[The study of new soft one-piece PHEMA keratoprosthesis implanted into alkali burned rabbits cornea].

OBJECTIVE: This study was to evaluate clinical results of two types soft one-piece keratoprosthesis (KPros) made of PHEMA implanted to alkali burned rabbit corneas. METHODS: Experimental study. Twelve pieces KPros were implanted alkali burned rabbit corneas(type I and II, 6 pieces respectively). The examinations including slit-lamp, fundus photography,B-ultrasound and ultrasound biomicroscopy (UBM) were carried out. RESULTS: All Kpros were stable and no complications happened including conjunctiva flap dehiscence, aqueous leak and infection. IOP were normal by finger touch. B-ultrasound show no retina detachment and UBM show synechia in most animals (10 case). Retroprosthetic membrane happened in type I (3 case) . Visible conjunctiva hyperplasia covering the optical region happened in all KPros. CONCLUSIONS: (1) The same material, integrated design, maybe eliminating the material interface problem. (2)Simple surgical skills. (3)IOP measurement is possible because of soft material. (4)KPro I do not need removal of the lens and vitrectomy. The projecting portion of KPro II may reduce the incidence of proliferative membrane. Long term effects need more cases and further observation.

[The expression level of MMP-2 and collagen of hydroxyapatite modified titanium for keratoprosthesis in the corneal stroma of rabbits].

OBJECTIVE: To investigate the expression level of metalloproteinases-2(MMP-2) and Collagen in a hydroxyapatite surfaced-modified of three Pan type titanium keratoprosthesis after that implanted into the corneal stroma of rabbits, further evaluate its biological compatibility. METHODS: Experimental study. Twenty-four New Zealand white rabbits, 2.0-2.5 kg, were respectively divided into three groups. Surgery was performed in right eye of all animals. skirt of HA-Ti and Ti were respectively inserted into the corneal stroma of rabbit of experimental group A and group B; only a sack was made without implantation in control group C . Cornea edema and corneal neovascularization were observed at scheduled times after operation; animals were sacrificed 2, 4 and 16 weeks after operation and their cornea was removed and examined under light microscopy; the surface of skirt was observed under scanning electron microscope. RESULTS: During the study period, all skirts were stable without infected, dissolved and excluded. Different degree of cornea edema and neovascularization was revealed after surgery. MMP-2 were absent in the normal corneal matrix. The expression level of MMP-2 in group A was higher than group C at all time points (F = 6.083, P < 0.05), and was increased than group B at 4th (F = 47.074, P < 0.01), and was increased than group C at 16th weeks too (F = 6.079, P < 0.05) . Corneal organization has a large green 4 weeks type III collagen and yellow red type I collagen, 16 weeks corneal mainly for bright red when within the collagen type I, still have a small amount of collagen type III. CONCLUSIONS: Rabbit cornea implanted HA-Ti skirts cause MMP-2 activation, continuous high expression didn't cause the cornea to dissolve; Collagen -III turned into collagen-I gradually in the extracellular matrix around the skirts. Hydroxyapatite modified titanium for Keratoprosthesis promoted the corneal neovascularization and improve the interfacial bio integration of skirt and host cornea.

Reactive Oxygen Species─Responsive Lipid Nanoparticles for Effective RNAi and Corneal Neovascularization Therapy.

Corneal neovascularization (CNV) is a common disease that affects the vision ability of more than 1 million people annually. Small interfering RNA (siRNA) delivery nanoparticle platforms are a promising therapeutic modality for CNV treatment. However, the efficient delivery of siRNA into cells and the effective release of siRNA from delivery vehicles in a particular cell type challenge effective RNAi clinical application for CNV suppression. This study reports the design of a novel reactive oxygen species (ROS)-responsive lipid nanoparticle for siRNA delivery into corneal lesions for enhanced RNAi as a potential CNV treatment. We demonstrated that lipid nanoparticles could efficiently deliver siRNA into human umbilical vein endothelial cells and release siRNA for enhanced gene silencing by using the upregulated ROS of CNV to promote lipid nanoparticle degradation. Moreover, the subconjunctival injection of siRNA nanocomplexes into corneal lesions effectively knocked down vascular endothelial growth factor expression and suppressed CNV formation in an alkali burn model. Thus, we believe that the strategy of using ROS-responsive lipid nanoparticles for enhanced RNAi in CNV could be further extended to a promising clinical therapeutic approach to attenuate CNV formation.

3D biomaterial P scaffolds carrying umbilical cord mesenchymal stem cells improve biointegration of keratoprosthesis.

Biointegration of a keratoprosthesis (KPro) is critical for the device stability and long-term retention. Biointegration of the KPro device and host tissue takes place between the surrounding corneal graft and the central optic (made by poly (methyl methacrylate)). Our previous clinical results showed that auricular cartilage reinforcement is able to enhance the KPro biointegration. However, the auricular cartilage is non-renewable and difficult to acquire. In this study, we developed a novel type of biomaterial using a three-dimensional porous polyethylene glycol acrylate scaffold (3D biological P-scaffold) carrier with chondrocytes differentiated from induced human umbilical cord mesenchymal stem cells (hUC-MSCs) and tested in rabbit corneas. The results showed hUC-MSCs bear stem cell properties and coule be induced into chondrocytes, P-scaffold is beneficial to the growth and differentiation of hUC-MSCs bothin vivoandin vitro. Besides, after implanting the P-scaffold into the corneal stroma, no serious immune rejection response, such as corneal ulcer or perforation were seen, suggested a good biocompatibility of P-scaffold with the corneal tissue. Moreover, after implanting P-scaffold in together with the differentiated chondrocytes into the rabbit corneal stroma, they significantly increased corneal thickness and strengthened the host cornea, and chondrocytes could stably persist inside the cornea. In summary, the 3D biological P-scaffold carrying differentiated hUC-MSCs could be the preferable material for KPro reinforcement.

[Biocompatible study of modified titanium skirt for keratoprosthesis].

OBJECTIVE: The research is engaged in developing an improved titanium skirt for keratoprosthesis, the aim of this study was to evaluate biocompatible of keratoprosthesis of novel design. METHODS: The pure titanium skirt for keratoprosthesis with three hands were first Sandblasted, and then bioactive hydroxyapatite coated on Sandblasted titanium for keratoprosthesis by a acid-alkali chemical treatment. A total of 18 New Zealand white rabbits and 18 alkali burned rabbit corneas were respectively divided into three groups (Group A, B and C; Group E, F and G) with simple random sampling methods. The modified titanium skirt was inserted into the corneal stroma of Group A and E, and then the control skirt was inserted into Group B and F. Group C and H did not insert skirt as surgery control. The interfacial biointegration of skirt/cornea were examined under light microscopy by HE, TEM and SEM. The transparent center was implanted to rabbit corneas with modified titanium skirt after 3 months. RESULTS: Dense hydroxyapatite coating was deposited on the Sandblasted specimens by an acid-alkali chemical treatment. The number of corneal fibroblasts increased significantly in Group A compared with Group B. The extracellular matrix deposited on the surface of modified titanium skirt was more dense and tight than that of control skirt. There was a significant difference in the shear force of skirt among groups A and B (t = 3.297, P < 0.05), E and F (t = 4.237, P < 0.05), and taking out the skirt in Group A and E were more difficulty than that in Group B and F after 3 months. The observation of the tissue sections of modified skirt inserted eyes revealed that there were cells and collagen-like fibres perpendicular to or at an angle to the rough interface and the cellular function was extremely active, in accordance with the results of TEM. Compared with healthy host tissue, skirt/cornea healing after alkali burn was impaired. CONCLUSIONS: Hydroxyapatite modified Sandblasted titanium skirt for keratoprosthesis can promote the interfacial biointegration of skirt and host cornea, no matter in healthy cornea or diseased cornea (alkali burn cornea). Hydroxyapatite coating improved the bioactivity of titanium.

J2 prolongs the corneal allograft survival through inhibition of the CD4+ T cell-mediated response in vivo.

In our previous report, we described a novel non-peptidic organic ligand of CD4 D1, designated J2, as a potential inhibitor of CD4 D1 and thus CD4-dependent T cell responses in vitro. In this work, we further used a murine model of corneal allograft rejection to determine its in vivo immunosuppressive activities. To mimic the situation in high-risk human eyes, the recipient mice corneas were all induced by intrastromal sutures to serve as neovascularized graft beds. J2 was administrated by mouth 3 h before transplantation and thereafter on consecutive 12 days. The results showed that J2 could significantly prolong the median survival time of the corneal allografts, compared to the untreated control group. And the subsequent functional assays, including T cell phenotype analysis, delayed-type hypersensitivity (DTH) and enzyme-linked immunospot (ELISPOT) assays revealed that the immunosuppressive activity of J2 was associated with its inhibitory effects on the CD4(+) T cells and these cells-mediated responses. All these results suggest that J2 is a potential lead for the development of new immunosuppressive agents to prevent the corneal allograft rejection.

[Biocompatibility of a special hydrogel orbital implant (PHEMA and MMA) in rabbits].

OBJECTIVE: To investigate the biocompatibility of a hydrogel orbital implant and its rate of vascularization in an experimental study. METHODS: The implant was made of copolymerization of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA). Thirty-two hydrogel orbital implants were implanted into the right orbit of 32 New Zealand albino rabbits. The rate of vascularization was examined by SPECT, immunohistochemistry and electron microscopy. RESULTS: Single Positron Emission Computerized Tomography (SPECT) examination showed that a radiotracer could be found locally concentrated on hydrogel orbital implants in vivo along with time. On light microscopy, implants fibro vascular tissue invade the pores of the hydrogel orbital implant after 2 weeks and gradually invaded the implant deeper and deeper from 4 to 8 weeks, almost all implants were fully vascularized after 12 weeks with a relatively spare inflammatory reaction. Only one case needed additional surgery. CONCLUSIONS: The hydrogel orbital implant has many advantages, such as well biocompatible, fast vascularization, simple operation and low complication of surgery. It is safe and applicable for clinical use as a new type of orbital implant material.

Biocompatibility of helicoidal multilamellar arginine-glycine-aspartic acid-functionalized silk biomaterials in a rabbit corneal model.

Silk proteins represent a unique choice in the selection of biomaterials that can be used for corneal tissue engineering and regenerative medical applications. We implanted helicoidal multilamellar arginine-glycine-aspartic acid-functionalized silk biomaterials into the corneal stroma of rabbits, and evaluated its biocompatibility. The corneal tissue was examined after routine hematoxylin-eosin staining, immunofluorescence for collagen I and III, and fibronectin, and scanning electron microscopy. The silk films maintained their integrity and transparency over the 180-day experimental period without causing immunogenic and neovascular responses or degradation of the rabbit corneal stroma. Collagen I increased, whereas Collagen III and fibronectin initially increased and then gradually decreased. The extracellular matrix deposited on the surface of the silk films, tightly adhered to the biomaterial. We have shown this kind of silk film graft has suitable biocompatibility with the corneal stroma and is an initial step for clinical trials to evaluate this material as a transplant biomaterial for keratoplasty tissue constructs.

Targeted delivery of chemically modified anti-miR-221 to hepatocellular carcinoma with negatively charged liposomes.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death. Gene therapy was established as a new strategy for treating HCC. To explore the potential delivery system to support the gene therapy of HCC, negatively charged liposomal delivery system was used to deliver miR-221 antisense oligonucleotide (anti-miR-221) to the transferrin (Tf) receptor over expressed HepG2 cells. The liposome exhibited a mean particle size of 122.5 nm, zeta potential of -15.74 mV, anti-miR-221 encapsulation efficiency of 70%, and excellent colloidal stability at 4°C. Anti-miR-221-encapsulated Tf-targeted liposome demonstrated a 15-fold higher delivery efficiency compared to nontargeted liposome in HepG2 cells in vitro. Anti-miR-221 Tf-targeted liposome effectively delivered anti-miR-221 to HepG2 cells, upregulated miR-221 target genes PTEN, P27(kip1), and TIMP3, and exhibited greater silencing efficiency over nontargeted anti-miR-221 liposome. After intravenous injection into HepG2 tumor-bearing xenografted mice with Cy3-labeled anti-miR-221 Tf-targeted liposome, Cy3-anti-miR-221 was successfully delivered to the tumor site and increased the expressions of PTEN, P27(kip1), and TIMP3. Our results demonstrate that the Tf-targeted negatively charged liposome could be a potential therapeutic modality in the gene therapy of human HCC.

An improved biofunction of titanium for keratoprosthesis by hydroxyapatite-coating.

Titanium framework keratoprosthesis has been commonly used in the severe corneal blindness, but the tissue melting occurred frequently around titanium. Since hydroxyapatite has been approved to possess a good tissue integration characteristic, nanostructured hydroxyapatite was coated on the surface of titanium through the aerosol deposition method. In this study, nanostructured hydroxyapatite coating was characterized by X-ray diffraction, scanning electron microscopy, atomic force microscopy, and auger electronic spectrometer. Biological evaluations were performed with rabbit cornea fibroblast in vitro and an animal model in vivo. The outcomes showed the coating had a grain-like surface topography and a good atomic mixed area with substrate. The rabbit cornea fibroblasts appeared a good adhesion on the surface of nanostructured hydroxyapatite in vitro. In the animal model, nanostructured hydroxyapatite-titanium implants were stably retained in the rabbit cornea, and by contrast, the corneal stroma became thinner anterior to the implants in the control. Therefore, our findings proved that nanostructured hydroxyapatite-titanium could not only provide an improved bond for substrate but also enhance the tissue integration with implants in host. As a promising material, nanostructured hydroxyapatite-titanium-based keratoprosthesis prepared by the aerosol deposition method could be utilized for the corneal blindness treatment.

A novel hydrolysis-resistant lipophilic folate derivative enables stable delivery of targeted liposomes in vivo.

Instability of targeting ligand is a roadblock towards successful development of folate targeted liposomes. Folate ligands have been linked to polyethylene glycol (PEG) and cholesterol by an amide bond to form folate-CONH-PEG-CONH-Cholesterol (F-CONH-PEG-CONH-Chol), which is subject to hydrolysis. To increase the stability of folate ligands and promote the long circulation and targeting effects, we synthesized a chemically stable lipophilic folate derivative, folate-CONH-PEG-NH-Cholesterol (F-CONH-PEG-NH-Chol), where the amide bond was replaced by a C-N bond, to deliver liposomal doxorubicin (Dox). Its physical stability, cellular uptake, cellular toxicity, pharmacokinetics, distribution, anti-tumor efficacy, and cardiac toxicity were investigated. Our results indicate that F-CONH-PEG-NH-Chol conjugated liposomes are taken up selectively by folate receptor-positive HeLa and KB cells. Compared with F-CONH-PEG-CONH-Chol with two carbonate linkages, F-CONH-PEG-NH-Chol better retained its drug entrapment efficiency and folate receptor-targeting activity during prolonged circulation. F-CONH-PEG-NH-Chol thus represents a physically stable and effective ligand for delivering folate receptor-targeted liposomes, with prolonged circulation time and efficient tissue distribution, as well as higher efficacy and less cardiac toxicity. Collectively, these results suggest that this novel conjugate can serve as a promising derivative for the delivery of anti-tumor therapeutic agents.

Novel liposomal gefitinib (L-GEF) formulations.

BACKGROUND: Gefitinib is a promising agent for the treatment of non-small cell lung cancer. The purpose of this study was to develop a novel liposomal formulation for gefitinib (L-GEF) to improve its therapeutic index. MATERIALS AND METHODS: Several L-GEF formulations were prepared and characterized for their physical chemical properties and cytotoxicity. The pharmacokinetic parameters of the liposomes were determined in mice. The effect of lipid composition, transmembrane pH gradient, and incorporation of hydroxypropyl-ß-cyclodextrin (HPßCD) on drug-loading efficiency, liposomal stability, and the rate of drug release were investigated. RESULTS: The L-GEF formulation composed of hydrogenated soy phosphatidylcholine (HSPC)/cholesterol (Chol)/monomethoxy polyethylene glycol 2000-distearoyl phosphatidyl-ethanolamine (mPEG-DSPE) encapsulating 0.3 M (NH4)2SO4 and 0.1 M HPßCD (L-GEF-HSPC), had a drug-loading efficiency (DLE) of 85.5%. In vitro release studies showed that gefitinib release from L-GEF-HSPC in the presence of human plasma was slow and exhibited non-Fickian kinetics. Pharmacokinetic study in mice after i.v. bolus administration of L-GEF-HSPC showed that the area under the plasma concentration time curve (AUC) for gefitinib was 32.41 µg·h /ml and six times that of free gefitinib. The elimination half life (t(1/2ß)) of L-GEF-HSPC was 7.29 h, while that of free gefitinib was 2.26 h. CONCLUSION: It was shown that gefitinib can be efficiently loaded into L-GEF-HSPC composed of HSPC/Chol/mPEG-DSPE (55/40/5 mol/mol) with 0.3 M (NH(4))(2)SO(4) and 0.1 M HPßCD as trapping agents. Compared with the free drug, L-GEF-HSPC had high drug loading, good stability, and long-circulating properties.