Yongming Bao

Esterification degree of fructose laurate exerted by Candida antarctica lipase B in organic solvents

Sugar esters of fatty acids have many applications as biocompatible and biodegradable emulsifiers, which are determined by their degrees of esterification (DE). Direct esterification of fructose with lauric acid in organic media used commercial immobilized Candida antarctica lipase B (CALB) was investigated for DE. Significant difference of DE was observed between 2-methyl-2-butanol (2M2B) and methyl ethyl ketone (MEK), as di-ester/mono-ester molar ratio of 1.05:1 in 2M2B and 2.79:1 in MEK. Fourier transform infrared (FTIR) spectra showed that the secondary structure of the enzyme binding mono-ester presented distinct difference in 2M2B and MEK. Contents of β-turn and antiparallel β-sheet of CALB in 2M2B were 26.9% and 16.2%, respectively, but 19.1% and 13.2% in MEK. To understand the relationship between the conformational changes and differences of DE, mono-ester and fatty acid were directly employed for synthesis of di-ester. The maximum initial velocity of di-ester synthesis in MEK was 0.59 mmolg(enzyme)(-1)h(-1), which was 2.19-fold as greater as that in 2M2B, indicating that CALB conformation in MEK was preferred for the synthesis of di-ester. These results demonstrated that the conformation of CALB binding mono-ester affected by organic solvents essentially determined DE.

New bifunctional-pullulan-based micelles with good biocompatibility for efficient co-delivery of cancer-suppressing p53 gene and doxorubicin to cancer cells

Combined treatment of drugs and therapeutic genes has emerged as a new modality of anticancer therapy. In this study, a new amphiphilic bifunctional pullulan derivative (named as PSP) containing stearic acid and low-molecular weight (1 kDa) branched polyethylenimine was prepared and evaluated as a nano-carrier for the co-delivery of a drug and gene for potential cancer therapy. The amphiphilic PSP could self-assemble into cationic core–shell nano-micelles in water, with a critical micelle concentration of around 58.9 mg L−1. PSP nanomicelles had an average size of 188.75 ± 3.18 nm, and a positive zeta potential of 17.83 ± 0.75 mV. The drug loading content and encapsulation efficiency of the PSP nanomicelles for doxorubicin (DOX), an anti-tumor drug, were about 5.10% and 56.07%, respectively, and DOX in PSP nanomicelles showed sustained release. The flow cytometry and confocal laser scanning microscopy showed that PSP/DOX nanomicelles could be successfully internalized by MCF-7 cells. The in vitro IC50 of PSP/DOX nanomicelles was slightly lower than that of free DOX against MCF-7 cells. Additionally, PSP nanomicelles condensed DNA efficiently to form compact structures, and induced comparable GFP gene expression level to Lipo2000 at N/P = 10 in gene transfection studies. In comparison with single DOX or p53 delivery, the co-delivery of DOX and therapy gene p53 using PSP micelles displayed higher cytotoxicity and induced a higher apoptosis rate of tumor cells in vitro. Moreover, PSP exhibited good blood compatibility and low cytotoxicity in the hemolysis and MTT assays, respectively. Altogether, PSP nanomicelles have a great potential in delivering hydrophobic anticancer drugs and therapeutic genes simultaneously for improved cancer therapy.

Biocompatible cationic pullulan-g-desoxycholic acid-g-PEI micelles used to co-deliver drug and gene for cancer therapy

The greatest crux in the combination of chemotherapy and gene therapy is the construction of a feasible and biocompatible carrier for loading the therapeutic drug and gene simultaneously. Here, a new amphiphilic bifunctional pullulan derivative (named as PDP) synthesized by grafting lipophilic desoxycholic acid and low-molecular weight (1kDa) branched polyethylenimine onto the backbone of pullulan was evaluated as a nano-carrier for the co-delivery of drug and gene for potential cancer therapy. PDP exhibited good blood compatibility and low cytotoxicity in the hemolysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. By self-assembly process, the amphiphilic PDP polymer formed cationic core-shell nanomicelles in aqueous solution with an average diameter of 160.8nm and a zeta potential of approximate 28mV. The PDP micelles had relative higher drug encapsulation efficiency (84.05%) and loading capacity (7.64%) for doxorubicin (DOX), an effective anti-tumor drug, demonstrating sustained drug release profile and good DNA-binding ability. The flow cytometry and confocal laser scanning microscopy showed that PDP/DOX micelles could be successfully internalized by MCF-7 cells, and presenting higher cytotoxicity against MCF-7 cells than that of free DOX. Furthermore, PDP micelles could efficiently transport tumor suppressor gene p53 into MCF-7 cells, and the expressed exogenous p53 protein induced MCF-7 cells to die. More excitedly, in comparison with single DOX or p53 delivery, the co-delivery of DOX and gene p53 using PDP micelles displayed higher cytotoxicity, induced higher apoptosis rate of tumor cells and blocked more effectively the migration of cancer cells in vitro. In tumor-bearing mice, co-delivery of DOX and p53 also exhibited enhanced antitumor efficacy as compared with single delivery of DOX or p53 alone. In summary, these results demonstrated that it is highly promising to use cationic PDP micelles for effectively co-delivering functional gene and chemotherapeutic agent, and thus improving antitumor efficacy and systemic toxicity.

Efficient mono-acylation of fructose by lipase-catalyzed esterification in ionic liquid co-solvents

Fructose monoesters are eco-friendly nonionic surfactants in various applications. Selective preparation of mono-acylated fructose is challenging due to the multiple hydroxyl sites available for acylation both chemically and enzymatically. Ionic liquids (ILs) have profound impacts not only on the reaction media but also on the catalytic properties of enzymes in the acylation process. In this study, utilizing an IL co-solvent system, selective synthesis of mono-acylated fructose with lauric acid catalyzed by immobilized Candida antarctica lipase B (CALB) was investigated. The imidazolium-based ILs selected as co-solvents with 2-methyl-2-butanol (2M2B) markedly improved the ratios of monolauroyl fructose in the presence of 60% [BMIM][TfO] (v/v) and 20% [BMIM][BF4] (v/v), in which the mono-acylated fructose was 85% and 78% respectively. Based on a Ping-Pong Bi-Bi model, a kinetic equation was fitted, by which the kinetic parameters revealed that the affinity between fructose and acyl-enzyme intermediate was enhanced. The inhibition effect of fructose on free enzyme was weakened in the presence of IL co-solvents. The conformation of CALB binding substrates also changed in the co-solvent system as demonstrated by Fourier transform infrared spectra. These results demonstrated that the variation of CALB kinetic characteristics was a crucial factor for the selectivity of mono-acylation in ILs/2M2B co-solvents.

PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro

Abstract Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical application. The PEGylation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEGylated hirudin were investigated. When the PEGylation reaction was conducted under 4°C after 10h, in the borate buffer at pH 8.5, with the molar ratio 250:1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactrvity of PEGylated hirudin was measured in vitro. Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.

Quercetin exerts synergetic anti-cancer activity with 10-hydroxy camptothecin

&NA; Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti‐cancer has not been fully investigated. Here, we show that Qu at low doses (≤10 &mgr;M) significantly enhances the inhibition of 10‐hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10 &mgr;M. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser139 of &ggr;H2A.X in MCF7 cells starts to increase significantly (P < 0.05) at 6 h when treated by the combination of 10 &mgr;M Qu and 0.62 &mgr;M HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12 h to 48 h via up‐regulation of p21 and induces apoptosis at 24 h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100 &mgr;M Z‐VAD‐FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single‐drug group. In summary, the synergic anti‐cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage. Graphical abstract Figure. No caption available.

Combination of baicalein and 10-hydroxy camptothecin exerts remarkable synergetic anti-cancer effects.

BACKGROUND 10-Hydroxy camptothecin (HCPT), a naturally occurring alkaloid, is a clinical drug for cancer chemotherapy. Baicalein (BA) is a flavonoid extracted from the root of Scutellaria baicalensis. The synergistic anti-cancer effect of BA and HCPT has not been reported. PURPOSE To explore whether and how BA enhances the anti-cancer effect of HCPT in BGC823 cells. METHODS Cell viability was measured by MTT assay. Apoptosis and cell cycle were analyzed through flow cytometry and western blotting analysis. DNA damage was determined by a comet assay. The activity of topoisomerase I (Topo I) was detected by the plasmid DNA relaxation assay. The synergistic anti-cancer effect of BA and HCPT in vivo was tested by BGC823 xenografted tumor model. RESULTS BA at non-toxic doses prominently enhanced the anti-cancer activities of HCPT in BGC823, MCF7 and SMMC7721 cells. Combination treatment of BA and HCPT induced BGC823 cells apoptosis mainly via intrinsic rather than extrinsic pathways, and preferentially arresting cell cycle in G1 and G2 phases with the aid of p21. Of note, p53, the upstream regulator of cell apoptosis and cycle, was increased by 5 folds in combination group. It helped to further trigger DNA damage and inhibit Topo I catalytic activity after combination treatment of BA and HCPT. Moreover, the BGC823 xenografted tumor growth rate in nude mice was repressed in a greater degree (P< 0.01) in the combinational group than the single-drug group. CONCLUSION HCPT and BA, a new and effective combination therapy, synergistically target Topo I and up-regulate p53 to induce cell apoptosis and cell cycle arrest.

Co-delivery of doxorubicin and shRNA of Beclin1 by folate receptor targeted pullulan-based multifunctional nanomicelles for combinational cancer therapy

Doxorubicin (DOX) is a widely-used effective antitumor agent. However, its clinical application is limited due to its side effects including anti-apoptotic defense of cancer cells caused by DOX-induced autophagy and deleterious effects in normal tissues. Therefore, in this study, a new folate (FA)-decorated amphiphilic bifunctional pullulan-based copolymer (named as FPDP) was developed as an efficient nano-carrier for the co-delivery of DOX and short hairpin RNA of Beclin1, a pivotal autophage-related gene, to enhance the anticancer effect of DOX by the blockade of the Beclin1 protein mediated autophagy process. In FPDP molecules, pullulan was modified with lipophilic desoxycholic acid for the formation of micelles, the introduced low molecular weight (1 kDa) branched polyethylenimine (PEI) was for shBeclin1 delivery, and folate (FA) was employed as the tumor-targeting group. FPDP micelles demonstrated an average diameter of 161.9 nm, good biocompatibility, applicable storage stability, excellent loading capacities for both DOX and shBeclin1 and a sustained drug release profile. In vitro cell culture experiments demonstrated that the uptake amount of FPDP/DOX micelles in folate receptor positive (FR+) HeLa cells was more than that in folate receptor negative (FR−) HepG2 cells, leading to significantly higher cytotoxicity against FR+ HeLa cells. The simultaneous co-delivery of shBeclin1 and DOX to HeLa cells with FPDP micelles led to efficient reduction in the expression level of Beclin1 as well as synergistic cell apoptotic induction. Furthermore, in vivo studies revealed superior antitumor efficacy of tumor-targeted FPDP/DOX/shBeclin1 in comparison with non-FR-targeted PDP micelles and free DOX. These results highlighted that co-delivery of DOX and shRNA of Beclin1 with FPDP micelles has the potential to overcome the limitations of DOX in clinical cancer therapy.

Specific light-up pullulan-based nanoparticles with reduction-triggered emission and activatable photoactivity for the imaging and photodynamic killing of cancer cells

Activatable photosensitizers that can be activated by cancer-associated stimuli have drawn increasing attention for simultaneous fluorescence imaging and photodynamic ablation of cancer cells. Here, we developed a cancer-cell specific photosensitizer nano-delivery system by synthesizing protoporphyrin IX (PpIX)-conjugated pullulan (P) with reducible disulfide bonds. The amphiphilic P-s-s-PpIX conjugate self-assembled in aqueous condition to form core-shell structured nanoparticles (P-s-s-PpIX NPs) with average size of 166nm, showing reduction-controllable stability. In in vitro, the photoactivity of P-s-s-PpIX NPs in an aqueous environment was significantly suppressed by the self-quenching effect, which kept P-s-s-PpIX NPs in a photo-inactive and quenched state. But in the presence of GSH, P-s-s-PpIX NPs quickly dissociated by reductive breakage of disulfide linkers, followed by the significant recovery of fluorescent emission and singlet oxygen generation. In MCF-7 cells, compared to non-reducible P-PpIX NPs with stable amide linkages, P-s-s-PpIX NPs displayed higher cytotoxicity and induced higher apoptosis rate of tumor cells with light irradiation treatment. As a result, the P-s-s-PpIX NPs may serve as an effective smart nanomedicine platform for specific light-up and reduction-triggered cancer imaging and photodynamic therapy with the prominently reduced damage to normal tissues and cells.

LotS/LotR/Clp, a novel signal pathway responding to temperature, modulating protease expression via c-di-GMP mediated manner in Stenotrophomonas maltophilia FF11

Stenotrophomonas maltophilia as one of increasing food spoilage bacteria and fish pathogens has become a threat to aquiculture industry. A major factor contributing to the success of bacterium is its outstanding ability to secrete protease at low temperatures. Here, a cAMP receptor like protein (Clp) shows a positive regulation on this protease, named S. maltophilia temperature-response protease (SmtP). Interestingly, a two-component system, comprising of LotS sensor and LotR regulator, for low-temperature response is also confirmed to modulate SmtP expression with similar effect to Clp. Evidence is presented that LotS/LotR modulates smtP (coding SmtP) expression via Clp: clp promoter activity was reduced significantly at low temperatures and protease activity was partially restored by Clp overexpressed in lotS or lotR deletion strain. Furthermore, as a Clp negative effector, the binding ability of c-di-GMP with Clp is not impacted by temperature. c-di-GMP level was increased in S. maltophilia growing at high temperature, but not exhibited significantly in lotR deleted strain, these indicate that LotR is required for temperature modulating c-di-GMP level, although the synthesis or degradation activity of c-di-GMP by LotR was not detected. These findings suggest that LotS/LotR/Clp play an important role in responding to temperature stimuli via c-di-GMP mediated manner.