TAT-surface modified acyclovir-loaded albumin nanoparticles as a novel ocular drug delivery system

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

Contents lists available at ScienceDirect

Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst

TAT-surface modified acyclovir-loaded albumin nanoparticles as a novel ocular drug delivery system

T

Panita Suwannoia, Mullika Chomnawangb, Amolnat Tunsirikongkonc, Angsuma Phongphisutthinanc, Christel C. Müller-Goymannd, Narong Sarisutaa,c,∗ a

Department of Manufacturing Pharmacy, Mahidol University, Bangkok, Thailand Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand c Division of Pharmaceutical Sciences, Faculty of Pharmacy, Thammasat University, Pathumthani, Thailand d Technische Universität Braunschweig, Institut für Pharmazeutische Technologie, Braunschweig 38106, Germany b

ARTICLE INFO

ABSTRACT

Keywords: Nanoparticles Acyclovir Bovine serum albumin Transcorneal permeability Ocular drug delivery TAT peptide

The aim of this study was to develop acyclovir (ACV)-loaded bovine serum albumin (BSA) nanoparticles (NPs) which were surface modified with transactivating transduction (TAT) peptide to improve the transcorneal drug delivery in treating viral related keratitis. The TAT-surface modifications were carried out by both conjugation (TAT-con) and coating (TAT-coat) techniques. Characterization of physicochemical properties and assessment of in vitro transcorneal permeation across human corneal epithelial (HCE-T) cell multilayers of prepared TATsurface modified ACV-BSA NPs were subsequently investigated. The prepared TAT-surface modified ACV-BSA NPs appeared to be spherical in shape and uniform in size of about 200 nm with surface charges ranging between −20 and −30 mV. Increasing TAT amount in TAT-coat ACV-BSA NPs resulted in an increased size of NPs as expected, but it was not the case for TAT-con ACV-BSA NPs. The prepared TAT-coat ACV-BSA NPs were shown to have less cytotoxic effects on HCE-T cells used in permeation studies than those of TAT-con ACV-BSA NPs and ACV solution. The in vitro transcorneal permeation results indicated that TAT-coat ACV-BSA NPs could bring about the highest ACV permeability as compared to ACV-BSA NPs and ACV solution. Such TAT-surface modified ACV-BSA NPs could be developed as novel ocular drug delivery systems.

1. Introduction The eye is an organ protected from exogenous substances and external stress by various barriers [1]. Ocular viral infections, for example herpes simplex virus (HSV) and cytomegalovirus (CMV), are opportunistic pathogens associated with significant morbidity and mortality in susceptible subjects such as immunocompromised patients [2–5]. Acyclovir (ACV), one of nucleoside analogs, has shown to be clinically effective against those viruses. The purine nucleoside analog acts selectively against viruses without causing substantial toxic effects on uninfected cells. However, due to its poor aqueous solubility and low lipophilicity, ACV exhibits low corneal permeability. As a result, ACV cannot be administered as a topical solution and is not very effective against ocular viral infections [4–6]. Several strategies have been used to improve ocular ACV bioavailability and therapeutic efficacy both by chemical [4] and pharmaceutical modifications, i.e. ointments [7], microspheres [8], nanospheres [9], liposomes [6,10], and nanoparticles [11]. Nanoparticles are solid colloidal particulate systems containing an

∗

active pharmaceutical ingredient (API). They have been designed as drug delivery systems based on numerous advantages including the controllability of particles size, surface charge, and morphology; the versatility to deliver a variety of API; the improvement of solubility and stability of entrapped drugs; and ability to be fabricated to obtain prolonged circulation time or even enhanced cellular uptake and targeting abilities [12]. In general, protein nanoparticles are very promising due to biocompatibility and biodegradability. Moreover, they can be prepared by using simple methods with mild conditions. According to their welldefined primary structure, protein-based nanoparticles could be surface-modified by various approaches including covalent attachment of API or ligand [12,13]. The presence of functional groups for example carboxylic and amino groups on albumin-based nanoparticles facilitates surface modification via several techniques such as conjugation, coating, or electrostatic adsorption. In the albumin-ligand combinations, the protein acts as a carrier for drug delivery while the ligand can be used for many reasons such as modifying the pharmacokinetics

Corresponding author. Department of Manufacturing Pharmacy, Mahidol University, Bangkok, Thailand. E-mail addresses: [email protected], [email protected] (N. Sarisuta).

https://doi.org/10.1016/j.jddst.2019.05.029 Received 14 December 2018; Received in revised form 21 April 2019; Accepted 14 May 2019 Available online 17 May 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

P. Suwannoi, et al.

parameters, enhancing the nanosystem stability, prolonging the circulation time, or targeting to site of action [12,14]. The modulation of the nanoparticle surface by appropriate material such as ligands or transporters which are relevant to create “molecular signature” could improve the cell interaction and determine the route of internalization pathway as well as the intracellular fate of nanoparticles [15]. A very effective class of ligand that can be conjugated onto the surface of nanoparticles in order to improve the membrane crossing efficiency and internalization into the cells includes cell penetrating peptides (CPPs). The HIV transactivator of transcription peptide (TAT) is the first sequence of CPPs, found in 1988, to be able to translocate through cell membranes and access into the cells [16,17]. TAT peptide has been employed to be part of drug delivery systems for quite some time such as TAT-modified liposomes [18–20], TAT-conjugated quantum dots [21], TAT-fusion proteins [17,22], lyophilisomes [23], and also nanoparticles [24]. Recently, TAT peptide has been widely studied based on its interesting characteristics in effectively transducing cells in various compartments of the eye. Therefore, TAT peptide is taken into account to be a potential molecular transporter when conjugated with any specific effector such as nanoparticles [17,22,25]. The aim of this study was to develop TAT-surface modified bovine serum albumin (BSA) nanoparticles (NPs) containing ACV as an ocular drug delivery system without using glutaraldehyde in the formulation in order to improve transcorneal drug delivery for viral infection treatment. Moreover, the physicochemical properties of the prepared TAT-surface modified ACV-loaded BSA nanoparticles as well as the in vitro permeation of ACV across human corneal cells was investigated.

Buckinghamshire, UK) and centrifugation (Biosan®, Riga, Latvia) at 1000 g for 30 min to remove excess ACV and BSA. The collected mixture was subsequently transferred to a round bottom flask, into which isotonic trehalose solution was added and ethanol was then removed by using a rotary evaporator (Eyela® N-1000 Series, Tokyo Rikakikai, Tokyo, Japan). The purified ACV-BSA NPs were finally adjusted to volume by SWI to make the final BSA concentration of 2.96 mg/mL, and kept at 4 °C in air-tight and light resistant containers until use. 3.2. TAT-surface modification of ACV-BSA NPs 3.2.1. TAT-conjugated ACV-BSA NPs (TAT-con ACV-BSA NPs) The conjugation of TAT peptide with ACV-BSA NPs was performed as described previously with some modifications [23]. 2.25 mL of prepared ACV-BSA NPs dispersion was firstly concentrated by using ultrafiltration unit (Vivaspin®, 100,000 MWCO, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and centrifugation (Biosan®, Riga, Latvia) at 1000 g for 30 min, after which it was adjusted to 1 mL with PBS to obtain 6.64 mg/mL BSA. These ACV-BSA NPs were subsequently activated by adding 0.382 mg of sulfo-GMBS, the heterobifunctional cross-linker, resulting in 10:1 molar ratio of sulfo-GMBS:BSA. The mixture was then incubated for 30 min at room temperature. The excess sulfo-GMBS was removed by using ultrafiltration unit (Vivaspin®, 30,000 MWCO, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and centrifugation (Scanspeed mini, Labogene®, Lynge, Denmark) at 1000 g for 30 min. The activated NPs dispersion was redispersed in PBS to 1 mL of the dispersion system and conjugated with 25 μL of 6.06 mg/mL TAT peptide aqueous solution to obtain TAT:BSA ratio by weight of 1:43. The mixture was allowed to incubate at room temperature for 30 min, after which it was purified by using ultrafiltration unit (Vivaspin®, 30,000 MWCO, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and centrifugation (Scanspeed mini, Labogene®, Lynge, Denmark) with SWI for four times at 1000 g for 30 min. The purified TAT-con ACV-BSA NPs were redispersed in isotonic trehalose solution to initial volume of 2.25 mL, and kept at 4 °C in air-tight and light resistant containers until use.

2. Materials 2.1. Chemicals Bovine serum albumin (BSA; fraction V) and fibronectin were purchased from Merck, Darmstadt, Germany. Acyclovir (ACV) micronized was obtained from Zhejiang Wuyi Pharmaceutical Factory, Jinhua, China. Cys-TAT (47–57) was received from Ana Spec, Fremont, CA, USA. Glacial acetic acid, ethanol, and acetonitrile were obtained from Labscan, Gliwice, Poland. Sodium hydroxide was from Carlo Erba, Valde-Reuil, France. Trehalose, dimethylsulfoxide (DMSO), N-γ-maleimidobutyryl-oxysulfosuccinimide ester (sulfo-GMBS), human recombinant insulin solution (5 mg/mL), and collagen from rat tail were from Sigma-Aldrich, St. Louis, MO, USA. Fetal bovine serum (FBS), Dulbecco's MEM/Ham's F-12 (1:1 mixture), and epidermal growth factor (EGF) were from Biochrom KG, Berlin, Germany. Trypsin-EDTA (0.05%) and antibiotics-antimycotic solution were from Gibco, Grand Island, NY, USA. EDTA was from MP Biomedicals, Solon, OH, USA. Bradford dye reagent was from Bio-Rad, CA, USA. All chemicals used were of analytical or reagent grade.

3.2.2. TAT-coated ACV-BSA NPs (TAT-coat ACV-BSA NPs) The coating of TAT peptide on ACV-BSA NPs was performed as described previously with some modifications [27]. Various quantities of prepared ACV-BSA NPs dispersion were mixed with 6.06 mg/mL TAT aqueous solution to obtain TAT:BSA ratios by weight of 1:80, 1:120, 1:160, and 1:320, and allowed to incubate at room temperature for 30 min. The TAT-coat ACV-BSA NPs were kept at 4 °C in air-tight and light resistant containers until use. 3.3. Physicochemical characterization

3. Methods

3.3.1. Microscopic appearance and morphology The morphology and surface structure of ACV-BSA NPs and TATsurface modified ACV-BSA NPs were examined employing field emission scanning electron microscope and energy dispersive X-Ray spectrometer (FESEM-EDS) (Model JSM-7610F, Jeol®, Tokyo, Japan) by injecting energetic electrons onto the sample specimen [26]. The samples were air-dried and covered with osmium tetroxide vapor approximately 1 h for border contrast, which were subsequently dehydrated with ethanol in series (30%, 50%, 70%, 90%, and 100%). The samples were dried using critical point drying and finally coated with a thin layer (approximately 20–30 nm) of gold. The magnification used was 40,000x.

3.1. Preparation of ACV-loaded BSA nanoparticles (ACV-BSA NPs) ACV-BSA NPs were prepared by desolvation method without using glutaraldehyde as previously described [26]. Briefly, BSA 100 mg and ACV 12.5 mg were dissolved together in 5 mL of sterile water for injection (SWI), and incubated at room temperature for 20 min, after which 30 mL of ethanol was submersibly pumped (Model 505S, Watson Marlow®, Wilmington, MA, USA) into the mixture through a 0.5-mm spinal needle at 15 mL/min with continuous stirring at 500 rpm. The mixture was then stirred for 10 min and subject to the hardening process by continuous stirring at 60 °C for 2 h (magnetic stirrer, C-MAG HS 7, IKA®, Staufen, Germany), and finally adjusted to volume with ethanol. The prepared ACV-BSA NPs were purified by using an ultrafiltration unit (Vivaspin®, 100,000 MWCO, GE Healthcare, Little Chalfont,

3.3.2. Particle size Particle size and polydispersity index (PdI) of ACV-BSA NPs and TAT-surface modified ACV-BSA NPs were determined by dynamic light scattering technique (DLS) (Zetasizer®, Nanoseries, Malvern, 625

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

P. Suwannoi, et al.

Worcestershire, UK) [26]. The samples were measured at 25 °C and a scattering angle of 173°. The technique is based on the light scattering of moving colloidal particles by Brownian motion in the surrounding liquid. The smaller particles move faster inversely in line with the decline of the autocorrelation function. The mean size of particles was calculated by using the Einstein equation. The measurements were done in triplicate.

3.4. Permeation studies across human corneal epithelial cell (HCE-T) multilayers 3.4.1. Cell culture Human corneal epithelial cell line (HCE-T) (Cell Bank, RIKEN BioResource Center, Lot No. 006, Ibaraki, Japan) was used as cell-based modeling of eye permeability epithelial barrier. The cell line is characterized as immortalized human corneal epithelial model in many terms of use, for example, passive transcellular and paracellular transport, transporter expression, and metabolic enzyme [28]. The cells were cultured in 25-cm2 tissue culture flask (Corning Costar®, Corning, NY, USA) at 37 °C in a humidified atmosphere containing 5% CO2 as described [26]. The culture medium consisted of DMEM/F12 (1:1 medium mixture of Dulbecco's modified eagle medium and Ham's F12 supplemented with 5% fetal bovine serum, 5 μg/mL insulin, 0.1 μg/mL epidermal growth factor, 1% antibiotic-antimycotic solution containing 10,000 units/mL of penicillin, 10,000 μg/mL of streptomycin, and 25 μg/mL of amphotericin B, and 0.5% DMSO as the growth medium), which was replaced three times per week. Having reached 90% confluence, the cell monolayers were incubated with EDTA solution for 3 min and then trypsinized with 0.05% trypsin in 0.02% EDTA solution at 37 °C for 7 min. The obtained cells were collected into centrifugation tubes, and subsequently spun 5 min at 1000 g. The pellets were resuspended in culture medium, and then continuously cultured in 25cm2 tissue culture flasks.

3.3.3. Zeta potential Zeta potentials of ACV-BSA NPs and TAT-surface modified ACV-BSA NPs were determined using the particle electrophoresis instrument (Zetasizer®, Nanoseries, Malvern, Worcestershire, UK) by measuring the direction and velocity of particle movement in the applied voltage of 150 V at 25 °C [26]. The mean zeta potential of particles was calculated by using the Helmholtz-Smoluchowsky equation. All measurements were performed in triplicate. 3.3.4. NPs recovery The percentage recovery of NPs was determined by using Bradford assay, which involves the binding of Coomassie Brilliant Blue G-250 dye to proteins. The amount of BSA in both NPs and filtrates collected from ultrafiltration unit (Vivaspin®, 100,000 MWCO, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and centrifugation (Biosan®, Riga, Latvia) at 1000 g for 30 min during purification process were examined. Firstly, 250 μL of Bradford dye reagent was added into each well of 96well plate followed by 5 μL of NPs dispersions or filtrates. Having incubated for 10 min at room temperature, the consequent increase in absorbance was measured at 595 nm by using a UV microplate reader (Tecan®, Infinite 200, Grödig, Austria). The NPs dispersion prior to purification process was diluted stepwise with isotonic trehalose solution to the given concentrations in order to create BSA calibration curve. Calibration curve with the coefficient of determination (r2) for linear regression of at least 0.999 was acceptable. The percentage yield was calculated as follows:

% Yiled =

Amount of albumin recovered × 100 Initial amount of albumin added

3.4.2. Cell viability assay Cytotoxic effects of ACV-BSA NPs and TAT-surface modified ACVBSA NPs on HCE-T cells were tested by using the MTT assay [26]. The assay of cell viability was based on the principle of mitochondrial conversion of a water-soluble tetrazolium salt [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; MTT] to the water-insoluble blue formazan product. HCE-T cells were seeded at a density of 1 × 105 per well in 24-well plate and cultivated over a period of 2 days prior to drug treatment. Culture medium was then removed and replaced with 500 μL ACV solution (0.02%), ACV-BSA NPs, or TAT-surface modified ACV-BSA NPs. Cells were then incubated for 3 h at 37 °C under 5% CO2. At the end of the treatment period, the supernatant was aspirated and the monolayer rinsed with PBS. Then 550 μL of medium containing 0.05% final concentration of MTT was added to each well, after which cells were incubated for 3 h at 37 °C. After MTT dye was removed, cells were rinsed with PBS and 1 mL of solubilization solution, which was composed of 0.36% w/w of 35% HCl, 0.27% w/w of sodium dodecyl sulfate, 8.82% w/w of distilled water, and 90.55% w/w of isopropanol, was then added to dissolve formazan crystals. Solubilization was allowed to complete upon mixing with an orbital shaker (Minishaker, BioSan®, PSU-2T, Riga, Latvia). Cell viability was assessed by measuring the absorbance at 570 nm using a microplate reader (Tecan®, Infinite 200, Grödig, Austria). The effect on cell viability of NPs was expressed as a percentage in comparison between treated cells and cells incubated with isotonic trehalose solution alone. The experiments were performed in triplicate.

(1)

3.3.5. Drug recovery The percentage of ACV recovery in BSA NPs was directly determined by dividing the amount of drug extracted from ACV-BSA NPs dispersed in isotonic trehalose solution by the initial amount of drug added into the system [26]. The extraction of ACV was done by treating the formulation with 1.0 N NaOH at 1:1 ratio by volume. After continuous stirring for 30 min with a magnetic stirrer, the mixture was then transferred to a centrifuge tube and centrifuged (Beckman®, Optima Max-XP, Indianapolis, USA) at 100,000 g for 30 min. The supernatant was then collected and the amount of drug was determined by HPLC method as previously described [26]. Duplicate measurements, at least, were performed for all samples. The percentage of ACV recovery and loading capacity were calculated as follows:

% Recovery =

Amount of drug recovered × 100 Initial amount of drug added

% Loading capacity =

Amount of drug recovered × 100 Amount of albumin recovered

3.4.3. Permeation studies The cultivation of the corneal epithelial model was performed on permeable, 3.0-μm pore size, polycarbonate filter inserts with a 12-mm diameter Transwell® (Corning Costar®, Corning, NY, USA) as previously described [26]. Prior to cultivation, 100 μL of an ethanolic solution of type I collagen, acid extracted from rat tail (1.5 mg/mL) and acidified with acetic acid, was cast onto the Transwell® plate. After evaporation of the solvent, 400 μL of an aqueous solution of fibronectin (10 μg/mL) was poured onto the collagen-coated filter and left for at least 30 min. The fibronectin solution was subsequently removed and the HCE-T cells suspended in the culture medium at a density of 1 × 105 per well were added. Upon obtaining confluence of the HCE-T cells, the monolayer was lifted to the air-liquid interface (ALI) by using a metal plate placed

(2) (3)

3.3.6. Determination of residual ethanol The residual amounts of ethanol in ACV-BSA NPs and TAT-surface modified ACV-BSA NPs were determined by using an ethanol assay kit (Amplite™, AAT Bioquest, Sunnyvale, CA, USA). The assay is based on the oxidation of ethanol by alcohol oxidase. The increase in absorbance was measured at 570 nm by using a UV microplate reader (Power Wave XS, BioTek®,Winooski, VT, USA). 626

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

P. Suwannoi, et al.

between insert and Transwell® in order to induce the cell differentiation and multilayered growth of the epithelium [26]. The integrity of the multilayered cells was evaluated by measuring transepithelial electrical resistance (TEER) value with an epithelial volt/ohmmeter (Millicell®ERS, Millipore, MA, USA). The cell inserts were used experimentally when the resistance reached values above 400 Ω cm2 [29]. The permeation experiments were performed at 37 °C directly in the Transwell® plate as mentioned [26]. Before applying the donor solutions, multilayered cells were rinsed and incubated with tempered Krebs-Ringer buffer (KRB) which was used as transport medium for 30 min at 37 °C, and then TEER value was measured. The transport medium was then removed from both sides of the multilayered cells and replaced by 400 μL of ACV solution, ACV-BSA NPs, or TAT-surface modified ACV-BSA NPs in the apical side and 2 mL of transport medium in the basolateral side. Aliquot 300-μL portions of transport medium were collected from acceptor compartment at various time intervals of 15, 30, 45, 60, 120 and 180 min after adding the donor solution, and analyzed by HPLC. The acceptor solution was replaced by the same volume of transport medium which had been equilibrated at the same temperature. During the experiment, the donor and acceptor solutions were agitated continuously with an orbital shaker (Sci Finetech, Seoul, Korea). Triplicate experiments were performed for all samples. The permeation coefficients (Papp [cm/s]) of ACV from each samples were determined by plotting the amount of the ACV permeated through the cultivated cells with respect to time. By the means of the steadystate flux (J [μg/(cm2 s)]) estimated from the linear ascent of the curve.

J=

dQ dt x A

(4)

The permeation coefficient, Papp, was calculated as:

Papp =

J C0

(5)

Where Q is the amount of ACV permeated through the multilayered cells, A is the area exposed and t is the exposure time. C0 is the initial concentration of the particular donor ACV at t = 0. The percentage of cumulative amount ACV permeated after experiments was calculated as:

%Cumulative =

Total amount of ACV in acceptor × 100 C0

(6)

3.5. Drug analysis The contents of ACV from permeation studies were analyzed by HPLC system equipped with a high-precision pump (LC-20AD, Shimadzu), UV–vis detector (SPD-20A, Shimadzu), and system controller (LC-20AD, Shimadzu). A C18 reverse-phase column (Mightysil RP-18 Aqua 250–4.6 (5 μm), Kanto Kagaku Co., Tokyo, Japan) was used at ambient temperature. The mobile phase consisted of 95% of 0.1% glacial acetic acid in water and 5% of acetonitrile, which was eluted at a flow rate of 1.0 mL/min. The sample injection volume was 40 μL and detection wavelength was 254 nm. The retention time was about 2.5 min [26]. Calibration curve with the coefficient of determination (r2) for linear regression of at least 0.999 was acceptable. The limit of detection (LOD) and limit of quantitation (LOQ) of the assay were determined based on the standard deviation of the response and the slope of the structured calibration curve. The LOD and LOQ were found to be 0.02 and 0.05 μg/mL, respectively.

4. Results and discussion

3.6. Data analysis

4.1. Results

Results were reported as mean ± standard deviation and analyzed using ANOVA test for variability followed by Tukey's test or Dunnett's test when significant differences were found. The p-value of less than 0.05 (p < 0.05) was considered to be statistically significant.

4.1.1. Physicochemical characterizations 4.1.1.1. Microscopic appearance and morphology. The scanning electron photomicrographs (SEM) of ACV-BSA NPs, TAT-con ACV-BSA NPs, and TAT-coat ACV-BSA NPs at TAT to BSA ratio by weight of 1:120

Fig. 1. Scanning electron photomicrographs (SEM) of ACV-BSA NPs (top), TATcon ACV-BSA NPs (middle), and TAT-coat ACV-BSA NPs at TAT to BSA ratio by weight of 1:120 (bottom). (40,000x magnification).

627

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

(0.09) (0.38) (0.16) (0.22)

appeared to be spherical in shape and nearly uniform in size (Fig. 1). The size of these NPs indicated by SEM was about 200 nm, which was subsequently confirmed by particle analyzer.

1.99 2.05 2.33 2.17

4.1.1.2. Particles size. The mean size and size distribution (PdI) of all formulations of ACV-BSA NPs were ranging from 173.0 ± 9.5 to 204.7 ± 15.5 nm, and 0.079 ± 0.023 to 0.226 ± 0.025, respectively (Table 1). It was observed that both size and size distribution of TAT-con ACV-BSA NPs and TAT-coat ACV-BSA NPs were increased significantly after TAT-surface modifications. Particularly, those of TAT-coat ACV-BSA NPs were found to increase with increasing TAT concentrations. All of those NPs were shown to be monodisperse system.

6.87 (0.96) 8.00 (2.27) 11.06 (0.79) 6.83 (0.72)

4.1.1.3. Zeta potential. The zeta potentials of all formulations of ACVBSA NPs were in the range of −22.8 ± 1.7 to −32.4 ± 0.7 mV (Table 1). The negative surface charges of TAT-coat ACV-BSA NPs were significantly lowered while that of TAT-con ACV-BSA NPs was not significantly different as compared to ACV-BSA NPs.

(0.29) (0.30)b (0.81)b (0.72)b

6.76 7.15 7.64 7.65

(0.04) (0.04)b (0.10)b (0.09)b

4.1.1.4. NPs and drug recovery. The average percentages recovery of all formulations of ACV-BSA NPs was found to be 99.21 ± 7.51% (n = 9). The values of drug recovery and loading capacity of all formulations of ACV-BSA NPs except TAT-con ACV-BSA NPs were found to be in the range of 51.58 ± 3.92% to 60.78 ± 0.72%, and 6.49 ± 0.49% to 7.65 ± 0.09%, respectively (Table 1). The residual amounts of ethanol in all formulations of ACV-BSA NPs were found to be less than 0.05%.

(1.09) (1.12) (3.03) (2.66)

53.68 56.82 60.68 60.78

4.1.2. Permeation studies across HCE-T cells multilayers 4.1.2.1. Cytotoxicity. The percentages of cell viability by MTT assay of HCE-T cells after being treated with ACV-BSA NPs and TAT-surface modified ACV-BSA NPs for 3 h were found to be in the range of 100.90 ± 5.92% to 143.84 ± 4.65% (Fig. 2). Meanwhile, those found for cells being treated with 0.02% ACV solution and isotonic trehalose solution were 109.67 ± 4.84% and 100.00 ± 10.56%, respectively. From the results, all formulations had no cytotoxicity effect on HCE-T cells and were considered to be safe for being tested in permeation studies. It was not surprised that high cell viability, above 100%, was found in those cells treated with all of ACV-BSA NPs and also 0.02% of ACV in isotonic trehalose solution, which was in accordance with the

Fig. 2. Cytotoxic effect pattern of HCE-T cells after being treated with ACV-BSA NPs, TAT-con ACV-BSA NPs, and TAT-coat ACV-BSA NPs at various ratios by weight of TAT to BSA (average from 3 determinations).

b

a

Average from 3 determinations. Significantly different from ACV-BSA NPs (p < 0.05, ANOVA, Dunnett).

199.73 211.41 225.79 226.15 (0.009) (0.016)b (0.016)b (0.016)b 0.100 0.108 0.145 0.165

ACV solution (0.02%) ACV-BSA NPs TAT-con ACV-BSA NPs (TAT:BSA = 1:43) TAT-coat ACV-BSA NPs (TAT:BSA = 1:320) (TAT:BSA = 1:160) (TAT:BSA = 1:120) (TAT:BSA = 1:80)

(9.8) (10.5) (7.9)b (15.5)b 177.3 182.8 190.8 204.7

−26.5 −26.3 −22.9 −22.8

b

0.226 (0.025)b 188.5 (7.4)b

−32.4 (0.7)

– 0.079 (0.023) – 173.0 (9.5)

– −31.1 (2.3)

(2.0) (2.0)b (1.9)b (1.7)b

– 0.07 (0.03)b 2.14 (0.76)

0.58 (0.20)b

–

2.77 (0.50) 2.39 (0.46) – 6.49 (0.49) 232.00 191.94 (14.58)

– 51.58 (3.92)

9.01 (3.45) 10.99 (1.33)

Papp ( × 10−6 cm/s) % Cumulative amount permeated after 3 h % Loading capacity % Recovery Particle size (nm)

PdI

Zeta potential (mV)

ACV recovery (μg/mL)

Mean (SD)a Formulation

Table 1 Particle size, polydispersity index (PdI), zeta potential, entrapment efficiency, loading capacity, % cumulative amount permeated after 3 h, and apparent permeability coefficient (Papp) of ACV-BSA NPs, TAT-con ACV-BSA NPs, and TAT-coat ACV-BSA NPs.

P. Suwannoi, et al.

628

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

P. Suwannoi, et al.

nanocarriers by using cell penetrating peptides (CPPs), a very interesting ligand which could improve the membrane crossing efficiency and internalization into the cells, can be achieved by both chemical and physical techniques [23,24,27,30,31]. The chemical surface modification of drug nanocarriers with ligands is based on chemical reaction between functional groups on their surfaces. However, if the functional groups on the drug nanocarriers or ligands are not promptly reactive for the chemical reaction, a heterobifunctional linker such as sulfo-GMBS [23], MBS [31], and NHS-PEG5000-maleimide [24] can be used to achieve the chemical surface modification. In this study, sulfo-GMBS was selected and used to activate the surface of BSA NPs. The succinimidyl ester functionality was conjugated to primary amine groups on the BSA NPs. After that, the maleimide functionality was used for conjugation to the free thiol of the cysteine residue coupled to TAT peptide as described above [23]. On the other hand, physical surface modification is based on electrostatic or ionic interactions between the NPs and the ligands [27,30]. The positive charge of the basic domain of TAT peptide could bring about the interaction with the negative charge of amino acid residues on those BSA NPs. After surface modifications of those prepared BSA NPs with TAT peptide, their physicochemical properties became altered as demonstrated in Table 1. Increasing TAT amount in TAT-coat ACV-BSA NPs resulted in an increased size of the NPs as expected, which were in accordance with previous reports [27,30]. It seemed however not to be the case for TAT-con ACV-BSA NPs. This might be due to the final amount of TAT on the NPs surface by conjugation which was lower than that by coating. The drug loss might occur following the purification steps in conjugation process, which were needed to get rid of salts from PBS used as the recommended conjugation medium [23]. Salt solutions are avoided being used as the ACV-BSA NPs medium due to ionic strength effect, leading to aggregation of NPs [26,32]. The surface charges of the TAT-surface modified ACV-BSA NPs significantly shifted to be less negative except the TAT-con ACV-BSA NPs. This might be due to the amount of TAT on the surface of NPs, which was analogous to the effect of TAT on particle size. This shift could be attributed to the interactions between the negatively charged amino acid residues of NPs and the positively charged amino acid residues of TAT peptide, leading to lower negative charge of NPs. Nevertheless, all of these TAT-surface modified NPs still exhibited a net negative surface charge possibly because of the exposed amino acid residues of NPs in neutral conditions of isotonic trehalose solution medium and also the unbound negative charge of TAT peptide [26,27]. Such typical change could be attributed to interaction between TAT and surface of NPs which was conformed to certain type of adsorption isotherm as described by the relationship between TAT concentration and both size and surface charge of NPs [33,34]. According to the abundance of drug binding sites present in the albumin molecule, it is prone to bind with varieties of drug to be loaded to BSA NPs. The mechanism of drug association to BSA NPs may be electrostatic adsorption of both positively and/or negatively charged molecules, depending on the content of charged amino acid residues in the albumin primary structure. Another proposed mechanism involved the covalent linkage between the drug molecule and the BSA matrix in BSA NPs. However, it should be noted that the most probable mechanism could be influenced by many factors such as the primary albumin structure, the drug properties, and also the production method as well as conditions [14,35]. By optimizing the production conditions of NPs, for example, drug loading incubation time, order of drug loading, or even drug to albumin ratio, the percentages of drug entrapment efficiency and loading capacity could be improved. The cytotoxicity test of the prepared ACV-BSA NPs was performed at 3 h to cover the permeation study period. The amounts of surviving cells after incubation with ACV in isotonic trehalose solution, ACV-BSA NPs, or TAT-surface modified ACV-BSA NPs were calculated by MTT assay. The results showed that cells treated with all of samples except ACV solution and TAT-con ACV-BSA NPs significantly increased in the

Fig. 3. Permeation profiles across HCE-T cell multilayers of TAT-coat ACV-BSA NPs at various ratios by weight of TAT to BSA.

previous study [26]. 4.1.2.2. Permeation studies. The permeation profiles across HCE-T cells multilayers of TAT-coat ACV-BSA NPs in Fig. 3 reveal that the formulation of TAT to ACV ratio by weight of 1:120 exhibited the highest one. Moreover, it can be seen in Fig. 4 that the formulation of TAT-coat ACV-BSA NPs could bring about the highest permeation profile, followed by those of ACV-BSA NPs and ACV solution, respectively. In contrast, the permeation profile of TAT-con ACV-BSA NPs was found to be far below those from other formulations because of the lower amount of drug recovery due to loss during conjugation process (Table 1). The cumulative percentages permeated after 3 h and apparent permeability coefficient (Papp) of ACV solution were found to be 9.01 ± 3.45% and 2.77 ± 0.50 × 10−6 cm/s, while those of all ACV-BSA NPs formulations except TAT-con ACV-BSA NPs were found to be in the range of 6.83 ± 0.72% to 11.06 ± 0.79% and 1.99 ± 0.09 × 10−6 cm/s to 2.39 ± 0.46 × 10−6 cm/s, respectively (Table 1). Since the permeated amount of ACV from TAT-con ACV-BSA NPs was drastically far below those from other formulations because of extremely low drug recovery, neither percentage of cumulative amount permeated after 3 h nor apparent permeability coefficient (Papp) were calculated. 4.2. Discussion The ACV-BSA NPs with the size of about 200 nm and monodisperse system could be produced by desolvation method without using glutaraldehyde in the formulation. The surface modification of drug

Fig. 4. Permeation profiles across HCE-T cell multilayers of ACV-BSA NPs, TATcon ACV-BSA NPs at TAT to BSA ratio by weight of 1:43, and TAT-coat ACVBSA NPs at TAT to BSA ratio by weight of 1:120 compared to ACV solution at the same concentration. 629

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

P. Suwannoi, et al.

measured amount of surviving cells compared to trehalose solution (p < 0.05, ANOVA, Dunnett). This might be explained by the fact that the MTT assay is based on measuring the mitochondrial enzyme succinyl dehydrogenase's activity which reflects the metabolic activity of the living cells. In particular, those cells after being treated with nutrient trehalose or even albumin might have an increasing metabolic activity as described elsewhere [26]. Comparing cell viabilities among cells treated with ACV-BSA NPs and TAT-surface modified ACV-BSA NPs, a slight cytotoxicity effect was found with TAT-con ACV-BSA NPs. Such cytotoxic effect might plausibly be caused by the heterobifunctional linker, sulfo-GMBS, having been used in the conjugation process. Although TAT-con ACV-BSA NPs exhibited a slight cytotoxicity effect on HCE-T cells compared to other formulations, they were considerably safe for being tested in permeation studies. The influence of BSA NPs and TAT-surface modified BSA NPs on the ACV permeability across a cell-based model of corneal barrier was investigated. Generally, the transcellular permeability of drug nanocarriers could be improved in association with their own particle sizes [36]. In particular, the nanoparticles of 100–200 nm size acquired the best properties for cellular uptake [31]. The enhanced transcorneal permeation of drug was attributed to their nanosize properties. Since TAT peptide has been widely reported in effectively transducing cells in various compartments of the eye [17,22,25], the permeation profile of TAT-coat ACV-BSA NPs in this study was shown to be considerably higher than that of ACV solution at the same concentration (Fig. 4). ACV itself is prone to permeate across the cornea by passive diffusion, while NPs were suggested to possibly permeate by endocytosis mechanism [37–41]. The permeation results showed that the TAT-coat ACV-BSA NPs at TAT to BSA ratio by weight of 1:120 appeared to be a good candidate for delivery system of ACV to the ocular tissue. This might be explained by the interaction between TAT peptide and negatively charged sulfated glycans at the cell surface such as heparan sulfate as described elsewhere [23,42,43]. Heparan sulfate was found in ocular tissue and also in corneal epithelial cells [44,45]. However, the permeation of ACV from TAT-con was much lower than those from other formulations due to low amount of entrapped ACV on NPs.

References [1] N. Kuno, S. Fujii, Recent advances in ocular drug delivery systems, Polymers 3 (1) (2011) 193–221. [2] K.K. Biron, Antiviral drugs for cytomegalovirus diseases, Antivir. Res. 71 (2–3) (2006) 154–163. [3] N.H. Barker, Ocular herpes simplex, BMJ Clin. Evid. 2008 (2008) 0707. [4] S. Katragadda, S. Gunda, S. Hariharan, A.K. Mitra, Ocular pharmacokinetics of acyclovir amino acid ester prodrugs in the anterior chamber: evaluation of their utility in treating ocular HSV infections, Int. J. Pharm. 359 (1–2) (2008) 15–24. [5] L. Zhu, H. Zhu, Ocular herpes: the pathophysiology, management and treatment of herpetic eye diseases, Virol. Sin. 29 (6) (2014) 327–342. [6] S.L. Law, K.J. Huang, C.H. Chiang, Acyclovir-containing liposomes for potential ocular delivery: corneal penetration and absorption, J. Control. Release 63 (1–2) (2000) 135–140. [7] M. Al-Ghabeish, X. Xu, Y.S.R. Krishnaiah, Z. Rahman, Y. Yang, M.A. Khan, Influence of drug loading and type of ointment base on the in vitro performance of acyclovir ophthalmic ointment, Int. J. Pharm. 495 (2) (2015) 783–791. [8] I. Genta, B. Conti, P. Perugini, F. Pavanetto, A. Spadaro, G. Puglisi, Bioadhesive microspheres for ophthalmic administration of acyclovir, J. Pharm. Pharmacol. 49 (8) (1997) 737–742. [9] C. Giannavola, C. Bucolo, A. Maltese, D. Paolino, M.A. Vandelli, G. Puglisi, et al., Influence of preparation conditions on acyclovir-loaded poly-d,l-lactic acid nanospheres and effect of PEG coating on ocular drug bioavailability, Pharm. Res. (N. Y.) 20 (4) (2003) 584–590. [10] P. Chetoni, S. Rossi, S. Burgalassi, D. Monti, S. Mariotti, M.F. Saettone, Comparison of liposome-encapsulated acyclovir with acyclovir ointment: ocular pharmacokinetics in rabbits, J. Ocul. Pharmacol. Ther. 20 (2) (2004) 169–177. [11] P. Noomwong, W. Ratanasak, A. Polnok, N. Sarisuta, Development of acyclovirloaded bovine serum albumin nanoparticles for ocular drug delivery, Int. J. Drug Deliv. 3 (4) (2011) 669–675. [12] W. Lohcharoenkal, L.Y. Wang, Y.C. Chen, Y. Rojanasakul, Protein nanoparticles as drug delivery carriers for cancer therapy, BioMed Res. Int. 2014 (2014) 180549. [13] C. Weber, C. Coester, J. Kreuter, K. Langer, Desolvation process and surface characterisation of protein nanoparticles, Int. J. Pharm. 194 (1) (2000) 91–102. [14] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Albumin-based nanoparticles as potential controlled release drug delivery systems, J. Control. Release 157 (2) (2012) 168–182. [15] Y. Diebold, M. Calonge, Applications of nanoparticles in ophthalmology, Prog. Retin. Eye Res. 29 (6) (2010) 596–609. [16] S.B. Fonseca, M.P. Pereira, S.O. Kelley, Recent advances in the use of cell-penetrating peptides for medical and biological applications, Adv. Drug Deliv. Rev. 61 (11) (2009) 953–964. [17] X. Guo, A.E.K. Hutcheon, J.D. Zieske, Transduction of functionally active TAT fusion proteins into cornea, Exp. Eye Res. 78 (5) (2004) 997–1005. [18] V.P. Torchilin, T.S. Levchenko, R. Rammohan, N. Volodina, B. PapahadjopoulosSternberg, G.G. D'Souza, Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes, Proc. Natl. Acad. Sci. U. S. A. 100 (4) (2003) 1972–1977. [19] M.M. Fretz, G.A. Koning, E. Mastrobattista, W. Jiskoot, G. Storm, OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis, Biochim. Biophys. Acta 1665 (1–2) (2004) 48–56. [20] V.P. Torchilin, R. Rammohan, V. Weissig, T.S. Levchenko, TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors, Proc. Natl. Acad. Sci. U. S. A. 98 (15) (2001) 8786–8791. [21] S. Santra, H. Yang, J.T. Stanley, P.H. Holloway, B.M. Moudgil, G. Walter, et al., Rapid and effective labeling of brain tissue using TAT-conjugated CdS:Mn/ZnS quantum dots, Chem. Commun. (25) (2005) 3144–3146. [22] Y. Wang, H. Lin, S. Lin, J. Qu, J. Xiao, Y. Huang, et al., Cell-penetrating peptide TAT-mediated delivery of acidic FGF to retina and protection against ischemia–reperfusion injury in rats, J. Cell Mol. Med. 14 (7) (2010) 1998–2005. [23] E. van Bracht, L.R.M. Versteegden, S. Stolle, W.P.R. Verdurmen, R. Woestenenk, R. Raave, et al., Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells, PLoS One 9 (11) (2014) e110813. [24] J. Look, N. Wilhelm, H. von Briesen, N. Noske, C. Günther, K. Langer, et al., Ligandmodified human serum albumin nanoparticles for enhanced gene delivery, Mol. Pharm. 12 (9) (2015) 3202–3213. [25] D.F. Schorderet, Vd Manzi, K. Canola, C. Bonny, Y. Arsenijevic, F.L. Munier, et al., D-TAT transporter as an ocular peptide delivery system, Clin. Exp. Ophthalmol. 33 (6) (2005) 628–635. [26] P. Suwannoi, M. Chomnawang, N. Sarisuta, S. Reichl, C.C. Müller-Goymann, Development of acyclovir-loaded albumin nanoparticles and improvement of acyclovir permeation across human corneal epithelial T cells, J. Ocul. Pharmacol. Ther. 33 (10) (2017) 743–752. [27] J. Patel, D. Galey, J. Jones, P. Ray, J.G. Woodward, A. Nath, et al., HIV-1 Tat-coated nanoparticles result in enhanced humoral immune responses and neutralizing antibodies compared to alum adjuvant, Vaccine 24 (17) (2006) 3564–3573. [28] I. Pepić, J. Lovrić, B. Cetina-Čižmek, S. Reichl, J. Filipović-Grčić, Toward the practical implementation of eye-related bioavailability prediction models, Drug Discov. Today 19 (1) (2014) 31–44. [29] E. Toropainen, V.P. Ranta, K.S. Vellonen, J. Palmgrén, A. Talvitie, M. Laavola, et al., Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model, Eur. J. Pharm. Sci. 20 (1) (2003) 99–106.

5. Conclusion The ACV-BSA NPs and TAT-surface modified ACV-BSA NPs could be prepared by desolvation method without using glutaraldehyde with the size of about 200 nm and surface charge of approximately −30 mV. The prepared ACV-BSA NPs and TAT-surface modified ACV-BSA NPs were shown to have no cytotoxicity effect on HCE-T cells used in permeation studies. The in vitro transcorneal permeation results indicated that TAT-coat ACV-BSA NPs could bring about the highest ACV permeation profile, followed by those of ACV-BSA NPs and ACV solution, respectively. Such TAT-surface modified ACV-BSA NPs could be developed as novel ocular drug delivery systems. Author disclosure statement No competing financial interests exist. Acknowledgments The financial support from the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/ 0357/2551) is gratefully acknowledged. The authors also wish to thank Dr. Kaoru Araki-Sasaki, JCHO Hoshigaoka Medical Center, Hirakata city, Japan for the kind donation of HCE-T cells, as well as Dr. Stephan Reichl, Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Braunschweig, Germany for support with the cell culture experiments. 630

Journal of Drug Delivery Science and Technology 52 (2019) 624–631

P. Suwannoi, et al. [30] Z. Cui, J. Patel, M. Tuzova, P. Ray, R. Phillips, J.G. Woodward, et al., Strong T cell type-1 immune responses to HIV-1 Tat (1-72) protein-coated nanoparticles, Vaccine 22 (20) (2004) 2631–2640. [31] S. Ji, J. Xu, B. Zhang, W. Yao, W. Xu, W. Wu, et al., RGD-conjugated albumin nanoparticles as a novel delivery vehicle in pancreatic cancer therapy, Cancer Biol. Ther. 13 (4) (2012) 206–215. [32] M. Kaasalainen, E. Mäkilä, J. Riikonen, M. Kovalainen, K. Järvinen, K.H. Herzig, et al., Effect of isotonic solutions and peptide adsorption on zeta potential of porous silicon nanoparticle drug delivery formulations, Int. J. Pharm. 431 (1–2) (2012) 230–236. [33] M. Dadparvar, S. Wagner, S. Wien, J. Kufleitner, F. Worek, H. von Briesen, et al., HI 6 human serum albumin nanoparticles–development and transport over an in vitro blood-brain barrier model, Toxicol. Lett. 206 (1) (2011) 60–66. [34] S.H. Brewer, W.R. Glomm, M.C. Johnson, M.K. Knag, S. Franzen, Probing BSA binding to citrate-coated gold nanoparticles and surfaces, Langmuir 21 (20) (2005) 9303–9307. [35] M. Merodio, A. Arnedo, M.J. Renedo, J.M. Irache, Ganciclovir-loaded albumin nanoparticles: characterization and in vitro release properties, Eur. J. Pharm. Sci. 12 (3) (2001) 251–259. [36] A. Hafner, J. Lovrić, M.D. Romić, M. Juretić, I. Pepić, B. Cetina-Čižmek, et al., Evaluation of cationic nanosystems with melatonin using an eye-related bioavailability prediction model, Eur. J. Pharm. Sci. 75 (2015) 142–150. [37] P. Calvo, J.L. Vila-Jato, M.J. Alonso, Comparative in vitro evaluation of several colloidal systems, nanoparticles, nanocapsules, and nanoemulsions, as ocular drug

carriers, J. Pharm. Sci. 85 (5) (1996) 530–536. [38] R.T. Addo, K.G. Yeboah, R.C. Siwale, A. Siddig, A. Jones, R.V. Ubale, et al., Formulation and characterization of atropine sulfate in albumin-chitosan microparticles for in vivo ocular drug delivery, J. Pharm. Sci. 104 (5) (2015) 1677–1690. [39] P. Calvo, C. Thomas, M.J. Alonso, J. Vila-Jato, J.R. Robinson, Study of the mechanism of interaction of poly(ε-caprolactone) nanocapsules with the cornea by confocal laser scanning microscopy, Int. J. Pharm. 103 (3) (1994) 283–291. [40] P. Calvo, M.J. Alonso, J.L. Vila-Jato, J.R. Robinson, Improved ocular bioavailability of indomethacin by novel ocular drug carriers, J. Pharm. Pharmacol. 48 (11) (1996) 1147–1152. [41] W. Schubert, P.G. Frank, B. Razani, D.S. Park, C.W. Chow, M.P. Lisanti, Caveolaedeficient endothelial cells show defects in the uptake and transport of albumin in vivo, J. Biol. Chem. 276 (52) (2001) 48619–48622. [42] J.M. Gump, S.F. Dowdy, TAT transduction: the molecular mechanism and therapeutic prospects, Trends Mol. Med. 13 (10) (2007) 443–448. [43] M. Tyagi, M. Rusnati, M. Presta, M. Giacca, Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans, J. Biol. Chem. 276 (5) (2001) 3254–3261. [44] V.J. Coulson-Thomas, S.H. Chang, L.K. Yeh, Y.M. Coulson-Thomas, Y. Yamaguchi, J. Esko, et al., Loss of corneal epithelial heparan sulfate leads to corneal degeneration and impaired wound healing, Investig. Ophthalmol. Vis. Sci. 56 (5) (2015) 3004–3014. [45] P.J. Park, D. Shukla, Role of heparan sulfate in ocular diseases, Exp. Eye Res. 110 (2013) 1–9.

631