In vivo culture of encapsulated endostatin-secreting Chinese hamster ovary cells for systemic tumor inhibition

Microencapsulation of recombinant cells is a novel alternative approach to tumor gene therapy. Therapeutic protein delivery can be sustained for systemic treatment of tumors because the recombinant cells are enclosed in microcapsules and the semipermeable membrane of the microcapsules protects the cells from host immune rejection and reduces the need for frequent injection. In this study, we describe a method to systemically inhibit tumor growth by in vivo culture of antiangiogenic endostatin-secreting Chinese hamster ovary (CHO) cells in microcapsules as small as 200 microm in diameter. Peritoneal administration of encapsulated endostatin-CHO cells inhibited melanoma growth to 66.4% and enhanced the survival of treated mice to 80% by 27 days posttreatment. Continuous systemic release of endostatin from microcapsules offers an effective therapeutic strategy to eradicate solid tumors.     

Tumor Anti-angiogenic Gene Therapy with Microencapsulated Recombinant CHO Cells

Microencapsulation of recombinant cells is a novel promising approach to tumor therapy in which therapeutic protein is sustainable and long-term delivered by microencapsulated cells. The semi-permeable membrane of microcapsule can protect cell from host’s immune rejection, increase the chemical stability of therapeutic protein and circumvent the problems of toxicity, limited half-lives and variation in circulating levels. Endostatin, a potent and specific angiogenesis inhibitor, could suppress the growth of primary and metastatic lesions in multiple murine tumor models. In this paper, APA microcapsules with high strength kept intact over 35 days and recombinant CHO cells kept the rapid proliferation viability and the continuous endostatin-expression function. The study of tumor treatment showed that the implantation of microencapsulated recombinant CHO cells decreased the neovascularization of tumor tissue by 59.4% and inhibited the B16 melanoma growth by 77.4%. Twenty days after tumor cell injection, 80% of animals treated with microencapsulated CHO-endo cells were alive compared to only 50% of animals in either control or mock control groups. Therefore, continuous delivery of endostatin from microencapsulated recombinant cells represents a feasible approach to tumor therapy.     

A novel 3-D model for cell culture and tissue engineering

A novel method of making microcapsules in a macrocapsule is demonstrated as a 3-D culture system in this article. Mouse embryonic stem (mES) cells as model cells were used in the 3-D culture space, and the cell viability and histological observation were conducted. Furthermore, Oct4 gene expression was evaluated for the undifferentiated status of mES cells in this 3-D model. The results showed that mES cells can grow in this 3-D model and retain their normal viability and morphology. This 3-D model allows mES cells to stay in the undifferentiated state better than 2-D culture systems. This work demonstrates a new 3-D tissue model which can provide an in vivo like microenvironment for non-differentiated mES cells with good immunoisolation. This approach may bridge the gap between traditional 2-D cell culture and animal models.     

Transferrin Receptor Targeted Lipopolyplexes for Delivery of Antisense Oligonucleotide G3139 in a Murine K562 Xenograft Model

Purpose  Transferrin (Tf) conjugated lipopolyplexes (LPs) carrying G3139, an antisense oligonucleotide for Bcl-2, were synthesized and evaluated in Tf receptor positive K562 erythroleukemia cells and then in a murine K562 xenograft model. Materials and Methods  Particle size and Zeta potentials of transferrin conjugated lipopolyplexs containing G3139 (Tf-LP-G3139) were measured by Dynamic Light Scattering and ZetaPALS. In vitro and in vivo sample’s Bcl-2 downregulation was analyzed using Western blot and tumor tissue samples also exhibited by immunohistochemistry method. For athymic mice bearing with K562 xenograft tumors, tumor growth inhibition and survival rate were investigated. Nanoparticle distribution in 3-D cell cluster was observed by Laser scan confocal microscopy. IL-12 production in the plasma was measured by ELISA kit. Results   In vitro, Tf-LP-G3139 was more effective in inducing down regulation of Bcl-2 in K562 cells than non-targeted LP-G3139, free G3139 and mismatched control ODN-G4126 in the same formulation. In vivo Tf-LP-G3139 was less effective than free G3139 in Bcl-2 down regulation. 3-D cell cluster model diffusion results indeed indicated limited penetration of the LPs into the cell cluster. Finally, the therapeutic efficacies of Tf-LP-G3139 and free G3139 were determined in the K562 xenograft model. Tf-LP-G3139 showed slower plasma clearance, higher AUC, and greater accumulation in the tumor compared to free G3139. In addition, Tf-LP-G3139 was found to be more effective in tumor growth inhibition and prolonging mouse survival than free G3139. This was associated with increased spleen weight and IL-12 production in the plasma. Conclusion  The role of the immune system in the therapeutic response obtained with the Tf-LPs is necessary and in vitro 3-D cell cluster model can be a potential tool to evaluate the nanoparticle distribution.     

低温挤出滚圆法制备银耳多糖微粒

目的：考察并优化低温挤出滚圆法制备银耳多糖微粒（TPP）的工艺条件。方法通过 L9（34）正交设计实验优化制剂工艺条件,制备直径小于190μm 微粒。结果载药量为65℅,稀释剂为65℅乙醇溶液, MCC 为主要辅料,挤出速度为50 r/ min,滚圆速度为1500 r/ min,滚圆时间为10 min,制备的微粒其平均粒径在115~190μm,圆整度、堆密度、总收率均较理想。结论应用挤出滚圆法制备 TPP,其工艺简便,稳定可行,适合小鼠药物药理学和毒理学研究的需要和大工业生产。     

A biodegradable, immunoprotective, dual nanoporous capsule for cell-based therapies

To demonstrate the transplantation of drug-secreting cells with immunoprotection, a biodegradable delivery device combining two nanoporous capsules is developed using secretory alkaline phosphatase gene (SEAP) transfected mouse embryonic stem (mES) cells as a model system. The outer capsule is a poly (ethylene glycol) (PEG)-coated poly (varepsilon-caprolactone) (PCL) chamber covered with a PEG grafted PCL nanoporous membrane made by phase inversion technique. SEAP gene transfected mES cells encapsulated in alginate-poly-l-lysine (AP) microcapsules are placed in the PCL capsule. Both nanoporous capsules showed good immunoprotection in the IgG solution. In microcapsules, mES cells could form a spheroid embryonic body (EB) and grow close to the microcapsule size. The secreted SEAP from encapsulated mES cells increased gradually to a maximum value before reaching a steady level, following the cell growth pattern in the microcapsule. Without microcapsules, mES cells only formed a monolayer in the large PCL capsule. The secreted SEAP release was very low. The integrated device showed a similar cell growth pattern to that in microcapsules alone, while the SEAP release rate could be regulated by the pore size of the large capsule. This integrated device can achieve multi-functionalities for cell-based therapy, i.e. a 3-D microenvironment provided by microcapsules for cell growth, superior immunoprotection and controllable release performance provided by the two nanoporous membranes, and good fibrosis prevention by PEG surface modification of the large capsule.     

The prolonged survival of fibroblasts with forced lipid catabolism in visceral fat following encapsulation in alginate-poly-L-lysine

Although alginate-poly-L-lysine (AP(L)) encapsulation of cells producing bioactive peptides has been widely tested, it is unknown whether AP(L) supports lasting catabolic functions of encapsulated cells in adipose tissue, which are required for obesity reduction. We tested functions of AP(L)-encapsulated fibroblasts isolated from wild-type (WT) and aldehyde dehydrogenase 1a1 knockout mice (KO), which resist obesity on a high-fat (HF) diet, have a higher metabolic rate, and express increased levels of thermogenic uncoupling protein-1 (Ucp1) in their deleterious visceral fat depots compared to WT mice. To enable in vivo detection and quantification, fibroblasts were stably transfected with green-fluorescent protein. WT- or KO-containing microcapsules were injected into two visceral depots of WT mice fed an HF diet. Eighty days after transplantation, microcapsules were located in vivo using magnetic resonance imaging. KO microcapsules prevented weight gain in obese WT mice compared to a mock- and WT capsule-injected groups on an HF diet. The weight loss in KO-treated mice corresponded to lipid reduction and induction of thermogenesis in the injected visceral fat. The non-treated subcutaneous fat was not altered. Our data suggest that the AP(L) polymer supports long-term catabolic functions of genetically-modified fibroblasts, which can be potentially used for depot-specific obesity treatment.