June 2011
Volume 52, Issue 7
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Nantotechnology and Regenerative Medicine  |   June 2011
A Lipid Nanoparticle System Improves siRNA Efficacy in RPE Cells and a Laser-Induced Murine CNV Model
Author Affiliations & Notes
  • Hong-an Liu
    From the Peking University Eye Center, Peking University Third Hospital, Peking University, Beijing, China; and
  • Yu-ling Liu
    From the Peking University Eye Center, Peking University Third Hospital, Peking University, Beijing, China; and
  • Zhi-zhong Ma
    From the Peking University Eye Center, Peking University Third Hospital, Peking University, Beijing, China; and
  • Jian-cheng Wang
    the State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China.
  • Qiang Zhang
    the State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China.
  • *Each of the following is a corresponding author: Yu-ling Liu, Peking University Eye Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China; yulingliu@medmail.com.cn. Qiang Zhang, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China; zqdodo@bjmu.edu.cn
Investigative Ophthalmology & Visual Science June 2011, Vol.52, 4789-4794. doi:10.1167/iovs.10-5891
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      Hong-an Liu, Yu-ling Liu, Zhi-zhong Ma, Jian-cheng Wang, Qiang Zhang; A Lipid Nanoparticle System Improves siRNA Efficacy in RPE Cells and a Laser-Induced Murine CNV Model. Invest. Ophthalmol. Vis. Sci. 2011;52(7):4789-4794. doi: 10.1167/iovs.10-5891.

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

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Abstract

Purpose.: To explore the possibility of the PEGylated liposome-protamine-hyaluronic acid nanoparticles (PEG-LPH-NP) loaded with siRNA (PEG-LPH-NP-S) in ARPE19 cells and a laser-induced rat model for the treatment of choroidal neovascularization (CNV).

Methods.: PEG-LPH-NP-S was characterized by dynamic light scattering and transmission electron microscopy (TEM). The encapsulation efficiency of siRNA in PEG-LPH-NP was analyzed by ultracentrifugation, whereas the protection of siRNA by PEG-LPH-NP was evaluated by electrophoresis. Human RPE cells (ARPE19) were used as the cell model for the studies of cellular uptake and the inhibition of VEGFR1 expression, visualized by a laser scanning confocal microscope. The area of CNV in the laser-induced rat model after intravitreous injection was measured. The distribution of the lipid nanoparticles in the retina after intravitreous administration was investigated by fluorescence microscopy. Finally, the TUNEL test and morphologic observation of the retina were conducted.

Results.: It was indicated that PEG-LPH-NP-S was approximately 132 nm in particle size with a positive charge of approximately 20 mV, whereas the encapsulation efficiency of siRNA in PEG-LPH-NP was >95%. PEG-LPH-NP could protect the siRNA load and could facilitate the intracellular delivery of fluorescein-labeled siRNA to ARPE19 cells. VEGFR1 expression in ARPE19 cells could be inhibited, and the CNV area in the murine model could be reduced more effectively by PEG-LPH-NP-S compared with naked siRNA and by PEG-LPH-NP with negative siRNA. It seems that the toxicity of PEG-LPH-NP-S on the rat retina is low, based on the results of TUNEL testing and morphologic observation.

Conclusions.: PEG-LPH-NP may be a promising lipid nanoparticle system for the siRNA treatment of CNV.

Age-related macular degeneration (AMD) is a leading cause of vision loss in the western world among people aged 50 and older. 1 Choroidal neovascularization (CNV) is a disorder that refers to the growth of new blood vessels originating from the choroids through a break in the Bruch's membrane into the subretinal pigment epithelium or the subretinal space. 2  
Recent studies using siRNA directed against VEGF or VEGF receptors in CNV animal models have shown promising results. 3 In a murine CNV model induced by laser, the subretinal injection of a mouse VEGF siRNA effectively inhibited the formation of CNV compared with the control GFP siRNA. 4 Moreover, the intravitreal or periocular injections of Sirna-027 (targeted to the human VEGFR1 mRNA) had been proved effectively. 5 The mechanisms of CNV reduction by siRNA is controversially discussed because it has been claimed that it was a TLR3-mediated, unspecific siRNA effect rather than a consequence of VEGFR knockdown. 6  
In spite of some successful reports on viral vectors in retinal gene delivery and therapy, several nonviral delivery systems are being explored, particularly in the case of retinitis pigmentosa. This is primarily because the viral vectors have crucial limitations such as the risk of insertional mutagenesis and adverse immune effects, 7 whereas the nonviral vectors have the profound advantage of being nonpathogenic and nonimmunogenic. 
Several different types of nonviral vectors have been investigated for gene silencing application, including the cationic lipids/liposomes complex, cationic polymer micelles, cationic dendrimers, and the vectors with cell penetrating peptides. 8 11 A common fact is that the net positive charges of these vectors facilitate the complex formation with the polyanionic nucleic acid and their interaction with the negatively charged cell membrane. For example, polyethylenimine was used in mice to deliver siRNA targeting the HEPR-2 receptor in murine models of ovarian carcinomas. 12 The 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP) was used for delivery of the siRNA targeting the endogenous tissues necrosis factor (TNF-α). 13 Recently, Huang et al. 14 developed a new formulation of a PEGylated cationic lipid vesicle to improve siRNA transfection for anticancer therapy with low toxicity, and this vector showed great potential for delivering siRNA to tumor cells. 
Therefore, the PEGylated cationic lipid vesicle may be useful in siRNA therapy for CNV. Based on this hypothesis, a lipid nanoparticle delivery system loaded with siRNA that targeted the human VEGFR1 mRNA was constructed and characterized. A human RPE cell line and a laser-induced murine CNV were used as the cell and pharmacodynamic models. In vitro and in vivo characteristics of such an ophthalmological delivery system were investigated to evaluate its possibility for the treatment of CNV. 
Materials and Methods
Materials
The siRNA (sense strand, 5′-CU GAG UUU AAA AGG CAC CCT T-3′; antisense strand, 5′-GGG UGC CUU UUA AAC UCA GTT-3′, targeted to the human VEGFR1 mRNA), 5 the negative control siRNA (sense strand, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; antisense strand, 5′-ACG UGA CAC GUU CGG AGA ATT-3′), and fluorescein-labeled siRNA (3′ end of the sense strand, FAM-siRNA) were obtained from GenePharma (Shanghai, China). Protamine sulfate (fraction X from salmon) and hyaluronic acid (HA) from Streptococcus equi were from Sigma-Aldrich (St. Louis, MO). DOTAP, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoetha-nolamine-N-c ammonium (DSPE-PEG2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 
Preparation of PEG-LPH-NP
PEG-LPH-NP-S was prepared according to Huang's method. 14 Briefly, small unilamellar cationic liposomes consisting of DOTAP and cholesterol (molar ratio, 1:2) were prepared by thin film hydration followed by membrane extrusion (100 nm × 10 times). To prepare the complex of (siRNA+HA)/protamine (PH-NP), 150 μL protamine (200 μg/mL) and 150 μL mixture of siRNA and HA (100–370μg/mL; weight ratio 1:1) were mixed in a 1.5-mL tube. The complex was allowed to stand at room temperature for 10 minutes, and then it was mixed with 12 to 250 μL prepared liposomes (total lipid concentration, 30 mM). This suspension was incubated with DSPE-PEG2000 at 50°C for 10 minutes. The final nanoparticle formulation (PEG-LPH-NP-S) was used within 20 minutes for the following experiments. The particle size and zeta potential were monitored by the dynamic light scattering method using the Malvern Zetasizer (Nano ZS; Malvern Instruments, Worcestershire, UK). PEG-LPH-NP with fluorescein-labeled siRNA and negative control siRNA were also prepared according to the same process. 
Transmission Electron Microscope
The morphology of PEG-LPH-NP-S was investigated with a transmission electron microscope (TEM; JEM-200CX; JEOL, Tokyo, Japan). 
Encapsulation Efficiency
Supernatants of various samples were obtained by ultracentrifugation at 10,000g for 30 minutes (HC-2064; USTC Chuangxin Co., Ltd., Hefei City, China). The supernatants were analyzed by a UV spectrophotometer (TU1901; Puxitongyong Company, Beijing, China). The supernatants of PEG-LPH-NP were served as a control. The supernatants of PEG-LPH-NP that were spiked with known amounts of siRNA were used to obtain the standard curve. The siRNA in the supernatants of PEG-LPH-NP-S (Wsupernatant) were determined, and their entrapment efficiency was calculated by the following equation, where Wtotal is the mount of siRNA added in the formulation: Encapsulation efficiency = (Wtotal − Wsupernatant)/Wtotal × 100%. 
Electrophoresis
Naked siRNA and PEG-LPH-NP-S were incubated separately in 50% fetal bovine serum at 37°C. At the indicated time-points (0, 10, 30, and 60 minutes, 3, 6, 12, 24, 36, and 48 hours), aliquots of samples were taken directly into gel loading buffer containing 3% SDS and were frozen in liquid nitrogen. After the final sample was frozen, siRNA was run on a 1% agarose gel and visualized by ethidium bromide staining. 
Cell Culture
Human ARPE19 cells (a spontaneously arising cell line from the RPE derived from a normal eye of a 19-year-old man) were obtained from American Type Culture Collection (Manassas, VA) and were used at passages 12 to 17. The cells were cultured 1:1 in Dulbecco modified Eagle's medium/F12 with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO2 to a confluent monolayer before they were harvested and treated. 
Intracellular Uptake of PEG-LPH-NP in ARPE19 Cells
Confocal fluorescence microscopy was used to compare the intracellular uptake of the lipid nanoparticles. The naked fluorescent dye labeled siRNA (FAM-siRNA; excitation/emission. 485/535 nm) was used as the control, and the PEG-LPH-NP loaded with 100 nM FAM-siRNA (PEG-LPH-NP-FS) was used instead of PEG-LPH-NP-S. ARPE19 cells were grown on glass-bottomed dish to 50% confluence and were incubated with PEG-LPH-NP-FS or control at 37°C for 1 hour and 2 hours, respectively. The cells were washed three times with cold PBS, fixed in 4% paraformaldehyde, and treated with Hoechst 33258 (excitation/emission, 364/460 nm) for 10 minutes for nuclei staining. Samples were embedded with antifade and analyzed using a laser scanning confocal microscope (LSM 510 Meta; Zeiss, Oberkochen, Germany). 
Expression of VEGFR1 Proteins in ARPE19 Cells
ARPE19 cells were grown on a glass-bottomed dish to 50% confluence and transfected with PEG-LPH-NP-S and naked siRNA in DMEM/F12 medium (without serum). The final concentration of siRNA was 100 nM, and the serum-free medium was used as the control. After 4 hours, the transfection medium was replaced with fresh complete medium, and cells were cultivated for 24 hours. The cells were then washed three times with cold PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature. The cells were blocked with 1% bovine serum albumin (BSA, in 0.1 M PBS) for 30 minutes, followed by incubation with the primary antibodies (1:200 dilution mouse anti-human monoclonal VEGFR1; Santa Cruz Biotechnology, Santa Cruz, CA) for 12 hours at 4°C. After washing, the cells were incubated with the secondary antibody (1:500 dilution FITC-goat anti-mouse IgG; ZhongShan Golden Bridge, Beijing, China) for 2 hours at 37°C, and then treated with Hoechst 33258 for 10 minutes for nuclei staining. Fluorescent images of cells were captured by laser scanning confocal microscopy using identical parameters and settings as described. 
Suppression of Laser-Induced CNV in Rats
Rats were treated in accordance with the recommendations of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, with the approval of the Institutional Authority for Laboratory Animal Care. Laser photocoagulation-induced rupture of Bruch's membrane was used to generate CNV. Briefly, 12 male Brown-Norway rats (weight range, 180–220 g; Vital Laboratory Animal Center, Beijing, China) were anesthetized with ketamine hydrochloride (100 mg/kg), and their pupils were dilated with 1% tropicamide. Four burns of 647 nm krypton laser (100-μm spot size, 0.1-second duration, 150 mW) were delivered to each retina with the slit-lamp delivery system of a photocoagulator (Coherent Novus Omni; Laser Labs Inc., Tampa, FL), and a handheld coverslip was used as contact lens. Burns were performed in the retinal blood vessel interspace, 1.5 to 2 PD (papilla diameter) from the optic nerve head. The production of a bubble at the time of laser burn indicating the rupture of Bruch's membrane is important for the experimental CNV model. Therefore, only burns in which a bubble was produced were included in the study. 
Through the intravitreous injections 1 day after laser photocoagulation, each group of rats (three animals or six eyes in each group, four groups; total, 12 rats) were treated with PEG-LPH-NP-S loaded with positive siRNA, naked positive siRNA, normal saline (NS), and PEG-LPH-NP loaded with negative control siRNA (PEG-LPH-NP-NC), respectively. The amount of siRNA was 1 μg/eye. 
Rats were euthanatized 14 days after laser treatment for the measurement of the area of CNV at Bruch's membrane rupture sites. At the end of 2 weeks, rats were perfused with fluorescein-labeled dextran (FITC-Dextran; mW, 2 × 106; Sigma), and choroidal flat mounts were prepared as described. 15 Elimination of burns that had not ruptured Bruch's membrane and caused large hemorrhages resulted in the following number of rupture sites for each group: PEG-LPH-NP-S, n = 18; naked siRNA, n = 20; PEG-LPH-NP- NC, n = 19; NS, n = 12. 
Distribution of PEG-LPH-NP in the Retina after Intravitreous Injection
Male Sprague-Dawley rats were purchased from Peking University Health Science Center (weight range, 180–200 g). Sprague-Dawley rats were anesthetized and intravitreously injected with PEG-LPH-NP-FS (containing 1 μg FAM-siRNA). NS was injected intravitreously as a control. Rats were euthanatized at different time points after injection, and eyes were frozen in optimal cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN). Frozen sections (7 μm) were fixed in 4% paraformaldehyde. After washing with 0.05 M PBS, the sections were covered with a standard glass coverslip and examined by fluorescence microscopy. 
Histologic Analysis of Rat Retina
PEG-LPH-NP-S (with 1 μg or 5 μg siRNA) and NS were injected intravitreously in the right and left eyes of Sprague-Dawley rats, respectively. The rats were killed 7 days and 14 days after injection. The rat eyes used for histologic analysis were frozen in OCT. Frozen sections (7 μm) were fixed in 4% paraformaldehyde. The sections were then mounted on glass slides and stained with hematoxylin and eosin. 
Apoptosis Analysis of Rat Retina
Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick ending-labeling method (TUNEL), according to the manufacturer's protocol (In Situ Cell Death Detection Kit, TMR red; Roche Applied Science, Mannheim, Germany). In short, tissue sections were fixed with fixation solution for 20 minutes at room temperature, washed with 0.05 M PBS, and incubated in permeabilization solution for 2 minutes on ice. Then a 50-μL reaction mixture was added to each sample. The slide was incubated in a humidified atmosphere for 60 minutes at 37°C in the dark and rinsed with PBS. Finally, the samples were embedded with antifade and analyzed under a fluorescence microscope. 
Statistical Analysis
One-way analyses of variance (ANOVA) were performed in statistical evaluation. P < 0.05 was considered significant. 
Results
Preparation and Characteristics of PEG-LPH-NP
The particle size and morphology of the optimized PEG-LPH-NP-S is shown in Figure 1. The particle size and zeta potential of final PEG-LPH-NP-S monitored by the dynamic light-scattering method was approximately 131.9 nm and 19.73 mV, respectively. The encapsulation efficiency of siRNA in PEG-LPH-NP was >95%. TEM examination confirmed that the particle size of the prepared PEG-LPH-NP-S was approximately 100 nm. In Figure 2, results show that the siRNA in PEG-LPH-NP was detectable for >48 hours in 50% fetal bovine serum at 37°C, whereas the naked siRNA could exist only for approximately 6 hours in the same condition. It was concluded that the PEG-LPH-NP could protect the siRNA loaded. 
Figure 1.
 
The morphology of PEG-LPH-NP-S. Transmission electron microscopy shows the size of PEG-LPH-NP-S to be approximately 100 nm. Scale bar, 0.2 μm.
Figure 1.
 
The morphology of PEG-LPH-NP-S. Transmission electron microscopy shows the size of PEG-LPH-NP-S to be approximately 100 nm. Scale bar, 0.2 μm.
Figure 2.
 
SiRNA protection effect of PEG-LPH-NP from serum. (A) PEG-LPH-NP-S was incubated in the presence of 50% fetal bovine serum at 37°C for 0, 10, 30, and 60 minutes and for 3, 6, 12, 24, 36, and 48 hours. (B) Naked siRNA as a control. The stability of siRNA in PEG-LPH-NP-S lasts for >48 hours. The naked siRNA can exist for only <6 hours.
Figure 2.
 
SiRNA protection effect of PEG-LPH-NP from serum. (A) PEG-LPH-NP-S was incubated in the presence of 50% fetal bovine serum at 37°C for 0, 10, 30, and 60 minutes and for 3, 6, 12, 24, 36, and 48 hours. (B) Naked siRNA as a control. The stability of siRNA in PEG-LPH-NP-S lasts for >48 hours. The naked siRNA can exist for only <6 hours.
Intracellular Uptake of PEG-LPH-NP in ARPE19 Cells
Figure 3 shows the observation of intracellular uptake of PEG-LPH-NP-FS in ARPE19 cells. One hour later, the FAM-siRNA in PEG-LPH-NP-FS group accumulated in the cytoplasm of the cells (Fig. 3A), whereas 2 hours later, more and more FAM-siRNA entered the cells and were distributed in the cytoplasm of the cells (Fig. 3B). On the other hand, after 2-hour incubation, the fluorescence in naked FAM-siRNA group was not observed in the ARPE19 cells (Fig. 3C). Therefore, PEG-LPH-NP could facilitate the intracellular delivery of siRNA into ARPE19 cells. 
Figure 3.
 
Intracellular uptake of PEG- LPH-NP in ARPE19. ARPE19 cells were grown on glass-bottomed dish to 50% confluence and incubated with PEG-LPH-NP-FS (containing 100 nM fluorescent dye-labeled siRNA [FAM-siRNA]) diluted in culture medium. (A) One hour later, FAM-siRNA was found accumulated in the cytoplasm of the cells. (B) Two hours later, more FAM-siRNA entered the cells and accumulated in the cytoplasm. (C) Naked FAM-siRNA was incubated for 2 hours; no FAM-siRNA was found in the cells. (green) FAM-siRNA. (blue) Nuclei. Scale bar, 100 μm.
Figure 3.
 
Intracellular uptake of PEG- LPH-NP in ARPE19. ARPE19 cells were grown on glass-bottomed dish to 50% confluence and incubated with PEG-LPH-NP-FS (containing 100 nM fluorescent dye-labeled siRNA [FAM-siRNA]) diluted in culture medium. (A) One hour later, FAM-siRNA was found accumulated in the cytoplasm of the cells. (B) Two hours later, more FAM-siRNA entered the cells and accumulated in the cytoplasm. (C) Naked FAM-siRNA was incubated for 2 hours; no FAM-siRNA was found in the cells. (green) FAM-siRNA. (blue) Nuclei. Scale bar, 100 μm.
Expression of VEGFR1 Proteins in ARPE19 Cells
ARPE19 cells were incubated with PEG-LPH-NP-S, naked siRNA, or free serum medium as the control. As shown in the Figure 4A, PEG-LPH-NP-S decreased VEGFR1 significantly compared with the naked siRNA (Fig. 4B) and the control group (Fig. 4C). This test indicated that siRNA loaded in PEG-LPH-NP was more effective than that of naked siRNA in vitro with regard to VEGFR1 inhibition. 
Figure 4.
 
Expression of VEGFR1 proteins in ARPE19. Fixed cells were immunolabeled with the monoclonal antibodies against VEGFR1 and were colabeled with Hoechst 33258. They were treated with (A) PEG-LPH-NP-S, (B) Naked siRNA, and (C) serum-free medium as a control. PEG-LPH-NP-S decreased VEGFR1 greatly compared with the naked siRNA and control. (green) VEGFR1. (blue) Nuclei. Scale bar, 100 μm.
Figure 4.
 
Expression of VEGFR1 proteins in ARPE19. Fixed cells were immunolabeled with the monoclonal antibodies against VEGFR1 and were colabeled with Hoechst 33258. They were treated with (A) PEG-LPH-NP-S, (B) Naked siRNA, and (C) serum-free medium as a control. PEG-LPH-NP-S decreased VEGFR1 greatly compared with the naked siRNA and control. (green) VEGFR1. (blue) Nuclei. Scale bar, 100 μm.
Suppression of Laser-Induced CNV in Rats
Treatment of Brown-Norway rats with naked siRNA (Fig. 5B) or PEG-LPH-NP-NC (Fig. 5C) 1 day after laser photocoagulation resulted in no significant difference in CNV area compared with the NS group (Fig. 5D). However, the PEG-LPH-NP-S group (Fig. 5A) demonstrated a significant decrease in the area of CNV in comparison with other groups. Reductions of CNV lesion size reached approximately 56% over that of the NS group (Fig. 5E). In other word, PEG-LPH-NP could facilitate the effect of siRNA on CNV reduction. 
Figure 5.
 
Intravitreous injection of PEG-LPH-NP-S suppresses CNV. Ruptures in Bruch's membrane were induced at four locations in each eye on day 1. Three rats (six eyes) in each group were treated with (A) PEG-LPH-NP-S, (B) naked siRNA, (C) PEG-LPH-NP-NC, and (D) NS by intravitreous injections 1 day after laser. siRNA, 1 μg/eye. (E) *P < 0.001, average CNV areas in the flat mounts of four treatment groups (AD) by one-way ANOVA. Scale bar, 50 μm.
Figure 5.
 
Intravitreous injection of PEG-LPH-NP-S suppresses CNV. Ruptures in Bruch's membrane were induced at four locations in each eye on day 1. Three rats (six eyes) in each group were treated with (A) PEG-LPH-NP-S, (B) naked siRNA, (C) PEG-LPH-NP-NC, and (D) NS by intravitreous injections 1 day after laser. siRNA, 1 μg/eye. (E) *P < 0.001, average CNV areas in the flat mounts of four treatment groups (AD) by one-way ANOVA. Scale bar, 50 μm.
Distribution of siRNA in the Retina after Intravitreous Injection
The distribution of PEG-LPH-NP-FS in the retina at various times after intravitreous injection is presented in Figure 6. It was found that 6 hours later, the fluorescence of FAM-siRNA was seen in the ganglion cell layer (Fig. 6B) and in the inner nuclear layer. At 24 hours after injection, most of the fluorescence was in the photoreceptor (Fig. 6C). These results demonstrated that PEG-LPH-NP could promote the distribution of more siRNA in the retina after intravitreous injection. 
Figure 6.
 
Distribution of PEG-LPH-NP in the retina at various times after intravitreous injection. PEG-LPH-NP-FS (with 1 μg FAM-siRNA) was injected into the vitreous cavity of mice. At 6 and 24 hours, mice were euthanatized, and ocular frozen sections were observed. (A) NS was injected as a control. (B) At 6 hours, fluorescence was limited to the ganglion cell layer, with some in the inner nuclear layer. (C) At 24 hours, there was widespread strong fluorescence in the photoreceptor layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 6.
 
Distribution of PEG-LPH-NP in the retina at various times after intravitreous injection. PEG-LPH-NP-FS (with 1 μg FAM-siRNA) was injected into the vitreous cavity of mice. At 6 and 24 hours, mice were euthanatized, and ocular frozen sections were observed. (A) NS was injected as a control. (B) At 6 hours, fluorescence was limited to the ganglion cell layer, with some in the inner nuclear layer. (C) At 24 hours, there was widespread strong fluorescence in the photoreceptor layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Histologic Analysis of Rat Retina
As shown in Figure 7, histologic changes were evaluated 7 days and 14 days after intravitreal injection of PEG-LPH-NP-S (with 1 μg or 5 μg siRNA). It was observed that the related retina was clear, without inflammatory cells in the vitreous, retina, or choroid. 
Figure 7.
 
Effect of PEG-LPH-NP-S on retinal structure. Histologic changes were evaluated at 7 days and 14 days after intravitreous injection of PEG-LPH-NP-S (with 1 μg or 5 μg siRNA). The sections were stained with hematoxylin and eosin. (A) NS. (B) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (D) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 7.
 
Effect of PEG-LPH-NP-S on retinal structure. Histologic changes were evaluated at 7 days and 14 days after intravitreous injection of PEG-LPH-NP-S (with 1 μg or 5 μg siRNA). The sections were stained with hematoxylin and eosin. (A) NS. (B) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (D) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Apoptosis Analysis of Rat Retina
Results of TUNEL assay of the rat retina treated with various siRNA formulations are shown in Figure 8. Seven and 14 days after PEG-LPH-NP-S injection with different amounts of siRNA, the number of apoptosis cells was not significantly increased compared with the control group. Taken together with histologic analysis results, it seems that the toxicity of PEG-LPH-NP-S in the rat retina was low. 
Figure 8.
 
TUNEL assay for the rat retinas treated with different amounts of PEG-LPH-NP-S. (A) Positive control. (B) Negative control. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (D) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (F) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 8.
 
TUNEL assay for the rat retinas treated with different amounts of PEG-LPH-NP-S. (A) Positive control. (B) Negative control. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (D) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (F) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Discussion
According to a previous report, 14 the formation of PEG-LPH-NP-S usually occurred through a self-assembling process mediated by charge-charge interaction between the cationic carriers and the anionic siRNA. In our study, the effects of different components and their ratios on the properties of lipid nanoparticles were investigated. For instance, it was observed that the particle size and zeta potential of the PEG-LPH-NP-S changed with the weight ratio of (siRNA+HA) and protamine. Large aggregates were found when the ratio increased to approximately 0.8. On the other hand, the amount of the cationic lipid (DOTAP) should be accurate; excess cationic lipid is reported to decrease siRNA delivery efficiency, possibly because of the competitive binding of empty cationic liposomes with the cells. 14 As seen in Figure 2, siRNA in PEG-LPH-NP was detectable for >48 hours in 50% fetal bovine serum at 37°C, suggesting that siRNA maintains its activity in the physiological condition, which is necessary for siRNA molecules to enter the cell and display their effect functionally. 
As we mentioned, CNV is a disorder that refers to the growth of new blood vessels into the retinal pigment epithelium. To be effective, siRNA or siRNA-loaded nanoparticles have to pass through the retina to reach the retinal pigment epithelium after intravitreous injection. That is why we used RPE cells as the cell model in vitro to determine whether PEG-LPH-NP could facilitate the intracellular delivery of siRNA into RPE cells. Our result gave a positive answer. A possible reason for the increased uptake of siRNA in the PEG-LPH-NP group might have been the increased interaction between the PEG-LPH-NP and the biomembrane because the proteins, proteoglycans, and glycerophosphates on the cell membrane are able to interact with the positively charged particles. 16,17 Additionally, PEG-LPH-NP offers a protective effect for siRNA, as proved in our test (Fig. 2). On the other hand, it has been observed that surface shielding of nonviral vectors by PEG chains enhances their stability in vivo and their transport across the biomembrane. This strategy has also been adopted for retinal DNA delivery after intravitreous administration. For instance, PEGylation has been explored in the delivery of lipoplexes to increase DNA stability in the vitreous, protect the liposomes from early degradation, and reduce toxicity. 18 The Pegaptanib (Macugen; Eyetech Inc., Nanuet, NY), an anti-VEGF aptamer linked to PEG, was the first aptamer therapy approved for humans in the treatment of CNV associated with AMD. 19 For all these reasons, it is reasonable that PEG-LPH-NP could facilitate the effect of siRNA in terms of the reduction of VEGFR1 expression in vitro, the decrease of the CNV area in vivo, and the increase of siRNA distribution in the retina. 
An interesting study is reported by Kleinman et al. 6 using different siRNA sequences to reduce the CNV area in a mouse model. It was indicated in their study that the suppression of mouse CNV is a generic property of siRNAs independent of sequence, target, and internalization, and siRNAs exert their antiangiogenic effect in mice not because of target knockdown but because of TLR3 activation. 6 In their report, 1 μg siRNA was found significantly to be decreased the CNV area, whereas 0.25 μg siRNA was not, after intravitreous administration to mice. In our study, we found that 1 μg naked siRNA could slightly decrease the CNV area, but there was no significance between the naked siRNA and the saline group after intravitreous injection to rats. We think that the different results between their test and our test may be due primarily to the different animal models. Kleinman et al. 6 used C57BL/6J mice, but we used Brown-Norway rats. Anatomically, mouse eyes are smaller than rat eyes; therefore, 1 μg siRNA in a mouse eye may result in higher concentrations than in the rat eye. Additionally, siRNAs may have a different half-time and a different penetration property to biomembrane in different animal models. On the whole, it seems reasonable for the different findings to have occurred between these two studies because the difference in animal models might be significant. In the Brown-Norway rat model, PEG-LPH-NP enhanced the efficacy of siRNA significantly compared with naked siRNA, revealing the advantage of nanoparticles prepared here (PEG-LPH-NP). 
We did not observe the significant retinal toxicity 1 week or 2 weeks after intravitreal injection of PEG-LPH-NP-S. HA is a naturally occurring polyanionic polysaccharide. It has been widely used in pharmaceuticals, and it is not immunostimulatory. Protamine is a naturally occurring polycation that condenses DNA in the head of spermatozoa. This small polycationic agent is expected to show the low probability of immunogenic responses in the target tissue caused by the absence of aromatic amino acids and a rigid structure. 20 The DOTAP and DSPE-PEG2000 are cationic, but they are biodegradable and have low immunogenicity. 13,19 In addition, a previous study proved that this kind of formulation does not induce significant production of IL-6 and IL-12. 14 The numbers of lipid nanoparticles that reached the retina might have been only parts of entire doses; most of the vehicles might have been in the vitreous body. 
In conclusion, it was found in our studies that PEG-LPH-NP could efficiently protect the siRNA load and could facilitate the intracellular delivery of siRNA, the expression inhibition of VEGFR1, and the reduction of CNV area in the rat model while demonstrating low toxicity in the rat retina. Generally, PEG-LPH-NP seems a promising lipid vector with which to deliver siRNA to the retina for the treatment of CNV. 
Footnotes
 Supported by the National Basic Research Program of China Grant 2009CB930300, State Key Projects Grant 2009ZX09310-001, and 863 Project Grant 2007AA021811.
Footnotes
 Disclosure: H. Liu, None; Y. Liu, None; Z. Ma, None; J. Wang, None; Q. Zhang, None
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Figure 1.
 
The morphology of PEG-LPH-NP-S. Transmission electron microscopy shows the size of PEG-LPH-NP-S to be approximately 100 nm. Scale bar, 0.2 μm.
Figure 1.
 
The morphology of PEG-LPH-NP-S. Transmission electron microscopy shows the size of PEG-LPH-NP-S to be approximately 100 nm. Scale bar, 0.2 μm.
Figure 2.
 
SiRNA protection effect of PEG-LPH-NP from serum. (A) PEG-LPH-NP-S was incubated in the presence of 50% fetal bovine serum at 37°C for 0, 10, 30, and 60 minutes and for 3, 6, 12, 24, 36, and 48 hours. (B) Naked siRNA as a control. The stability of siRNA in PEG-LPH-NP-S lasts for >48 hours. The naked siRNA can exist for only <6 hours.
Figure 2.
 
SiRNA protection effect of PEG-LPH-NP from serum. (A) PEG-LPH-NP-S was incubated in the presence of 50% fetal bovine serum at 37°C for 0, 10, 30, and 60 minutes and for 3, 6, 12, 24, 36, and 48 hours. (B) Naked siRNA as a control. The stability of siRNA in PEG-LPH-NP-S lasts for >48 hours. The naked siRNA can exist for only <6 hours.
Figure 3.
 
Intracellular uptake of PEG- LPH-NP in ARPE19. ARPE19 cells were grown on glass-bottomed dish to 50% confluence and incubated with PEG-LPH-NP-FS (containing 100 nM fluorescent dye-labeled siRNA [FAM-siRNA]) diluted in culture medium. (A) One hour later, FAM-siRNA was found accumulated in the cytoplasm of the cells. (B) Two hours later, more FAM-siRNA entered the cells and accumulated in the cytoplasm. (C) Naked FAM-siRNA was incubated for 2 hours; no FAM-siRNA was found in the cells. (green) FAM-siRNA. (blue) Nuclei. Scale bar, 100 μm.
Figure 3.
 
Intracellular uptake of PEG- LPH-NP in ARPE19. ARPE19 cells were grown on glass-bottomed dish to 50% confluence and incubated with PEG-LPH-NP-FS (containing 100 nM fluorescent dye-labeled siRNA [FAM-siRNA]) diluted in culture medium. (A) One hour later, FAM-siRNA was found accumulated in the cytoplasm of the cells. (B) Two hours later, more FAM-siRNA entered the cells and accumulated in the cytoplasm. (C) Naked FAM-siRNA was incubated for 2 hours; no FAM-siRNA was found in the cells. (green) FAM-siRNA. (blue) Nuclei. Scale bar, 100 μm.
Figure 4.
 
Expression of VEGFR1 proteins in ARPE19. Fixed cells were immunolabeled with the monoclonal antibodies against VEGFR1 and were colabeled with Hoechst 33258. They were treated with (A) PEG-LPH-NP-S, (B) Naked siRNA, and (C) serum-free medium as a control. PEG-LPH-NP-S decreased VEGFR1 greatly compared with the naked siRNA and control. (green) VEGFR1. (blue) Nuclei. Scale bar, 100 μm.
Figure 4.
 
Expression of VEGFR1 proteins in ARPE19. Fixed cells were immunolabeled with the monoclonal antibodies against VEGFR1 and were colabeled with Hoechst 33258. They were treated with (A) PEG-LPH-NP-S, (B) Naked siRNA, and (C) serum-free medium as a control. PEG-LPH-NP-S decreased VEGFR1 greatly compared with the naked siRNA and control. (green) VEGFR1. (blue) Nuclei. Scale bar, 100 μm.
Figure 5.
 
Intravitreous injection of PEG-LPH-NP-S suppresses CNV. Ruptures in Bruch's membrane were induced at four locations in each eye on day 1. Three rats (six eyes) in each group were treated with (A) PEG-LPH-NP-S, (B) naked siRNA, (C) PEG-LPH-NP-NC, and (D) NS by intravitreous injections 1 day after laser. siRNA, 1 μg/eye. (E) *P < 0.001, average CNV areas in the flat mounts of four treatment groups (AD) by one-way ANOVA. Scale bar, 50 μm.
Figure 5.
 
Intravitreous injection of PEG-LPH-NP-S suppresses CNV. Ruptures in Bruch's membrane were induced at four locations in each eye on day 1. Three rats (six eyes) in each group were treated with (A) PEG-LPH-NP-S, (B) naked siRNA, (C) PEG-LPH-NP-NC, and (D) NS by intravitreous injections 1 day after laser. siRNA, 1 μg/eye. (E) *P < 0.001, average CNV areas in the flat mounts of four treatment groups (AD) by one-way ANOVA. Scale bar, 50 μm.
Figure 6.
 
Distribution of PEG-LPH-NP in the retina at various times after intravitreous injection. PEG-LPH-NP-FS (with 1 μg FAM-siRNA) was injected into the vitreous cavity of mice. At 6 and 24 hours, mice were euthanatized, and ocular frozen sections were observed. (A) NS was injected as a control. (B) At 6 hours, fluorescence was limited to the ganglion cell layer, with some in the inner nuclear layer. (C) At 24 hours, there was widespread strong fluorescence in the photoreceptor layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 6.
 
Distribution of PEG-LPH-NP in the retina at various times after intravitreous injection. PEG-LPH-NP-FS (with 1 μg FAM-siRNA) was injected into the vitreous cavity of mice. At 6 and 24 hours, mice were euthanatized, and ocular frozen sections were observed. (A) NS was injected as a control. (B) At 6 hours, fluorescence was limited to the ganglion cell layer, with some in the inner nuclear layer. (C) At 24 hours, there was widespread strong fluorescence in the photoreceptor layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 7.
 
Effect of PEG-LPH-NP-S on retinal structure. Histologic changes were evaluated at 7 days and 14 days after intravitreous injection of PEG-LPH-NP-S (with 1 μg or 5 μg siRNA). The sections were stained with hematoxylin and eosin. (A) NS. (B) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (D) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 7.
 
Effect of PEG-LPH-NP-S on retinal structure. Histologic changes were evaluated at 7 days and 14 days after intravitreous injection of PEG-LPH-NP-S (with 1 μg or 5 μg siRNA). The sections were stained with hematoxylin and eosin. (A) NS. (B) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (D) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 8.
 
TUNEL assay for the rat retinas treated with different amounts of PEG-LPH-NP-S. (A) Positive control. (B) Negative control. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (D) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (F) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
Figure 8.
 
TUNEL assay for the rat retinas treated with different amounts of PEG-LPH-NP-S. (A) Positive control. (B) Negative control. (C) PEG-LPH-NP-S (with 1 μg siRNA) for 7 days. (D) PEG-LPH-NP-S (with 1 μg siRNA) for 14 days. (E) PEG-LPH-NP-S (with 5 μg siRNA) for 7 days. (F) PEG-LPH-NP-S (with 5 μg siRNA) for 14 days. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.
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