A biocompatible and cathepsin B sensitive nanoscale system of dendritic polyHPMA-gemcitabine prodrug enhances antitumor activity markedly.

In an attempt to improve the therapeutic indices of gemcitabine (GEM), a prodrug was designed by conjugating GEM with a stimuli-responsive dendritic polyHPMA copolymer (dendritic polyHPMA-GEM) and synthesized using the one-pot method of RAFT polymerization. The prodrug with dendritic architectures was able to aggregate and form stable nanoscale systems in the order of 46 nm. The high molecular weight (HMW, 168 kDa) dendritic prodrug could biodegrade into segments of low molecular weight (LMW, 29 kDa) for excretion. The prodrug demonstrates enzyme-responsive drug release features; over 95% GEM was released from the carrier in the presence of cathepsin B within 3 h. Investigation of the cellular mechanism underlying the dendritic prodrug suggests that cytotoxicity is associated with cellular uptake and cell apoptosis. The prodrug shows good hemocompatibility and in vivo biosafety. In particular, the dendritic polymer prodrug displays high accumulation within tumors and markedly improved in vivo antitumor activity in the 4T1 murine breast cancer model compared to free GEM. The in vivo antitumor activities are characterized by a marked suppression in tumor volumes indicating much higher tumor growth inhibition (TGI, 83%) than that in GEM treatment (TGI, 36%). In addition, some tumors were eliminated. The tumor xenograft immunohistochemistry study clearly indicates that tumor apoptosis occurs through antiangiogenic effects. These results suggest that the stimuli-responsive dendritic polymer-gemcitabine has great potential as an efficient anticancer agent.

Preparation, Characterization and Application of a Molecularly Imprinted Polymer for Selective Recognition of Sulpiride.

A novel molecular imprinting polymer (MIP) was prepared by bulk polymerization using sulpiride as the template molecule, itaconic acid (ITA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the crosslinker. The formation of the MIP was determined as the molar ratio of sulpiride-ITA-EGDMA of 1:4:15 by single-factor experiments. The MIP showed good adsorption property with imprinting factor α of 5.36 and maximum adsorption capacity of 61.13 µmol/g, and was characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR) and surface area analysis. With the structural analogs (amisulpride, tiapride, lidocaine and cisapride) and small molecules containing a mono-functional group (p-toluenesulfonamide, formamide and 1-methylpyrrolidine) as substrates, static adsorption, kinetic adsorption, and rebinding experiments were also performed to investigate the selective adsorption ability, kinetic characteristic, and recognition mechanism of the MIP. A serial study suggested that the highly selective recognition ability of the MIP mainly depended on binding sites provided by N-functional groups of amide and amine. Moreover, the MIP as solid-phase extractant was successfully applied to extraction of sulpiride from the mixed solution (consisted of p-toluenesulfonamide, sulfamethoxazole, sulfanilamide, p-nitroaniline, acetanilide and trimethoprim) and serum sample, and extraction recoveries ranged from 81.57% to 86.63%. The tentative tests of drug release in stimulated intestinal fluid (pH 6.8) demonstrated that the tablet with the MIP-sulpiride could obviously inhibit sulpiride release rate. Thus, ITA-based MIP is an efficient and promising alternative to solid-phase adsorbent for extraction of sulpiride and removal of interferences in biosample analysis, and could be used as a potential carrier for controlled drug release.

Extraction and characterization of polysaccharides from Semen Cassiae by microwave-assisted aqueous two-phase extraction coupled with spectroscopy and HPLC.

A novel and rapid method for simultaneous extraction and separation of the different polysaccharides from Semen Cassiae (SC) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) in a one-step procedure. Using ethanol/ammonium sulfate system as a multiphase solvent, the effects of MAATPE on the extraction of polysaccharides from SC such as the composition of the ATPS, extraction time, temperature and solvent-to-material ratio were investigated by UV-vis analysis. Under the optimum conditions, the yields of polysaccharides were 4.49% for the top phase, 8.80% for the bottom phase and 13.29% for total polysaccharides, respectively. Compared with heating solvent extraction and ultrasonic assisted extraction, MAATPE exhibited the higher extraction yields in shorter time. Fourier-transform infrared spectra showed that two polysaccharides extracted from SC to the top and bottom phases by MAATPE were different from each other in their chemical structures. Through acid hydrolysis and PMP derivatization prior to HPLC, analytical results by indicated that a polysaccharide of the top phases was a relatively homogeneous homepolysaccharide composed of dominant gucose glucose while that of the bottom phase was a water-soluble heteropolysaccharide with multiple components of glucose, xylose, arabinose, galactose, mannose and glucuronic acid. Molar ratios of monosaccharides were 95.13:4.27:0.60 of glucose: arabinose: galactose for the polysaccharide from the top phase and 62.96:14.07:6.67: 6.67:5.19:4.44 of glucose: xylose: arabinose: galactose: mannose: glucuronic acid for that from the bottom phase, respectively. The mechanism for MAATPE process was also discussed in detail. MAATPE with the aid of microwave and the selectivity of the ATPS not only improved yields of the extraction, but also obtained a variety of polysaccharides. Hence, it was proved as a green, efficient and promising alternative to simultaneous extraction of polysaccharides from SC.

Folate-Modified Poly(malic acid) Graft Polymeric Nanoparticles for Targeted Delivery of Doxorubicin: Synthesis, Characterization and Folate Receptor Expressed Cell Specificity.

A novel amphiphilic biodegradable cholesterol and poly(ethylene glycol)-folate grafted poly(α,ß-malic acid) (PMA-g-Chol/PEG-FA) was synthesized and characterized as self-assembled nanoparticles for targeted delivery of doxorubicin (DOX). The nanoparticles showed extremely low critical aggregation concentrations (CAC), appropriate zeta potential, narrow size distribution, good stability in serum conditions and negligible toxicity. After encapsulation'of DOX, PMA-g-Chol/PEG-FA nanoparticles showed significantly reduced cell viability (up to 30% for Hela and 27% for 4T1 cells) compared with the non-targeted ones on carcinoma cells with different levels of folate receptor (FR) expression. While no difference was detected on HEK293 cells (FR receptor negative) between the two nanoparticles. Addition of extra free folate obviously decreased the cellular mortality and inhibited the cellular uptake of targeted nanoparticles. In the Hela/HEK293 co-culture model, folate conjugated nanoparticles showed specific affiliation with Hela cells other than HEK293 cells, indicating good targeting property of the delivery system. As detected from ex vivo fluorescent imaging, PMA-g-Chol/PEG-FA nanoparticles could accumulate at tumor site with higher selectivity compared to PMA-g-Chol/PEG nanoparticles and DOX x HCl. In vivo antitumor studies confirmed the significant tumor inhibition efficacy of drug-loaded PMA-g-Chol/PEG-FA nanoparticles with lower toxicity to normal tissues than DOX x HCI at the same dosage.

Enantioseparation of Racemic Flurbiprofen by Aqueous Two-Phase Extraction With Binary Chiral Selectors of L-dioctyl Tartrate and L-tryptophan.

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two-phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two-phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L-dioctyl tartrate and L-tryptophan, which were screened from amino acids, ß-cyclodextrin derivatives, and L-tartrate esters. Factors such as the amounts of L-dioctyl tartrate and L-tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L-dioctyl tartrate, 80 mg; L-tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L-dioctyl tartrate and L-tryptophan, which enantioselectively recognized R- and S-enantiomers in top and bottom phases, respectively. Compared to conventional liquid-liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers.

Sustained PDGF-BB release from PHBHHx loaded nanoparticles in 3D hydrogel/stem cell model.

This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering.

Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo.

Breast cancer stem cells (BCSCs) are pan-resistant to different anticancer agents and responsible for cancer relapse. Disulfiram (DS), an antialcoholism drug, targets CSCs and reverses pan-chemoresistance. The anticancer application of DS is limited by its very short half-life in the bloodstream. This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo. The relationship between hypoxia and CSCs was examined by in vitro comparison of BC cells cultured in spheroid and hypoxic conditions. To determine the importance of NFκB activation in bridging hypoxia and CSC-related pan-resistance, the CSC characters and drug sensitivity in BC cell lines were observed in NFκB p65 transfected cell lines. The effect of Lipo-DS on the NFκB pathway, CSCs and chemosensitivity was investigated in vitro and in vivo. The spheroid cultured BC cells manifested CSC characteristics and pan-resistance to anticancer drugs. This was related to the hypoxic condition in the spheres. Hypoxia induced activation of NFκB and chemoresistance. Transfection of BC cells with NFκB p65 also induced CSC characters and pan-resistance. Lipo-DS blocked NFκB activation and specifically targeted CSCs in vitro. Lipo-DS also targeted the CSC population in vivo and showed very strong anticancer efficacy. Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed. Hypoxia induced NFκB activation is responsible for stemness and chemoresistance in BCSCs. Lipo-DS targets NFκB pathway and CSCs. Further study may translate DS into cancer therapeutics.

Effects of pH-sensitive chain length on release of doxorubicin from mPEG-b-PH-b-PLLA nanoparticles.

BACKGROUND: Two methoxyl poly(ethylene glycol)-poly(L-histidine)-poly(L-lactide) (mPEG-PH-PLLA) triblock copolymers with different poly(L-histidine) chain lengths were synthesized. The morphology and biocompatibility of these self-assembled nanoparticles was investigated. METHODS: Doxorubicin, an antitumor drug, was trapped in the nanoparticles to explore their drug-release behavior. The drug-loaded nanoparticles were incubated with HepG2 cells to evaluate their antitumor efficacy in vitro. The effects of poly(L-histidine) chain length on the properties, drug-release behavior, and antitumor efficiency of the nanoparticles were investigated. RESULTS: The nanoparticles were pH-sensitive. The mean diameters of the two types of mPEG-PH- PLLA nanoparticle were less than 200 nm when the pH values were 5.0 and 7.4. The nanoparticles were nontoxic to NIH 3T3 fibroblasts and HepG2 cells. The release of doxorubicin at pH 5.0 was much faster than that at pH 7.4. The release rate of mPEG(45)-PH(15)-PLLA(82) nanoparticles was much faster than that of mPEG(45)-PH(30)-PLLA(82) nanoparticles at pH 5.0. CONCLUSION: The inhibition effect of mPEG(45)-PH(15)-PLLA(82) nanoparticles on the growth of HepG2 cells was greater than that of mPEG(45)-PH(30)-PLLA(82) nanoparticles when the concentration of encapsulated doxorubicin was less than 15 µg/mL.

Stabilization of pH-sensitive mPEG-PH-PLA nanoparticles by stereocomplexation between enantiomeric polylactides.

Methoxy poly(ethylene glycol)-poly(L-histidine)-poly(lactide) (mPEG(45)-PH(30)-PLA(82)) triblock copolymers self-assemble into nanoparticles by sterocomplexation. The properties of the stereocomplex nanoparticles including morphology, stability, and biocompatibility are investigated. The results reveal that the stereocomplexation between PLLA and PDLA segments could prevent the aggregation of the nanoparticles when the pH value is around 6.8. The mean diameter of the stereocomplex nanoparticles is stabilized at about 100 nm when the pH values are changed from 7.9 to 5.0. The cytotoxicity of the stereocomplex nanoparticles is evaluated, and the results demonstrate that the stereocomplexation could decrease the cytotoxicity of the PDLA segments.