Mingxi Qiao

Self-assembled pH-responsive hyaluronic acid–g-poly(l-histidine) copolymer micelles for targeted intracellular delivery of doxorubicin

Hyaluronic acid (HA) was conjugated with hydrophobic poly(l-histidine) (PHis) to prepare a pH-responsive and tumor-targeted copolymer, hyaluronic acid-g-poly(l-histidine) (HA-PHis), for use as a carrier for anti-cancer drugs. The effect of the degree of substitution (DS) on the pH-responsive behaviour of HA-PHis copolymer micelles was confirmed by studies of particles of different sizes. In vitro drug release studies demonstrated that doxorubicin (DOX) was released from HA-PHis micelles in a pH-dependent manner. In vitro cytotoxicity assays showed that all the blank micelles were nontoxic. However, MTT assay against Michigan Cancer Foundation-7 (MCF-7) cells (overexpressed CD44 receptors) showed that DOX-loaded micelles with a low PHis DS were highly cytotoxic. Cellular uptake experiments revealed that these pH-responsive HA-PHis micelles taken up in great amounts by receptor-mediated endocytosis and DOX were efficiently delivered into cytosol. Moreover, micelles with the lowest DS exhibited the highest degree of cellular uptake, which indicated that the micelles were internalized into cells via CD44 receptor-mediated endocytosis and the carboxylic groups of HA are the active binding sites for CD44 receptors. Endocytosis inhibition experiments and confocal images demonstrated that HA-PHis micelles were internalized into cells mainly via clathrin-mediated endocytosis and delivered to lysosomes, triggering release of DOX into the cytoplasm. These results confirm that the biocompatible pH-responsive HA-PHis micelles are a promising nanosystem for the intracellular targeted delivery of DOX.

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.

pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity: preparation and in vitro evaluation

pH-sensitive self-aggregated nanoparticles (SNPs), based on amphiphilic deoxycholic acid (DOCA) modified carboxymethyl chitosan (DCMC), were prepared for delivery of the anticancer drug doxorubicin (DOX). DCMCs with different degrees of substitution (DS) of DOCA were initially synthesized and characterized. Based on self-aggregation, DCMC formed nanoparticles with size ranging from 87 to 174 nm. The critical aggregation concentration (CAC) decreased on increasing the DS of DOCA. Moreover, the DCMC SNPs showed an acidic pH-induced aggregation and deformation behavior. The DOX-loaded SNPs ([D]NP) exhibited a sustained drug release manner, which could be accelerated by an acidic pH, but delayed by a higher DS of DOCA. Antitumor efficacy results showed that [D]NP could suppress both sensitive and resistant MCF-7 cells effectively in a dose- and time-dependent manner. The enhanced cellular uptake and greater retention of [D]NP in drug-resistant cells, as evidenced by confocal microscopy and flow cytometry, contributed to a superior efficacy of [D]NP over free DOX. These results suggest the potential of DCMC SNPs as carriers for the hydrophobic drug DOX for effective cancer therapy against drug-resistant tumors.

A pH-sensitive gene delivery system based on folic acid-PEG-chitosan – PAMAM-plasmid DNA complexes for cancer cell targeting

In this study, pH-sensitive biomaterials coated polymer/DNA nanocomplexes containing a high mobility group box 1 (HMGB1) were developed as an efficient non-viral gene delivery system. HMGB1 is a family of endogenous molecules that contains nuclear locating sequences (NSL). Polyethylene glycol tethered carboxylated chitosan modified with folic acid (FA-PEG-CCTS) was synthesized and its buffering capacity was determined by acid-base titration. A pH-sensitive core-shell system FA-PEG-CCTS/PAMAM/HMGB1/pDNA nanocomplexes (FPCPHDs), was prepared and characterized. Electrophoresis showed that FPCPHDs were resistant to heparin replacement and DNase I digestion. FPCPHDs exhibited only minor toxic effects on HepG2 and KB cells. The results of both luciferase activity assay and RFP fluorescence intensity analysis showed that FPCPHDs enhanced gene transfection and expression in KB cells. Moreover, gene transfection and expression in KB cells were inhibited by free folic acid. Intracellular trafficking of FPCPHDs in KB cells showed that FPCPHDs could rapidly escape from endo-lysosomes and become exclusively located in the nucleus at 3 h post transfection. In addition, FPCPHDs exhibited increased red fluorescence protein (RFP) expression at the tumor site of S180 xenograft nude mice. All results suggest that FPCPHDs is an efficient approach to improve the transfection and expression efficiency in most FR-positive cancer cells.

pH-sensitive nanoparticles of poly(l-histidine)–poly(lactide-co-glycolide)–tocopheryl polyethylene glycol succinate for anti-tumor drug delivery

A novel pH-sensitive polymer, poly(L-histidine)-poly(lactide-co-glycolide)-tocopheryl polyethylene glycol succinate (PLH-PLGA-TPGS), was synthesized to design a biocompatible drug delivery system for cancer chemotherapy. The structure of the PLH-PLGA-TPGS copolymer was confirmed by (1)H-NMR, FTIR and GPC. The apparent pKa of the PLH-PLGA-TPGS copolymer was calculated to be 6.33 according to the acid-base titration curve. The doxorubicin (DOX)-loaded nanoparticles (PLH-PLGA-TPGS nanoparticles and PLGA-TPGS nanoparticles) and corresponding blank nanoparticles were prepared by a co-solvent evaporation method. The blank PLH-PLGA-TPGS nanoparticles showed an acidic pH-induced increase in particle size. The DOX-loaded nanoparticles based on PLH-PLGA-TPGS showed a pH-triggered drug-release behavior under acidic conditions. The results of in vitro cytotoxicity experiment on MCF-7 and MCF-7/ADR cells showed that the DOX-loaded PLH-PLGA-TPGS nanoparticles resulted in lower cell viability versus the PLGA-TPGS nanoparticles and free DOX solution. Confocal laser scanning microscopy images showed that DOX-loaded PLH-PLGA-TPGS nanoparticles were internalized by MCF-7/ADR cells after 1 and 4h incubation and most of them accumulated in lysosomes to accelerate DOX release under acidic conditions. In summary, the PLH-PLGA-TPGS nanoparticles have great potential to be used as carriers for anti-tumor drug delivery.

Gold nanorods/mesoporous silica-based nanocomposite as theranostic agents for targeting near-infrared imaging and photothermal therapy induced with laser

Photothermal therapy (PTT) is widely regarded as a promising technology for cancer treatment. Gold nanorods (GNRs), as excellent PTT agent candidates, have shown high-performance photothermal conversion ability under laser irradiation, yet two major obstacles to their clinical application are the lack of selective accumulation in the target site following systemic administration and the greatly reduced photothermal conversion efficiency caused by self-aggregating in aqueous environment. Herein, we demonstrate that tLyp-1 peptide-functionalized, indocyanine green (ICG)-containing mesoporous silica-coated GNRs (I-TMSG) possessed dual-function as tumor cells-targeting near-infrared (NIR) fluorescent probe and PTT agents. The construction of the nanostructure began with synthesis of GNRs by seed-mediated growth method, followed by the coating of mesoporous silica, the chemical conjugation of PEG and tLyp-1 peptide, and the enclosure of ICG as an NIR imaging agent in the mesoporous. The as-prepared nanoparticles could shield the GNRs against their self-aggregation, improve the stability of ICG, and exhibit negligible dark cytotoxicity. More importantly, such a theranostic nanocomposite could realize the combination of GNRs-based photothermal ablation under NIR illumination, ICG-mediated fluorescent imaging, and tLyp-1-enabled more easy endocytosis into breast cancer cells. All in all, I-TMSG nanoparticles, in our opinion, possessed the strong potential to realize the effective diagnosis and PTT treatment of human mammary cancer.

Reversing multidrug resistance by intracellular delivery of Pluronic® P85 unimers.

Pluronics have been demonstrated as excellent multidrug resistance (MDR) reversal agent in the form of unimers rather than micelles. However, the effective intracellular delivery of Pluronic(®) unimers to MDR cancer cells still remains a big challenge. To address this issue, a mixed micellar system based mainly on the pH-sensitive copolymer of poly (L-histidine)-poly (D,L-lactide)-polyethyleneglycol-poly (D,L-lactide)-poly (L-histidine) (PHis-PLA-PEG-PLA-PHis) and Pluronic(®) F127, some of which was conjugated with folate, was constructed to intracellularly deliver the unimers of Pluronic(®) P85 to MDR cells. The folate-mediated endosomal pH-sensitive mixed micelles (pHendoSM-P85/f) were prepared by a thin-film hydration method, by which Pluronic(®) P85 unimers and doxorubicin (DOX) were incoporated into the mixed micelles. The incorporation of Pluronic(®) P85 unimers was investigated by the surface tension test. The results indicated that the Pluronic(®) P85 unimers probably first inserted into the binary mixed micelles and then formed a triple-component mixed micelles with Pluronic(®) F127 and PHis-PLA-PEG-PLA-PHis as the loading content increased. Further analyzed with flow cytometry, confocal laser scanning microscopy (CLSM) and MTT assay, the micelles with inserted Pluronic(®) P85 unimers demonstrated much more cellular uptake and higher cytotoxicity against MDR cells than the triple-component mixed micelles and plain Pluronic(®) micelles. The enhanced MDR reversal effect was attributed to the successful intracellular delivery of Pluronic(®) P85 unimers to the MDR cells, which was confirmed by the subcellular colocalization of Pluronic(®) P85 unimers with mitochondria, the decreased ATP energy and mitochondrial membrane potential (MP) in the MCF-7/ADR cells. The pHendoSM-P85/f/DOX also demonstrated more dramatic antitumor efficiency and remarkable reduction of ATP energy in the MDR cells in tumors than the control formulations. The intracellular delivery of Pluronic(®) P85 unimers to the MDR cells based on the targeted and endosomal pH triggerd release mixed micelles has been demonstrated as a promising approach to reverse MDR.

Thermo- and pH-responsive copolymers based on PLGA-PEG-PLGA and poly(L-histidine): synthesis and in vitro characterization of copolymer micelles.

A series of novel thermo- and pH-responsive block copolymers of PHis-PLGA-PEG-PLGA-PHis composed of poly(ethylene glycol) (PEG), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-histidine) (PHis) were synthesized and used for the construction of stimuli-responsive copolymer micelles. The starting polymers of PLGA-PEG-PLGA and PHis were synthesized by ring-opening polymerization of dl-lactide and glycolide with PEG as an initiator and L-histidine N-carboxylanhydride with isopropylamine as an initiator, respectively. The final copolymer was obtained by the coupling reaction of PHis with PLGA-PEG-PLGA. The copolymer micelles were constructed to have an inner core consisting of two hydrophobic blocks (PLGA and deprotonated PHis) and an outer hydrophilic PEG shell. The temperature- and pH-induced structure changes of the micelles were characterized by an alteration in particle size, a decrease in pyrene fluorescence intensity, and a variation of (1)H NMR spectra in D2O. It was speculated that the hydrophobic-hydrophilic transitions of PEG and PHis in response to temperature and pH variations accounted for the destabilization of micelles. In vitro release profiles, cell cytotoxicity and intracellular location studies further confirmed the temperature- and pH-responsive properties of the copolymer micelles. These results demonstrate the potential of the developed copolymers to be stimuli-responsive carriers for targeted delivery of anti-cancer drugs.

Preparation, Characterization, and Evaluation of Liposomal Ferulic Acid In Vitro and In Vivo

In the present study, various gradients were evaluated for efficient loading of weak acid into liposomes. Several salt gradients showed efficient loading of ferulic acid (FA) into liposomes and the optimized conditions were established in calcium acetate gradient method to obtain 80.2 ± 5.2% entrapment efficiency (EE). Unilamellar vesicles were observed in micrographs and liposomal FA showed good stability. 80% of FA was released from liposomes within 5 h in vitro. There is a novel finding in this study: that drugs could be entrapped with a high solubility in the intraliposomal buffer in contrast to the low solubility in the extraliposomal buffer. The results of body distribution in rats indicated that liposomes could improve the body distribution of FA. For FA liposome, the concentration of FA in brain was two-fold higher than that of free FA. Liposomal FA was a promising approach to improve the body distribution of FA.

Preparation, characterization, cellular uptake and evaluation in vivo of solid lipid nanoparticles loaded with cucurbitacin B

In this work, solid lipid nanoparticles loaded with cucurbitacin B (Cu B-SLNs) were prepared. It was found that the concentration of poloxamer 188 and soybean lecithin had effects on the mean particle size and size distribution. The zeta potentials were around −33 mV. In vitro release studies showed a sustained release after a burst release. Internalization of Cu B into HepG2 cells could be enhanced by the encapsulation of SLN matrix. The IC50 values of Cu B-SLNs were lower than that of Cu B solution. Both free Cu B and Cu B-SLNs had effectively inhibited the tumor growth and displayed a dose-dependent anti-tumor efficacy. Cu B-SLNs at a dose of 0.11 mg/kg produced the greatest anti-tumor effects (53.3%), which was significant higher than Cu B solution (31.5%, p < 0.05). Cu B-SLNs showed a longer MRT in vivo. The AUC of Cu B-SLNs for tumor increased 3.5 –fold when compared to Cu B solution. The targeting efficiency of Cu B-SLNs was 1.94 times higher in liver as compared to that of Cu B solution. These results indicated that Cu-B SLNs could passively target the tumor with EPR effect, improve the therapeutic efficacy of Cu B, and reduce the doses.