Assessing the Potential for DrugâNanoparticle Surface Interactions To

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Assessing the potential for drug-nanoparticle surface interactions to improve drug penetration into the skin X. J. Cai, A. Woods, P. Mesquida, and S. A. Jones Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00032 • Publication Date (Web): 04 Mar 2016 Downloaded from http://pubs.acs.org on March 11, 2016

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Molecular Pharmaceutics

Assessing the potential for drug-nanoparticle surface interactions to improve drug penetration into the skin X. J. Cai, A. Woods, P. Mesquida, S.A. Jones*

King’s College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH

*Corresponding author: Dr. S. A. Jones. King’s College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH. Tel: +44 (0)207 848 4843. Fax: +44 (0)207 848 4800. E-mail: [email protected]

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Table of contents/ Abstract graphic


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Abstract

There is continued debate as to how nanomaterials enhance the passive diffusion of drugs through the skin. This study examined if drug-nanoparticle surface interactions, which occurred during topical application, had the capability to enhance percutaneous penetration. Atomic force microscopy force adhesion measurements were used to demonstrate that a model drug, tetracaine, strongly adsorbed to the surface of a negatively charged carboxyl-modified polystyrene nanoparticle (Nano ) through both its methyl and amine functionalities (up to a 6 and 16 fold greater adhesion force respectively compared the CH3 – CH3 control). These drug-particle adhesion forces were significantly reduced (p < 0.05) to values that were lower than the CH3 – CH3 control measurements when tetracaine interacted with a silica nanoparticle (Nano ). This reduction in adhesion was attributed to the lower surface charge of the Nano (ca. – 23 mV) compared to the Nano (ca. -40 mV), which diminished the electrostatic interactions between +ve amine of tetracaine and the –ve particle. Mixing Nano with tetracaine on the skin retarded percutaneous drug penetration compared to the control (tetracaine saturated solution without nanoparticles), but the Nano , which still adsorbed the tetracaine, produced a 3.6-fold enhancement in percutaneous penetration compared to the same control. These data demonstrated the capability of moderate nanoparticle surface interactions that occurred within the application vehicle to promote drug percutaneous penetration.

Keywords: nanoparticles; tetracaine; skin; topical; drug delivery; AFM.


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Introduction

A number of different nanomaterials have been used in products that are applied topically to the skin

1, 2

. This has generated a large body of literature that has

suggested that nanomaterials can be used to optimise the colour and odour of a topical product

3, 4

, control the release of chemicals from the product

5, 6

and provide

protection for important components in the product through particle encapsulation 7, 8. There are also a number of published studies that have demonstrated that using nanomaterials in topical products can enhance the percutaneous penetration of therapeutic agents

9-11

. However, it has proven problematic to gain a clear

understanding of the mechanism by which this occurs 12.

It is possible that small flexible nanomaterials that are produced from lipids could enhance drug penetration into the skin by carrying a drug payload directly into the barrier

9-11

. However, it is unlikely that such a mechanism of action applies to solid

nanomaterials with a size of greater than 10 nm, i.e., the nanomaterials most regularly employed in drug delivery studies, because they are too large to pass into the skin at a rate that would aid the delivery of a drug into the local tissue

13-15

. Alternative

mechanisms of action for the penetration enhancement properties of nanoparticles include: barrier disruption

12, 16

, drug supersaturation

17, 18

provision of a drug reservoir to prevent drug depletion

, skin occlusion 7, 19 and the

20, 21

. However, each of these

modes of action have only found support from one or two previously published studies, which suggests that there is still further work to be done to understand the way in which nanoparticles act to enhance drug penetration when used in topical formulations applied to the skin.


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One important element to consider when nanoparticles are combined with a drug in a topical formulation is the surface interactions of the particulate with the drug. This is especially important if the therapeutic agent applied to the skin is surface active. For amphiphilic drugs, the drug-nanoparticle surface interactions are probably more important than the other potential penetration enhancing effects aforementioned because the drug nanoparticle surface interactions may alter the drug-drug interaction equilibrium 22-25. When the drug-drug interactions are strong in topical pharmaceutical preparations, aggregate structures can be formed 26. These aggregates exhibit different physiochemical properties in solution compared to the non-aggregated form of the molecule and this can influence the permeation of the drug through a membrane 27-30. Ueda et al. (2011) showed the interactions between nanomaterials and drug aggregates enhanced drug penetration into a model membrane. However, the link between drug aggregation, nanomaterial surface interactions and percutaneous penetration has not been investigated in terms of drug delivery to the skin.

The aim of this study was to examine if the interactions that occurred between a nanoparticle surface and a drug when they were co-administered to the skin conferred the capability to enhance percutaneous penetration. This represented a very different approach to try and understand the capacity of nanomaterials enhance penetration into the skin because it did not attempt to encapsulate the drug or adsorb it on to the surface prior to administration. The study used tetracaine as the model drug to measure percutaneous penetration and drug-nanoparticle adsorption in an aqueous vehicle set at pH 4 and pH 8. Two different pH vehicles were used in order to assess the effects of changing the drug ionisation upon nanoparticle interactions. At these two pHs, Marvin Sketch (Chem Axon, Ltd) predicted that 100 % of the tertiary amine


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would be ionised and 23 % of the secondary amine would be ionized at pH 4; whereas 72 % of the tertiary amine would be ionised and 28 % of tetracaine would be unionised at pH 8 (pKa’s at the secondary and tertiary amine of 3.41 and 8.24 respectively at 32 °C). Negatively charged carboxyl-modified polystyrene nanoparticles ( Nano ) and silica nanoparticles (Nano ) were used to represent two different nanoparticle surfaces with which the model drug tetracaine could interact. The Nano presented a mixture of COO- and SO42- groups on the particle surface and the Nano presented SiOH- groups. Whilst both were predicted to display an overall negative charge it was anticipated that the charge density on the two surfaces would be different. Two different nanoparticle core materials were selected because preliminary work had suggested this was required to obtain a significant difference between the surface properties of the materials. We did not employ positively charged nanoparticles in the study as we hypothesised that the positively charged tetracaine would need a negative surface with which to interact. The nanoparticle-drug interactions were studied using fluorescence spectroscopy, adsorption studies and atomic force spectroscopy (AFM). The effects of mixing tetracaine with the two nanoparticles upon drug permeation through two membranes, the porcine epidermis and silicone, were assessed. Two membranes were studied in order to understand the delivery of the drug through a barrier with and without the presence of the follicular transport route because the follicular transport route has previously been shown play a role in the transport of aggregated drugs such as tetracaine when they are presented to the barrier topically

15, 20, 32

. The study also

compared two drug-nanoparticle skin application protocols. The first involved instant mixing of the drug with nanoparticles upon the barrier and the second mixed the drug and the nanoparticles, incubated them for a period of time, and then applied the


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mixture to the membrane. Through the comparison of these two administration protocols the effect of the nanoparticle-drug mixing equilibrium on transport was determined.

Materials and Methods

Materials

Tetracaine free base (≥ 98%), hydrochloric acid, sodium chloride, anhydrous sodium phosphate dibasic, 0.1 % (w/v) poly-L-lysine solution, 1-octadecanethiol (11Mercaptoundecyl)-N,N-N-trimethylammonium bromide and ethyl alcohol (200 Proof, A.C.S Reagent) were purchased from Sigma Aldrich, UK.

Carboxyl-modified

polystyrene nanoparticles, Nano , with a diameter of 60 nm (PCO2N) were sourced from 2BScientific, UK. Silica nanoparticles, Nano , with a diameter of 200 nm (Psi-0,2) were obtained from Kisker Biotech GmbH and Co., Germany. Ultrapure water (18.2 MΩ) was used throughout this study unless stated otherwise. Centrifugal filters (Amicon Ultra 0.5 mL) were purchased from Millipore Limited, UK. Phosphate-buffered saline tablets were supplied by Oxoid Limited, UK. Acetonitrile, methanol and water (high-performance liquid chromatography (HPLC) grade) were obtained from Fisher Scientific International , UK. Sheets of silicone membrane with a thickness of 0.12 mm were purchased from GBUK Healthcare, UK. Whatman no .1 filters, UK were used. Mica surfaces were bought from Agar Scientific, Elektron Technology UK Limited. Gold-coated Si3N4 V-shaped contact mode cantilevers with integrated pyramidal tips (Model: NPG10) were obtained from Bruker, France.


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Test sample preparation

Tetracaine solutions were prepared and adjusted to pH 4.0 and 8.0 using hydrochloric acid and equilibrated at 32 °C unless stated otherwise. Solutions were stirred for at least 24 h and the pH rechecked prior to analysis to ensure they were at equilibrium. The vehicle containing the nanoparticles was corrected to the necessary pH using hydrochloric acid prior to mixing with tetracaine solutions.

Particle size and zeta potential analysis

The nanoparticle size was analysed by photon correlation spectroscopy (Malvern Nanoseries Zetasizer ZEN3600, Malvern Instruments Ltd, UK). Detection of the light scattering signal was achieved at a 173 ° backscattering angle with samples equilibrated at 32 °C using water as a dispersant (refractive index 1.33, viscosity 0.8872 cP). Each measurement comprised of 10-14 runs. The zeta potential of the nanoparticles was recorded using the same Malvern Nanoseries Zetasizer instrument using zeta potential capillary cells. Prior to the zeta potential measurements, the samples were prepared in 10 mM NaCl to normalise the sample osmolality. The measured electrophoretic mobility was converted into zeta potential using Smoluchowski’s formula. Each measurement comprised of 50 to 100 runs. Triplicates of each sample were assessed. In an attempt to estimate the pKa at the nanoparticle surfaces and determine the isoelectric point a zeta potential titration was performed by measuring the zeta potential of the particles at pH units between pH 1 to 11. The pH of the solution was adjusted using hydrochloric acid. A logarithm curve was fitted


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onto the zeta potential titration cure using the OriginPro software (OriginPro version 8.6, OriginLab Corporation, US) to calculate the isoelectric point and the pKa.

Tetracaine transport studies

A concentration of 0.001 g/mL of Nano and 6.289 g/mL of Nano , which provided an equivalent surface area to volume ratio of 9.43x10-10:1 µm2/mL, were added to 10 µM tetracaine solutions to investigate the effects of nanomaterials on tetracaine transport. The vehicle of the nanoparticles, used in the transport studies to test if there was any penetration effect of the solution alone, was obtained by using a centrifugal filter with nominal molecular weight limit of 100 kDa at 1500 g for 10 min (Biofuge pico, Heraeus Instruments, Germany).

Silicone membrane was selected as the confluent barrier without pores and porcine epidermis was selected as the confluent barrier with pores in the form of hair follicles and sweat glands. The porcine skin was still considered to be confluent as pores are invaginations of the epidermis wherein there is a continuity of the epithelia and hence even in these regions the barrier function is maintained. However, the pores can trap the nanosized material and hence they may influence the drug permeation in these studies. The porcine skin was prepared from fresh white adult porcine ears that were obtained from a local abattoir (Evans and Sons, UK). Damaged ears were discarded and the remaining ears were washed with deionized water. After patting dry, the visible hairs were trimmed carefully. The epidermal layer of the porcine skin was isolated from the full thickness tissue, which included the dermis, by heat separation 31

. This involved immersing the full thickness porcine tissue in deionised water at 60


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°C for 1 min. The skin was removed from the water placed on a corkboard with the dermal side down and the epidermis was carefully separated from the dermis with tweezers. The separated epidermis was washed with deionized water and floated on filter paper to remove it from the liquid. The skin sample on the filter paper was patted dry with tissue and wrapped in aluminium foil prior to storage at - 20 °C (maximum of up to 1 month 32). The samples were thawed before use.

The transport studies were carried out using upright individually calibrated Franz diffusion cells with an average surface area of 2.1 ± 0.1 cm2 and an average receptor compartment volume of 9.2 ± 0.5 mL. The barrier was cut into appropriately sized pieces, mounted between two chambers of the glass diffusion cell and the two chambers, which were held in position with parafilm. A 13 mm magnetic flea was placed in the receiver chamber. The cell was inverted and filled with previously filtered and sonicated receiver fluid. In the silicone membrane studies two receiver fluids were employed. Both solutions were aqueous and adjusted to either pH 4 or pH 8 with HCl. Phosphate buffered saline (pH 7.4) was employed as a receiver fluid for the porcine epidermis transport studies to mimic the skin environment. The transport studies were performed on a submergible magnetic stirrer plate which was situated in a pre-heated water bath set at 37 °C to provide a membrane surface temperature of 32 °C. After cell equilibration for 1 h, the cells were checked for leaks by inversion and visual inspection for back diffusion. Infinite doses of tetracaine mixtures (1 mL) were applied uniformly to the surface of each membrane and the donor compartment was covered with parafilm to minimise donor phase evaporation. A 0.5 mL aliquot of the electrolyte/nanoparticle mixture was added to 0.5 mL of 20 µM of the tetracaine solution at the 0 h time point after correcting the suspension medium pH to 4 or 8.


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The final tetracaine concentration in the nanoparticle–tetracaine mixtures was 10 µM. HPLC grade water was added in place of the additives in the control system. There was no significant difference between the drug permeation of equilibrated tetracaine solutions from those where additives were added at the same time as application to the skin (mixed immediately prior to application, Table S1). The direct mixing of the drug and nanoparticles on the surface of the skin using two pump sprays was the preferred application method throughout the study as it minimised any potential interference from chemical or physical instability as a consequence of forming the mixture. At predetermined time intervals, 1 mL aliquots were removed from the receiver phase and replaced with fresh receiver fluid to keep the liquid volume in the receiver compartment constant. The collected samples were analysed by HPLC. A total of 5 replicates of each experiment were performed and cumulative amounts of drug (ng) penetrating the unit surface of the membrane area (cm2) were corrected for sample removal and plotted against time (h). The steady-state flux (J) was calculated from the slope of the linear portion of the curve (R2 ≥ 0.98), using at least 4 points with values above the assay limit of detection (LOD). The permeability coefficient of tetracaine was calculated using equation 1 33:

J =

(Equation 1)

where J represents the flux, kp is the permeability coefficient of the permeant across the membrane and Cv is the concentration of the drug in the vehicle. The enhancement ratio (ER) due to the various additives were determined using the following equation:

ER =

(Equation 2)


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where J1 and J2 are the steady-state transmembrane transport rate of tetracaine from the tetracaine and tetracaine-nanoparticle mixture respectively.

Tetracaine quantification

The quantification of tetracaine was performed using a reverse-phase HPLC system consisting of a pump with autosampler (Hewett-Packard series 1050, Agilent Technologies UK Ltd., UK) connected to a fluorescence detector (Shimadzu detector RF-551, Shimadzu corp., Japan). The system was controlled via a computer with Chromeleon software (Dionex Corp., USA), which was also used to record and interpret the analytical data. The mobile phase comprised acetonitrile-methanolacetate buffer (0.1 M) (25:25:50 (v/v), pH 5.1) set at a flow rate of 1.0 mL.min-1. Tetracaine was separated using a Luna 3 µm C18(2) (150 X 4.6 mm) stationary phase (Phenomenex, UK) at room temperature with a 100 µL injection volume and the fluorescence detection at an excitation wavelength of 310 nm and an emission wavelength of 372 nm. The retention time for tetracaine was 4.2 min. The calibration curves were constructed on the basis of the peak area measurements using standard solutions of known tetracaine concentrations dissolved in an identical fluid as the receiver phase for the transport studies. The assay was shown to be “fit for purpose” in terms of sensitivity (LOD – 4.08 ng/mL, n=25), precision (6 % CV, acceptable due the low concentration range employed), and linearity (R2 ≥ 0.99).

Physical adsorption


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Nanoparticles were added to tetracaine solutions at time point 0 h and at several subsequent time points (0.25, 0.50. 0.75, 1, 3, 5, 24, 48 h), 500 µL of the tetracainenanoparticle mixtures were removed and centrifuged through a centrifugal filter with nominal molecular weight limit of 100 kDa at 1500 g for 10 min (Biofuge pico, Heraeus Instruments, Germany). A 300 µL aliquot of the centrifuged liquid phase was transferred into vials. The analyte was dissolved in a 1:2 ratio. v/v with acetonitrile and unbound tetracaine concentrations were determined using the HPLC assay. Tetracaine-NP binding was calculated using equation 3:

C (%) = X 100 %

(Equation 3)

where C is the concentration of the bound tetracaine, C is the unbound tetracaine analysed at the time point and C! is the initial tetracaine.

Fluorescence spectroscopy

Fluorescence emission spectra were recorded using a fluorescence spectrometer fitted with a Xenon pulse lamp (Varian Cary Eclipse Fluorescence Spectrometer, Agilent Technologies, UK). Quartz fluorescence cells (Helima Fluorescence Cell, Helima UK Ltd., UK) with a 3 mm path length were used. Excitation and emission slits were fixed at 5 nm. In all measurements, the excitation wavelength was set at 310 nm. The samples were scanned from 320 to 450 nm at a wavelength scan rate of 120 nm/min with a PMT detector gain of 600 V. The data were smoothed with a Savitzky Golay function filter size 25 using the Cary Eclipse software. The experiments were performed at a temperature of 32 °C. Analysis of the spectrum was conducted using


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OriginPro software (OriginPro version 8.6, OriginLab Corporation, US). The nanoparticles gave a small signal in the 340 – 420 region of the spectrum but as the particles were not fluorescent, this was probably due light scattering caused by the nanoparticles (Fig. S2). The small signal generated by the nanoparticles was subtracted from the measurements to focus the results on the tetracaine fluorescence.

Atomic force microscopy (AFM)

Adhesion measurements and AFM images were obtained using a Nanoscope V Dimension Icon atomic force microscope (Digital Instruments, UK). To mimic the conditions in the Franz cell experiments, the substrates were analyzed using a fluid cell set up that employed the liquid submerged samples, which were all housed in a petri dish. The fluid cell was washed with detergent water and rinsed with ultrapure water before drying with a steam of nitrogen gas prior to the start of every experiment. The same individual AFM tip was used in all experiments in order to cancel out possible variations caused by different tip properties such as tip size, etc. The HCl in the fluids did not seem to influence the particles according to their morphology (see figure S6 in the supplementary material).

The AFM images of the Nano and Nano were taken after deposition on freshly cleaved mica surfaces. To facilitate nanoparticle deposition an aqueous 0.1 % (w/v) poly-L-lysine solution was applied to mica prior to the addition of the nanoparticles. The functionalized materials employed in the force adhesion AFM measurements were produced using a gold-thiol coating procedure. 1-octadecanethiol and (11-Mercaptoundecyl)-N,N-N-trimethylammonium bromide were selected as the


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thiols because they produced functional groups on the surface of the materials which best mimicked the two terminuses of the tetracaine molecule. The material functionalisation was achieved by incubating the gold-coated AFM materials (i.e., AFM substrates and AFM cantilevers) in freshly prepared 3 mM solution of the chosen thiol, which was dissolved in ethyl alcohol overnight in a sealed petri dish. After incubation, the substrates and AFM probes were washed thoroughly with ethanol. The substrates were dried with nitrogen while the AFM probes were allowed to dry in air.

Prior to making the force-adhesion measurements the spring constant of the cantilevers was determined as follows: First, the photodetector sensitivity was calibrated by determining the slope of the repulsive branch of a force curve on a hard surface (photodetector signal versus z-scanner displacement plot on mica). Then, thermal-tuning was performed using the corresponding Nanoscope programme function to determine the actual spring constants of the cantilevers. Gold-coated Si3N4 V-shaped contact mode cantilevers with integrated pyramidal tips (Model: NPG10) were used to obtain the force measurements. The nominal spring constant specified in the cantilever datasheets was 0.35 N/m and the measured spring constants were (0.39 ± 0.01) N/m. Three independent measurements of 210 force curves were obtained from 10 randomly distributed areas on the sample surfaces to ensure representative sampling. Average adhesion force values were determined by fitting a Gaussian profile to the histogram of the forces using OriginPro software. Sessile drop contact angles were captured by a high resolution camera (Photron Ultima APX Monochrome, UK) and analysed using Image J with a Dropsnake plug-in to confirm the formation of surface modified gold substrates (Table S3 and Fig. S4). In addition,


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force interactions between the tip and CH3 substrates were measured before each sample substrate to confirm that the tip was not significantly modified during the sequential measurement of the different samples (Table S5).

Statistical Analysis

All values were expressed as their mean ± standard deviation (SD). The statistical analysis of data was performed using the statistical package for social sciences, SPSS version 21, (IBM Corp., USA) with a significance level of 0.05. The normality (Sapiro-Wilk) and homogeneity of variances (Levene’s test) of the data were assessed prior to statistical analysis. Transport data were analysed statistically using one-way analysis of variance (ANOVA) tests for normally distributed data and a nonparametric Kruskal-Wallis tests for non-Gaussian distributed data. Post hoc comparisons of the means of individual groups were performed when appropriate using Dunnett’s test for normal distributed data and Games Howell test for nonGaussian distributed data.

For all pair-wise comparison of means, Student’s

independent t-test or Mann-Whitney test was applied. Data were presented using OriginPro software (OriginPro version 8.6, OriginLab Corporation, US).

Results

Particle characterization

The mean diameter of the Nano obtained from the cross-sectional analysis of AFM (36.8 ± 7.3 nm) (Fig. S6) was significantly smaller (independent t-test, p0.05) to those obtained using photon correlation spectroscopy (242.6 ± 2.6 nm) (Table 1). Silica nanoparticles in an aqueous environment are known to have hydroxyl surface groups 34

. The zeta potential data for the Nano suggested they had a pKa of 4.03 and an

isoelectric point of 2.00 (Fig. S7). In an aqueous solution set at pH 4, the surface charge of the Nano was significantly less negative (independent t-test, p

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