A Novel Vector System for Gene Transfer into the ...

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Current Eye Research, 33:839–848, 2008 c Informa Healthcare USA, Inc. Copyright ISSN: 0271-3683 print / 1460-2202 online DOI: 10.1080/02713680802382963

A Novel Vector System for Gene Transfer into the Cornea Using a Partially Dried Plasmid Expressing 18 Basic Fibroblast Growth Factor–Synthetic Amphiphile INTeraction-18 (SAINT-18) Complex Chien-Neng Kuo Department of Ophthalmology, Chang Gung Memorial Hospital, Chiayi, Taiwan, and Chang Gung University College of Medicine and Chang Gung Institute of Technology, Taiwan, R.O.C. Lin-Cheng Yang Gene Therapy Laboratory, E-DA Hospital, I-Shou University, Kaohsiung Hsien, Taiwan, R.O.C. Cheng-Ta Yang Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi, Taiwan, R.O.C. Miao-Fen Chen Department of Radiation Oncology, Chang Gung Memorial Hospital, Chiayi, Taiwan, and Chang Gung University College of Medicine and Chang Gung Institute of Technology, Taiwan, R.O.C. Chien-Hsiung Lai Department of Ophthalmology, Chang Gung Memorial Hospital, Chiayi, Taiwan, and Chang Gung University College of Medicine and Chang Gung Institute of Technology, Taiwan, R.O.C. Yi-Hao Chen Department of Ophthalmology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, and Chang Gung University College of Medicine, Taiwan, R.O.C. Ching-Hsein Chen Graduate Institute of Biomedical and Biopharmaceutical Sciences, College of Life Sciences, National Chiayi University, Chiayi, Taiwan Chi-Hung Chen Graduate Institute of Food Science and Biopharmaceutics, National Chiayi University, Chiayi, Taiwan Pei-Chang Wu, Hsi-Kung Kou, and Jen-Chia,Tsai Department of Ophthalmology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, and Chang Gung University College of Medicine, Taiwan, R.O.C. Chia-Hui Hung Department of Dermatology, Chia-Yi Christian Hospital, Taiwan, R.O.C.

Received 19 October 2007 Accepted 31 July 2008 Correspondence: Dr. Chia-Hui Hung, Chia-Yi Christian Hospital Dermatology, 539 Chung Hsiao RD. Chia-Yi, Taiwan, R.O.C. E-mail: [email protected]

ABSTRACT Purpose: We describe a novel vector system of nonviral gene transfer into the cornea using a partially dried form of a plasmid expressing 18-kDa basic fibroblast growth factor (p-bFGF)–synthetic amphiphile INTeraction-18 (SAINT-18) complex. Methods: Corneal neovascularization (NV) was evaluated in 48 eyes of Sprague-Dawley rats after implantation of SAINT-18 containing 2 μg of plasmid-expressing green fluorescent protein (p-GFP; control group), 0.2 μg, 2 μg, or 20 μg of p-bFGF from day 0 to day 60. bFGF protein expression was analyzed by Western blotting and immunohistochemistry. Results: The p-bFGF–SAINT-18 complex induced dose-dependent corneal neovascularization, which reached a maximum on days 15–21 in the 20-μg p-bFGF group, days 12–18 in the 2-μg p-bFGF group, and on days 9–15 in the 0.2-μg p-bFGF group, and then regressed progressively. No NV was observed in the p-GFP group. Conclusions: This noninflammatory corneal transfection model using partially dried p-bFGF–SAINT-18 complex allows precise localization of tranfection reagents for producing corneal neovascularization. KEYWORDS angiogenesis; cornea; fibroblast growth factor; nonviral vector; SAINT

INTRODUCTION Neovascularization (NV), the formation of new vascular structures, is a normal process that accompanies tissue growth, reproduction, and repair of damaged tissue during the process of wound healing. Two possible overlapping mechanisms may be involved in neovascularization processes: vasculogenesis, which is the formation of new blood vessels from bone marrow-derived angioblasts, mainly during embryogenesis; and angiogenesis, which is the formation of new vessels from preexisting vascular structures. The latter is the common mechanism in tumor growth and in corneal and retinal disorders, when the balance between angiogenic and anti-angiogenic factors is tilted towards the angiogenic factors.1−4 Therapeutic angiogenesis represents an attempt to relieve inadequate 839


blood flow by directing growth and proliferation of blood vessels. NV is a complex process that involves multiple growth factors, receptors, extracellular matrix glycoproteins, intracellular and extracellular signaling pathways, and local bone-marrow-derived constituent cells.5,6 Several substances are used for inducing corneal neovascularization, such as vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-α and β, and endotoxin.7,8 The basic fibroblast growth factor (bFGF) that was utilized in this study is used extensively in corneal angiogenesis models and is believed to be a major factor in the induction of corneal angiogenesis.9 The fibroblast growth factor (FGF) family encompasses 23 structurally related heparin-binding peptides that are widely expressed in developing and adult tissues during cellular differentiation, angiogenesis, mitogenesis, and wound repair. Moreover, it is widely recognized to act directly on endothelial cells, accelerate cellular migration, and maintain cell proliferation as well as cell mobility.10 FGF function is mediated by binding to its cognate receptors (FGFR 1, 2, 3, and 4). FGF 1 is expressed in the normal corneal epithelium. FGF 2, on the other hand, is upregulated after injury and has been detected in keratocyte-vascular endothelial cell co-culture.11 Damaged corneal epithelium actively produces bFGF, whereas uninjured corneas contain very low levels of the growth factor.12 For gene therapy to be effective, at least three conditions must be satisfied: (1) a functional gene must be placed in an appropriate vector, (2) the gene must be transferred to the nucleus after internalization, and (3) the gene must integrate in the endogenous genome where it has to be translated and transcribed.13 Viral and nonviral vectors containing bFGF cDNA are widely used for gene therapy because of their potential transfection efficiency and capacity to express genes in cells.14−16 Viral vectors are natural gene-delivery systems, and adenovirus is the most effective and the most frequently used viral vector for gene transfer into the corneal endothelium. In contrast to the viral gene delivery systems, there are new classes of pharmaceutical agents that have potential utility in the treatment of human diseases. These gene-delivery vehicles have many inherent advantages over viral vectors in terms of safety, immunogenicity, and commercial availability.16−18 Various nonviral transfection methods, such as membrane integrins, polyamidoamine dendrimers, and electric pulse, have been tested C.-N. Kuo et al.

FIGURE 1 Molecular structure of SAINT-18 (1-methyl-4-(cis-9dioleyl)methylpyridinium-chloride).

for transfection into the corneal endothelium.16,19−21 In this study, we used SAINT-18 (1-methyl-4-(cis9-dioleyl)methylpyridinium-chloride), a cationic synthetic amphiphile, as the delivery system (Fig. 1). This nonvirus-mediated expression of the secreted form of p-bFGF successfully induces corneal angiogenesis in vivo. An important feature of pyridinium-derived amphiphiles is their relatively low toxicity to the cells tested; toxicity is a major drawback in the application of many amphiphiles that have been used thus far.22 We created NV of the cornea, because the cornea’s extreme visibility, transparency, accessibility, avascularity, and highly ordered structure make it unique among all tissues of the body. This is highly advantageous in facilitating biomicroscopic grading of neovascularization responses to topical applications of test agents. In this study, we report a new corneal transgenic angiogenesis model. Therapeutic angiogenesis will likely be offered as an adjunct to conventional revascularization strategies in subsets of patients whose tissues are “suboptimally” revascularized using conventional techniques; it might also evolve into a stand-alone treatment for some patients with diseases that are not “revascularizable,” such as ischemic heart disease.23−25 It is difficult to localize a liquefied plasmid expressing 18-kDa basic fibroblast growth factor (pbFGF)–SAINT-18 complex after it has been transfected into limited regions of experimental tissue. Therefore, in this study, we examined the efficacy of inducing corneal angiogenesis using a partially dried form of p-bFGF–SAINT-18 complex transfected to the corneal stroma (30◦ –45◦ , fan-shaped, central-peripheral, corneal intrastromal lamellar pocket) at ambient temperature in vivo.

MATERIALS AND METHODS Animals Male Sprague-Dawley rats (300–350 g; NSC Animal Center, Taiwan) were used in this study. All protocols and the treatment of animals were in accordance with the Association for Research in Vision and 840

Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.


Naked DNA Vector The bFGF-2 and GFP expression vectors pCMVbFGF-2 and pCMV-GFP, respectively, were kindly provided by Dr. MH Tai (Department of Medical Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan). The plasmids were purified commercially by Clone-E Therapeutics, Inc (Kaohsiung, Taiwan) and were endotoxin free. DNA was produced according to the proprietary process established by Clone-E Therapeutics. Briefly, E. coli (DH5) cells carrying the plasmid were grown in an ampicillin-containing medium in a 5– l fermentor. The fermentation broth was subjected to a series of purification steps, including complete cell lysis, anion exchange, and gel filtration chromatography. The purified plasmids were dialyzed against a formulation buffer (saline, pH 7.0) and were quantified using UV absorbance. Agarose gel analysis showed primarily supercoiled plasmids with a small amount of nicked plasmids.

p-DNA-SAINT-18 Complex Preparation The SAINT-18 (1-methyl-4-(cis-9-dioleyl)methylpyridinium-chloride) (Fig. 1) delivery system (Synvolux Therapeutics, BV, Netherlands) is based on a cationic pyridinium head group, showing excellent biocompatibility. Each vial was “ready to use” and contained 2 ml of SAINT-18 in water at a concentration of 0.75 mM. Before use, SAINT-18 was vortexed thoroughly to minimize micelle size, thereby increasing complexing efficacy. The relative amounts of p-DNA to carrier for p-DNA-SAINT-18 (1 μl of SAINT-18 per 1 μg p-DNA phosphate) were allowed to form at room temperature. To minimize the loss of liquid reagents during transfection into the corneal tissue, the complex was partially dried using a SpeedVac (SpeedVac Model SC110+ VLP120 Oil Vacuum Pump, Savant Instruments, Inc., Farmingdale, NY, USA) at ambient temperature for 60–90 min after the p-bFGF (20 μg, 2 μg, or 0.2 μg) and p-GFP (2 μg) were complexed with SAINT-18. The partially dried form of the complex was extracted from the Eppendorff tube by curettage and prepared for immediate implantation into the corneal pocket. 841

Corneal Pocket Assay All surgical procedures were performed using sterile techniques. The corneal pocket implantation performed in this study was a modification of a previously described technique.7,26−28 The rats were placed under general anesthesia with 3% isoflurane in an O2 /room air mixture (1:1). As additional topical anesthesia, 0.4% benoxinate hydrochloride (Novesin, Ciba Vision, Hettlingen, Switzerland) was applied to the corneal surface. The eyes were proptosed by grasping the temporal limboconjunctival epithelium with a jeweler’s forceps, and a 30◦ –45◦ , fan-shaped central-peripheral, corneal intrastromal lamellar pocket (middle stroma depth; the inlet was 0.7–1.0 mm; the radius was 1.5–2.0 mm) was dissected with a surgical blade (Paragon No. 11, Maersk Medical, Sheffield, UK) and an ophthalmic slit knife (Alcon Inc., Fort Worth, TX, USA). The pocket was extended 1.5 mm from the limbus (Fig. 2). The partially dried form of the p-bFGF–SAINT-18 complex (20 μg, 2 μg, or 0.2 μg) was implanted immediately into the corneal stromal pocket in each eye using forceps and a blade. Topical antibiotic ointment (0.3% gentamicin; Alcon, Cusi, Spain) was applied to the corneal surface to reduce irritation and prevent infection.

Visualization and Quantification of Corneal Neovascularization Examinations were made with a dissecting microscope and results were photographed. While the rats were under anesthesia, the eyes were proptosed and the maximum vessel length and width of the neovascularization region were measured with calipers. Photographs obtained during the corneal angiogenesis assay were taken at a resolution of 640 × 480 pixels using a digital Nikon CoolPix 995 camera (Nikon, Japan). The operator was masked to the treatment group from which each cornea was derived. Areas containing blood vessels were traced on the computer monitor (FT Data Systems, Stanton, CA, USA). The area within the trace was calculated using image analysis software (Enhance 3.0; MicroFrontier, Des Moines, IO, USA) and was reported in square millimeters with determinations confirmed at 40× magnification. Three independent observers conducted masked assessments. p-bFGF–SAINT-18 in Corneal Gene Transfer


FIGURE 2 (A) In order to control the localization of the partially dried (gelled) p-DNA-SAINT-18 complex, a 30◦ –45◦ , fan-shaped, centralperipheral corneal intrastromal lamellar pocket (middle stroma depth; inlet = 0.7–1.0 mm; radius = 1.5–2.0 mm) was dissected with a surgical blade (Paragon No. 11; Maersk Medical, Sheffield, UK) and an ophthalmic slit knife (Alcon Inc., Fort Worth, TX, USA). The pocket was extended 1.5 mm from the limbus. (B) Central-peripheral, corneal, intrastromal lamellar pockets were created in vivo.

Analysis of 18-kDa bFGF Protein Expression On day 15 after transfection in the 2-μg p-bFGF group, two rats were killed with an overdose of thiopental sodium, and fresh corneas were removed along the contour of neovascularization using scissors. These specimens were homogenized by sonication in ice-cold lysis buffer (50 mM Tris, pH 7.5; 150 mM NaCl, 2% Triton X-100, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin), and then centrifuged at 50,000 × g for 30 min at 4◦ C. The protein content of the supernatant was determined using the Bio-Rad Protein Assay system. An equal volume of sample buffer (2% sodium dodecylsulfate [SDS], 10% glycerol, 0.1% bromophenol blue, 2% 2-mercaptoethanol, 50 mM Tris-HCl, pH 7.2) was added to the sample. Proteins were separated by electrophoresis (NuPAGE Electrophoresis; Invitrogen, San Diego, CA, USA) on 15% SDS-polyacrylamide gels at 120 V for 90 min. They were then transferred to polyvinylidene difluoride membranes (0.45 μM pore size; Immobilon-P, Millipore) in transfer buffer (50 mM Tris-HCl, 380 mM glycine, 1% SDS, 20% methanol) at 50 V for 60 min. The membrane was blocked with 5% nonfat dry milk in Tween-20 and Tris-buffered saline (TTBS; 0.1% Tween-20, 20 mM Tris-HCl, 137 mM NaCl, pH 7.4) for 60 min at room temperature. The membrane was then incubated with anti-bFGF antibody (purified anti-human FGF-basic antibody; C.-N. Kuo et al.

BioLegend, San Diego, CA, USA) for 90 min at room temperature. Each blot was washed three times for 10 min in TTBS, blocked with 5% nonfat dry milk in TTBS, and then incubated with horseradish-peroxidaseconjugated secondary antibody (1:1000; Transduction Laboratories) for 1 hr at room temperature. Antibody labeling was detected by chemiluminescence (ECL, Amersham). Colored molecular-weight standards were run in parallel on each gel. The housekeeping geneα-tubulin was used as the control.

Histological Examination On day 15 after transfection in the 2-μg p-bFGF group, the rats were killed with an overdose of thiopental sodium; their eyes were then enucleated, fixed in paraformaldehyde, and frozen at −70◦ C in OCT (optic cutting temperature) compound. Corneas were excised and cryostatically cut into 3- to 8-μm sections for hematoxylin and eosin staining and immunohistochemistry. Excised corneal tissues for immunohistochemistry were fixed in Bouin’s solution and embedded in paraffin. After the different sections were cut (3 μm) and mounted on slides coated with poly-L-lysine, they were immunostained (ImmunoBlot, Invitrogen) with anti-bFGF antibody (purified anti-human FGF-basic antibody; BioLegend, San Diego, CA, USA) and anti-rat HLADR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). bFGF immunoreactivity was visualized 842

with DAB chromogen (3,3’-diaminobenzidine tetrahydrochloride) using a polymer detection system (Zymed, San Francisco, CA, USA). Tissues were counterstained with hematoxylin (blue).

Statistical Analyses


Repeated measures analysis of variance (SPSS version 10.0 for Windows; SPSS Inc., Chicago, IL, USA) and the Bonferroni post hoc test were used to analyze differences in the length and area of corneal neovascularization between pairs of groups. Length and area were measured every 3 days from day 0 to day 60. The values for each measure were recorded as separate variables and were defined as within-subject factors. The assigned groups were defined as between-subject factors. A value of p ≤ 0.05 was considered statistically significant.

RESULTS Biomicroscopic Examination of Corneal Neovascularization Forty-eight rats (48 eyes) were divided equally into three experimental groups (20-, 2-, and 0.2-μg p-bFGF

groups) and one control group (2-μg p-GFP). Biomicroscopic examinations revealed that the corneal epithelium healed within 24 hr of surgery. Corneal edema and limbal injection were noted in all corneas. Limbal vessels began sprouting into the cornea on postoperative day 3. Corneal neovascularization was induced in a dose-dependent manner by the partially dried form of the p-bFGF–SAINT-18 complex. Neovascularization reached a maximum on days 15–21 in the 20-μg p-bFGF group, days 12–18 in 2-μg p-bFGF group, and days 9– 15 in the 0.2-μg p-bFGF group, and then regressed progressively. The neovascularization response was intense, localized, and reproducible. Maximal growth of neovascularization is shown in Figure 3. Compared to the control group, neovascularization occurred in the 20μg p-bFGF, 2-μg p-bFGF, and 0.2-μg p-bFGF groups. However, the standard deviation of length and area were high in the 0.2-μg p-bFGF group (e.g., day 9, 2506 ± 3016 × 10−4 mm; day 12, 2517 ± 3550 × 10−4 mm) (Fig. 3). Data from the repeated measures ANOVA for the four groups were compared. The results of length data were F = 567.16, p < 0.001 between the 2-μg pGFP group and the 2-μg p-bFGF and 20-μg p-bFGF

FIGURE 3 Slit lamp photographs of Sprague-Dawley rat corneas showing maximum corneal neovascularization for each group after dilatation with tropicamide 1% + phenylephrine 10% ophthalmic solution. (1) Control group on day 15 (2-μg p-GFP); (2) 0.2-μg p-bFGF group on day 12; (3) 2-μg p-bFGF group on day 15; (4) 20-μg p-bFGF group on day 18. *Partially dried p-DNA–SAINT-18 complex.

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p-bFGF–SAINT-18 in Corneal Gene Transfer


FIGURE 4 The partially dried form of the p-bFGF–SAINT-18 complex produced dose-dependent corneal neovascularization; the mean length (A) and area (B) of neovascularization reached a maximum on days 9–15 in the 0.2-μg p-bFGF group, days 12–18 in the 2-μg p-bFGF group, and days 15–21 in the 20-μg p-bFGF group, and then regressed progressively.

groups, but p = 0.056 between the 2-μg p-GFP and 0.2-μg p-bFGF groups. The results of area data were F = 94.87, p < 0.001 between the 2-μg p-GFP group and the 2-μg p-bFGF and 20-μg p-bFGF groups, but p =1.000 between the 2-μg p-GFP and 0.2-μg p-bFGF groups (Fig. 4).

anti-human FGF-basic antibody [BioLegend]), but were absent from the control sample proteins. When similar amounts of plasmid expressing 2- and 20-μg bFGF without SAINT-18 were implanted into the corneal micropockets, these bands were absent in the control groups (Fig. 5).

Western Blot Analysis

Histology

On day 15 after transfection (2-μg p-bFGF group), two rats were killed with an overdose of sodium thiopental and corneal samples were removed along the contour of neovascularization. Levels of 18-kDa bFGF protein were estimated by Western blot analysis. The α-tubulin band was used as the control for normalization. The bFGF bands (molecular weight approximately 18 kDa) were detected in protein from the groups treated with 20-, 2-, or 0.2-μg p-bFGF–SAINT-18 using rabbit anti-human bFGF polyclonal antibody (purified

The reporter gene (for green fluorescein protein expression) was expressed within the corneal intrastromal layer and in keratocytes (Fig. 6A; SAINT-18(+)), but not in the control group (Fig. 6A; SAINT-18(–)). A vast number of capillaries appeared in the corneal stroma, which displayed cells, edema, a mononuclear inflammatory response, and numerous vascular lumens running from the limbal blood vessels up to the 18-kDa bFGF implant (Fig. 6B; SAINT-18(+)). Some inflammatory reaction was noted, but no lumens in the control

C.-N. Kuo et al.

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FIGURE 5 (A) Levels of 18 kDa bFGF protein were estimated by Western blot analysis. The α-tubulin band served as the normal control; bFGF bands (molecular weight approximately 18 kDa) produced with 20, 2, 0.2 μg p-bFGF-encoded plasmids. This band is absent in the 2 μg p-GFP sample. (B) Similar amount of plasmid expressing 2 and 20 μg bFGF without SAINT-18 had been implanted into the corneal micropockets; these bands were absent at the control groups.

group. Immune staining (anti-rat HLA-DR antibody) of macrophages and other inflammatory cells, such as lymphocytes indicated inflammation (Fig. 6C). Some inflammatory cells were noted in the control group, but the reactions were milder. Immunohistochemistry staining with anti-human bFGF antibody was positive for bFGF in the implanted region; keratocytes and epithelial cells were stained with rabbit anti-bFGF polyclonal antibody (purified anti-human FGF-basic antibody [BioLegend]) (Fig. 6D)., but not in the control group.

DISCUSSION The primary purpose of this study was to develop a novel and accurate method of gene transfer into the corneal stroma in vivo to induce angiogenesis using a nonviral vector, a partially dried p-bFGF–SAINT-18 complex, at room temperature. Our results showed 845

that three doses of p-bFGF(20 μg, 2 μg, and 0.2 μg)– SAINT-18 complexes induced corneal angiogenesis (Figs. 3 and 4). One of the major determinants for successful gene therapy is the use of a gene delivery technique. Several nonviral and viral vectors have been tested in pre-clinical models for ocular gene delivery. Of the nonviral vectors, (Lys)(16)-based reducible polycation/DNA/fusogenic peptide particles are reported to be highly effective for gene delivery to both cell lines and post-mitotic corneal endothelium.29 Adenoviral vectors encoding the interleukin-12p40 gene30 and the nerve growth factor gene31 prevent corneal allograft rejection. Several angiogenic factors including bFGF,32 vascular endothelial growth factor (VEGF), and transforming growth factors (TGF)-α and −β play vital roles in corneal neovascularization.3 Several anti-angiogenic factors, such as angiostatin, endostatin,33−35 and pigment epithelium-derived factor (PEDF),36 could play roles in the control of corneal neovascularization. The regulation of corneal angiogenesis is a complex process that involves the equilibrium between pro- and antiangiogenetic factors. Loss of equilibrium between these factors results in abnormal corneal angiogenesis.37 In this study, the effects of our nonviral vector system differed between each group; neovascularization reached a maximum on days 15–21 in the 20-μg p-bFGF group, days 12–18 in the 2-μg p-bFGF group, and days 9–15 in the 0.2-μg p-bFGF group, and then regressed progressively. The transgenic outcomes were longer in our study than in the studies by Wu et al,35 who used 90 ng of bFGF, and Kenyon et al,7 who used (180 ng of VEGF). Both of those studies revealed that corneal neovascularization reached a maximum on days 5 and 6. However, our technique involving surgical procedures does not allow for long-term treatment of eye disease, but results in rather limited expression of the transgene. To preclude the possibility of interference by intrinsic bFGF during surgery, 2 μg of p-GFP–SAINT-18 was used in the control group; no neovascularization was observed in this group. p-GFP was used as a control in this study because it could be directly imaged using a fluorescence microscope. GFP is a powerful marker for assaying the quantity of a particular protein in a cell. The Bonferroni test indicated significant differences in length and area of neovascularization between the 2-μg p-GFP, 2-μg p-bFGF, and 20-μg p-bFGF groups; however, the test revealed no significant differences between the 2-μg p-GFP and 0.2-μg p-bFGF groups (p > 0.05). According to these results, if we wished p-bFGF–SAINT-18 in Corneal Gene Transfer


FIGURE 6 Corneal angiogenesis in vivo induced by partially dried p-bFGF–SAINT-18 complex (2 μg) in rats. Fifteen days after administration, the corneal tissue was histologically evaluated. (A) Green fluorescein protein was expressed within the corneal intrastromal layer and keratocytes, but not in the control group. (B) The intervening stroma displayed cells, edema, a mononuclear inflammatory response, and numerous vascular lumens (indicated by arrows) after hematoxylin and eosin staining. Some inflammatory reaction was noted, but no lumens in the control group (100× magnification). (C) Inflammation was detected by the presence of some macrophages and other inflammatory cells such as lymphocyte by immune staining (anti-rat HLA-DR antibody). Some inflammatory cells were noted in the control group, but the reactions were milder. (D) Immunohistochemistry anti-human bFGF antibody was positive in the implanted region; keratocytes and epithelial cells (indicated by arrows) were stained with purified anti-human basic-FGF antibody (BioLegend), but not in the control group.

to obtain a stable corneal neovascularization model, a dose of at least 2 μg of p-bFGF would be needed to induce corneal neovascularization at the limbus. In our animal model, we chose to study the cornea because of its accessibility and avascularity, factors that facilitate the biomicroscopic grading of neovascularization responses to topical applications. Several angiogenesis models involving corneal intrastromal implantation are described, including sustained-release pellets containing bFGF protein and sucrafate into rabbit eyes,38 and even the injection of interleukin-2 into the mouse cornea.7,39 Serial observations and measurements of neovascularization response in localized sectors, or the inhibition thereof, can be readily and noninvasively documented with slit lamp biomicroscopy. The cornea has five layers, including the epithelium, Bowman’s membrane, the stromal layer, Descemet’s membrane, and the endothelium layer. In our study, we created a corneal intrastromal lamellar pocket, dissecting through the epithelium, Bowman’s membrane, and stromal layer. ImmunohisC.-N. Kuo et al.

tochemistry (anti-human bFGF antibody) was positive in the implanted region. In contrast, gene expression in other areas of the corneas was not significant. Chollet et al.40 demonstrated that necrotic areas were always observed in the livers of animals that received high doses of DNA-polyethylenimine complexes. Additionally, shock was observed in minutes following the intravenous injection. We also noted that greater corneal edema and limbal injection occurred after implantation of DNA-PEI complex in a previous study.15 On the contrary, Audouy,41 and Hosper et al.42 showed that p-DNA-SAINT-18 complexes were safe, low-toxicity vehicles for gene transfer, and efficient for in vivo gene delivery. Thus, we changed the nonviral vector to SAINT-18 for this study. In order to reduce the volume of implanted reagents, we partially dehydrated the p-bFGF–SAINT-18 complex using a SpeedVac at ambient temperature for 10– 90 min (the range was adjusted for different doses: 0.2 μg, 2 μg, or 20 μg of p-bFGF). The partially dried form of the complex was then stored in an ice bath until 846


the corneal pocket was complete. The implant was then inserted immediately into the corneal pocket and rehydrated with tissue fluid. Our results showed that the partially dried form of the p-bFGF–SAINT-18 complex produced by partial drying without freezing was transfected successfully and subsequently induced corneal neovascularization (Figs. 3 and 4). Therefore, using partially dried formulations appears to be an effective approach for stabilizing and storing these plasmids. One of the limitations of this study was the lack of control over localizing the partially dried form of the vector, especially during implantation into the corneal tissue. We believe that creating partially dried p-DNASAINT-18 complexes and dissecting a 30◦ –45◦ , fanshaped, central-peripheral, corneal intrastromal lamellar pocket could increase the accuracy of delivery. To our knowledge, no previous work investigated the implantation of partially dried forms of p-bFGF–SAINT-18 complexes into the corneal stroma. We strongly recommend this novel animal model of angiogenesis. In this study, we demonstrate efficacy of a nonviral gene delivery method that uses transfection mixtures prepared from SAINT-18 and plasmid vector expressing GFP marker gene or bFGF. The bFGF gene is known to induce corneal neovascularization in the eye in vivo and has been selected to study the dose-dependent effects of dried SAINT-18-DNA complex. In conclusion, the transfection mixture made of dried SAINT-18-plasmid complex can be used to express genes in the cornea.

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ACKNOWLEDGMENTS This work was supported by a project grant from Chang Gung Memorial Hospital Grand CMRP and the National Science Council Grant (NMRP and genome project). Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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