Cationized Gelatin Microspheres Research Articles

The Transfer of the Hepatocyte Growth Factor Gene by Macrophages Ameliorates the Progression of Peritoneal Fibrosis in Mice.

Growing evidence indicates that hepatocyte growth factor (HGF) possesses potent antifibrotic activity. Furthermore, macrophages migrate to inflamed sites and have been linked to the progression of fibrosis. In this study, we utilized macrophages as vehicles to express and deliver the HGF gene and investigated whether macrophages carrying the HGF expression vector (HGF-M) could suppress peritoneal fibrosis development in mice. We obtained macrophages from the peritoneal cavity of mice stimulated with 3% thioglycollate and used cationized gelatin microspheres (CGMs) to produce HGF expression vector-gelatin complexes. Macrophages phagocytosed these CGMs, and gene transfer into macrophages was confirmed in vitro. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) for three weeks; seven days after the first CG injection, HGF-M was administered intravenously. Transplantation of HGF-M significantly suppressed submesothelial thickening and reduced type III collagen expression. Moreover, in the HGF-M-treated group, the number of α-smooth muscle actin- and TGF-β-positive cells were significantly lower in the peritoneum, and ultrafiltration was preserved. Our results indicated that the transplantation of HGF-M prevented the progression of peritoneal fibrosis and indicated that this novel gene therapy using macrophages may have potential for treating peritoneal fibrosis.

Comparative study in physico-chemical properties of gelatin derivatives and their microspheres as carriers for controlled release of green tea's extract

Two types of gelatin derivatives were prepared from type A and type B gelatin, respectively. Cationized gelatin was produced by conjugation of ethylenediamine into type A gelatin using carbodiimide reaction. Anionized gelatin was produced by conjugation of succinic anhydride to type B gelatin. Characteristics of four gelatin types and their microspheres fabricated by water-in-oil emulsion and glutaraldehyde crosslinking were evaluated. Cationized gelatin showed the highest positive charge and free amino group concentration due to the ethylenediamine conjugation, resulting in the highest crosslinking percentage and slowest degradation rate of cationized gelatin microspheres. Type A, B and anionized gelatin which had lower concentration of free amino groups produced the low crosslinking and fast-degrading microspheres. These gelatin microspheres were introduced as carrier for controlled release of epigallocatechin 3-gallate (EGCG), a high antioxidant green tea's extract. The cationized gelatin microspheres showed prolonged release of EGCG due to the slow degradation and ionic attraction with the negative-charged EGCG. The release of EGCG from type A, B and anionized gelatin microspheres was accelerated as a result of diffusion and microsphere's swelling mechanisms. These EGCG-loaded gelatin microspheres can be further studied as injectable drug for localized treatment of inflammation-related diseases with minimal invasion.

HSP47 siRNA conjugated with cationized gelatin microspheres suppresses peritoneal fibrosis in mice

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for the biosynthesis and secretion of collagen and is expressed in the fibrotic peritoneum. In the present study, we evaluated the efficacy of HSP47 small interfering RNA (siRNA) to suppress the development of peritoneal fibrosis induced by chlorhexidine gluconate in mice. We initially confirmed that biodegradable cationized gelatin microspheres (CGMs) containing HSP47 siRNA could continuously release siRNA over 21days as a result of microsphere degradation. We then determined that a single injection of CGMs incorporating HSP47 siRNA suppressed collagen expression and macrophage infiltration, thereby preventing peritoneal fibrosis. Therefore, we suggest that this controlled-release technology using HSP47 siRNA is a potential treatment for peritoneal fibrosis. Additionally, RNA interference combined with CGMs as a drug-delivery system may lead to new strategies for knocking down specific genes in vivo.

Controlled Release of Matrix Metalloproteinase-1 Plasmid DNA Prevents Left Ventricular Remodeling in Chronic Myocardial Infarction of Rats

The present study investigated whether administration of controlled release matrix metalloproteinase-1 (MMP-1) plasmid DNA prevents left ventricular (LV) remodeling in a rat chronic myocardial infarction (MI) model. Rats with a moderate-sized MI were randomized to 2 groups: injection of phosphate buffered saline (PBS) containing microspheres into the peri-infarct area (MI group, n=14) and injection of cationized gelatin microspheres incorporating MMP-1 plasmid DNA (MI+MMP-1 group, 50 microg MMP-1/20 microl; n=14). As a control group (n=14), rats received neither the coronary artery ligation nor the injection of PBS. Echocardiography, cardiac catheterization and histological studies were performed. At 2 and 4 weeks after the treatment, the MI+MMP-1 group had smaller LV end-diastolic and end-systolic dimensions, better fractional area change and smaller akinetic areas than the MI group. The LV end-systolic elastance and time constant of isovolumic relaxation were also better in the MI+MMP-1 group compared with the MI group 4 weeks after the treatment. Fibrosis evaluated with Masson's trichrome staining was less in the MI+MMP-1 group than the MI group. Gelatin microspheres for the controlled release of MMP-1 plasmid DNA are promising for improving cardiac remodeling and function when they are administered during the chronic phase of MI.

Controlled Delivery of T-box21 Small Interfering RNA Ameliorates Autoimmune Alopecia (Alopecia Areata) in a C3H/HeJ Mouse Model

Autoimmune alopecia (alopecia areata) is considered to be triggered by a collapse of immune privilege in hair follicles. Here we confirmed that infiltrating CD4 T lymphocytes around hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells, suggesting a dominant role of Th1 cells in the alopecic lesion. Given this finding, we sought to elucidate the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant interleukin-4 and neutralizing anti-interferon-gamma antibody. We found that local injections of both interleukin-4 and neutralizing anti-interferon-gamma antibody effectively treated alopecia in C3H/HeJ mice. Results from immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction demonstrated that intralesional injection of interleukin-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon-gamma mRNA expression in the affected alopecic skin. Furthermore, Th1 transcription factor T-box21 small interfering RNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. Taken together, the use of gelatin-small interfering RNA conjugates promises to be a novel, efficient, and safe tool as an alternative gene therapy for the treatment of various human diseases. To our knowledge, this is the first report of effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease.

Suppression of Renal Tubulointerstitial Fibrosis by Small Interfering RNA Targeting Heat Shock Protein 47

Background/Aim: Unilateral ureteral obstruction (UUO) is a well-established model for tubulointerstitial fibrosis. During the progression of tubulointerstitial fibrosis, upregulation of collagen synthesis and subsequent accumulation of collagen were observed in the tubulointerstitial area. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and plays an essential role in regulating collagen synthesis. We designed small interfering RNA (siRNA) sequences for HSP47 mRNA to examine whether HSP47 is involved in the progression of renal tubulointerstitial fibrosis in a mouse UUO model. Methods: The HSP47 siRNA was injected once via the ureter at the time of UUO preparation. We also applied a new gene delivery system for siRNA using cationized gelatin microspheres. The kidneys were harvested 7 and 14 days after UUO. The HSP47 and type I, III, and IV collagen expression levels were analyzed by immunohistochemistry and Western blotting. Results: Seven days after UUO, the expression levels of HSP47 and type I, III, and IV collagens were markedly upregulated in obstructed kidneys or green fluorescent protein siRNA treated obstructed kidneys. HSP47 siRNA injection significantly reduced the protein expression levels and significantly diminished the accompanying interstitial fibrosis. Moreover, cationized gelatin microspheres as a delivery system enhanced and lengthened the antifibrotic effect of HSP47 siRNA. Conclusions: Our results indicate that HSP47 is a candidate target for the prevention of tubulointerstitial fibrosis and that selective blockade of the HSP47 expression by using siRNA could be a potentially useful therapeutic approach for patients with renal disease.

Gene Transfer Using Nonviral Delivery Systems

In peritoneal dialysis, loss of peritoneal function is a major factor in treatment failure. The alterations in peritoneal function are related to structural changes in the peritoneal membrane, including peritoneal sclerosis with increased extracellular matrix. Although peritoneal sclerosis is considered reversible to some extent through peritoneal rest, which improves peritoneal function and facilitates morphological changes, there has been no therapeutic intervention and no drug against the development and progression of peritoneal sclerosis. Using recent biotechnological advances in genetic engineering, a strategy based on genetic modification of the peritoneal membrane could be a potential therapeutic maneuver against peritoneal sclerosis and peritoneal membrane failure. Before this gene therapy may be applied clinically, a safe and effective gene delivery system as well as the selection of a gene therapy method must be established. There are presently two kinds of gene transfer vectors: viral and nonviral. Viral vectors are used mainly as a gene delivery system in the field of continuous ambulatory peritoneal dialysis research; however, they have several problems such as immunogenicity and toxicity. On the other hand, nonviral vectors have several advantages over viral vectors. We review here gene transfer using nonviral vector systems in the peritoneum: electroporation, liposomes, and cationized gelatin microspheres. In the field of peritoneal dialysis, gene therapy research using nonviral vectors is presently limited. Improvement in delivery methods together with an intelligent design of targeted genes has brought about large degrees of enhancement in the efficiency, specificity, and temporal control of nonviral vectors.

Characterization of DNA release from composites of oligo(poly(ethylene glycol) fumarate) and cationized gelatin microspheres in vitro

This research investigates the release of plasmid DNA from novel hydrogel composites of oligo(poly(ethylene glycol) fumarate) (OPF) and cationized gelatin microspheres (CGMS), as well as the swelling and degradation of these materials in vitro. The release of total DNA and of double-stranded DNA was measured fluorescently, and the swelling properties and polymer mass loss of the hydrogels were assessed. Further, the structural integrity of the released DNA was determined through electrophoresis. It was found that plasmid DNA can be released in a sustained fashion over the course of up to 49-140 days in vitro from hydrogels of OPF synthesized from poly(ethylene glycol) of nominal molecular weights of 10 kDa and 3 kDa, respectively, with the release kinetics depending upon the material composition and the method of DNA loading. Released DNA was predominately double-stranded DNA (dsDNA) in structure and of the open-circular conformation. The results suggest that DNA release from hydrogel composites of OPF and CGMS is dominated by the degradation of the OPF component of the gels. Electrophoresis results indicate that the released DNA retains suitable conformation for potential bioactivity over the course of at least 63 days of release. Thus, these studies demonstrate the potential of composites of OPF and CGMS in controlled gene delivery applications.

Evaluation of bone regeneration by DNA release from composites of oligo(poly(ethylene glycol) fumarate) and cationized gelatin microspheres in a critical‐sized calvarial defect

This research examines the bone formation response to release of plasmid DNA encoding human Bone Morphogenetic Protein-2 from hydrogel composites consisting of cationized gelatin microspheres (CGMS) embedded within a crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel network in a critical-sized rat cranial defect model after 30 days. Four composite groups were investigated: (1) composites with 10 microg DNA loaded into the CGMS phase, (2) composites with 10 microg DNA loaded into the OPF phase, (3) composites with 100 microg DNA loaded into the OPF phase, and (4) composites without DNA (material control). Light microscopy revealed no enhancement in bone formation for groups releasing plasmid DNA, relative to the material control group. Limited formation of new bone was observed from the defect margins and within the defect for some samples. The hydrogels swelled appreciably and fragmentation of the implants was noted to varying degrees among samples within groups, with a presence of inflammatory cells related to the degree of fragmentation. The lack of enhancement in bone formation indicates that the release of plasmid DNA from the composites was not sufficient to elicit a bone regeneration response.

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma

Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.

In vivo release of plasmid DNA from composites of oligo(poly(ethylene glycol)fumarate) and cationized gelatin microspheres

Composites of cationized gelatin microspheres (CGMS), crosslinked with either 3 mM or 6 mM glutaraldehyde solution, and a novel hydrogel material, oligo(poly(ethylene glycol)fumarate) (OPF) were fabricated and investigated toward prolonging the release of plasmid DNA in vivo relative to the constituent materials. The composites and constituent materials were investigated in a subcutaneous murine model to assess the release of 125I-labeled plasmid DNA and 125I-labeled cationized gelatin in vivo. The time profiles of the radioactivity remaining were employed to compare the profiles of DNA release and cationized gelatin degradation. Both composite formulations (incorporating either 3 mM or 6 mM CGMS) prolonged the bioavailability of plasmid DNA relative to both injected plasmid DNA solution and the respective non-embedded cationized gelatin microspheres. Injected plasmid DNA solution persisted in the subject for only 7–10 days, whereas the persistence of DNA from composites of OPF and either 3 mM or 6 mM CGMS extended to at least day 42. The 3 mM and 6 mM CGMS each increased the persistence of DNA slightly, relative to injection of DNA solution, to between 28 and 35 days. Interestingly, the release profile of plasmid DNA from composites was not significantly different from the release of DNA from OPF alone. The release of plasmid DNA from the composites was in accord with the degradation of the microspheres within the OPF. These results show that composites of OPF and cationized gelatin microspheres are able to prolong the availability of plasmid DNA in vivo relative to cationized gelatin microspheres alone and provide a promising candidate material for the sustained, controlled release of plasmid DNA.

Controlled Release Technology Suppresses the Progression of Disseminated Pancreatic Cancer Cells

NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as a potent angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release of NK4 plasmid DNA in suppressing tumor growth. Controlled release by a biodegradable hydrogel enabled the NK4 plasmid DNA to enhance the tumor suppression effects. Biodegradable microspheres of cationized gelatin were prepared for the controlled release of a NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA were subcutaneously injected to tumor-bearing mice to evaluate the suppressive effects on tumor angiogenesis and growth. The cationized gelatin microspheres incorporating NK4 plasmid DNA could release over 28 days. When the cationized gelatin microspheres incorporating NK4 plasmid DNA were injected into the subcutaneous tissue of mice intraperitoneally inoculated with pancreatic cancer cells, their survival time period was prolonged. Tumor growth was suppressed to a significantly greater extent than free NK4 plasmid DNA. The controlled release of NK4 plasmid DNA suppressed angiogenesis and increased cell apoptosis in the tumor tissue, while it enhanced and prolonged the serum level of NK4 protein. We conclude that the controlled release technology was promising to enhance the tumor suppression effects of NK4 plasmid DNA.

Suppression of the progress of disseminated pancreatic cancer cells by NK4 plasmid DNA released from cationized gelatin microspheres.

NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as a potent angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release of NK4 plasmid DNA in suppressing the tumor growth. Controlled release by a biodegradable hydrogel enabled the NK4 plasmid DNA to exert the tumor suppression effects. Biodegradable cationized gelatin microspheres were prepared for the controlled release of an NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA were subcutaneously injected to tumor-bearing mice to evaluate the suppressive effects on tumor angiogenesis and growth. The cationized gelatin microspheres incorporating NK4 plasmid DNA could release over 28 days as a result of microspheres degradation. The injection of cationized gelatin microspheres incorporating NK4 plasmid DNA into the subcutaneous tissue of mice inoculated with pancreatic cancer cells prolonged their survival time period. An increase in the tumor number was suppressed to a significantly greater extent than free NK4 plasmid DNA. The controlled release of NK4 plasmid DNA suppressed angiogenesis and increased the cell apoptosis in the tumor tissue while it enhanced and prolonged the NK4 protein level in the blood circulation. We conclude that the controlled release technology is promising to enhance the tumor suppression effects of NK4 plasmid DNA.

Suppression of tumor metastasis by NK4 plasmid DNA released from cationized gelatin.

NK4, composed of the NH(2)-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as an HGF-antagonist and angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release formulation of NK4 plasmid DNA in suppressing the tumor growth, and lung metastasis. Biodegradable cationized gelatin microspheres were prepared for the controlled release of an NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA could continuously release plasmid DNA over 28 days as a result of microspheres degradation following the subcutaneous injection. The injection of cationized gelatin microspheres incorporating NK4 plasmid DNA into the subcutaneous tissue significantly prolonged the survival time period of the mice bearing Lewis lung carcinoma tumor. Increases in the tumor volume and the number of lung metastatic nodules of NK4 plasmid DNA release group were suppressed to a significantly greater extent than that of solution-injected group (77.4 and 64.0%, respectively). The number of blood vessels and the apoptosis cells in the tumor tissue were significantly suppressed (80.4%) and increased (127.3%) against free NK4 plasmid DNA-injected group. Thus, the controlled release of NK4 plasmid DNA augmented angiogenesis suppression and apoptosis of tumor cells, which resulted in suppressed tumor growth. We conclude that this controlled release technology is promising to enhance the tumor suppression achieved by gene expression of NK4.