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.     

Micellar carriers based on block copolymers of poly(epsilon-caprolactone) and poly(ethylene glycol) for doxorubicin delivery.

Diblock copolymers of poly(epsilon-caprolactone) (PCL) and monomethoxy poly(ethylene glycol) (MPEG) with various compositions were synthesized. The amphiphilic block copolymers self-assembled into nanoscopic micelles and their hydrophobic cores encapsulated doxorubicin (DOX) in aqueous solutions. The micelle diameter increased from 22.9 to 104.9 nm with the increasing PCL block length (2.5-24.7 kDa) in the copolymer composition. Hemolytic studies showed that free DOX caused 11% hemolysis at 200 microg ml(-1), while no hemolysis was detected with DOX-loaded micelles at the same drug concentration. An in vitro study at 37 degrees C demonstrated that DOX-release from micelles at pH 5.0 was much faster than that at pH 7.4. Confocal laser scanning microscopy (CLSM) demonstrated that DOX-loaded micelles accumulated mostly in cytoplasm instead of cell nuclei, in contrast to free DOX. Consistent with the in vitro release and CLSM results, a cytotoxicity study demonstrated that DOX-loaded micelles exhibited time-delayed cytotoxicity in human MCF-7 breast cancer cells.     

Polymer micelles with cross-linked polyanion core for delivery of a cationic drug doxorubicin

Polymer micelles with cross-linked ionic cores were prepared by using block ionomer complexes of poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMA) copolymer and divalent metal cations as templates. Doxorubicin (DOX), an anthracycline anticancer drug, was successfully incorporated into the ionic cores of such micelles via electrostatic interactions. A substantial drug loading level (up to 50 w/w%) was achieved and it was strongly dependent on the structure of the cross-linked micelles and pH. The drug-loaded micelles were stable in aqueous dispersions exhibiting no aggregation or precipitation for a prolonged period of time. The DOX-loaded polymer micelles exhibited noticeable pH-sensitive behavior with accelerated release of DOX in acidic environment due to the protonation of carboxylic groups in the cores of the micelles. The attempt to protect the DOX-loaded core with the polycationic substances resulted in the decrease of loading efficacy and had a slight effect on the release characteristics of the micelles. The DOX-loaded polymer micelles exhibited a potent cytotoxicity against human A2780 ovarian carcinoma cells. These results point to a potential of novel polymer micelles with cross-linked ionic cores to be attractive carriers for the delivery of DOX.     

Core cross-linked block ionomer micelles as pH-responsive carriers for cis-diamminedichloroplatinum(II)

Benefits of the frequently prescribed platinum (II) chemotherapy drugs are compromised by undesirable side effects, poor pharmacokinetics and development of drug resistance. Polymer micelles derived from amphiphillic block copolymers, offer a novel macromolecular platform for carrier based delivery of such compounds. Soft polymeric nanocarriers were synthesized by template-assisted method involving condensation of the poly(ethylene oxide)-b-polymethacrylate anions by metal ions into core-shell block ionomer complex micelles followed by chemical cross-linking of the polyion chains in the micelle cores. The resulting micelles can efficiently incorporate cisplatin with a high loading capacity (up to 42% w/w). Core cross-linking stabilized the micelles against structural disintegration and prevented premature drug release. The reversible cisplatin entrapment involved the carboxylate groups of the micellar core. The drug was released in a pH-responsive manner, without loss of its biological activity. The stable cross-linked polymer micelles can potentially improve platinum (II) drug disposition with improved therapeutic potential.     

Doxorubicin loaded pH-sensitive polymeric micelles for reversal of resistant MCF-7 tumor.

In order to overcome multidrug resistance in solid tumors, doxorubicin (DOX) loaded pH-sensitive micelles of which surface was decorated with folate (PHSM/f) were evaluated both in vitro and in vivo experiments. PHSM/f were fabricated from a mixture of two block copolymers of poly(L-histidine) (M(n): 5K)-b-PEG (M(n): 2K)-folate (polyHis/PEG-folate) (75 wt.%) and poly(L-lactic acid) (M(n): 3K)-b-PEG (M(n): 2K)-folate (PLLA/PEG-folate) (25 wt.%). The PHSM/f showed more than 90% cytotoxicity of DOX resistant MCF-7 (MCF-7/DOX(R)) when cultured with PHSM/f at a concentration of 10 microg/ml DOX. The result was interpreted by a sequential event of active internalization of PHSM/f via folate-receptor mediated endocytosis and ionization of His residues which result in micelle destabilization and probably disturbance of endosomal membranes. This potential mechanism may endow the drug carriers to bypass Pgp efflux pump and sequestration of DOX in acidic intracellular compartments, yielding high cytotyoxicity. Experimental evaluation of tumor regression was carried out in a small animal model bearing s.c. MCF-7 or MCF-7/DOX(R) xenografts. The tumor (MCF-7/DOX) volumes of mice treated with PHSM/f were significantly less than control groups treated with free DOX or similar micelles but without folate (PHSM). In the MCF-7/DOX(R) xenograft model, the accumulated DOX level of PHSM/f in solid tumors was 20 times higher than free DOX group, and 3 times higher than PHSM group. The results demonstrate that PHSM/f is a viable means for treating drug resistant tumors.     

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.     

Micelles of methoxy poly(ethylene oxide)-b-poly(epsilon-caprolactone) as vehicles for the solubilization and controlled delivery of cyclosporine A.

The commercial formulation of Cyclosporine A (CsA) for intravenous administration contains Cremophor EL, a low molecular weight surfactant known to be toxic. In this study, micelles of methoxy poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) were investigated as alternative vehicles for the solubilization and delivery of CsA. PEO-b-PCL block copolymers having identical PEO chain lengths and PCL molecular weights of 5000, 13,000, or 24,000 g mol(-)(1) were synthesized and assembled into polymeric micelles using a co-solvent evaporation method. PEO-b-PCL micelles were then compared to Cremophor EL micelles for their functional properties in drug delivery including micellar size, thermodynamic stability, core viscosity, CsA encapsulation, and in vitro CsA release. Among different PCL block lengths, optimum solubilization was achieved by utilizing polymeric micelles having a PCL block of 13,000 g mol(-)(1). CsA reached an aqueous solubility of 1.3 mg/mL in the presence of PEO-b-PCL micelles. This concentration is comparable to injectable CsA levels in its Cremophor EL formulation (0.5-2.5 mg/mL). In contrast to the Cremophor EL formulation, the in vitro rate of CsA release was significantly sustained by the polymeric micellar carrier. Within 12 h, only 5.8% of CsA was released from polymeric micelles while Cremophor EL micelles released 77% of their drug content. Accordingly, viscosity of the PEO-b-PCL micellar core was found to be significantly higher than Cremophor EL micelles. The results points to a potential for PEO-b-PCL micelles as nanoscopic drug carriers for efficient solubilization and controlled delivery of CsA.     

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.     

Novel reduction-sensitive micelles for triggered intracellular drug release

Novel reduction-sensitive micelles based on poly(ethylene oxide)-b-poly(N-methacryloyl-N′-(t-butyloxycarbonyl)cystamine) (PEO-b-PMABC) diblock copolymers were developed and applied for triggered intracellular drug release. PEO-b-PMABC block copolymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of MABC with dithioester-capped PEO as macroRAFT agent. Gel permeation chromatography (GPC) and ¹H NMR analysis showed that the copolymers have controlled compositions and molecular weights, indicating the living nature of polymerization. These copolymers were self-assembled into micelles. The physicochemical characteristics and reduction-sensitivity of the resultant micelles were investigated by fluorescence measurement, transmission electron microscopy (TEM), and dynamic light scattering (DLS). The results showed that PEO-b-PMABC micelles are stable at normal physiologic condition but readily cleaved into free copolymers under reducing environment. In vitro release of doxorubicin (DOX) and cell experiments showed that the drug-loaded PEO-b-PMABC micelles accomplished much faster drug release under reducing condition and higher anticancer efficacy as compared to the control without reduction-sensitivity, indicating great potential of PEO-b-PMABC micelles for efficient intracellular drug delivery.     

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.     

ATRP-based synthesis of a pH-sensitive amphiphilic block polymer and its self-assembled micelles with hollow mesoporous silica as DOX carriers for controlled drug release

The atom transfer radical polymerization (ATRP)-based synthesis of a pH-sensitive fluorescent polymer (PSDMA-b-POEGMA) was successfully prepared using 3,6-dibromo-isobutyramide acridine (DIA), an initiator with a fluorescent chromophore, to initiate a lipophilic monomer 2-styryl-1,3-dioxan-5-yl methacrylate (SDMA) and a hydrophilic monomer oligo(ethylene glycol) methyl ether (OEGMA), which contained a cinnamic aldehyde acetal structure. With the addition of hollow mesoporous silicon (HMS@C18), the pH-sensitive core–shell nanoparticles (HMS@C18@PSDMA-b-POEGMA) were developed via a self-assembly process as carriers for the anticancer drug doxorubicin (DOX) for drug loading and controlled release. The nanocomposites showed a higher drug loading capacity which was much higher than that observed using common micelles. At the same time, the polymer coated on the surface of the nanoparticles contains the fluorescent segment of an initiator, which can be used for fluorescence contrast of the cells. The nanocomposite carrier selectively inhibits human melanoma cell A375 relative to human normal fibroblasts GM. The in vitro results suggested that a smart pH sensitive nanoparticles drug delivery system was successfully prepared for potential applications in cancer diagnosis and therapy.

A pH-sensitive core–shell nanoparticle (HMS@C18@PSDMA-b-POEGMA) was developed via a self-assembly process as the carrier of anticancer drug doxorubicin (DOX) for drug loading and controlled release.     

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.     

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-conjugated methoxy poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric micelles for tumor-targeted drug delivery.

Amphiphilic block copolymers composed of methoxy poly(ethylene glycol) (MPEG) and poly(epsilon-caprolactone) (PCL) were synthesized and then conjugated with folic acid to produce a folate-receptor-targeted drug carrier for tumor-specific drug delivery. Folate-conjugated MPEG/PCL micelles containing the anticancer drug paclitaxel were prepared by micelle formation in aqueous medium. The size of the folate-conjugated MPEG/PCL micelles formed was about 50-130 nm, depending on the molecular weight of block copolymers, and was maintained at less than 150 nm even after loading with paclitaxel. The in vitro release profile of the paclitaxel from the MPEG/PCL micelles exhibited no initial burst release and showed sustained release. Paclitaxel-loaded folate-conjugated MPEG/PCL micelles (PFOL50) exhibited much higher cytotoxicity for cancer cells, such as MCF-7 and HeLa cells, than MPEG/PCL micelles without the folate group (PMEP50). Confocal image analysis revealed that fluorescent paclitaxel-loaded PFOL50 micelles were endocytosed into MCF-7 cells through the interaction with overexpressed folate receptors on the surface of the cancer cells.     

Ultrasound-triggered drug release and enhanced anticancer effect of doxorubicin-loaded poly(D,L-lactide-co-glycolide)-methoxy-poly(ethylene glycol) nanodroplets

A novel ultrasound-responsive doxorubicin (DOX)-loaded nanoparticulate system was prepared in this study. The DOX-loaded polymeric micelles were first prepared using poly(D,L-lactide-co-glycolide)-methoxy-poly(ethylene glycol) (PLGA-mPEG) with a high encapsulation efficiency of 89.2%. After filling with perfluoropentane (boiling point 29°C), the micelles were transformed into nanodroplets that were stable as a result of the PEG shell. The nanodroplets were transformed into nanobubbles at 37°C, and little drug was released if no ultrasound was exerted. Ultrasound-triggered drug release, with pH dependency, was shown. The DOX release percentage was 9.59% at pH 6.5 (also appeared in tumor) and only 2.22% at pH 7.4 after sonicating for 0.5 min at 37°C. The tumor inhibitory rate of Group III (DOX-loaded nanodroplets combined with ultrasound) was 84.3%, more than that of Group II (DOX-loaded nanodroplets), which was 60.4%. Moreover, the nanodroplets showed much lower toxicity than free drugs. The novel nanodroplets could be a promising anticancer drug delivery system.     

Polymeric micelles with a pH-responsive structure as intracellular drug carriers.

Polymeric micelles based on poly(L-lactide)-b-poly(2-ethyl-2-oxazoline)-b-poly(L-lactide) (PLLA-PEOz-PLLA) ABA triblock copolymers were designed as intracellular drug carriers. The PLLA-PEOz-PLLA micelles adopt a "flower-like" arrangement with A-blocks at the core and a B-block on the shell under neutral condition. The deformation of the core-shell structure is then promoted by the aggregation of PEOzs due to the formation of inter- and intra-hydrogen bonding between protonated nitrogen and carbonyl groups. The experiments on in vitro release have confirmed that the release of doxorubicin (DOX) from micelles was successfully inhibited at pH 7.4. In contrast, an accelerated release of DOX from micelles was observed at acidic conditions. The results of growth inhibition assay indicated that the cell-killing rate of DOX-loaded micelles gradually approached that of free DOX as increasing the concentration and the incubation time. The overlay of fluorescent images on CLSM observation clearly demonstrated the colocalization of DOX with acidic compartments, suggesting that the drug release was successfully triggered in the acidic organelles by means of micelle deformation.     

Tumor pH-responsive flower-like micelles of poly(l-lactic acid)-b-poly(ethylene glycol)-b-poly(l-histidine)

Polymeric micelles were constructed from poly(l-lactic acid) (PLA; M_n 3K)-b-poly(ethylene glycol) (PEG; M_n 2K)-b-poly(l-histidine) (polyHis; M_n 5K) as a tumor pH-specific anticancer drug carrier. Micelles (particle diameter: ∼ 80 nm; critical micelle concentration (CMC): 2 μg/ml) formed by dialysis of the polymer solution in dimethylsulfoxide (DMSO) against pH 8.0 aqueous solution, are assumed to have a flower-like assembly of PLA and polyHis blocks in the core and PEG block as the shell. The pH-sensitivity of the micelles originates from the deformation of the micellar core due to the ionization of polyHis at a slightly acidic pH. However, the co-presence of pH-insensitive lipophilic PLA block in the core prevented disintegration of the micelles and caused swelling/aggregation. A fluorescence probe study showed that the polarity of pyrene retained in the micelles increased as pH was decreased from 7.4 to 6.6, indicating a change to a more hydrophilic environment in the micelles. Considering that the size increased up to 580 nm at pH 6.6 from 80 nm at pH 7.4 and that the transmittance of micellar solution increased with decreasing pH, the micelles were not dissociated but rather swollen/aggregated. Interestingly, the subsequent decline of pyrene polarity below pH 6.6 suggested re-self-assembly of the block copolymers, most likely forming a PLA block core while polyHis block relocation to the surface. Consequently, these pH-dependent physical changes of the PLA-b-PEG-b-polyHis micelles provide a mechanism for triggered drug release from the micelles triggered by the small change in pH (pH 7.2–6.5).     

Hyaluronic acid hydrogel as Nogo-66 receptor antibody delivery system for the repairing of injured rat brain: in vitro.

Nogo-66 and NgR are important receptors inhibiting neuronal regeneration and therefore are targets for treating CNS injury. Antagonists of this receptor including blocking antibodies are potential therapeutic agents for CNS axonal injuries such as spinal cord and brain trauma. A new antibody (IgG) releasing system has been developed by covalently attaching IgG to the biodegradable hyaluronic acid (HA) hydrogel via the hydrolytically unstable hydrazone linkage, aiming to deliver the antibody of CNS regeneration inhibitors to the injured brain. In this paper we describe the synthesis, physico-chemical characteristics and test results of biological activity of antibody released from hyluronic acid hydrogel. To form the conjugates the antibody is attached to the polymer backbone using a condensation reaction between aldehyde group of the antibody and hydrazide group of the HA hydrogel. Furthermore, pH sensitive linkage-hydrozone has been formed between hydrogel and antibody. The amount of conjugated antibodies can reach 135 microg antibody/mg hydrogel in the dry state. At low pH, the antibodies released quite fast. However, the antibodies released much slower in neutral and alkaline environment. The bioactivity of antibody released from hydrogel was retained as demonstrated by indirect immunofluorescence technique.     

HPMA copolymer conjugates with reduced anti-CD20 antibody for cell-specific drug targeting. I. Synthesis and in vitro evaluation of binding efficacy and cytostatic activity

Synthesis, physicochemical and biological properties and preliminary anticancer activity of new star-shaped polymer–doxorubicin (DOX) conjugates targeted with anti-CD20 monoclonal antibody were investigated. Mild reduction of antibody (Ab) with dithiothreitol (DTT) resulted in introduction of thiol groups into Ab. Polymer precursors used for the synthesis of the conjugates were based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with a functional group at the polymer chain end. The copolymers were linked to the thiol groups of the reduced Ab via one-point attachment forming a star-shaped structure with central antibody surrounded by hydrophilic polymer chains. Neither reduction nor polymer modification of Ab influenced binding activity of the Ab to its specific cancer cell membrane antigen as it was confirmed in vitro by standard flow cytometry. The anticancer drug DOX was attached to the HPMA copolymer chain in an Ab–polymer system via a pH–labile hydrazone linkage or via an oligopeptide sequence degradable by lysosomal enzymes. Such Ab–polymer–DOX conjugates were fairly stable in aqueous solution at pH 7.4 and the drug was readily released in mildly acid environment at pH 5–5.5 by hydrolysis of hydrazone bond or more slowly by enzymolysis with lysosomal enzymes. The cytostatic activity of the anti-CD20 monoclonal Ab-targeted conjugates tested on several CD20-positive or negative human and mouse cancer cell lines confirmed considerable targeting capacity of the monoclonal Ab after its binding to the polymer carrier. New method of synthesis of star antibody-targeted polymer–drug conjugates with pH-controlled drug release described in this paper opens new perspectives for development of new therapeutics intended for cancer therapy.