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�:	Title: Intraocular biocompatibility of gold-nanoparticles.
Running Title: Biocompatibility of gold-nanoparticles. 
Authors: 
Jeffrey L. Olson, MD1*.			jeffrey.olson@ucdenver.edu
Raul Velez-Montoya, MD1. 			raul.velez-montoya@ucdenver.edu
Nicole Nghiem, BA2.				nicole.nghiem@ucdenver.edu
David A. Ammar, PhD1.			david.ammar@ucdenver.edu
Naresh Mandava, MD1. 			naresh.mandava@ucdenver.edu
Conrad R. Stoldt, PhD3.   			conrad.stoldt@colorado.edu
Affiliations:
Department of Ophthalmology, University of Colorado School of Medicine, Aurora CO 80045, US. 
University of Colorado School of Medicine, Aurora CO 80045, US.
Department of Mechanical Engineering, University of Colorado Boulder, Boulder CO 80302, US.
Corresponding Author: Jeffrey L. Olson, MD. Address: 1675 Aurora Court, Aurora CO 80045. Phone: (720) 848.2500. Fax: (720) 848.5014. Email: jeffrey.olson@ucdenver.edu
Author Contributions: Olson JL, Ammar DA and Velez-Montoya R: Contributed equally to this work. Jeffrey JL, Ammar DA, Mandava N and Velez-Montoya R designed the study. Velez-Montoya R and Nghiem N performed the research. Velez-Montoya R, Olson JL and Amman DA analyzed the data. Velez-Montoya R and Olson JL wrote the paper. 
Word Count: 3,878
A preliminary version of this study has never been presented as a paper or poster or submitted for publication. The authors (JO, NM & CRS) have a patent application pending for the disclosed device.  The rest of the authors do not have financial or economic interest on the publication of this manuscript. There were no grants or funds allocated to the completion of this study. The authors state that they have full control of all primary data and they agree to allow The Journal of Ocular Pharmacology and Therapeutics to review their data upon request.


Abstract.
Background: to assess the in vitro biocompatibility of colloidal gold nanoparticles (CGN) and to describe the effect of CGN over the normal electrical activity of the retina on an in vivo rat model.  
Material and Methods: Retinal pigment epithelium cells (ARPE-19) were cultured to confluence. Three wells were incubated with a mixture of CGN conjugated to goat anti-mouse IgG and three were incubated with culture media as a control. After 2 days, media was aspirated and incubated again with 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide for 2 hrs. The absorbance of each well was measured by spectrophotometry at a 540nm wavelength, subtracting the background measurement at 670nm. A total of 16 eyes of eight brown Norway rats were used, divided in two groups of eight eyes each; Right eyes received an intravitreal injection of 1�M/5�L CGN suspension at baseline. An electroretinogram (ERG) was done at baseline and then again after six weeks. A two-sample t-test was used as statistical method. 
Results: Absorbance signal for the CGN Group was 89.06% with respect to the reference values of 100% for untreated controls. There was no statistical difference between groups (p=0.1). The ERG yielded no statistically significant difference between groups at any time point for any of the five steps of the ERG. 
Conclusion: The in vitro viability of ARPE-19 cells seems to not be affected by addition of 5nm CGN to the culture, with no significant morphological changes. The normal electrical activity of the retina seems not be disturbed by the intravitreal injection of CNG.

Key Words: Gold Nanoparticles, biocompatibility, Quantum dots, Retina Pigment Epithelium, Electroretinogram. 


Introduction: 
Nanotechnology is a relative new science that studies new way to manipulate matter on an atomic and molecular level scale[1, 2]. Its rapid evolution has generated different nanoscale-sized materials, being the metal-based ones the most interesting and promising among them[3]. The unique physical and chemical properties of metal nanomaterials (due to the high quantity of surface situated atoms and high area/volume relation) have made its application in engineering, chemistry, biology and medicine feasible[3-5].  

Since ancient times, humans have been fascinated by gold (Au) and its unique beauty, malleability and resistance to chemical modification[4]. The first description of the use of Au as a therapeutic agent is dated to the Chinese in 2500BC[4]. Since then, multiple medical properties have been assigned to Au with various degrees of success[4]. Au is a remarkable material in nanoscale. In biomedical research, the most often used types of Au nanoparticles are: nanorods, nanospheres and nanoshells[4, 5]. Their possible applications include non-invasive bioimaging, detection and regulation of DNA[6], drug delivery systems[7, 8], cancer diagnosis and treatment[9-11], bacterial screening and even as a bactericide, independent on antibiotic action[3, 12]. Although Au nanoparticles seem to be nontoxic and biocompatible despite ability to enter all cell types, little is known about local tolerability in ocular tissue[13]. Therefore studies aimed to increase the information about the consequences as well as the effects of Au nanoparticles on ocular tissue in order to allow safe clinical trials are desirable. 

Nanoparticles in a solution are called colloids[4]. Colloidal Au nanoparticles (CGN) are prepared from the chemical reduction of an aqueous Au salt solution. Often, the particle morphology and size is controlled by the addition of one or more surfactants during the synthesis[14]. The Au colloid size distribution can be further narrowed by centrifugation, yielding monodispersive particle size distributions with diameters in the nanometer range[14]. The moderately reactive Au surface permits facile modification of the nanoparticle with a variety of different interface chemistries using charge attraction, hydrophobic interaction or chemical binding processes[15, 16]. For biological applications, the CGN are commonly conjugated to proteins and other ligands via sulfur groups, which spontaneously form covalent bonds with the Au surface[4]. 

There has been an increasing interest in the ophthalmic use of CGN because of its anti-angiogenic properties to treat diseases such as diabetic retinopathy, macular degeneration, and retinopathy of prematurity[13, 17]. Bakri et al previously reported that intravitreal administration of gold nanoparticles at 67�M/0.1ml and 670�M/0.1ml had no signs of retinal or optic nerve toxicity by light microscopy[13]. The aim of this study is to expand the available evidence regarding intraocular biocompatibility of CGN by describing the effect of adding CGN to in vitro cultures of retinal pigment epithelium (RPE) cells. The study also aims to describe the effect of CGN over the normal electrical activity of the retina as well, on an in vivo rat model.

Material and methods: 
The study was reviewed and approved by the University of Colorado Institutional Animal Care and Use Committee. All procedures were performed according to the statement for the use of animals in ophthalmic and visual research from the Association of Research of Vision and Ophthalmology (ARVO). The study was fully conducted at the University of Colorado facilities. There were no funds allocated for the realization of this study. 



RPE cell biocompatibility  assay: 
ARPE-19 cells (American Type culture Collections, Manassas, VA) were cultured using a 1:1 mixture (vol/vol) of Dulbecco�s modified Eagle�s and Ham�s nutrient mixture F-12 medium (DMEM F-12; Thermo Scientific HyClone, Logan, UT) with 4mM glutamine, 10% Fetal Bovine Serum (FBS; Gibco, Carlsbad, CA), Penicillin, and Streptomycin in a humidified incubator, kept at 37�C in a 5% CO2 environment. The media was changed every 2 to 3 days[18, 19].   

ARPE cells were allowed to grow to confluence in standard 96-well culture plates. Three wells of cells were then exposed to two hundred �L of a 1:3 mixture of 5nm CGN conjugated to goat anti-mouse IgG antibodies (colloidal volume <12%; SPI-MarkTM; SPI Supplies, West Chester, PA) and culture media. Three other wells were incubated with culture media and served as controls (all cultures were conducted in triplicates).  

After 2 days, media was aspirated from each well and 100�L of 0.05% 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO) in DMEM F-12 was added to each well and incubated at 37�C in a 5% CO2 environment for 2 hrs. Then, all media was removed from the wells and 100�L of dimethyl sulfoxide (DMSO 99.7%; Sigma-Aldrich, St. Louis, MO) was added and incubated under the same conditions as before for 10 minutes.

The absorbance of each well was measured at a 540nm wavelength, subtracting the background measurement at 670nm using a SynergyTM4 Microplate Reader  and Gen5 Data analysis software (BioTek Instruments Inc, Winooski, VT). 


Electroretinograms (ERG): 
Eight brown Norway rats (sixteen eyes) were used for the study. All animals were approximately 2 months of age at baseline testing. The animals' eyes were divided into two groups: All right eyes received the intraocular CGN (1�M/5�L CGN suspension) and all left eyes served as controls (CGN vs. Controls). 
 ERGs were performed at baseline prior intravitreal injections of CGN and then again after six weeks.  Both dark- and light-adapted ERGs were recorded. Prior to recording, the animals were anesthetized using Ketamine (80mg/kg) and Xylazine (5-10mg/kg) and their pupils dilated with 1% tropicamide/2% phenylephrine drops[20]. The cornea was additionally anesthetized using 1% tetracaine drops and coated with 1% methylcellulose. Using a handheld Ganzfeld stimulator bilateral ERGs were recorded simultaneously with DTL Plus electrodes (Diagnosys, Lowell, MA) in contact with the ocular surface through a coating of 1% methylcellulose[20].   Monopolar needle electrodes (Advena, Hereford, UK) were placed in the cheek (reference) and the tail (ground). 

The ERG protocol was the same for all animals and it was as follows: Step 1) dark-adapted rod response: single Flash intensity of -24dB, low cut filter of 0.3Hz and High cut filter of 500Hz, Gain: �500�v and time between stimulus of at least 5s. Step 2) dark-adapted maximal combined response: Single flash intensity of 0dB, low cut filter of 0.3Hz and High cut filter of 500Hz, Gain: �1250�v and time between stimulus of at least 10s. Step 3) dark-adapted oscillatory potentials: Single flash intensity of 0dB, low cut filter of 0.3Hz and High cut filter of 500Hz, Gain: �1250�v. Step 4) light-adapted single-flash cone response: Single flash intensity of 0dB, White background light intensity of 30cd/m2, low cut filter of 0.3Hz and High cut filter of 500Hz, Gain: �250�v and time between stimulus of at least 10s. Step 5) light-adapted 30Hz flicker response: flash intensity of 0dB, rate of 30.3Hz, background light intensity of 30cd/m2, low cut filter of 0.3Hz and High cut filter of 500Hz, Gain: �250�v and time between stimulus of 0.033s. All animals went into a dark adaptation phase of at least 30 minutes before step 1 to 3 and a light adaptation phase of at least 10 minutes before step 4 and 5. The LKC Epic 4000 electroretinography system (LKC Technologies, Gaithersburg, MD) was used for data filtering, acquisition, and averaging. A complete photopic, scotopic and flicker response were recorded each time[20, 21]. Every measurement was recorded a minimum of five times. 

The b-wave amplitude was measured for steps 1 (scotopic -25 dB), 2 (scotopic 0 dB), and 4 (photopic 0 dB).  The amplitude of the b-wave is determined by measuring the difference from the peak of the a-wave (if present) to the peak of the b-wave or from the baseline to the peak of the b-wave. 
For steps 3 (oscillatory potentials) and step 5 (30 Hz flicker) the measurements are automated by the EmWin software (LKC Technologies, Gaithersburg, MD), using automated root mean square for two-frequency bands in five horizontal and four vertical locations for the oscillatory potentials calculation.

Intravitreal Injection: 
After baseline ERG was recorded and with the animal under anesthesia (see above), right eyes were prepped with topical povidone iodine (10%). The procedure was made under a slit lamp and using micro forceps to help stabilize the eye. After the limbus was visualized, the conjunctiva and sclera was pierced with a 30 gauge needle 2mm posterior to the limbus, and a 1�M/5�L CGN suspension was injected with a 32 gauge needle into the mid-vitreous cavity. After the procedure, topical instillation of analgesics (diclofenac 0.1%) and antibiotics (tobramicyn ointment 3.0mg) were given as a single dose. The animals were monitored every 4-5 minutes while under anesthesia by observing any response to a toe or skin pinch until complete recovery. The animals were monitored daily for the first five days following injection for any signs of infection or alteration in behavior.  All the animals were then evaluated six weeks post-injection. Animals with traumatic crystalline lens puncture, vitreous hemorrhage or retinal detachment (as a complication of the intravitreal injection) were excluded from analysis.  

Statistical analyses: 
 Statistical analyses were made using Excel (Excel 2007; Microsoft Corp., Redmond, WA). A two sample t-test for means of two unpaired samples, with a p value of less than 0.05 for statistical significance, was used to compare the absorbance�s signals of the cell cultures. A two-sample t-test for means of two paired samples, with a p value of less than 0.05 for statistical significance was used to compare the differences of the means of the ERG recordings. Sample size was determined by using a power analysis, assuming a type I error (alpha) of 0.05 and a power of 80% for the test to detect a difference of 20% between groups, which yielded a minimum sample size of 7 per group.

Results: 
CGN in ARPE cell culture: 
After normalizing the MTT absorbance signal to untreated controls, absorbance signal from the CGN group was 89% � 0.33% (average � standard deviation) with respect to the reference value 100.0% � 0.34% for untreated controls (Figure 1: Control versus CGNs incubated ARPE-19 wells absorbances chart. There were no statistical differences between the two groups. SD: Standard deviation). There was no statistical difference between the absorbances of the groups (p=0.1). (Figure 2: Comparative pictures between control and CGN treated ARPE-19 wells, after and before incubation with CGNs (Nikon Eclipse TS100 microscope with the Nikon DS-Fi1 camera; Nikon Metrology Inc, Irvine CA, at 10x and no filter). It is important to note that despite the general cell morphology on the CGNs incubated ARPE-19 wells is preserved, the cells looks larger due to increased vacuolization) Figure 2 depict the microphotographs of two representative wells before and after the incubation with the CGN. 

ERG: 
None of the animals were excluded from analysis due to traumatic cataract, vitreous hemorrhage or retinal detachment. There was no statistically significant difference between the control and CGN injections groups, at any time point for any of the five steps of the electroretinogram.  For the scotopic -25 dB test, the average b-wave amplitude was 78.9�V in the control group and 62.8�V in the CGN group (t-test for paired samples, p = 0.05) at baseline.  At the end of six weeks, the average b-wave amplitudes were 70.5�V for the controls and 56.7�V for the CGN group (p= 0.118).  In the maximal combined test (scotopic 0 dB), the baseline readings were 180.1�V in the controls and 167.8�V in the CGN group (p=0.22). After six weeks, the readings were 165.5 �V in the controls and 159.1�V in the CGN group (p= 0.14).  Measuring oscillatory potentials at baseline, the control group averaged 47.2 �V and the CGN group 36.8�V (p=0.11).  After six weeks, oscillatory potentials measured 39.8�V and 34.8�V in the control and CGN groups respectively (p = 0.77).  Looking at the light-adapted portion of the testing, the baseline readings for the photopic 0 dB b-wave testing were 61.5�V and 56.2 �V (p = 0.59) and after six weeks were 38.3�V and 39.3�V (p = 0.93), respectively.  Lastly, the baseline flicker readings were 14.4�V and 13.5�V in the CGN and control groups (p = 0.74) and after six weeks 9.5�V and 6.2�V (p = 0.27).  As can be seen from figure 3 (Graphical depiction of ERG amplitudes for both the nanogold and control groups at baseline and six weeks following intravitreal injection.  There was no statistically significant difference between the control and nanogold groups on any of the five steps of the ERG at baseline or at six weeks after injection.  There is a general trend for an overall decrease in the amplitudes in both groups over time, which is consistent with normal aging changes.  (Error bars represent +/- Standard error of measurement). There was a tendency for the overall amplitudes to decrease slightly within each group between the baseline and 6 week testing, but this difference was equal between the two groups and represents the normal decrease in ERG amplitude with increasing age.
Discussion: 
The fast evolution of nanotechnology in recent years has allowed the development of new materials with very promising properties like gold nanoparticles.  CGNs are unique tools with a size of 100nm or less. They have unique optical, electronic and structural properties that allow them to be used in a wide range of scientific fields[22]. The interest for the use of CGN as therapeutic agent due to its antiangiogenic and anti-inflammatory properties is increasing. Their potential application in ophthalmology for the treatment of ophthalmic diseases such as macular degeneration and diabetic retinopathy is a logic and viable research option to pursue[13, 17].  CGNs can also be easily conjugated to peptides, antibodies, and a variety of other organic molecules which make them extremely effective as imaging contrast and potentially as drugs delivery vessels[3, 4]. In ophthalmology, CGN could potentially be used to deliver genetic material to treat inherited diseases such as Leber�s congenital amaurosis and certain forms of retinitis pigmentosa[23, 24]. Besides the fact that CGNs can enter all cells types, when they are in the range of 20nm when administered systemically, they can also cross the blood-brain barrier and distribute into all retina layers, representing a new drug delivery paradigm and biocarrier for retinal diseases[25].  

In the present study, we evaluated the biocompatibility of 5nm CGN with ARPE cells by analyzing the specific absorbance of the cells by spectrophotometry. We compared the viability after exposing the cells to a known concentration of CGNs to a control of cells in fresh culture media. Cultured RPE cells exposed to CGN show a slight decrease in viability compared against the control (Figure 1), and these cells seem to have increased vacuolization (Figure 2). Although we are not entirely sure about the reasons why this happens, it is possible that it is because CGNs uptake by the ARPE cells. Cell density appears similar between CGN and control treatment, suggesting there is no evidence of cell death.  The decrease in MTT absorbance, therefore, may reflect reduced cellular metabolic activity instead of cell death.  It should be noted that the concentration of CGN used here is extremely high compared to that used in vivo. These findings support the notion that CGNs are probably nontoxic for RPE cells and that can be use safely in the eye. The current study also demonstrates that CGNs can be injected intravitreally in a rat model with no major disturbances on the retinal electrical activity as evidenced by normal electroretinographic recording in the study group. Nevertheless, the specific details about the dynamic behavior of CGN within the eye and migration rate (by dose concentration) still needs to be studied in greater detail as this may alter the results observed in this study.

In vivo studies with CGN have shown antiangiogenic properties by inhibiting the activity of heparin-binding glycoproteins[26]. They also inhibit vascular endothelial growth factor 165 and basic fibroblast growth factor by inhibiting vascular endothelial growth factor receptor 2 phosphorylation, intracellular calcium release and migration and RhoA activation[26]. CGN was also successfully used as a contrast in X-ray CT in imaging of tumor vasculature in mice[27]. Chitosan reduced CGN have proved to be a viable way to improve transmucosal insulin absorption[28]. The ease with which CGNs can be conjugated with chemotherapeutic drugs and the possibility of its photothermal activation has opened a new door for cancer treatment and diagnosis[10, 11]. Regarding specific toxicities, it is known that metal poisoning can have deleterious effect over renal and hepatic function. In a study in where renal and liver function were determined after CGN administration, there were no significant differences between serum levels of creatinine, bilirubin, blood urea nitrogen, alkaline phosphatases, alanine aminotransferase and aspartate aminotransferase between the CGNs group and the control group[26]. In the eye specifically, Bakri et al investigated the histological effect in the retina and optic nerve by means of light microscopy. Their result showed that there is no evidence of retina or optic nerve toxicity even after 1 month of follow-up and relative high doses of CGNs[13].  

In summary, the in vitro viability of ARPE 19 cells seems to not be affected by the addition of CGNs to the culture media, with no significant morphological changes. There was no evidence of electrophysiologic disturbances after intravitreal injection of CGNs as evidenced by a normal electrical activity of the retina in a rat model.

References: 
1.	Barroso MM (2011) Quantum dots in cell biology. J Histochem Cytochem 59:237-251
2.	Chiang A,Haller JA (2010) Vitreoretinal disease in the coming decade. Curr Opin Ophthalmol 21:197-202
3.	Chekman IS, Ulberg ZR, Gorchakova NO, Nebesna TY, Gruzina TG, et al. (2011) The prospects of medical application of metal-based nanoparticles and nanomaterials. Lik Sprava 3-21
4.	Rippel RA,Seifalian AM (2011) Gold revolution--gold nanoparticles for modern medicine and surgery. J Nanosci Nanotechnol 11:3740-3748
5.	Jain KK (2010) Advances in the field of nanooncology. BMC Med 8:83
6.	Csaki A, Moller R, Straube W, Kohler JM,Fritzsche W (2001) DNA monolayer on gold substrates characterized by nanoparticle labeling and scanning force microscopy. Nucleic Acids Res 29:E81
7.	Guo S, Huang Y, Jiang Q, Sun Y, Deng L, et al. (2010) Enhanced gene delivery and siRNA silencing by gold nanoparticles coated with charge-reversal polyelectrolyte. ACS Nano 4:5505-5511
8.	Kim CK, Ghosh P,Rotello VM (2009) Multimodal drug delivery using gold nanoparticles. Nanoscale 1:61-67
9.	Tan A, De La Pena H,Seifalian AM (2010) The application of exosomes as a nanoscale cancer vaccine. Int J Nanomedicine 5:889-900
10.	Thaxton CS, Elghanian R, Thomas AD, Stoeva SI, Lee JS, et al. (2009) Nanoparticle-based bio-barcode assay redefines "undetectable" PSA and biochemical recurrence after radical prostatectomy. Proc Natl Acad Sci U S A 106:18437-18442
11.	Fadeel B, Kasemo B, Malmsten M,Stromme M (2010) Nanomedicine: reshaping clinical practice. J Intern Med 267:2-8
12.	Donoghue HD,Holton J (2009) Intestinal tuberculosis. Curr Opin Infect Dis 22:490-496
13.	Bakri SJ, Pulido JS, Mukherjee P, Marler RJ,Mukhopadhyay D (2008) Absence of histologic retinal toxicity of intravitreal nanogold in a rabbit model. Retina 28:147-149
14.	Chen J, Wiley BJ,Xia Y (2007) One-Dimensional Nanostructures of Metals:	  Large-Scale Synthesis and Some Potential Applications. Langmuir 23:4120-4129
15.	Dreaden EC, Mackey MA, Huang X, Kang B,El-Sayed MA (2011) Beating cancer in multiple ways using nanogold. Chem Soc Rev 40:3391-3404
16.	Powell RD,Hainfeld JF (2011) Preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices. Micron 42:163-174
17.	Kim JH, Kim MH, Jo DH, Yu YS,Lee TG (2011) The inhibition of retinal neovascularization by gold nanoparticles via suppression of VEGFR-2 activation. Biomaterials 32:1865-1871
18.	Ablonczy Z, Dahrouj M, Tang PH, Liu Y, Sambamurti K, et al. (2011) Human retinal pigment epithelium cells as functional models for the RPE in vivo. Invest Ophthalmol Vis Sci 52:8614-8620
19.	Narayanan R, Mungcal JK, Kenney MC, Seigel GM,Kuppermann BD (2006) Toxicity of triamcinolone acetonide on retinal neurosensory and pigment epithelial cells. Invest Ophthalmol Vis Sci 47:722-728
20.	Rosolen S, Kolomiets B, Varela O,Picaud S (2008) Retinal electrophysiology for toxicology studies: Applications and limits of ERG in animals and ex vivo recordings. Experimental and Toxicologic Pathology 60:17-32
21.	Marmor MF, Fulton AB, Holder GE, Miyake Y, Brigell M, et al. (2009) ISCEV Standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol 118:69-77
22.	Sandhiya S, Dkhar SA,Surendiran A (2009) Emerging trends of nanomedicine--an overview. Fundam Clin Pharmacol 23:263-269
23.	Lukianova-Hleb EY, Samaniego AP, Wen J, Metelitsa LS, Chang CC, et al. (2011) Selective gene transfection of individual cells in vitro with plasmonic nanobubbles. J Control Release 152:286-293
24.	Prow T, Smith JN, Grebe R, Salazar JH, Wang N, et al. (2006) Construction, gene delivery, and expression of DNA tethered nanoparticles. Mol Vis 12:606-615
25.	Kim JH, Kim KW, Kim MH,Yu YS (2009) Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity. Nanotechnology 20:505101
26.	Mukherjee P, Bhattacharya R, Wang P, Wang L, Basu S, et al. (2005) Antiangiogenic properties of gold nanoparticles. Clin Cancer Res 11:3530-3534
27.	Cai QY, Kim SH, Choi KS, Kim SY, Byun SJ, et al. (2007) Colloidal gold nanoparticles as a blood-pool contrast agent for X-ray computed tomography in mice. Invest Radiol 42:797-806
28.	Bhumkar DR, Joshi HM, Sastry M,Pokharkar VB (2007) Chitosan reduced gold nanoparticles as novel carriers for transmucosal delivery of insulin. Pharm Res 24:1415-1426







Legends and Footnotes: 
Figure 1: ARPE-19 cell absorbance results.
Figure 2: Comparative ARPE-19 cell cultures of treated and untreated cells with CGN.
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