Synthesis,antitumor activity and molecular mechanism of doxorubicin conjugated trimethyl-chitosan polymeric micelle loading Beclin1 siRNA for drug-resisted bladder cancer therapy

Herein, we describe a convenient approach for the preparation of a polymeric micelle using doxorubicin (DOX) conjugated trimethyl-chitosan (TMC) with Beclin-1 siRNA (Si-Beclin1/DOX-TMC). This micelle displayed a potent capacity for autophagy inhibition and reversed drug-resistance to DOX in BIU-87/ADR cell lines. The Si-Beclin1/DOX-TMC micelle was highly cytotoxic to both drug-sensitive BIU-87 and drug-resistant BIU-87/ADR cells. Its capacity to reverse drug-resistance was dependent upon upregulation of autophagy levels in BIU-87/ADR cells. DOX was conjugated to TMC via a pH-sensitive Schiff base, which responded to the acidic lysosome microenvironment and resulted in the cytoplasmic release of DOX. The structure of DOX conjugation to the TMC polymeric micelle was characterized by NMR, GPC, TEM and DLS. DOX release profiles in different pH environment were determined by HPLC. Cellular uptake, changes to nuclei morphology and formation of autophagosomes were observed using a fluorescence microscope. Finally, in vivo antitumor activity of systemic Si-Beclin1/DOX-TMC micelle administration was evaluated in BIU-87/ADR xenograft models and Si-Beclin1/DOX-TMC micelles showed significantly suppressed tumor growth.

Herein, we describe a convenient approach for the preparation of a polymeric micelle using doxorubicin (DOX) conjugated trimethyl-chitosan (TMC) with Beclin-1 siRNA (Si-Beclin1/DOX-TMC).     

Polymeric micelle for tumor pH and folate-mediated targeting.

Novel pH-sensitive polymeric mixed micelles composed of poly(L-histidine) (polyHis; M(w) 5000)/PEG (M(n) 2000) and poly(L-lactic acid) (PLLA) (M(n) 3000)/PEG (M(n) 2000) block copolymers with or without folate conjugation were prepared by diafiltration. The micelles were investigated for pH-dependent drug release, folate receptor-mediated internalization and cytotoxicity using MCF-7 cells in vitro. The polyHis/PEG micelles showed accelerated adriamycin release as the pH decreased from 8.0. When the cumulative release for 24 h was plotted as a function of pH, the gradual transition in release rate appeared in a pH range from 8.0 to 6.8. In order to tailor the triggering pH of the polymeric micelles to the more acidic extracellular pH of tumors, while improving the micelle stability at pH 7.4, the PLLA/PEG block copolymer was blended with polyHis/PEG to form mixed micelles. Blending shifted the triggering pH to a lower value. Depending on the amount of PLLA/PEG, the mixed micelles were destabilized in the pH range of 7.2-6.6 (triggering pH for adriamycin release). When the mixed micelles were conjugated with folic acid, the in vitro results demonstrated that the micelles were more effective in tumor cell kill due to accelerated drug release and folate receptor-mediated tumor uptake. In addition, after internalization polyHis was found to be effective for cytosolic ADR delivery by virtue of fusogenic activity. This approach is expected to be useful for treatment of solid tumors in vivo.     

Folate receptor targeted biodegradable polymeric doxorubicin micelles.

Biodegradable polymeric micelles, self-assembled from a di-block copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG), were prepared to achieve folate receptor targeted delivery of doxorubicin (DOX). In the di-block copolymer structure of PLGA-b-PEG, DOX was chemically conjugated to a terminal end of PLGA to produce DOX-PLGA-mPEG, and folate was separately conjugated to a terminal end of PEG to produce PLGA-PEG-FOL. The two di-block copolymers with different functional moieties at their chains ends were physically mixed with free base DOX in an aqueous solution to form mixed micelles. It was expected that folate moieties were exposed on the micellar surface, while DOX was physically and chemically entrapped in the core of micelles. Flow cytometry and confocal image analysis revealed that folate conjugated mixed micelles exhibited far greater extent of cellular uptake than folate unconjugated micelles against KB cells over-expressing folate receptors on the surface. They also showed higher cytotoxicity than DOX, suggesting that folate receptor medicated endocytosis of the micelles played an important role in transporting an increased amount of DOX within cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that when systemically administered, tumor volume was significantly regressed. Biodistribution studies also indicated that an increased amount of DOX was accumulated in the tumor tissue.     

Novel biomimetic polymersomes as polymer therapeutics for drug delivery.

Novel amphiphilic diblock copolymers, cholesterol-end-capped poly(2-methacryloyloxyethyl phosphorylcholine) (CMPC), which have poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) as hydrophilic segment and cholesterol as hydrophobic segment, was specially designed as drug delivery systems. Fluorescence probe technique and transmission electron microscope (TEM) characterizations indicated that this novel amphiphilic copolymer formed micelles structure in water and the critical micelle concentration (CMC) was determined to be 1.57 x 10(-7) mol/l. A commercial obtained polymeric amphiphiles, Cholesterol end capped PEO (CPEO), which had a similar structure with CMPC, was used as a control in the cytotoxicity test. While CPEO showed obvious cytotoxicity, cytotoxicity of this novel amphiphiles was not observed as indicated by cell culture. Anti-cancer drug adriamycin (ADR) was incorporated into the micelles by oil-in-water method. The size of the drug-containing micelles was less than 200 nm, and the size distribution of the drug-containing micelles showed a narrow and monodisperse unimodal pattern. The release rate of ADR from the nanosphere was slow and the release continued over 7 days and the release rate decreased with the increase of molecular weights of the copolymer and the amount of the drug entrapped. These experimental results suggested that the nanoparticles prepared from CMPC block copolymers could be a good candidate for injectable drug delivery carrier.     

Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles: their pharmaceutical characteristics and biological significance.

Doxorubicin (DOX) was physically loaded into micelles prepared from poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) block copolymer (PEG-PBLA) by an o/w emulsion method with a substantial drug loading level (15 to 20 w/w%). DOX-loaded micelles were narrowly distributed in size with diameters of approximately 50-70 nm. Dimer derivatives of DOX as well as DOX itself were revealed to be entrapped in the micelle, the former seems to improve micelle stability due to its low water solubility and possible interaction with benzyl residues of PBLA segments through pi-pi stacking. Release of DOX compounds from the micelles proceeded in two stages: an initial rapid release was followed by a stage of slow and long-lasting release of DOX. Acceleration of DOX release can be obtained by lowering the surrounding pH from 7.4 to 5.0, suggesting a pH-sensitive release of DOX from the micelles. A remarkable improvement in blood circulation of DOX was achieved by use of PEG-PBLA micelle as a carrier presumably due to the reduced reticuloendothelial system uptake of the micelles through a steric stabilization mechanism. Finally, DOX loaded in the micelle showed a considerably higher antitumor activity compared to free DOX against mouse C26 tumor by i.v. injection, indicating a promising feature for PEG-PBLA micelle as a long-circulating carrier system useful in modulated drug delivery.     

Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer.

Biodegradable polymeric micelles containing doxorubicin in the core region were prepared from a di-block copolymer composed of doxorubicin-conjugated poly(DL-lactic-co-glycolic acid) (PLGA) and polyethyleneglycol (PEG). The di-block copolymer of PLGA-PEG was first synthesized and the primary amino group of doxorubicin was then conjugated to the terminal hydroxyl group of PLGA, which had been pre-activated using p-nitrophenyl chloroformate. The resulting polymeric micelles in aqueous solution were characterized by measurement of size, drug loading, and critical micelle concentration. The micelles containing chemically-conjugated doxorubicin exhibited a more sustained release profile than PEG-PLGA micelles containing physically-entrapped doxorubicin. The cytotoxic activity of the micelles against HepG2 cells was greater than free doxorubicin, suggesting that the micelles containing conjugated doxorubicin were more effectively taken up cellularly, by an endocytosis mechanism rather than by passive diffusion. Confocal microscopic observation and flow cytometry analysis supported the enhanced cellular uptake of the micelles.     

Process design for efficient and controlled drug incorporation into polymeric micelle carrier systems.

For the efficient and well-controlled incorporation of the anti cancer drug adriamycin (ADR) into the inner core of a thermo-responsive polymeric micelle carrier system, we have analyzed and optimized the incorporation procedure in this paper. A dialysis method was used for preparing the micelle solution and ADR incorporation simultaneously. Quantities of ADR and triethylamine (TEA) were varied and the effects of their quantities were analyzed. Solvent composition at the starting time of dialysis was also varied. The initial dialysis condition, solvent with 40% water, brought about the largest amount and yield of ADR incorporation. With the initial 40% water content, it was considered that the block polymers formed a micelle-like association with a swollen hydrophobic core. This swollen core may be suitable for a large amount of ADR incorporation, since this core, swollen by an organic solvent-water mixture, is expected to show a liquid-state character to allow ADR molecules entry into the cores. By starting the dialysis procedure at this 40% water content, this swollen core suitable for the ADR incorporation is considered to be maintained for a much longer period than a case starting with a polymer-ADR solution in a solvent with a water content of less than 40%, and, therefore, ADR is expected to be incorporated efficiently. Preparation temperature of 20-25 degrees C was found to provide the most effective ADR incorporation in this thermo-responsive polymeric micelle system. These results indicate that the efficient incorporation of ADR can be achieved in consideration of the dynamic micelle formation and drug incorporation processes.     

Enhanced antitumor effect of camptothecin loaded in long-circulating polymeric micelles.

A water-insoluble antitumor agent, camptothecin (CPT) was successfully incorporated into polymeric micelles formed from poly(ethylene glycol)-poly(benzyl aspartate) block copolymers (CPT-loaded polymeric micelles). Antitumor effects and biodistribution of CPT-loaded micelles were evaluated in mice subcutaneously transplanted by colon 26 tumor cells. Tumor growth was significantly inhibited after a single i.v. injection of CPT-loaded polymeric micelles at doses of either 15 or 30 mg/kg. Efficacy of a single high-dose injection was comparable to low dose multiple injections. CPT loaded in polymeric micelles showed prolonged blood circulation and higher accumulation in tumors compared with CPT in solution. Polymeric micelle systems offer a stable and effective platform for cancer chemotherapy with CPT.     

Structure and design of polymeric surfactant-based drug delivery systems.

The review concentrates on the use of polymeric micelles as pharmaceutical carriers. Micellization of biologically active substances is a general phenomenon that increases the bioavailability of lipophilic drugs and nutrients. Currently used low-molecular-weight pharmaceutical surfactants have low toxicity and high solubilization power towards poorly soluble pharmaceuticals. However, micelles made of such surfactants usually have relatively high critical micelle concentration (CMC) and are unstable upon strong dilution (for example, with the blood volume upon intravenous administration). On the other hand, amphiphilic block co-polymers are also known to form spherical micelles in solution. These micelles have very high solubilization capacity and rather low CMC value that makes them very stable in vivo. Amphiphilic block co-polymers suitable for micelle preparation are described and various types of polymeric micelles are considered as well as mechanisms of their formation, factors influencing their stability and disintegration, their loading capacity towards various poorly soluble pharmaceuticals, and their therapeutic potential. The basic mechanisms underlying micelle longevity and steric protection in vivo are considered with a special emphasis on long circulating drug delivery systems. Advantages and disadvantages of micelles when compared with other drug delivery systems are considered. New polymer-lipid amphiphilic compounds such as diacyillipid-polyethylene glycol, are described and discussed. These compounds are very attractive from a practical point of view, since they easily micellize yielding extremely stable micelles with very high loading capacity. Micelle passive accumulation in the areas with leaky vasculature (tumors, infarct zones) is discussed as an important physiology-based mechanism of drug delivery into certain target zones. Targeted polymeric micelles prepared by using thermo- or pH-sensitive components or by attaching specific targeted moieties (such as antibodies) to their outer surface are described as well as their preparation and some in vivo properties. The fast growing field of diagnostic micelles is analyzed. Polymeric micelles are considered loaded with various agents for gamma, magnetic resonance, and computed tomography imaging. Their in vitro and in vivo properties are discussed and the results of the initial animal experiments are presented.     

Thermo-responsive drug delivery from polymeric micelles constructed using block copolymers of poly(N-isopropylacrylamide) and poly(butylmethacrylate).

To achieve a combination of spatial specificity in a passive manner with a stimuli-responsive targeting mechanism, a temperature-responsive polymeric micelle is prepared using block copolymers of (poly(N-isopropylacrylamide-b-butylmethacrylate) (PIPAAm-PBMA)). The micelle inner core formed by self-aggregates of PBMA segments successfully loaded with a drug (adriamycin), and the outer shell of PIPAAm chains played a role of stabilization and initiation of micellar thermo-response. Optimum conditions were investigated for the micelle formation and drug loading into the inner cores in a view of micellar stability and function as drug carriers. Outer shell hydrophilicity that prevents inner core interaction with biocomponents and other micelles can be suddenly switched to hydrophobic at a specific site by local temperature increase beyond the LCST (lower critical solution temperature) (32.5 degrees C). These micelles showed reversible structural changes allowing drug release upon heating/cooling thermal fluctuations through the LCST. Polymeric micelles incorporated with adriamycin showed a dramatic thermo-responsive on/off switching behavior for both drug release and in vitro cytotoxicity according to the temperature responsive structural changes of a micellar shell structure. The reversible and sensitive thermo-response of the micelle opens up opportunities to construct a novel drug delivery system in conjunction with localized hyperthermia.     

Nanoscopic micelle delivery improves the photophysical properties and efficacy of photodynamic therapy of protoporphyrin IX

Nanodelivery systems have shown considerable promise in increasing the solubility and delivery efficiency of hydrophobic photosensitizers for photodynamic therapy (PDT) applications. In this study, we report the preparation and characterization of polymeric micelles that incorporate protoporphyrin IX (PpIX), a potent photosensitizer, using non-covalent encapsulation and covalent conjugation methods. Depending on the incorporation method and PpIX loading percentage, PpIX existed as a monomer, dimer or aggregate in the micelle core. The PpIX state directly affected the fluorescence intensity and ¹O₂ generation efficiency of the resulting micelles in aqueous solution. Micelles with lower PpIX loading density (e.g. 0.2%) showed brighter fluorescence and higher ¹O₂ yield than those with higher PpIX loading density (e.g. 4%) in solution. However, PDT efficacy in H2009 lung cancer cells showed an opposite trend. In particular, 4% PpIX-conjugated micelles demonstrated the largest PDT therapeutic window, as indicated by the highest phototoxicity and relatively low dark toxicity. Results from this study contribute to the fundamental understanding of nanoscopic structure-property relationships of micelle-delivered PpIX and establish a viable micelle formulation (i.e. 4% PpIX-conjugated micelles) for in vivo evaluation of antitumor efficacy.     

Heparin conjugated polymeric micelle for long-term delivery of basic fibroblast growth factor.

Heparin conjugated amphiphilic block copolymer, Tetronic-PCL-heparin (TCH), was developed and its polymeric micelles (PMs) were prepared as an injectable vehicle for long-term delivery of bFGF, which is one of the heparin-binding growth factors (HBGF). TCH PMs were fabricated by a single emulsion and solvent evaporation method. The structural properties of TCH were confirmed by (1)H NMR, FT-IR and GPC. The contents of bound heparin were 0.44 micro g/micro g and the heparin activity by APTT assay was 43.6% when compared to free heparin. The critical micelle concentration (CMC) of TCH PMs was approximately 0.11 g/l. The diameter of TC micelle was approximately 25 nm and its size after conjugation of heparin was increased to 114 nm due to the heparin molecules on the shell of the micelle. The bFGF loading amount of TCH PMs was considerably higher than that of TC, caused by specific interactions between heparin and bFGF. In vitro study, bFGF was released from TCH PMs in a controlled manner over 2 months. The results demonstrated that TCH PMs become a novel candidate for the long-term delivery of various growth factors with heparin-binding domain in tissue engineering.     

In vivo gene delivery into ocular tissues by eye drops of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles.

The primary objective of this study was to investigate the feasibility of using PEO-PPO-PEO non-ionic copolymeric micelles as a carrier for eye-drop gene delivery of plasmid DNA with lacZ gene in vivo. Using pyrene fluorescence probe methods, zeta potential, and dynamic light scattering test (DLS), the ability of micelle formation of these block copolymers with plasmid was studied. Gene expressions were visualized by both the quality of enzymatic color reaction using X-gal staining and by the quantification of the substrate chlorophenol red galactopyranoside (CPRG) in enucleated eyes on day 2 after gene transfer. In addition, microscopy to identify the types of cell showing uptake and expression of the transferred gene was used. We found that the block polymeric micelles were formed above 0.1% (w/v) of block copolymer with a size of 160 nm and a zeta potential of -4.4 mV. After 2 days of topically delivery three times a day, the most intense gene expression was observed on days 2 and 3. Reporter expression was detected around the iris, sclera, conjunctiva, and lateral rectus muscle of rabbit eyes and also in the intraocular tissues of nude mice upon in vivo topical application for 48 h with a DNA/polymeric micelle formulation. Furthermore, after two enhancement treatments, the transport mechanisms of the block copolymeric micelles were found through endocytosis in tissues by enhancement through the tight junction pathway. Thus, efficient and stable transfer of the functional gene could be achieved with PEO-PPO-PEO polymeric micelles through topical delivery in mice and rabbits. These in vivo experiments indicate the possible potential use of block copolymers for DNA transfer.     

Amphiphilic triblock copolymer poly(p-dioxanone-co-L-lactide)-block-poly(ethylene glycol), enhancement of gene expression and inhibition of lung metastasis by aerosol delivery

We describe the development of an aerosol system for topical gene delivery to the lungs of C57BL/6 mice. This system is based on the combination of the commercial cationic lipid Lipofectin with a novel amphiphilic triblock copolymer, poly(p-dioxanone-co-L-lactide)-block-poly(ethylene glycol) (PPDO/PLLA-b-PEG, and abbreviated in the text as polymeric micelles). After optimizing conditions for DNA delivery to the lungs of mice using the combination of polymeric micelles with Lipofectin and LacZ DNA, we used the Lipofectin/polymeric micelle system to deliver the tumor suppressor gene PTEN to the lungs of C57BL/6 mice bearing the B16-F10 melanoma. Lipofectin/PTEN/polymeric micelles significantly improved gene expression of PTEN in the lungs of mice with no evidence of cell toxicity or acute inflammation. Importantly, lung metastasis, as measured by lung weight, was significantly reduced (P<0.001), as were total tumor foci in the lungs (P<0.001) and size of individual tumor nodules in animals treated with Lipofectin/PTEN/polymeric micelles compared with control animals. Survival time was also extended. These results suggest that the Lipofectin/polymeric micelle system is appropriate for enhancing gene delivery in vivo and that it can be applied as a non-invasive gene therapy for lung cancer.     

Doxorubicin-conjugated biodegradable polymeric micelles having acid-cleavable linkages.

Doxorubicin was chemically conjugated to the terminal end of a di-block copolymer composed of poly(L-lactic acid) (PLLA) and methoxy-poly(ethylene glycol) (mPEG) via two acid-cleavable linkages. A hydrazone bond and a cis-acotinyl bond were formed between doxorubicin and the terminal group of PLLA segment in the block copolymer. Doxorubicin-conjugated PLLA-mPEG di-block copolymers self-assembled to form micelles in aqueous solution. The doxorubicin-conjugated micelles were about 89.1 nm in diameter and their critical micelle concentration was 1.3 microg/ml. These values were comparable with those of unconjugated micelles. In an acidic condition, the conjugated doxorubicin in the hydrazone linkage was readily cleaved, releasing doxorubicin in an intact structure. Doxorubicin-conjugated PLLA-mPEG micelles were more potent in cell cytotoxicity than free doxorubicin, suggesting that they were more easily taken up within cells with concomitant rapid release of cleaved doxorubicin into the cytoplasm from acidic endosomes.     

TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors.

A novel drug targeting system for acidic solid tumors has been developed based on ultra pH-sensitive polymer and cell penetrating TAT. The delivery system consisted of two components: 1) A polymeric micelle that has a hydrophobic core made of poly(l-lactic acid) (PLLA) and a hydrophilic shell consisting of polyethylene glycol (PEG) conjugated to TAT (TAT micelle), 2) an ultra pH-sensitive diblock copolymer of poly(methacryloyl sulfadimethoxine) (PSD) and PEG (PSD-b-PEG). The anionic PSD is complexed with cationic TAT of the micelles to achieve the final carrier, which could systemically shield the micelles and expose them at slightly acidic tumor pH. TAT micelles had particle sizes between 20 and 45 nm and their critical micelle concentrations were 3.5 mg/l to 5.5 mg/l. The TAT micelles, upon mixing with pH-sensitive PSD-b-PEG, showed a slight increase in particle size between pH 8.0 and 6.8 (60-90 nm), indicating complexation. As the pH was decreased (pH 6.6 to 6.0) two populations were observed, one that of normal TAT micelles (45 nm) and the other of aggregated hydrophobic PSD-b-PEG. Zeta potential measurements showed similar trend substantiating the shielding/deshielding process. Flow cytometry and confocal microscopy showed significantly higher uptake of TAT micelles at pH 6.6 compared to pH 7.4 indicating shielding at normal pH and deshielding at tumor pH. The confocal microscopy indicated that the TAT not only translocates into the cells but is also seen on the surface of the nucleus. These results strongly indicate that the above micelles would be able to target any hydrophobic drug near the nucleus.     

Molecular design of biodegradable polymeric micelles for temperature-responsive drug release.

We designed thermo-responsive and biodegradable polymeric micelles for an ideal drug delivery system whose target sites are where external stimuli selectively release drugs from the polymeric micelles. The thermo-responsive micelles formed from block copolymers that were composed both of a hydrophobic block and a thermo-responsive block. Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) showing a lower critical solution temperature (LCST) around 40 degrees C was synthesized for the thermo-responsive block, while biodegradable poly(D,L-lactide), poly(epsilon-caprolactone), or poly(D,L-lactide-co-epsilon-caprolactone) was used for the hydrophobic block. By changing both the block lengths of the poly(D,L-lactide)-containing block copolymers, physical parameters such as micelle diameter and critical micelle concentration were varied. On the other hand, the choice of the hydrophobic block was revealed to be critical in relation to both on the thermo-responsive release of the incorporated anti-cancer drug, doxorubicin, and the temperature-dependent change of the hydrophobicity of the micelles' inner core. One polymeric micelle composition successfully exhibited rapid and thermo-responsive drug release while possessing a biodegradable character.     

A polymeric micelle MRI contrast agent with changeable relaxivity.

Polymeric micelles were formed from cationic polymers (polyallylamine or protamine) and anionic block copolymers (poly(ethylene glycol)-b-poly(aspartic acid) derivative) that bound Gd ions providing high contrasts in Magnetic Resonance Imaging (MRI) by shortening the T(1) longitudinal relaxation time of protons of water. The Gd-binding block copolymer alone showed high relaxivity (T(1)-shortening ability) values from 10 to 11 mol(-1) s(-1), while the polymeric micelles exhibited low relaxivity values from 2.1 to 3.6 mol(-1) s(-1). These findings point to the feasibility of a novel MRI contrast agent that selectively provides high contrasts at solid tumor sites owing to a dissociation of the micelle structures, while selective delivery to the tumor sites is achieved in the polymeric micelle form.     

Polycaprolactone-b-poly(ethylene oxide) copolymer micelles as a delivery vehicle for dihydrotestosterone.

Block copolymer micelles formed from copolymers of poly(caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) were investigated as a drug delivery vehicle for dihydrotestosterone (DHT). The physical parameters of the PCL-b-PEO micelle-incorporated DHT were measured, including the loading capacity of the micelles for DHT, the apparent partition coefficient of DHT between the micelles and the external medium and the kinetics of the release of DHT from the micelle solution. The MTT survival assay was used to assess the in vitro biocompatibility of PCL-b-PEO micelles in HeLa cell cultures. The biological activity of the micelle-incorporated DHT was evaluated in HeLa cells which had been co-transfected with the expression vectors for the androgen receptor and the MMTV-LUC reporter gene. The PCL-b-PEO micelles were found to have a high loading capacity for DHT and the release profile of the drug from the micelle solution was found to be a slow steady release which continued over a 1-month period. The biological activity of the micelle-incorporated DHT was found to be fully retained.