Feasibility of using a bone-targeted, macromolecular delivery system coupled with prostaglandin E(1) to promote bone formation in aged, estrogen-deficient rats.

PURPOSE: Macromolecular delivery systems have therapeutic uses because of their ability to deliver and release drugs to specific tissues. The uptake and localization of HPMA copolymers using Asp(8) as the bone-targeting moiety was determined in aged, ovariectomized (ovx) rats. PGE(1) was attached via a cathepsin K-sensitive linkage to HPMA copolymer-Asp(8) conjugate and was tested to determine if it could promote bone formation. MATERIALS AND METHODS: The uptake of FITC-labeled HPMA copolymer-Asp(8) conjugate (P-Asp(8)-FITC) on bone surfaces was compared with the mineralization marker, tetracycline. Then a targeted PGE(1)-HPMA copolymer conjugate (P-Asp(8)-FITC-PGE(1)) was given as a single injection and its effects on bone formation were measured 4 weeks later. RESULTS: P-Asp(8)-FITC preferentially deposited on resorption surfaces, unlike tetracycline. A single injection of P-Asp(8)-FITC-PGE(1) resulted in greater indices of bone formation in aged, ovx rats. CONCLUSIONS: HPMA copolymers can be targeted to bone surfaces using Asp(8), with preferential uptake on resorption surfaces. Additionally, PGE(1) attached to the Asp(8)-targeted HPMA copolymers and given by a single injection resulted in greater bone formation measured 4 weeks later. This initial in vivo study suggests that macromolecular delivery systems targeted to bone may offer some therapeutic opportunities and advantages for the treatment of skeletal diseases.

Enhanced antitumor activity of combinations of free and HPMA copolymer-bound drugs.

The synergism in anticancer effect toward human renal carcinoma A498 cells by binary combinations of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound anticancer drugs, SOS thiophene (SOS), doxorubicin (DOX), and mesochlorin e6 monoethylenediamine (Mce6), was evaluated. The combination index (CI) analysis was used to quantify the synergism, antagonism, and additive effects. Both free drugs and HPMA copolymer conjugates, when used as single agents or in combination, exhibited cytotoxic activities against A498 cells, as determined using a modified MTT assay. As single agents, SOS and P-GFLG-SOS (HPMA copolymer conjugates containing SOS bound via glycylphenylalanylleucylglycine [GFLG] spacer) were significantly more effective than the other agents evaluated. The synergistic effects ranked in the order SOS+DOX>P-GFLG-DOX+P-GFLG-Mce6 approximately DOX+Mce6>P-GFLG-SOS+P-GFLG-DOX approximately SOS+Mce6>P-GFLG-SOS+P-GFLG-Mce6. The combination of SOS+DOX proved to be synergistic over all cell growth inhibition levels. All other combinations exhibited synergism in a wide range of drug effect levels. The SOS+Mce6 and P-GFLG-SOS+P-GFLG-Mce6 combinations displayed synergism up to drug affected fraction (fa) values of about 0.8 and reached slight antagonism and nearly additivity at fa=0.95, respectively. However, all other combinations were synergistic up to fa<0.9 and were additive at higher fa values. The observations that most combinations produced synergistic effects will be important for clinical translation.

Liberation of doxorubicin from HPMA copolymer conjugate is essential for the induction of cell cycle arrest and nuclear fragmentation in ovarian carcinoma cells.

Despite intensive study, the molecular mechanism for cell toxicity of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin remains unclear. Moreover, the ability of the released drug to accumulate in the nucleus has also been questioned. We have hypothesized that the pattern of cell cycle progression is a useful indicator for the presence of free doxorubicin in the nucleus and its interaction with nuclear DNA. The effects of HPMA copolymer-bound doxorubicin on cell cycle progression were evaluated in this study in cultured human ovarian cancer A2780 cells. We determined that P-GFLG-DOX, but not P-GG-DOX, initiates cell cycle arrest and nuclear fragmentation in the same manner as free DOX, but with a time-delay. Our data indicate that drug release from the conjugate is required for the apoptotic activity associated with the conjugate.

Cathepsin K inhibitor-polymer conjugates: potential drugs for the treatment of osteoporosis and rheumatoid arthritis.

The role of the newly discovered cysteine protease, cathepsin K, in osteoporosis and rheumatoid arthritis is reviewed. The current development of cathepsin K inhibitors and their targeted delivery using synthetic polymer carriers are discussed. Future challenges and possible strategies to improve these delivery systems are addressed.

Design of a multivalent galactoside ligand for selective targeting of HPMA copolymer-doxorubicin conjugates to human colon cancer cells.

N-(2-hydroxypropyl)methacrylamide (HPMA)-based copolymers have been shown to be efficient carriers for anticancer drugs because of their versatile chemistry and good biocompatibility. As demonstrated with hepatocytes, targeting efficacy of anticancer drugs could be further improved when the drug (doxorubicin) was conjugated to HPMA copolymers with biorecognisable groups, such as simple carbohydrates. The present study was devised to learn whether the cluster (multivalent) construction of carbohydrate residues could improve the targeting capability of HPMA copolymer-doxorubicin (DOX) conjugates towards human colon adenocarcinoma cells. DOX was linked via a lysosomally degradable tetrapeptide sequence to HPMA copolymers bearing galactosamine (GalN), lactose (Lac), or multivalent galactose residues (TriGal) to produce targetable polymeric drug carriers. The effect of the type of sugar moiety and its three-dimensional cluster arrangement on biorecognition by three human colon-adenocarcinoma cell lines was studied. The role of galectin-3 in the biorecognition of HPMA copolymer conjugates was explored. Biorecognition of the targetable (glycoside-bearing) conjugates decreased their IC(50) doses in comparison to the non-targetable (non-glycosylated) conjugates. The biorecognition of the TriGal-containing HPMA copolymer-doxorubicin conjugate by the cells was superior with concomitant decrease of its IC(50) doses. It is suggested that the increased cytotoxicity of the glycosylated HPMA-copolymer-DOX conjugates toward human colon-adenocarcinoma cells was caused by their biorecognition and effective internalisation via receptor-mediated endocytosis. All three human colon adenocarcinoma cell lines tested, Colo-205, SW-480 and SW-620, expressed the galectin-3 protein and the galectin-3-specific RNA. However, contrary to expectation, Colo-205 cells did not express a detectable amount of galectin-3 on the cell surface. This suggests that the binding of the glycoside-bearing HPMA copolymer-DOX conjugates to the cells was mediated not only by galectin-3. We conclude that targeting of the anticancer agent, doxorubicin, using HPMA copolymer conjugates bearing multivalent galactoside residues can improve their cytotoxicity.

The influence of a colonic microbiota on HPMA copolymer lectin conjugates binding in rodent intestine.

Germ-free (GF) animals lack a colonic microflora like that seen in conventional (CV) animals. Bacterial presence plays a role in the development of glycoproteins in the gastrointestinal (GI) tract; the absence of a microbiota has been seen to suppress the production of certain glycoproteins and glycolipids. Binding patterns of lectins are modified when glycoprotein structures are altered (e.g., during development or disease). Little information on lectin binding patterns in mature GF animals is available. We examined the binding of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) [P(HPMA)-(WGA-FITC)] and FITC-labeled peanut agglutinin (PNA) [P(HPMA)-(PNA-FITC)] in CV and GF mouse colon with and without neuraminidase pretreatment. Anti-Thomsen-Friedenreich (TF) antigen (a development and disease-related glycoprotein) antibody binding was also examined in these tissues. Subtle differences were seen in the binding patterns between CV and GF animals. CV animals showed strong P(HPMA)-(WGA-FITC) binding in goblet cells, but minimal P(HPMA)-(PNA-FITC) binding was visible. In GF animals, luminal surface binding of P(HPMA)-(WGA-FITC) was visible, and goblet cell binding of P(HPMA)-(PNA-FITC) was seen. These subtle changes suggest that altered glycoprotein expression occurred under GF conditions.

Enhanced biorecognition and internalization of HPMA copolymers containing multiple or multivalent carbohydrate side-chains by human hepatocarcinoma cells.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing pendant saccharide moieties (galactosamine, lactose, and triantennary galactose) were synthesized. The relationship between the content of saccharide moieties and three-dimensional arrangement of galactose residues and their biorecognition and internalization by human hepatocarcinoma HepG2 cells was investigated. The results obtained clearly indicated preferential binding of the trivalent galactose and the lactose-containing copolymers to these cells. The higher the saccharide moieties content in HPMA copolymers, the higher the levels of binding. The biorecognition of the glycosylated HPMA copolymers by HepG2 cells was inhibited by free lactose. The data on the internalization and subcellular trafficking of HPMA copolymer conjugates obtained by confocal fluorescence microscopy correlated well with the flow cytometric analysis of their biorecognition by target cells. Structural features of the glycosides responsible for the specific recognition of the HPMA copolymers have been identified. The results underline the potential of glycosylated HPMA copolymers for delivery of pharmaceutical agents to hepatocarcinoma cells.

Improved synthesis and evaluation of 17-substituted aminoalkylgeldanamycin derivatives applicable to drug delivery systems.

The 17-methoxy group of geldanamycin was substituted with 1,3-diaminopropane and 1,3-diamino-2-hydroxypropane to introduce a primary amino group useful for conjugation with targeting moieties and drug carriers. We have developed a procedure that has provided improved yield and reproducibility of the syntheses. Both geldanamycin derivatives demonstrated antiproliferative activity towards the human ovarian carcinoma cell line, A2780.

Water soluble polymers in tumor targeted delivery.

The rationales for the use of water soluble polymers for anticancer drug delivery include: the potential to overcome some forms of multidrug resistance, preferential accumulation in solid tumors due to enhanced permeability and retention (EPR) effect, biorecognizability, and targetability. The utility of a novel paradigm for the treatment of ovarian carcinoma in an experimental animal model, which combines chemotherapy and photodynamic therapy with polymer-bound anticancer drugs is explained. Research and clinical applications as well as directions for the future development of macromolecular therapeutics are discussed.

Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates.

Geldanamycin (GDM) is a benzoquinone ansamycin antibiotic with anticancer activity. The use of drug delivery systems based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing lysosomally degradable oligopeptide (GFLG) spacers results in an increased therapeutic efficacy of anticancer drugs. The objective of this study was to synthesize HPMA copolymer-GDM conjugates with anticancer activity and reduced toxic side-effect of the compound. 17-(3-Aminopropylamino)-17-demethoxygeldanamycin (AP-GDM) was synthesized and converted into a polymerizable GDM derivative, N-methacryloylglycylphenylalanylglycyl-17-(3-aminopropylamino)-17-demethoxygeldanamycin [MA-GFLG-(AP-GDM)]. The structures of AP-GDM and MA-GFLG-(AP-GDM) were validated by mass spectroscopy, elemental analysis, and two-dimensional nuclear magnetic resonance. MA-GFLG-(AP-GDM) was copolymerized with HPMA and N-methacryloyglycylglycine p-nitrophenylester by radical precipitation polymerization. Water-soluble HPMA copolymer-AP-GDM conjugates (M(r)=16 kDa) were obtained. Monoclonal antibody OV-TL16, which recognizes the OA-3 antigen expressed on the OVCAR-3 human ovarian carcinoma cell line, was optionally attached to the HPMA copolymer-AP-GDM conjugate. Cytotoxicity of polymer-bound AP-GDM (both targeted and non-targeted) was determined using OVCAR-3 and another human ovarian carcinoma cell line, A2780. The HPMA copolymer-AP-GDM conjugate was cytotoxic toward A2780 cells. Attachment of OV-TL16 antibody enhanced cytotoxicity of the conjugate toward OVCAR-3 cells.

Combination chemotherapy and photodynamic therapy of targetable N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin/mesochlorin e(6)-OV-TL 16 antibody immunoconjugates.

The aim of this study was to evaluate the combination chemotherapy and photodynamic therapy of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin (DOX) and mesochlorin e(6) (Mce(6)) targeted with an OV-TL 16 monoclonal antibody (P-DOX-Ab and P-Mce(6)-Ab, respectively) in nude mice bearing human ovarian OVCAR-3 carcinoma xenografts. P-DOX-Ab and P-Mce(6)-Ab were synthesized by first conjugating DOX or Mce(6) to an HPMA copolymer precursor (Mw=21000), then reacting with OV-TL 16 antibody. The immunoconjugates were purified by size exclusion chromatography on Superose 6 column and analyzed. The Mce(6) concentration in tissues was determined by a fluorescence assay. Eighteen hours after administration, the tumors received a light dose of 220 J/cm(2) from a KTP 650-nm dye-laser. P-DOX-Ab and P-Mce(6)-Ab had polymer:drug:protein weight ratios of 32:3:62 and 26:2:72, corresponding to polymer:drug:protein molecular ratios of approximately 4:14:1 and 3:8:1, respectively. The biodistribution results indicated that the percentage of total administered dose of Mce(6) in tumors reached approximately 1% for the nontargeted conjugate at 18 h after administration, while that of P-Mce(6)-Ab was approximately 13 times higher. Nude mice bearing OVCAR-3 xenografts that received one i.v. dose of P-DOX-Ab (2.2 mg/kg DOX equivalent) and P-Mce(6)-Ab (1.5 mg/kg Mce(6) equivalent) with light irradiation achieved a xenograft cure rate of more than 60%. The incorporation of OV-TL 16 antibody dramatically enhanced the accumulation in tumors with a concomitant increase in the therapeutic efficacy of P-DOX-Ab and P-Mce(6)-Ab in combination therapy, which may probably be attributed to both antibody targeting and enhanced permeability and retention (EPR) effects.

Preparation and biological evaluation of polymerizable antibody Fab' fragment targeted polymeric drug delivery system.

A new polymerizable antibody Fab' fragment with a PEG spacer (MA-PEG-Fab') was prepared from OV-TL 16 antibody, specific against the OA-3 antigen expressed on most human ovarian carcinomas. The MA-PEG-Fab' possessed a higher reactivity in the copolymerization with N-(2-hydroxypropyl)methacrylamide (HPMA) than the polymerizable Fab' fragment MA-Fab' with a short spacer. The MA-PEG-Fab' was copolymerized with HPMA and MA-Gly-Phe-Leu-Gly-Mce(6) producing an Fab' targeted HPMA copolymer-Mce(6) conjugate. The number and weight average molecular weights of the copolymer were 164000 and 271000 Da, respectively. About two MA-PEG-Fab' fragments per chain were incorporated in the copolymer conjugates. Preliminary in vivo antitumor studies indicated that the Fab' targeted conjugates showed a higher efficacy of tumor growth inhibition in nude mice than the non-targeted conjugate.

The effects of subcellular localization of N-(2-hydroxypropyl)methacrylamide copolymer-Mce(6) conjugates in a human ovarian carcinoma.

Photosensitizers, light-sensitive compounds, become activated upon illumination with a specific wavelength of light generating cytotoxic oxygen species. Due to the short half-life of singlet oxygen, the subcellular site of localization and excitation affects the type of cellular damage produced as well as cellular responses to different types of photodamage created within the cell. Here, we investigated the effects of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e(6) monoethylenediamine (Mce(6)) conjugates localized to different subcellular compartments. Temperature was utilized to achieve subcellular localization of conjugates and subcellular fractionation was performed to confirm localization patterns of HPMA copolymer-Mce(6) conjugates. Cytotoxicity studies suggest plasma membrane and late endosomes were more sensitive to photodamage than lysosomal compartments as observed by an approximate 2-fold decrease in the IC(50) compared to lysosomally accumulated conjugate. Releasing Mce(6) from the polymer backbone within lysosomal compartments significantly lowered the IC(50) when compared to HPMA copolymer conjugates with Mce6 bound via a nondegradable linkage. These differences will prove useful in the future design of HPMA copolymer-Mce(6) conjugates for the treatment of ovarian cancer.

Potential of lectin-N-(2-hydroxypropyl)methacrylamide copolymer-drug conjugates for the treatment of pre-cancerous conditions.

N-(2-Hydroxypropyl)methacrylamide (HPMA)-lectin (wheat germ agglutinin (WGA), peanut agglutinin (PNA)) drug conjugates for treatment of the pre-cancerous conditions ulcerative colitis and Barrett's esophagus are being developed. Cell-surface glycoproteins that are altered in disease and development bind lectins. PNA binds alpha-lactose and the Thomsen-Friedenreich (TF) antigen, a disease- and development-associated glycoprotein. PNA incorporation in conjugates may allow for preferential delivery to diseased over healthy tissues. Conjugates were prepared by attaching lectins to HPMA copolymers via an amide linkage. Frontal affinity chromatography was used to measure dissociation constants (K(d)) of free and conjugated lectins. Animal models of colitis (DSS, TNBS/EtOH) were developed. Human biopsy specimens were obtained. Free and HPMA copolymer-conjugated FITC-labeled lectin and anti-TF antigen antibody binding patterns were examined in normal neonatal, adult and diseased rodent tissues and normal and diseased human tissues. K(d) values of free and conjugated lectins were similar ( approximately 10(-5) M(-1)). Free and conjugated lectins had comparable binding patterns. In health, strong WGA binding was seen in goblet cells; PNA binding was minimal, occurring only in the supranuclear goblet cell region. In disease, WGA binding was not altered, but PNA binding was increased in both human and rodent tissues; entire goblets bound the lectin. Anti-TF antigen antibody binding was minimal, but did overlap with PNA binding patterns both in normal and diseased tissues. Conjugation of lectins to HPMA copolymers does not affect binding affinity. Alterations in glycoprotein structures in development and disease resulted in modified lectin binding patterns. In development and disease, the PNA binding seen was to the TF antigen and other lactose-containing glycoproteins. The results suggest that site-specific delivery of therapeutic agents such as cyclosporin A (CsA) for ulcerative colitis and mesochlorin e(6) for Barrett's esophagus may be achieved. P(HPMA)-lectin-CsA conjugates have been prepared and preliminary in vivo studies are underway.

Biorecognizable HPMA copolymer-drug conjugates for colon-specific delivery of 9-aminocamptothecin.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates for colon-specific delivery of 9-aminocamptothecin (9-AC) were designed. They hold 9-AC bound via spacers containing amino acid residues and aromatic azo bonds. In vitro release profiles of 9-AC from HPMA copolymer conjugates were evaluated under artificial conditions that simulated large intestinal azoreductase and peptidase activities. The studies indicated that the azo bond was reduced first, followed by the release of unmodified 9-AC from the 9-AC containing fragment by peptidases. Release profiles depended on the chemical structure of the peptide part of the spacer. Conjugates containing leucylalanine showed high colon-specific release of 9-AC when compared to alanine containing conjugates. It appears that the studied conjugates are suitable as colon-specific drug delivery systems.

Preliminary evaluation of caspases-dependent apoptosis signaling pathways of free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

The purpose of the study was to examine the role of caspases in signaling pathways of apoptosis induced by free doxorubicin (DOX) and HPMA copolymer-bound DOX (P(GFLG)-DOX) in human ovarian carcinoma cells. Sensitive A2780 and DOX resistant A2780/AD cells were exposed to different doses of drugs within 12, 18, 24 and 36 h. Caspase activity, expression of genes encoding human caspases 1-10, Apaf-1 and bcl-2 proteins and apoptosis were studied. In sensitive cells both free and P(GFLG)-DOX activated caspases 3, 7 and 9. In addition, P(GFLG)-DOX activated caspases 6 and 8. In resistant cells apoptosis induced by free DOX depended on the activation of caspases 2, 7 and 9, while caspase 3 was not involved; this explains the low degree of apoptosis induced by free DOX in resistant cells. P(GFLG)-DOX triggered the additional caspases 3, 6 and 8. A more pronounced degree of caspase activation and apoptosis after the action of P(GFLG)-DOX depended on the inhibition of bcl-2-encoded cellular defensive mechanisms and a more significant activation of Apaf-1. It was concluded that HPMA copolymer-bound DOX induced additional caspase-dependent apoptosis signaling pathways and the degree of the induction was higher, which led to more pronounced apoptosis when compared to free DOX.

Modification of cyclosporin A and conjugation of its derivative to HPMA copolymers.

Cyclosporin A (CsA) was epoxidized with m-chloroperoxybenzoic acid in the presence of sodium carbonate or with tert-butyl hydroperoxide in the presence of dioxomolybdenum iminodiethanoxide. The CsA epoxide was not stable and rearranged into a compound with a more stable five-member ring structure. An amino group containing cyclosporin A derivative (CsA amine) was obtained by the reaction of CsA epoxide with excess ethylenediamine. The yield of the CsA amine was 30--40% based on the CsA. An HPMA copolymer--CsA conjugate was prepared by the reaction of the CsA amine with an HPMA and MA-Gly-Phe-Leu-Gly-ONp copolymer. The content of CsA amine in the conjugate was 8.7 wt %. The CsA amine was released from the copolymer by enzymatic hydrolysis with papain.

Biodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates in nude mice.

The aim of this study was to evaluate the influence of the molecular weight (mol. wt) of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (DOX) conjugates (P-DOX) on biodistribution and therapeutic efficacy in nu/nu mice bearing human ovarian carcinoma OVCAR-3 xenografts. Copolymerisation of HPMA, a polymerisable derivative of DOX (N-methacryloylglycylphenylalanylleucylglycyl doxorubicin) and a newly designed crosslinking agent, N(2),N(5)-bis(N-methacryloylglycylphenylalanyl-leucylglycyl)ornithine methyl ester monomers resulted in novel, high mol. wt, branched, water-soluble P-DOX containing lysosomally degradable oligopeptide sequences as crosslinks and side-chains terminated in DOX. Four conjugates with mol. wt of 22, 160, 895 and 1230 kDa were prepared. The results indicated that the half-life in blood and the elimination rate from the tumour were up to 28 times longer and 25 times slower, respectively, for P-DOX (mol. wt=1230 kDa) than for free DOX. Treatment with P-DOX (mol. wt > or = 160 kDa) inhibited tumour growth more efficiently than that of 22 kDa P-DOX or free DOX (P<0.02) at a 2.2 mg/kg DOX equivalent dose. In conclusion, the administration of long circulating P-DOX resulted in enhanced tumour accumulation with a concomitant increase in therapeutic efficacy.

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in Hep G2 cells.

The development of macromolecules as drugs and drug carriers requires knowledge of their fate in cells. To this end, we studied the internalization and subcellular Fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in Hep G2 (human hepatocellular carcinoma) cells. Semiquantitative fluorometry confirmed that galactose was an effective ligand for receptor-mediated endocytosis for Hep G2 cells. The rate of internalization of a galactose-targeted copolymer was almost 2 orders of magnitude larger than that of the nontargeted copolymer. Confocal fluorescent microscopy of both fixed and live cells revealed that the polymer entered the cells by endocytosis. After longer incubation times (typically >8 hours), polymer escaped from small vesicles and distributed throughout the cytoplasm and nuclei of the cells. Polymer that entered the cytoplasm tended to accumulate in the nucleus. Microinjection of the HPMA copolymers into cells' cytoplasm and nuclei indicated that the polymers partitioned to the nucleus. The data from fixed cells was confirmed by microscopy of live, viable cells. To examine the effect of the fluorescent dye on the intracellular fate, polymers with fluorescein, Oregon Green 488, Lissamine rhodamine B, and doxorubicin were tested; no significant differences were observed.

Time- and concentration-dependent apoptosis and necrosis induced by free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

A2780 sensitive and A2780/AD doxorubicin (DOX) resistant human ovarian carcinoma cells were exposed to different concentrations (0.25, 0.5, 1, 5 and 10xIC(50)) of free and HPMA copolymer-bound DOX for 12, 24, 36, 48, 60 and 72 h. Apoptosis and necrosis were evaluated using the FITC-conjugated annexin V and propidium iodide staining. The data obtained showed that the induction of apoptosis and necrosis by both free DOX and HPMA copolymer-bound DOX were time- and concentration-dependent. The data also showed significant differences between the drugs. It was found that: (i) under the action of HPMA copolymer-bound doxorubicin the alterations in the plasma membrane permeability preceded disturbances in cellular metabolism; (ii) HPMA copolymer-bound doxorubicin kills the cells mainly by necrosis; (iii) HPMA copolymer-bound doxorubicin is a more effective anticancer drug than free doxorubicin.