Aflibercept Nanoformulation Inhibits VEGF Expression in Ocular In Vitro Model: A Preliminary Report

Age-related macular degeneration (AMD) is one of the leading causes of blindness in the United States, affecting approximately 11 million patients. AMD is caused primarily by an upregulation of vascular endothelial growth factor (VEGF). In recent years, aflibercept injections have been used to combat VEGF. However, this treatment requires frequent intravitreal injections, leading to low patient compliance and several adverse side effects including scarring, increased intraocular pressure, and retinal detachment. Polymeric nanoparticles have demonstrated the ability to deliver a sustained release of drug, thereby reducing the necessary injection frequency. Aflibercept (AFL) was encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) via double emulsion diffusion. Scanning electron microscopy showed the NPs were spherical and dynamic light scattering demonstrated that they were uniformly distributed (PDI < 1). The encapsulation efficiency and drug loading were 75.76% and 7.76% respectively. In vitro release studies showed a sustained release of drug; 75% of drug was released by the NPs in seven days compared to the full payload released in 24 h by the AFL solution. Future ocular in vivo studies are needed to confirm the biological effects of the NPs. Preliminary studies of the proposed aflibercept NPs demonstrated high encapsulation efficiency, a sustained drug release profile, and ideal physical characteristics for AMD treatment. This drug delivery system is an excellent candidate for further characterization using an ocular neovascularization in vivo model.


Introduction
A decrease in visual acuity can lead to alterations in the activities of daily living, quality of life, self-care, and mental health of an individual. A disease commonly responsible for this visual impairment is age-related macular degeneration (AMD). AMD is a degenerative disease of the macula and it affects the part of the retina responsible for sharp, detailed central vision. AMD is the most prominent cause of vision loss in people 50 years and older in North America and it is expected to affect 7.5 million people by 2020 in the United States [1]. Several factors influence the prognosis and progression of AMD, including genetic variation, living environment, and lifestyle [2]. polymer PLGA. The NPs were then characterized for their physical characteristics, encapsulation efficiency, and sustained release profile. The cytotoxicity and efficacy of the NPs in human retinal pigment epithelial (ARPE-19) cells were determined via MTT assay and ELISA. By doing so, it was demonstrated that a high encapsulation efficiency, uniform size distribution and sustained drug release were achieved by the nanoformulation of the protein AFL.

Nanoparticle Preparation
The double-emulsion diffusion method was used to prepare AFL NPs (Figure 1). One milligram of AFL (25 µL stock solution) was added to 100 µL 1× PBS and a separate solution of 4.5 mg PLGA was prepared in 1 mL dichloromethane (DCM). These solutions were combined and emulsified using sonication at 9.5 W for approximately 30 s. After the dropwise addition of 2 mL 1% w/v PVA, the suspension was emulsified via sonication at 9.5 W for another 30 s. The emulsion was stirred at 500 rpm overnight at 4 • C to allow diffusion. The emulsion was split into two fractions and centrifuged at 20,000× g at 4 • C for 15 min. Each pellet was then resuspended in 1 mL 2% w/v mannitol. Blank NPs were prepared similarly excluding the addition of AFL. demonstrated that a high encapsulation efficiency, uniform size distribution and sustained drug release were achieved by the nanoformulation of the protein AFL.

Nanoparticle Preparation
The double-emulsion diffusion method was used to prepare AFL NPs (Figure 1). One milligram of AFL (25 µL stock solution) was added to 100 µL 1× PBS and a separate solution of 4.5 mg PLGA was prepared in 1 mL dichloromethane (DCM). These solutions were combined and emulsified using sonication at 9.5 W for approximately 30 s. After the dropwise addition of 2 mL 1% w/v PVA, the suspension was emulsified via sonication at 9.5 W for another 30 s. The emulsion was stirred at 500 rpm overnight at 4 °C to allow diffusion. The emulsion was split into two fractions and centrifuged at 20,000× g at 4 °C for 15 min. Each pellet was then resuspended in 1 mL 2% w/v mannitol. Blank NPs were prepared similarly excluding the addition of AFL.

Nanoparticle Characterization
The size and polydispersity index (PDI) of both the AFL and blank NPs were analyzed via dynamic light scattering (DLS) using a Wyatt DynaPro plate reader (Wyatt Technology Corporation,

Nanoparticle Characterization
The size and polydispersity index (PDI) of both the AFL and blank NPs were analyzed via dynamic light scattering (DLS) using a Wyatt DynaPro plate reader (Wyatt Technology Corporation, Santa Barbara, CA, USA). The NPs were diluted 1:200 in filtered deionized water to meet equipment specifications and analyzed in triplicate.

Scanning Electron Microscopy (SEM)
The NPs were viewed via SEM using a JOEL JSM-6490LV (JOEL Industries, Tokyo, Japan). Samples were diluted 1:10 in filtered deionized water and adhered to aluminum cylinders using a carbon polymer adhesive. All images were obtained using a 5 kV acceleration voltage.

Encapsulation Efficiency and Drug Loading
The encapsulation efficiency (EE) and drug loading (DL) of the NP formulation was calculated using the concentration of drug present in a 1 mL sample of the NPs. After centrifugation at 15,000× g for 5 min, the NPs were suspended in methanol and placed at 4 • C overnight to dissolve the NPs. Both this sample and a standard curve of AFL in methanol were prepared using the Pierce BCA protein assay (Thermo Scientific, Waltham, MA, USA) and analyzed via VIS spectroscopy at 562 nm on a BioTek Synergy H4 plate reader (BioTek Instruments Inc., Winooski, VT, USA). The standard curve was used to convert the absorbance of the NP sample to a concentration. EE and DL were calculated as follows: %EE = (Mass of entrapped drug)/(Total mass of drug) × 100% %DL = (Mass of entrapped drug)/(Mass of entrapped drug + Mass of polymer) × 100%.

In Vitro Release Studies
An in vitro release study was conducted according to a previously reported method [19]. Dialysis membrane cassettes were soaked in 1× PBS at 4 • C. Approximately 500 µL of AFL-NPs or AFL solution were inserted into the membrane and suspended in 100 mL PBS at 37 • C. Aliquots were removed and replaced with preheated PBS at regular intervals over a period of seven days. The collected samples were analyzed via BCA Protein Assay and read using spectroscopy at 562 nm with the Synergy H4 plate reader (Biotek Industries, Inc., Winooski, VT, USA). These values were compared to a calibration curve of AFL in 1× PBS to determine the cumulative percent of drug released at each interval.

Cytotoxicity
The cytotoxicity of the AFL-loaded and blank NPs in ARPE-19 cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) assay [12]. ARPE-19 cells were seeded in a 48-well plate and incubated at 37 • C with 5% CO 2 for 48 h to achieve confluence. Cell culture media was aspirated and the cells were treated with each of the NP formulations at final concentrations of 0.5 µM and 1 µM. After a 24-h incubation period, 300 µL of a 0.5 mg/mL solution of MTT reagent previously prepared in DMEM were added to each well. After another four hours of incubation, the MTT reagent was removed and 300 µL of DMSO were added to end the reaction. After shaking briefly, the plate was analyzed at 570 nm using a BioTek Synergy H4 plate reader (BioTek Instruments Inc., Winooski, VT, USA) to determine the relative amounts of live cells present in the sample. These values were reported as a percentage of the untreated control.

VEGF-A Inhibition
ARPE-19 cells were seeded in a 48-well plate and incubated at 37 • C with 5% CO 2 to achieve confluence and treated with each of the NP formulations at a final concentration of 0.5 µM. After 72 h, the expression of VEGF-A was quantified via enzyme-linked immunosorbent assay (ELISA) (Human VEGFA ELISA kit, Thermo Scientific, Waltham, MA, USA). Media samples were collected and analyzed using the Synergy H4 plate reader (Biotek Industries, Inc., Winooski, VT, USA) at 450 nm and 550 nm. VEGF-A expression was calculated as follows: %Expression = (absorbance 450 nm − absorbance 550 nm)/(Control absorbance 450 nm − Control absorbance 550 nm) × 100%.

Statistical Analysis
Statistical analyses were carried out using GraphPad Prism 5, Version 5.02. Comparisons of each concentration of NP treatment, on both cell viability and VEGF-A expression, were completed using two-way ANOVA with Bonferroni post-tests to compare each treatment to the control, as well as other concentrations (if applicable). All results are written as mean values ± SD.

Nanoparticle Characterization
The diameter and PDI of the AFL and blank NPs were found in triplicate via DLS. AFL-NPs were larger than their respective blanks ( Table 1). The PDI was consistently less than 1, indicating a uniform size distribution (Figure 2A).

VEGF-A Inhibition
ARPE-19 cells were seeded in a 48-well plate and incubated at 37 °C with 5% CO2 to achieve confluence and treated with each of the NP formulations at a final concentration of 0.5 µM. After 72 h, the expression of VEGF-A was quantified via enzyme-linked immunosorbent assay (ELISA) (Human VEGFA ELISA kit, Thermo Scientific, Waltham, MA, USA). Media samples were collected and analyzed using the Synergy H4 plate reader (Biotek Industries, Inc., Winooski, VT, USA) at 450 nm and 550 nm. VEGF-A expression was calculated as follows: %Expression = (absorbance 450 nm − absorbance 550 nm)/(Control absorbance 450 nm − Control absorbance 550 nm) × 100%.

Statistical Analysis
Statistical analyses were carried out using GraphPad Prism 5, Version 5.02. Comparisons of each concentration of NP treatment, on both cell viability and VEGF-A expression, were completed using two-way ANOVA with Bonferroni post-tests to compare each treatment to the control, as well as other concentrations (if applicable). All results are written as mean values ± SD.

Nanoparticle Characterization
The diameter and PDI of the AFL and blank NPs were found in triplicate via DLS. AFL-NPs were larger than their respective blanks (Table 1). The PDI was consistently less than 1, indicating a uniform size distribution (Figure 2A).

Scanning Electron Microscopy (SEM)
AFL and blank NPs were visualized using SEM (Figure 3). Inspection showed that the NPs were spherical. The size and uniformity of the NPs corroborated with data determined via DLS.

Scanning Electron Microscopy (SEM)
AFL and blank NPs were visualized using SEM (Figure 3). Inspection showed that the NPs were spherical. The size and uniformity of the NPs corroborated with data determined via DLS.

Encapsulation Efficiency and Drug Loading
The EE and DL of each NP formulation was determined via VIS spectroscopy after analysis with the BCA protein assay. A standard curve of AFL in methanol was prepared to determine the concentration of AFL present in the NP fraction (r 2 = 0.9247) (not shown). The NPs demonstrated an EE of 75.76 ± 2.59% and DL of 7.76 ± 0.24% (Table 1).

In Vitro Release
The rate of drug release from the NP formulation versus AFL in free solution was examined over seven days in 100 mL 1× PBS at 37 °C. AFL was fully released by solution within 24 h. Comparatively, the NPs released 74.49% in seven days. An initial burst release is demonstrated from the NPs in the first 2 h ( Figure 2B).

Cytotoxicity
The cytotoxicity of blank NPs, AFL NPs and AFL solution to ARPE-19 cells were determined via MTT assay. After 24 hours, 0.5 µM AFL NPs and blank NPs reduced cell viability in ARPE-19 cells by 30.11 ± 0.344% and 27.24 ± 5.22%, respectively compared to 17.29 ± 4.44% reduction caused by AFL. The 1 µM treatments of AFL NPs and blank NPs resulted in a 41.29 ± 10.99% and 40.87 ± 7.15% reduction in cell viability, and the AFL reduced viability by 21.16 ± 9.37%. The differences between the two concentrations of each treatment were considered insignificant (p < 0.05). Both NPs at each concentration showed significant toxicity compared to the control (p < 0.001). However, the AFLloaded NPs showed no significant difference in cytotoxicity compared to the blank NPs ( Figure 4A).

VEGF-A Inhibition
The effect of AFL and AFL NPs on the VEGF-A expression of ARPE-19 cells was examined via ELISA. After 72 h, VEGF-A was not significantly reduced by 0.5 µM AFL solution or AFL NPs. AFL solution reduced VEGF-A expression by 21.7 ± 16.4% and AFL NPs reduced expression by 0.6 ± 11.3% ( Figure 4B). It must be noted that, though AFL solution is fully released into the surrounding media within 72 h, AFL NPs take over seven days to fully release ( Figure 2B).

Encapsulation Efficiency and Drug Loading
The EE and DL of each NP formulation was determined via VIS spectroscopy after analysis with the BCA protein assay. A standard curve of AFL in methanol was prepared to determine the concentration of AFL present in the NP fraction (r 2 = 0.9247) (not shown). The NPs demonstrated an EE of 75.76 ± 2.59% and DL of 7.76 ± 0.24% (Table 1).

In Vitro Release
The rate of drug release from the NP formulation versus AFL in free solution was examined over seven days in 100 mL 1× PBS at 37 • C. AFL was fully released by solution within 24 h. Comparatively, the NPs released 74.49% in seven days. An initial burst release is demonstrated from the NPs in the first 2 h ( Figure 2B).

Cytotoxicity
The cytotoxicity of blank NPs, AFL NPs and AFL solution to ARPE-19 cells were determined via MTT assay. After 24 hours, 0.5 µM AFL NPs and blank NPs reduced cell viability in ARPE-19 cells by 30.11 ± 0.344% and 27.24 ± 5.22%, respectively compared to 17.29 ± 4.44% reduction caused by AFL. The 1 µM treatments of AFL NPs and blank NPs resulted in a 41.29 ± 10.99% and 40.87 ± 7.15% reduction in cell viability, and the AFL reduced viability by 21.16 ± 9.37%. The differences between the two concentrations of each treatment were considered insignificant (p < 0.05). Both NPs at each concentration showed significant toxicity compared to the control (p < 0.001). However, the AFL-loaded NPs showed no significant difference in cytotoxicity compared to the blank NPs ( Figure 4A).

VEGF-A Inhibition
The effect of AFL and AFL NPs on the VEGF-A expression of ARPE-19 cells was examined via ELISA. After 72 h, VEGF-A was not significantly reduced by 0.5 µM AFL solution or AFL NPs. AFL solution reduced VEGF-A expression by 21.7 ± 16.4% and AFL NPs reduced expression by 0.6 ± 11.3% ( Figure 4B). It must be noted that, though AFL solution is fully released into the surrounding media within 72 h, AFL NPs take over seven days to fully release ( Figure 2B).

Discussion
The double emulsion diffusion method was used to prepare AFL NPs. Particle size data revealed that the drug-loaded NPs were 73 nm larger in diameter than similarly prepared blank NPs, confirming the presence of the drug. A NP of approximately 200 nm in diameter is ideal to localize to retinal pigment epithelial (RPE) cells; this is indeed demonstrated by the AFL NPs (Table 1). Additionally, the PDI of each formulation remained below one, demonstrating a uniform size distribution (Figure 2A). SEM micrographs showed that the NPs possessed a spherical morphology (Figure 3). Spherical NPs possess a high surface area to volume ratio compared to other shapes (rod, cube, etc.) resulting in a more reactive surface and ultimately more opportunities to produce a therapeutic effect [20]. Furthermore, the NPs demonstrated a Gaussian size distribution (0.2 PDI) thereby reducing any variability in this effect among the individual NPs (Figure 2A). Protein assay showed that the NPs encapsulated 75.76 ± 2.59% of the drug used in NP preparation and 7.76 ± 0.24% of the NP mass consisted of drug. As drug loading is a ratio of drug mass to NP total mass, this value may be increased by adding a higher ratio of drug to polymer during NP preparation. However, it has been previously noted that these changes have adverse effects on shape uniformity [21]. These results were consistently reproducible (data not shown) and corroborated with previously published studies [21][22][23].
Cytotoxicity studies demonstrated that free AFL is non-toxic. After 24 h, 0.5 µM and 1 µM concentrations of both AFL NPs and blank NPs significantly reduced cell viability in ARPE-19 cells (p < 0.001). However, there was no significant difference in the toxicity of the AFL NPs compared to blank NPs at each concentration ( Figure 4A), meaning that this toxicity is due to the use of NPs rather than any inherent cytotoxicity of AFL. This may be due to the method of in vitro testing; if NPs settled to the flat bottom of the wells any cells trapped underneath would be suffocated. A similar scenario was observed by Irfan et al. as silica NPs produced sedimentation during an LDH assay [24]. They suggest that this is due to dose concentration and aggregation. Perhaps varying these factors would produce different results. The eye's natural circulation prevents this adverse effect in vivo.
After 72 h, VEGF-A was significantly reduced in ARPE-19 cells by 0.5 µM AFL solution but not by the same concentration of AFL NPs ( Figure 4B). It must be noted that, though AFL solution is fully released into the surrounding media within 72 h, AFL NPs take over seven days to fully release ( Figure 2B). In vitro release studies showed that in 72 h, less than 57% of AFL was released into the surrounding media ( Figure 2B). The sustained release properties of the NPs account for the low VEGF-A reduction by the AFL NPs. Hirani

Discussion
The double emulsion diffusion method was used to prepare AFL NPs. Particle size data revealed that the drug-loaded NPs were 73 nm larger in diameter than similarly prepared blank NPs, confirming the presence of the drug. A NP of approximately 200 nm in diameter is ideal to localize to retinal pigment epithelial (RPE) cells; this is indeed demonstrated by the AFL NPs (Table 1). Additionally, the PDI of each formulation remained below one, demonstrating a uniform size distribution (Figure 2A). SEM micrographs showed that the NPs possessed a spherical morphology (Figure 3). Spherical NPs possess a high surface area to volume ratio compared to other shapes (rod, cube, etc.) resulting in a more reactive surface and ultimately more opportunities to produce a therapeutic effect [20]. Furthermore, the NPs demonstrated a Gaussian size distribution (0.2 PDI) thereby reducing any variability in this effect among the individual NPs (Figure 2A). Protein assay showed that the NPs encapsulated 75.76 ± 2.59% of the drug used in NP preparation and 7.76 ± 0.24% of the NP mass consisted of drug. As drug loading is a ratio of drug mass to NP total mass, this value may be increased by adding a higher ratio of drug to polymer during NP preparation. However, it has been previously noted that these changes have adverse effects on shape uniformity [21]. These results were consistently reproducible (data not shown) and corroborated with previously published studies [21][22][23].
Cytotoxicity studies demonstrated that free AFL is non-toxic. After 24 h, 0.5 µM and 1 µM concentrations of both AFL NPs and blank NPs significantly reduced cell viability in ARPE-19 cells (p < 0.001). However, there was no significant difference in the toxicity of the AFL NPs compared to blank NPs at each concentration ( Figure 4A), meaning that this toxicity is due to the use of NPs rather than any inherent cytotoxicity of AFL. This may be due to the method of in vitro testing; if NPs settled to the flat bottom of the wells any cells trapped underneath would be suffocated. A similar scenario was observed by Irfan et al. as silica NPs produced sedimentation during an LDH assay [24]. They suggest that this is due to dose concentration and aggregation. Perhaps varying these factors would produce different results. The eye's natural circulation prevents this adverse effect in vivo.
After 72 h, VEGF-A was significantly reduced in ARPE-19 cells by 0.5 µM AFL solution but not by the same concentration of AFL NPs ( Figure 4B). It must be noted that, though AFL solution is fully released into the surrounding media within 72 h, AFL NPs take over seven days to fully release ( Figure 2B). In vitro release studies showed that in 72 h, less than 57% of AFL was released into the surrounding media ( Figure 2B). The sustained release properties of the NPs account for the low VEGF-A reduction by the AFL NPs. Hirani et al. reported a similar effect with triamcinolone acetonide. More potent inhibitory properties were demonstrated after 72 h compared to 12 h of treatment [12]. PLGA NPs formulated by Patel et al. illustrated a similar in vitro release profile with 25% of drug released after 48 h [25]. These sustained release properties allow the NPs to deliver a more powerful effect in vivo.
The current study demonstrates the potential of a novel nanoformulation containing AFL for the inhibition of VEGF in vitro. Further in vitro studies will be needed to demonstrate VEGF inhibition by the nanoformulation as well as AFL solution. Future in vivo studies will be carried out in a laser-induced choroidal neovascularization mouse model. The efficacy, toxicity, biodistribution and pharmacokinetics will be examined in this manner. Further clinical studies may be conducted in a human population to analyze the total effect of the nanoformulation. In summary, our nanoformulation of AFL shows potential as a protein drug delivery vehicle for the amelioration of ocular neovascularization in vitro.

Conclusions
Polymeric NPs were prepared for the sustained delivery of the protein aflibercept. The NPs were approximately 200 nm in diameter; an appropriate size for ocular therapeutics. The NPs were uniformly distributed and possessed a spherical morphology. The NPs demonstrated a sustained drug release over seven days and were not significantly more toxic to ARPE-19 cells than similarly prepared blank NPs. VEGF-A reduction in ARPE-19 cells was not significant after 72 h due to the sustained release property of the NPs. Future in vivo studies are needed to determine the effect of the NPs on live animal tissue as well as the clinical viability of the NPs in humans. Overall, aflibercept NPs demonstrated promising properties as a protein delivery vehicle for the future treatment of AMD and other neovascular conditions.