Nanovesicles loaded with a TGF-ß receptor 1 inhibitor overcome immune resistance to potentiate cancer immunotherapy.

The immune-excluded tumors (IETs) show limited response to current immunotherapy due to intrinsic and adaptive immune resistance. In this study, it is identified that inhibition of transforming growth factor-ß (TGF-ß) receptor 1 can relieve tumor fibrosis, thus facilitating the recruitment of tumor-infiltrating T lymphocytes. Subsequently, a nanovesicle is constructed for tumor-specific co-delivery of a TGF-ß inhibitor (LY2157299, LY) and the photosensitizer pyropheophorbide a (PPa). The LY-loaded nanovesicles suppress tumor fibrosis to promote intratumoral infiltration of T lymphocytes. Furthermore, PPa chelated with gadolinium ion is capable of fluorescence, photoacoustic and magnetic resonance triple-modal imaging-guided photodynamic therapy, to induce immunogenic death of tumor cells and elicit antitumor immunity in preclinical cancer models in female mice. These nanovesicles are further armored with a lipophilic prodrug of the bromodomain-containing protein 4 inhibitor (i.e., JQ1) to abolish programmed death ligand 1 expression of tumor cells and overcome adaptive immune resistance. This study may pave the way for nanomedicine-based immunotherapy of the IETs.

The interaction of folate-modified Bletilla striata polysaccharide-based micelle with bovine serum albumin.

We fabricated an amphiphilic folate-modified Bletilla striata polysaccharide (FA-BSP-SA) copolymer that exhibited good biocompatibility and superior antitumor effects. This study investigated the affinity between FA-BSP-SA and bovine serum albumin (BSA) via multispetroscopic approaches. Changes in the morphology and particle size showed that FA-BSP-SA formed a blurry "protein corona". Stern-Volmer equation demonstrated that FA-BSP-SA micelles decreased the fluorescence of BSA via static quenching. The measurement results of thermodynamic parameters (entropy change, enthalpy change, and Gibbs free energy) suggested that the binding between FA-BSP-SA and BSA was spontaneous in which Van der Waals forces and hydrogen bonding played major roles. The results from synchronous fluorescence, circular dichroism, and UV spectra also revealed that BSA conformation was slightly altered by decreasing α-helical contents. In addition, the antitumor effects in vitro of Dox@FA-BSP-SA micelles and the cellular uptake behavior of micelles in 4T1 cells were decreased after incubating with BSA.

Doxorubicin-loaded folate-mediated pH-responsive micelle based on Bletilla striata polysaccharide: Release mechanism, cellular uptake mechanism, distribution, pharmacokinetics, and antitumor effects.

This study evaluated the potential of folate (FA)-mediated and stearic acid (SA) modified Bletilla striata polysaccharide (FA-BSP-SA) copolymer as the vehicle for targeted delivery of anticancer drugs to tumor tissues and enhanced antitumor efficacy. The critical aggregation concentration, morphology, particle size, and zeta potential of micelles were increased with the reduction of pH values. The complex between doxorubicin (Dox) hydrochloride and sodium cholate via electrostatic interaction was fabricated and then directly encapsulated into FA-BSP-SA micelles. Dox in micelles existed in the status of amorphism. The Dox/FA-BSP-SA micelles demonstrated pH-responsive release behavior under the combination of diffusion and erosion mechanism. They could clearly strengthen the cellular uptake of Dox and inhibit the proliferation and the migration of tumor cells compared with the Dox/BSP-SA micelles and the free Dox. The Dox/FA-BSP-SA micelles were further delivered to lysosomes, mainly due to clathrin-mediated endocytosis. The FA-BSP-SA micelles distinctly improved the absolute bioavailability of Dox compared with the free Dox and the Dox/BSP-SA micelles (p < 0.01) and prolong the mean residence time. The Dox/FA-BSP-SA micelles significantly increased the drug enrichment in the tumor sites and enhanced the antitumor effects in vivo. Taken together, the FA-BSP-SA micelle could be exploited as a potential platform for targeting anticancer drug delivery.

Synergistic effects of antitumor efficacy via mixed nano-size micelles of multifunctional Bletilla striata polysaccharide-based copolymer and D-α-tocopheryl polyethylene glycol succinate.

To evaluate if mixed micelles of Dox@FA-BSP-SA/TPGS can allow for the superior antitumor efficiency than Dox@FA-BSP-SA micelles. The complex of doxorubicin (Dox) and sodium cholate was encapsulated into the mixed micelles composed of folate-mediated stearic acid-modified Bletilla striata polysaccharide (FA-BSP-SA) and D-α-tocopheryl polyethylene glycol succinate (TPGS). Its average particle size increased whereas load capacity (LC) and encapsulation efficiency (EE) decreased with the increase of TPGS mass ratio in the mixed micelles. The changes of morphology, particle size and doxorubicin release in vitro demonstrated the pH sensitivity of micelles. FA-BSP-SA/TPGS mixed micelle exhibited average particle size of 147.3 nm, LC of 14.4% and EE of 91.9% for doxorubicin at the weight ratio of 3: 1. The doxorubicin release rate of micelles was faster in pH 5.0 media compared with that in pH 6.0 and 7.4 media. The cytotoxicity in vitro and antitumor efficacy in vivo results of Dox@FA-BSP-SA/TPGS micelle were more superior to that of free doxorubicin and Dox@FA-BSP-SA single micelle. For Dox@FA-BSP-SA/TPGS micelle, the clathrin-mediated endocytosis was the dominant mechanism of intracellular uptake. The FA-BSP-SA/TPGS mixed micelle may be a promising drug delivery system for cancer chemotherapy.

Bio-responsive Bletilla striata polysaccharide-based micelles for enhancing intracellular docetaxel delivery.

The aim of this study was to design a pH- and redox-dual responsive Bletilla striata polysaccharide (BSP)-based copolymer to enhance anti-tumor drugs release at tumor sites and improve the therapeutic effect. The copolymer was synthesized using stearic acid (SA) and cystamine via a disulfide linkage and characterized using 1H-Nuclear Magnetic Resonance spectroscopy and Fourier Transform Infrared spectroscopy. The BSP-ss-SA copolymer could self-assemble into micelle in an aqueous environment and could encapsulate docetaxel therein. Its inhibitory effects on HepG2 cells and 4 T1 cells were determined. Besides, the anti-cancer effects in vivo and histopathological study of 4 T1-bearing tumor mice were also evaluated. Docetaxel-loaded BSP-ss-SA micelles showed significant pH-sensitive release behavior, supplying a greater drug release percentage in pH 5.0 media compared to pH 7.4 media. BSP-ss-SA micelles exhibited a clear redox-responsive release property in pH 7.4 media whereas the similar cumulative release percentage of docetaxel from BSP-ss-SA micelles in pH 5.0 media in the presence and absence of DL-dithiothreitol. The Docetaxel-loaded BSP-ss-SA micelles clearly inhibited the proliferation of HepG2 and 4 T1 cells compared with docetaxel solution. The results of MTT and histopathological study indicated that BSP-ss-SA copolymer exhibited good blood compatibility. The BSP-ss-SA copolymer may be used as carriers to deliver anti-tumor drugs to special tumor tissues.

Interactions of Self-Assembled Bletilla Striata Polysaccharide Nanoparticles with Bovine Serum Albumin and Biodistribution of Its Docetaxel-Loaded Nanoparticles.

: Amphiphilic copolymers of stearic acid (SA)-modified Bletilla striata polysaccharides (BSPs-SA) with three different degrees of substitution (DSs) were synthesized. The effects of DS values on the properties of BSPs-SA nanoparticles were evaluated. Drug state, cytotoxicity, and histological studies were carried out. The affinity ability of bovine serum albumin (BSA) and the BSPs-SA nanoparticles was also characterized utilizing ultraviolet and fluorescence spectroscopy. Besides, the bioavailability and tissue distribution of docetaxel (DTX)-loaded BSPs-SA nanoparticles were also assessed. The results demonstrated that the DS increase of the hydrophobic stearic acid segment increased the negative charge, encapsulation efficiency, and drug-loading capacity while decreasing the critical aggregation concentration value as well as the release rate of docetaxel from the nanoparticles. Docetaxel was encapsulated in nanoparticles at the small molecules or had an amorphous status. The inhibitory capability of DTX-loaded BSPs-SA nanoparticles against 4T1 tumor cells was superior to that of Duopafei®. The ultraviolet and fluorescence results exhibited a strong binding affinity between BSPs-SA nanoparticles and bovine serum albumin, but the conformation of bovine serum albumin was not altered. Additionally, the area under the concentration⁻time curve (AUC0⁻∞) of DTX-loaded BSPs-SA nanoparticles was about 1.42-fold higher compared with Duopafei® in tumor-bearing mice. Docetaxel levels of DTX-loaded BSPs-SA nanoparticles in some organs changed, and more docetaxel accumulated in the liver, spleen, and the tumor compared with Duopafei®. The experimental results provided a theoretical guidance for further applications of BSPs-SA conjugates as nanocarriers for delivering anticancer drugs.

In vitro characterization of pH-sensitive Bletilla Striata polysaccharide copolymer micelles and enhanced tumour suppression in vivo.

OBJECTIVES: A system of stearic acid (SA)-modified Bletilla striata polysaccharide (BSP) micelles was developed for the targeted delivery of docetaxel (DTX) as a model anticancer drug (DTX-SA-BSP). METHODS: Particle size, zeta potential and DTX release in vitro were measured in release media at different pH values. Quantitative cellular uptake, cytotoxicity assay in vitro and antitumour efficacy in vivo were also evaluated. Cell apoptosis was assessed by flow cytometry. KEY FINDINGS: DTX-SA-BSP copolymer micelles displayed pH-dependent properties in the respects of particle size, zeta potential and in vitro release behaviour ranging from pH 5.0 to pH 7.4. DTX-SA-BSP copolymer micelles showed higher release rate at pH 5.0 than that at pH 6.0 and 7.4. In vitro cytotoxic effect of DTX-SA-BSP copolymer micelles was higher than that of DTX injection. The results of high-performance liquid chromatography determination confirmed that DTX cellular uptake of micelles was enhanced compared with that of DTX injection. Anticancer activity in vivo further confirmed the enhanced tumour targeting and anticancer efficacy of DTX-SA-BSP copolymer micelles. CONCLUSIONS: The above results show that DTX-SA-BSP copolymer micelles have pH sensitivity. SA-BSP copolymers are a promising carrier for delivering hydrophobic anticancer drugs.

Evaluation of the cytotoxicity and intestinal absorption of a self-emulsifying drug delivery system containing sodium taurocholate.

Currently, many surfactants used in self-emulsifying drug delivery systems (SMEDDS) can cause gastrointestinal mucosal irritation and systemic toxicity. In the present study, SMEDDS were loaded with pueraria flavones, using sodium taurocholate to replace polyoxyl 40 dydrogenated castor oil (Cremophor® RH 40) as the surfactant (PF-SMEDDSNR) to reduce the toxicity of SMEDDS using Cremophor® RH 40 as the surfactant (PF-SMEDDSR). The absorption rate constants (Ka) and intestinal permeability coefficients (Peff) were measured. The effects of P-glycoprotein inhibitor (verapamil), adenosine triphosphate (ATP) inhibitor (2,4-dinitrophenol), and carrier inhibitor on Ka and Peff values in the ileum were determined. Biological safety was also evaluated. The Ka and Peff values increased for PF-solution concentrations of 200µg/ml>100µg/ml>400µg/ml in individual segments of the intestines. The results indicated that Peff values of PF-SMEDDSNR were distinctly higher than those of SMEDDS loaded with pueraria flavones using Cremophor®RH 40 as the surfactant (PF-SMEDDSR) and PF-solution in four intestinal segments. However, the Ka values of PF-SMEDDSNR were higher only in the jejunum and ileum segments compared with those of PF-SMEDDSR and PF-solution. The Ka and Peff values without verapamil were significantly lower than those with verapamil. 2,4-Dinitrophenol had no effect on Ka and Peff values. The Ka and Peff values of PF-SMEDDSNR significantly decreased after perfusing B-SMEDDSNR for 1h prior to the study. The cell viabilities after exposure to SMEDDSNR were higher than those of SMEDDSR in the range of 81-324µg/ml. Lactate dehydrogenase release from cells treated with PF-SMEDDSNR or B-SMEDDSNR was significantly lower than that from cells treated with PF-SMEDDSR or B-SMEDDSR at surfactant concentrations of 243 and 324µg/ml. However, there were no differences with SMEDDS treatment at surfactant concentrations of 0-162µg/ml. Hence, we conclude that SMEDDS using sodium taurocholate as the surfactant can reduce the toxicity of SMEDDS, meanwhile, maintain the characteristics of SMEDDS, and enhance intestinal absorption.

In vitro and in vivo evaluation of docetaxel-loaded stearic acid-modified Bletilla striata polysaccharide copolymer micelles.

Bletilla striata polysaccharides (BSPs) have been used in pharmaceutical and biomedical industry, the aim of the present study was to explore a BSPs amphiphilic derivative to overcome its application limit as poorly water-soluble drug carriers due to water-soluble polymers. Stearic acid (SA) was selected as a hydrophobic block to modify B. striata polysaccharides (SA-BSPs). Docetaxel (DTX)-loaded SA-BSPs (DTX-SA-BSPs) copolymer micelles were prepared and characterized. The DTX release percentage in vitro and DTX concentration in vivo was carried out by using high performance liquid chromatography. HepG2 and HeLa cells were subjected to MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazonium bromide) assay to evaluate the cell viability. In vitro evaluation of copolymer micelles showed higher drug encapsulation and loading capacity. The release percentage of DTX from DTX-SA-BSPs copolymer micelles and docetaxel injection was 66.93 ± 1.79% and 97.06 ± 1.56% in 2 days, respectively. The DTX-SA-BSPs copolymer micelles exhibited a sustained release of DTX. A 50% increase in growth inhibition was observed for HepG2 cells treated with DTX-SA-BSPs copolymer micelles as compared to those treated with docetaxel injection for 72 h. DTX-SA-BSPs copolymer micelles presented a similar growth inhibition effect on Hela cells. Furthermore, absolute bioavailability of DTX-SA-BSPs copolymer micelles was shown to be 1.39-fold higher than that of docetaxel injection. Therefore, SA-BSPs copolymer micelles may be used as potential biocompatible polymers for cancer chemotherapy.

Docetaxel-Loaded Self-Assembly Stearic Acid-Modified Bletilla striata Polysaccharide Micelles and Their Anticancer Effect: Preparation, Characterization, Cellular Uptake and In Vitro Evaluation.

Poorly soluble drugs have low bioavailability after oral administration, thereby hindering effective drug delivery. A novel drug-delivery system of docetaxel (DTX)-based stearic acid (SA)-modified Bletilla striata polysaccharides (BSPs) copolymers was successfully developed. Particle size, zeta potential, encapsulation efficiency (EE), and loading capacity (LC) were determined. The DTX release percentage in vitro was determined using high performance liquid chromatography (HPLC). The hemolysis and in vitro anticancer activity were studied. Cellular uptake and apoptotic rate were measured using flow cytometry assay. Particle size, zeta potential, EE and LC were 125.30 ± 1.89 nm, -26.92 ± 0.18 mV, 86.6% ± 0.17%, and 14.8% ± 0.13%, respectively. The anticancer activities of DTX-SA-BSPs copolymer micelles against HepG2, HeLa, SW480, and MCF-7 (83.7% ± 1.0%, 54.5% ± 4.2%, 48.5% ± 4.2%, and 59.8% ± 1.4%, respectively) were superior to that of docetaxel injection (39.2% ± 1.1%, 44.5% ± 5.3%, 38.5% ± 5.4%, and 49.8% ± 2.9%, respectively) at 0.5 µg/mL drug concentration. The DTX release percentage of DTX-SA-BSPs copolymer micelles and docetaxel injection were 66.93% ± 1.79% and 97.06% ± 1.56% in two days, respectively. Cellular uptake of DTX-FITC-SA-BSPs copolymer micelles in cells had a time-dependent relation. Apoptotic rate of DTX-SA-BSPs copolymer micelles and docetaxel injection were 73.48% and 69.64%, respectively. The SA-BSPs copolymer showed good hemocompatibility. Therefore, SA-BSPs copolymer can be used as a carrier for delivering hydrophobic drugs.