, followed by 25 µL of pH 10.5 glycine buffer (0.1 M glycine, 0.1 M NaCl). The optical densities at 570 nm were obtained using a SpectraMAX 190 microplate reader (Molecular Devices). Optical densities measured for wells containing cells that received no polymer or drug were considered to represent 100% growth. Laser Scanning Confocal Microscopy. MDA-MB231 cells (4.8 × 105 cells per well) were seeded onto a six-well plate containing collagen I-coated coverslips (round, 2.2 cm diameter, BD Biosciences). After incubation overnight (37 °C, 5% CO2), the medium was carefully aspirated and replaced with 2.0 mL of medium (DMEM, 10% FBS, 1% penicillin-streptomycin) containing 1.0 µg/ mL DOX equiv of DOX-loaded micelles or free DOX. The cells were incubated for 24 h, and then the medium was carefully removed. The wells were each rinsed twice with 3 mL of PBS. Two milliliters of 1% paraformaldehyde was added, and the cells were fixed for 10 min. The solution was aspirated, the wells were rinsed three times with PBS, and then the coverslips were transferred to glass slides. Images were obtained using a confocal laser scanning microscope (LSM 510, Carl Ziess Inc.) using a 40× objective and an excitation wavelength of 543 nm (He-Ne laser).

pH-Responsive Doxorubicin-Loaded Micelles

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Figure 1. Hydrolysis of acetals on the dendrimer periphery of the micelle-forming copolymer 1 leads to a solubility change designed to disrupt micelle formation and trigger the release of drug. RESULTS AND DISCUSSION

Preparation of DOX-Loaded Micelles. Copolymer 1 (Figure 1), composed of a 10 000 MW PEO block and a third generation polyester dendrimer with cyclic acetals of 2,4,6-trimethoxybenzaldehyde attached to the periphery was prepared as previously reported (27). As illustrated in Figure 1, hydrolysis of the cyclic acetals on the dendrimer periphery releases 2,4,6-trimethoxybenzaldehyde (2) and reveals polar 1,3-diol moieties on the dendrimer periphery to provide 3 after complete hydrolysis. This change in solubility of the dendrimer block is designed to trigger the disruption of the micelle and release of drug. Of the various polymers prepared in previous studies, 1 was selected for development of a DOX delivery system because of the ideal micelle size of 27 nm, a CMC of 40 mg/L, and the relatively rapid rate of micelle degradation (27). DOX was loaded into micelles of 1 by an oil/water emulsion method similar to that described previously by Kataoka and co-workers (31, 36). Chloroform was used as the organic phase, and 3 equiv of NEt3 was used relative to DOX, as the drug is known to partition most effectively into the chloroform phase and into the micelle upon deprotonation of the glycosidic amine (31). Using 30 wt % of DOX relative to copolymer, a drug loading of approximately 12 wt % was typically obtained after centrifugation to remove insoluble free DOX particles and ultrafiltration to remove soluble unencapsulated DOX. DOX loadings were determined according to absorbance using UV-visible spectroscopy (37). As shown in Figure 2, that DOX is encapsulated in the micelles is supported by the significant attenuation in fluorescence (>20 times) observed for DOX-loaded micelles relative to a solution of free DOX of the same absorbance. This fluorescence attenuation has been observed previously for micellar DOX solutions and is attributed to fluorescence selfquenching of the drug molecules, which are within close proximity at the micelle core (36, 37). Hydrolysis of Acetals at the Micelle Core. A cyclic acetal composed of 2,4,6-trimethoxybenzaldehyde and the 1,3-diol moiety of serinol was selected as the pH-sensitive

Figure 2. Fluorescence quenching of DOX-loaded micelles compared to free DOX (normalized for absorbance).

linkage for this system. While cyclic acetals are known to hydrolyze quite slowly in comparison to noncyclic acetals (39), the introduction of electron-donating methoxy groups at the ortho and para positions on the aromatic ring accelerates the hydrolysis rate significantly. We have also found that when these acetals are incorporated into the core block of a micelle-forming copolymer, the hydrolysis half-life is strongly dependent on the chemical environment at the micelle core. For example, while the half-life for a water-soluble low molecular weight model compound containing the same acetal is 1 h at pH 5, and several days at pH 7.4 (27), the rate is slowed to 50% hydrolysis after 4 h at pH 5.0 for micelles of copolymer 1 (27). This rate can be accelerated by the incorporation of hydrophilic spacers between the acetal and the dendrimer periphery that make the micelle core more hydrophilic, or slowed further by use

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Figure 3. Hydrolysis rates of the acetals in DOX-loaded micelles of copolymer 1 at pH’s ranging from 4 to 7.4.

of an even more hydrophobic linkage between the acetal and dendrimer or increasing the generation of the hydrophobic dendrimer block, making the dendrimer core more hydrophobic (27). Therefore, we were interested in the effect on the hydrolysis rate of incorporating DOX into the micelle core. The acetal hydrolysis rates at pH’s ranging from 4 to 7.4 were investigated for the DOXloaded micelles of copolymer 1. Production of 2,4,6trimethoxybenzaldehyde upon hydrolysis was detected by its absorbance at 290 nm. As shown in Figure 3, the amount of hydrolysis at pH 7.4 is quite negligible over a period of 24 h, while approximately 20% hydrolysis occurs at pH 6 over the same time period. At pH 4.0, the rate is rapid, with 50% hydrolysis after less than 30 min and at pH 5.0 the rate is very similar to that of the empty micelles with 50% hydrolysis after approximately 3 h. Therefore, the incorporation of DOX into the micelle core has only a small effect on the acetal hydrolysis rate. This may be because the hydrophobic aromatic portion of the DOX molecule is balanced by the hydrophilic glycosidic fragment, thus giving only a small change in the overall polarity of the micelle core. pH-Dependent Release of DOX from the Micelles. The release of DOX from the micelles at different pH’s

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was investigated using a dialysis procedure. A solution of the DOX-loaded micelles in a dialysis cassette was dialyzed against 100 mM buffer of the desired pH and the solution in the cassette was sampled at various times over a 72 h period to determine the amount of DOX remaining in the micelles. As shown in Figure 4a, a significant amount of DOX is released in a pH-dependent manner in the pH range of 4.0-6.0, while at pH 7.4 the system is very stable with less than 10% of the DOX released over 72 h. As a control, the release of free DOX from a dialysis cassette was investigated at pH 5.0 and pH 7.4. As illustrated in Figure 4b, the release rates are very similar at both pH’s, with essentially complete release after less than 10 h. This is consistent with the expected rate of diffusion for low molecular weight molecules across the dialysis membrane. Additionally, the release of DOX from micelles formed from copolymer 4 (Figure 5a) was also investigated as a control. In contrast to the highly sensitive cyclic acetals on the dendrimer periphery of 1, the simple benzylidene acetals of 4 do not undergo hydrolysis at mildly acidic pH (40). Copolymer 4 was prepared as previously reported (35), and the micelles were prepared and loaded with DOX as described above for micelles of copolymer 1. As shown in Figure 5b, the release rates of DOX from these micelles at both pH 5.0 and pH 7.4 are significant, with somewhat faster release at pH 5.0 as observed previously by Kataoka and coworkers using a pH-insensitive micelle system based on a PEO-poly(benzylasparate) block copolymer (31). These results indicate that the release of DOX from micelles is generally pH-dependent to some degree due to the increased aqueous solubility of DOX at mildly acid pH; However, the pH-sensitive micelle system based on copolymer 1 shows a stronger dependence of the release rate on pH (i.e. a greater difference between the rates at pH 7.4 and pH 5.0), suggesting that the hydrolysis of the pH-sensitive acetals likely plays a role. In addition, the increased stability of the pH-sensitive system at pH 7.4 is advantageous so that DOX will not be released during blood circulation, thus avoiding the undesirable organ accumulation and toxicity associated with the free drug. The slower release rate at pH 7.4 for micelles of 1 versus 4 highlights the fact that subtle changes in the polymer structure have a significant effect on the compatibility

Figure 4. (a) pH-Dependent release of DOX from micelles of copolymer 1. (b) Release of free DOX from a dialysis cassette.

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Figure 5. (a) Chemical structure of copolymer 4 forming micelles that are not pH-sensitive at mildly acidic pH. (b) Release of DOX from the pH-insensitive micelles.

Figure 6. Dynamic light scattering measurements on micelles of copolymer 1: (a) Degradation of empty micelles at pH 5.0. (b) Disruption of DOX-loaded micelles at pH 5.0. (c) Stability of DOX-loaded micelles at pH 7.4.

of the micelle core with the drug, thus altering the drug’s overall release rate, and making the design of ideal model systems challenging. While exhibiting high stability under neutral conditions, the release of DOX from micelles of copolymer 1 at pH 5.0 is somewhat faster than that reported for other micelle systems where DOX is covalently bound to the polymer by a pH-sensitive linkage (22, 23). Therefore, overall these results indicate that the pH-sensitive DOX-loaded micelles of 1 have characteristics ideal for the selective release of DOX in mildly acidic physiological environments. Investigation of Micelle Degradation by Dynamic Light Scattering. Although DOX is steadily released from the micelles of copolymer 1 at mildly acidic pH, the release rates are generally slower than the acetal hydrolysis rates. Furthermore, even after 100% acetal hydrolysis, not all of the DOX is immediately released. Therefore, we were interested in probing the degradation of the DOX-loaded micelles by dynamic light scattering (DLS) to determine the time dependence of the micelle size. While the empty micelles have an initial volume average diameter of 27 nm, the DOX-loaded micelles are somewhat larger with a diameter of 35 nm and a small fraction of aggregates in the size range of 200-400 nm is observed. As shown in Figure 6a, the size of the empty

micelles decreases over several h at pH 5.0, consistent with the disintegration of the micelles into soluble copolymer molecules. In contrast, over several h at pH 5.0, the size of the DOX-loaded micelles increases and the fraction of aggregates in the population becomes greater as shown in Figure 6b. This aggregation probably occurs upon disruption of the micelles due to acetal hydrolysis and is facilitated by the tendency of DOX to form aggregates by π-stacking. The lack of appearance of a population with a size less than 10 nm indicates that after acetal hydrolysis, the copolymers are probably associated with the aggregates rather than being dissociated in solution. It is likely that some DOX release occurs during rearrangement of the micelles to aggregates, while the remainder of the release over a longer time period occurs from the aggregates and is assisted by the increased water solubility of DOX at acidic pH upon protonation of the glycosidic amine. At pH 7.4, the micelles are stable over several days, with no significant changes in the size distribution over this time period (Figure 6c). In Vitro Investigation of DOX-Loaded Micelles. Preliminary in vitro studies were conducted to compare the toxicities of the empty micelles, DOX-loaded micelles, and free DOX in MDA-MB-231 breast cancer cells using

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Figure 7. In vitro toxicity of (a) empty micelles and (b) free DOX and DOX-loaded micelles of copolymer 1 with MDA-MB-231 cells after a 72 h incubation (MTT assay).

Figure 8. Laser scanning confocal microscopy images of (a) DOX and (b) DOX-loaded pH-sensitive micelles incubated with MDAMB-231 cells for 24 h.

the MTT assay (38). As shown in Figure 7a, the empty micelles of copolymer 1 are nontoxic at concentrations up to 1 mg/mL, with an IC50 of approximately 2 mg/mL. The toxicity of the DOX-loaded micelles was investigated at a constant, nontoxic copolymer concentration of 0.2 mg/ mL, and the concentration of DOX was varied. As shown in Figure 7b, above 1 µg/mL DOX equiv, the DOX-loaded micelles were toxic and an IC50 of approximately 3 µg/ mL was determined. For comparison, the toxicity of free DOX was also investigated and the IC50 was found to be about 0.8 µg/mL. The fact that the micelle formulation is toxic, with an IC50 on the same order of magnitude as free DOX is consistent with the release of free and active DOX in the cells and is encouraging for the therapeutic potential of the system. The somewhat lower toxicity of the micelle system may result from the gradual release of DOX within the cell and from differences in the released drug’s cellular localization relative to the free drug. However, the potential for the selective accumulation of the micelle system in tumor tissue by the enhanced permeation and retention effect may enhance its overall therapeutic efficacy in vivo relative to free DOX (5).

The intracellular localization of both free DOX and DOX-loaded micelles was investigated using confocal microscopy. As shown in Figure 8a, cells exposed to free DOX show significant accumulation of DOX in the nucleus after 24 h, consistent with the results of previous studies (41). In contrast, cells exposed to DOX-loaded pHsensitive micelles have a punctate fluorescence that is concentrated in the cytoplasm after 24 h as shown in Figure 8b. These observations are important for several reasons. First, the absence of DOX fluorescence in the nucleus suggests that the micelles are stable in the presence of cells and serum-containing cell medium, as the rapid destabilization of the micelles in the extracellular environment and subsequent release of DOX outside the cell would be expected to result in an image similar to that observed for free DOX. In addition, the fluorescence in the cytoplasm suggests that the DOXloaded micelles are indeed taken up by cells, and its punctate nature is consistent with the localization of the drug in subcellular organelles. While it is not possible with these data to determine the precise mechanism of cellular uptake or localization of the DOX-loaded micelles or the released drug, comparisons with similar images

pH-Responsive Doxorubicin-Loaded Micelles

previously reported for both pH-sensitive DOX-loaded micelles (23) and polymer-DOX conjugates (42) suggest that the fluorescence may be associated with organelles such as lysosomes, mitochondria, and the golgi apparatus. While one important mechanism of action of DOX involves DNA intercalation (43), other mechanisms have also been proposed, including the inhibition of mitochondrial function (44), thus explaining the toxicity of this and other delivery systems that do not lead to the accumulation of DOX in the nucleus. More in vitro studies with these DOX-loaded pH-sensitive micelles will be necessary in order to further explore their mechanism of cell uptake and intracellular trafficking. CONCLUSIONS

In summary, the anticancer drug doxorubicin was encapsulated in pH-sensitive micelles formed from a PEO-dendritic polyester copolymer with acid-labile acetal groups on the core-forming dendrimer periphery. It was found that the acetal groups undergo hydrolysis and that DOX is selectively released at acidic pH’s such as those encountered in tumor tissue and in endocytic vesicles including endosomes and lysosomes. In comparison with a non-pH-sensitive micelle control, the release of DOX from the pH-sensitive system was much more dependent on pH, although the release of DOX at acidic pH is clearly to some degree due to its increased aqueous solubility. Dynamic light scattering studies revealed that while the pH-sensitive micelles are stable at pH 7.4, acetal hydrolysis at acidic pH results in disruption of the micelles and rearrangment to form larger aggregates. In vitro toxicity studies revealed that the empty micelles are relatively nontoxic, while DOX encapsulated in the micelles has a toxicity quite similar to that of the free drug. Laser scanning confocal microscopy images support the localization of DOX in intracellular organelles in contrast to free DOX, which is localized in the cell nucleus after 24 h. This suggests that the DOX-loaded micelles are indeed taken up by cells and that the mechanism of action of the released drug may differ from that of the free drug. Future in vitro and in vivo studies will further explore the biological properties of these micelles. Thus far, these new pH-responsive drug carriers show promise for the controlled release of therapeutics in mildly acidic physiological environments. ACKNOWLEDGMENT

We thank the National Institute of Health (GM 65361 and EB 002047) and the U.S. Department of Energy (DEAC03-765F00098) for support of this research. We thank Ann Fisher and Young Kwon for help with in vitro studies. The Center for New Directions in Organic Synthesis is supported by Bristol-Myers Squibb as a Sponsoring Member and Novartis Pharma as Supporting Member. LITERATURE CITED (1) Kataoka, K., Harada, A., and Nagasaki, Y. (2001) Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Delivery Rev. 47, 113131. (2) Lavasanifar, A., Samuel, J., and Kwon, G. S. (2002) Poly(ethylene oxide)-block-poly(L-amino acid) micelles for drug delivery. Adv. Drug Delivery Rev. 54, 169-190. (3) Kabanov, A. V., Batrakova, E. V., and Alakhov, V. Y. (2002) Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery. J. Controlled Release 82, 189-212. (4) Kwon, G. S., and Okano, T. (1996) Polymeric micelles as new drug carriers. Adv. Drug Delivery Rev. 21, 107-116.

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