Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors

The aim of this study was to exploit the possibility of combination of active targeting function of folic acid by folate receptor-mediated endocytosis and overcoming multidrug resistance (MDR) by Pluronic block copolymers to promote drug delivery to MDR tumor following intravenous administration with paclitaxel (PTX) as model drug. Folic acid functionalized Pluronic P123/F127 mixed micelles encapsulating PTX (FPF-PTX) was firstly developed and tested in vitro and in vivo, while PTX-loaded Pluronic P123/F127 mixed micelles (PF-PTX) and Taxol were used as control. FPF-PTX was about 20 nm in diameter with spherical shape and high encapsulation efficiency. Cellular uptake of FPF-PTX was found to be higher than that of PF-PTX due to the folate receptor-mediated endocytosis effect. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies also revealed that FPF-PTX was more potent than those of PF-PTX and Taxol. In vivo pharmacokinetic study in rats showed that the polymeric micelles significantly enhanced the bioavailability of PTX (～3 fold) than Taxol. Moreover, in BALB/c mice bearing KBv MDR tumor xenografts, stronger antitumor efficacy was shown in FPF-PTX group, with good correlation between in vitro and in vivo. In conclusion, folate-conjugated Pluronic micelles could be a potential vehicle for delivering hydrophobic chemotherapeutic drugs to MDR tumors.     

Synergistic effect of folate-mediated targeting and verapamil-mediated P-gp inhibition with paclitaxel -polymer micelles to overcome multi-drug resistance

Multidrug resistance (MDR) in tumor cells is a significant obstacle for successful cancer chemotherapy. Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) is a key factor contributing to the development of tumor drug resistance. Verapamil (VRP), a P-gp inhibitor, has been reported to be able to reverse completely the resistance caused by P-gp. For optimal synergy, the drug and inhibitor combination may need to be temporally colocalized in the tumor cells. Herein, we investigated the effectiveness of simultaneous and targeted delivery of anticancer drug, paclitaxel (PTX), along with VRP, using DOMC-FA micelles to overcome tumor drug resistance. The floate-functionalized dual agent loaded micelles resulted in the similar cytotoxicity to PTX-loaded micelles/free VRP combination and co-administration of two single-agent loaded micelles, which was higher than that of PTX-loaded micelles. Enhanced therapeutic efficacy of dual agent micelles could be ascribe to increased accumulation of PTX in drug-resistant tumor cells. We suggest that the synergistic effect of folate receptor-mediated internalization and VRP-mediated overcoming MDR could be beneficial in treatment of MDR solid tumors by targeting delivery of micellar PTX into tumor cells. As a result, the difunctional micelle systems is a very promising approach to overcome tumor drug resistance.     

Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin

P-glycoprotein (P-gp) mediated drug efflux has been recognized as a key factor contributing to the multidrug resistance (MDR) in tumor cells. To address this issue, a new pH-sensitive mixed copolymer micelles system composed of hyaluronic acid-g-poly(l-histidine) (HA-PHis) and d-α-tocopheryl polyethylene glycol 2000 (TPGS2k) copolymers was developed to co-deliver doxorubicin (DOX) and TPGS2k into drug-resistant breast cancer MCF-7 cells (MCF-7/ADR). The DOX-loaded HA-PHis/TPGS2k mixed micelles (HPHM/TPGS2k) were characterized to have a unimodal size distribution, high DOX loading content and a pH dependent drug release profile due to the protonation of poly(l-histidine). As compared to HA-PHis micelles (HPHM), the HPHM/TPGS2k showed higher and comparable cytotoxicity against MCF-7/ADR cells and MCF-7 cells, respectively. The enhanced MDR reversal effect was attributed to the higher amount of cellular uptake of HPHM/TPGS2k in MCF-7/ADR cells than HPHM, arising from the inhibition of P-gp mediated drug efflux by TPGS2k. The measurements of P-gp expression level and mitochondrial membrane potential indicate that the blank HPHM/TPGS2k inhibited P-gp activity by reducing mitochondrial membrane potential and depletion of ATP but without inhibition of P-gp expression. In vivo study of micelles in tumor-bearing mice indicate that HPHM/TPGS2k could reach the tumor site more effectively than HPHM. The pH-sensitive mixed micelles system has been demonstrated to be a promising approach for overcoming the MDR.     

Reversal of multidrug resistance by co-delivery of paclitaxel and lonidamine using a TPGS and hyaluronic acid dual-functionalized liposome for cancer treatment

Multidrug resistance (MDR) remains the primary issue in cancer therapy, which is characterized by the overexpressed P-glycoprotein (P-gp)-included efflux pump or the upregulated anti-apoptotic proteins. In this study, a D-alpha-tocopheryl poly (ethylene glycol 1000) succinate (TPGS) and hyaluronic acid (HA) dual-functionalized cationic liposome containing a synthetic cationic lipid, 1,5-dioctadecyl-N-histidyl-l-glutamate (HG2C₁₈) was developed for co-delivery of a small-molecule chemotherapeutic drug, paclitaxel (PTX) with a chemosensitizing agent, lonidamine (LND) to treat the MDR cancer. It was demonstrated that the HG2C₁₈ lipid contributes to the endo-lysosomal escape of the liposome following internalization for efficient intracellular delivery. The TPGS component was confirmed able to elevate the intracellular accumulation of PTX by inhibiting the P-gp efflux, and to facilitate the mitochondrial-targeting of the liposome. The intracellularly released LND suppressed the intracellular ATP production by interfering with the mitochondrial function for enhanced P-gp inhibition, and additionally, sensitized the MDR breast cancer (MCF-7/MDR) cells to PTX for promoted induction of apoptosis through a synergistic effect. Functionalized with the outer HA shell, the liposome preferentially accumulated at the tumor site and showed a superior antitumor efficacy in the xenograft MCF-7/MDR tumor mice models. These findings suggest that this dual-functional liposome for co-delivery of a cytotoxic drug and an MDR modulator provides a promising strategy for reversal of MDR in cancer treatment.     

Addition of TPGS to folate-conjugated polymer micelles for selective tumor targeting

Folate-conjugated polymer micelles poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) was fabricated to encapsulate anticancer drug doxorubicin for targeting delivery to cancer cells with overexpression of folate receptors. To increase therapeutic effect, D-alpha-tocopheryl polyethylene glycol succinate (TPGS) was added during the micelles preparation. The physicochemical study showed that the mixed micelles of PLGA-PEG-FOL and TPGS formed a homogeneous population. The addition of TPGS did not result in much variation in the micellar size, surface charge, and drug encapsulation efficiency. The cellular uptake study showed that mixed micelles with TPGS had higher cellular uptake compared with the ones without TPGS to drug-resistant cancer cells. These mixed micelles also selectively increased the cytotoxicity of drug on cancer cells but exhibited minimal cytotoxic enhancement on normal fibroblasts. Furthermore, the accumulation of rhodamine study showed that the mixed micelles with TPGS increased the cellular uptake of drugs on Caco-2 cells. This indicates that TPGS in the mixed micelles may act as P-glycoprotein inhibitor to reduce drug efflux. This new formulation with TPGS may have dual functions of folate-mediated targeting and multidrug resistance inhibition and can be promising in improving the therapeutic efficacy of polymer micellar targeting delivery system.     

Shell-crosslinked Pluronic L121 micelles as a drug delivery vehicle

Pluronic block copolymers (PBCs) have been shown to reverse multidrug resistance (MDR) by inhibiting the P-glycoprotein (P-gp) pump in cancer cells. One of the problems encountered with the use of PBCs is that the micelles disassociate at low concentrations. The study focused on the stabilization of PBC L121 micelles by the formation of crosslinks within their outer shells. To form crosslinks, the two terminal alcohols on L121 were first chemically converted into aldehydes (L121-CHO) using the Dess-Martin periodinane. Diamine compounds were then used to bridge the converted aldehyde termini on L121-CHO via conjugated Schiff bases. After crosslinking, the morphology of the L121 micelles remained spherical in shape and the mean particle sizes of the micelles before and after crosslinking were comparable (100nm). After exposure of MDR KBv cells to free rhodamine-123 (R123), the accumulation of R123 in cells was limited due to the function of P-gp. In contrast, crosslinking of L121 micelles within their outer shells significantly reduced their critical micelle concentration and greatly enhanced their stability, while maintaining their ability to inhibit P-gp function in resistant cells. The results indicated that the L121 micelles with shell crosslinks may be useful as a drug delivery vehicle for cancer chemotherapy.     

TPGS-stabilized NaYbF4:Er upconversion nanoparticles for dual-modal fluorescent/CT imaging and anticancer drug delivery to overcome multi-drug resistance

Multi-drug resistance (MDR) is a major cause of failure in cancer chemotherapy. Tocopheryl polyethylene glycol 1000 succinate (TPGS) has been extensively investigated for overcoming MDR in cancer therapy because of its ability to inhibit P-glycoprotein (P-gp). In this work, TPGS was for the first time used as a new surface modifier to functionalize NaYbF₄:Er upconversion nanoparticles (UNCPs) and endowed the as-prepared products (TPGS-UCNPs) with excellent water-solubility, P-gp inhibition capability and imaging-guided drug delivery property. After the chemotherapeutic drug (doxorubicin, DOX) loading, the as-formed composites (TPGS-UCNPs-DOX) exhibited potent killing ability for DOX-resistant MCF-7 cells. Flow-cytometric assessment and Western blot assay showed that the TPGS-UCNPs could potently decrease the P-gp expression and facilitate the intracellular drug accumulation, thus achieving MDR reversal. Moreover, considering that UCNPs process efficient upconversion emission and Yb element contained in UCNPs has strong X-ray attenuation ability, the as-obtained composite could also serve as a dual-modal probe for upconversion luminescence (UCL) imaging and X-ray computed tomography (CT) imaging, making them promising for imaging-guided cancer therapy.     

Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas

Combinations of drugs promoting anti-angiogenesis and apoptosis effects are meaningful for cancer therapy. In the present study, dual peptides-modified liposomes were designed by attaching two receptor-specific peptides, specifically low-density lipoprotein receptor-related protein receptor (Angiopep-2) and neuropilin-1 receptor (tLyP-1) for brain tumor targeting and tumor penetration. Vascular endothelial growth factor (VEGF) siRNA and chemotherapeutic docetaxel (DTX) were chosen as the two payloads because VEGF is closely associated with angiogenesis, and DTX can kill tumor cells efficiently. Binding to glioma cells, co-delivery of siRNA and DTX in human glioblastoma cells (U87 MG) and murine brain microvascular endothelial cells (BMVEC), VEGF gene silencing, antiproliferation and anti-tumor effects of the dual peptides-modified liposomes were evaluated in vitro and in vivo. The dual peptides-modified liposomes persisted the binding ability to glioma cells, enhanced the internalization via specific receptor mediated endocytosis and tissue penetration, thus the dual peptides-modified liposomes loading VEGF siRNA and DTX possessed stimulative gene silencing and antiproliferation activity compared with non-modified and single peptide-modified liposomes. The co-delivery research revealed different intracellular behavior of hydrophilic large molecular and lipophilic small molecule, the former involves endocytosis and subsequent escape of endosome/lysosomes, while the latter experiences passive diffusion of lipophilic small drugs after its release. Furthermore, the dual peptides-modified liposomes showed superiority in anti-tumor efficacy, combination of anti-angiogenesis by VEGF siRNA and apoptosis effects by DTX, after both intratumor and system application against mice with U87 MG tumors, and the treatment did not activate system-associated toxicity or the innate immune response. Combination with the dual peptides-guided tumor homing and penetration, the dual peptides-modified liposomes provide a strategy for effective targeting delivery of siRNA and DTX into the glioma cell and inhibition of tumor growth in a synergistic manner.     

Star-shape copolymer of lysine-linked di-tocopherol polyethylene glycol 2000 succinate for doxorubicin delivery with reversal of multidrug resistance

A star-shape copolymer of nanostructure-forming material, P-glycoprotein (P-gp) reversible inhibitor and anticancer enhancer, lysine-linked di-tocopherol polyethylene glycol 2000 succinate (PLV(2K)), was synthesized to overcome multidrug resistance (MDR) in cancer chemotherapy. The critical micellar concentration of PLV(2K) was as low as 1.14?μg/mL, which can endow nanoassemblies good physical stability. Doxorubicin (DOX) was encapsulated into the hydrophobic core of PLV(2K) (PLV(2K)-DOX), with encapsulation efficiency as high as 94.5% and a particle size of 16.4?nm. DOX released from PLV(2K)-DOX nanomicelles was pH-dependent, which ensures micelles stable in blood circulation and releases DOX within tumor cells. Facilitated by the cytotoxicity and uncompetitive P-gp ATPase inhibition by PLV(2K), PLV(2K)-DOX showed greater cytotoxicity compared with DOX solution with increased intracellular accumulation in resistant MCF-7/Adr cells. PLV(2K)-DOX nanomicelles were uptaken into MCF-7/Adr cells via macropinocytosis and caveolae-mediated endocytosis, which further facilitate escapement of P-gp efflux. The anticancer efficacy in?vivo was evaluated in 4T1-bearing mice and inhibition of tumor by PLV(2K)-DOX was more effective than TPGS-DOX and DOX solution. In summary, PLV(2K) copolymer has striking functions such as uncompetitive P-gp ATPase reversible inhibitor and anticancer efficacy, and could be a promising nanocarrier in improving the chemotherapy of hydrophobic anticancer drugs.     

Restoration of chemosensitivity by multifunctional micelles mediated by P-gp siRNA to reverse MDR

One of the main obstacles in tumor therapy is multiple drug resistance (MDR) and an underlying mechanism of MDR is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins, especially P-glycoprotein (P-gp). In the synergistic treatment of siRNA and anti-cancer drug doxorubicin, it is crucial that both the siRNA and doxorubicin are simultaneously delivered to the tumor cells and the siRNA can fleetly down-regulate P-g before doxorubicin inactivates the P-gp and is pumped out. Herein, a type of micelles comprising a polycationic PEI-CyD shell to condense the siRNA and hydrophobic core to package doxorubicin is reported. The structure of the polymer is determined by ¹H NMR, FT-IR, DSC, and XRD and the micelles are characterized by DLS, 2D-NOESY NMR, and TEM to study the self-assembly of the micelles with siRNA and drugs. In vitro studies demonstrate controlled release and temporal enhancement of the therapeutic efficacy of P-gp siRNA and doxorubicin. Release of siRNA down-regulates the mRNA and protein levels of P-gp in the MCF-7/ADR cell lines effectively and the accumulated doxorubicin facilitates apoptosis of the cells to reverse MDR. Moreover, in vivo research reveals that the siRNA and doxorubicin loaded micelles induce tumor cell apoptosis and inhibit the growth of MDR tumor. The western blotting and RT-PCR results illustrate that the synergistic treatment of siRNA and doxorubicin leads to efficient reduction of the P-gp expression as well as cell apoptotic induction in MDR tumors at a small dosage of 0.5 mg/kg. The micelles have large clinical potential in drug/RNAi synergistic treatment via restoration of the chemosensitivity in MDR cancer therapy.     

The use of mitochondrial targeting resveratrol liposomes modified with a dequalinium polyethylene glycol-distearoylphosphatidyl ethanolamine conjugate to induce apoptosis in resistant lung cancer cells

Intrinsic multidrug resistance (MDR) of cancers remains a major obstacle to successful chemotherapy. A dequalinium polyethylene glycol-distearoylphosphatidylethanolamine (DQA-PEG(2000)-DSPE) conjugate was synthesized as a mitochondriotropic molecule, and mitochondrial targeting resveratrol liposomes were developed by modifying DQA-PEG(2000)-DSPE on the surface of liposomes for overcoming the resistance. Evaluations were performed on the human lung adenocarcinoma A549 cells and resistant A549/cDDP cells, A549 and A549/cDDP tumor spheroids as well as the xenografted resistant A549/cDDP cancers in nude mice. The yield of DQA-PEG(2000)-DSPE conjugate synthesized was about 87% and the particle size of mitochondrial targeting resveratrol liposomes was approximately 70 nm. The mitochondrial targeting liposomes significantly enhanced the cellular uptake, and selectively accumulated into mitochondria when encapsulating coumarin as the fluorescent probe. Furthermore, mitochondrial targeting resveratrol liposomes induced apoptosis of both non-resistant and resistant cancer cells by dissipating mitochondria membrane potential, releasing cytochrome c and increasing the activities of caspase 9 and 3. They also exhibited significant antitumor efficacy in two kinds of cancer cells, in tumor spheroids by penetrating deeply into the core, and in xenografted resistant A549/cDDP cancers in nude mice. Mitochondrial targeting resveratrol liposomes co-treating with vinorelbine liposomes significantly enhanced the anticancer efficacy against the resistant A549/cDDP cells. In conclusion, mitochondrial targeting resveratrol liposomes would provide a potential strategy to treat the intrinsic resistant lung cancers by inducing apoptosis via mitochondria signaling pathway.     

Tumor accumulation and antitumor efficacy of docetaxel-loaded core-shell-corona micelles with shell-specific redox-responsive cross-links

A robust core-shell-corona micelle bearing redox-responsive shell-specific cross-links was evaluated as a carrier of docetaxel (DTX) for cancer therapy. The polymer micelles of poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-phenylalanine) (PEG-PLys-PPhe) in the aqueous phase provided the three distinct functional domains: the PEG outer corona for prolonged circulation, the PLys middle shell for disulfide cross-linking, and the PPhe inner core for DTX loading. The shell cross-linking was performed by the reaction of disulfide-containing cross-linkers with Lys moieties in the middle shells. The shell cross-linking did not change the micelle size or the spherical morphology. The shell cross-linked micelles exhibited enhanced serum stability. The DTX release from the DTX-loaded disulfide cross-linked micelles (DTX-SSCLM) was facilitated by increasing the concentration of glutathione (GSH). At an intracellular GSH level, DTX release was facilitated due to the reductive cleavage of the disulfide cross-links in the shell domains. The in vivo tissue distribution and tumor accumulation of the DTX-SSCLM that were labeled with a near-infrared fluorescence (NIRF) dye, Cy5.5, were monitored in MDA-MB231 tumor-bearing mice. Non-invasive real-time optical imaging results indicated that the DTX-SSCLM exhibited enhanced tumor specificity due to the prolonged stable circulation in blood and the enhanced permeation and retention (EPR) effect compared with the DTX-loaded non-cross-linked micelles (DTX-NCLM). The DTX-SSCLM exhibited enhanced therapeutic efficacy in tumor-bearing mice compared with free DTX and DTX-NCLM. The domain-specific shell cross-linking that is described in this work may serve as a useful guidance for enhancing the antitumor therapeutic efficacy of various polymer micelles and nano-aggregates.     

CD3AK、LAK对KB、KBv200细胞株耐药逆转前后的杀伤作用

目的　探讨免疫活性细胞对肿瘤细胞的杀伤活性与mdr1的关系。方法　利用特异性切割mdr1的ribozyme为工具 ,以表达mdr1的耐药细胞株KBv2 0 0 为靶细胞 ,采用脂质体转染技术 ,将含ribozyme的质粒pHβApr 1neo/ 5mR3及空载体pHβApr 1neo导入KBv2 0 0 及其亲本KB细胞内 ,运用North ernblotting、免疫组化方法观察ribozyme对mdr1mRNA及P 170的影响 ,采用MTT法检测了CD3AK、LAK对KB、KBv2 0 0 细胞株耐药逆转前后杀伤活性的变化。结果　含ribozyme的质粒pHβApr 1neo/ 5mR3及空载体pHβApr 1neo可以在KB、KBv2 0 0 细胞中稳定表达 ,ribozyme可以特异性地切割mdr1,导致KBv2 0 0 / 5mR3的mdr1mRNA含量下降 ,P 170表达减低 ,LAK和CD3AK对KBv2 0 0 的杀伤活性较对KB的强 ,MDR逆转后杀伤活性降至敏感株水平 ,LAK和CD3AK对KB/ 5mR3、KB/vec、KBv2 0 0 /vec的杀伤活性也较KB及KBv2 0 0 / 5mR3强。结论　mdr1 ribozyme在细胞内具有一定的逆转肿瘤多药抗性的生物学效应 ,LAK和CD3AK对MDR肿瘤细胞的杀伤活性与P糖蛋白表达相关     

转染肿瘤坏死因子-α及MDR1反义RNA逆转乳腺癌多药耐药的研究

目的：转染肿瘤坏死因子-α(TNF-α)cDNA和多药耐药基因(MDR1)的反义RNA到乳腺癌耐药细胞株MCF-7/ADR中进行表达，并观察它们在乳腺癌耐药逆转中的作用。 方法：应用RT-PCR和DNA重组技术构建反义绿色荧光蛋白pEGFP-MDR1融合蛋白表达载体和红色荧光蛋白pDsRed2-TNF-α融合蛋白表达载体，分别和同时导入乳腺癌耐药细胞株MCF-7/ADR中进行表达，检测转染前后细胞的生长曲线、细胞凋亡程度、MDR1-mRNA和P糖蛋白（P-gp）表达情况及对ADR敏感性的变化。 结果：转染后的细胞生长明显减慢，凋亡率显著增加，MDR1-mRNA和P糖蛋白（P-gp）表达明显降低，对ADR的耐药性明显下降，敏感性增加。 结论：联合运用不同的逆转耐药机制，将TNF-α cDNA及MDR1反义RNA分别或同时导入乳腺癌耐药细胞中，能有效达到逆转耐药的目的。     

Doxorubicin-loaded,charge reversible,folate modified HPMA copolymer conjugates for active cancer cell targeting

Although folate exhibits many advantages over other targeting ligands, it has one major defect: poor water solubility. Once it was conjugated to hydrophilic drug carrier such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, the hydrophobic folate may be buried inside the random polymer coil and not exposed to be accessible to its receptor on the cell surface, thus losing its active targeting ability. To address this folate dilemma, the positive charge was introduced in the present study. The obtained cationic folate-functionalized HPMA copolymers exhibited a synergistic enhancing effect on cellular uptake by folate receptor (FR) positive Hela cells via electrostatic absorptive endocytosis and folate receptor-mediated endocytosis, with the involvement of multiple internalization pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and energy-dependent endocytosis. As demonstrated in binding efficiency study, the FR antibody bound to 71.2% of tested cells in the competition with neutral folate modified HPMA copolymers, while the FR antibody-bounded cells decreased to only 34.0% in competition with cationic folate modified HPMA copolymers, indicating that the positively charge could probably amplify the binding efficiency of folate to its receptor due to close proximity of the conjugates to the cell surface by the electronic adhesion. In addition, the cell uptake study on FR negative A549 cells also confirmed the specific role of folate as targeting ligand. Then, to avoid non-specific binding by positive charge in the circulation, the charge shielding/deshielding approach was further employed. With selective hydrolysis of the charge shielding groups 2,3-dimethylmaleic anhydride (DMA) at tumor extracellular pH 6.8, the conjugates underwent a quick charge-reversible process with more than 80% DMA cleavage within 2 h and endocytosed into the endo/lysosomes much more rapidly than at physiological pH 7.4. And then the drug release was triggered by the cleavage of hydrazone spacer at another level of pH 5 in endo/lysosomal compartment. Furthermore, the anticancer activity results showed that Dox-loaded, charge-switchable, folate modified HPMA copolymer conjugates could indeed lead to enhanced cytotoxicity, stronger apoptosis and greater tumor spheroid inhibition towards Hela cells, indicating the great potential feasibility of this multiple responsive drug delivery system.     

Cellular internalization of doxorubicin loaded star-shaped micelles with hydrophilic zwitterionic sulfobetaine segments

Four arm star-shaped poly(ε-caprolactone)-b-poly((N,N-diethylaminoethyl methacrylate)-r-(N-(3-sulfopropyl)-N-methacryloxyethy-N,N-diethylammoniumbetaine)) (4sPCLDEAS) micelles were loaded with anticancer drug doxorubicin to track their endocytosis in Hela cancer cell line. The effects of mean diameters and surface charges of the drug loaded micelles on the cellular uptake were studied in details. The results demonstrated that the internalization of micelles was both time and energy dependent process. Endocytic pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis were all involved in the internalization; caveolae-mediated endocytosis was the main pathway for the internalization of 4sPCLDEAS micelles. The assays for cell apoptosis and growth inhibition of tumor spheroids identified that these doxorubicin loaded micelles could induce cell apoptosis and inhibit tumor spheroids growth efficiently, which was even equal to free DOX·HCl. This study provided a rational design strategy for fabricating diverse micellar drug delivery systems with high anticancer efficiency.     

Biofunctionalized polymer-lipid supported mesoporous silica nanoparticles for release of chemotherapeutics in multidrug resistant cancer cells

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. A polymer-lipid supported mesoporous silica nanoparticle (PLS-MSNs) is described here to facilitate intracellular delivery of anticancer drug and enhance the antitumor efficacy against MDR breast cancer cells. By coating MSNs with a synthetic dual-functional polymer-lipid material P123-DOPE, the supported membrane acted as an intact barrier against the escape of encapsulated drugs before reaching the target cells, leading to depolymerization and triggered storm release of loaded irinotecan (CPT-11) in acidic endosomal pH of tumor cells. In addition, P123-DOPE can inhibit breast cancer resistance protein (BCPR) mediated CPT-11 efflux in drug resistant MCF-7/BCRP breast cancer cells, thus acting as a “door blocker”. Compared to free CPT-11, PLS-MSNs resulted in a maximum increase in the intracellular CPT-11 concentration (12.9-fold), had 7.1-fold higher cytotoxicity and processed a stronger cell cycle arrest in MCF-7/BCRP cells. Moreover, CPT-11 loaded PLS-MSNs showed high therapeutic performance and low toxicity in BALB/c nude mice bearing drug resistant breast tumors, with an inhibition rate of 81.2% compared to free CPT-11 treatment group. The reported PLS-MSNs provide promising applicability in future preclinical and clinical MDR cancer treatment.     

Multifunctional nanoassemblies for vincristine sulfate delivery to overcome multidrug resistance by escaping P-glycoprotein mediated efflux

Multifunctional nanoassemblies (MNAs) were successfully developed for controlled delivery of water-soluble cationic vincristine sulfate (VCR) to overcome multidrug resistance (MDR). The incorporation of anionic small molecule of phosphatidylserine (PS) significantly enhanced the encapsulation efficiency of VCR in MNAs up to 94.4% by electrostatic interaction. Obvious sustained-release characteristics were found in VCR-loaded MNAs (VCR-MNAs) as the cumulative release of VCR was 83.2% at 96?h, and burst-release was effectively diminished. In vivo pharmacokinetics in rats following intravenous administration demonstrated that VCR-MNAs had higher AUC and longer t(1/2) than VCR solution (VCR-Sol). To investigate the MDR reversal effect and clarify the possible mechanism induced by MNAs, the cytotoxicity, cellular uptake and uptake mechanism experiments were performed in MCF-7 and P-glycoprotein over-expressing MCF-7/Adr cells, respectively. Compared with VCR-Sol, VCR-MNAs efficiently enhanced the cytotoxicity to 36.5-fold by increasing the cellular accumulation of VCR (12.6-fold higher) in MCF-7/Adr cells. The results of endocytosis inhibition experiment proved that VCR-MNAs were uptaken into the resistant cancer cells by clathrin- and caveolae-mediated endocytosis pathways, which escaped the efflux induced by P-gp transporter and thereby overcame the MDR of VCR.     

Docetaxel-carboxymethylcellulose nanoparticles target cells via a SPARC and albumin dependent mechanism

Cellax, a polymer-docetaxel (DTX) conjugate that self-assembled into 120 nm particles, displayed significant enhancements in safety and efficacy over native DTX across a number of primary and metastatic tumor models. Despite these exciting preclinical data, the underlying mechanism of delivery of Cellax remains elusive. Herein, we demonstrated that serum albumin efficiently adsorbed onto the Cellax particles with a 4-fold increased avidity compared to native DTX, and the uptake of Cellax by cells was primarily driven by an albumin and SPARC (secreted protein acidic and rich in cysteine, an albumin binder) dependent internalization mechanism. In the SPARC-positive cells, a >2-fold increase in cellular internalization of Cellax was observed in the presence of albumin. In the SPARC-negative cells, no difference in Cellax internalization was observed in the presence or absence of albumin. Evaluation of the internalization mechanism using endocytotic inhibitors revealed that Cellax was internalized predominantly via a clathrin-mediated endocytotic mechanism. Upon internalization, it was demonstrated that Cellax was entrapped within the endo-lysosomal and autophagosomal compartments. Analysis of the tumor SPARC level with tumor growth inhibition of Cellax in a panel of tumor models revealed a positive and linear correlation (R² > 0.9). Thus, this albumin and SPARC-dependent pathway for Cellax delivery to tumors was confirmed both in vitro and in vivo.     

Folate-functionalized polymeric micelles for tumor targeted delivery of a potent multidrug-resistance modulator FG020326

To overcome multidrug resistance (MDR) existing in tumor chemotherapy, polymeric micelles encoded with folic acid on the micelle surface were prepared with the encapsulation of a potent MDR modulator, FG020326. The micelles were fabricated from diblock copolymers of poly(ethylene glycol) (PEG) and biodegradable poly(epsilon-caprolactone) (PCL) with folate attached to the distal ends of PEG chains. The folate-conjugated copolymers, folate-PEG-PCL, were synthesized by multistep chemical reactions. First, allyl-terminated copolymer (allyl-PEG-PCL) was synthesized through a ring-opening polymerization of epsilon-caprolactone in bulk employing monoallyl-PEG as a macroinitiator. Second, the allyl terminal groups of copolymers were converted into primary amino groups by a radical addition reaction, followed by conjugation of the carboxylic group of folic acid. In vitro studies at 37 degrees C demonstrated that FG020326 release from micelles at pH 5.0 was faster than that at pH 7.4. Cytotoxicity studies with MTT assays indicated that folate-functionalized and FG020326-loaded micelles resensitized the cells approximately five times more than their folate-free counterparts (p < 0.01) in human KB(v200) cells treated with vincristine (VCR). The in vitro Rhodamine 123 efflux experiment using MDR KB(v200) cells revealed that when cells were pretreated with folate-attached and FG020326-loaded micelles, the P-glycoprotein (P-gp) drug efflux function was significantly inhibited.