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.     

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.     

In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates.

Doxorubicin was chemically conjugated to a terminal end group of poly(D,L-lactic-co-glycolic acid) [PLGA] by an ester linkage and the doxorubicin-PLGA conjugate was formulated into nanoparticles. A carboxylic acid end group of PLGA was conjugated to a primary hydroxyl group of doxorubicin. The primary amine group of doxorubicin was protected during the conjugation process and then deprotected. The nanoparticles containing the conjugate exhibited sustained doxorubicin release profiles over a 1-month period, whereas those containing unconjugated free doxorubicin showed a rapid doxorubicin release within 5 days. Doxorubicin release patterns could be controlled by conjugating doxorubicin to PLGA having different molecular weights. The conjugated doxorubicin nanoparticles showed increased uptake within a HepG2 cell line, which was quantitated by a flow cytometry and visualized by confocal microscopy. The nanoparticles exhibited slightly lower IC(50) value against the HepG2 cell line compared to that of free doxorubicin. In vivo anti-tumor activity assay also showed that a single injection of the nanoparticles had comparable activity to that of free doxorubicin administered by daily injection. The conjugation approach of doxorubicin to PLGA was potentially useful for the formulation of nanoparticles that requires targeting for cancer cells as well as sustained release at the site.     

HPMA copolymer-bound doxorubicin targeted to tumor-specific antigen of BCL1 mouse B cell leukemia.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin and targeted with B1 monoclonal antibody (mAb) to BCL1 leukemia cells was synthesised and tested in vitro and in vivo. BCL1 leukemia growing in syngenic Balb/c mice was selected as a tumor model system. B1 mAb recognising the idiotype of surface IgM on BCL1 cells was used as a targeting moiety. Both B1 mAb and doxorubicin were conjugated to HPMA copolymer carrier by aminolysis through a tetrapeptidic Gly-Phe(D,L)-Leu-Gly spacer to ensure the intracellular delivery and controlled release of the drug. B1 mAb-targeted conjugate was shown to possess strictly tumor-specific binding capacity to target BCL1 cells in vitro. A similar conjugate, but containing human nonspecific Ig (HuIg) instead of B1 mAb, failed to bind to BCL1 cells. In vitro, B1 mAb-targeted conjugate demonstrated 40-fold higher cytotoxic effect than nontargeted or human nonspecific Ig-containing HPMA copolymer-bound doxorubicin. Conjugate targeted with B1 mAb was also shown to bind to target BCL1 cells in vivo. B1 mAb-targeted conjugate was shown to be more efficient in the treatment of established BCL1 leukemia than free doxorubicin, nontargeted and human nonspecific Ig-containing conjugate. Antibody-targeted polymeric drugs are thus promising conjugates for cancer treatment.     

N-isopropylacrylamide copolymers for the preparation of pH-sensitive liposomes and polymeric micelles.

Hydrophobically-modified copolymers of N-isopropylacrylamide bearing a pH-sensitive moiety were investigated for the preparation of pH-responsive liposomes and polymeric micelles. The copolymers having the hydrophobic anchor randomly distributed within the polymeric chain were found to more efficiently destabilize egg phosphatidylcholine (EPC)/cholesterol liposomes than the alkyl terminated polymers. Release of both a highly-water soluble fluorescent contents marker, pyranine, and an amphipathic cytotoxic anti-cancer drug, doxorubicin, from copolymer-modified liposomes was shown to be dependent on pH, the concentration of copolymer, the presence of other polymers such as polyethylene glycol, and the method of preparation. Both polymers were able to partially stabilize EPC liposomes in human serum. These polymers were found to self-assemble to form micelles. The critical association concentration was low (9--34 mg/l) and influenced by the position of the alkyl chains. In phosphate buffered saline, the micelles had a bimodal size distribution with the predominant population having a mean diameter of 35 nm. The polymeric micelles were studied as a delivery system for the photosensitizer aluminum chloride phthalocyanine, (AlClPc), currently evaluated in photodynamic therapy. pH-Responsive polymeric micelles loaded with AlClPc were found to exhibit increased cytotoxicity against EMT-6 mouse mammary cells in vitro than the control Cremophor EL formulation.     

Characterization of physical entrapment and chemical conjugation of adriamycin in polymeric micelles and their design for in vivo delivery to a solid tumor

An anticancer drug adriamycin (ADR) was incorporated into polymeric micelles forming from poly(ethylene glycol)-poly(aspartic acid) block copolymer by chemical conjugation and physical entrapment. Structural stability of the polymeric micelles was found to be dependent on both the contents of chemically conjugated and physically entrapped ADR. The polymeric micelle with high contents of the chemically conjugated ADR and the physically entrapped ADR expressed very high in vivo antitumor activity against murine C 26 tumor, while the polymeric micelle with only the chemically conjugated ADR showed negligible in vivo activity. This indicates that the physically entrapped ADR played a major role in antitumor activity in vivo. For the polymeric micelle with the high ADR contents, it was found that a dimer of adriamycin molecules formed and that this dimer was physically entrapped in the inner core of the micelle as well as intact ADR.     

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.     

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).     

Properties of HPMA copolymer-doxorubicin conjugates with pH-controlled activation: effect of polymer chain modification.

Various conjugates of anticancer drug doxorubicin (DOX) covalently attached via hydrolytically degradable hydrazone bond to water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer drug carriers were synthesized. Three types of precursors containing either positively or negatively charged groups or a hydrophobic substituent were employed. In vitro incubation of the conjugates in buffers showed relative stability at pH 7.4 (modelling blood) and a fast DOX release at pH 5 (modelling intracellular environment). The presence of carboxylic groups in the copolymer structure resulted in an increase in the DOX release rate of 15-20% while no effect of the introduction of positively charged groups was observed if compared with the unmodified conjugate. Self-assembling of the oleoyl groups-containing conjugate led into formation of polymeric micelles with high apparent molecular weight (M(w)=170,000) in aqueous solution and resulted in a decrease in the DOX release rate of approximately 20%. The cytostatic activity of the conjugates tested on several cancer cell lines was comparable with that of free DOX.HCl, depending on the sensitivity of a particular cell line to DOX. All the conjugates showed a much higher antitumour activity in vivo than the free drug tested in mice bearing EL4 T-cell lymphoma and treated using the therapeutic regime of drug administration. The highest activity (100% long-term survivors) exhibited polymer-DOX conjugate containing negatively charged GFLG sequences.     

Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery.

The preparation, release and in vitro cytotoxicity of a novel polymeric micellar formulation of paclitaxel (PTX) were investigated. The micelles consisted of an AB block copolymer of poly(N-(2-hydroxypropyl) methacrylamide lactate) and poly(ethylene glycol) (pHPMAmDL-b-PEG). Taking advantage of the thermosensitivity of pHPMAmDL-b-PEG, the loading was done by simply mixing of a small volume of a concentrated PTX solution in ethanol and an aqueous polymer solution and subsequent heating of the resulting solution above the critical micelle temperature of the polymer. PTX could be almost quantitatively loaded in the micelles up to 2 mg/mL. By dynamic light scattering and cryo-transmission electron microscopy, it was shown that PTX-loaded micelles have a mean size around 60 nm with narrow size distribution. At pH 8.8 and 37 degrees C, PTX-loaded micelles destabilized within 10 h due to the hydrolysis of the lactic acid side group of the pHPMAmDL. Because the hydrolysis of the lactic acid side groups is first order in hydroxyl ion concentration, the micelles were stable for about 200 h at physiological conditions. The presence of serum proteins did not have an adverse effect on the stability of the micelles during at least 15 h. Interestingly, the dissolution kinetics of pHPMAmDL-b-PEG micelles was retarded by incorporation of PTX, indicating a strong interaction between PTX and the pHPMAmDL block. The PTX-loaded micelles showed a release of the incorporated 70% of PTX during 20 h at 37 degrees C and at pH 7.4. PTX-loaded pHPMAmDL-b-PEG micelles showed comparable in vitro cytotoxicity against B16F10 cells compared to the Taxol standard formulation containing Cremophor EL, while pHPMAmDL-b-PEG micelles without PTX were far less toxic than the Cremophor EL vehicle. Confocal laser-scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) analysis of fluorescently labelled micelles showed that pHPMAmDL-b-PEG micelles were internalized by the B16F10 cells. The present results suggest that pHPMAmDL-b-PEG block copolymer micelles are a promising delivery system for the parenteral administration of PTX.     

Self-assembled siRNA-PLGA conjugate micelles for gene silencing

Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was conjugated to the 3′ end of small interfering RNA (siRNA) via a disulfide bond to synthesize siRNA-PLGA hybrid conjugates. siRNA-PLGA conjugates were spontaneously self-assembled to form a spherical core/shell type micellar structure of ~ 20 nm in an aqueous environment, probably by hydrophobic interaction of PLGA blocks in the core surrounded by an siRNA shell layer. When linear polyethylenimine was added to the siRNA-PLGA micelles in aqueous solution, stable siRNA-PLGA/LPEI micelles with a size of ~ 30 nm were produced via ionic complexation between siRNA and LPEI in the outer shell. The cationic siRNA-PLGA/LPEI micelles showed superior intracellular uptake and enhanced gene silencing effect, compared to naked siRNA/LPEI complexes. The hybrid micelle structure based on siRNA and PLGA can be potentially used as an efficient siRNA delivery system for gene silencing.     

Folate receptor mediated intracellular protein delivery using PLL-PEG-FOL conjugate.

To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins or other bioactive macromolecules into a specific cell, a di-block copolymer conjugate, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL), was synthesized. The PLL-PEG-FOL conjugate was physically complexed with fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) in an aqueous phase by ionic interactions. Cellular uptake of PLL-PEG-FOL/FITC-BSA complexes was greatly enhanced against a folate receptor over-expressing cell line (KB cells) compared to a folate receptor deficient cell line (A549 cells). The presence of an excess amount of free folate (1 mM) in the medium inhibited the intracellular delivery of PLL-PEG-FOL/FITC-BSA complexes. This suggests that the enhanced cellular uptake of FITC-BSA by KB cells in a specific manner was attributed to folate receptor-mediated endocytosis of the complexes having folate moieties on the surface. The PLL-PEG-FOL di-block copolymer could be potentially applied for intracellular delivery of a wide range of other biological active agents that have negative charges on the surface.     

Hydrotropic agents for study of in vitro paclitaxel release from polymeric micelles.

A new experimental method for in vitro release studies of poorly soluble drugs from polymeric micelle systems was developed using a hydrotropic agent, sodium salicylate. It is difficult to maintain a good sink condition for poorly water-soluble drugs, such as paclitaxel (PTX), because of their low aqueous solubility. In this study, a good sink condition for PTX was achieved by using aqueous sodium salicylate solution which solubilized more than 10 times the total amount of PTX incorporated in polymeric micelles. Sodium salicylate at 1 M concentration increased the aqueous PTX solubility by 100 times without destroying the micellar structure of poly(ethylene glycol)-block-poly(phenylalanine) (PEG-b-PPhe) copolymer. PTX was continuously released from PEG-b-PPhe micelles in the hydrotropic release medium. The hydrotropic solution presents a simple method for studying in vitro release behavior of poorly soluble drugs from polymeric micelles in aqueous media.     

Micelle-like nanoparticles of star-branched PEO-PLA copolymers as chemotherapeutic carrier.

Four-armed (star-branched) block copolymers of l-PLA and PEO were synthesized using ring opening polymerization with different LA/EO ratio. Micellar aggregates were prepared from these block copolymers and characterized. Some surface segregation of PEG was found : the extent depends on the state of the material (whether it is in film or particle form), as well as on molecular geometry. The degradation behavior of star-shaped copolymer was studied over a three week period and compared to its linear counterpart. Anti-cancer drugs 5-FU and paclitaxel were loaded into the micellar nanoparticles. The drug release profile showed that the release of paclitaxel from these polymers could be controlled over 2 weeks. The kinetics of drug release for star-branched, tri- and di-block copolymers were compared. The micelles from star-shaped branch showed more complete release of drug than the diblock copolymers; also, the lower hydrodynamic radius of star-shaped polymers may result in better clearance of the carrier polymer from the body.     

Indomethacin-loaded polymeric nanocarriers based on amphiphilic polyphosphazenes with poly (N-isopropylacrylamide) and ethyl tryptophan as side groups: Preparation, in vitro and in vivo evaluation.

The effects of copolymer composition, drug structure and initial drug feed on drug loading of polymeric micelles based on amphiphilic polyphosphazenes were investigated. It was found that the drug loading capacity of micelles based on this type of amphiphilic copolymers was mainly determined by copolymer composition and the chemical structure of drug. In addition to the compatibility between drug and micellar core, hydrogen bonding interaction between drug and hydrophilic corona may significantly influence drug loading as well. In vitro drug release in 0.1 M PBS (pH 7.4) suggested that indomethacin (IND) in the micelles was released through Fickian diffusion, and no significant difference in release rate was observed for micelles based on copolymers with various EtTrp content. Compared with in vitro IND release profile, in vivo pharmacokinetic study after subcutaneous administration provides a more sustained release behavior. Additionally, in comparison with free drug solution at the same dose, IND concentration in rat plasma showed a prolonged retention when the drug was delivered through polymeric micelles. In vivo pharmacodynamic study based on both carrageenan-induced acute and complete Freund's adjuvant-induced adjuvant arthritis model indicated that sustained therapeutic efficacy could be achieved through intraarticular injection of IND-loaded micelles. Most importantly, local delivery of IND can avoid the severe gastrointestinal stimulation, which was frequently associated with oral administration.     

DPD simulations on morphologies and structures of blank PLGA-b-PEG-b-PLGA polymeric micelles and docetaxel-loaded PLGA-b-PEG-b-PLGA polymeric micelles

Dissipative particle dynamics (DPD) simulation was used to study the morphologies and structures of blank (no drug) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PLGA-b-PEG-b-PLGA) polymeric micelles and the docetaxel (Dtx)-loaded PLGA-b-PEG-b-PLGA polymeric micelles. We focused on the influences of PLGA-b-PEG-b-PLGA copolymer concentration, composition, Dtx drug content and the shear rate on morphologies and structures of the micelles. Our simulations show that the PLGA-b-PEG-b-PLGA copolymers in the aqueous solutions could aggregate and form blank micelles while Dtx drug and PLGA-b-PEG-b-PLGA could aggregate and form drug-loaded micelles. Under different PLGA-b-PEG-b-PLGA concentrations and drug content, the blank and drug-loaded micelles are observed as spherical, onionlike, columnar, and lamellar structures. The onionlike structures are comprised of the PEG hydrophilic core, the PLGA hydrophobic middle layer, and the PEG hydrophilic shell. As the structure of micelles varies from a spherical core–shell structure to a core–middle layer–shell onionlike structure, the distribution of the Dtx drugs diffuses from the core to the PLGA middle layer of the aggregate. In addition, the drug release process of the Dtx-loaded micelles under shear flow is also simulated. And the results show that the spherical micelles turn into a columnar structure under a shear rate from 0.2 to 3.4. When the shear rate increases to 3.5, the Dtx drugs released gradually increase until all are released with time evolution. These findings illustrate the dependence of the structural morphologies on the detailed molecular parameters of PLGA-b-PEG-b-PLGA and Dtx.

Dissipative particle dynamics simulation was used to study the morphologies and structures of blank (no drug) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) polymeric micelles and the docetaxel-loaded polymeric micelles.     

Block copolymer micelles: preparation, characterization and application in drug delivery.

Block copolymer micelles are generally formed by the self-assembly of either amphiphilic or oppositely charged copolymers in aqueous medium. The hydrophilic and hydrophobic blocks form the corona and the core of the micelles, respectively. The presence of a nonionic water-soluble shell as well as the scale (10-100 nm) of polymeric micelles are expected to restrict their uptake by the mononuclear phagocyte system and allow for passive targeting of cancerous or inflamed tissues through the enhanced permeation and retention effect. Research in the field has been increasingly focused on achieving enhanced stability of the micellar assembly, prolonged circulation times and controlled release of the drug for optimal targeting. With that in mind, our group has developed a range of block copolymers for various applications, including amphiphilic micelles for passive targeting of chemotherapeutic agents and environment-sensitive micelles for the oral delivery of poorly bioavailable compounds. Here, we propose to review the innovations in block copolymer synthesis, polymeric micelle preparation and characterization, as well as the relevance of these developments to the field of biomedical research.     

Polymeric micellar pH-sensitive drug delivery system for doxorubicin.

A novel polymeric micellar pH-sensitive system for delivery of doxorubicin (DOX) is described. Polymeric micelles were prepared by self-assembly of amphiphilic diblock copolymers in aqueous solutions. The copolymers consist of a biocompatible hydrophilic poly(ethylene oxide) (PEO) block and a hydrophobic block containing covalently bound anthracycline antibiotic DOX. The starting block copolymers poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PEO-PAGE) with a very narrow molecular weight distribution (Mw/Mn ca. 1.05) were prepared by anionic ring opening polymerization using sodium salt of poly(ethylene oxide) monomethyl ether as macroinitiator and allyl glycidyl ether as functional monomer. The copolymers were covalently modified via reactive double bonds by the addition of methyl sulfanylacetate. The resulting ester subsequently reacted with hydrazine hydrate yielding polymer hydrazide. The hydrazide was coupled with DOX yielding pH-sensitive hydrazone bonds between the drug and carrier. The resulting conjugate containing ca. 3 wt.% DOX forms micelles with Rh(a)=104 nm in phosphate-buffered saline. After incubation in buffers at 37 degrees C DOX was released faster at pH 5.0 (close to pH in endosomes; 43% DOX released within 24 h) than at pH 7.4 (pH of blood plasma; 16% DOX released within 24 h). Cleavage of hydrazone bonds between DOX and carrier continues even after plateau in the DOX release from micelles incubated in aqueous solutions is reached.     

Distribution kinetics of a micelle-forming block copolymer Pluronic P85

Pluronic block copolymers, micelle-forming polymeric surfactants, are currently being evaluated in chemotherapy clinical trials in combination with doxorubicin to treat multidrug-resistant (MDR) tumors. This study examines the pharmacokinetics and biodistribution of Pluronic P85 (P85), a potent inhibitor of P-glycoprotein (Pgp). P85 was radioactively labeled and administered intravenously (i.v.) to mice. The concentration of the copolymer was varied to examine the effects of micelle formation on the distribution kinetics. The main pharmacokinetic parameters (the area under the curve, half-life, clearance, mean residence time, and volume of distribution) were determined. The results suggest that half-life of P85 varies from 60 to 90 h, depending on its aggregation state. Formation of micelles decreased the uptake of the block copolymer in the liver. However, it had no effect on the total clearance, suggesting that the elimination of P85 was controlled by the renal elimination of P85 unimers and not by the rate of micelle disposition or disintegration. The total clearance value suggests that a significant portion of P85 is reabsorbed back into the blood, probably through the kidney's tubular membranes.     

Poly( -histidine)–PEG block copolymer micelles and pH-induced destabilization

Poly(L-histidine)-poly(ethylene glycol) diblock copolymers (polyHis-b-PEG) were prepared and used for the construction of polymeric micelles responding to local pH changes in the body. PolyHis was synthesized by ring opening polymerization of L-histidine N-carboxyanhydride, the imidazole amine group of which was protected by the dinitrophenyl group. The resulting polymer (M(n): 5,000 g/mole) was coupled to poly(ethylene glycol) (M(n): 2,000 g/mole) via an amide linkage using the dicyclohexyl carbodiimide and N-hydroxysuccinimide-mediated reaction. The block copolymer in dimethyl sulfoxide formed polymeric micelles on diafiltration against a borate buffer at pH 8. Dynamic light scattering and atomic force microscopy showed the micelles were spherical, diameter approximately 114 nm, with a unimodal distribution. The critical micelle concentration (CMC) at pH 8.0 was 2.3 mg/l. The CMC increased markedly on decreasing the pH of the diafiltration medium below 7.2. Micelles prepared at pH 8.0 were gradually destabilized below pH 7.4, as evidenced by a slight increase in light transmittance, an alteration in size distribution, and a decrease in the pyrene fluorescence intensity. It was concluded that the ionization of the polyHis block forming the micelle core determined the pH-dependent CMC and stability. After further optimization of the pH-sensitivity, pH-sensitive micelles are expected to have application for solid tumor treatment, exploiting the fact that most solid tumors have an acidic extracellular pH.