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

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Abstract

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) (Mn: 5K)-b-PEG (Mn: 2K)-folate (polyHis/PEG-folate) (75 wt.%) and poly(l-lactic acid) (Mn: 3K)-b-PEG (Mn: 2K)-folate (PLLA/PEG-folate) (25 wt.%). The PHSM/f showed more than 90% cytotoxicity of DOX resistant MCF-7 (MCF-7/DOXR) when cultured with PHSM/f at a concentration of 10 μg/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/DOXR xenografts. The tumor (MCF-7/DOXR) 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/DOXR 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.

Introduction

The resistance of cancer cells to multiple structurally unrelated chemotherapeutic drugs has been termed ‘multidrug resistance (MDR).’ Clinical resistance of certain cancers to cytotoxic agents has been observed from the beginning of chemotherapy. The degree of resistance widely depends on the type of cancers. In past years, various mechanisms of multidrug resistance have been proposed, including the expression of multidrug efflux pumps (P-glycoprotein (Pgp) [1] or multidrug resistance-associated proteins (MRP1) [2]), altered glutathione metabolism [3], reduced activity of topoisomerase II [4], and various changes in cellular proteins [5], [6], [7] and mechanisms [8]. Various evidences strongly supported that the expression of Pgp on the cell membrane is associated with drug-resistance in cancer. An attempt to circumvent Pgp-based MDR in cancer chemotherapy has utilized Pgp blocking agents (MDR modulators or reverters) [9], [10], [11], which can be co-administered with the anticancer drug. This approach is, however, often associated with exacerbated toxicity of the anticancer drugs and limited its utility because of the blockade of the excretory functions of Pgp expressed in healthy tissues such as liver and kidney, which markedly reduces anticancer drug clearance.

Acidic intracellular organelles are known to participate in resistance to chemotherapeutic drugs. Parental drug-sensitive cells are characterized to have rather acidic, diffuse pH profile inside cells, while multidrug resistant cells develop more acidic organelles (recycling endosome, lysosome, and trans-Golgi network) and more alkaline cytosolic and nucleoplasmic pH [12], [13], [14].

Since most anticancer drugs are in an ionizable form, the pH of extracellular matrix and intracellular compartments is a critical factor in determining intracellular drug distribution. For instance, weakly basic drugs accumulate more in an acidic phase, where the ionized form predominates, because an ionized drug cannot permeate through cellular or subcellular membranes. Drug sequestration followed by extrusion into the external medium is another major mechanism for MDR for weakly acidic drugs [13], [15].

It is interesting to note that transfection of Pgp into certain sensitive cells is often coupled with increased cytosolic pH. However, this is not the case for MCF-7 cell line. The transfection did not influence the internal pH but induced 20-fold increased resistance. When the same cell line was selected from an escalated doxorubicin treatment schedule, the cells presented the overexpression of Pgp together with typical MDR pH profile and increased the resistance 980-fold. These observations indicate that the activity of efflux pump combined with drug sequestration induced by pH gradient and exocytosis mechanism seem to be most influential factors in MDR [16]. Thus, this intracellular pH effect must be taken into account in the carrier design to avoid sequestration effect and to improve drug availability to the cytosol and nucleus [15].

To overcome drug resistance, several polymeric carrier approaches have been proposed. When the resistant cells were treated with a cytotoxic agent with certain nonionic PEG/PPO/PEG triblock copolymer (Poloxamer) surfactants below the critical micelle concentration, the cytotoxicity of the drug was significantly improved [17], [18]. The recent findings suggest that the block copolymer reduced ATP production in the resistant cells with no noticeable change in ATP production in sensitive cells. The low ATP production in the resistant cells decreases the Pgp activity. However, the functional mechanism of the block copolymers for low ATP production has not yet been elucidated. The doxobubicin loaded micelles formed from similar block copolymers were also partially effective for resistant cells [19].

The receptor mediated-endocytosis of liposomal drug has been regarded as a primary method for bypassing Pgp [20], [21]. Liposomes decorated with endogenous ligands such as folic acid, transferrin, and lipoprotein bind to cancer cells via corresponding receptors that are overexpressed on the cell surface, followed by internalization and killing of resistant cells. Similar approaches have been used in a gene transfection study.

Few investigators have addressed the MDR cell drug sequestration problem in drug carrier design. In order to overcome MDR, our approach is based on pH-sensitive polymeric micelles conjugated with folate. If the carrier simultaneously exhibits both a triggered release in early endosomes (∼pH 6) and endosomal disruption after internalization, it may provide higher concentrations of the drug in the cytosol and the nucleus (potential drug acting sites). Such a system would be an effective delivery mode, particularly for multidrug-resistant (MDR) cancer cells.

Poly(l-histidine) (polyHis) is known to have an endosomal membrane disruption activity induced by a “proton sponge” mechanism of the imidazole groups [22], [23]. Thus, when the polymeric micelle system with the polyHis core is employed, it would become more effective mode for cytosolic delivery of anticancer drug. However, the difficulty in controlling the molecular weight of polyHis and limited solubility of high molecular weight (MW) polymer in organic solvents has restricted the fabrication and applications of polyHis-based hydrophobic-drug carriers.

We synthesized the block copolymer of polyHis (Mn: 5K)-b-poly(ethylene glycol) (PEG) (Mn: 2K) which can be dissolved in acidic aqueous solution (pH below 7.0) and organic solvents (DMSO and DMF), and fabricated a pH-sensitive polymeric micelle from the copolymer [24], [25]. The micelles showed a pH-induced destabilization. At pH above 7.4, the micelles are stable and they are destabilized below pH 7.0. In order to have more dramatic pH-transition around the physiological pH, a mixed micelle composed of polyHis-b-PEG-folate (75 wt.%) and poly(l-lactide) (PLLA)-b-PEG-folate (25 wt.%) was also developed. PLLA-b-PEG-folate was introduced to further stabilize the micelle and to control the destabilizing pH. The mixed micelles showed a pH-dependent DOX release behavior. Although the amount of released DOX for 24 h was about 30–35 wt.% in the pH range of 7.0–7.4, at pH 6.8 it reached almost 70 wt.%. During this process, it was also observed that the drug or polymer was distributed in the cytosol and nucleus in a sensitive breast cancer cell line (MCF-7) [25]. These properties are thought to provide a promising delivery mode for MDR tumors and MCF-7/DOXR cell line was selected for tests after stepwise exposure to 0.001–10 μg/ml of free DOX as reported [1], [26], [27], [28], [29].

Section snippets

Materials

Tetrazolium salt MTT, l-glutamine, n-propyl galate, glycerol, RPMI-1640, EDTA, Na2B4O7, NaN3, triethylamine (TEA), bovine serum albumin (BSA), goat anti-mouse IgG fluorescein-conjugate, sodium dodecyl sulfate, H2SO4, EDTA, estrone, and doxorubicin·HCl (DOX·HCl) were purchased from Sigma Chemical Co. (St. Louis, USA). Triethylamine, H2O2, formaldehyde, methanol, chloroform, isopropyl alcohol, and dimethylsulfoxide (DMSO) were provided from J. T. Baker (Deventer, Netherlands). Solvable was

Results

The micelle size of PHSM/f, as a typical example, was 65±12 nm (a mean±SD) with the monodistribution in size. The micelle shape was spherical as visualized in AFM photograph as in our previous publication [25].

Discussion

The detailed cytotoxity results against MCF-7 cells of the micelles were reported varying the micelle composition and pH in a previous publication [25]. Briefly, with the sensitive-type MCF-7 cells the DOX-dose dependent cytotoxicity curve of PHSM/f at pH 7.4 was close to free DOX. The cell viability was inversely proportional to log[DOX], reaching cell viability 20–30% at 10 μg/ml DOX. All other micelles of PHSM, PHIM/f, and PHIM showed 70–80% viability at a DOX concentration of 10 μg/ml.

Conclusion

DOX loaded pH-sensitive polymeric micelle (PHSM/f) was equally effective for suppressing both sensitive and MDR tumors of MCF-7 as demonstrated by in vitro and in vivo experiments and the micelle treatment caused minimal weight loss in animals like pH-insensitive micelles. This study suggests that the combined mechanisms of EPR effect, active internalization, endosomal-triggered release, and drug escape from endosomes are potential working mechanisms.

Acknowledgement

This work was supported by NIH CA101850.

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