Tumor Microenvironmental pH and Enzyme Dual Responsive Polymer-Liposomes for Synergistic Treatment of Cancer Immuno-Chemotherapy.

Despite recent advances in tumor treatment through cancer immunotherapy, the efficacy of this approach remains to be improved. Looking forward to high rates of objective clinical response, cancer immunotherapy combined with chemotherapy has gained increasing attention recently. Here, we constructed liposomes with matrix metalloproteinases (MMPs) responsive moiety and PD-L1 inhibitor conjugate combine with low dose chemotherapy to achieve enhanced antitumor efficacy. Upon introduction of the pH-responsive polymer to LPDp, the coassembly could be almost stable in physiological conditions and tumor microenvironments and release the loaded cargos at the lysosome. MMP-2 enzyme extracellularly secreted by the B16F10 cells could cleave the cross-linker and liberate the PD-L1 inhibitor effectively disrupting the PD-1/PD-L1 interaction in vitro. Low dose DOX encapsulated in the LPDp was capable of sensitizing B16F10 cells to CTLs by inducing overexpression of M6PR on tumor cell membranes. In comparison with free PD-L1 inhibitor, LPDp improved the biodistribution and on-demand release of the peptide inhibitor in tumor regions following administration. LPDp achieved the optimal tumor suppression efficiency (∼78.7%), which demonstrated the significantly enhanced antitumor effect ( P < 0.01) than that of LPp (∼57.5%) as well as that of LD (<40%), attributing to synergistic contribution from the substantial increase in M6PR expression on tumor cells and the blockade of immune checkpoints. This strategy provides a strong rationale for combining standard-of-care chemotherapy with relative nontoxic and high specific immunotherapy.

3-D culture of human umbilical vein endothelial cells with reversible thermosensitive hydroxybutyl chitosan hydrogel.

The aim of this study was to present a non-trypsin 3D cell culture method with a reversible thermosensitive HBCS hydrogel. In this study, hydroxybutyl chitosan (HBCS) was synthesized by grafting hydroxybutyl groups on chitosan molecule chains. The prepared HBCS was water-soluble, and the reversible phase transformation temperature was 26 °C. Scanning electron microscope images illuminated the 3-D network of hydrogel formed irregular porous structure which ranged from 50-250 µm. Cell viability assay indicated that HBCS solution could promote the proliferation of human umbilical vein endothelial cells (HUVECs), and the boost of proliferation was enhanced with the increase of HBCS concentration. HBCS had no harm to the nitric oxide (NO) synthesis functionality of HUVECs. HUVECs could grow and reproduce inside the hydrogel, and showed good vitality after 14-days culture. Meanwhile, cells cultured inside the hydrogel could be passaged successively through the reversible phase transformation process of HBCS. The results revealed that HBCS have the potential to be used for 3-D cell culture without the use of trypsin.

Self-assembled polymeric nanoparticles based on oleic acid-grafted chitosan oligosaccharide: biocompatibility, protein adsorption and cellular uptake.

Oleic acid-grafted chitosan oligosaccharide (OA-g-CSO) was synthesized to prepare self-assembled nanoparticles by sonication at physiological pH value (7.4). The nanoparticles appeared to be spherical in shape with a diameter of 158.1 ± 64.3 nm. The biocompatibility of OA-g-CSO nanoparticles was evaluated in vitro via MTT assay and hemolysis test. The nanoparticles showed no cytotoxicity to mouse embryo fibroblasts and the hemolysis rates came well within permissible limits (<2 %) in the tested conditions. When incubated with bovine calf serum, the protein adsorption on the surface of OA-g-CSO nanoparticles was concentration-dependent, and the amount of bovine serum albumin was in the highest proportion of the total amount of adsorbed proteins. Cellular uptake rate was evaluated by incubating fluorescence labeled OA-g-CSO nanoparticles with human lung carcinoma cells (A549). OA-g-CSO nanoparticles could be taken up by A549 cells, and the uptake rates increased with incubation time and particle concentration.

Preparation and evaluation of oleoyl-carboxymethy-chitosan (OCMCS) nanoparticles as oral protein carriers.

Oleoyl-carboxymethy chitosan (OCMCS) nanoparticles based on chitosan with different molecular weights (50, 170 and 820 kDa) were prepared by self-assembled method. The nanoparticles had spherical shape, positive surface charges and the mean diameters were 157.4, 274.1 and 396.7 nm, respectively. FITC-labeled OCMCS nanoparticles were internalized via the intestinal mucosa and observed in liver, spleen, intestine and heart following oral deliverance to carps (Cyprinus carpio). Extracellular products (ECPs) of Aeromonas hydrophila as microbial antigen was efficiently loaded to form OCMCS-ECPs nanoparticles and shown to be sustained release in PBS. Significantly higher (P < 0.05) antigen-specific antibodies were detected in serum after orally immunized with OCMCS-ECPs nanoparticles than that immunized with ECPs alone and non-immunized in control group in carps. These results implied that amphiphilic modified chitosan nanoparticles had great potential to be applied as carriers for the oral administration of protein drugs.

Investigation of polymeric amphiphilic nanoparticles as antitumor drug carriers.

In this paper, polymeric amphiphilic nanoparticles based on oleoyl-chitosan (OCH) with different degrees of substitution (DS, 5%, 11% and 27%) were prepared by Oil/Water emulsification method. Mean diameters of the nanoparticles were 327.4 nm, 255.3 nm and 192.6 nm, respectively. Doxorubicin (DOX) was efficiently loaded into OCH nanoparticles and provided a sustained released after a burst release in PBS. These nanoparticles showed no cytotoxicity to mouse embryo fibroblasts (MEF) and low hemolysis rates (<5%). The results of SDS-PAGE indicated that bovine calf serum (BCS) adsorption on OCH nanoparticles was inhibited by smaller particle size. Cellular uptake was evaluated by incubating fluorescence labeled OCH nanoparticles with human lung carcinoma cells (A549) and mouse macrophages (RAW264.7). Cellular uptake of OCH nanoparticles was time--and concentration--dependent. Finding the appropriate incubation time and concentration of OCH nanoparticles used as drug carriers might decrease phagocytic uptake, increase cancer cell uptake and ultimately improve therapeutic efficiency of antitumor therapeutic agents.

Biocompatibility study of theophylline/chitosan/beta-cyclodextrin microspheres as pulmonary delivery carriers.

To evaluate the biocompatibility of the theophylline/chitosan/beta-cyclodextrin microspheres, which has a potential application in pulmonary delivery system. The detection of LDH and protein in BALF was examined acute cell toxicity, hemolysis test was carried out to estimate blood toxicity; Micronucleus Test was reckoned to identify genotoxicity, MTT assay was used to evaluate in vitro cytotoxicity, and muscle implantation investigated the tissue biocompatibility. The results demonstrated that the total contents of protein and LDH in BALF were not significantly different from that of normal group. The experiments showed that the cytotoxicity was depended on the concentration and had no cytoxicity at low concentration and no hemolysis activity. The micronucleus frequency of MS B was 0.99 per thousand, which showed no genotoxic effects either. The results of implantation showed that the microspheres had no effect on hemoglobin and no toxicity in the liver and kidney. The inflammations of muscle tissue were not significantly different from that of operative suture, therefore, the MS B possess high good biocompatibility and can be applied in pulmonary sustained release systems.

Injectable thermosensitive hydrogel based on chitosan and quaternized chitosan and the biomedical properties.

A novel injectable thermosensitive hydrogel (CS-HTCC/alpha beta-GP) was successfully designed and prepared using chitosan (CS), quaternized chitosan (HTCC) and alpha,beta-glycerophosphate (alpha,beta-GP) without any additional chemical stimulus. The gelation point of CS-HTCC/alpha beta-GP can be set at a temperature close to normal body temperature or other temperature above 25 degrees C. The transition process can be controlled by adjusting the weight ratio of CS to HTCC, or different final concentration of alpha,beta-GP. The optimum formulation is (CS + HTCC) (2% w/v), CS/HTCC (5/1 w/w) and alpha,beta-GP 8.33% or 9.09% (w/v), where the sol-gel transition time was 3 min at 37 degrees C. The drug released over 3 h from the CS-HTCC/alpha,beta-GP thermosensitive hydrogel in artificial saliva pH 6.8. In addition, CS-HTCC/alpha,beta-GP thermosensitive hydrogel exhibited stronger antibacterial activity towards two periodontal pathogens (Porphyromonas gingivalis, P.g and Prevotella intermedia, P.i). CS-HTCC/alpha, beta-GP thermosensitive hydrogel was a considerable candidate as a local drug delivery system for periodontal treatment.

Mussel-inspired antibacterial polydopamine/chitosan/temperature-responsive hydrogels for rapid hemostasis.

The aim of this study was to develop an effective wound dressing using a temperature-responsive hydroxybutyl chitosan (HBC) based hydrogel. The HBC - chitosan (CS) - dopamine (HCS-DOPA) composite hydrogels were prepared by the dopamine self-polymerization at different concentrations (0, 0.5, 1.0 and 2.0 mg/mL), termed as HCS, HCS-DOPA-0.5, HCS-DOPA-1 and HCS-DOPA-2, respectively. The gelling characteristic of HBC hydrogel was not influenced by composite CS and DOPA. The HCS-DOPA composite hydrogels were non-cytotoxic to mouse fibroblast cells (L929), and induced under 5.0% hemolysis rate. In vitro antibacterial studies, composite HCS-DOPA-2 hydrogels exhibited lasting inhibition to S. aureus >8 h. The whole blood test in vitro demonstrated that blood clotting time treated with HCS-DOPA-2 composite hydrogels was shortened to 95.6 s compared with that of HCS in vitro hemostasis. The results suggested that HCS-DOPA-2 composite hydrogels could be applied as a promising wound dressing for hemostasis in vitro.

Inducing sustained release and improving oral bioavailability of curcumin via chitosan derivatives-coated liposomes.

Liposomes (LPs), a delivery vehicle for stabilizing drugs, the characteristics of being easy to aggregate and fuse limit its application. Polymer coating is a promising way to tackle these issues. In this study, the potential of carboxymethyl chitosan (CMCS) and quaternary ammonium chitosan (TMC)-coated liposomes (CMCS/TMC-LPs) for improving the oral delivery capacity of curcumin (CUR) was explored. CMCS/TMC-LPs were prepared by electrostatic adsorption in a layer-by-layer manner. CMCS/TMC-LPs were spherical and had not obvious change in particle size and morphology after storage at 4 °C for 7 and 14 days. CMCS/TMC-LPs possessed favorable gastric acid tolerance (the cumulative drug release rate <10%) due to stable structure. The hemolysis test and Cell Counting Kit-8 (CCK8) assay appeared satisfactory biocompatibility of CMCS/TMC-LPs. The pharmacokinetics exhibited that oral absolute bioavailability of CUR loaded CMCS/TMC-LPs was about 38%, which was around 6 folds and 3 folds higher than CUR loaded LPs and CUR loaded TMC-LPs, respectively. The in vivo experiments showed that CMCS/TMC-LPs could prolong the retention time of CUR in systemic circulation and generate high level of CUR in liver, spleen and lung. Thus, CMCS/TMC-LPs may be a promising carrier for improving the efficacy and safety of orally administered drugs.

The temperature-responsive hydroxybutyl chitosan hydrogels with polydopamine coating for cell sheet transplantation.

The aim of this study was to develop an effective cell sheet translocation method using a cell adhesive and temperature-responsive hydroxybutyl chitosan hydrogel (HBC). The polydopamine (PD)-coated HBC hydrogels were prepared by the dopamine self-polymerization on the surface of HBC hydrogel with different coating time, termed as P30, P60 and P120, respectively. Gelling property of HBC was not affected by PD coating. The PD-coated HBC hydrogels promoted the attachment and proliferation of mouse fibroblast cells (L929) and human umbilical vein endothelial cells (HUVECs), and allowed formation of monolayer cell sheet. In vitro translocation of HUVECs sheet could be obtained successively through phase transition of PD coated HBC hydrogel from gel to sol, and the cells sheet transferred from P30 hydrogel to a round cell coverglass maintained relatively complete monolayer and normal cell morphology. The results showed that P30 hydrogel has the potential to be used for cell transplantation therapy.

In vitro and in vivo evaluation of mucoadhensiveness of chitosan/cellulose acetate multimicrospheres.

Chitosan/cellulose acetate multimicrospheres (CCAM) with or without ranitidine (RT) were prepared by the method of W/O/W emulsion with no toxic reagents and had the size interval of 200-280 microm. The angles of repose were only a little more than 30 degrees and the maximum angles of one-plane-critical-stability (OPCS phi) were about 20 degrees . The CCAM had good suspension ability for the tapped density of CCAM was less than 0.127g/mL. The pH value affected the swelling ability of CCAM and the relative humidity had little effect on the characteristics of CCAM when it was not more than 75%. The CCAM system had good effect on the controlled release of RT in vitro and the release rate was almost 60% during 48 h. Furthermore the release of RT was not affected by pH value of release medium. The mucoadhesive tests showed that CCAM could retain in gastrointestinal tract for an extended period of time. There were 53.7% of CCAM remained in stomach after administered for 2(1/2) h and 98.9% of CCAM remained in stomach and small intestine after administered for 3(1/2) h. These results suggest that CCAM is a useful dosage form targeting the gastric mucosa or prolonging gastric residence time as a multiple-unit mucoadhesive system.

Cellulose acetate/chitosan multimicrospheres preparation and ranitidine hydrochloride release in vitro.

A noval cellulose acetate/chitosan multimicrospheres (CACM) was prepared by the method of w/o/w emulsion. The concentration of cellulose acetate (CA) and the ratio of CA/chitosan (CS) had influence on the CACM size, and appearance. Ranitidine hydrochloride loading, and releasing efficiency in vitro were investigated. The optimal condition for preparation of the microspheres was CA concentration at 2% and the ratio of CA/CS at 3/1. The microspheres size was 200-350 microm. The appearance of microspheres was spherical, porous, and nonaggregated. The highest loading efficiency was 21%. The ranitidine release from the CACM was 40% during 48 hr in buffers.

The effect of carboxymethyl-chitosan on proliferation and collagen secretion of normal and keloid skin fibroblasts.

In this study, different molecular weight CM-chitosans were prepared and the effects on the growth and collagen secretion of normal skin fibroblasts and keloid fibroblasts were investigated in vitro. CM-chitosan promoted the proliferation of the normal skin fibroblast significantly but inhibited the proliferation of keloid fibroblast. The higher CM-chitosan concentration had a higher initial effect and the lower CM-chitosan concentration had a longer affecting time to the normal skin fibroblast. The lower molecular weight CM-chitosan had significant twofold activities. The CM-chitosan could reduce the ratio of type I/III collagen in keloid fibroblast by inhibiting the secretion of collagen type I; and had no effect on the secretion of types I and III collagen in the normal skin fibroblast.

Molecular affinity and permeability of different molecular weight chitosan membranes.

Membranes were prepared from chitosan with different molecular weights by a casting method. The molecular affinity and permeability of the membranes for sodium chloride, glucose, tyrosine, and bovine serum protein were measured at 4 degrees C and pH 7. The molecular permeability of the chitosan membranes was inversely related to molecular weight. All prepared membranes showed a molecular affinity to bovine serum protein. The higher the molecular weight of chitosan membrane, the higher the affinity and the lower permeability of the membrane to bovine serum protein.

Dynamic disordering of liposomal cocktails and the spatio-temporal favorable release of cargoes to circumvent drug resistance.

Multidrug resistance (MDR) has been a major impediment to the success of cancer chemotherapy. Extensive efforts have been devoted to the development of drug delivery systems using nanotechnology to reverse MDR in cancer. However, the spontaneous release of drug payloads was always a slow process, which leads to the low intracellular drug concentration resulting in consequent drug insensitivity. To circumvent this limitation, we described a liposomal cocktail (LMDHV) constructed by a pH-responsive molecule (i.e., malachite green carbinol base (MG)) and liposome conjugated with Her-2 antibody for codelivery of doxorubicin (DOX) and verapamil (VER) to suppress drug resistance in Her-2 positive breast cancer. MG inserted in the bilayer as pH responders greatly contributed to the destabilization of the vesicle membrane in low pH, followed by the rapid release of the payloads. LMDHV showed 6-fold reversal efficiency in DOX resistant breast cancer owing to the efficient tumor targeting delivery and rapid burst release of drug intracellularly. Compared to tumor inhibition ratio of treated groups by free DOX (32.4 ± 7.4%), our designed kinetically favorable drug release system exhibited significantly (P < 0.01) enhanced tumor inhibition ratio up to 83.9 ± 12.5%, which is attributed to the remarkably increased drug concentration in cells. The spatio-temporal favorable release of drugs resulted in synergistic inhibition of tumor growth in xenografts. We envision that this new type of liposomal cocktail might be potentially utilized to circumvent drug resistance in the future.

Molecular structural transformation regulated dynamic disordering of supramolecular vesicles as pH-responsive drug release systems.

The spontaneous release of drug payloads in the whole body always results in the compromised drug bioavailability and ultimate therapeutic efficacy. To achieve enhanced therapeutic efficacy and reduced side effects, pH-responsive targeted drug delivery systems have been studied due to their enhanced tumor accumulation and controllable maximum drug release feature. The present study described a co-assembly constructed by a pH responsive molecule (i.e., malachite green carbinol base (MG)) and liposome for highly efficient doxorubicin (DOX) release in tumor cells (MG-DOX⊂L). The structural transformation of MG effectively regulates the drug release profile in acidic environment. The pH-responsive sensitivity of co-assembly can be fine-tuned by altering the mixing ratios of building blocks with pH responders (i.e., MG molecules). MG-DOX⊂L was beneficial for the DOX release at pH5.0 and showed a higher cytotoxicity in KB cells owing to the pH-responsive drug release in acidic organelles following endocytosis pathway. In vivo tumor targetability and growth inhibition were evaluated in KB cell-xenografted nude mice. We have demonstrated that effective tumor growth inhibition in vivo is attributed to the synergistic contributions from highly efficient cellular entry and responsive intracellular release of DOX from MG-DOX⊂L.

Preparation and hydrolytic erosion of differently structured PLGA nanoparticles with chitosan modification.

Two types of poly-lactic-co-glycolic acid (PLGA) based nanoparticles were prepared by surface modification (C-NPs) and homogeneous modification (G-NPs) with chitosan. The naked PLGA NPs were served as control. These nanoparticles all showed regularly spherical shape with mean diameters as 191.3 ± 3.6 nm, 211.9 ± 13.2 nm and 187.5 ± 17.6 nm for PLGA NPs, C-NPs and G-NPs, respectively. Their zeta potentials were -22.4 ± 1.31 mV, -8.7 ± 0.45 mV, -3.1 ± 0.12 mV, respectively. Both C-NPs (15.3% PLGA remained after 2 weeks) and G-NPs (3.7% PLGA remained after 2 weeks) had higher hydrolysis rate than PLGA NPs (18.4% PLGA remained after 6 weeks), with G-NPs showing the highest rate in hydrolysis due to the incorporation of chitosan and its self-assembled structure. Self-assembling properties and controllable biodegradability of G-NPs indicated that it could be a promising drug delivery carrier for tumor drug delivery.

Biocompatibility, cellular uptake and biodistribution of the polymeric amphiphilic nanoparticles as oral drug carriers.

Oleoyl-carboxymethyl-chitosan (OCMCS) was synthesized and were soluble at neutral pH. The critical micelle concentration (CMC) of OCMCS in deionized water was 0.021 mg/ml. OCMCS nanoparticles were successfully prepared via self-assembly with mean diameter of 215.34 nm, zeta potential of 19.26 mV and an almost spherical shape as determined by electron microscopy. The OCMCS nanoparticles showed low erythrocyte membrane-damaging effect. The MTT survival assay indicated no significant cytotoxicity to Caco-2 cells and MEFs cells. The uptake of FITC labeled OCMCS nanoparticles by Caco-2 cells was confirmed via confocal laser scanning microscope (CLSM). In vivo toxicity assays were performed via histopathological evaluation, and no specific anatomical pathological changes or tissue damage was observed in the tissues of carps. The extent of tissue distribution and retention following oral administration of FITC-OCMCS nanoparticles was analyzed for 3 days. After 3 days, the nanoparticles remained detectable in the muscle, heart, kidney, liver, intestine, and spleen. The results showed that 34.32% of the particles were localized in the liver, 18.79% in the kidney, and 17.36% in the heart. The lowest percentage was observed in the muscle. These results implied that OCMCS nanoparticles had great potential to be applied as safe carriers for the oral administration of protein drugs.

Preparation of biocompatible chitosan grafted poly(lactic acid) nanoparticles.

Chitosan grafted poly(lactic acid) (CS-g-PLA) copolymer was synthesized and characterized by FT-IR and elemental analysis. The degree of poly(lactic acid) substitution on chitosan was 1.90 ± 0.04%. The critical aggregation concentration of CS-g-PLA in distilled water was 0.17 mg/ml. Three methods of preparing CS-g-PLA nanoparticles (diafiltration method, ultrasonication method and diafiltration combined with ultrasonication method) were investigated and their effect was compared. Of the three methods, diafiltration combined with ultrasonication method produced nanoparticles with optimal property in terms of size and morphology, with size ranging from 133 to 352 nm and zeta potential from 36 to 43 mV. Also, the hemolytic activity and cytotoxicity of the CS-g-PLA based nanoparticles was tested, and results showed low hemolysis rate (<5%) and no significant cytotoxicity effect of these nanoparticles.

A facile method for preparing biodegradable chitosan derivatives with low grafting degree of poly(lactic acid).

Chemical modification of chitosan by grafting with PLA (CS-g-PLA) was developed via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) mediated coupling reaction. The introduction of PLA disrupted the crystalline structure of chitosan, improved its solubility and thermal stability. Low degree of PLA substitution showed better degradation efficiency than chitosan and PLA. Weight loss of CS-g-PLA6 and CS-g-PLA4 was 87% and 94%, respectively, in 7 days enzymatic degradation study. CS-g-PLA2 was totally degraded in 1 day. Self-assembly behavior was studied using pyrene fluorescence dye technique and found to be PLA grafting level dependent. CS-g-PLA with low grafting degree showed hydrophilic, self-assembling properties and controllable biodegradability that may widen its applications.