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.     

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.     

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.     

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.     

Evaluation of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy.

Thermally responsive elastin-like polypeptides (ELPs) were synthesized by recombinant DNA techniques and conjugated to doxorubicin through an acid-labile hydrazone bond to enable release of the drug in the acidic environment of lysosomes. The thermal properties, intracellular localization and cytotoxicity of the conjugate were investigated in this study. The conjugation procedure resulted in a mixed population of free ELP and ELP-doxorubicin (ELP-dox) conjugates that exhibit a broader transition than the parent ELP. A simple centrifugation procedure was developed to purify the ELP-dox conjugate from other reactants and resulted in a sharper thermal transition, similar to the parent ELP. The ELP was endocytosed by squamous cell carcinoma cells (FaDu) and trafficked into lysosomes, as observed by the colocalization of the ELP with a lysosome-specific dye through confocal fluorescence microscopy. Interestingly, both the ELP-dox conjugate and free drug exhibited near equivalent in vitro cytotoxicity, although their subcellular localization was significantly different. The free drug was largely concentrated in the nucleus, while the conjugate was dispersed throughout the cytoplasm with limited nuclear accumulation. These differences are significant because they suggest a different mechanism of cytotoxicity for the conjugate as compared with the free drug.     

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.     

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 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.     

Modification of the rates of chain cleavage of poly(ϵ-caprolactone) and related polyesters in the solid state

The rates of chain cleavage of poly(ϵ-caprolactone) (PCL), poly (l-lactic acid) (PLLA), and a 3:7 copolymer of glycolic acid and l-lactic acid (PGLA) as films in water, alcohols, and acidic and basic reagents were determined by measurements of intrinsic viscosities and molecular weights. Partial ethoxylation of the carboxylic acid end groups of PCL reduced the rate of chain cleavage in water, consistent with an autocatalytic mechanism. The firstorder rate constants for hydrolysis of PCL, PLLA, and PGLA were 7.5 × 10⁻⁵, 7.4 × 10⁻⁵, and 1.2 × 10⁻³ h⁻¹, respectively. Ethanol, pentanol, 5% oleic acid, decylamine, and tributylamine increased the rate of chain cleavage of PCL but not that of PLLA. The rate of erosion but not the rate of chain cleavage of PGLA was enhanced by these reagents, with the exception of pentanol. Changes in the morphology of the polymers during degradation were characterized by differential scanning calorimetry. The origin of the more rapid rate of hydrolysis of PGLA, relative to PCL and PLLA, appears to be the intrinsic reactivity of the glycolate linkage.     

Structural optimization of a "smart" doxorubicin-polypeptide conjugate for thermally targeted delivery to solid tumors.

A thermoresponsive, genetically engineered, elastin-like polypeptide (ELP) containing a C-terminal cysteine residue was synthesized and purified by inverse transition cycling (ITC) and conjugated to doxorubicin (Dox) molecules through four different pH-sensitive, maleimide-activated, hydrazone linkers. The efficiency of Dox activation, conjugation ratios to ELP and biophysical characterization-hydrodynamic radius (Rh) and the temperature transition kinetics-of the ELP-Dox conjugates and pH-mediated release of Dox were quantified in this study. Conjugation ratios of the maleimide-activated Dox to the thiol group of a unique cysteine in the ELP were close to unity. The Rh of the conjugate increased as the linker length between the ELP backbone and Dox was increased. The linker structure and length had little effect on the Tt of the ELP-Dox conjugates, as all conjugates exhibited Tt's that were similar to the native ELP. However, the ELP-Dox conjugates with longer linkers exhibited slower transition kinetics compared to the ELP-Dox conjugates with shorter linkers. The highest release of the ELP-Dox conjugate by cleavage of the hydrazone bond at pH 4 was nearly 80% over 72 h and was exhibited by the conjugate with the shortest linker.     

HPMA copolymers containing doxorubicin bound by a proteolytically or hydrolytically cleavable bond: comparison of biological properties in vitro.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin bound either by a proteolytically degradable bond (non-targeted PK1 or targeted with alpha-CD71 mAb) or by a hydrolytically degradable bond were synthesised and tested in vivo for various biological properties. Mouse 38C13 B-cell lympoma was used as a well established and defined cell line for this study. 38C13 cells are sensitive to free doxorubicin and IC50 was very low, about 0.014 microM. PK1 showed a strongly decreased cytostatic effect, IC50 being 12.6 microM. alpha-CD71 targeted conjugate, which can be considered as an antibody-targeted form of PK1, had IC50 0.358 microM. HPMA copolymer with doxorubicin bound via a hydrolytically sensitive bond (HYD conjugate) showed a high cytostatic effect with IC50 about 0.052 microM. We demonstrated that HYD conjugate inhibited DNA synthesis and induced p21(Waf1/Cip1) protein expression (p21(Waf1/Cip1) is cyclin-dependent kinase inhibitor which blocks cell cycle progression) as quickly as free doxorubicin, whereas PK1 acted much more slowly. Similarly, apoptosis induction measured by Annexin V binding and Caspase 3 activity was detected later after incubation of cells with PK1 or alpha-CD71 targeted conjugate. Apoptosis was manifested by elevation of bax and bad mRNA levels, which was much more rapid and intense in the case of free doxorubicin and HYD conjugate. Expression of antiapoptotic genes as well as cyclin-dependent kinases was surprisingly not affected.     

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.     

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.     

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 targeted quantum dot-mucin 1 aptamer-doxorubicin conjugate for imaging and treatment of cancer

In this study, we report the design and delivery of a tumor-targeted, pH-responsive quantum dot-mucin1 aptamer-doxorubicin (QD-MUC1-DOX) conjugate for the chemotherapy of ovarian cancer. To achieve active cancer targeting, QD was conjugated with a DNA aptamer specific for mutated MUC1 mucin overexpressed in many cancer cells including ovarian carcinoma. DOX was attached to QD via a pH-sensitive hydrazone bond in order to provide the stability of the complex in systemic circulation and drug release in acidic environment inside cancer cells. The data show that this bond is stable at neutral and slightly basic pH and undergoes rapid hydrolysis in mildly acidic pH. Confocal microscopy and in vivo imaging studies show that the developed QD-MUC1-DOX conjugate had higher cytotoxicity than free DOX in multidrug resistant cancer cells and preferentially accumulated in ovarian tumor. Data obtained demonstrate a high potential of the proposed conjugate in treatment of multidrug resistant ovarian cancer.     

pH-Responsive hyperbranched polypeptides based on Schiff bases as drug carriers for reducing toxicity of chemotherapy

Polymeric micelles have great potential in drug delivery systems because of their multifunctional adjustability, excellent stability, and biocompatibility. To further increase the drug loading efficiency and controlled release ability, a pH-responsive hyperbranched copolymer methoxy poly(ethylene glycol)-b-polyethyleneimine-poly(Nε-Cbz-l-lysine) (MPEG-PEI-PBLL) was synthesized successfully. MPEG-PEI-NH₂ was synthesized to initiate the ring-opening polymerization of benzyloxycarbonyl substituted lysine N-carboxyanhydride (Z-lys NCA). The introduction of Schiff bases in the polymer make it possible to respond to the variation of pH values, which cleaved at pH 5.0 while stable at pH 7.4. As the polymer was amphiphilic, MPEG-PEI-PBLL could self-assemble into micelles. Owing to the introduction of PEI, which make the copolymer hyperbranched, the pH-responsive micelles could efficiently encapsulate theranostic agents, such as doxorubicin (DOX) for chemotherapy and NIRF dye DiD for in vivo near-infrared (NIR) imaging. The drug delivery system prolonged the drug circulation time in blood and allowed the drug accumulate effectively at the tumor site. Following the guidance, the DOX was applied in chemotherapy to achieve cancer therapeutic efficiency. All the results demonstrate that the polymer micelles have great potential for cancer theranostics.

Polymeric micelles have great potential in drug delivery systems because of their multifunctional adjustability, excellent stability, and biocompatibility.     

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 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.     

HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity.

Data on the synthesis, physicochemical characterisation and in vitro and in vivo biological properties of the new, nontargeted or antibody-targeted polymer-doxorubicin conjugates designed as anticancer drugs are presented. In the conjugates, the anticancer drug doxorubicin (DOX) is attached to the polymer carrier via a simple hydrolytically labile spacer containing either a hydrazone bond or cis-aconitic acid residue. In vitro incubation of the conjugates in buffers led to a fast DOX release from the polymer at pH 5 (modelling intracellular environment) while at pH 7.4 (modelling blood) the conjugates are relatively stable. Cytotoxicity of the conjugates to T cell lymphoma EL4 depended on the detailed structure of the spacer and the method used for antibody attachment and was much higher compared with the effect of similar classic conjugates (DOX attached to the polymer via enzymatically degradable spacer). In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the hydrazone conjugates containing only DOX was significantly enhanced (T cell lymphoma EL4, C57BL/10 mice) in comparison with free DOX or classic PK1, the PHPMA-DOX conjugate clinically tested at present. Increasing the molecular weight of the polymer carrier resulted in a more pronounced in vivo antitumor effect. Antibody-targeted conjugates with DOX bound via hydrazone bond exhibited even more extensive inhibition of the tumor growth with some long-term survivors. No survivors were observed after treatment of mice with free DOX or the nontargeted PHPMA-DOX conjugate.