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Nantotechnology and Regenerative Medicine  |   December 2013
Combination of Targeted PDT and Anti-VEGF Therapy for Rat CNV by RGD-Modified Liposomal Photocyanine and Sorafenib
Author Affiliations & Notes
  • Jia-lin Wang
    Peking University Eye Center, Peking University Third Hospital, Beijing, People's Republic of China
  • Yi Xi
    Peking University Eye Center, Peking University Third Hospital, Beijing, People's Republic of China
  • Yu-ling Liu
    Peking University Eye Center, Peking University Third Hospital, Beijing, People's Republic of China
  • Zhao-hui Wang
    State Key Laboratory of Natural and Biomimotic Drugs, School of Pharmaceutical Science, Peking University, Beijing, People's Republic of China
  • Qiang Zhang
    State Key Laboratory of Natural and Biomimotic Drugs, School of Pharmaceutical Science, Peking University, Beijing, People's Republic of China
  • Correspondence: Yu-ling Liu, Peking University Eye Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, P. R. China; yulingliu@medmail.com.cn
  • Qiang Zhang, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, P. R. China; zqdodo@bjmu.edu.cn
Investigative Ophthalmology & Visual Science December 2013, Vol.54, 7983-7989. doi:10.1167/iovs.13-13068
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      Jia-lin Wang, Yi Xi, Yu-ling Liu, Zhao-hui Wang, Qiang Zhang; Combination of Targeted PDT and Anti-VEGF Therapy for Rat CNV by RGD-Modified Liposomal Photocyanine and Sorafenib. Invest. Ophthalmol. Vis. Sci. 2013;54(13):7983-7989. doi: 10.1167/iovs.13-13068.

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

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Abstract

Purpose.: To achieve a combination therapy of targeted PDT and anti-VEGF for choroidal neovascularization (CNV).

Methods.: Arg-Gly-Asp (RGD)-modified liposomes encapsulating photocyanine and sorafenib (RGD-SSL-[P]-[S]) were prepared and characterized. Drug concentration in RGD-SSL-[P]-[S] and irradiation time were optimized on ARPE-19 and HUVEC cells in vitro. A laser-induced CNV rat model was used to assess the efficacy of this targeted combinational system. The effect of RGD-SSL-[P]-[S] on the retinal structure of BN rats was also examined.

Results.: The particle size of RGD-SSL-[P]-[S] was approximately 100 nm, with a encapsulation efficiency more than 90% for both photocyanine and sorafenib. From RGD-SSL-[P]-[S], the release rate of photocyanine was approximately 22%, whereas that of sorafeinb was approximately 40% at 48 hours. With the optimal drug concentration and irradiation time, RGD-SSL-[P]-[S] exhibited cytotoxicity only to HUVEC without obvious damage to normal ARPE-19 cells. Rats treated with RGD-SSL-[P]-[S] showed the least CNV area and fluorescein leakage in fluorescein fundus angiography. RGD-SSL-[P]-[S] was also found safe to the rat retina.

Conclusions.: Combination of targeted PDT and anti-VEGF might be an effective therapy for CNV.

Introduction
Choroidal neovascularization (CNV) causes severe and rapidly progressing vision loss, especially in the exudative form of AMD patients. 1,2 Photodynamic therapy (PDT) is a promising CNV treatment. 3 In photodynamic therapy, a photosensitive dye, verteporfin, which is believed to preferentially accumulate in active new vessels, is infused intravenously and then activated by a low-energy visible laser (689 nm). The resultant reaction produces localized thrombosis of the new vessels. The treatment is repeated every 3 months as required. Significant improvement in the proportion of patients with stable or improved vision occurs in those with classic CNV. 
However, on PDT treatment for CNV secondary to AMD, the expression of VEGF is increased, leading to the regrowth of the neovasculature. 4 Therefore, blocking VEGF (anti-VEGF therapy) seems beneficial for the treatment of CNV, in terms of reducing the retreatment rate and improving visual outcome. Previous study revealed that intravitreal injection of ranibizumab in combination with PDT produced a greater reduction in angiographic leakage than PDT alone. 5,6 But, repeated intravitreal injections are intolerant, and a better therapy is needed. 
Photocyanine, a new drug candidate, has been tested in the first phase of a clinical trial in China. It represents excellent photodynamic activity, selectivity to the focus of disease, and hypotoxicity. 710 Sorafenib, an active multikinase inhibitor available orally, can inhibit VEGF. These two drugs were used here as the models of photosensitive dye and VEGF inhibitor, respectively. 
Sterically stabilized liposomes (SSL) can passively accumulate into CNV tissue due to the effect of enhanced permeability and retention (EPR). 11,12 Liposomes modified with suitable ligand may realize site-specific delivery to the target tissue. Recent studies show that integrin (arginine-glycine-aspartic acid [RGD]-dependent receptor) is overexpressed in CNV endothelial cells and CNV-related RPE cells. 13  
In this article, to establish a therapy system combining both targeted PDT and anti-VEGF treatment, we designed an RGD-modified liposome system loaded with both photocyanine and sorafenib (RGD-SSL-[P]-[S]). One potential advantage of this system is the surface modification with RGD peptide, which may enhance intracellular drug uptake by HUVEC cells (human umbilical vein endothelial cells). Another benefit from the combination therapy is that it may improve therapy efficacy. For the proof of concept, the RGD-SSL-[P]-[S] was constructed and characterized, followed by the evaluation on optimal concentration of RGD-SSL-[P]-[S] and optimal irradiation time by 3-(4, 5-dimethylthiazol-2-yl)-2,5-dipherltetrazdium bromide (MTT) in vitro. Finally, a laser-induced CNV rat model was used to assess the efficacy of this targeted combination system in vivo. 
Materials and Methods
This section is in accordance with institutional review board approval. RGD was purchased from Zhongkeyaguang Biotechnology Co. (Beijing, China). Hydrogenated soy phosphatidylcholine (HSPC) was supplied by Lipoids GmbH (Hanover, DE). Cholesterol (CHOL), polyethylene glycol-distearoylphostearoylphosphatitylethanolamine (DSPE-PEG2000), and DSPE-PEG-NHS (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly (ethylene-glycol)]-hydroxyl succinamide, PEG molecular weight [MW] = 2000) were purchased from NDF (Tokyo, Japan). Fluorescein-labeled dextran (FITC-Dextran, MW = 2 × 106) and collagenase type I and MTT were purchased from Sigma-Aldrich (St. Louis, MO). Photocyanine was supplied by Longhua Pharmaceutical Co. (Fujian, China). Sorafenib was supplied by Peking University Pharmaceutical Sciences School (Beijing, China). 
Synthesis of RGD-PEG-DSPE
RGD and DSPE-PEG-NHS in the molar ratio of 1:2 were dissolved in dimethylformamide (DMF), 14 stirring continuously at room temperature for 36 hours. Following reacting for 10 hours, triethylamine was added to adjust the pH to a slight alkaline condition. 15,16 The reaction end point was traced by the thin layer chromatography (TLC) method until the spot of RGD disappeared completely. The reaction product was dialyzed against distilled water for 48 hours to remove all impurities and was then lyophilized. 
Preparation and Characterization of Liposomes
Accurately weighed amounts of materials (HSPC/CHOL/DSPE-PEG/RGD-PEG-DSPE/photocyanine/sorafenib: 15/3.87/2/3.3/0.4/1.0, mg/mg) for RGD-SSL and (HSPC/CHOL/DSPE-PEG/photocyanine/sorafenib: 15/3.87/4/0.4/1.0, mg/mg) for SSL were dissolved in chloroform and methanol (1:2, vol/vol) in a pear-shaped flask, and dried into a transparent thin film on a rotary evaporator under vacuum at 40°C. The resulting lipid film was then hydrated with 2 mL PBS at room temperature by sonication in the water bath for 10 minutes, and sonicated in the water bath at 50°C for 60 minutes. The suspensions were successively extruded through a 200-nm filter three times. 
To prepare photocyanine liposomes, the liposomes were made with the same method as in the previous paragraph, excluding the addition of sorafenib. Similarly, to prepare sorafenib liposomes, the liposomes were made with the same method, excluding the addition of photocyanine. 
The particle size, polydispersity index (PDI), and zeta potential of photocyanine-loaded liposomes SSL-[P], sorafenib-loaded liposomes SSL-[S], and RGD-modified photocyanine and sorafenib-loaded liposomes RGD-SSL-[P]-[S] were monitored by the dynamic light-scattering method using a Malvern Zetasizer (Nano ZS; Malvern Instruments, Worcestershire, UK). The morphology of RGD-SSL-[P]-[S] was investigated with a transmission electron microscope (JEM-200CX; JEOL, Tokyo, Japan). 
Based on a methodology study using a UV method, which eliminated the interference of liposomes, photocyanine in the liposomal samples was analyzed with a UV spectrophotometer (TU1901 spectrophotometer; Purkinje General Instrument Co., Ltd., Beijing, China) in DMF at 675 nm. Sorafenib in the liposomal samples was analyzed in acetonitrile at 265 nm, similar to previous report. 17  
For encapsulation efficiency measurement, a 100-μL liposome suspension was diluted to 10 mL with PBS. Another 100-μL liposome suspension was passed through a Sephadex G50 gel-filtration column (Pharmacia Biotech; Piscataway, NJ) and collected liposomes were diluted to 10 mL with PBS. The supernatants were analyzed with a UV spectrophotometer (Purkinje General Instrument Co., Ltd.). The encapsulation efficiency was calculated with the formula: Encapsulation efficiency% = (the measured amount of drug after gel-filtration/the measured amount before gel-filtration) ×100%. 
Lipsomes were stored at 4°C for 28 days under light protection. The particle size and drug loading of liposomes were evaluated. 
The leakage of drugs from liposomes was monitored by dialysis (MWCO 14000; JingKeHongDa Biotechnology Co., Ltd., Beijing, China) against cell culture media containing 20% fetal bovine serum (FBS) for 48 hours at 37°C with gentle shaking. At designated time points, 100-μL aliquots were withdrawn from the incubation medium and replaced with equal volume of the fresh medium. At the end of 48 hours, the dialysis bags were cut open and 2 mL 10% Triton X-100 was mixed thoroughly with the release medium. The concentrations of photocyanine and sorafinib were then measured by UV spectrophotometer at 675 nm dissolved in DMF and at 265 nm dissolved in acetonitrile, respectively. 
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured with the method we previously described. 18 A human RPE cell line (ARPE-19; American Type Culture Collection, Manassas, VA) was used at passages 12 to 16 and cultured with the method we previously described. 19  
In Vitro Cytotoxicity in the Dark Assay
Cytotoxicitiy of RGD-SSL-[P]-[S] was tested on ARPE-19 and HUVEC cells. In brief, each well of 96-well plates was seeded with 5000 cells and incubated for 24 hours. The cells were then exposed to serial concentrations of two drugs (photocyanine 5 × 10−8 M, 1 × 10−7 M, 5 × 10−7 M, 1 × 10−6 M, 5 × 10−6 M, 1 × 10−5 M; combining with sorafenib 4 × 10−5 M, 8 × 10−5 M, 1.6 ×10−4 M, 3.2 × 10−4 M , 6.4 × 10−4 M, 1.28 × 10−3 M) of RGD-SSL-[P]-[S] diluted in culture medium. After that, all steps were kept in the dark (with red light bubble lighting). After culturing for 24 hours at 37°C, the cell monolayer was washed with PBS. Then, 20 μL MTT solution (5 mg/mL) was added to each well, followed by incubating for another 4 hours and the absorbance was read on a Sunrise Absorbance Microplate Reader (TECAN, Melbourne, Australia) at a wavelength of 490 nm. The survival percentage was calculated using the following formula: Survival% = (A490 nm for the treated cells/A490 nm for the control cells). The experiment was carried out in triplicate. The data reported represent the means of triplicate measurement. 
Phototoxicity of the Cells
Phototoxicity of RGD-SSL-[P]-[S] was tested on ARPE-19 and HUVEC cells. The cells were seeded onto 96-well culture plates at a density of 5000 cells per well and grown in the incubator at 37°C for 24 hours. Then the cells were exposed to the concentration chosen by cytotoxicity in the dark experiment diluted in the culture medium. All steps were kept in the dark (with red light bubble lighting). After culturing for 24 hours at 37°C, new culture medium replaced the old medium. The output wavelength of 670 nm nonthermal diode (LD-670; LNCT12PF; Panasonic, Bizen, Japan) was exposed above the 96-well plate at room temperature (irradiation time 20 seconds, 40 seconds, 80 seconds, 160 seconds, 320 seconds; lighting power density 200 mW/cm2). After culturing for 24 hours at 37°C, the cells were disposed, as in the cytotoxicity in the dark experiment. 
In Vivo Therapeutic Efficacy
The therapeutic efficacy of liposomes was investigated in male Brown Norway (BN) rats (180–220 g; Vital Laboratory Animal Center, Beijing, China). Briefly, BN rats were weighed and randomly divided into different treatment groups (n = 5). Laser-induced CNV rats were prepared according to the method of Liu et al. 20 The spots for laser treatment were located between major retinal vessels. Disruption of Bruch's membrance was confirmed by central bubble formation. 21  
According to clinical dosage of photocyanine, 0.2 mg/kg was used in our research, the corresponding sorafenib dosage was 0.43 to 0.45 mg/kg. Seven days after laser photocoagulation, each group of rats (n = 5) was tail vein injected with normal saline (NS), SSL-[P], RGD-SSL-[P], SSL-[P]-[S], and RGD-SSL-[P]-[S]. Then, 10 to 15 minutes after giving the drugs, a nonthermal diode was used to irradiate whole rat retina (irradiation time 240 seconds, light power density 200 mW/cm2, laser energy 50 J/cm2). At the end of 2 weeks, rats under deep anesthesia were perfused with FITC-Dextran, and choroidal flat mounts were calculated using a method similar to that used in a previous report. 22 Elimination of burns that had not ruptured Bruch's membrane and caused a large hemorrhage resulted in the following number of rupture sites for analysis in each group: (n > 3). 
The CNV was observed by fluorescein fundus angiography (FFA) 1 and 2 weeks after photocoagulation of different therapy groups. The method was prepared according to the method of Wang et al. 19  
The Effect of RGD-SSL-[P]-[S] on the Retinal Structure of BN Rats
The histological changes of the retinal structure of BN rats after treatment with NS and RGD-SSL-[P]-[S] were evaluated. The rats were divided into an NS group and RGD-SSL-[P]-[S] group. The rats were killed 14 days after photocoagulation. The sections from the rat eyes were made, stained, and examined as usual. 
Statistical
Statistical evaluations were performed using one-way ANOVA. A P value less than 0.05 was considered to be significant. 
Results
Synthesis of RGD-PEG-DSPE and the Preparation of Liposomes
RGD was successfully conjugated to the DSPE-PEG-NHS under the conditions of reaction described in our previous report. Approximately 50% of input DSPE-PEG-NHS in the reaction system was conjugated with RGD peptide. 16  
Characterization of Liposomes
As shown in the Table, the particle sizes of various liposomes were approximately 100 nm (PDI < 0.3), and all liposomes were slightly negatively charged. The encapsulation efficiency for photocyanine was larger than 91%, whereas that of sorafenib was between 82% and 99% for different formulations. These data indicated that the modification of RGD and the co-encapsulation of both drugs did not significantly affect the particle size and zeta potentials, but sometimes did impact the encapsulation efficiency. 
Table
 
Characteristics of the Prepared Liposomes (n = 3)
Table
 
Characteristics of the Prepared Liposomes (n = 3)
Liposomes Average Particle Size, nm Polydispersity, PDI Zeta Potential, mV Entrapment Efficiency of P, % Entrapment Efficiency of S, %
SSL-[P] 99.13 ± 2.52 0.216 ± 0.021 −2.4 ± 0.27 93.40 ± 0.36
RGD-SSL-[P] 101.08 ± 1.83 0.297 ± 0.013 −3.12 ± 0.38 91.86 ± 0.22*
SSL-[S] 94.48 ± 3.28 0.258 ± 0.038 −2.93 ± 0.23 98.98 ± 0.83
RGD-SSL-[S] 98.39 ± 1.99 0.290 ± 0.049 −3.43 ± 0.41 98.02 ± 0.64
SSL-[P]-[S] 100.58 ± 2.09 0.280 ± 0.0313 −3.40 ± 0.66 93.98 ± 0.98 86.17 ± 0.52
RGD-SSL-[P]-[S] 99.69 ± 3.10 0.305 ± 0.0247 −2.94 ± 0.20 93.66 ± 0.71 82.35 ± 1.49†
Transmission electron miscroscopy in Figure 1 showed that the particle size of RGD-SSL-[P]-[S] was approximately 100 nm. The membrane structure of this kind of liposomes can be clearly seen in Figure 1
Figure 1
 
The morphology of RGD-SSL-[P]-[S] under transmission electron microscope. The size of RGD-SSL-[P]-[S] was approximately 90 nm. Scale bar: 100 nm.
Figure 1
 
The morphology of RGD-SSL-[P]-[S] under transmission electron microscope. The size of RGD-SSL-[P]-[S] was approximately 90 nm. Scale bar: 100 nm.
It is clear from Figure 2A that the particle size of SSL-[P]-[S] and RGD-SSL-[P]-[S] demonstrated no significant difference within 28 days. Also, there were no significant changes found in the encapsulation efficiency of photocyanine and sorafenib, respectively (Figs. 2B, 2C) with the same period of time. 
Figure 2
 
Stability study of liposomal dispersion stored at 4°C for 28 days. (A) Change of particle size. (B) Change of sorafenib loading. (C) Change of photocyanine loading.
Figure 2
 
Stability study of liposomal dispersion stored at 4°C for 28 days. (A) Change of particle size. (B) Change of sorafenib loading. (C) Change of photocyanine loading.
As seen in Figure 3, photocyanine and sorafenib released from SSL-[P]-[S] at a rate comparable to those from RGD-SSL-[P]-[S]; however, sorafenib released much faster than photocyanine in both targeting and nontargeting liposomes. The release rate of photocyanine (Fig. 3A) at 48 hours from SSL-[P]-[S] or RGD-SSL-[P]-[S] was approximately 22%, whereas that of sorafenib (Fig. 3B) from two liposome systems was approximately 40% at 48 hours. There was no significant difference in the release rate between SSL-[P]-[S] and RGD-SSL-[P]-[S] (Fig. 2A, P = 0.801; Fig. 2B, P = 0.813), revealing the little effect from the peptide modification. 
Figure 3
 
Release rates of (A) photocyanine and (B) sorafenib from SSL-[P]-[S] and RGD-SSL-[P]-[S] in cell culture media containing 20% FBS (n = 3).
Figure 3
 
Release rates of (A) photocyanine and (B) sorafenib from SSL-[P]-[S] and RGD-SSL-[P]-[S] in cell culture media containing 20% FBS (n = 3).
Cytotoxicity in the Dark to HUVEC and ARPE-19 Cells
Figure 4A shows the inhibition effects of RGD-SSL-[P]-[S] at various concentrations against ARPE-19 cells. No significant difference was found between the first four drug groups and the control group. The group of 5 × 10−6 M photocyanine/6.4 × 10−4 M sorafenib, which had demonstrated cytotoxicity in the dark, showed an obviously lower survival rate compared with the control group (P < 0.05); the 1 × 10−5 M/1.28 × 10−3 M group showed the lowest survival rate compared with the control group (P < 0.001). Figure 4B shows the inhibition effects of RGD-SSL-[P]-[S] at various concentrations against HUVEC cells; the 1 × 10−6 M/3.2 × 10−4 M group showed certain cytotoxicity with a survival rate below 90% (P < 0.05 versus the control group). With the increase of drug concentration, cytotoxicity obviously increased. Generally, the 1 × 10−6 M/3.2 × 10−4 M group might be the optimal concentration for RGD-SSL-[P]-[S], because under this level, its cytotoxicity to HUVEC and ARPE-19 cells was obvious (significant) and unobvious (not significant), respectively. 
Figure 4
 
Inhibitory effects to ARPE-19 cells (A) and HUVEC cells (B) after applying various concentrations of RGD-SSL-[P]-[S] in the dark by MTT assay (12-hour incorporation, 24-hour incubation).
Figure 4
 
Inhibitory effects to ARPE-19 cells (A) and HUVEC cells (B) after applying various concentrations of RGD-SSL-[P]-[S] in the dark by MTT assay (12-hour incorporation, 24-hour incubation).
PDT on HUVEC and ARPE-19 Cell Models
Figure 5A shows the inhibition effects of RGD-SSL-[P]-[S] to ARPE-19 cells under different irradiation times. At the 240-second irradiation time, the survival rate of ARPE-19 cells was above 90%, compared with the control group (P ≥ 0.05). When the irradiation time increased to 360 seconds, the survival rate of ARPE-19 cells decreased, suggesting obvious phototoxicity compared with the control group (P ≤ 0.05). Figure 5B shows the inhibition effects of RGD-SSL-[P]-[S] to HUVEC cells under different irradiation times. At the 20-second irradiation time, there was no significant difference in survival rate between the RGD-SSL-[P]-[S] and control groups. With the extension of irradiation time, the stronger phototoxicity on cells could be seen. In conclusion, 240 seconds might be the optimal irradiation time, as the cytotoxicity of RGD-SSL-[P]-[S] to HUVEC and ARPE-19 cells at this irradiation time was obvious (significant) and unobvious (not significant), respectively. 
Figure 5
 
Phototoxicity of RGD-SSL-[P]-[S] delivered in liposomes on ARPE-19 cells (A) and HUVEC cells (B) under light determined by MTT assay (12-hour incorporation irradiation, 24-hour incubation).
Figure 5
 
Phototoxicity of RGD-SSL-[P]-[S] delivered in liposomes on ARPE-19 cells (A) and HUVEC cells (B) under light determined by MTT assay (12-hour incorporation irradiation, 24-hour incubation).
Combination Therapy With Both Targeted PDT and Anti-VEGF Drug In Vivo
Representative choroidal flat mounts are presented in Figure 6. Quantification of choroidal flat mounts is given in Figure 6F. All therapy groups significantly decreased the CNV area compared with the NS group (P < 0.05), whereas the RGD-SSL-[P]-[S] most reduced this value (P < 0.001). The CNV area of SSL-[P]-[S] and RGD-SSL-[P]-[S] groups was less than that of SSL-[P] and RGD-SSL-[P] groups (P < 0.05). The CNV area of E group (RGD-SSL-[P]-[S] group) was significantly less than that of SSL-[P]-[S] group (P < 0.05). 
Figure 6
 
Effect of NS (A), SSL-[P] (B), RGD-SSL-[P] (C), SSL-[P]-[S] (D), and RGD-SSL-[P]-[S] (E) on the size of the CNV complex. Representative choroidal flat mounts with FITC-dextran–perfused vessels (green). (F) Quantification of the choroidal flat mounts. Data are reported as the mean ± SD.
Figure 6
 
Effect of NS (A), SSL-[P] (B), RGD-SSL-[P] (C), SSL-[P]-[S] (D), and RGD-SSL-[P]-[S] (E) on the size of the CNV complex. Representative choroidal flat mounts with FITC-dextran–perfused vessels (green). (F) Quantification of the choroidal flat mounts. Data are reported as the mean ± SD.
Figure 7 shows the representative images of CNV staining and the leakage on fluorescein angiogram 2 weeks after laser photocoagulation. The effect of NS, SSL-[P], RGD-SSL-[P], SSL-[P]-[S], and RGD-SSL-[P]-[S] on the size of the CNV complex was similar to those observed in Figure 6. It indicated that there were obvious differences in CNV area and fluorescein leakage in FFA between the NS group and other therapy groups. Rats treated with RGD-SSL-[P]-[S] showed the least CNV area and fluorescein leakage in FFA. 
Figure 7
 
Laser-induced CNV on fluorescein angiogram. Representative images of CNV staining and leakage on fluorescein angiogram 2 weeks after laser photocoagulation. (A) NS, (B) SSL-[P], (C) RGD-SSL-[P], (D) SSL-[P]-[S], and (E) RGD-SSL-[P]-[S].
Figure 7
 
Laser-induced CNV on fluorescein angiogram. Representative images of CNV staining and leakage on fluorescein angiogram 2 weeks after laser photocoagulation. (A) NS, (B) SSL-[P], (C) RGD-SSL-[P], (D) SSL-[P]-[S], and (E) RGD-SSL-[P]-[S].
The Effect of RGD-SSL-[P]-[S] on the Retinal Structure of BN Rats
Figure 8 summarizes the effect of RGD-SSL-[P]-[S] on retinal structure in BN rats 14 days after photocoagulation. Compared with the NS group, the RGD-SSL-[P]-[S] group showed a smaller CNV area. It was observed that the other tissues without CNV were clear, with no inflammatory cells. This also indicated the safety of RGD-SSL-[P]-[S] to the rat eye, especially the retina. 
Figure 8
 
Effect of RGD-SSL-[P]-[S] on a retinal structure in BN rats. Histological changes were evaluated 14 days after laser photocoagulation. (A) NS, (B) RGD-SSL-[P]-[S].
Figure 8
 
Effect of RGD-SSL-[P]-[S] on a retinal structure in BN rats. Histological changes were evaluated 14 days after laser photocoagulation. (A) NS, (B) RGD-SSL-[P]-[S].
Discussion
Combination therapy refers to using multiple therapies to treat a single disease and this strategy is used often in cancer therapy. For the treatment of ocular neovascularization, including CNV, there is now a great deal of interest and increasing competition to develop a new and more efficacious combination therapy. For instance, PDT was reported to combine with immunosuppression and VEGF inhibitor, respectively, and some studies are already in clinical trials with satisfactory therapeutic effect. 23  
Targeted PDT is believed to be the next generation of PDT. It promises to increase specificity and potency, and improve drug pharmacokinetics, thus better delivering photosensitive dye or PDT drug while retaining its other benefits. Although PDT itself is a selective modality, it can be further enhanced by combining other targeted strategies, including the use of home peptides to modify the drug delivery system of photosensitizers. Targeted PDT is still at its beginning, with only a few reports on antibody-targeted PDT for tumor treatment. 24  
The RGD sequence is the cell attachment site of a large number of adhesive cell surface proteins, and nearly half of the more than 20 known integrins that serve as receptors for RGD recognize and bind this sequence. So RGD is often used as the homing peptide to promote the intracellular delivery of related drugs in different therapies. 25 In our previous study, we found that the modification with RGD on the surface of liposomes enhanced their cell uptake in HUVECs, 16 laying the foundation for the design of RGD-SSL-[P]-[S] in this report. 
As listed in the Table and Figures, there was no significant difference between SSL-[P]-[S] and RGD-SSL-[P]-[S] in terms of particle size, surface potential, encapsulation ratio, and drug release rate, suggesting little effect from the peptide modification. This is favorable for the comparison of the active targeted delivery system and passive targeted delivery system. 
At 48 hours, the release rate of photocyanine from SSL-[P]-[S] and RGD-SSL-[P]-[S] was approximately 22%, whereas that of sorafenib from two liposome systems was approximately 40% (Fig. 3). Although the distribution of liposomes in vivo may be somehow a dynamic process, it is reported that the drug distribution in tissues after intravenous injection has usually reached its maximum at approximately 1 to 2 hours. 26 So it means that during the phase of fast distribution, only a few drugs were released from the lipid nanocarriers. 
To obtain better efficacy, we optimize the drug concentrations as well as the irradiation time. As shown in Figure 4, at the concentration of 1 × 10−6 M photocyanine/3.2 × 10−4 M sorafenib, RGD-SSL-[P]-[S] exhibited obvious cytotoxicity to only HUVECs. With an irradiation time of 240 seconds at the same drug levels as above, again RGD-SSL-[P]-[S] showed cytotoxicity only to HUVECs (Fig. 5). This is vital, as normal cells might be well protected under these conditions, and that is what a targeted delivery system is all about. 
In general, integrin-targeted liposomes that incorporate both photocyanine and sorafeinb were developed and investigated here for the treatment of CNV. The targeted system and its control were found to be qualified in terms of particle size, encapsulation efficiency, and drug release rate. Under the optimal conditions, RGD-SSL-[P]-[S] exhibited toxicity only to HUVECs with neovascularization, without obvious damage to normal ARPE-19 cells. In vivo studies observed that RGD-SSL-[P]-[S] presented the least CNV area and fluorescein leakage in FFA, whereas it was safe to the rat retina. The improved therapy for CNV might come from the targeted PDT, anti-VEGF treatment, EPR effect, and so on. 
Acknowledgments
Supported by projects from the National Science Foundation (81130059), Ministry of Science and Technology of China (2009CB930300 and 2009ZX09310-001), and Innovation Team of the Ministry of Education (BMU20110263). 
Disclosure: J.-L. Wang, None; Y. Xi, None; Y.-L. Liu, None; Z.-H. Wang, None; Q. Zhang, None 
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Footnotes
 J-LW and YX contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
The morphology of RGD-SSL-[P]-[S] under transmission electron microscope. The size of RGD-SSL-[P]-[S] was approximately 90 nm. Scale bar: 100 nm.
Figure 1
 
The morphology of RGD-SSL-[P]-[S] under transmission electron microscope. The size of RGD-SSL-[P]-[S] was approximately 90 nm. Scale bar: 100 nm.
Figure 2
 
Stability study of liposomal dispersion stored at 4°C for 28 days. (A) Change of particle size. (B) Change of sorafenib loading. (C) Change of photocyanine loading.
Figure 2
 
Stability study of liposomal dispersion stored at 4°C for 28 days. (A) Change of particle size. (B) Change of sorafenib loading. (C) Change of photocyanine loading.
Figure 3
 
Release rates of (A) photocyanine and (B) sorafenib from SSL-[P]-[S] and RGD-SSL-[P]-[S] in cell culture media containing 20% FBS (n = 3).
Figure 3
 
Release rates of (A) photocyanine and (B) sorafenib from SSL-[P]-[S] and RGD-SSL-[P]-[S] in cell culture media containing 20% FBS (n = 3).
Figure 4
 
Inhibitory effects to ARPE-19 cells (A) and HUVEC cells (B) after applying various concentrations of RGD-SSL-[P]-[S] in the dark by MTT assay (12-hour incorporation, 24-hour incubation).
Figure 4
 
Inhibitory effects to ARPE-19 cells (A) and HUVEC cells (B) after applying various concentrations of RGD-SSL-[P]-[S] in the dark by MTT assay (12-hour incorporation, 24-hour incubation).
Figure 5
 
Phototoxicity of RGD-SSL-[P]-[S] delivered in liposomes on ARPE-19 cells (A) and HUVEC cells (B) under light determined by MTT assay (12-hour incorporation irradiation, 24-hour incubation).
Figure 5
 
Phototoxicity of RGD-SSL-[P]-[S] delivered in liposomes on ARPE-19 cells (A) and HUVEC cells (B) under light determined by MTT assay (12-hour incorporation irradiation, 24-hour incubation).
Figure 6
 
Effect of NS (A), SSL-[P] (B), RGD-SSL-[P] (C), SSL-[P]-[S] (D), and RGD-SSL-[P]-[S] (E) on the size of the CNV complex. Representative choroidal flat mounts with FITC-dextran–perfused vessels (green). (F) Quantification of the choroidal flat mounts. Data are reported as the mean ± SD.
Figure 6
 
Effect of NS (A), SSL-[P] (B), RGD-SSL-[P] (C), SSL-[P]-[S] (D), and RGD-SSL-[P]-[S] (E) on the size of the CNV complex. Representative choroidal flat mounts with FITC-dextran–perfused vessels (green). (F) Quantification of the choroidal flat mounts. Data are reported as the mean ± SD.
Figure 7
 
Laser-induced CNV on fluorescein angiogram. Representative images of CNV staining and leakage on fluorescein angiogram 2 weeks after laser photocoagulation. (A) NS, (B) SSL-[P], (C) RGD-SSL-[P], (D) SSL-[P]-[S], and (E) RGD-SSL-[P]-[S].
Figure 7
 
Laser-induced CNV on fluorescein angiogram. Representative images of CNV staining and leakage on fluorescein angiogram 2 weeks after laser photocoagulation. (A) NS, (B) SSL-[P], (C) RGD-SSL-[P], (D) SSL-[P]-[S], and (E) RGD-SSL-[P]-[S].
Figure 8
 
Effect of RGD-SSL-[P]-[S] on a retinal structure in BN rats. Histological changes were evaluated 14 days after laser photocoagulation. (A) NS, (B) RGD-SSL-[P]-[S].
Figure 8
 
Effect of RGD-SSL-[P]-[S] on a retinal structure in BN rats. Histological changes were evaluated 14 days after laser photocoagulation. (A) NS, (B) RGD-SSL-[P]-[S].
Table
 
Characteristics of the Prepared Liposomes (n = 3)
Table
 
Characteristics of the Prepared Liposomes (n = 3)
Liposomes Average Particle Size, nm Polydispersity, PDI Zeta Potential, mV Entrapment Efficiency of P, % Entrapment Efficiency of S, %
SSL-[P] 99.13 ± 2.52 0.216 ± 0.021 −2.4 ± 0.27 93.40 ± 0.36
RGD-SSL-[P] 101.08 ± 1.83 0.297 ± 0.013 −3.12 ± 0.38 91.86 ± 0.22*
SSL-[S] 94.48 ± 3.28 0.258 ± 0.038 −2.93 ± 0.23 98.98 ± 0.83
RGD-SSL-[S] 98.39 ± 1.99 0.290 ± 0.049 −3.43 ± 0.41 98.02 ± 0.64
SSL-[P]-[S] 100.58 ± 2.09 0.280 ± 0.0313 −3.40 ± 0.66 93.98 ± 0.98 86.17 ± 0.52
RGD-SSL-[P]-[S] 99.69 ± 3.10 0.305 ± 0.0247 −2.94 ± 0.20 93.66 ± 0.71 82.35 ± 1.49†
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