Study on mechanism of elemene reversing tumor multidrug resistance based on luminescence pharmacokinetics in tumor cells in vitro and in vivo

While elemene (ELE) can reverse tumor multidrug resistance (MDR), the mechanisms for ELE reversing MDR remain unclear. Numerous studies have suggested that the efflux functionality of ATP-binding cassette (ABC) transporters, not their quantity, is more relevant to tumor MDR. However, no appropriate methods exist for real-time detection of the intracellular drug efflux caused by ABC transporters in vitro, especially in vivo, which hinders the examination of MDR reversal mechanisms. This study directly investigates the correlation between efflux functionality of ABC transporters and MDR reversal via ELE, using d-luciferin potassium salt (d-luc) as the chemotherapeutic substitute to study the intracellular drug efflux. Here, a luciferase reporter assay system combined with bioluminescence imaging confirmed that the efflux of d-luc from MCF-7/DOX^Fluc cells in vitro and in vivo was significantly reduced by ELE and when combined with Doxorubicin (DOX), ELE showed a synergistically anti-tumor effect in vitro and in vivo. Additionally, the luminescence pharmacokinetics of d-luc in MCF-7/DOX^Fluc cells and pharmacodynamics of the combined ELE and DOX in vivo showed a great correlation, implying that d-luc might be used as a probe to study ABC transporters-mediated efflux in order to explore mechanisms of traditional Chinese medicines reversing MDR.

The correlation between efflux functionality of ATP-binding cassette transporters and tumor multidrug resistance reversing via elemene was investigated using bioluminescence imaging (BLI) technology and luciferase reporter gene technology.     

CD44-targeted hyaluronic acid–curcumin reverses chemotherapeutics resistance by inhibiting P-gp and anti-apoptotic pathways

Chemotherapeutic drug resistance poses a great challenge in cancer therapy. Drug efflux and anti-apoptotic processes are the two most common mechanisms leading to chemotherapy resistance. In this study, we focused on the applicability of curcumin (CUR) as a sensitizer for chemotherapeutics (doxorubicin [DOX] as the model drug) modified with hyaluronic acid (HA) as an effective therapeutic strategy against multidrug resistance (MDR) in cancer cells. We constructed an HA–CUR/DOX delivery system measuring approximately 180 nm with superior encapsulation efficacy and serum stabilities. In vitro, we found that HA modification could facilitate the efficient delivery of chemotherapeutics through CD44 receptor-mediated targeted delivery. MTT assay results confirmed that the combination of CUR and DOX/paclitaxel (PTX) had a significant synergistic effect and significantly reversed MDR. Further experiments including real-time polymerase chain reaction and western blotting proved that the main mechanisms by which CUR reversed MDR in tumor cells were inhibiting the expression and activity of P-glycoprotein (P-gp) and inducing apoptosis through mitochondrial pathway. Taken together, our new engineered tumor-targeting nanoparticle delivery system may have the potential for overcoming MDR in cancer.

Chemotherapeutic drug resistance poses a great challenge in cancer therapy.     

Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers

Cancers are frequently chemoresistant because of overexpression of P-glycoprotein. Two different approaches to improve cancer treatment are currently being investigated in clinical trials: inhibition of P-glycoprotein function by reversing agents, and alleviation of leukocytopenia by MDR1 gene transfer to normal bone marrow of patients. We report here that retroviral vectors encoding a mutant P-glycoprotein (MDR1-F983A) protect hematopoietic cells from anticancer drugs even in the presence of trans-(E)-flupentixol, an inhibitor of P-glycoprotein. Transfer of either mutant or wild-type MDR1 to K562 erythroleukemia cells or primary murine bone marrow resulted in reduced accumulation of daunomycin and vinblastine because of increased drug efflux.trans-(E)-Flupentixol at concentrations up to 10 microM failed to reverse drug efflux mediated by the product of the mutant MDR1 while wild-type P-glycoprotein was inhibited. In the presence of 2 microM trans-(E)-flupentixol chemoresistance to daunomycin was circumvented only in K562 cells transduced with wild-type, but not with mutant, MDR1. Moreover, drug resistance of KB-8-5 epidermoid cancer cells, which express the wild-type MDR1 gene at levels comparable to clinical specimens from multidrug-resistant cancers, was fully overcome in the presence of trans-(E)-flupentixol. Vectors expressing mutant P-glycoprotein may help improve chemotherapy by allowing safe dose intensification under conditions in which multidrug-resistant cancers are rendered drug sensitive by reversing agents.     

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.     

ABCG2/BCRP: Specific and Nonspecific Modulators

Multidrug resistance (MDR) in cancer cells is the development of resistance to a variety of structurally and functionally nonrelated anticancer drugs. This phenomenon has become a major obstacle to cancer chemotherapy seriously affecting the clinical outcome. MDR is associated with increased drug efflux from cells mediated by an energy‐dependent mechanism involving the ATP‐binding cassette (ABC) transporters, mainly P‐glycoprotein (ABCB1), the MDR‐associated protein‐1 (ABCC1), and the breast cancer resistance protein (ABCG2). The first two transporters have been widely studied already and reviews summarized the results. The ABCG2 protein has been a subject of intense study since its discovery as its overexpression has been detected in resistant cell lines in numerous types of human cancers. To date, a long list of modulators of ABCG2 exists and continues to increase. However, little is known about the clinical consequences of ABCG2 modulation. This makes the design of novel, potent, and nontoxic inhibitors of this efflux protein a major challenge to reverse MDR and thereby increase the success of chemotherapy. The aim of the present review is to describe and highlight specific and nonspecific modulators of ABCG2 reported to date based on the selectivity of the compounds, as many of them are effective against one or more ABC transport proteins.     

A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system

The objectives of this study were to evaluate the potential of a polymer-lipid hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein (Pgp) substrate, in Pgp-overexpressing cancer cell lines and to explore the underlying mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer (Dox-PLN) were prepared using an ultrasound method. Two Pgp-overexpressing breast cancer cell lines (a human cell line, MDA435/LCC6/MDR1, and a mouse cell line, EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-overexpressing cell lines with Dox-PLNs resulted in significantly enhanced Dox uptake and more substantial increases in drug retention after the end of treatment compared with free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-PLN form to MDA435/LCC6/MDR1 cells. Endocytosis inhibition studies revealed that phagocytosis is an important pathway in the membrane permeability of the nanoparticles. These findings suggest that some of the Dox physically associated with the nanoparticles bypass the membrane-associated Pgp when delivered as Dox-PLNs, and in this form, the drug is better retained within the Pgp-overexpressing cells than the free drug. The present study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing tumor cells using lipid-based nanoparticle formulations.     

一株耐阿霉素小鼠白血病L1210细胞系的建立及耐药机制的研究

肿瘤细胞耐药是肿瘤病人治疗失败的主要原因。通过连续递增阿霉素浓度的方法,筛选出一株对阿霉素的耐药性增高91.2倍的小鼠白血病L1210细胞系(L1210/DOX)。该细胞与L1210敏感细胞的增殖速度相似,倍增时间分别为15和15.5h。电子显微镜结果显示L1210/DOX细胞的形状和大小仍保持典型的白血病细胞态,与敏感株相似。该耐药细胞系与高三尖杉酯碱和长春新碱有交叉耐药,其半数抑制浓度(IC_(50))分别为敏感株的31.4和33.4倍,但对烷化剂马法兰无交叉耐药,呈多药耐药性。细胞内药物浓度检测的结果为阿霉素在L1210/DOX细胞内蓄积浓度仅为敏感细胞的9％。斑点杂交的结果显示L1210/DOX的多药耐药基因(MDR-1)mRNA水平显著高于敏感株。然而,二者的谷胱甘肽S-转移酶活性无显著差异。以上结果提示MDR-1基因的过度表达在L1210/DOX细胞耐药中起着重要作用;L1210/DOX细胞可经DBA/2小鼠传代,将成为体内耐药的小鼠模型。     

Chitosan nanoparticles as delivery systems for doxorubicin.

The aim of this paper was to evaluate the potential of chitosan nanoparticles as carriers for the anthracycline drug, doxorubicin (DOX). The challenge was to entrap a cationic, hydrophilic molecule into nanoparticles formed by ionic gelation of the positively charged polysaccharide chitosan. To achieve this objective, we attempted to mask the positive charge of DOX by complexing it with the polyanion, dextran sulfate. This modification doubled DOX encapsulation efficiency relative to controls and enabled real loadings up to 4.0 wt.% DOX. Separately, we investigated the possibility of forming a complex between chitosan and DOX prior to the formation of the particles. Despite the low complexation efficiency, no dissociation of the complex was observed upon formation of the nanoparticles. Fluorimetric analysis of the drug released in vitro showed an initial release phase, the intensity of which was dependent on the association mode, followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated that those containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed to chitosan before nanoparticle formation showed slightly decreased activity. Additionally, confocal studies showed that DOX was not released in the cell culture medium but entered the cells while remaining associated to the nanoparticles. In conclusion, these preliminary studies showed the feasibility of chitosan nanoparticles to entrap the basic drug DOX and to deliver it into the cells in its active form.     

SIRB,sans iron oxide rhodamine B,a novel cross‐linked dextran nanoparticle,labels human neuroprogenitor and SH‐SY5Y neuroblastoma cells and serves as a USPIO cell labeling control

This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross‐linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post‐labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH‐SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH‐SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterisation and tumour targeting of PEGylated polylysine dendrimers bearing doxorubicin via a pH labile linker

Polylysine dendrimers have potential as biodegradable vectors for the delivery of cytotoxic drugs to solid tumours. Here, the cytotoxicity, drug release and tumour targeting properties of Generation 5 PEGylated polylysine dendrimers comprising an outer generation of l-lysine or succinimyldipropyldiamine (SPN) and containing doxorubicin (DOX) linked through an acid labile 4-(hydrazinosulfonyl) benzoic acid (HSBA) linker have been characterised. Less than 10% of the DOX load was released from LYS or SPN dendrimers in pH 7.4 buffer over 3 days. In contrast approximately 100% release was evident at pH 5. The DOX-conjugated dendrimers also retained similar cytotoxic properties to free DOX in in vitro cell culture studies (presumably as a result of in situ liberation of free DOX). The clearance patterns of the DOX conjugated SPN and all-lysine dendrimers were similar to the equivalent non-DOX conjugated systems, however the SPN dendrimers showed reduced metabolic lability and increased uptake into RES organs when compared to the equivalent all-lysine dendrimers. In vivo assessment of the DOX-conjugated, PEGylated polylysine dendrimers (both SPN and LYS constructs) in rats bearing Walker 256 tumours revealed higher uptake into tumour tissue when compared with control tissue such as muscle (~ 8 fold) and heart (~ 3 fold). The data suggest that polylysine dendrimers containing DOX conjugated via an acid labile HSBA linker may provide a mechanism to target the delivery of DOX to tumours.     

Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells

P-glycoprotein (Pgp), a membrane transporter encoded by the MDR1 gene in human cells, mediates drug efflux from cells, and it plays a major role in causing multidrug resistance (MDR). Confocal microscopy was used to study in vitro and in vivo drug accumulation, net uptake and efflux, and MDR modulation by P-glycoprotein inhibitors in MDR1-transduced human MDA-MB-435mdr (MDR) cancer cells. The MDR cells were approximately 9-fold more resistant to the anticancer drug doxorubicin than their parental wild-type MDA-MB-435wt (WT) cells. Doxorubicin accumulation in the MDR cells was only 19% of that in the WT cells. The net uptake of doxorubicin in the nuclei of the MDR cells was 2-fold lower than that in the nuclei of the WT cells. Pgp inhibitors verapamil, cyclosporine A, or PSC833 increased doxorubicin accumulation in the MDR cells up to 79%, and it reversed drug resistance in these cells. In living animals, doxorubicin accumulation in MDA-MB-435mdr xenograft tumors was 68% of that in the wild-type tumors. Administration of verapamil, cyclosporine A, or PSC833 before doxorubicin treatment of the animals increased doxorubicin accumulation in the MDR tumors up to 94%. These studies have added direct in vitro and in vivo information on the capacity of the transporter protein Pgp to efflux doxorubicin and on the reversal of MDR by Pgp inhibitors in resistant cancer cells.     

Chemical molecular‐based approach to overcome multidrug resistance in cancer by targeting P‐glycoprotein (P‐gp)

Multidrug resistance (MDR) remains one of the major impediments for efficacious cancer chemotherapy. Increased efflux of multiple chemotherapeutic drugs by transmembrane ATP‐binding cassette (ABC) transporter superfamily is considered one of the primary causes for cancer MDR, in which the role of P‐glycoprotein (P‐gp/ABCB1) has been most well‐established. The clinical co‐administration of P‐gp drug efflux inhibitors, in combination with anticancer drugs which are P‐gp transport substrates, was considered to be a treatment modality to surmount MDR in anticancer therapy by blocking P‐gp‐mediated multidrug efflux. Extensive attempts have been carried out to screen for sets of nontoxic, selective, and efficacious P‐gp efflux inhibitors. In this review, we highlight the recent achievements in drug design, characterization, structure–activity relationship (SAR) studies, and mechanisms of action of the newly synthetic, potent small molecules P‐gp inhibitors in the past 5 years. The development of P‐gp inhibitors will increase our knowledge of the mechanisms and functions of P‐gp‐mediated drug efflux which will benefit drug discovery and clinical cancer therapeutics where P‐gp transporter overexpression has been implicated in MDR.     

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.     

Enhanced chemotherapy of cancer using pH-sensitive mesoporous silica nanoparticles to antagonize P-glycoprotein-mediated drug resistance

Multidrug resistance (MDR) is the major clinical obstacle in the management of cancer by chemotherapy. Overexpression of ATP-dependent efflux transporter P-glycoprotein (PGP) is a key factor contributing to multidrug resistance of cancer cells. The purpose of the present study was to use the endosomal pH-sensitive MSN (mesoporous silica nanoparticles; MSN-Hydrazone-Dox) for controlled release of doxorubicin (Dox) in an attempt to overcome the PGP-mediated MDR. In vitro cell culture studies indicate that uptake of MSN-Hydrazone-Dox by the human uterine sarcoma MES-SA/Dox-resistant tumor (MES-SA/Dx-5) cell occurs through endocytosis, thus bypassing the efflux pump resistance. This improves the efficacy of the drug and leads to significant cytotoxicity and DNA fragmentation evidenced by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA laddering assays. In vivo studies show that the intratumor injection of MSN-Hydrazone-Dox induces significant apoptosis of MES-SA/Dox-resistant cancer cells. This is validated by active caspase-3 immunohistochemical analysis. However, MSN-Hydrazone, without doxorubicin conjugation, cannot induce apoptosis in vitro and in vivo. In conclusion, both in vitro and in vivo studies show that MSN could serve as an efficient nanocarrier entering cell avidly via endocytosis, thus bypassing the PGP efflux pump to compromise the PGP-mediated MDR. MSN-Hydrazone-Dox could further respond to endosomal acidic pH to release doxorubicin in a sustained manner. Besides the cell study, this is the first report that successfully shows the therapeutic efficacy of using MSN against MDR cancer in vivo.     

Doxorubicin-loaded mesoporous magnetic nanoparticles to induce apoptosis in breast cancer cells

Selective targeting of chemotherapeutic drugs toward the cancer cells overcomes the limitations involved in chemotherapy. Ideally, targeted delivery system holds great potential in cancer therapy due to specific release of drug in the cancer tissues. In this regard, DOX-loaded chitosan coated mesoporous magnetic nanoparticles (DOX-CMMN) were prepared and evaluated for its physicochemical and biological characteristics. Nanosized magnetic nanoparticles were observed with a high loading capacity for DOX. The drug-loaded nanoparticles exhibited a controlled and sustained release of drug without any burst release phenomenon. The DOX-DMMN showed a concentration-dependent cell proliferation inhibitory action against breast cancer cells. The blank nanoparticles showed excellent biocompatibility with cell viability >85% at the maximum tested concentration. Our results showed that chitosan coated magnetic system has high potential for breast cancer targeting under an alternating current magnetic field (ACMF). The present study showed that magnetic nanoparticles can be targeted to tumor cells under the presence of oscillating magnetic field. The combined effect of chemotherapy and thermotherapy can have a promising clinical potential for the treatment of breast cancer.     

Amelioration of doxorubicin-induced cardiotoxicity by an anticancer-antioxidant dual-function compound, HO-3867

Doxorubicin (DOX) is a drug commonly used for the treatment of cancer. The development of resistance to DOX is common, and high cumulative doses cause potentially lethal cardiac side effects. HO-3867 (3,5-bis(4-fluorobenzylidene)-1-[(2,2,5,5-tetramethyl-2,5-dihydro-1-hydroxy-pyrrol-3-yl)methyl]piperidin-4-one), a synthetic curcumin analog, has been shown to exhibit both anticancer and cardioprotective effects. However, its cardioprotection in the setting of a conventional cancer therapy has not been established. This work investigated the use of HO-3867 and DOX to achieve a complementary outcome, i.e., increased toxicity toward cancer cells, and reduced cardiac toxicity. Combination treatment was investigated using DOX-resistant MCF-7 breast cancer cells [MCF-7 multidrug-resistant (MDR)] and BALB/c mice. Lower doses of HO-3867 and DOX (5 and 2.5 μM, respectively) reduced viability of MCF-7 MDR cells to an extent significantly greater than that when either drug was used alone, an effect equivalent to that induced by exposure to 50 μM DOX. In normal cardiac cells, the loss of viability from combination treatment was significantly lower than that induced by 50 μM DOX. Increases in apoptotic markers, e.g., cleaved caspase-3, and decreases in fatty acid synthase and pAkt expressions were observed by Western blotting. Mice treated with both HO-3867 and DOX showed significant improvement in cardiac functional parameters compared with mice treated with DOX alone. Reduced expression of Bcl-2 and pAkt was observed in mice treated with DOX alone, whereas mice given combination treatment showed levels similar to control. The study indicates that combination treatment of HO-3867 and DOX is a viable option for treatment of cancer with reduced cardiotoxic side effects.     

环孢霉素A对K562／DOX细胞药物积聚和外排的影响

目的：寻找克服肿瘤细胞多药耐药的方法。方法：以环孢霉素Ａ（ＣｓＡ）作为耐药逆转剂，用ＭＴＴ法体外药物敏感试验，观察ＣｓＡ对多药耐药白血病细胞株Ｋ５６２／ＤＯＸ的药物敏感性、细胞内药物的积聚和外排的影响。结果：ＣｓＡ可增强阿霉素（ＤＯＸ）对Ｋ５６２／ＤＯＸ的细胞毒作用，且存在剂量依赖关系。ＣｓＡ≥２μｇ／ｍｌ能较明显提高Ｋ５６２／ＤＯＸ对ＤＯＸ的敏感性。用２μｇ／ｍｌＣｓＡ处理后，Ｋ５６２／ＤＯＸ细胞内ＤＯＸ外排速度明显减慢，ＤＯＸ含量仅减少１２．３％，但对Ｋ５６２细胞内药物外排无影响。结论：ＣｓＡ能有效地减慢Ｋ５６２／ＤＯＸ细胞内ＤＯＸ外排速度，增加细胞内ＤＯＸ积聚。ＣｓＡ对Ｋ５６２细胞药物敏感性、细胞内药物外排和积聚均无影响。     

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.