A Simple Preparation Method of Gelatin Hydrogels Incorporating Cisplatin for Sustained Release.

The objective of this study was to develop a new preparation method for cisplatin (CDDP)-incorporated gelatin hydrogels without using chemical crosslinking nor a vacuum heating instrument for dehydrothermal crosslinking. By simply mixing CDDP and gelatin, CDDP-crosslinked gelatin hydrogels (CCGH) were prepared. CDDP functions as a crosslinking agent of gelatin to form the gelatin hydrogel. Simultaneously, CDDP is incorporated into the gelatin hydrogel as a controlled release carrier. CDDP's in vitro and in vivo anticancer efficacy after incorporation into CCGH was evaluated. In the in vitro system, the CDDP was released gradually due to CCGH degradation with an initial burst release of approximately 16%. CDDP metal-coordinated with the degraded fragment of gelatin was released from CCGH with maintaining the anticancer activity. After intraperitoneal administration of CCGH, CDDP was detected in the blood circulation while its toxicity was low. Following intraperitoneal administration of CCGH in a murine peritoneal dissemination model of human gastric cancer MKN45-Luc cell line, the survival time was significantly prolonged compared with free CDDP solution. It is concluded that CCGH prepared by the CDDP-based crosslinking of gelatin is an excellent sustained release system of CDDP to achieve superior anticancer effects with minimal side effects compared with free CDDP solution.

Pioglitazone-incorporated microspheres targeting macrophage polarization alleviates cardiac dysfunction after myocardial infarction.

OBJECTIVES: Excessive and chronic inflammation after a myocardial infarction (MI) is associated with left ventricular remodelling and impaired cardiac function. Among inflammatory cells, macrophages play a critical role in polarizing proinflammatory M1 or the reparative M2 subtype. Pioglitazone (PGZ) is reported to regulate macrophage polarization to the M2 subtype. Our goal was to validate the therapeutic effects and the mechanisms of PGZ utilizing a drug delivery system. METHODS: Poly L-lactic-co-glycolic acid microspheres (MS) incorporating PGZ were prepared. To validate the therapeutic potential of PGZ-MS, Sprague-Dawley rats were subjected to permanent left coronary artery ligation to induce an MI. Placebo-MS (100 µg) or PGZ-MS (100 µg) was injected to the infarct region just after induction. Cardiac function and size were assessed by echocardiography. At 28 days after surgery, the rats were sacrificed, and the excised hearts were evaluated histologically. RESULTS: Sustained release of PGZ from the PGZ-MS was confirmed in vitro. PGZ-MS significantly rehabilitated cardiac dysfunction after an MI (fractional shortening: MI vs MI+placebo-MS vs MI+PGZ-MS, 24.4 ± 1.1 vs 24.3 ± 1.6 vs 32.2 ± 1.4%; P = 0.0035) with reverse remodelling. Immunohistochemical analyses revealed that PGZ-MS enhanced macrophage polarization (ratio of M2 subtype: 0.39 ± 0.03 vs 0.42 ± 0.02 vs 0.54 ± 0.02; P = 0.0004) and attenuated apoptosis of cardiomyocytes in the ischaemic border zone. CONCLUSIONS: We confirmed macrophage polarization by sustained release of PGZ, which resulted in amelioration of adverse left ventricular remodelling and cardiac dysfunction. Drug delivery system-based macrophage polarization might serve as a promising strategy in cardiac regenerative therapy for ischaemic heart disease. (241 words).

Transplantation of human iPSC-derived muscle stem cells in the diaphragm of Duchenne muscular dystrophy model mice.

Duchenne muscular dystrophy (DMD) is an intractable genetic muscular disorder characterized by the loss of DYSTROPHIN. The restoration of DYSTROPHIN is expected to be a curative therapy for DMD. Because muscle stem cells (MuSCs) can regenerate damaged myofibers with full-length DYSTROPHIN in vivo, their transplantation is being explored as such a therapy. As for the transplanted cells, primary satellite cells have been considered, but donor shortage limits their clinical application. We previously developed a protocol that differentiates induced pluripotent stem cells (iPSCs) to MuSCs (iMuSCs). To ameliorate the respiratory function of DMD patients, cell transplantation to the diaphragm is necessary but difficult, because the diaphragm is thin and rapidly moves. In the present study, we explored the transplantation of iMuSCs into the diaphragm. First, we show direct cell injection into the diaphragm of mouse was feasible. Then, to enhance the engraftment of the transplanted cells in a rapidly moving diaphragm, we mixed polymer solutions of hyaluronic acid, alginate and gelatin to the cell suspension, finding a solution of 20% dissolved hyaluronic acid and 80% dissolved gelatin improved the engraftment. Thus, we established a method for cell transplantation into mouse diaphragm and show that an injectable hyaluronic acid-gelatin solution enables the engraftment of iMuSCs in the diaphragm.

Efficient cell transplantation combining injectable hydrogels with control release of growth factors.

INTRODUCTION: The objective of this study is to investigate the effect of gelatin microspheres incorporating growth factors on the therapeutic efficacy in cell transplantation. The strength of this study is to combine gelatin hydrogel microspheres incorporating basic fibroblast growth factor and platelet growth factor mixture (GM/GF) with bioabsorbable injectable hydrogels (iGel) for transplantation of adipose-derived stem cells (ASCs). METHODS: The rats ASCs suspended in various solutions were transplanted in masseter muscle. Rats were euthanized 2, 7, 14 days after injection for measurement of the number of ASCs retention in the muscle and morphological evaluation of muscle fibers and the inflammation of the injected tissue by histologic and immunofluorescent stain. RESULTS: Following the injection into the skeletal muscle, the GM/GF allowed the growth factors to release at the injection site over one week. When ASCs were transplanted into skeletal muscle using iGel incorporating GM/GF (iGel+GM/GF), the number of cells grafted was significantly high compared with other control groups. Moreover, for the groups to which GM/GF was added, the cells transplanted survived, and the Myo-D expression of a myoblast marker was observed at the region of cells transplanted. CONCLUSIONS: The growth factors released for a long time likely enhance the proliferative and differentiative capacity of cells. The simple combination with iGel and GM/GF allowed ASCs to enhance their survival at the injected site and consequently achieve improved therapeutic efficacy in cell transplantation.

A Co-Culture System of Three-Dimensional Tumor-Associated Macrophages and Three-Dimensional Cancer-Associated Fibroblasts Combined with Biomolecule Release for Cancer Cell Migration.

The objective of this study is to design a cancer invasion model by making use of cancer-associated fibroblasts (CAF) or tumor-associated macrophages (TAM) and gelatin hydrogel microspheres (GM) for the sustained release of drugs. The GM containing adenosine (A) (GM-A) were prepared and cultured with TAM to obtain three-dimensional (3D) TAM aggregates incorporating GM-A (3D TAM-GM-A). The GM-A incorporation enabled TAM to enhance the secretion level of vascular endothelial growth factor. When co-cultured with HepG2 liver cancer cells in an invasion assay, the 3D TAM-GM-A promoted the invasion rate of cancer cells. In addition, the E-cadherin expression level decreased to a significantly greater extent compared with that co-cultured with TAM aggregates incorporating GM, whereas the significantly higher expression of N-cadherin and Vimentin was observed. This indicates that the epithelial-mesenchymal transition event was induced. The GM containing transforming growth factor-ß1 (TGF-ß1) were prepared to incorporate into 3D CAF (3D CAF-GM-TGF-ß1). Following a co-culture of mixed 3D CAF-GM-TGF-ß1 and 3D TAM-GM-A and every HepG2, MCF-7 breast cancer cell, or WA-hT lung cancer cell, the invasion rate of every cancer cell enhanced depending on the mixing ratio of 3D TAM-GM-A and 3D CAF-GM-TGF-ß1. The amount of matrix metalloproteinase-2 (MMP-2) secreted also enhanced, and the enhancement was well corresponded with that of cancer cell invasion rate. The higher MMP secretion assists the breakdown of basement membrane, leading to the higher rate of cancer cell invasion. This model is a promising 3D culture system to evaluate the invasion ability of various cancer cells in vitro. Impact statement This study proposes a cell culture system to enhance the tumor-associated macrophage function based on the combination of three-dimensional (3D) cell aggregates and gelatin hydrogel microspheres (GM) for adenosine delivery. An additional combination of 3D cancer-associated fibroblasts incorporating GM containing transforming growth factor-ß1 allowed cancer cells to enhance their invasion rate. This co-culture system is promising to evaluate the ability of cancer cell invasion for anticancer drug screening.

Gelatin hydrogels with eicosapentaenoic acid can prevent osteoarthritis progression in vivo in a mouse model.

Eicosapentanoic acid (EPA) is an antioxidant and omega-3 polyunsaturated fatty acid that reduces inflammatory cytokine production. Gelatin hydrogel can be used as a carrier of a physiologically active substance that release it gradually for an average of ~3 weeks. Therefore, this study aimed to clarify the effect of EPA-incorporating gelatin hydrogels on osteoarthritis (OA) progression in vivo. Ten-week-old male C57BL/6J mice were randomly divided into six groups (n = 6): Sham, destabilization of the medial meniscus (DMM), Corn: DMM + 2 µL corn oil, EPA injection alone (EPA-I): DMM + 2 µL corn oil + 125 µg/µL EPA, Gel: DMM + gelatin hydrogels, and EPA-G: DMM + 125 µg/µL EPA-incorporating gelatin hydrogels. The mice were euthanized at 8 weeks after DMM or Sham surgery, and subjected to histological evaluation. Matrix-metalloproteinases-3 (MMP-3), MMP-13, interleukin-1ß (IL-1ß), p-IKK α/ß, CD86, and CD163 protein expression in the synovial cartilage was detected by immunohistochemical staining. F4/80 expression was also assessed using the F4/80 score of macrophage. Histological score was significantly lower in EPA-G than in EPA-I. MMP-3-, MMP-13-, IL-1ß-, and p-IKK α/ß-positive cell ratio was significantly lower in EPA-G than in EPA-I. However, CD86- and CD163-positive cell ratio was not significantly different between EPA-I and EPA-G. The average-sum F4/80 score of macrophage in EPA-G was significantly lower than that in EPA-I. EPA-incorporating gelatin hydrogels were shown to prevent OA progression in vivo more effectively than EPA injection alone. Our results suggested that intra-articular administration of controlled-release EPA can be a new therapeutic approach for treating OA.

Combination therapy of oncolytic herpes simplex virus HF10 and bevacizumab against experimental model of human breast carcinoma xenograft.

Breast cancer is one of the most common and feared cancers faced by women. The prognosis of patients with advanced or recurrent breast cancer remains poor despite refinements in multimodality therapies involving chemotherapeutic and hormonal agents. Multimodal therapy with more specific and effective strategy is urgently needed. The oncolytic herpes simplex virus (HSV) has potential to become a new effective treatment option because of its broad host range and tumor selective viral distribution. Bevacizumab is a monoclonal antibody against VEGFA, which inhibits angiogenesis and therefore tumor growth. Our approach to enhance the antitumor effect of the oncolytic HSV is to combine oncolytic HSV HF10 and bevacizumab in the treatment of breast cancer. Our results showed that bevacizumab enhanced viral distribution as well as tumor hypoxia and expanded the population of apoptotic cells and therefore induced a synergistic antitumor effect. HF10 is expected to be a promising agent in combination with bevacizumab in the anticancer treatment.

Phase I Dose-escalation Clinical Trial of HF10 Oncolytic Herpes Virus in 17 Japanese Patients with Advanced Cancer.

Oncolytic virus therapy is a promising new therapeutic method, one of an eagerly anticipated class of biological therapies against cancer. There are many different classes of oncolytic virus. One of these, herpes oncolytic virus, is strongly oncolytic and has a large DNA genome as 150k bp. HF10 is a spontaneous mutant of herpes simplex virus -1 (HSV-1) that replicates within tumors and destroys cancers without damaging normal tissue and organs. Clinical trials of HF10 are underway in Japan and the United States. The first pilot study of HF10 was initiated in Japan in 2003. This study examined the safety and efficacy of HF10 in the treatment of breast cancer and head and neck cancers; the trial also included careful dose escalation studies. In 2005, a clinical trial using HF10 to treat pancreatic cancer was initiated. screened In this Japanese study, 17 patients received HF10 in their tumor sites. A clinical trial in the United States is also ongoing to evaluate safety, tolerability and evidence of antitumor activity in patients with refractory superficial solid tumors. Here, we report the evaluation of the 17 patients treated in Japan. Among the patients, 6 had recurrent breast cancer, 3 had recurrent head and neck cancer, and 8 had non-resectable pancreatic cancer. No severe adverse side effects have been observed, and some therapeutic potential has been reported based on pathological findings, tumor markers, and diagnostic radiography. Those results should encourage further clinical trials of HF10 around the world.