Facile Noninvasive Retinal Gene Delivery Enabled by Penetratin

Jul 11, 2016 - The eyelids were gently pulled up and closed to disperse the complexes or plasmid on the corneal surface. Each formulation was administ...
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Facile Noninvasive Retinal Gene Delivery Enabled by Penetratin Chang Liu,† Kuan Jiang,† Lingyu Tai,†,‡ Yu Liu,† Gang Wei,*,† Weiyue Lu,† and Weisan Pan‡ †

Key Laboratory of Smart Drug Delivery, Ministry of Education; Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China ‡ School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China ABSTRACT: Gene delivery to the posterior segment of the eye is severely hindered by the impermeability of defensive barriers; therefore, in clinical settings, genomic medicines are mainly administered by intravitreal injection. We previously found that penetratin could transport the covalently conjugated fluorophore to the fundus oculi by topical instillation. In this study, gene delivery systems enabled by penetratin were designed based on electrostatic binding to target the retina via a noninvasive administration route and prepared with red fluorescent protein plasmid (pRFP) and/or poly(amidoamine) dendrimer of low molecular weight (G3 PAMAM). Formulation optimization, structure confirmation, and characterization were subsequently conducted. Penetratin alone showed limited ability to condense the plasmid but had powerful uptake and transfection by corneal and conjunctival cells. G3 PAMAM was nontoxic to the ocular cells, and when introduced into the penetratin-incorporated complex, the plasmid was condensed more compactly. Therefore, further improved cellular uptake and transfection were observed. After being instilled in the conjunctival sac of rats, the intact complexes penetrated rapidly from the ocular surface into the fundus and resided in the retina for more than 8 h, which resulted in efficient expression of RFP in the posterior segment. Intraocular distribution of the complexes suggested that the plasmids were absorbed into the eyes through a noncorneal pathway during which penetratin played a crucial role. This study provides a facile and friendly approach for intraocular gene delivery and is an important step toward the development of noninvasive gene therapy for posterior segment diseases. KEYWORDS: gene delivery, posterior segment of the eye, penetratin, PAMAM, physical complex

1. INTRODUCTION Intraocular neovascular diseases (INDs) primarily locating in the posterior segment of the eye, including retinopathy of prematurity in children, proliferative diabetic retinopathy in mid-adults, and age-related macular degeneration in the elderly, are the leading causes of irreversible visual loss and blindness worldwide.1−3 Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of INDs and has been regarded as a main therapeutic target.4 Currently, the interest of applying DNA- or RNA-based gene therapy for antagonism of VEGF and inhibition of neovascularization is growing in ophthalmology clinics.5 These gene drugs are difficult to penetrate into the eyes by noninvasive pathways because of their large size and strong hydrophilicity compared to that of other chemical molecules. Intravitreal injection is the major administration route for genomic medicines, which has a rather low patient compliance and a high risk of inducing infection or impairment of the retina, lens, and vitreous.6,7 Thus, development of noninvasive delivery to the posterior segment becomes key for these types of macromolecular pharmaceuticals. Cell-penetrating peptides (CPPs) are positively charged peptides of generally less than 30 amino acid residues with the capability of being easily internalized into cells by a receptorindependent pathway.8 When covalently conjugated with CPPs, bulky cargos such as proteins, genes, and even nanoparticles © XXXX American Chemical Society

can be successfully transferred across formidable membrane barriers.9 TAT is the most well-known CPP, derived from HIV transactivator of transcription. Fusion proteins composed of TAT with acidic fibroblast growth factor (FGF)10 or endostatin,11 which are generated by genetic engineering, exhibited the capability of penetrating into the eyes and were found distributed in the retina following topical administration. Nowadays, more attention has been paid to nanoscale drug delivery systems due to their inherent advantages, such as protection for macromolecules, enhanced permeability, controlled release, and so on.12,13 CPPs have also been applied to construct nanoparticles for ocular drug delivery. For instance, the small arginine-rich peptide protamine was employed to modify the solid lipid nanoparticles for gene therapy of retinoschisis. Incorporating protamine into the nanoparticles greatly promoted the in vitro expression of retinoschisin in the retinal pigment epithelia cell line.14 Kumar-Singh and colleagues designed a novel peptide for ocular delivery (POD) based on the glycosaminoglycan-binding domain of FGF to transport genes and cell factors into the eyes via subretinal or intravitreal injection.15−17 The common conReceived: April 16, 2016 Accepted: July 11, 2016

A

DOI: 10.1021/acsami.6b04551 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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2. MATERIALS AND METHODS

clusion drawn from the published works suggests that when the CPP-mediated vectors, especially those nanoparticles, were topically administered as eye drops, gene distribution and transfection were detectable in the cornea but rarely in the posterior segment. We previously found that, among several frequently reported CPPs, penetratin, a peptide derived from antennapedia homeoprotein, showed an outstanding ocular permeability over other peptides, including TAT and protamine of low molecular weight, and could deliver a conjugated fluorescence probe to the fundus oculi via topical instillation.18 Penetratin has been reported as one of a few gene delivery vectors for various purposes,19,20 but the resultant effects were controversial. For example, cationic micelles composed of linoleic acid and penetratin dual-functionalized chitosan-condensed pDNA to polyplexes, which exhibited much higher transfection in cellular models compared with the unmodified chitosan.21 In contrast, other researchers revealed that the conjugates of penetratin and peptide nucleic acid or siRNA showed only minimal or no activity upon gene expression.22,23 Furthermore, studies on penetratin-mediated gene delivery for ophthalmic applications has been rarely reported to date; therefore, whether penetratin-modified nanoparticles could deliver their cargos into the eye is still unknown. Poly(amidoamine) (PAMAM) dendrimer is also a nonviral gene vector. As a cationic polymer, PAMAM possesses strong gene condensation and protection ability24−27 along with the ability to induce cellular uptake and endosomal escape, which is known as proton sponge effect.28,29 The branched concentric layers of the dendrimers are referred to as “generations”. Generally, the endocytosis capability for PAMAM of low generations is poor.30 In contrast, fourth- and fifth-generation PAMAMs have been applied as ophthalmic vehicles for treatment of ocular diseases.31−33 For example, a G4 PAMAM-based gene delivery vector was reported to improve the in vitro transfection efficiency of plasmid DNA in the retinal pigment epithelium cells.34 Unfortunately, cationic dendrimers of high generations have strong cytotoxicity; thus, their utilization is limited by safety issues.35−37 In the present study, we designed a noninvasive gene delivery system targeting to the posterior segment of the eye. Penetratin, according to our previous work, was selected to condense the model gene, red fluorescent protein plasmid (pRFP), into a physical complex to promote ocular absorption. The third generation (G3) PAMAM, a branched cationic polymer of low molecular weight and with theoretically lower cytotoxicity, was additionally introduced in the complex for enhancement on gene condensation and endosomal escape. The charge ratio and N/P ratio among different materials were optimized, resulting in two physical complexes with different structures: penetratin-condensed pRFP and penetratinPAMAM double-condensed pRFP. In vitro uptake and transfection on corneal and conjunctival cell lines were performed for a preliminary assessment. Confocal images illustrated the internalization process of the complex formulations. In vivo distribution and gene transfection of the optimized complexes were evaluated after topical application in the rat eyes. We hope the novel gene delivery systems transport bioactive macromolecules from the ocular surface to the fundus and consequently improve patient compliance by substituting topical instillation for intravitreal injection.

2.1. Materials. Fetal calf serum (FCS), Dulbecco’s Modified Eagle Medium (DMEM), LysoTracker Red DND-99, and TOTO-3 fluorescence dye were bought from Life Technologies (Karlsruhe, Germany). The pDsRED-N1 strain was kindly donated by Professor Chen Jiang (School of Pharmacy, Fudan University). Plasmid Mega Kits was purchased from Qiagen (Hilden, Germany). Tryptone, yeast extract, and agar were purchased from Oxoid (Basingstoke, U.K.). Ampicillin and kanamycin were bought from Sinopharm (Shanghai, China). PAMAM (G3, MW = 6908, 32 NH2 groups per molecule), 3(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), ethidium bromide (EtBr), and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co. (St. Louis, MO, United States). Penetratin was synthesized by ChinaPeptides Co., Ltd. (Shanghai, China). The C-terminal of penetratin was conjugated with 5-carboxyfluorescein (FAM) via an additional lysine residue (amino acid sequence is RQIKIWFQNRRMKWKKK-FAM). The molecular masses of all peptides were verified by electrospray ionization mass spectrometry. Artificial tear fluid was composed of 116.02 mmol/L NaCl, 25.95 mmol/L NaHCO3, 18.51 mmol/L KCl, and 0.65 mmol/L CaCl2·H2O in deionized water. 2.2. Cell Lines and Animals. Human conjunctival epithelial cells (NHC) were propagated at 37 °C in a 5% CO2 humidified atmosphere and a DMEM medium supplemented with 10% FCS, 4 mmol/L Lglutamine, 100 μg/mL streptomycin, and 100 units/mL penicillin. Spontaneously derived human corneal epithelial cells (SDHCEC) donated by Zhongshan Ophthalmic Center, Sun Yat-sen University (Guangzhou, China) were cultured in the same medium but with the addition of 5 mg/mL insulin, 10 ng/mL human EGF, and 1% (w/v) hydrocortisone. Male Sprague−Dawley rats (17 weeks old) were purchased from the Experimental Animal Center, Fudan University. The rats were maintained in laboratory conditions (22 ± 2 °C and a 12 h light/dark cycle) for 1 week prior to experiments. Our animal experiments complied with protocols approved by the Ethical Committee, Fudan University. 2.3. Cytotoxicity of G3 PAMAM on Ocular Cell Lines. The cytotoxicity of G3 PAMAM on ocular cell lines was evaluated using an MTT assay. NHC and SDHCEC cells were separately seeded on 96well plates at 5 × 103 cells per well and cultured for 24 h in a 5% CO2 humidified atmosphere and DMEM at 37 °C. Then, the culture medium was refreshed with 200 μL DMEM containing different concentrations of PAMAM and 10% FCS. After 24 h of incubation, the cells were rinsed with a phosphate buffer solution (PBS, pH 7.4) 3 times and cultured for another 24 h in fresh DMEM medium. MTT agent was subsequently added in each well at a concentration of 0.5 mg/mL and incubated with the cells at 37 °C for 4 h. Finally, 200 μL of dimethyl sulfoxide was employed to dissolve formazan, whose absorbance was determined with a microplate reader (Power Wave XS, Bio-TEK, United States) at 490 nm. GraphPad Prism 5 software was used to generate the survival curve and calculate IC50. 2.4. Preparation and Characterization of Physical Complexes. 2.4.1. Preparation of the pRFP/P Complex. The plasmid was extracted from the pDsRED-N1 strain using Qiagen Plasmid Mega Kits. To prepare the pRFP-penetratin complexes (pRFP/P), penetratin at different concentrations in 60 μL of artificial tear fluid was mixed with 20 μL of the pRFP solution to produce a final DNA concentration of 0.05 mg/mL followed by vortexing for 30 s and then incubation for 1 h at 37 °C. The formation of pRFP/P was evaluated by a gel retardation assay using 1% agarose gel electrophoresis. pRFP was stained with GelRed (Biotium, United States) and visualized using a fluorescence imaging system (FluorChem FC3, Alpha Innotech, United States). Double confirmation was applied by EtBr assay. Solutions of the complexes (500 μL) were mixed with 1 μL of the EtBr solution for a final EtBr concentration of 0.02 μg/mL. After being vortexed for 30 s, the solution was transferred to a black-background 96-well plate. The fluorescence intensity was measured by a fluorescence spectrophotometer (Hitachi, F7000, Japan). B

DOI: 10.1021/acsami.6b04551 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces 2.4.2. Preparation of the pRFP/PAMAM/P Complex. Preparation of the pRFP/PAMAM/penetratin complex (pRFP/PAMAM/P) includes two steps: preparation of the primary complex pRFP/ PAMAM and extra incorporation with penetratin. PAMAM at different concentrations in 60 μL of artificial tear fluid was mixed with 20 μL of pRFP solution to produce a final DNA concentration of 0.05 mg/mL followed by vortexing for 30 s and then incubation for 1 h at 37 °C. On the basis of the complex pRFP/PAMAM, penetratin of different concentrations was subsequently added at a certain charge ratio to pRFP followed by vortexing for 30 s and incubation for 1 h at 37 °C. The formation of pRFP/PAMAM and pRFP/PAMAM/P was also evaluated by gel retardation assay. In the abbreviation of the complex pRFP/Pn or pRFP/PAMAM/Pn, n stands for the selected charge ratio of penetratin to pRFP, unless noted otherwise. 2.4.3. Entrapment Efficiency of Penetratin in the Complexes. Formation of complexes pRFP/P and pRFP/PAMAM/P was further confirmed by centrifugal ultrafiltration. FAM-labeled penetratin was used to prepare the complexes. Solutions of the complexes (500 μL) were added into an ultrafiltration centrifuge tube (MW cutoff = 10 kDa, Millipore, United States). After centrifugation for 15 min at 3000 r/min, the lower ultrafiltrate was transferred to a black-background 96well plate and measured by a fluorescence spectrophotometer. The percentage of penetratin bound in the complexes was calculated by the following equation:

Bound penetratin% = (1 − Cultrafiltrate/C total) × 100%

investigate their colocalization with lysosomes and distribution in cytoplasm. SDHCEC cells were seeded at 5 × 104 cells per well in 60 mm Petri dishes (Corning, Manassas, United States) and grown for 4 days prior to the experiment. The medium was substituted with 1 mL DMEM containing pRFP, pRFP/P20, or pRFP/PAMAM/P20 (equal to 2 μg pRFP) and 10% FCS. The cells were incubated at 37 °C with pRFP or the complexes for 1, 2, 4, and 12 h and then rinsed with icecold PBS containing 75 mmol/L sodium azide and 1000 IU/mL heparin 3 times. After cell fixation by 4% paraformaldehyde for 15 min, LysoTracker Red DND-99 (excitation at 577 nm and emission at 590 nm) was applied to label lysosomes and DAPI for cell nuclei. Each well was immersed in 1 mL buffered glycerin. Cells were visualized with the multiphoton confocal microscope (Zeiss, 710, Germany) at 37 °C with a 100× oil immersion lens. 2.7. In Vivo Ocular Distribution. After topical administration, distribution of pRFP and the complexes in the eyes was investigated on 54 healthy rats. Ophthalmological examination on rat eyes was conducted before the experiment to make sure that no ocular abnormalities were observed. FAM-labeled penetratin and TOTO-3labeled plasmid were used to prepare the complexes. Artificial lacrimal fluid (7 μL) used to dissolve the FAM and TOTO-3 double-labeled complexes (containing 2 μg of pRFP) or 2 μg of TOTO-3-labeled nude pRFP was instilled into the conjunctival sac of rats every 10 min 3 times. The eyelids were gently pulled up and closed to disperse the complexes or plasmid on the corneal surface. Each formulation was administered to a single eye of 18 rats. After the last administration, 3 rats were sacrificed at 10 and 30 min and 1, 2, 4, and 8 h. Eyeballs were denucleated and immersed in Davidson’s solutions for 30 min. Dehydration was performed overnight in 30% sucrose solution followed by preparation of DAPI-stained frozen sections. Afterward, observations on the sections were implemented using a fluorescence microscope (DMI4000 B, LEICA, Germany). 2.8. In Vivo RFP Expression. Visual evaluation on the transfection efficacy of the complexes was performed in 12 healthy rats. Artificial lacrimal fluid (7 μL) used to dissolve the complexes (containing 2 μg of pRFP) or 2 μg of nude pRFP was instilled into the conjunctival sac of rats every 8 h for 3 continuous days. Each formulation was administered to a single eye of 3 rats. The rats were sacrificed 3 days after the last instillation. The DAPI-stained frozen sections of the treated eyes were subsequently prepared as mentioned above. A fluorescence microscope was used to observe the RFP expression.

(1)

Where Cultrafiltrate is the concentration of penetratin in the lower ultrafiltrate and Ctotal is the total concentration of penetratin added in the system. 2.4.4. Size, Morphology, Stability, and Cytotoxicity of the Complexes. The size and morphology of the complexes were estimated by transmission electron microscopy (TEM, Philips, CM120, the Netherlands). Briefly, 5 μL of each sample was adhered onto glow-discharged carbon-coated grids for 2 min. The remaining liquid was dried by an infrared lamp. Samples were visualized under the microscope operating at an accelerating voltage of 200 keV in the bright-field image mode. The particle sizes and ζ-potential of the complexes were measured by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, U.K.). The stability of the complexes under eye temperature (34 °C) was also characterized by the variation in particle size. The cytotoxicity of the complex pRFP/PAMAM/P20 was assayed by the MTT method as described above. 2.5. In Vitro RFP Expression. To quantify the RFP expression on the ocular cell lines, flow cytometry assays were implemented. FAMlabeled penetratin was used to prepare the complexes. NHC and SDHCEC cells were separately seeded on 12-well plates at 1 × 104 cells per well and incubated in a 5% CO2 humidified atmosphere and DMEM at 37 °C for 4 days. Then, 1 mL of DMEM containing different complexes equal to 2 μg of pRFP and 10% FCS was substituted for the medium. The dose was determined according to the results of the MTT assays, and 2 μg of nude pRFP was added as a negative control. The cells was incubated with the complexes at 37 °C for 12 h followed by rinsing 3 times with ice-cold PBS containing 75 mmol/L sodium azide and 1000 IU/mL heparin to remove cations bound on the external cell surface.38,39 Further incubation for 48 h in DMEM containing 10% FCS was performed. After rinsing and trypsinization, the cells were transferred into 1.5 mL Eppendorf centrifugal tubes and suspended in a 500 μL DMEM solution containing 10% FCS and kept in an ice bath in the dark until analysis. The number of RFP-positive cells and mean fluorescence intensities of RFP (RFP-A), FAM-positive cells, and FAM (FAM-A) were assayed by a flow cytometer (BD Bioscience, United States). Flow Jo software (Tree Star Inc., San Carlos, United States) was utilized to analyze the results. 2.6. Intracellular Distribution of the Complexes. The endocytosis process of the complexes was evaluated by confocal assay. TOTO-3 (excitation at 642 nm and emission at 660 nm) was premixed with pRFP before preparation of the complexes to label the plasmid. FAM-labeled penetratin was used to prepare the complexes. Nude pRFP, pRFP/P20, and pRFP/PAMAM/P20 were selected to

3. RESULTS 3.1. Cytotoxicity of PAMAM on NHC and SDHCEC Cell Lines. The survival curves measured by MTT assays (Figure 1) illustrate that G3 PAMAM is almost nontoxic after being exposed to ocular cells for 24 h, resulting in the IC50 on both SDHCEC and NHC cells much higher than 1 mg/mL. The conjunctival cells seem to tolerate PAMAM better than corneal cells because the survival rate of NHC cells exposed to 1 mg/

Figure 1. In vitro cytotoxicity of G3 PAMAM on NHC and SDHCEC cells (n = 5). C

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Figure 2. Schematic diagram of the penetratin-incorporated complex pRFP/P (A(a)) and the PAMAM-penetratin-incorporated complex pRFP/ PAMAM/P (B(a)). (A) Agarose gel electrophoresis image (b) and ethidium bromide assay (c) of pRFP/P complexes at various charge ratios. (B) Agarose gel electrophoresis image of the primary complexes pRFP/PAMAM at various N/P ratios (b), and agarose gel electrophoresis image of the secondary complexes pRFP/PAMAM/P at various charge ratios between pRFP and penetratin (c).

mL PAMAM was significantly higher (p < 0.05). Cytotoxicity of penetratin was evaluated in our previous work.18 3.2. Preparation and Characterization of Physical Complexes. 3.2.1. Charge Ratio of pRFP/P. Agarose gel electrophoresis and EtBr assays were simultaneously performed to screen the charge ratio between pRFP and penetratin. EtBr binds with the plasmid, inserting into the major groove of the DNA helix and emitting fluorescence, while the free EtBr molecule has no fluorescence emission.40 GelRed functions in a similar way with EtBr.41 Both of these two reagents can be used for quantitative determination of free pRFP. As illustrated in the schematic diagram of the complex composition (Figure 2A(a)), penetratin bound with pRFP by electrostatic interaction, forming nest-like nanoparticles. The electrophoresis results (Figure 2A(b)) showed that, when the charge ratio of penetratin to pRFP was higher than 2:1, most of the negative charges from the plasmid were sheltered, and therefore, no bright band of the free pRFP was observed. However, the results of the EtBr assays indicated that approximately 10% of the plasmids remained uncondensed when the charge ratio of

penetratin to plasmid was lower than 5:1 because weak fluorescence emitted from those EtBr binding with free pRFP was still detectable (Figure 2A(c)). The plasmids were not completely entrapped in the complexes until the charge ratio increased to 10:1. 3.2.2. N/P Ratio and Charge Ratio of pRFP/PAMAM/P. Agarose gel electrophoresis was also used to screen the N/P ratio between PAMAM and pRFP, which formed the primary complex pRFP/PAMAM. The screening range was set from 1:1 to 10:1. For further binding with penetratin, negative charges in the primary complex should not be totally sheltered (Figure 2B(a)). The electrophoresis result (Figure 2B(b)) showed that when N/P was lower than 5:1, the pRFP was not completely condensed. On the other hand, for better endosomal escape in cells, PAMAM should be added at a higher level in the complex. Thus, the N/P ratio of the primary complex was set at 5:1. After the addition of penetratin in the primary complex solution, the secondary complex pRFP/PAMAM/P formed, as shown in Figure 2B(a). It could be seen from the electrophoresis result (Figure 2B(c)) that, when the charge ratio of D

DOI: 10.1021/acsami.6b04551 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Table 1. Characterization of the Selected Physical Complexes (n = 3)a abbreviation

composition

bound penetratin (%)

particle size (nm)

PDI

ζ-potential (mV)

pRFP/P20 pRFP/PAMAM pRFP/PAMAM/P20

pRFP + penetratin pRFP + PAMAM pRFP + PAMAM + penetratin

25.00 ± 4.43

419.7 ± 11.4 260.7 ± 10.6 154.8 ± 8.9*

0.210 ± 0.002 0.115 ± 0.003 0.167 ± 0.007

24.3 ± 1.6 21.9 ± 0.2 29.9 ± 1.0*

42.50 ± 3.30*

a

The asterisk indicates a statistically significant difference (p < 0.01) calculated using a t-test compared with that of complexes pRFP/P20 and pRFP/ PAMAM.

Figure 3. (A) Morphology of the complexes pRFP/P20 (a and b), pRFP/PAMAM (c and d), and pRFP/PAMAM/P20 (e and f) observed by TEM. Scale bars are 100 nm for a, b, c, and d and are 50 nm for e and f. (B) The particle size and distribution of the complexes measured by DLS. (C) The particle size stability of the complexes under eye temperature (34 °C). The asterisk indicates a statistically significant difference (p < 0.01) calculated using a t-test compared with the initial particle size (n = 3). (D) The cytotoxicity of the complex pRFP/PAMAM/P20 on SDHCEC and NHC cells assayed by the MTT method (n = 5).

formed nanoscale particles with a relatively low polydispersity index (PDI). Complex pRFP/P20 was the biggest one in particle size compared with the others, whose diameter was around 420 nm. The particle size of complex pRFP/PAMAM was smaller and approximately 260 nm. Incorporation of penetratin further condensed the plasmid, and the particle size of the resultant complex pRFP/PAMAM/P20 reduced to around 150 nm. The morphology of complexes pRFP/P20, pRFP/PAMAM, and pRFP/PAMAM/P20 was observed by TEM, as illustrated in Figure 3A. All of the complexes showed a discrete spherical morphology. The complexes pRFP/P20 and pRFP/PAMAM seem looser in structure compared to pRFP/PAMAM/P20. The particle sizes observed by TEM were consistent with those measured by DLS, as shown in Figure 3B. The ζ-potentials of the tested complexes ranged from approximately 20 to 30 mV, among which the lowest one was about 22 mV for pRFP/PAMAM (Table 1). In contrast, the ζpotential of pRFP/PAMAM/P20 significantly increased to 30 mV (p < 0.01), indicating positively charged penetratin was further bound with the primary complex. The stability of these complexes is illustrated in Figure 3C with the change in particle size. When incubated under eye temperature, the particle sizes of complexes pRFP/P20 and pRFP/PAMAM increased with time, particularly pRFP/PAMAM, which significantly changed

penetratin to pRFP was higher than 8:1, the negative charges were completely sheltered. Studies on the complexes formed by TAT and plasmid for transfection of mammalian cells suggested that, at higher charge ratios (for example 20:1), excesses of TAT peptide enhanced the efficiency of cellular transfection.42 Accordingly, in this work, we chose 20:1 as the charge ratio between penetratin and pRFP for a better transfection effect. The two different complexes were therefore designated as pRFP/P20 and pRFP/PAMAM/P20, respectively. 3.2.3. Validation of Complex Formation. Formation of the complexes pRFP/P20 and pRFP/PAMAM/P20 was assessed by centrifugal ultrafiltration. As listed in Table 1, about 25% penetratin was tightly bound in the complex pRFP/P20, which was consistent with the result of agarose gel electrophoresis. Approximately 17.5% more penetratin was entrapped in the complex pRFP/PAMAM/P20 than in pRFP/P20, implying that these two complexes condensed plasmid in a different way and therefore had different structures. 3.2.4. Characterization of the Complexes. According to the results of the gel retardation and EtBr assays, we selected three different complexes for characterization: pRFP/P20 (containing penetratin alone), pRFP/PAMAM (containing PAMAM alone), and pRFP/PAMAM/P20 (containing both penetratin and PAMAM). From the results of the particle size measurements (Table 1), it can be clearly seen that all formulations E

DOI: 10.1021/acsami.6b04551 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. Flow cytometry analysis on SDHCEC and NHC cells. The cells were incubated with various complexes equal to 2 μg of pRFP in serumcontaining DMEM medium at 37 °C for 12 h. The diagrams show a combination of fluorescence intensity from both FAM and RFP. The horizontal coordinate corresponds to the mean fluorescence intensity from FAM (FAM-A), while the vertical coordinate corresponds to the mean fluorescence intensity from RFP (RFP-A). The percentage numbers in each diagram stand for the cell proportions in quadrant 2, the region of double-positive fluorescence. (A) Complexes pRFP/P: Effects of penetratin contents on the cellular uptake and transfection efficacy. (B) Complexes pRFP/ PAMAM/P: Effects of both PAMAM and penetratin on the cellular uptake and transfection efficacy at different preparation stages.

fluorescence intensity from both FAM and RFP. The percentage numbers stand for the cell proportions of doublepositive fluorescence (DPF) in the top-right regions (Q2 quadrant) of each diagram. The nude plasmid rarely induced any RFP expression in both cell lines, showing no significant difference compared with the untreated cells. For the pRFP/P treated cells (Figure 4A), when the charge ratio of penetratin to plasmid (pRFP/P5) reached 5:1, the proportion of DPF cells was lower than 4% in both cell lines. When the charge ratio increased to 20:1, the transfection efficacy was dramatically improved. The proportions of DPF cells were approximately 67% in SDHCEC cells and 54% in

within 4 h (p < 0.01). It is worth noting that the particle size of pRFP/PAMAM/P20 remained stable over 24 h, indicating a relatively good stability against aggregation due to its compact structure and high charge. Furthermore, for the complex pRFP/ PAMAM/P20, no cytotoxicity was observed on either corneal or conjunctival cells under the double dose for in vitro and in vivo applications (Figure 3D). 3.3. In Vitro RFP Expression. Flow cytometry assays on SDHCEC and NHC cells were applied to perform a quantitative evaluation of complex uptake and RFP expression. The penetratin used to prepare all of the complexes was labeled by FAM. The diagrams in Figure 4 show a combination of F

DOI: 10.1021/acsami.6b04551 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 5. Confocal images of the SDHCEC cells after being incubated with nude plasmid (upper panel) or the complexes pRFP/PAMAM/P20 (middle panel) and pRFP/P20 (lower panel) for 1, 2, 4, and 12 h. The complexes double-labeled by FAM and TOTO-3 contained 2 μg of pRFP. To avoid color confusions, the images were merged separately, and pseudocolors were assigned for enhanced visualization. In all images labeled A: blue, DAPI-labeled cell nuclei; green, FAM-labeled penetratin; red, LysoTracker Red DND-99-labeled lysosomes; yellow, overlays of green and red areas. In all images labeled B: blue, DAPI-labeled cell nuclei; red, LysoTracker Red DND-99-labeled lysosomes; yellow, TOTO-3-labeled pRFP; bright yellow, overlays of red and yellow areas. The gray arrows point out the enlarged lysosomes filled with penetratin or plasmid. Scale bar = 10 μm.

NHC cells, illustrating a strong uptake-enhancing effect by incorporating more penetratin. In another group, G3 PAMAM was further introduced in the complex (Figure 4B). For the primary complex pRFP/PAMAM formed by pRFP and PAMAM (N/P = 5), almost no RFP expression was observed. After a small amount of penetratin was added into the primary complex (pRFP/PAMAM/P5, charge ratio = 5), the proportion of DPF cells slightly increased but was still lower than 8%. Similar to the complex pRFP/P,

when the charge ratio of penetratin to pRFP (pRFP/PAMAM/ P20) increased to 20:1, the uptake and transfection efficacy were remarkably improved. Especially, the DPF proportion accounted for 73.1% in the SDHCEC cells. However, less than 21% of NHC cells located in the DPF region after being exposed to the complex pRFP/PAMAM/P20. The compatibility of SDHCEC cells with the complexes seemed better than that of the NHC cells. G

DOI: 10.1021/acsami.6b04551 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 6. Fluorescence images of corneas and retinae of rat bulbus oculi that illustrate distribution and elimination of penetratin and pRFP after topical administration of nude plasmid (A), the complexes pRFP/P20 (B), or pRFP/PAMAM/P20 (C). Artificial lacrimal fluid (7 μL) to dissolve the FAM and TOTO-3 double-labeled complexes (containing 2 μg of pRFP) or 2 μg of TOTO-3-labeled nude pRFP was instilled into the conjunctival sac of rats every 10 min 3 times. Blue, DAPI-labeled cell nuclei; green, FAM-labeled penetratin; red, TOTO-3-labeled pRFP. The arrow points to weak red fluorescence located on the endothelium of the cornea. Images show both of the two complexes penetrated into the posterior segment (retina) but rarely in the anterior segment (cornea). Scale bar = 50 μm.

attributed to the unique function of penetratin instead of the higher cationic charge. Taken together, pRFP/P20 and pRFP/ PAMAM/P20 significantly promoted nanoparticle internalization and gene expression and were therefore chosen for further evaluation. 3.4. Intracellular Distribution of the Complexes. The endocytosis process of nude plasmid pRFP and two selected complexes (pRFP/P20 and pRFP/PAMAM/P20) was ob-

It is worth noting that under the same charge ratio (1:5 between negatively charged plasmid to positively charged penetratin or PAMAM) pRFP/P5 exhibited obviously higher transfection efficiency in both SDHCEC and NHC cells compared with pRFP/PAMAM. Moreover, the ζ-potential of pRFP/P5 was 6.9 ± 0.1 mV (n = 5), which was much lower than that of pRFP/PAMAM (Table 1). These results suggest that the higher transfection efficiency of the complex was H

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ACS Applied Materials & Interfaces served in SEHCEC cells. Confocal images (Figure 5) illustrate the intracellular distribution of the fluorescence-labeled pRFP and penetratin. The cells treated with nude pRFP did not show any yellow fluorescence (upper panel), which means no TOTO-3-labeled plasmids penetrated into the cells within 12 h. In the pRFP/PAMAM/P20-treated cells (middle panel), both plasmids and penetratin permeated into the cytoplasm and gathered in the lysosomes. After incubation for 4 h, distinct light yellow spots can be observed (as pointed by the gray arrows), revealing the enlargement of lysosomes. It should be noted that most of plasmids were colocalized with penetratin in the cytoplasm, which suggests that the complexes remained intact during uptake and facilitated further delivery of plasmid into the nucleus. In the cells exposed to the complex pRFP/P20 (lower panel), plasmids dispersed evenly in the cytoplasm, while penetratin concentrated in the lysosomes, as seen from the yellow spots overlaid by green and red fluorescence (pointed out by the gray arrows). The plasmids were not colocalized with penetratin, suggesting that the complexes were partially disintegrated during uptake. The different intracellular distribution resulting from these two complexes implies that they had diverse uptake and escape mechanisms when being endocytosed by cells. 3.5. In Vivo Ocular Distribution. Intraocular distribution of penetratin and plasmid was inspected in the anterior segments (cornea) and posterior segments (retina) of the rat eyes subjected to topical administration. The plasmid was labeled by TOTO-3 and emitted red fluorescence, while penetratin was modified by FAM and emitted green fluorescence. Micrographs of the nude plasmid-treated eyes revealed no fluorescence distributed in either the cornea or retina (Figure 6A). These images also prove that ocular tissues do not have background fluorescence at the wavelengths we detected. In contrast, the ocular tissues treated with both pRFP/P20 or pRFP/PAMAM/P20 presented obvious fluorescence, which was mainly visable in the posterior segments of the eyes but was rarely distributed in the anterior segments (Figures 6B and C). Fluorescence from the eyes treated by complex pRFP/P20 could be clearly seen, while it was generally weaker compared to that treated by the PAMAM-containing formulation. After instillation of the complex pRFP/PAMAM/P20, penetratin together with pRFP reached the posterior segments in merely 10 min (Figure 6C). The fluorescence intensity in the retina peaked during 1 to 2 h and then gradually faded with time. Nevertheless, obvious fluorescence could also be seen until 8 h post-application, suggesting both penetratin and pRFP resided in the retina persistently. Fluorescence images of the cornea and retina treated by pRFP/PAMAM/P20 were amplified to illustrate the detailed ocular distribution of penetratin and pRFP (Figure 7). In the cornea, only few green spots were found in the epithelium, while red fluorescence was absent. However, it is interesting that 8 h later, weak red fluorescence emerged on the endothelium of the cornea, as pointed by the arrow (Figure 6C). In the retina, the photoreceptor segments (PRS) and retinal pigment epithelium (RPE) showed the most intensive green and red fluorescence, and the next were the ganglion cell layer (GCL), inner plexiform layers (IPL), and outer plexiform layer (OPL), followed by the choroid (Chor). By contrast, only scattered fluorescence appeared in the inner nuclear layer (INL) and outer nuclear layer (ONL).

Figure 7. Amplified fluorescent images of corneas and retinae that illustrate the distribution of penetratin and pRFP in rat bulbus oculi after topical administration of pRFP/PAMAM/P20 for 1 h. Blue, DAPI-labeled cell nuclei; green, FAM-labeled penetratin; red, TOTO3-labeled pRFP. The general retina structure includes the ganglion cell layer (GCL), inner plexiform layers (IPL), outer plexiform layer (OPL), inner nuclear layer (INL), outer nuclear layer (ONL), photoreceptor segments (PRS), retinal pigment epithelium (RPE), and choroid (Chor). Scale bar = 50 μm.

3.6. In Vivo Gene Expression. Gene expression of the two selected complexes was evaluated on rats. Complexes pRFP/ P20 and pRFP/PAMAM/P20 were instilled in conjunctival sac every 8 h constantly for 3 days, and the bulbus oculi was enucleated 3 days after the last administration. Expression of RFP in the eyes is shown in Figure 8. Barely any red fluorescence could be found in the corneas whether they were untreated or exposed to the plasmid-containing formulations. Nude plasmid did not transfect the retina by topical application, whereas both the selected complexes did well. Fluorescence in the posterior segments of the eyes treated by pRFP/PAMAM/ P20 was obviously stronger than that treated by pRFP/P20. The transfection occurred mostly in photoreceptor segments (PRS), inner plexiform layers (IPL) and outer plexiform layer (OPL), which was highly consistent with the in vivo results of plasmid distribution in the eyes.

4. DISCUSSION Besides the inherent nature of nucleic acids, ocular gene therapy through topical administration has many absorptive barriers such as tear turnover, impermeability of the cornea, and nasolacrimal drainage, resulting in poor bioavailability.43 Recent strategies of gene delivery to the posterior ocular segment mainly focus on intravitreal injection, which usually leads to low patient compliance.7 CPPs are employed in the gene delivery systems targeting tumors, the brain-blood barrier, and other tissues.9,44 Few ocular gene delivery systems mediated by CPPs have been reported, and most of them were based on complicated chemical conjugation or could not reach the posterior segment of the eye by topical administration. We previously knew that penetratin could permeate from the ocular surface to the fundus; therefore, in this work, we attempted to formulate a gene complex with penetratin to target the retina. To reduce the loss of positively charged penetratin and condense genes more efficiently, a cationic dendrimer was used to construct the primary complex, and then penetratin was noncovalently modified on the surface of I

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Figure 8. Fluorescence images of corneas and retinae of rat bulbus oculi that illustrate RFP expression after topical administration of nude plasmid and the complexes. The complexes containing 2 μg of pRFP were instilled in the conjunctival sac every 8 h constantly for 3 days, and the bulbus oculi was enucleated 3 days after the last administration. Blue, DAPI-labeled cell nuclei; red, expressed RFP. Scale bar = 50 μm.

SDHCEC cells were relatively weak compared to that of pRFP/ PAMAM/P due to the different particle sizes and internalization processes between the two complexes. After the two complexes were instilled in the conjunctival sac, penetratin and plasmid reached the fundus oculi together (Figures 6 and 7), which suggested that penetratin played a crucial role in gene delivery. Additionally, the complexes resided in the retina for longer than 8 h, providing plenty of time for the plasmid to induce transfection (Figure 8). Intraocular distribution of the complexes demonstrated that they penetrated into the retina but not much into the cornea, even though they could be taken up by corneal cells in vitro. When tissue characteristics are taken into consideration, the cornea has a compact epithelial layer and is continually being rinsed by tears. For most drugs, especially hydrophilic macromolecules, it is difficult to permeate into the cornea.50,51 By contrast, the sclera has a relatively looser tissue structure, wider intercellular space, and much larger surface area for drugs to permeate,52,53 and hence it is a potential absorption pathway for hydrophilic molecules and even macromolecules.54 As seen from Figures 7 and 8, the reporter gene was mainly distributed and transfected in PRS, IPL, and OPL. These three layers are not so dense in the whole retina and therefore are more amiable for uptake and transfection.55,56 Thus, our hypothesis is that the complexes penetrated into the eye via a noncorneal route and were probably absorbed through the sclera after topical application. Some phenomena observed during this study supported the hypothesis as well. For example, in Figure 6C, there was no fluorescence emitted in the corneal endothelium and stroma until 8 h after application of the complex pRFP/PAMAM/P20, as pointed out by the arrows. If the complex penetrated into the eye via the corneal route, the epithelium would be the first lighted. However, this did not occur. The anterior chamber is filled with aqueous humor, which flows and forms convection in the eye.57 The complexes distributed in the retina could reach the corneal endothelium and stroma following the aqueous humor circulation. This is consistent with our hypothesis that the nanoscale complexes were absorbed via a noncorneal route into the eye. Additionally, fluorescence distribution in the ocular appendages, choroid, and muscular tissues provided further evidence for our hypothesis. Although the complexes facilitated transportation of the reporter gene to the posterior segment of the eye, the therapeutic efficiency of functional genes delivered by this system still needs to be evaluated in model animals.

the resultant nanoparticles. G3 PAMAM is an ideal candidate to precondense the plasmid due to low content of free amino groups. More importantly, it is safe to the eye, as seen from Figure 1. The preparation of the complex is very simple and is done just by mixing the plasmid in turn with PAMAM and penetratin. The complex was as successful as we thought in delivering the plasmid to the posterior segment of the eye. Unexpectedly, we found that the binding of penetratin with the primary complex was stronger than with the nude plasmid (Figure 2). Although PAMAM would disturb the electrostatic interaction between plasmid and penetratin, from the increased bound penetratin and the reduced particle size (Table 1 and Figure 3), it could be deduced that the complex became more compact after incorporation of PAMAM. The complex pRFP/ PAMAM/P20 might not be held together by only electrostatic interaction, as occurred in the complex pRFP/P20. PAMAM dendrimers have hydrophobic internal cavities, which were reported to interact with the hydrophobic amino acid tryptophan.45 Furthermore, tryptophan-containing protein could also bind to PAMAM dendrimers.46 Penetratin possesses two tryptophan residues; therefore, hydrophobic interaction would be involved in the formation of the complex pRFP/ PAMAM/P20. The binding between the hydrophobic amino acids of penetratin with the interior of PAMAM is supposed to be powerful to form tightly condensed nanoparticles.47 The achieved complexes pRFP/P20 and pRFP/PAMAM/ P20 showed significantly improved cellular uptake and transfection compared with that of the nude plasmid. Flow cytometry analysis in Figure 4 shows that the green fluorescence of penetratin and the red fluorescence of RFP correlate with each other closely and present a linear distribution, which means the cells that took up penetratin were transfected by the reporter gene pRFP as well. These diagrams essentially suggest that penetratin and pRFP were internalized together in the complex form. The confocal images in Figure 5 illustrate that the two complexes behaved dissimilarly in the corneal cells. pRFP/PAMAM/P was entrapped by lysosomes and induced lysosome enlargement, which could be explained by the proton sponge effect of PAMAM,48,49 producing bright spots in the cells (pointed out by the arrows). In the pRFP/P treated cells, only penetratin localized in the lysosomes, while the plasmids dispersed in the cytoplasm, which probably was attributed to the incompact structure of the complex pRFP/P. Although the plasmids escaped from the lysosomes, it is hard to further penetrate into the nuclei. The uptake and transfection of pRFP/P in the J

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(10) Wang, Y.; Lin, H.; Lin, S.; Qu, J.; Xiao, J.; Huang, Y.; Xiao, Y.; Fu, X.; Yang, Y.; Li, X. Cell-penetrating Peptide TAT-mediated Delivery of Acidic FGF to Retina and Protection against Ischemiareperfusion Injury in Rats. J. Cell. Mol. Med. 2010, 14, 1998−2005. (11) Zhang, X.; Li, Y.; Cheng, Y.; Tan, H.; Li, Z.; Qu, Y.; Mu, G.; Wang, F. Tat PTD-endostatin: A Novel Anti-angiogenesis Protein with Ocular Barrier Permeability via Eye-drops. Biochim. Biophys. Acta, Gen. Subj. 2015, 1850, 1140−1149. (12) Li, X.; Zhang, Z.; Li, J.; Sun, S.; Weng, Y.; Chen, H. Diclofenac/ Biodegradable Polymer Micelles for Ocular Applications. Nanoscale 2012, 4, 4667−4673. (13) Kumar, A.; Chen, F.; Mozhi, A.; Zhang, X.; Zhao, Y.; Xue, X.; Hao, Y.; Zhang, X.; Wang, P. C.; Liang, X. J. Innovative Pharmaceutical Development Based on Unique Properties of Nanoscale Delivery Formulation. Nanoscale 2013, 5, 8307−8325. (14) Delgado, D.; del Pozo-Rodríguez, P. A.; Solinis, M. A.; AvilesTriqueros, M.; Weber, B. H.; Fernandez, E.; Gascon, A. R. Dextran and Protamine-based Solid Lipid Nanoparticles as Potential Vectors for the Treatment of X-linked Juvenile Retinoschisis. Hum. Gene Ther. 2012, 23, 345−355. (15) Johnson, L. N.; Cashman, S. M.; Kumar-Singh, R. Cellpenetrating Peptide for Enhanced Delivery of Nucleic Acids and Drugs to Ocular Tissues including Retina and Cornea. Mol. Ther. 2008, 16, 107−114. (16) Read, S. P.; Cashman, S. M.; Kumar-Singh, R. A Poly(ethylene) Glycolylated Peptide for Ocular Delivery Compacts DNA into Nanoparticles for Gene Delivery to Post-mitotic Tissues in Vivo. J. Gene Med. 2010, 12, 86−96. (17) Johnson, L. N.; Cashman, S. M.; Read, S. P.; Kumar-Singh, R. Cell Penetrating Peptide POD Mediates Delivery of Recombinant Proteins to Retina, Cornea and Skin. Vision Res. 2010, 50, 686−697. (18) Liu, C.; Tai, L.; Zhang, W.; Wei, G.; Pan, W.; Lu, W. Penetratin, A Potentially Powerful Absorption Enhancer for Noninvasive Intraocular Drug Delivery. Mol. Pharmaceutics 2014, 11, 1218−1227. (19) Christiaens, B.; Dubruel, P.; Grooten, J.; Goethals, M.; Vandekerckhove, J.; Schacht, E.; Rosseneu, M. Enhancement of Polymethacrylate-mediated Gene Delivery by Penetratin. Eur. J. Pharm. Sci. 2005, 24, 525−537. (20) Nigatu, A. S.; Vupputuri, S.; Flynn, N.; Neely, B. J.; Ramsey, J. D. Evaluation of Cell-penetrating Peptide/Adenovirus Particles for Transduction of CAR-negative Cells. J. Pharm. Sci. 2013, 102, 1981− 1993. (21) Layek, B.; Singh, J. Cell Penetrating Peptide Conjugated Polymeric Micelles as A High Performance Versatile Nonviral Gene Carrier. Biomacromolecules 2013, 14, 4071−4081. (22) Bendifallah, N.; Rasmussen, F. W.; Zachar, V.; Ebbesen, P.; Nielsen, P. E.; Koppelhus, U. Evaluation of Cell-penetrating Peptides (CPPs) as Vehicles for Intracellular Delivery of Antisense Peptide Nucleic Acid (PNA). Bioconjugate Chem. 2006, 17, 750−758. (23) Moschos, S. A.; Jones, S. W.; Perry, M. M.; Williams, A. E.; Erjefalt, J. S.; Turner, J. J.; Barnes, P. J.; Sproat, B. S.; Gait, M. J.; Lindsay, M. A. Lung Delivery Studies Using siRNA Conjugated to TAT(48−60) and Penetratin Reveal Peptide Induced Reduction in Gene Expression and Induction of Innate Immunity. Bioconjugate Chem. 2007, 18, 1450−1459. (24) Navarro, G.; Maiwald, G.; Haase, R.; Rogach, A. L.; Wagner, E.; de Ilarduya, C. T.; Ogris, M. Low Generation PAMAM Dendrimer and CpG Free Plasmids Allow Targeted and Extended Transgene Expression in Tumors after Systemic Delivery. J. Controlled Release 2010, 146, 99−105. (25) Liu, X.; Wu, J.; Yammine, M.; Zhou, J.; Posocco, P.; Viel, S.; Liu, C.; Ziarelli, F.; Fermeglia, M.; Pricl, S.; Victorero, G.; Nguyen, C.; Erbacher, P.; Behr, J.; Peng, L. Structurally Flexible Triethanolamine Core PAMAM Dendrimers Are Effective Nanovectors for DNA Transfection in Vitro and in Vivo to the Mouse Thymus. Bioconjugate Chem. 2011, 22, 2461−2473. (26) Conti, D. S.; Brewer, D.; Grashik, J.; Avasarala, S.; Da Rocha, S. R. P. Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol

5. CONCLUSION In summary, physical complexes for intraocular gene delivery were constructed utilizing electrostatic interactions between plasmid DNA and penetratin. Introduction of PAMAM of a low molecular weight into the complex condensed the plasmid more compactly and produced nanoparticles with an approximate size of 150 nm. The complexes were easy to prepare and showed outstanding permeability and transfection capability to ocular cells with low cytotoxicity. Most attractively, when instilled in the conjunctival sac, the complexes delivered the plasmid to the posterior ocular segment rapidly and expressed fluorescence protein in the retina. The proofs collected in this work suggested that the complexes penetrated into the eye through a noncorneal route during which penetratin played a key role in delivering genes to the fundus oculi. As a noninvasive delivery system, the complex provides an easy and friendly approach for gene therapy of intraocular diseases.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], Tel.: +86 21 51980091, Fax: +86 21 51980090. Author Contributions

C.L. and K.J. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Yong Gan (Shanghai Institute of Materia Medica) for kindly donating the NHC cells and Prof. Ying Liu (Zhongshan Ophthalmic Center, Sun Yat-sen University) for providing the SDHCEC cells. This study was supported by the National Natural Science Fund of China (Grants 81172994 and 81573358).

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REFERENCES

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