Inhibiting Multidrug Resistance with Transferrin-Targeted Polymersomes through Optimization of Ligand Density.

Transferrin-conjugated polymersomes, transferrin-biotin/avidin/biotin-Pluronic F127-poly(lactic acid) (Tf-F127-PLA), were successfully prepared through a biotin-avidin bridging technique to study their ability to inhibit multidrug resistance of cancer cells. Hydrophilic doxorubicin (DOX) was selected as the model drug to be loaded into Tf-F127-PLA polymersomes. DOX loaded in Tf-F127-PLA polymersomes was released fast initially, followed by a slow release. The effect of the transferrin ligand density of Tf-F127-PLA/DOX polymersomes on their targeting properties was studied by both cytotoxicity and cellular uptake assays against A549 lung cancer cells. It was shown that Tf-F127-PLA/DOX polymersomes had better targeting ability than nontargeted drug-loaded polymersomes. Furthermore, Tf-F127-PLA/DOX polymersomes with 2% Tf molar content have more effective antitumor activity and a higher cellular uptake than those with 4 and 5% Tf molar content. 2% Tf-F127-PLA/DOX polymersomes also exhibited better anticancer ability in multidrug resistant cancer cells A549/ADR than nontargeted PLA-F127-PLA/DOX polymersomes. It was further proved that the endocytosis of polymersomes by A549/ADR cells was an energy-dependent endocytosis process, which was related to clathrin, macrocytosis, and caveolin. Also, the endocytosis of Tf-F127-PLA/DOX polymersomes was proven to be mediated by the transferrin receptor.

Synergistic antitumor effect of folate-targeted Pluronic™ F-127/poly(lactic acid) polymersomes for codelivery of doxorubicin and paclitaxel.

Aim: Folate-targeted Pluronic™ F-127/poly(lactic acid) (FA-F127-PLA) polymersomes were used as codelivery carriers of doxorubicin hydrochloride (DOX) and paclitaxel (PTX) to achieve a targeted synergistic antitumor effect. Materials & methods: The cytotoxicity of PTX/DOX polymersomes against OVCAR-3 cells was determined by methyl thiazolyl tetrazolium assay. The cellular uptake of PTX/DOX polymersomes was examined by HPLC and micro-bicinchoninic acid techniques. Results: The polymersomes showed a bilayer core-shell structure with negative charge and good dispersion. PTX1/DOX5 polymersomes with a mass ratio of PTX to DOX of 1:5 showed the best synergistic effect and the highest cellular uptake. Conclusion: FA-F127-PLA polymersomes have the great promise for codelivery of multiple chemotherapeutics to achieve a targeted antitumor synergistic effect.

Hydrophilic doxorubicin hydrochloride (DOX) and hydrophobic paclitaxel (PTX) are two well-known anticancer drugs. Coadministration of DOX and PTX as a free drug cocktail has been widely used in clinical treatment to further improve their anticancer effect. However, this free drug cocktail often causes a lot of side effects such as cardiotoxicity and nephrotoxicity. In order to reduce the side effects of the drug cocktail and enhance their targeted delivery, folic acid-targeted Pluronic™ F-127 / poly(lactic acid) (FA-F127-PLA) polymersomes were used to load the drug cocktail. Both the cytotoxicity and cellular uptake data showed that PTX/DOX coloaded FA-F127-PLA polymersomes had better synergistic anticancer ability than a DOX and PTX free-drug cocktail.

Co-Delivery of paclitaxel and doxorubicin in folate-Targeted pluronic/ploy (D,L-lactide-b-glycolide) polymersomes.

Drugs with different solubility can be selectively embedded into polymersomes with the hydrophilic core and hydrophobic bilayer. Novel folate-targeted Pluronic/poly (D,L-lactide-b-glycolide) polymersomes were constructed and used for the co-delivery of paclitaxel (PTX) and doxorubicin (DOX) to improve their inhibitory effect over cancer cells. The particle size of blank polymersomes was mainly distributed below 125 nm. The release of PTX and DOX from polymersomes showed an initial burst release followed by a sustained and slow release. The in vitro cytotoxicity data showed that the targeted co-loaded polymersomes (PTX&DOX FA-Ps) exhibited better inhibitory effect than single-loaded polymersomes and free drugs did. Furthermore, PTX&DOX FA-Ps showed the synergistic therapeutic effect over OVCAR-3 cancer cells. The cellular uptake results also showed that folate modified polymersomes had excellent targeting performance. Therefore, the folate-targeted Pluronic/poly (D,L-lactide-b-glycolide) polymersomes have potential application value as novel drug carriers to co-deliver PTX and DOX.

Transferrin/folate dual-targeting Pluronic F127/poly(lactic acid) polymersomes for effective anticancer drug delivery.

A novel dual-targeting Pluronic/poly(lactic acid) polymersome containing transferrin and folic acid ligands (Tf/FA-F127-PLA) has been designed to study its application in the targeted drug delivery system. Both biotin and folic acid conjugated Biotin/FA-F127-PLA polymersomes (Ps) were prepared as the precursor. The dual-targeting behaviors of Tf/FA-F127-PLA over C6 glioma cells were then fulfilled through connecting the precursor with biotinylated transferrin by using a three-step biotin-avidin technique. Paclitaxel (PTX) was loaded successfully into Biotin/FA-F127-PLA and showed a burst release followed by a slow-release process in vitro. It was also obtained that Tf/FA-F127-PLA had higher cytotoxicity and cellular uptake amount than non-targeted and single-targeted Ps did. These results could be because more PTX-loaded Tf/FA-F127-PLA Ps entered C6 cells through both FA-folate receptor (FR) and Tf-transferrin receptor (TfR) specific affinity and thus possessed the better anti-tumor ability. It was further proved that the uptake of Ps by C6 cells was through the endocytosis related to clathrin, caveolae, lysosome, etc. Furthermore, it was demonstrated that the uptake of dual-targeting Tf/FA-F127-PLA Ps by C6 cells was related to the endocytosis mediated by both FR and TfR. These findings indicated that dual-targeting Tf/FA-F127-PLA Ps could be a potential carrier in targeted drug delivery systems.

Study on the Influencing Factors of Hypoglycemic Effect of Folate Targeted Polymersomes Encapsulating Insulin.

PURPOSE: To study the effects of the density of folic acid (FA) on the hypoglycemic ability of FA-targeted polymersomes as oral insulin carriers. Also to study the change of the hypoglycemic effect of FA-targeted mixed polymersomes added with various mass ratio of d-α-tocopherol polyethylene glycol 1000 succinate (TPGS). METHODS: The FA-targeted polymersomes with different FA molar contents were prepared. The in vitro insulin release experiments in different media for FA-targeted polymersomes with various FA contents were studied. Their quantitative cellular uptake in Caco-2 cells was examined. The in vivo hypoglycemic activity of FA-targeted polymersomes was also studied with diabetic rats. The polymersomes with the optimal FA molar content was chosen to prepare mixed polymersomes with various TPGS contents. RESULTS: Among insulin-loaded FA-targeted polymersomes with four different FA molar contents, insulin-loaded polymersomes with 10% FA molar content (insulin-loaded 10%FA-Ps) showed the hightest cellular uptake and the best hypoglycemic response. In addition, the insulin-loaded FA-Ps/TPGS5:1 mixed polymersomes exhibited higher cellular uptake and better hypoglycemic response than the other two insulin-loaded mixed polymersomes adding TPGS did. CONCLUSIONS: FA-Ps/TPGS5:1 could be a promising formulation for the oral administration of insulin.

Folate-conjugated pluronic/polylactic acid polymersomes for oral delivery of paclitaxel.

Cancer chemotherapy and the patient's life will be more convenient if oral administration of anti-cancer drugs can be achieved. The feasibility of folate-targeted Pluronic F127/polylactic acid (FA-F127-PLA) polymersomes as the oral delivery carriers of paclitaxel (PTX) has been explored in this study. PTX loaded in FA-F127-PLA and PLA-F127-PLA polymersomes showed biphasic release behaviors in simulated gastric and intestinal fluids. PTX loaded in FA-F127-PLA polymersomes exhibited higher cytotoxicity and cellular uptake than PTX loaded in PLA-F127-PLA polymersomes. In vivo pharmacokinetic studies in rats showed that oral PTX loaded in FA-F127-PLA polymersomes had a higher bioavailability than oral PTX loaded in PLA-F127-PLA polymersomes. D-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS or Vitamin E TPGS) was also added to the FA-F127-PLA polymersomes as an optimization agent. Compared with PTX-loaded FA-F127-PLA polymersome, PTX-loaded FA-F127-PLA/TPGS mixed polymersomes showed even better cytotoxic ability, more cellular uptake and higher bioavailability. The above results indicate that FA-F127-PLA and FA-F127-PLA/TPGS mixed polymersomes could be good candidates for the oral delivery carrier of anti-cancer drugs.

Effect of the Folate Ligand Density on the Targeting Property of Folated-Conjugated Polymeric Nanoparticles.

Targeted drug delivery systems have attracted increasing attention due to their ability for delivering anticancer drugs selectively to tumor cells. Folic acid (FA)-conjugated targeted block copolymers, FA-Pluronic-polycaprolactone (FA-Pluronic-PCL) are synthesized in this study. The anticancer drug paclitaxel (PTX) is loaded in FA-Pluronic-PCL nanoparticles by nanoprecipitation method. The in vitro release of PTX from FA-Pluronic-PCL nanoparticles shows slow and sustained release behaviors. The effect of FA ligand density of FA-Pluronic-PCL nanoparticles on their targeting properties is examined by both cytotoxicity and fluorescence methods. It is shown that FA-Pluronic-PCL nanoparticles indicated better targeting ability than non-targeted PCL-Pluronic-PCL nanoparticles. Furthermore, FA-F127-PCL nanoparticle with 10% FA molar content has more effective antitumor activity and higher cellular uptake than those with 50% and 91% FA molar content. These results prove that FA-F127-PCL nanoparticle with 10% FA molar content can be a better candidate as the drug carrier in targeted drug delivery systems.

The targeting properties of folate-conjugated Pluronic F127/poly (lactic-co-glycolic) nanoparticles.

Novel Folated Pluronic F127/poly (lactic-co-glycolic) (FA-F127-PLGA) and PLGA-F127-PLGA block copolymer were synthesized and nanoparticles self-assembled from these two copolymers were prepared by dialysis method. Paclitaxel (PTX) was successfully encapsulated in these two nanoparticles. According to in vitro release studies of PTX-loaded nanoparticles, after an initial burst release during the first 11h, the entrapped PTX released slowly in the following 82h. The cytotoxicity studies demonstrated that the in vitro antitumor effect of PTX could be improved by encapsulating PTX into PLGA-F127-PLGA nanoparticles. Moreover, folate-targeted FA-F127-PLGA nanoparticles were more effective than PLGA-F127-PLGA when delivering PTX in folate receptor overexpressing OVCAR-3 cells, which mainly due to the FA-receptor-meditated endocytosis. As the treatment time became longer, the targeting effects were more obvious. The targeting effect of FA-F127-PLGA nanoparticles was also investigated in vitro by measuring the cellular uptake of the nanoparticles. The results showed that FA-F127-PLGA nanoparticles were more easily to be uptaken by OVCAR-3 cells in comparison with PLGA-F127-PLGA nanoparticles. In vivo pharmacokinetic studies indicated that FA-F127-PLGA nanoparticles prolong the circulation time of PTX in plasma, and delay the blood clearance of PTX. These results indicated that Folated FA-F127-PLGA could be a potential carrier in long-term PTX delivery.

Enhanced effect of folated pluronic F87-PLA/TPGS mixed micelles on targeted delivery of paclitaxel.

Targeted drug delivery systems have great potential to overcome the side effect and improve the bioavailability of conventional anticancer drugs. In order to further improve the antitumor efficacy of paclitaxel (PTX) loaded in folated Pluronic F87/poly(lactic acid) (FA-F87-PLA) micelles, D-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS or Vitamin E TPGS) were added into FA-F87-PLA to form FA-F87-PLA/TPGS mixed micelles. The LE of PTX-loaded mixed micelles (13.5%) was highest in the mass ratio 5 to 3 of FA-F87-PLA to TPGS. The in vitro cytotoxicity assays indicated that the IC50 values for free PTX injections, PTX-loaded FA-F87-PLA micelles and PTX-loaded FA-F87-PLA/TPGS mixed micelles after 72h of incubation were 1.52, 0.42 and 0.037mg/L, respectively. The quantitative cellular uptake of coumarin 6-loaded FA-F87-PLA/TPGS and FA-F87-PLA micelles showed that the cellular uptake efficiency of mixed micelles was higher for 2 and 4h incubation, respectively. In vivo pharmacokinetic studies found that the AUC of PTX-loaded FA-F87-PLA/TPGS mixed micelles is almost 1.4 times of that of PTX-loaded FA-F87-PLA micelles. The decreased particle size and inhibition of P-glycoprotein effect induced by the addition of TPGS could result in enhancing the cellular uptake and improving the antitumor efficiency of PTX-loaded FA-F87-PLA/TPGS mixed micelles.

A review of biodegradable polymeric systems for oral insulin delivery.

Currently, repeated routine subcutaneous injections of insulin are the standard treatment for insulin-dependent diabetic patients. However, patients' poor compliance for injections often fails to achieve the stable concentration of blood glucose. As a protein drug, the oral bioavailability of insulin is low due to many physiological reasons. Several carriers, such as macromolecules and liposomes have been used to deliver drugs in vivo. In this review article, the gastrointestinal barriers of oral insulin administration are described. Strategies for increasing the bioavailability of oral insulin, such absorption enhancers, enzyme inhibitors, enteric coatings are also introduced. The potential absorption mechanisms of insulin-loaded nanoparticles across the intestinal epithelium, including intestinal lymphatic route, transcellular route and paracellular route are discussed in this review. Natural polymers, such as chitosan and its derivates, alginate derivatives, γ-PGA-based materials and starch-based nanoparticles have been exploited for oral insulin delivery; synthetic polymers, such as PLGA, PLA, PCL and PEA have also been developed for oral administration of insulin. This review focuses on recent advances in using biodegradable natural and synthetic polymers for oral insulin delivery along with their future prospects.

Pluronic P85/poly(lactic acid) vesicles as novel carrier for oral insulin delivery.

Poly(lactic acid)-b-Pluronic-b-poly(lactic acid) (PLA-P85-PLA) vesicles were developed as novel carrier for oral insulin delivery. PLA-P85-PLA block copolymer was synthesized by ring opening polymerization of the monomer l-lactide using Pluronic copolymer P85 as the initiator. Insulin-loaded PLA-P85-PLA vesicles were prepared by dialysis method and the mean diameter of insulin-loaded PLA-P85-PLA vesicles was determined to be 178 nm. The cytotoxicity studies using human ovarian cancer cells OVCAR-3 indicate that PLA-P85-PLA block copolymer has good biocompatibility. Both in vitro and in vivo release behavior of insulin loaded in PLA-P85-PLA vesicles were studied. It was observed that insulin was released out gradually from PLA-P85-PLA vesicles and almost all insulin was released out 7.5h later. More importantly, for the oral administration of insulin-loaded PLA-P85-PLA vesicles at insulin doses of 200 IU/kg, the minimum blood glucose concentration was observed in the diabetic mice test after 2.5h, which was 15% of initial glucose level. Furthermore, the blood glucose concentration increased slowly to 31.8% of initial blood glucose concentration after 10.5h and was maintained at this level for at least an additional 14h (32.5% of initial blood glucose concentration at 24.5h). These results proved that PLA-P85-PLA vesicles could be promising polymeric carriers for oral insulin delivery application due to their sustained and enhanced hypoglycemic effect.

In vitro &in vivo targeting behaviors of biotinylated Pluronic F127/poly(lactic acid) nanoparticles through biotin-avidin interaction.

Biotinylated Pluronic F127/poly(lactic acid) block copolymers (B-F127-PLA) were successfully synthesized previously by our group. In the present study, the release behaviors of paclitaxel-loaded B-F127-PLA nanoparticles and their targeting properties to human ovarian carcinoma cells were investigated. Paclitaxel (pac) loaded in B-F127-PLA nanoparticles shows an initial burst release in the first 6h and followed by a slow release. The in vitro targeting behaviors of B-F127-PLA nanoparticles against human ovarian cancer cells (OVCAR-3, SKOV-3) were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) tests and fluorescence microscopy (FM) technique. Targeting was based on a three-step biotin-avidin targeting approach using biotinylated anti-CA125 antibody specific for the CA-125 antigen that is highly expressed on OVCAR-3 cells but not expressed on SKOV-3 cells. MTT results show that the anticancer effect of paclitaxel in B-F127-PLA nanoparticles over OVCAR-3 cells was stronger than that over SKOV-3 cells, indicating that B-F127-PLA nanoparticles were delivered more effectively to OVCAR-3 cells than to SKOV-3 cells. The targeting behaviors of B-F127-PLA nanoparticles were further confirmed by FM technique. The intracellular distribution of B-F127-PLA nanoparticles was also studied using a triple-labeling method. It was observed that B-F127-PLA nanoparticles are mainly localized within the cytoplasm of OVCAR-3 cells. The in vivo antitumor efficacy of pac-loaded B-F127-PLA nanoparticles by three-step method as measured by change in tumor volume of OVCAR-3 implanted in Balb/C nude mice was greater than that by one-step method.

Active targeting behaviors of biotinylated pluronic/poly(lactic acid) nanoparticles in vitro through three-step biotin-avidin interaction.

In order to prepare targeted drug carriers, previously a biotin group has been attached by our group to the end of Pluronic F87/poly(lactic acid) and Pluronic P85/poly(lactic acid) block co-polymers to obtain B-F87-PLA and B-P85-PLA, respectively. In this paper, the active targeting properties of B-F87-PLA and B-P85-PLA nanoparticles in vitro were investigated through a three-step biotin-avidin interaction by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) tests and fluorescence microscopy (FM). Two kinds of human ovarian cancer cells (OVCAR-3 and SKOV-3) and paclitaxel were chosen for the cytotoxicity tests. CA-125 antigen is over-expressed on OVCAR-3 cells but not on SKOV-3 cells. The loading and release behavior of paclitaxel loaded in B-Pluronic-PLA nanoparticles were also studied. Paclitaxel loaded in both B-F87-PLA and B-P85-PLA nanoparticles shows an initial rapid release followed by a slow release period. Compared with SKOV-3 cells, the cytotoxicity results implied that paclitaxel-loaded B-Pluronic-PLA nanoparticles were delivered more effectively to OVCAR-3 cells due to the specific interaction between the biotin groups on the surface of B-Pluronic-PLA nanoparticles and the avidin/biotinylated MAb X306/CA-125 antigen complexes on the surface of OVCAR-3 cells. The active targeting properties of B-F87-PLA nanoparticles were further confirmed by FM.

Vesicles from Pluronic/poly(lactic acid) block copolymers as new carriers for oral insulin delivery.

The morphologies of poly(lactic acid)-b-Pluronic-b-poly(lactic acid) (PLA-F127-PLA) aggregates in aqueous solutions were reported previously to be vesicular nano-particles by our group. In the present study, we seek to investigate the feasibility of using PLA-F127-PLA vesicles as oral delivery carrier for insulin. Both in vitro and in vivo release behavior of insulin loaded in PLA-F127-PLA vesicles were studied. A biphasic release behavior was observed for the in vitro release of insulin from PLAF127-29 vesicles. More importantly, it was found in the diabetic mice tests that the blood glucose concentration of oral insulin-loaded PLAF127-29 vesicles decreased from 18.5 to 5.3 mmol/L within 4.5 h and the minimum blood glucose concentration (about 4.5 mmol/L) was achieved after about 5 h. Furthermore, the blood glucose concentration was maintained at this level for at least an additional 18.5 h. These results proved that PLA-F127-PLA vesicles could be promising polymeric carriers for oral insulin delivery application due to their prolonged hypoglycemic effect.

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone).

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.