Penetratin, a Potentially Powerful Absorption Enhancer for

Article pubs.acs.org/molecularpharmaceutics

Penetratin, a Potentially Powerful Absorption Enhancer for Noninvasive Intraocular Drug Delivery Chang Liu,†,‡ Lingyu Tai,†,‡,§ Wenjian Zhang,† Gang Wei,*,† Weisan Pan,§ and Weiyue Lu† †

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 S Supporting Information *

ABSTRACT: Intraocular drug delivery is extraordinarily hampered by the impermeability of defensive barriers of the eye. In this study, the ocular permeability of fluorophore-labeled cell-penetrating peptides (CPPs), including penetratin, TAT, low molecular weight protamine, and poly(arginine)8, was investigated based on multilevel evaluations. The human conjunctival epithelial cell (NHC) was exposed to various CPPs to determine the cytotoxicity and cellular uptake. Ex vivo studies with rabbit cornea were performed using side-by-side diffusion chambers to evaluate the apparent permeability coefficients and acute tissue tolerance of the CPP candidates. Among all examined CPPs, penetratin shows an outstanding cellular uptake, by increasing more than 16 and 25 times at low and high concentrations, compared to the control peptide poly(serine)8 respectively. Additionally, the permeability of penetratin across excised cornea is 87.5 times higher in comparison with poly(serine)8. More importantly, after instilled in the conjunctival sac of rat eyes, fluorophore-labeled penetratin displayed a rapid and wide distribution in both anterior and posterior segment of the eye, and could be observed in the corneal epithelium and retina lasting for at least 6 h. Interestingly, penetratin showed the lowest ocular cell and tissue toxicities among all examined CPPs. The high ocular permeability of penetratin could be attributed to its amphipathicity and spatial conformation determined by circular dichroism. Taken together, these data demonstrate that penetratin is potentially useful as an absorption enhancer for intraocular drug delivery. KEYWORDS: cell-penetrating peptide, penetratin, ocular drug delivery, permeability, toxicity

1. INTRODUCTION Cornea, conjunctiva, and tear film are main barriers protecting the eye from noxious substances. The epithelia of cornea and conjunctiva seal the ocular tissues with intercellular tight junctional complexes, presenting a relative impermeability for both xenobiotics and therapeutic agents.1−3 The mucous tear film entraps microorganisms, particles, and debris and eliminates them with continual secretion through the nasolacrimal drainage. This efficient system commendably prevents pathogens from invasion; however, it also rejects helpful substances curing diseases of the anterior surfaces and the inner compartments. Thus, these barriers are difficult to cross for many drugs, especially hydrophilic macromolecules such as genes and most peptides and proteins. Furthermore, to deliver drugs to the posterior segment of eyes, intraocular injection is required, which scarcely results in patient compliance. Ocular absorption enhancers in topical instillation are perceived as promising strategies for noninvasive drug delivery.4 However, the use of many absorption enhancers like cholate and fusidate is limited by their toxicity and irritation.4,5 Cell-penetrating peptides (CPPs) are positively charged amino acid sequences of usually less than 30 residues in length from multiple sources that can help deliver micro- and macromolecules into living cells.6 Bulky cargos such as proteins, genes, © 2014 American Chemical Society

nanoparticles, liposomes, and micelles are successfully transferred across the cell membrane facilitated by CPPs.7 They demonstrate prominent advantages including low toxicity, biocompatibility, biodegradability, and ease of synthesis compared with other absorption enhancers and polymeric carriers.8,9 The mechanism of membrane penetration has not been completely explained though extensive studies were performed. It is acknowledged that various endocytic pathways coexist during the cellular internalization including endocytosis, direct translocation, and pathways regulated by clathrin, caveolin, and flotillin, despite the influence of temperature, concentration, and size of the cargo. In addition, the penetration efficiency is closely related to the cationic residues which may interact with the negatively charged moieties of proteoglycans on the surface of cellular membrane.10−12 CPP-mediated transport in ophthalmological application has not been studied as extensively as oral administration and intravenous injection.13−17 A novel synthetic CPP composed of four repeat ARKKAAKA units and a total of 35 amino acid residues, Received: Revised: Accepted: Published: 1218

November 12, 2013 January 14, 2014 February 12, 2014 February 12, 2014 dx.doi.org/10.1021/mp400681n | Mol. Pharmaceutics 2014, 11, 1218−1227

Molecular Pharmaceutics

Article

Table 1. A Summary of Molecular Information and Characterization of the CPP Candidates and the Control Peptidea

a

name

abbreviation

sequencesb

Mw (Da)

pI

charge

TAT penetratin poly(arginine)8 low molecular weight protamine poly(serine)8

TAT penetratin R8 protamine S8

GRKKRRQRRRPPQK-FAM RQIKIWFQNRRMKWKKK-FAM RRRRRRRRK-FAM VSRRRRRRGGRRRRK-FAM SSSSSSSSK-FAM

2205.5 2733.3 1754.0 2366.7 1201.1

12.7 12.3 12.8 13.0 8.5

+9 +8 +9 +11 +1

Mw: molecular weight. pI: isoelectric point. bThe hydrophobic amino acids are underlined.

named peptide for ocular delivery (POD), has been explored to deliver gene medicines, chemotherapeutic drugs, and cell factors to ocular tissues including retina and cornea.14 A recent report described solid lipid nanoparticles (SLN) modified by protamine as gene vectors for the treatment of X-linked juvenile retinoschisis.16 Introduction of protamine onto the surface of SLN significantly improved the expression of retinoschisin and enhanced green fluorescent protein (EGFP) in ARPE-19 cells. Other research on TAT-mediated delivery of acidic fibroblast growth factor (aFGF) to retina revealed the protection effect against rats’ ischemia reperfusion.17 However, it is still unclear whether there are distinctions in permeability between so many CPPs when administered to the eye. If any, which one is more suitable for ocular application and what is the possible reason resulting in the difference become questions worthy to be investigated. Therefore, a closer comparison of CPPs’ safety and efficacy on ocular permeability and absorption is necessary. The purpose of this study is to screen a library to find a CPP as an effective absorption enhancer for intraocular administration, which will be useful for noninvasive delivery of bioactive macromolecule into eyes. Depending on the sources, CPPs can be classified into different types, such as natural protein transduction domains and chemically synthesized peptides. Four different CPPs, including TAT (a fragment of transcriptiontransactivating protein from HIV virus), penetratin (a peptide derived from a nonviral protein), low molecular weight protamine (a shortened substitute of protamine in clinical heparin neutralization), and a totally artificial peptide poly(arginine)8 (R8), were selected and modified with a fluorophore. According to previously published reports,18 the highly efficient translocation capacities of these peptides have been observed in a variety of cell lines with minimal toxicity. Another common feature of these peptides is that they are small in molecular weight and simple in molecular structure, so that the factors affecting on their permeability are easily elucidated. In vitro uptake and cytotoxicity of the CPP candidates on conjunctiva cell lines were performed for a preliminary optimization. Ex vivo permeability and acute tissue tolerance of these CPPs were subsequently confirmed with excised rabbit cornea. In vivo distribution of the optimized CPP was evaluated by topical application in the rat eyes. Finally, elucidation of the diversity of CPPs on the ocular permeability is attempted from the perspective of difference in their spatial conformation.

Figure 1. Schematic representation of the side-by-side diffusion cell device.

of all the CPP candidates were conjugated via a lysine residue with 5-carboxyfluorescein (FAM) as a fluorophore. An electroneutral pseudopeptide poly(serine)8-Lys-FAM (S8) was assigned as a control peptide. Amino acid sequence and characterization of these peptide molecules are summarized in Table 1. 2.3. Animals. Male Sprague−Dawley rats (17 weeks old) and male albino rabbits (3.0 kg) were obtained from the Experimental Animal Center of Fudan University and maintained at 22 ± 2 °C on a 12 h light−dark cycle with access to food and water ad libitum. The animals used for the experiments were treated according to protocols that were evaluated and approved by the Ethical Committee of Fudan University. The animals were acclimatized to laboratory conditions for 1 week prior to experiments. 2.4. In Vitro Experiments Using NHC Cell Lines. 2.4.1. Cell Culture. Wong-Kilbourne-derived human conjunctival epithelial cells (NHC) (ATCC CCL-20.2, USA) were propagated in DMEM supplemented with 10% FCS, 4 mM stable glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2 humidified atmosphere. 2.4.2. Cytotoxicity. The cytotoxicity of fluorophore-labeled CPPs and the control peptide was evaluated using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.20 NHC cells were harvested at the logarithmic growth phase and seeded on 96 well plates at a cell density of 5 × 103 cells/well, and they were incubated at 37 °C in DMEM medium and 5% CO2 humidified atmosphere for 24 h. The culture medium was then replaced with 200 μL of DMEM containing 10% FCS and different peptides at a series of concentrations. After NHC cells were exposed to various peptides for 12 h, the cells were washed 3 times with phosphate buffer solution (PBS), followed by further culture in DMEM medium for 24 h. Cells were then incubated with MTT agent in a concentration of 0.5 mg/mL in each well, for 4 h at 37 °C. Finally, formazan was extracted with 200 μL of dimethyl sulfoxide, and absorbance was measured at 490 nm using a microplate reader (Power Wave XS, Bio-TEK, USA). The survival curve and IC50 were illustrated and calculated using GraphPad Prism 5 software.

2. EXPERIMENTAL SECTION 2.1. Reagents. Fetal calf serum (FCS), antibiotics, and Dulbecco’s modified Eagle medium (DMEM) were purchased from Life Technologies (Karlsruhe, Germany). Artificial tear fluid was composed of 6.78 g/L NaCl, 1.38 g/L KCl, 2.18 g/L NaHCO3, and 0.084 g/L CaCl2·H2O in water.19 All other chemicals used in this research were of analytical grade. 2.2. CPP candidates. All the CPP candidates were synthesized by GL Biochem Ltd. (Shanghai, China). The N-terminals 1219

dx.doi.org/10.1021/mp400681n | Mol. Pharmaceutics 2014, 11, 1218−1227


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2.4.3. Cellular Internalization. NHC cells were seeded on 6 well plates at a cell density of 5 × 104 cells/well. After incubation at 37 °C in DMEM medium and 5% CO2 humidified atmosphere for 4 days, the medium was replaced by 2 mL of DMEM containing 10% FCS and different peptides at a concentration of 285 μmol/L (the quantitative value was chosen based on the results of MTT assay). Incubation of NHC cells with peptides was then carried out for 0.5, 1, 2, and 4 h at 37 °C, followed by a washing step with PBS containing 1000 IU/mL heparin and 75 mmol/L sodium azide 3 times on ice, to remove extracellular bound peptides.21,22 Cells in each well were immersed in 1 mL of PBS and immediately subjected to fluorescence microscopy imaging using Leica DM4000B automated upright microscope system (Leica, Germany). To quantify the cellular uptake of penetratin and the control peptide, flow cytometry experiments were implemented. NHC cells were treated as described above. After incubation for 4 days, the medium was replaced by 2 mL of DMEM containing 10% FCS and penetratin or S8 at low and high concentrations (57 μmol/L and 285 μmol/L, respectively). NHC cells were exposed to penetratin or S8 for 1 and 4 h at 37 °C. Subsequently, the cells were incubated with 1000 IU/mL heparin to remove any extracellular adsorbed peptides. After being washed and trypsinized, NHC cells were suspended in 500 μL of DMEM solution containing 10% FCS and were transferred into Eppendorf centrifugal tubes on ice in the dark until analysis. The amount of FAM positive cells and mean fluorescence intensity (Fm) (excitation at 488 nm and emission at 508 nm) were determined using a flow cytometer (BD Bioscience, USA). Results were analyzed using FlowJo software (Treestar Inc., San Carlos, CA, USA). 2.5. Ex Vivo Permeability Experiments with Rabbit Cornea. Permeability of CPPs through the rabbit cornea was determined using a side-by-side diffusion cell device (TK-6H1, Kaikai Science and Technology Co., Ltd., Shanghai, China). The diameter of the diffusional opening is 10.25 mm, generating a diffusional area of 0.825 cm2. The rabbits were sacrificed by an injection of a fatal dose of chloral hydrate via marginal ear vein. The corneas were removed and gently washed with artificial tear fluid. Each cornea was carefully placed vertically between the diffusion cells with endothelial surface contacting the acceptor solution (shown in Figure 1). The acceptor solution was composed of 3 mL of blank artificial tear fluid with a mixture of O2/CO2 (95:5) bubbled throughout the experiment, while the donor side contained 57 μmol/L various peptides in the same medium of equal volume. The solution in the diffusion cells was preheated and maintained at 34 ± 0.5 °C by a circulation heating system. An aliquot of 500 μL of sample was extracted at time intervals of 0.5 h during a 4 h period from the acceptor cell, immediately followed by a supplement of equivalent blank artificial tear fluid. The fluorescence intensity of the extracted samples was determined by a fluorescence spectrophotometer (Hitachi, F7000, Japan) (excitation at 488 nm and emission at 508 nm) and adopted to calculate their corresponding apparent permeability coefficients (Papp, cm·s−1). As an important parameter to toxicological evaluation, hydration value (ΔH) levels of the corneas were investigated following the permeability test. After detachment from the diffusion cells, the remaining scleral tissue was cut away from the corneas, and each cornea was divided into two halves. One half of the cornea was accurately weighed before and after a drying process at 60 °C for 48 h. The other half of the cornea was made into frozen section and dyed separately with hematoxylin−eosin

(HE) and 4′,6-diamidino-2-phenylindole (DAPI) for a further observation by the inverted fluorescence microscope (DMI 4000B, LEICA, Germany).23 2.6. In Vivo Ocular Distribution Studies. Rats were used to investigate peptide distribution in eyeballs after topical administration. An aliquot of 5 μL of penetratin- or S8-dissolved artificial tear fluid was instilled into the conjunctival sac of rats three times with 10 min intervals. The concentration of peptide was 570 μmol/L, 10-fold over the concentration used in the ex vivo test. The peptide was dispersed on the corneal surface by gently pulling up and closing the eyelids. The rats were sacrificed 10 min, 30 min, 1 h, 2 h, 4 h, and 6 h after the last administration. Eyeballs were harvested and fixed in the Davidson’s solutions for 30 min. An overnight dehydration was implemented in 30% sucrose solution before DAPI-dyed frozen section production.24,25 An inverted fluorescence microscope determination was carried out afterward. 2.7. Circular Dichroism. Circular dichroism (CD) was measured on a Chirascan spectropolarimeter (Applied Photophysics, U.K.) in a 0.1 cm quartz cuvette. The peptides were dissolved in water to a concentration of 0.1 mg/mL. The scan wavelength ranged from 190 to 260 nm with a 0.5 nm step, 1 nm bandwidth at 20 °C. Every spectrum was collected over 10 scans to make an average. The measured ellipticity was converted to a mean residue molar ellipticity (θ) (deg·cm2·dg−1) after background signal correction. The CPP candidates and the control peptide were determined in triplicate.26,27

3. RESULTS 3.1. Cytotoxicity of CPPs to NHC Cell Line. To evaluate the cytotoxicity, NHC cells were cultured with different peptide solutions for 12 h and cell viability was measured by an MTT assay. The survival curve illustrates that all CPPs show rarely cytotoxic effects with a concentration lower than 0.3 mM (Figure 2).

Figure 2. Cell viability of NHC cells following 12 h incubation with increasing concentrations of various peptides as determined by the MTT assay (n = 5, mean ± SD). Results are expressed as percent of the positive control values obtained from cells incubated with culture medium without peptides.

Overall, the most cytotoxic CPPs are R8 and protamine with an average IC50 of approximate 0.7 mM; TAT shows an IC50 of about 2 mM, similar to 2.7 mM of the control peptide S8; the least cytotoxicity was observed on penetratin, with an IC50 considerably higher than 2.5 mM, and did not demonstrate any effects on the viability of NHC cells under the tested concentration range (Table 2). 1220



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Table 2. IC50 of NHC Cells Determined by the MTT Assay (n = 5) IC50 (M)

penetratin

TAT

R8

protamine

S8

>2.50 × 10−3a

1.96 × 10−3

7.33 × 10−4

6.76 × 10−4

2.73 × 10−3

a

IC50 of penetratin on NHC cells could not be calculated since no obvious inhibition on cell viability was observed even under the highest tested concentration.

3.4. Corneal Permeability. 3.4.1. Apparent Permeability Coefficients of CPPs. The apparent permeability coefficients (Papp, cm·s−1) were calculated using the following equation: Papp =

ΔQ Δt ·c0·A ·3600

where ΔQ/Δt is changes in the amount of the peptides in moles with time, represented by the slope of the linear part of the permeability curve (Figure 5A); c0 is the initial concentration of the peptides in the donor chamber; A is the surface area of the mounted cornea exposed to the solutions, taken to be 0.9 cm2; and 3600 is a conversion factor to a unit of second.28 As shown in the permeability curves (Figure 5A), CPP permeation across the excised corneas is a linear process depending on diffusion time. The observed apparent permeability coefficients of penetratin and TAT during the 4 h period were significantly higher than that of the negative control S8 group (Figure 5B, Table 4). Particularly, Papp of the penetratin group was 87.5-fold higher compared to that of the S8 group, which indicates distinctive corneal permeation ability. Moreover, TAT, protamine, and R8 also showed moderate properties in penetrating the excised corneas compared with S8 (31.8-, 17.6-, and 16.3-fold, respectively) (Figure 5B, Table 4). 3.4.2. Acute Tissue Tolerance of Rabbit Corneas. The hydration value (ΔH) levels of rabbit cornea were computed with the equation below: m − mt ΔH = 0 × 100% m0

Figure 3. Time-dependent uptake of FAM-labeled peptides by NHC cells. Representative images of fluorescence microscope illustrating NHC cells exposed to 285 μmol/L various CPP candidates or the control peptide S8 for 0.5 h, 1 h, 2 h, and 4 h. Scale bar, 200 μm.

3.2. Cellular Uptake of CPPs by NHC Cells. Effects of different CPPs and various incubation time on the cellular uptake were characterized by images of fluorescence microscope (Figure 3). Based on the cytotoxicity results, this study was performed with a CPP concentration of 285 μmol/L. Cells treated with the control peptide S8 showed normal morphological details with barely green fluorescence in 4 h incubation as expected. Generally, NHC cells preserved a regular morphology similar to the untreated status after incubating with CPP solutions, indicating that no damage was induced by exposure to the CPPs for 4 h. As shown in Figure 3, FAM-labeled CPPs saturated the cells nonspecifically with a time dependent trend. Among them, penetratin exhibited an outstanding conjunctiva cell uptake evidenced by an intense fluorescent image. TAT was also absorbed abundantly and obviously presented inside the cells as early as 30 min after incubation. Protamine and R8 faded next to the above two CPPs, but the signs were still significantly higher than the negative control S8 group. 3.3. Quantitative Uptake of Penetratin by NHC Cells. A further quantitative determination of penetratin uptake was carried out by a flow cytometry assay with different incubation time and concentrations (Figures 4A and 4B). Flow cytometric analyses revealed that the cells treated with FAMlabeled penetratin displayed an average fluorescence significantly higher (P < 0.001) than that of the untreated cells and the cells treated with FAM-labeled S8. With an increasing incubation time and concentration, the average fluorescence of penetratin group was 28- to 153-fold higher than that of the untreated group and about 16- to 29-fold higher than that of the S8 group (Figure 4C, Table 3). Interestingly, the S8 group also showed significant, but modest, increase in fluorescence (1.7- to 6.1-fold compared with the untreated group), which did accord with the microscope images.

where m0 is the weight of the detached moist cornea and mt is the weight of its desiccative remaining. The hydration values of CPP-treated rabbit corneas showed no significant difference with that of the untreated cornea, which demonstrates that they are almost free of ex vivo tissue toxicity under the tested concentration (Figure 6). Especially, the hydration value of the penetratin group was quite close to that of the untreated control, and even better than that of the S8 group. Comparison of the corneal hydration values of the peptides demonstrates a similar ranking in accordance with the results of cytotoxicity evaluation (Table 2). The HE staining sections of corneas after permeation experiment corroborated the results, in which all the corneal epithelium preserved their integrity with no leakage (Supporting Information, Figure S1). 3.4.3. Distribution of CPP in Excised Rabbit Corneas. After exposure to blank artificial tear fluid in the permeation experiment, frozen sections of untreated rabbit corneas showed no fluorescence as expected (Figure 7). The sections of S8 treated cornea emitted weak fluorescent signals, which is much less than that of the CPP-treated corneas. In sections of CPP groups, green fluorescence was densely localized in the epithelium and sparsely presented throughout the corneal stroma. The fluorescent pattern observed in CPP-treated corneas was intense and nonspecifically distributed along the corneas. The fluorescent intensity ranking was consistent with the results of cellular uptake and ex vivo corneal permeability. The penetratin group was notably stronger in fluorescence, followed by TAT, 1221



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Figure 4. Flow cytometry analysis of (A) penetratin and (B) S8 uptake by NHC cells and (C) the histogram plot of the mean fluorescence intensity (Fm). Untreated control (a), cells treated with 57 μmol/L penetratin (Penetratin-L) for (b) 1 h and (c) 4 h, cells treated with 285 μmol/L penetratin (Penetratin-H) for (d) 1 h and (e) 4 h, cells treated with 57 μmol/L S8 (S8-L) for (f) 1 h and (g) 4 h, and cells treated with 285 μmol/L S8 (S8-H) for (h) 1 h and (i) 4 h were analyzed and quantified by their fluorescence emission (excitation at 488 nm and emission 508 nm). The symbol ** indicates P < 0.001 as determined by a two-way RM ANOVA test.

plexiform layers (IPL), and outer plexiform layer (OPL). A faintish dendritic pattern of fluorescence extended into the inner nuclear layer (INL) and outer nuclear layer (ONL). The subretinal space containing the photoreceptor segments (PRS) and the retinal pigment epithelium (RPE) showed an intense sparkle in the rods and cones compared with that in the choroid (Chor) (Figure 8B). There was no evidence of retinal damage of the lens capsule caused by administration of peptides in any frozen sections. Only 10 min after application, the FAM-labeled penetratin had reached both anterior and posterior segments of the eyeballs (Figure 8C). The intensity of fluorescence peaked at 30 min and receded gradually with increasing time. Moderate fluorescence could still be observed as long as 6 h, which suggested a long lasting retention for penetratin in the ocular tissues. In contrast, weak fluorescence emerged in the sections from S8-treated eyes and died out rapidly in about 2 h (Figure 8D). 3.6. Circular Dichroism. Circular dichroism of the examined CPPs and S8 was applied to reveal the difference in secondary

protamine, and R8 groups. Sporadically, some aggregated CPPs remained on the surface of corneal epithelium which formed bright spots. 3.5. In Vivo Ocular Distribution. To inspect the in vivo ocular distribution and pharmacokinetics of penetratin after topical administration, anterior segment (cornea) and posterior segment (retina) of frozen bulbus oculi sections with different sacrifice timings were observed separately by fluorescent microscope. Sections of untreated eyes revealed no fluorescence (Supporting Information, Figure S2). Occasionally in experimental rats, control specimens of contralateral cornea and retina emitted dim fluorescence though much weaker than that of penetratin- or S8-treated eyes. In vivo distribution of the peptides in the cornea after topical administration coincides with the ex vivo results (Figure 8A). Fluorescence was again noted in the endothelium, stroma, and especially epithelium. The pattern of fluorescence in retina is moderate and uniform in ganglion cell layer (GCL), inner 1222



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Table 3. The Mean Fluorescence Intensity (Fm) of Penetratin- and S8-Treated NHC Cells with Different Incubation Time and Concentration (n = 3) no.

peptide

concn (μmol/L)

T (h)

Fm

a b c d e f g h i

none penetratin penetratin penetratin penetratin S8 S8 S8 S8

57 57 285 285 57 57 285 285

1 4 1 4 1 4 1 4

451 ± 18 12667 ± 1566 25529 ± 2870 48603 ± 64 69291 ± 1187 780 ± 14 880 ± 45 1845 ± 16 2745 ± 22

enhancement ratio to S8a 16.2 29.0 26.3 25.2

a Enhancement ratio to S8 is calculated by comparing the mean fluorescence intensity (Fm) of penetratin group with that of S8 group at the corresponding concentration and incubation time.

Figure 6. The hydration value (ΔH) levels of the peptide-treated rabbit corneas. All the samples point to no significant difference with the untreated control (n = 3, mean ± SD).

Figure 7. Fluorescent images of the frozen sections of the corneas treated with blank artificial tear fluid (Untreated), or artificial tear fluid containing FAM-labeled peptides at a concentration of 57 μmol/L after the 4 h permeation experiment. For each group, the fluorescent images were merged with DAPI-stained corneas. Scale bar, 50 μm.

structure between these peptides, which may cause diversity in their abilities of ocular penetration (Figure 9). S8 established a complete random-coil conformation with an extremely weak negative band just below 200 nm. The CPPs were dominated by random structure mixed with different proportions of polyproline type II (PPII) structure.29 CD spectra of protamine and R8 were characterized by a negative band around 200 nm and by a weaker positive band approximately at 215 nm, which represented a nearly typical random coil. The negative band of TAT is more intense but without obvious blue shift compared to the CD spectra of protamine and R8, appearing to be greater structural content. Penetratin exhibited a strong negative band around 205 nm and a weak positive band at 225 nm, coinciding exactly with the typical PPII structure.30

4. DISCUSSION Topical application of ophthalmic drugs obtains an unsatisfactory ocular absorption due to impermeability of the cornea, tear turnover, nasolacrimal drainage, systemic absorption, and other factors.31 Recent strategies for noninvasive approaches to optimize the ophthalmic drug delivery focus on penetration enhancers, prodrugs, bioadhesive agents, and colloidal systems.32

Figure 5. Permeability curves (A) and the calculated apparent permeability coefficients (Papp) (B) of the CPP candidates and the control peptide S8 across excised rabbit corneas (n = 3, mean ± SD). Penetratin showed impressive permeability in the ex vivo experiment with a steeper slope than the other peptides. Papp of TAT also make a significant increase by more than 10-folds than S8. The symbol ** indicates P < 0.001 and * indicates P < 0.05 as determined by a Tukey’s multiple comparison test. 1223



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Table 4. The Apparent Permeability Coefficients (Papp) of Four CPP Candidates and the Control Peptide S8 (n = 3) Papp (×106/cm·s−1) enhancement ratio to S8a a

penetratin

TAT

protamine

R8

S8

10.5 ± 2.20 87.5

3.81 ± 0.27 31.8

2.11 ± 1.25 17.6

1.95 ± 0.63 16.3

0.12 ± 0.07

Enhancement ratio to S8 is calculated by comparing the apparent permeability coefficients (Papp) of the CPPs with that of S8.

Figure 8. Fluorescent merged images with DAPI-stained corneas and retinae from penetratin and poly(serine)8 (S8) treated rats. High magnification micrographs illustrate the distribution of fluorescence in the cornea (A) and in the retina (B). The fluorescent intensity decay of penetratin group (C) and S8 group (D) is shown time-dependently. An aliquot of 5 μL of artificial tear fluid containing 570 μmol/L penetratin or S8 was instilled in the conjunctival sac of rats three times with 10 min intervals. The general retina structure contains 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, 200 μm for A and B, 50 μm for C and D.

peptides by chemical synthesis can easily reach hundreds of grams, therefore the cost of CPP contained formulation is expected to be economical. Our findings demonstrate that the four examined CPPs and the control peptide showed a consistent tendency of permeability in different assays, which in order is penetratin > TAT > protamine ≈ R8 ≫ S8. In cellular uptake, the fluorescence intensity in CPP-treated NHC cells increased with time and penetratin exhibited a remarkable intense fluorescent image. These data drew our attention to penetratin, which is the

CPPs, an emerging kind of absorption enhancers, have been applied to various drug delivery systems promoting oral absorption of macromolecules, targeting brain−blood barrier, tumors, and other locations.7,33 Though several CPP-based ocular delivery systems have been established, a systemic evaluation of penetration efficiency of CPPs has not been reported.15,34,35 In this study, we made a multilevel comparison among some of the most concerned CPPs. The selected CPPs were minipeptides composed of fewer than 16 amino acid residues. Yield of the 1224



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too faint to interfere with observation of the distribution of the fluorophore-labeled peptides. Cornea and conjunctiva represent the principal penetration pathways into the anterior and posterior chamber, respectively. In this work, human conjunctival epithelial cells (NHC) were used in the cellular internalization experiment and rabbit corneas were used in the ex vivo permeability experiments. All the results of the above studies showed observable CPP transportation. As a consequence, it is supposed that both conjunctival and corneal pathways are involved in the penetration process. The density of positive charge on a CPP molecule was once considered as a decisive factor for membrane penetration.42,43 This hypothesis is partially true, because the permeability of the control peptide S8 is 1 order of magnitude weaker compared to all examined CPPs, although the molecular weight of S8 is the smallest one among all the peptides. S8 is composed of polar amino acid serine, having the least positive charge and an isoelectric point close to neutral pH (Table 1). Therefore, positive charge indeed plays an important role in membrane transportation. Interestingly, in this study, penetratin is not the most positively charged molecule compared to the other three CPPs (Table 1). Cationization seems to be a prerequisite but not the only determining factor for ocular permeability. As seen from the CD spectra, the distinctive property of penetratin in ocular permeation is possibly correlated with its amphipathicity and spatial structure. It was reported that neither peptide helicity nor amphipathicity is essential for brain cell internalization.44,45 Nevertheless, these two factors make sense in ocular penetration. The intricate structure of cornea, conjunctiva, and sclera restricts the delivery of both hydrophilic and hydrophobic molecules.46 Penetratin is the only peptide containing hydrophobic aromatic residues (2 tryptophans and 1 phenylalanine) in the sequence (Table 1), and it therefore possesses the strongest lipophilicity among the tested CPPs. Amphipathic penetratin can express hydrophilicity in the tear film, meanwhile exhibiting lipophilicity when contacting the corneal epithelium. The PPII helix structure of penetratin also provides more hydrogen-bonding sites so that the peptide could form more effective interference with the cellular membrane.27 Even minimal sequence mutations on penetratin, such as substitution of tryptophan residues, would impact on peptide/lipid interactions and reduce translocation in living cells.47,48 All the results imply that, in addition to the highly cationic nature, both amphipathicity and spatial conformation are crucial for the prominent ocular permeation of penetratin.

Figure 9. Circular dichroism spectra of the CPP candidates and S8. The scan wavelength ranged from 190 to 260 nm.

first CPP derived from a nonviral natural protein.36 Flow cytometry results for the best candidate, penetratin, showed a 16−29-fold increase in fluorescence over S8 (Table 3). In the following ex vivo assay, penetratin also displayed a remarkable permeation property across the excised cornea of rabbits (Table 4). The Papp of TAT, protamine, and R8 is approximately 10−6 cm·s−1, decuple that of S8. By contrast, penetratin possessed the coefficient almost 88 times over S8, which is close to that of some small molecule drugs.37,38 CPP mediated complex and conjugation have been proved feasible in delivering various cargoes by different administration routines.14,17,39,40 The excellent corneal permeability of penetratin has promising prospects in ophthalmic drug delivery. Since we have modified the CPPs with a fluorophore (FAM), other low molecular weight bioactive agents, or even some larger complexes, would be able to be translocated into the eye through the same pathway as well. In the cytotoxicity experiments, we did not obtain an accurate IC50 for penetratin. The concentration of applied penetratin reached as high as 30 mM, while the cell viability was still around 100%. High concentration of penetratin did not inhibit the cellular activity. IC50 of other CPPs are slightly lower than that of S8, suggesting mild toxicities by cellular membrane perturbation during the trafficking process.41 The hydration value of cornea (ΔH) is an important indicator for tissue irritation and damage. In ex vivo test, ΔH of all groups including the examined CPPs and control peptide is about 85%, slightly higher than the regulation value 76−83%.5 However, no significant difference was observed between dosing groups and the blank artificial tear fluid treated group (85 ± 3.5%). The HE stained sections proved that these corneal epithelia preserve an intact structure all along without any leakage (Supporting Information, Figure S1). In vivo distribution in rat eyeballs illustrated an extensive and rapid penetration of penetratin in cornea and retina. The fluorophore-labeled peptide reached anterior and posterior segments of the eyeballs in 10 min and remained detectable for over 6 h, revealing the possible application of penetratin in treatment of posterior segment diseases. Specimens of contralateral cornea and retina, which were treated with blank artificial tear fluid as negative control, occasionally emitted dim fluorescence probably caused by the endogenous fluorescent substances or nasolacrimal fluid diffusion. However, the autofluorescence was

5. CONCLUSION In summary, this work demonstrated that among the examined CPP candidates penetratin showed an outstanding property in both high permeability and low toxicity to ocular tissues. As a potential permeation enhancer for ophthalmic use, penetratin can be conjugated with bioactive agents, like the fluorescent probe in the present work, or modified onto various delivery systems. The redundant positive charges can also shelter some electronegative biomolecules, for example gene, to improve their absorption. Furthermore, penetratin exerts a rapid and wide distribution, especially in the posterior segment of the eye, which can be utilized to establish a vector for noninvasive intraocular drug delivery via topical administration. The powerful ocular permeability of penetratin could be attributed to its amphipathicity and PPII helix structure, both of which were different from the other examined CPPs. Further investigations on the penetration mechanism of penetratin are ongoing. 1225



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ASSOCIATED CONTENT

S Supporting Information *

Figures depicting HE stained sections of corneas after ex vivo permeation experiment and fluorescent images of contralateral cornea and retina of rats treated with blank artificial tear fluid as control. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Key Laboratory of Smart Drug Delivery, Ministry of Education; Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China. Tel: +86 21 51980091. Fax: +86 21 51980090. E-mail: weigang@ shmu.edu.cn. Author Contributions ‡

Equally contributing authors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Institute of Applied Physics of Shanghai, for technical assistance of CD analysis; Shanghai Institute of Materia Medica, for kindly providing the Wong-Kilbourne-derived human conjunctival epithelial cells; Yuchen Zhang, PhD, University of Kansas Medical Center, for review of the article. This study was supported by funding from the National Natural Science Fund of China (Grant No. 81172994), National Science and Technology Major Project (2012ZX09304004), and Grants from Shanghai Science and Technology Committee (11431921200).

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ABBREVIATIONS USED CPPs, cell-penetrating peptides; NHC, Wong-Kilbourne-derived human conjunctival epithelial cells; SLN, solid lipid nanoparticles; R8, poly(arginine)8; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle medium; FAM, 5-carboxyfluorescein; S8, poly(serine)8-Lys-FAM; PBS, phosphate buffer solution; Fm, fluorescence intensity; Papp, apparent permeability coefficients; ΔH, hydration value; HE, hematoxylin−eosin; DAPI, 4′, 6diamidino-2-phenylindole; CD, circular dichroism; GCL, ganglion cell layer; IPL, inner plexiform layers; OPL, outer plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRS, photoreceptor segments; RPE, retinal pigment epithelium; Chor, choroid; PPII, polyproline type II structure

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Penetratin, a Potentially Powerful Absorption Enhancer for

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