July 2007
Volume 48, Issue 7
Free
Retinal Cell Biology  |   July 2007
Prolonged Protective Effect of Basic Fibroblast Growth Factor–Impregnated Nanoparticles in Royal College of Surgeons Rats
Author Affiliations
  • Tsutomu Sakai
    From the Department of Ophthalmology, Jikei University School of Medicine, Tokyo, Japan; and the
  • Noriyuki Kuno
    Research and Development Center, Santen Pharmaceutical Co., Ltd., Nara, Japan.
  • Fumihiko Takamatsu
    Research and Development Center, Santen Pharmaceutical Co., Ltd., Nara, Japan.
  • Erika Kimura
    Research and Development Center, Santen Pharmaceutical Co., Ltd., Nara, Japan.
  • Hideo Kohno
    From the Department of Ophthalmology, Jikei University School of Medicine, Tokyo, Japan; and the
  • Kiichiro Okano
    From the Department of Ophthalmology, Jikei University School of Medicine, Tokyo, Japan; and the
  • Kenji Kitahara
    From the Department of Ophthalmology, Jikei University School of Medicine, Tokyo, Japan; and the
Investigative Ophthalmology & Visual Science July 2007, Vol.48, 3381-3387. doi:10.1167/iovs.06-1242
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      Tsutomu Sakai, Noriyuki Kuno, Fumihiko Takamatsu, Erika Kimura, Hideo Kohno, Kiichiro Okano, Kenji Kitahara; Prolonged Protective Effect of Basic Fibroblast Growth Factor–Impregnated Nanoparticles in Royal College of Surgeons Rats. Invest. Ophthalmol. Vis. Sci. 2007;48(7):3381-3387. doi: 10.1167/iovs.06-1242.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the protective effect of intravitreal injection of basic fibroblast growth factor-impregnated nanoparticles (bFGF-NPs) against photoreceptor degeneration in Royal College of Surgeons (RCS) rats.

methods. Three-week-old RCS rats received intravitreal injection of PBS, blank NPs, bFGF (2.5 μg), or bFGF-NPs (2.5 μg). Eyes were assessed by morphologic, immunohistochemical, and physiological analyses for the following 8 weeks. Cell death was examined using the TUNEL assay, and bFGF protein levels in the retina were measured by Western blot analysis. Rhodamine (Rh)-labeled bFGF-NPs were injected intravitreally and visualized by confocal microscopy to determine the localization of the nanoparticles in the retina.

results. Intravitreally injected Rh-labeled bFGF-NPs were found in the outer nuclear layer 6 and 8 weeks after injection. ERG a- and b-wave amplitudes in bFGF-NP–treated retinas were greater than amplitudes in retinas receiving other treatment. Immunocytochemical analysis showed consistently greater opsin preservation in bFGF-NP–treated retinas, and a significantly higher number of photoreceptors and significantly fewer TUNEL-positive cells were present after bFGF-NP treatment than after bFGF treatment. Western blot analysis showed a significant increase in the bFGF level in bFGF-NP–treated retinas.

conclusions. The results suggest that intravitreally injected bFGF-NPs prevent photoreceptor degeneration by inhibiting apoptosis in the RCS rat retina because of targeting and sustained release of bFGF. This novel drug delivery system for bFGF may serve as a potential short-term treatment for photoreceptor degeneration in humans.

Basic fibroblast growth factor (bFGF) has well-documented protective neurotrophic activity in rats with photoreceptor degeneration. 1 2 3 4 5 6 However, although a single intravitreal or subretinal injection of bFGF delays photoreceptor degeneration, such treatment does not allow long-term rescue of photoreceptors because of the short half-life of the trophic factor. Therefore, recent “rescue” studies using bFGF have focused on efficient approaches for continuous delivery; for example, gene therapy with viral vectors has been found useful in preventing the progression of photoreceptor degeneration in rats. 7 8 9 The prolonged effect of bFGF is presumably beneficial; however, several problems remain to be solved for potential human applications. In particular, some viral vectors are biologically unsafe because of severe immune reactions or oncogenic effects, and subretinal injection for bFGF gene transfer has potential risks for retinal detachment, proliferative vitreoretinopathy, and choroidal neovascularization. Thus, it is important to develop a delivery system that permits enhanced localization of bFGF at the target site and permits sustained drug release without causing serious complications. 
Various biodegradable polymeric particles have been investigated for increasing bioavailability and prolonging controlled release of bioactive molecules. 10 Among these polymers, the gelatin matrix is a promising carrier system for controlled drug delivery of polypeptides or proteins. 11 12 Binding of therapeutic drugs to a gelatin polymer provides the drug with long-term protection from enzymatic degradation and thereby enhances its effectiveness. 13 Furthermore, because gelatin is a nontoxic and noncarcinogenic material of low antigenicity, it has the potential for sustained delivery of polypeptides or proteins in clinical use. 14  
Nanoparticles (NPs), which are nanometer-sized spherical polymeric particles made of natural or synthetic polymers, have also been examined as technology for the improvement of pharmacologic and therapeutic properties of various drugs. 15 16 The therapeutic advantages associated with NPs include relative ease of crossing tissue barriers and provision of active doses. Studies on intraocular kinetics of biodegradable NPs after injection into the vitreous cavity have indicated that NPs can be used to deliver drugs to the retina 17 ; the results suggest that biodegradable NPs carrying incorporated or conjugated drugs may serve as new therapies for vitreoretinal disorders because of efficient targeting and sustained drug retention. 
We hypothesized that intravitreal injection of gelatin NPs with bFGF would increase the efficacy of bFGF in preventing photoreceptor degeneration through improved targeting and sustained release. In this study, we examined the protective effect of intravitreally injected bFGF-NPs against photoreceptor degeneration in Royal College of Surgeons (RCS) rats. 
Materials and Methods
Animals and Anesthesia
RCS rats obtained from CLEA Japan (Tokyo, Japan) were used in the study. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were anesthetized with a mixture (1:1) of ketamine hydrochloride (10 mg/kg; Wako Pure Chemicals Industries, Ltd., Osaka, Japan) and xylazine hydrochloride (4 mg/kg; Wako). 
Preparation of bFGF-NPs
bFGF-NPs were prepared as follows, using acidic gelatin isolated from bovine bone collagen by an alkaline process (S-4; Nitta Gelatin, Osaka, Japan; isoelectric point: 5.0; MWt 99,000 Da) and human recombinant bFGF (PeproTech Inc., Rocky Hill, NJ). Cross-linked gelatin NPs were prepared through a dehydrothermal process and ultraviolet irradiation of pre-prepared non–cross-linked gelatin particles. In brief, gelatin aqueous solution (100 mg/mL, 0.2 mL) was added to preheated olive oil (5 mL) at 40°C, followed by agitation for 1 minute. The resultant emulsion was cooled in crushed ice to allow solidification of aqueous gelatin droplets. Acetone was then added to the emulsion, and stirring was continued for 1 hour at 4°C. Resultant particles were washed three times with acetone and recovered by centrifugation at 3500 rpm and 4°C for 5 minutes. Non–cross-linked gelatin particles were dried in a desiccator at 4°C and then placed in a glass dish and heated at 160°C for 72 hours. Gelatin particles were then exposed to ultraviolet irradiation for 30 minutes, after which the particles were suspended in distilled water and filtrated using a membrane filter (pore size, 1 μm). The filtrate was freeze-dried, and cross-linked NPs were obtained. The NP diameter was measured by dynamic light scattering (NICOMP-370; Particle Sizing Systems, Santa Barbara, CA); the average diameter measured approximately 585 nm. 
bFGF was incorporated into the gelatin NPs by the dropping of 5 mg/mL bFGF solution (20 μL) onto 2 mg freeze-dried gelatin NPs, which were then left to stand at 4°C for 12 hours. The solution (20 μL) was completely absorbed into the NPs during swelling because its volume was less than that required theoretically for equilibrative swelling of the NPs. 
In Vivo Evaluation for Intraocular Kinetics of NPs
Gelatin NPs were radioiodinated using iodine I 125 (125I)–Bolton-Hunter reagent. Briefly, 100 μL 125I-Bolton-Hunter reagent solution in anhydrous benzene (NEX120H; PerkinElmer, Boston, MA) was bubbled with dry nitrogen gas until benzene evaporation was complete. Then, 200 μL of 0.1 M sodium borate-buffered solution (pH 8.5) was added to the dried reagent to prepare an aqueous solution of 125I-Bolton-Hunter reagent, and 10 mg freeze-dried, cross-linked gelatin NP was impregnated with this solution. Five microliters of the suspension of 125I-labeled, cross-linked gelatin NPs in PBS (50 μg/μL) was injected into the vitreous of each male Wistar rat (n = 28; postnatal week 5; Charles River Japan, Yokohama, Japan). At predetermined intervals, rats were killed with ether, and eyeballs were enucleated. The remaining radioactivity in the enucleated eyeball was determined by a gamma counter (Cobra II Series Auto-Gamma Counting System; Packard Instrument Co., Meriden, CT). 
Localization of Rhodamine-Labeled bFGF-NPs
Rhodamine-B isothiocyanate (RITC; Sigma-Aldrich, St. Louis, MO) was conjugated to freeze-dried, cross-linked gelatin NPs. In brief, 25 mg freeze-dried cross-linked gelatin NPs were rehydrated in 1 mL carbonate–bicarbonate solution (pH 9.5). Then, 1.38 mg RITC in 1 mL dimethyl sulfoxide was added to this suspension and kept at room temperature for 3 hours. The resultant suspension was washed with a mixture of similar volumes of dimethyl sulfoxide and distilled water four times and with distilled water three times. Finally, rhodamine-labeled, cross-linked gelatin NPs (Rh-NPs) were stored after freeze-drying. bFGF was incorporated into Rh-NPs by the dropping of 5 mg/mL of bFGF solution (20 μL) onto 2 mg freeze-dried Rh-NPs, which were then left to stand at 4°C for 12 hours. 
RCS rat received intravitreal injection of 5 μL Rh-labeled bFGF-NPs and were killed 6 or 8 weeks after injection (n = 3 for each time period). Eyes were removed, and agarose-embedded sections of 100-μm thickness were cut on a vibratome (Leica VT1000S; Leica Microsystems, Heerbrugg, Switzerland). These sections were immediately observed by confocal microscopy (Laser Scanning System LSM510; Carl Zeiss Meditech, Oberkochen, Germany), with images obtained by differential interference contrast. 
Intravitreal Injection of bFGF-NPs, bFGF, Blank NPs, and PBS
Intravitreal injection of the right eyes was performed through the pars plana with 5 μL bFGF-NPs (containing 2.5 μg bFGF), bFGF (2.5 μg), or blank NPs on day 21 (n = 15 in each group), when photoreceptor degeneration was just beginning (Fig. 1) . Left eyes were uninjected or received PBS (pH 7.4; Wako) as the control. Injections were performed with a syringe (80001; Hamilton Co., Reno, NV) with a 30-gauge needle while viewing the eye under a microscope. Animals with hemorrhage or cataract were excluded. The detailed experimental protocol is shown in Figure 1
Electroretinographic Analysis
Scotopic ERGs were recorded in both eyes simultaneously with a Ganzfeld bowl. Rats were dark adapted overnight and were prepared under dim red light before anesthetization with single intraperitoneal injections of ketamine and xylazine. Pupils were dilated by topical 0.5% tropicamide and 0.5% phenylephrine, and the cornea was anesthetized with topical 0.4% oxybuprocaine hydrochloride. 
Gold wire loops were placed on the center of the cornea, reference electrodes were placed subcutaneously under each eye, and a ground electrode was inserted into the tail. ERGs were elicited with 10-ms flashes of white light, and responses were recorded (Synax ER1100; NEC San-ei Instruments, Tokyo, Japan). The a-wave amplitudes were measured from the prestimulus baseline to the bottom of the a-wave, and b-wave amplitudes were measured from the a-wave peak to the most positive peak. 
Histopathology and Immunocytochemistry
Rats were killed with sodium pentobarbital (intravenously; Wako) 4, 6, or 8 weeks after injection (n = 4 for each time period), and enucleated eyes were immersion fixed for 10 minutes in 4% paraformaldehyde in a sodium cacodylate buffer (0.1 N; pH 7.4; Wako). After the cornea and lens were removed, the eyecup was cut in half. Half the tissue was stored in the fixative solution, and small areas of retina were excised and embedded in low melting point agarose (Sigma-Aldrich) for immunocytochemical analysis by confocal microscopy. Embedded sections were cut on a vibratome and blocked overnight in normal donkey serum (1:20; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 4°C. Sections were then incubated with primary antibodies overnight at 4°C on a rotator. Primary antibodies used in this study were a mouse monoclonal antibody (mAb) to rod opsin (1:400; Chemicon, Temecula, CA), a mouse mAb to vimentin (1:400; Sigma), a rabbit polyclonal Ab (pAb) to glial fibrillary acidic protein (1:400; GFAP; Dako, Glostrup, Denmark), and a rabbit pAb to medium/long wavelength-sensitive (M/L) cone opsin (1:200; Chemicon). All antibody solutions were made in PBTA (0.1 M PBS) containing 0.5% bovine serum albumin (BSA; Fisher Scientific, Pittsburgh, PA), 0.1% Triton X-100 (Boehringer-Mannheim, Indianapolis, IN), and 0.1% sodium azide (Sigma-Aldrich). After the sections were rinsed in PBTA, they were incubated with donkey anti–mouse IgG conjugated to the fluorochrome Cy3 (GFAP, M/L cone opsin) and donkey anti–rabbit IgG conjugated to the fluorochrome Cy2 (rod opsin, vimentin; Jackson ImmunoResearch Laboratories) overnight at 4°C on a rotator. Sections were mounted in mounting medium for fluorescence (Vectashield; Vector Laboratories, Inc., Burlingame, CA) and were viewed using a laser scanning confocal microscope. 
For high-resolution transmitted light microscopy analysis, the other half of the eyecup was immersion fixed in 1% glutaraldehyde (Wako) and 1% paraformaldehyde (Wako) in a sodium phosphate buffer (0.086 M; pH 7.3) overnight at 4°C and then was fixed in phosphate-buffered osmium tetroxide (2%; Nisshin-EM Co., Tokyo, Japan) for 1 hour and embedded in Epoxy resin (Nisshin-EM Co.). Samples were sectioned at 1 μm and were stained with toluidine blue (Wako). 
The number of photoreceptor cells was counted using a standardized approach adapted from a previously described protocol. 18 Three 50-μm sampling bins were established for each of six retinal regions: superior peripheral, superior equatorial, superior central, inferior central, inferior equatorial, and inferior peripheral. Three sections were examined for each eye, giving a total of nine samples per region and 54 samples per eye. 
TUNEL Assay
Detection of dying (apoptotic) cells was achieved using a terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay, as described previously. 19 The agarose-embedded sections were used in this assay. After the sections were rinsed three times in PBS, they were immersed in 70% alcohol for 30 minutes, followed by washing in double-distilled water. They were incubated in 1% citrate and 1% Triton in PBS at 4°C for 4 minutes and, after further washing, were placed in terminal deoxynucleotidyl transferase (TdT) buffer (Boehringer Mannheim, Indianapolis, IN) at room temperature for 30 minutes. They were then incubated with TdT enzyme and 2 μM biotinylated deoxyuridine triphosphate (dUTP; Boehringer Mannheim) at 37°C for 120 minutes. Finally, the sections were washed in SSC (150 mM sodium chloride and 15 mM sodium citrate, pH 7.4) for 15 minutes, rinsed with PBS, and examined with a confocal microscope. 
TUNEL-positive cells were counted in four areas of each section from four different eyes. The percentage of dying photoreceptor cells was expressed as the number of TUNEL-positive cells divided by the total number of nuclei in the outer nuclear layer (ONL). Nuclear counts were normalized using the number of cells per millimeter of retinal length. 
Western Blot Analysis
Freshly isolated rat retinas were frozen in liquid nitrogen. They were then homogenized in 1× lysis buffer (Reporter Lysis 5× Buffer; Promega, Madison, WI) and kept at 4°C for 1 hour. The protein concentration was determined using a BCA kit (Micro BCA Assay Reagent Kit; Pierce Chemical, Rockford, IL). Five micrograms of protein from each sample were fractionated on a 15% SDS-PAGE with protein markers (BMA ProSieve Color Protein Markers; BioWhittaker Molecular Applications, Rockland, ME). After electrophoresis, proteins were transferred to a nitrocellulose membrane. The blot was incubated in blocking solution (5% milk protein, 0.1% Tween-20 in PBS) for 2 hours, followed by incubation with the anti–FGF2 clone bFM-2 mouse monoclonal IgG (1:1000; Upstate Biotechnology, Lake Placid, NY) in blocking buffer at 4°C overnight. The membrane was then incubated with goat anti–mouse IgG conjugated with horseradish peroxidase (1:10,000; Chemicon International) in blocking buffer for 1 hour at room temperature. Antibody detection was performed with enhanced chemiluminescence (Chemiluminescence System and SuperSignal West Pico Chemiluminescent Substrate; Pierce Chemical). Densitometric analysis was performed with Image J software (available by FTP at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). 
Statistical Analysis
All data are shown as mean ± SD. Data were analyzed using a nonparametric Mann-Whitney U test, and P values < 0.05 were considered statistically significant. 
Results
Long-term Delivery of NPs in Rat Vitreous
The time course for the remaining radioactivity in rat vitreous after intravitreal injection of 125I-labeled cross-linked gelatin NPs is shown in Figure 2 . A burst was observed within the first 24 hours, and radioactivity from NPs in the vitreous was sustained over 30 days. 
Long-term Retention of bFGF-NPs in the Retina
Several Rh-labeled bFGF-NPs were observed in the retina 6 weeks after injection (Fig. 3A) . These NPs also remained in the retina 8 weeks after administration because diffuse red staining was observed in the inner nuclear layer (INL), ONL, outer segment debris zone, and retinal pigment epithelium (RPE; Fig. 3B ). NPs may well be in the numerous microglia/macrophages that occupy these layers. 
Effects of bFGF-NPs on Morphology
Representative histopathologic features of the superior retina of RCS rats in week 8 after injection are shown in Figure 4 . In the blank NP-treated eyes, a remarkable decrease in the total nuclei in the ONL was observed (Figs. 4C 4D) , and disruption of the inner and outer segments of all surviving photoreceptors occurred in all areas. The morphology of eyes treated with bFGF-NPs is illustrated in Figures 4A and 4B . Dramatic changes are apparent in comparison with blank NP-treated eyes. In all areas, a greater number of nuclei remained in the ONL, and the inner segment morphology was disrupted to a lesser extent than in blank NP-treated eyes. The ONL in all areas of bFGF-NP–treated eyes was much thicker than in blank NP-treated eyes. 
The data in Figure 5show how intravitreal injection of bFGF-NPs affected the actual number of nuclei in the ONL based on cell counts in semithin resin sections. The number of nuclei in the ONL was significantly greater in bFGF-NP–treated retinas (P < 0.05) 8 weeks (Fig. 5)after injection, and the effects were dramatic in all sampling areas. 
Immunohistochemical Evaluation Using Confocal Microscopy
Immunohistochemical results for retinas collected 8 weeks after injection from rats treated with blank NPs, bFGF, and bFGF-NPs are shown in Figure 6 . In the blank NP-treated retinas, rod and cone opsin was present in only small amounts in the collapsed rods and cones in the outer segment, and GFAP was expressed in activated Müller cells (Figs. 6A 6D) . Enhanced activation of Müller cells was also observed in bFGF-treated retinas (Figs. 6B 6E) . In contrast, rats treated with bFGF-NPs maintained rod and cone opsin expression and showed less GFAP expression and greatly reduced Müller cell activation (Figs. 6C 6F) . In Figures 6D 6E 6F , some light immunoreactivity with the M/L cone opsin antibody occurred in retinal ganglion cells and INL cells, but this was the same in all preparations. 
Effects of bFGF-NPs on Physiology
bFGF-NP–treated retinas showed significant preservation of a- and b-wave amplitudes 6 weeks after injection, compared with bFGF-treated retinas (a-wave, P < 0.005; b-wave, P < 0.05; Figs. 7A 7B ). Mean amplitudes of a- and b-waves in bFGF-NP–treated retinas 8 weeks after injection were 6.8 and 32.9 μV, respectively, whereas no ERG responses were found in PBS-, blank NP-, and bFGF-treated retinas after 8 weeks. 
Effects of bFGF-NPs on Photoreceptor Cell Death
Using agarose-embedded sections, TUNEL data for the effects of intravitreally injected bFGF-NPs were determined 4 weeks (Figs. 8A 8B 8C)and 8 weeks after injection (Figs. 8E 8F 8G) , and these data were used to determine the percentage of TUNEL-positive cells in the ONL after these time periods (Figs. 8D 8H) . In blank NP-treated retinas, the percentage of TUNEL-positive cells in the ONL was much greater (Figs. 8D 8H) , but in bFGF-NP-treated retinas there was a significant reduction compared with bFGF-treated retinas in the percentage of cells in the ONL that were TUNEL positive (Fig. 8D , P < 0.05; Fig. 8H , P < 0.01). 
Effects of bFGF-NP on bFGF Expression in the Retina
To determine whether the effect of bFGF-NPs on photoreceptor rescue resulted from an increase in the bFGF level, the amount of bFGF in the retina was measured, through Western blot analysis, with semiquantification using the anti–bFGF antibody. Treatment with bFGF-NPs resulted in a 2.7-fold increase in retinal bFGF levels (P < 0.001) compared with retinas treated with bFGF, suggesting that intravitreal injection of bFGF-NP leads to increased bFGF levels in the retina of RCS rats for at least 6 weeks after injection (Fig. 9)
Discussion
Intravitreal injection of bFGF has been shown to rescue rodent photoreceptors from genetic degeneration and the damaging effect of light. 1 2 3 4 5 6 7 8 9 In this study, our goal was to test the ability of bFGF encapsulated in gelatin NPs to rescue photoreceptors in a well-established model of photoreceptor degeneration and to determine whether NP-based delivery enhances the protective effects of bFGF. 
Our data for the intraocular kinetics of intravitreally injected fluorescence-labeled bFGF-NPs show that the NPs can be delivered to photoreceptors directly and continuously. This result prompted us to determine whether this system can be used as an effective drug delivery system for bFGF for the prevention of photoreceptor degeneration. We found a significantly increased number of cells in the ONL and a preservation of a- and b-wave amplitudes in the ERG studies in bFGF-NP–treated retinas 6 and 8 weeks after injection. These observations suggest that intravitreal injection of bFGF-NPs prevented photoreceptor degeneration in RCS rats morphologically and functionally, and this conclusion was supported by the effect on photoreceptor apoptosis. Data collected in week 8 after injection suggested that bFGF-NP treatment did not maintain significant functional preservation at this time; rather, the maintenance of morphology was evident, including a reduction in photoreceptor apoptosis. Although the average amplitude of the a-wave or b-wave 8 weeks after injection did not differ significantly between bFGF-NP–treated retinas and those receiving other treatment, we believe that the differences in morphology 8 weeks after injection are important. Taken together, the results suggest that a single intravitreal injection of bFGF-NP has a significant protective effect on photoreceptor degeneration, morphologically and functionally, through the inhibition of apoptosis in RCS rat retinas because of effective targeting and sustained release of bFGF. 
Positively charged bFGF is electrostatically complexed with negatively charged acidic gelatin chains. If an environmental change, such as increased ionic strength, occurs, the complexed bFGF will be released from the bFGF–gelatin complex. Even if such an environmental change does not take place, enzymatic degradation of the gelatin matrix itself will also lead to bFGF release. Because the latter is more likely to happen in vivo than the former, it is thought that bFGF is preferably released by enzymatic degradation of gelatin matrix. Therefore, the present findings may suggest that bFGF is probably released from the NPs with degraded gelatin fragments in the retina. 
Western blot analysis showed increased bFGF levels in bFGF-NP–treated retinas, and the release of endogenous bFGF from intracellular or extracellular sources may be an integral part of natural protection of photoreceptors. Although localization of bFGF was not examined, photoreceptor preservation did occur after intravitreal injection of bFGF-NPs, suggesting that this novel drug delivery system (DDS) might contribute to prolonged protection of photoreceptors by increasing the level of bFGF in the RCS rat retina. 
Delivery of bFGF for photoreceptor rescue has been performed by intravitreal or subretinal injection of the native protein or through introduction of the bFGF cDNA gene in a viral vector. 1 2 3 4 5 6 7 8 9 To maintain therapeutic levels of bFGF in vivo, subretinal injection of a viral vector encoding bFGF would be ideal; however, this method has several problems, including uncontrolled drug delivery, a difficult injection technique, limited retinal distribution, and biological safety concerns. Our novel DDS for bFGF enables targeting to photoreceptors, controlled drug delivery in all regions of the retina, a simple injection technique, and biological safety. In fact, we found a protective effect on photoreceptors in all retinal regions without serious complications, suggesting that this approach to the delivery of bFGF will improve the therapeutic efficacy for photoreceptor degeneration without causing serious complications. Furthermore, to the best of our knowledge, a single intravitreal injection of bFGF-NP had a longer protective effect than did any other drug in the RCS rat. This result may allow the use of the DDS in short-term photoreceptor rescue for future patient treatment. 
Our morphologic data show reduced labeling with an antibody to GFAP in bFGF-NP–treated eyes, leading to the conclusion that activation of Müller glial cells in bFGF-NP–treated eyes was lower than in bFGF-treated eyes. Additionally, adverse effects such as cataract, epiretinal membrane formation, and corneal neovascularization were not observed in bFGF-NP–treated eyes. Although we have not determined whether the lack of these effects was the result of encapsulation, penetration into the retina, or slow release of bFGF, our DDS for bFGF may reduce the serious adverse effects of bFGF, and this will be advantageous if intravitreally injected bFGF-NPs are to be used therapeutically. 
In conclusion, our results show that intravitreal administration of bFGF-NPs effectively prevents photoreceptor degeneration in RCS rats without causing serious complications because of the targeting and sustained release of bFGF in situ. bFGF-NPs may have advantages clinically because the safety of gelatin has been clinically proven. 20 Thus, intravitreal administration of bFGF-NPs is a strong candidate as a new therapeutic strategy for photoreceptor degeneration. However, several important issues remain to be addressed. In particular, we have no data indicating a dose–response relationship for single or multiple injections of bFGF. In addition, when it comes to potential human applications, further sustained long-term delivery of drug would be necessary to stop the progression of chronic photoreceptor degenerative diseases, such as retinitis pigmentosa. We selected bFGF for conjugation to NPs simply because the molecule has been well documented as an agent for photoreceptor rescue 1 2 3 4 5 6 7 8 9 and its receptors are present in photoreceptors 21 22 23 24 ; other factors may be equally or more potent than bFGF. 25 26 27 It will be important to pursue these questions in the development of new therapies for photoreceptor degeneration. 
 
Figure 1.
 
Experimental protocol.
Figure 1.
 
Experimental protocol.
Figure 2.
 
Time course of radioactivity remaining after intravitreal injection of 125I-labeled cross-linked gelatin nanoparticles in rat vitreous.
Figure 2.
 
Time course of radioactivity remaining after intravitreal injection of 125I-labeled cross-linked gelatin nanoparticles in rat vitreous.
Figure 3.
 
Representative confocal images (A, B) of the retina of RCS rats. Images of the retina of an RCS rat 6 (A) and 8 (B) weeks after injection of Rh-encapsulated bFGF-NPs are shown. Note the presence of the NPs within the retina in Rh-encapsulated bFGF-NP–injected RCS rat at 6 weeks and up to 8 weeks. NPs were observed in the IPL, ONL, and outer segment debris zone at 6 weeks. At 8 weeks after injection, the NPs were in the ONL, outer segment debris zone, and RPE apical surface. IPL, inner plexiform layer. Arrowheads: NPs. Scale bar, 50 μm.
Figure 3.
 
Representative confocal images (A, B) of the retina of RCS rats. Images of the retina of an RCS rat 6 (A) and 8 (B) weeks after injection of Rh-encapsulated bFGF-NPs are shown. Note the presence of the NPs within the retina in Rh-encapsulated bFGF-NP–injected RCS rat at 6 weeks and up to 8 weeks. NPs were observed in the IPL, ONL, and outer segment debris zone at 6 weeks. At 8 weeks after injection, the NPs were in the ONL, outer segment debris zone, and RPE apical surface. IPL, inner plexiform layer. Arrowheads: NPs. Scale bar, 50 μm.
Figure 4.
 
Morphologic rescue of the superior retina of RCS rats. Representative photographs 8 weeks after injection with bFGF-NPs (A, B) and blank NPs (C, D). Note the preservation of the ONL in the bFGF-NP–treated retina compared with the blank NP-treated retina. Higher magnification better showed the respective differences. Original magnification, ×100. Scale bar, 50 μm.
Figure 4.
 
Morphologic rescue of the superior retina of RCS rats. Representative photographs 8 weeks after injection with bFGF-NPs (A, B) and blank NPs (C, D). Note the preservation of the ONL in the bFGF-NP–treated retina compared with the blank NP-treated retina. Higher magnification better showed the respective differences. Original magnification, ×100. Scale bar, 50 μm.
Figure 5.
 
Mean number of photoreceptors per 50 μm for all treatment groups of RCS rats 8 weeks after injection. Note that the number of photoreceptors in bFGF-NP–treated rats was significantly larger than that in bFGF-treated rats in all regions. Data represent mean ± SD (n = 5, each group). *P < 0.05 (bFGF-NPs vs bFGF).
Figure 5.
 
Mean number of photoreceptors per 50 μm for all treatment groups of RCS rats 8 weeks after injection. Note that the number of photoreceptors in bFGF-NP–treated rats was significantly larger than that in bFGF-treated rats in all regions. Data represent mean ± SD (n = 5, each group). *P < 0.05 (bFGF-NPs vs bFGF).
Figure 6.
 
Immunohistochemistry of the retina of RCS rats 8 weeks after injection. Representative results of blank NP-treated (A, D), bFGF-treated (B, E), and bFGF-NP–treated (C, F) retinas double immunostained with the antibody to (AC) rod opsin (green) and GFAP (red) or (DF) vimentin (green) and M/L cone opsin (red). Note the remaining rod and M/L cone opsin in bFGF-NP–treated retinas, though the most intense M/L cone opsin labeling in the bFGF-NP–treated retinas appears to be in the outer segment debris zone. Rats treated with bFGF showed intense labeling of GFAP and vimentin in Müller cells. Scale bar, 100 μm.
Figure 6.
 
Immunohistochemistry of the retina of RCS rats 8 weeks after injection. Representative results of blank NP-treated (A, D), bFGF-treated (B, E), and bFGF-NP–treated (C, F) retinas double immunostained with the antibody to (AC) rod opsin (green) and GFAP (red) or (DF) vimentin (green) and M/L cone opsin (red). Note the remaining rod and M/L cone opsin in bFGF-NP–treated retinas, though the most intense M/L cone opsin labeling in the bFGF-NP–treated retinas appears to be in the outer segment debris zone. Rats treated with bFGF showed intense labeling of GFAP and vimentin in Müller cells. Scale bar, 100 μm.
Figure 7.
 
Effects of bFGF-NPs on scotopic ERG in RCS rats 6 weeks after injection. (A) Representative ERGs from baseline, PBS-, blank NP-, bFGF-, and bFGF-NP–treated retinas. Amplitudes of a- and b-waves were measured and plotted (B, C). Note that bFGF-NP–treated eyes show significantly increased a- and b-wave amplitudes compared with bFGF-treated eyes (a-wave, *P < 0.005; b-wave, **P < 0.05). Data represent mean ± SD (n = 8, each group).
Figure 7.
 
Effects of bFGF-NPs on scotopic ERG in RCS rats 6 weeks after injection. (A) Representative ERGs from baseline, PBS-, blank NP-, bFGF-, and bFGF-NP–treated retinas. Amplitudes of a- and b-waves were measured and plotted (B, C). Note that bFGF-NP–treated eyes show significantly increased a- and b-wave amplitudes compared with bFGF-treated eyes (a-wave, *P < 0.005; b-wave, **P < 0.05). Data represent mean ± SD (n = 8, each group).
Figure 8.
 
Fluorescence photomicrographs of retinal sections from TUNEL assay at 4 (AC) and 8 weeks (EG) after injection. (A, E) Blank NP-treated retina; (B, F) bFGF-treated retina; (C, G) bFGF-NP–treated retina. The percentage of TUNEL-positive cells in the ONL is smaller in the bFGF-NP–treated retina than in the bFGF-treated retina 4 and 8 weeks after injection (D, H). Scale bar, 50 μm.
Figure 8.
 
Fluorescence photomicrographs of retinal sections from TUNEL assay at 4 (AC) and 8 weeks (EG) after injection. (A, E) Blank NP-treated retina; (B, F) bFGF-treated retina; (C, G) bFGF-NP–treated retina. The percentage of TUNEL-positive cells in the ONL is smaller in the bFGF-NP–treated retina than in the bFGF-treated retina 4 and 8 weeks after injection (D, H). Scale bar, 50 μm.
Figure 9.
 
Expression of bFGF protein in 9-week-old RCS rats by Western blot analysis. bFGF expression level was greater in the bFGF-NP–treated retina than in the bFGF-treated retina.
Figure 9.
 
Expression of bFGF protein in 9-week-old RCS rats by Western blot analysis. bFGF expression level was greater in the bFGF-NP–treated retina than in the bFGF-treated retina.
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Figure 1.
 
Experimental protocol.
Figure 1.
 
Experimental protocol.
Figure 2.
 
Time course of radioactivity remaining after intravitreal injection of 125I-labeled cross-linked gelatin nanoparticles in rat vitreous.
Figure 2.
 
Time course of radioactivity remaining after intravitreal injection of 125I-labeled cross-linked gelatin nanoparticles in rat vitreous.
Figure 3.
 
Representative confocal images (A, B) of the retina of RCS rats. Images of the retina of an RCS rat 6 (A) and 8 (B) weeks after injection of Rh-encapsulated bFGF-NPs are shown. Note the presence of the NPs within the retina in Rh-encapsulated bFGF-NP–injected RCS rat at 6 weeks and up to 8 weeks. NPs were observed in the IPL, ONL, and outer segment debris zone at 6 weeks. At 8 weeks after injection, the NPs were in the ONL, outer segment debris zone, and RPE apical surface. IPL, inner plexiform layer. Arrowheads: NPs. Scale bar, 50 μm.
Figure 3.
 
Representative confocal images (A, B) of the retina of RCS rats. Images of the retina of an RCS rat 6 (A) and 8 (B) weeks after injection of Rh-encapsulated bFGF-NPs are shown. Note the presence of the NPs within the retina in Rh-encapsulated bFGF-NP–injected RCS rat at 6 weeks and up to 8 weeks. NPs were observed in the IPL, ONL, and outer segment debris zone at 6 weeks. At 8 weeks after injection, the NPs were in the ONL, outer segment debris zone, and RPE apical surface. IPL, inner plexiform layer. Arrowheads: NPs. Scale bar, 50 μm.
Figure 4.
 
Morphologic rescue of the superior retina of RCS rats. Representative photographs 8 weeks after injection with bFGF-NPs (A, B) and blank NPs (C, D). Note the preservation of the ONL in the bFGF-NP–treated retina compared with the blank NP-treated retina. Higher magnification better showed the respective differences. Original magnification, ×100. Scale bar, 50 μm.
Figure 4.
 
Morphologic rescue of the superior retina of RCS rats. Representative photographs 8 weeks after injection with bFGF-NPs (A, B) and blank NPs (C, D). Note the preservation of the ONL in the bFGF-NP–treated retina compared with the blank NP-treated retina. Higher magnification better showed the respective differences. Original magnification, ×100. Scale bar, 50 μm.
Figure 5.
 
Mean number of photoreceptors per 50 μm for all treatment groups of RCS rats 8 weeks after injection. Note that the number of photoreceptors in bFGF-NP–treated rats was significantly larger than that in bFGF-treated rats in all regions. Data represent mean ± SD (n = 5, each group). *P < 0.05 (bFGF-NPs vs bFGF).
Figure 5.
 
Mean number of photoreceptors per 50 μm for all treatment groups of RCS rats 8 weeks after injection. Note that the number of photoreceptors in bFGF-NP–treated rats was significantly larger than that in bFGF-treated rats in all regions. Data represent mean ± SD (n = 5, each group). *P < 0.05 (bFGF-NPs vs bFGF).
Figure 6.
 
Immunohistochemistry of the retina of RCS rats 8 weeks after injection. Representative results of blank NP-treated (A, D), bFGF-treated (B, E), and bFGF-NP–treated (C, F) retinas double immunostained with the antibody to (AC) rod opsin (green) and GFAP (red) or (DF) vimentin (green) and M/L cone opsin (red). Note the remaining rod and M/L cone opsin in bFGF-NP–treated retinas, though the most intense M/L cone opsin labeling in the bFGF-NP–treated retinas appears to be in the outer segment debris zone. Rats treated with bFGF showed intense labeling of GFAP and vimentin in Müller cells. Scale bar, 100 μm.
Figure 6.
 
Immunohistochemistry of the retina of RCS rats 8 weeks after injection. Representative results of blank NP-treated (A, D), bFGF-treated (B, E), and bFGF-NP–treated (C, F) retinas double immunostained with the antibody to (AC) rod opsin (green) and GFAP (red) or (DF) vimentin (green) and M/L cone opsin (red). Note the remaining rod and M/L cone opsin in bFGF-NP–treated retinas, though the most intense M/L cone opsin labeling in the bFGF-NP–treated retinas appears to be in the outer segment debris zone. Rats treated with bFGF showed intense labeling of GFAP and vimentin in Müller cells. Scale bar, 100 μm.
Figure 7.
 
Effects of bFGF-NPs on scotopic ERG in RCS rats 6 weeks after injection. (A) Representative ERGs from baseline, PBS-, blank NP-, bFGF-, and bFGF-NP–treated retinas. Amplitudes of a- and b-waves were measured and plotted (B, C). Note that bFGF-NP–treated eyes show significantly increased a- and b-wave amplitudes compared with bFGF-treated eyes (a-wave, *P < 0.005; b-wave, **P < 0.05). Data represent mean ± SD (n = 8, each group).
Figure 7.
 
Effects of bFGF-NPs on scotopic ERG in RCS rats 6 weeks after injection. (A) Representative ERGs from baseline, PBS-, blank NP-, bFGF-, and bFGF-NP–treated retinas. Amplitudes of a- and b-waves were measured and plotted (B, C). Note that bFGF-NP–treated eyes show significantly increased a- and b-wave amplitudes compared with bFGF-treated eyes (a-wave, *P < 0.005; b-wave, **P < 0.05). Data represent mean ± SD (n = 8, each group).
Figure 8.
 
Fluorescence photomicrographs of retinal sections from TUNEL assay at 4 (AC) and 8 weeks (EG) after injection. (A, E) Blank NP-treated retina; (B, F) bFGF-treated retina; (C, G) bFGF-NP–treated retina. The percentage of TUNEL-positive cells in the ONL is smaller in the bFGF-NP–treated retina than in the bFGF-treated retina 4 and 8 weeks after injection (D, H). Scale bar, 50 μm.
Figure 8.
 
Fluorescence photomicrographs of retinal sections from TUNEL assay at 4 (AC) and 8 weeks (EG) after injection. (A, E) Blank NP-treated retina; (B, F) bFGF-treated retina; (C, G) bFGF-NP–treated retina. The percentage of TUNEL-positive cells in the ONL is smaller in the bFGF-NP–treated retina than in the bFGF-treated retina 4 and 8 weeks after injection (D, H). Scale bar, 50 μm.
Figure 9.
 
Expression of bFGF protein in 9-week-old RCS rats by Western blot analysis. bFGF expression level was greater in the bFGF-NP–treated retina than in the bFGF-treated retina.
Figure 9.
 
Expression of bFGF protein in 9-week-old RCS rats by Western blot analysis. bFGF expression level was greater in the bFGF-NP–treated retina than in the bFGF-treated retina.
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