Preparation and Characterization of Cellulose Ether Liposomes for the Inhibition of Prion Formation in Prion-Infected Cells

Prion accumulation in the brain and lymphoreticular system causes fatal neurodegenerative diseases. Our previous study revealed that cellulose ethers (CE) have anti-prion activities in vivo and in prion-infected cells when administered at high doses. This study aims to improve the bioavailability of a representative CE using a liposomal formulation and characterized CE-loaded liposomes in cultured cells. The liposomal formulation reduced the EC₅₀ dose of CE by <1/200-fold in prion-infected cells. Compared to empty liposomes, CE-loaded liposomes were taken up much more highly by prion-infected cells and less by macrophage-like cells. Phosphatidylserine modification reduced the uptake of CE-loaded liposomes in prion-infected cells and did not change the anti-prion activity, whereas increased the uptake in macrophage-like cells. Polyethylene glycol modification reduced the uptake of CE-loaded liposomes in both types of cells and reduced the anti-prion activity in prion-infected cells. These results suggest that a liposomal formulation of CE is more practical than unformulated CE and showed that the CE-loaded liposome uptake levels in prion-infected cells were not associated with anti-prion activity. Although further improvement of the stealth function against phagocytic cells is needed, the liposomal formulation is useful to improve CE efficacy and elucidate the mechanism of CE action.     

Dual-Targeting Nanoparticles: Codelivery of Curcumin and 5-Fluorouracil for Synergistic Treatment of Hepatocarcinoma

Chemotherapy has been the standard for cancer therapy, but the nonspecific cytotoxicity of chemotherapeutic agents and drug resistance of tumor cells has limited its efficacy. However, multidrug combination therapy and targeting therapy have resulted in enhanced anticancer effects and have become increasingly important strategies in clinical applications. In this study, a biotin-/lactobionic acid–modified poly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly(ethylene glycol) (BLPP) copolymer was synthesized, and curcumin- and 5-fluorouracil-loaded nanoparticles (BLPPNPs/C + F) were prepared to enhance the treatment of hepatocellular carcinoma. Blank BLPPNPs were shown to have great biocompatibility via both in vitro and in vivo studies. Good targeting of tumor cells of BLPPNPs was confirmed by flow cytometry, fluorescence microscopy, and biodistribution. The synergistic anticancer effects of BLPPNPs/C + F were demonstrated by cytotoxicity and animal studies, while western blotting was used to further verify the synergistic effect of curcumin and 5-fluorouracil. The dual-targeting and drug-loaded codelivery nanosystem demonstrated higher cellular uptake and stronger cytotoxicity for tumor cells. Therefore, these dual-targeting NPs are a promising codelivery carrier that could be made available for cellular targeting of anticancer drugs to achieve better intracellular delivery and synergistic anticancer efficacy.     

包载多西他赛的聚合物胶束抑制小鼠乳腺癌转移作用研究

目的 考察包载多西他赛的聚合物胶束抑制小鼠乳腺癌转移效果。方法 采用薄膜分散法制备两种包载多西他赛（docetaxel,DTX）的聚合物胶束：普通胶束（DSPE-mPEG₂₀₀₀-Micelles,DM）和含聚乙二醇维生素E琥珀酸酯（D-α-tocopheryl polyethylene glycol 1000 succinate,TPGS₁₀₀₀）的聚合物胶束（TPGS₁₀₀₀/DSPE-mPEG₂₀₀₀-Micelles,TDM）。评价胶束包封率、载药量、粒径和zeta电位。采用尾静脉注射4T1/Luc细胞建立小鼠肺转移模型,评价胶束治疗后的肺部肿瘤生物发光强度和结节数量。结果 所制备的载多西他赛聚合物胶束包封率>85%,载药量约为3%,粒径约为20 nm,zeta电位约为 -4 mV,且TDM包封率和载药量更高,粒径更小。两种聚合物胶束均可降低乳腺癌肺转移模型小鼠肺部肿瘤生物发光强度、减少肺部结节数量,且TDM作用更强。结论 含TPGS₁₀₀₀的包载多西他赛的聚合物胶束TDM具有较好的抑制小鼠乳腺癌转移的作用。     

Specific VEGF sequestering to biomaterials: Influence of serum stability

Vascular endothelial growth factor (VEGF) was originally discovered as a tumor-derived factor that is able to induce endothelial cell behavior associated with angiogenesis. It has been implicated during wound healing for the induction of endothelial cell proliferation, tube formation and blood vessel remodeling. However, previous investigations into the biological effect of VEGF concluded that a particular range of growth factor concentrations are required for healthy vasculature to form, motivating recent studies to regulate VEGF activity via molecular sequestering to biomaterials. Numerous VEGF sequestering strategies have been developed, and they have typically relied on extracellular matrix mimicking moieties that are not specific for VEGF and can affect many growth factors simultaneously. We describe here a strategy for efficient, specific VEGF sequestering with poly(ethylene glycol) (PEG) microspheres, using peptides designed to mimic VEGF receptor type 2 (VEGFR2). By immobilizing two distinct peptides with different serum stabilities, we examined the effect of serum on the specific interaction between peptide-containing PEG microspheres and VEGF. We addressed the hypothesis that VEGF sequestering in serum-containing solutions would be influenced by the serum stability of the VEGF-binding peptide. We further hypothesized that soluble VEGF could be sequestered in serum-containing cell culture media, resulting in decreased VEGF-dependent proliferation of human umbilical vein endothelial cells. We show that soluble VEGF concentration can be effectively regulated in serum-containing environments via specific molecular sequestering, which suggests potential clinical applications.     

Covalent immobilization of stem cell factor and stromal derived factor 1α for in vitro culture of hematopoietic progenitor cells

Hematopoietic stem cells (HSCs) are currently utilized in the treatment of blood diseases, but widespread application of HSC therapeutics has been hindered by the limited availability of HSCs. With a better understanding of the HSC microenvironment and the ability to precisely recapitulate its components, we may be able to gain control of HSC behavior. In this work we developed a novel, biomimetic PEG hydrogel material as a substrate for this purpose and tested its potential with an anchorage-independent hematopoietic cell line, 32D clone 3 cells. We immobilized a fibronectin-derived adhesive peptide sequence, RGDS; a cytokine critical in HSC self-renewal, stem cell factor (SCF); and a chemokine important in HSC homing and lodging, stromal derived factor 1α (SDF1α), onto the surfaces of poly(ethylene glycol) (PEG) hydrogels. To evaluate the system’s capabilities, we observed the effects of the biomolecules on 32D cell adhesion and morphology. We demonstrated that the incorporation of RGDS onto the surfaces promotes 32D cell adhesion in a dose-dependent fashion. We also observed an additive response in adhesion on surfaces with RGDS in combination with either SCF or SDF1α. In addition, the average cell area increased and circularity decreased on gel surfaces containing immobilized SCF or SDF1α, indicating enhanced cell spreading. By recapitulating aspects of the HSC microenvironment using a PEG hydrogel scaffold, we have shown the ability to control the adhesion and spreading of the 32D cells and demonstrated the potential of the system for the culture of primary hematopoietic cell populations.     

Continuous gradient scaffolds for rapid screening of cell–material interactions and interfacial tissue regeneration

In tissue engineering, the physical and chemical properties of the scaffold mediates cell behavior, including regeneration. Thus a strategy that permits rapid screening of cell–scaffold interactions is critical. Herein, we have prepared eight “hybrid” hydrogel scaffolds in the form of continuous gradients such that a single scaffold contains spatially varied properties. These scaffolds are based on combining an inorganic macromer (methacrylated star polydimethylsiloxane, PDMS_star-MA) and organic macromer (poly(ethylene glycol)diacrylate, PEG-DA) as well as both aqueous and organic fabrication solvents. Having previously demonstrated its bioactivity and osteoinductivity, PDMS_star-MA is a particularly powerful component to incorporate into instructive gradient scaffolds based on PEG-DA. The following parameters were varied to produce the different gradients or gradual transitions in: (1) the wt.% ratio of PDMS_star-MA to PEG-DA macromers, (2) the total wt.% macromer concentration, (3) the number average molecular weight (M_n) of PEG-DA and (4) the M_n of PDMS_star-MA. Upon dividing each scaffold into four “zones” perpendicular to the gradient, we were able to demonstrate the spatial variation in morphology, bioactivity, swelling and modulus. Among these gradient scaffolds are those in which swelling and modulus are conveniently decoupled. In addition to rapid screening of cell–material interactions, these scaffolds are well suited for regeneration of interfacial tissues (e.g. osteochondral tissues) that transition from one tissue type to another.     

聚乙二醇去细胞瓣复合支架联合人主动脉瓣膜间质细胞体外构建组织工程心脏瓣膜

目的:运用聚乙二醇(PEG)交联猪去细胞瓣,共价接枝GRGDSPC多肽(甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸),并联合人主动脉瓣膜间质细胞(HAVICs)体外构建组织工程心脏瓣膜。方法:通过迈克尔加成反应,将去细胞瓣支架上引入的巯基与枝化状PEG末端丙烯酰基共价结合,使PEG交联去细胞瓣,并行生物力学检测。再利用PEG-去细胞瓣支架上未饱和的丙烯酰基与GRGDSPC多肽末端半胱氨酸的巯基发生迈克尔加成反应,对PEG-去细胞瓣支架行GRGDSPC多肽共价修饰,免疫荧光测定GRGDSPC多肽修饰效果。在RGD-PEG-去细胞瓣复合支架上种植HAVICs,缝制于生物反应器内,体外构建心脏瓣膜。接种2、4、8h后,通过细胞计数观察细胞粘附情况。体外培养10d后行形态学观察和DNA含量测定。结果:力学测试结果显示,PEG-去细胞瓣支架的最大抗张强度较单纯去细胞瓣支架明显提高[(7.53±0.32)Mpa∶(5.65±0.28)Mpa],差异有统计学意义(P<0.05),与天然主动脉瓣的差异无统计学意义(P>0.05)。免疫荧光检测显示,GRGDSPC多肽可有效地与PEG-去细胞瓣共价接枝。不同时段细胞粘附计数及形态学观察提示,RGD-PEG-去细胞瓣复合支架明显促进HAVICs的粘附,并可在瓣膜表面形成连续单细胞层。DNA含量测定提示,RGD-PEG-去细胞瓣复合支架DNA含量明显增高(P<0.05)。结论:PEG交联去细胞瓣可明显改善瓣膜支架力学性能,GRGDSPC多肽接枝的PEG-去细胞瓣复合支架,可促进种子细胞的粘附,改善瓣膜支架生物学性能。