Fluorouracil-Loaded Gold Nanoparticles for the Treatment of Skin

Apr 27, 2018 - Fluorouracil (5-FU) is an antimetabolite drug used in the treatment of various malignancies, such as colon and skin cancers. However, i...
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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Fluorouracil-Loaded Gold Nanoparticles for the Treatment of Skin Cancer: Development, in Vitro Characterization, and in Vivo Evaluation in a Mouse Skin Cancer Xenograft Model Mohamed A. Safwat,†,‡ Ghareb M. Soliman,*,†,⊥ Douaa Sayed,§ and Mohamed A. Attia† †

Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt Department of Pharmaceutics, Faculty of Pharmacy, South Valley University, Qena 83523, Egypt § Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University, Assiut 71526, Egypt ⊥ Department of Pharmaceutics, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia ‡

ABSTRACT: Fluorouracil (5-FU) is an antimetabolite drug used in the treatment of various malignancies, such as colon and skin cancers. However, its systemic administration results in severe side effects. Topical 5-FU delivery for the treatment of skin cancer could circumvent these shortcomings, but it is limited by the drug poor permeability through the skin. To enhance 5-FU efficacy against skin cancer and reduce its systemic side effects, it was loaded into a gold nanoparticle (GNP)-based topical delivery system. 5-FU was loaded onto GNPs capped with CTAB through ionic interactions between 5-FU and CTAB. GNPs were prepared at different 5-FU/CTAB molar ratios and evaluated using different techniques. GNP stability and drug release were studied as a function of salt concentration and solution pH. Optimum 5-FU/CTAB-GNPs were incorporated into gel and cream bases, and their ex vivo permeability was evaluated in mice dorsal skin. The in vivo anticancer efficacy of the same preparations was evaluated in A431 tumor-bearing mice. The GNPs had spherical shape and a size of ∼16−150 nm. Maximum 5FU entrapment was achieved at 5-FU/CTAB molar ratio of 1:1 and pH 11.5. Drug release from GNPs was sustained and pHdependent. 5-FU GNP gel and cream had around 2-fold higher permeability through mice skin compared with free 5-FU gel and cream formulations. Further, in vivo studies in a mouse model having A431 skin cancer cells implanted in the subcutaneous space showed that the GNP gel and cream achieved 6.8- and 18.4-fold lower tumor volume compared with the untreated control, respectively. These results confirm the potential of topical 5-FU/CTAB-GNPs to enhance drug efficacy against skin cancer. KEYWORDS: gold nanoparticles, 5-fluorouracil, cetyltrimethylammonium bromide, skin cancer

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malignancies, such as colon, breast, and skin cancers.6 5-FU causes apoptosis of rapidly dividing cancerous cells as a consequence of inhibiting thymidylate synthase enzyme and limited synthesis of thymidine, a vital nucleoside for DNA replication.7 However, 5-FU oral administration results in erratic and variable bioavailability of 0 to 80%, which mandates intravenous administration.8 The latter results in alarming toxic effects of gastrointestinal, hematological, neural, cardiac, and

INTRODUCTION Skin cancer is one of the most prevalent cancers in the United States and other regions of the world.1 Skin malignancy is manifested in many forms, such as melanoma, squamous cell carcinoma, and basal cell carcinoma. The most common cause of skin cancer is the prolonged exposure to direct sunlight, which results in substantial cellular proliferation and keratinocyte destruction.2 Common treatment protocols of skin cancers include surgical excision or radiation therapy.3,4 In addition, chemotherapy might be a better alternative in some situations where surgery is contraindicated or not feasible.5 5-Fluorouracil (5-FU) is one of the oldest and most effective anticancer drugs with broad spectrum of activity against several © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

January 16, 2018 April 1, 2018 April 27, 2018 April 27, 2018 DOI: 10.1021/acs.molpharmaceut.8b00047 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

5-FU was loaded through electrostatic interactions between CTAB cationic charges and 5-FU anionic ones. Aliquots of 5FU aqueous solution (1.5 × 10−2 M) were added in a dropwise manner while mixing by vortex to aqueous CTAB-GNPs solution (5 mL, CTAB/gold chloride molar ratio of 44:1) to obtain solutions having 5-FU/CTAB molar ratios ranging from 0:1 to 4:1. The mixtures were stirred for 30 min after adjusting their pH to 9.5. 5-FU/CTAB-GNPs were separated by centrifugation (14,000 rpm, 30 min, 25 °C). The concentration of free (unbound) 5-FU in the supernatant was assayed spectrophotometrically at 266 nm. 5-FU entrapment efficiency (EE%) was calculated from eq 1. UV−vis spectra of samples were recorded on a UV−vis Spectrophotometer (Shimadzu50−02, Kyoto, Japan) in the range of 500−700 nm.

dermatological origin due to off-target cytotoxic effects of 5FU.9 Topical 5-FU delivery would circumvent these systemic side effects and enhance drug efficacy against skin cancer through maximization of drug concentration at the target tissues. However, 5-FU suffers from poor skin permeability due to its hydrophilic nature and unfavorable hydrophilic/lipophilic balance.10,11 Several delivery systems have been exploited to enhance 5-FU skin permeability including microemulsions, niosomes, nanogels, solid lipid nanoparticles, microneedles, and gold nanoparticles.10,12−16 Gold nanoparticles (GNPs) are currently under intensive investigation for drug delivery applications due to their potential to maximize anticancer drug efficacy and to minimize off-target side effects.17−19 GNPs’ unique optical and photothermal properties in addition to the straightforward functionalization of their surface make them extremely promising tools in cancer chemotherapy.20,21 Moreover, they enhanced the transdermal delivery of several drugs.22,23 GNPs also showed promising results in photothermal therapy and photodynamic therapy of skin cancer.24,25 For instance, Zn(II)-phthalocyanine disulfide bound to GNPs showed about 2-fold better accumulation in amelanotic melanoma and more effective photodynamic therapy compared with free drug.26 Despite these interesting results, careful literature review shows no studies on enhancing 5-FU efficacy against skin cancer using GNPs. To bridge this gap, we have recently shown that 5-FU loaded into GNPs capped with benzalkonium chloride or poly(ethylene imine) had about 2-fold higher permeability through mice skin compared with the drug aqueous solution.16 However, the in vivo efficacy of these nanoparticles against skin cancer was not evaluated. The aim of this study was to prepare GNPs capped with cetyltrimethylammonium bromide (CTAB) and test their ability to increase 5-FU loading capacity and enhance its efficacy against skin cancer. The cationic surfactant CTAB served as a stabilizer for GNPs, as well as a ligand to load 5-FU through electrostatic interactions between 5-FU anionic charges and CTAB cationic ones. An optimized 5-FU/CTAB-GNP formulation was incorporated into cream and gel bases, and their ex vivo drug permeability through mice skin was tested. Anticancer efficacy of 5-FU/CTAB-GNPs cream and gel was tested in vivo in a mouse skin cancer model.

5‐FU EE (%) total 5‐FU added initially − 5‐FU in the supernatant = total 5‐FU added initially × 100

(1)

The effect of pH on 5-FU entrapment efficiency was evaluated by adjusting the pH of 5-FU/CTAB-GNPs (5-FU/ CTAB molar ratio of 1:1, pH 9.5) to 5, 7, 8.5, 10.5, and 11.5 using either 1 N HCl or 1 N NaOH. The mixtures were left for 60 min, and drug entrapment efficiency was determined as described above. Ionic Strength Studies. Different volumes of sodium chloride stock solution (2.5 M) were added to 5-FU/CTABGNPs solution (5-FU/CTAB molar ratio of 1:1, pH 11.5) to have salt concentration of 50−300 mM. The samples were magnetically stirred for 5 min, and 5-FU entrapment efficiency was determined as described above. GNPs particle size, polydispersity index, and zeta potential were measured on a Malvern ZetaSizer (Nano-ZS, Malvern Instruments, Worcestershire, UK). UV−vis spectra in the 500−700 nm range were also recorded. TEM Measurements. GNPs size and morphology were characterized on a high-resolution transmission electron microscope (HR-TEM, Tecnai G20, FEI, Netherland) equipped with eagle CCD camera with (4k × 4k) image resolution at an acceleration voltage of 200 kV. TEM samples were prepared by adding 100 μL of the aqueous GNP solutions onto a TEM copper grid. The samples were allowed to dry overnight at room temperature. Particle Size, Polydispersity Index, and Zeta Potential Measurements. 5-FU/CTAB-GNPs (5-FU/CTAB molar ratios of 0:1 to 4:1, pH 11.5) were characterized for particle size, polydispersity index, and zeta potential at 25 °C using a Malvern ZetaSizer (Nano-ZS, Malvern Instruments, Worcestershire, UK). The instrument had a 4 mW helium/neon laser (λ = 633 nm) and a thermoelectric temperature controller. Zeta potential was calculated from the electrophoretic mobility values using Smoluchowski equation. The measurements were done in triplicate and are shown as mean ± SD. Stability Study of 5-FU-Loaded GNPs. 5-FU/CTABGNPs (5-FU/CTAB molar ratio of 0.6:1) were prepared as described above at pH 11.5 and aliquots were stored for 4 months in tightly closed containers at 4 °C and at room temperature. At different time intervals, aliquots were withdrawn, and their particle size, zeta potential, drug entrapment efficiency (%) and UV−vis spectra were characterized as described above.

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EXPERIMENTAL SECTION Materials. 5-Fluorouracil was purchased from Alfa Aesar (Ward Hill, MA). Gold chloride (HAuCl4) was purchased from Electron Microscopy Sciences Co. (Hatfield, PA). Sodium borohydride (NaBH4) and Pluronic F-127 were purchased from Sigma-Aldrich Co. (St. Louis, MO). CTAB and stearic acid were obtained from El-Nasr Chemical Co. (Cairo, Egypt). Dialysis membranes (MWCO 3.5 kDa) were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). All other chemicals were of reagent grade and used as received. Synthesis of CTAB-GNPs and 5-FU Loading. CTAB aqueous solution (5 mL, 2.4 × 10−2 M) was mixed with HAuCl4 aqueous solution (5 mL, 5.6 × 10−4 M) followed by the addition of ice-cold NaBH4 (1 mL, 1 × 10−2 M) under magnetic stirring. The color of the solution turned reddish brown. Next, the solution was vigorously stirred for 2 min, and it was kept at 25 °C without further stirring for 2 h.27 CTABcoated GNPs were separated by centrifugation at 14,000 rpm using a Microlitre centrifuge, Micro 200R, Germany for 1 h at 25 °C. B

DOI: 10.1021/acs.molpharmaceut.8b00047 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Incorporation of 5-FU/CTAB-GNPs into Gel and Cream Bases. Selected 5-FU/CTAB-GNPs (5-FU/CTAB molar ratio of 1:1, pH 11.5) were incorporated into Pluronic F127 gel and vanishing cream bases to facilitate their application on the skin. Pluronic F127 gel (25% w/w) was prepared by the cold method.28 The required amount of the polymer was dispersed in distilled water and left in a refrigerator overnight until it became a clear solution. 5-FU/CTAB-GNPs solution containing 100 mg of 5-FU was added and thoroughly mixed with Pluronic gel so that the final gel weight was 10 g and final 5-FU concentration was 1% w/w. Vanishing cream was prepared by melting stearic acid (15% w/w) on a nonboiling water bath followed by the addition of aqueous phase containing dissolved KOH (0.7% w/w) and glycerin (5% w/w). The aqueous phase was added to the oily phase gradually with trituration, and the mixture was removed from the water bath and mixed with CTAB-GNPs loaded with 100 mg of 5-FU to prepare 10 g of medicated cream. The mixture was triturated until congealing. Gel and cream containing 1% w/w free drug were also prepared and used as a control. Viscosity Measurement. The viscosity of different cream and gel formulations was determined at room temperature using a Brookfield (Brookfield, Middleboro, MA, USA) DV-III ultra viscometer (RV model) using T bar spindle T-D 94 at a spindle speed of 50 rpm. In Vitro 5-FU Release Studies. Drug-loaded CTAB-GNPs solution (2 mL containing 10 mg of 5-FU, 5-FU/CTAB molar ratio of 1:1, pH 11.5) was introduced into a dialysis bag membrane and transferred to screw capped tubes containing 50 mL of phosphate buffer pH 3, 5, 7, or 8. The tubes were immersed in a water bath shaker maintained at a temperature of 37 °C and a speed of 50 rpm. Samples were withdrawn from the release medium at predefined time intervals and replaced by the same volume of fresh release medium kept at 37 °C. 5-FU concentration in the withdrawn samples was determined spectrophotometrically at 266 nm. Cumulative percent of 5FU released was plotted vs time. For 5-FU/CTAB-GNPs gel and cream, a one-gram sample containing 10 mg of 5-FU was introduced into the dialysis bag membrane. The membranes were transferred to screw-capped tubes containing 50 mL of phosphate buffer pH 7.4 and treated as above. The experiments were run in triplicate, and results are shown as mean ± SD. Kinetic Analysis of Release Data. Drug release data was fitted using different mathematical models. The models used were zero order, first order, Higuchi diffusion model, Baker− Lonsdale model, Hixson−Crowell cube root law, and Korsmeyer−Peppas equation.29−34 The appropriate model was selected based on the highest correlation coefficient (R2). Ex Vivo Skin Permeation Study. Full thickness C57BL/6 mice dorsal skin was used to study the ex vivo permeability of 5FU from different formulations according to previously reported procedures.14 Hair was carefully removed using an electric clipper 24 h before the experiment. The mice were sacrificed, and their dorsal skin was separated from the underlying connective tissue with scalpel. The harvested skins were stored at −20 °C and used within one month. Next, a specimen of the skin (diffusion area: 5 cm2) was mounted onto a Franz diffusion cell (PermeGear, Inc., Hellertown, PA) with the epidermis side facing upward. Phosphate buffer (10 mM, pH 7.4, 16 mL) was added to the receiver chamber and maintained at 37 °C. The donor chamber was loaded with 5FU/CTAB-GNPs gel or cream (1 g equivalent to 10 mg of 5FU) and covered with parafilm to prevent evaporation. 5-FU/

CTAB-GNPs (2 mL, 10 mg of 5-FU) and 5-FU/CTAB complex solution (2 mL, 10 mg of 5-FU) were similarly treated and used as a control. At predefined time intervals, samples (1 mL) were taken from the receiver chamber and replaced with an equal volume of fresh medium maintained at 37 °C. Drug concentration in the samples was determined spectrophotometrically at λmax = 266 nm. Each experiment was run in triplicate. The cumulative amount of 5-FU permeated through skin (μg/cm2) was calculated and plotted vs time. The flux (Jss) was calculated from the slope of the linear regression line.35 The curves had good linearity as indicated by their high correlation coefficient (0.968−0.995). The apparent permeability coefficient (Papp) was calculated using the following equation:

Papp =

Jss C0

(2)

where Jss is the flux and C0 is the initial 5-FU concentration. Enhancement factor (EF) (%) was calculated from the following equation:36 EF (%) =

Jss(A) × 100 Jss(B)

(3)

where Jss(A) is the flux obtained for the tested preparation while Jss(B) is the flux of free 5-FU solution. In Vivo Anticancer Studies. In vivo anticancer efficacy of different 5-FU preparations was tested in male C57BL/6 mice (8−10 weeks old) according to previously reported procedures with slight modifications.14 Experiments were carried out according to the animal ethics guidelines approved by Assiut University, Egypt. Hair in the lower dorsal skin of anesthetized mice was trimmed using an electric clipper. Human epidermoid carcinoma A431 cells were obtained from VACSERA Company (Giza, Egypt). Each mouse was injected subcutaneously with 400,000 cells in 400 μL of phosphate buffer in the hair-trimmed area on day 0. On day 24 when palpable tumors were developed, mice were randomly divided into five groups (n = 5). The first group received no treatment and served as a negative control. The other four groups received, respectively, free 5-FU gel, free 5-FU cream, 5-FU/CTAB-GNPs gel, and 5FU/CTAB-GNPs cream. 5-FU concentration in all preparations was 1% w/w. The cream or gel formulation (100 mg) was applied on the area (∼1 cm × 2 cm) where the tumor cells were injected using a spatula, once daily for 28 consecutive days. Tumor growth was monitored, and tumor size was measured using a digital caliper. Tumor volume was calculated based on the following equation:14 tumor volume (mm 3) =

length × width × width 2

(4)

On the last day of the study (day 52), mice were euthanized, and tumor tissues were collected and weighed. Tumor tissues were fixed with formalin solution (10%) for 48 h and then transferred to 70% ethanol until sections were prepared following paraffin embedding. The sections were stained using hematoxylin−eosin (H&E). Slides were scanned, and images were taken using the Scan Scope XT (Aperio Technologies, Vista, CA, USA). Statistical Analysis. All experiments were run in triplicate, and the results are shown as mean ± SD. Data analysis was carried out using GraphPad Prism software version 5 C

DOI: 10.1021/acs.molpharmaceut.8b00047 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. (A) Schematic diagram for GNPs synthesis and 5-FU loading using CTAB. (B) UV−vis spectra of CTAB-GNPs and 5-FU/CTAB-GNPs (5-FU/CTAB molar ratio of 1:1 at pH 11.5). (C) TEM image of 5-FU/CTAB-GNPs (5-FU/CTAB molar ratio of 1:1 at pH 11.5).

(GraphPad Software Inc., La Jolla, CA). Statistical significance was defined at p < 0.05. Data was analyzed using one-way analysis of variance (ANOVA) with Newman−Keuls method as a posthoc test.

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RESULTS AND DISCUSSION Synthesis of CTAB-Capped GNPs. GNPs were prepared using NaBH4 as a reducing agent due to its ability to produce Table 1. Influence of 5-FU/CTAB Molar Ratio on Size and Zeta Potential of GNPs Prepared at pH 11.5 molar ratio of 5-FU: CTAB 0:1 0.6:1 1:1 1.3:1 2:1 4:1 0.6:1a

particle size (nm) 14.22 16.02 16.02 17.44 39.69 148.92 19.04

± ± ± ± ± ± ±

0.42 0.22 0.36 0.74 0.53 1.52 1.02

PDIb 0.12 0.18 0.18 0.29 0.37 0.50 0.28

± ± ± ± ± ± ±

0.01 0.02 0.01 0.07 0.09 0.14 0.01

zeta potential (mV) +49.61 +47.81 +44.52 +38.84 +14.11 +3.99 +33.65

± ± ± ± ± ± ±

1.32 0.43 0.61 1.42 1.17 0.44 1.71

Figure 2. Drug entrapment efficiency as a function of 5-FU/CTAB molar ratio and solution pH.

nanoparticles of small size and good stability.37 CTAB was chosen as a stabilizer for GNPs due to its ability to form a bilayer structure around GNPs through binding between GNPs surface and CTAB head groups (Figure 1).38 Results show that CTAB was an essential stabilizer for GNPs since there was

a

GNP solution after the addition of 150 mM sodium chloride. bPDI, polydispersity index. All results are the mean ± SD (n = 3). D

DOI: 10.1021/acs.molpharmaceut.8b00047 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 3. (A) Effect of salt concentration on the entrapment efficiency of 5-FU onto CTAB-GNPs. (B) Effect of salt concentration on the UV−vis spectra of 5-FU/CTAB-GNPs (5-FU/CTAB molar ratio of 1:1 at pH 11.5).

Table 2. Influence of Storage at 4 °C and Room Temperature (RT) on the Particle Size, Zeta Potential, and Entrapment Efficiency (EE%) of 5-FU/CTAB-GNPs (5-FU/CTAB Molar Ratio of 0.6:1 at pH 11.5) storage time (month) parameters particle size zeta potential EE (%)

temp (°C) 4 RT 4 RT 4 RT

initial 16.02 16.02 +47.81 +47.81 70.50 70.50

± ± ± ± ± ±

1 0.22 0.22 0.43 0.43 1.15 1.15

16.23 16.30 +47.41 +47.22 69.61 69.23

± ± ± ± ± ±

2 0.55 0.32 0.22 0.41 0.62 0.52

17.11 17.42 +47.13 +46.92 69.49 69.11

± ± ± ± ± ±

3 0.41 0.16 0.14 0.34 0.47 0.12

17.24 17.46 +46.62 +46.56 68.33 68.15

4

± ± ± ± ± ±

0.43 0.74 0.53 0.84 1.02 0.66

17.45 17.62 +46.52 +46.40 68.31 67.98

± ± ± ± ± ±

0.36 0.29 0.71 0.62 0.77 1.22

facilitate 5-FU loading through ionic interactions between 5-FU anionic charges and cationic ones of CTAB. CTAB is a quaternary ammonium compound offering permanent cationic charges that are not affected by solution pH.41 At pH above 8.5, 5-FU carries negative charges, while CTAB carries positive ones, which favors ionic interactions between them (Figure 1A).42 Ionic interactions of 5-FU with CTAB-GNPs resulted in a slight red shift in the GNPs SPR peaks from 525.0 ± 1.0 to 527.0 ± 1.0 nm, presumably due to GNPs size increase upon 5FU loading (Figure 1B).43,44 GNPs size measurement by DLS confirmed size increase from 14.22 ± 0.42 to 16.02 ± 0.22 nm upon increasing 5-FU/CTAB molar ratio from 0:1 to 0.6:1 (Table 1). These results confirm that CTAB served two functions: a stabilizing agent for GNPs and a ligand for 5-FU loading. Successful synthesis of GNPs was also ascertained by TEM imaging where the nanoparticles appear as discrete units having uniform shape and free of aggregation (Figure 1C). Size obtained from this image was 15.10 ± 3.00 nm, which is in good agreement with that obtained from DLS measurements. Effect of pH on 5-FU Entrapment Efficiency. In order to achieve maximum 5-FU entrapment efficiency, the nanoparticles were prepared at different pH values and different 5FU/CTAB molar ratios. 5-FU pKa values are 8 and 13, which result in higher drug ionization at pH ≥ 8.45,46 5-FU entrapment efficiency was very low (less than 2%) at pH less than 8, possibly due to minimal drug ionization and, in turn, limited ionic interactions between 5-FU and CTAB (Figure 2). By contrast, there was a significant increase in 5-FU entrapment efficiency upon pH increase from 8.5 to 11.5 (p < 0.05) (Figure 2). CTAB charge density is not affected by pH since it is a quaternary ammonium compound, whereas 5-FU anionic charge density increases with pH leading to higher density of ionic interactions and, consequently, better drug entrapment.47

Figure 4. UV−vis spectra of 5-FU/CTAB-GNPs after 4 months of storage at temperature 4 °C and at room temperature.

rapid aggregation for the nanoparticles prepared using NaBH4 only. In addition, there was no specific surface plasmon resonance (SPR) peaks for the nanoparticles prepared with NaBH4 only (Figure 1). Contrarily, there was a specific SPR absorption band at 525.0 ± 1.0 nm for the nanoparticles stabilized by CTAB (Figure 1). CTAB-coated gold nanorods had a cytotoxic effect to human skin HaCaT keratinocyte cells, which was attributed to the free CTAB in solution.39 To limit this cytotoxic effect, we used centrifugation to separate CTABGNPs from free CTAB. Another centrifugation step was also used after 5-FU loading to further limit the concentration of free CTAB in the final preparation. 5-FU Loading. Our previous studies indicated that direct loading of 5-FU onto GNPs surface was not feasible and resulted in very low drug loading capacity.16,40 Accordingly, GNPs were decorated with a quaternary surfactant, CTAB, to E

DOI: 10.1021/acs.molpharmaceut.8b00047 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 5. (A) Cumulative percent of 5-FU released from CTAB-GNP solutions in phosphate buffer at different pH values compared with free 5-FU aqueous solution. (B) Cumulative percent of 5-FU released from gel and cream preparations in phosphate buffer 7.4. 5-FU concentration in all preparations was 1% w/w. 5-FU/CTAB-GNPs in all preparations had 5-FU/CTAB molar ratio of 1:1.

Table 3. Viscosity of Different 5-FU Gel and Cream Preparations formula

viscosity (cP)

free 5-FU gel free 5-FU cream 5-FU/CTAB-GNPs gel 5-FU/CTAB-GNPs cream

86500 190250 86250 191420

± ± ± ±

1520 3620 2130 3450

Next, the nanoparticles were prepared at different 5-FU/CTAB molar ratios, and their drug entrapment efficiency was determined. Figure 2 shows that, at any given pH, 5-FU entrapment efficiency remained almost constant with the increase in the 5-FU/CTAB molar ratio up to a ratio of 1:1, after which it started to decrease. This is possibly attributed to saturation of CTAB with the drug where the available cationic charges became insufficient to fully complex 5-FU. Similar results were reported earlier for lysozyme/sodium dodecyl sulfate complex loaded into lipid−polymer hybrid nanoparticles.48 Maximum 5-FU entrapment efficiency at 5-FU/CTAB molar ratio of 1:1 was 70.0 ± 1.1% for the nanoparticles prepared at pH 11.5 (Figure 2). Based on these findings, these nanoparticles were chosen for further investigations. Ionic Strength Studies. Figure 3 shows that the drug entrapment efficiency decreased insignificantly upon adding 50 mM NaCl (p > 0.05). Salt concentrations above 50 mM resulted in significant decrease in drug entrapment efficiency (p < 0.05). For instance, 5-FU entrapment efficiency decreased

Figure 6. Ex vivo permeation of 5-FU through mice skin from different formulations.

from 70.0 ± 1.7% to 61.0 ± 2.1% for 5-FU/CTAB-GNPs when the salt concentration was increased from 0 to 300 mM. Monovalent salts, such as sodium chloride, were previously shown to weaken ionic interactions leading to reduced entrapment efficiency for drugs loaded by ionic interactions.49,50 Interestingly, increasing salt concentration to 150 mM (physiological salt concentration) decreased drug entrapment efficiency to 64.0 ± 1.1%. Although this decrease in drug entrapment efficiency is significant (p < 0.05), it is much smaller than that observed previously for 5-FU loaded into glutathione-GNPs and thioglycolic acid-GNPs (∼50%).40 This

Table 4. Kinetic Assessment of Drug Release Data from Different GNP Formulations at Different pH Values According to Various Kinetic Models correlation coefficient (R2) formula

pH

zero

first

Higuchi

Hixon

Baker

Korsmeyer−Peppas release exponent (n)

5-FU/CTAB-GNPs 5-FU/CTAB-GNPs 5-FU/CTAB-GNPs 5-FU/CTAB-GNPs Free 5-FU gel Free 5-FU cream 5-FU/GNP gel 5-FU/GNP cream

3 5 7 8 7.4 7.4 7.4 7.4

0.859 0.820 0.956 0.977 0.955 0.981 0.995 0.988

0.754 0.783 0.893 0.926 0.930 0.913 0.974 0.961

0.972 0.950 0.980 0.958 0.998 0.992 0.985 0.947

0.991 0.943 0.995 0.991 0.968 0.949 0.992 0.975

0.998 0.985 0.985 0.973 0.956 0.933 0.934 0.907

0.810 0.681 1.084 0.933 0.941 0.943 0.981 0.689

F

DOI: 10.1021/acs.molpharmaceut.8b00047 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 5. Ex Vivo Skin Permeation Parameters of 5-FU Incorporated in Different Preparations preparation

J (μg·cm−2·h−1)

P × 10−3 (cm·h−1)

EF (%)

Free 5-FU solution 5-FU/CTAB solution 5-FU/CTABGNPs Free 5-FU gel 5-FU/CTAB-GNP gel Free 5-FU cream 5-FU/CTAB-GNP cream

28.46 ± 1.32 39.22 ± 0.20

5.68 ± 0.26 7.84 ± 0.40

137.97 ± 5.70

57.52 ± 0.56

11.50 ± 0.10

202.33 ± 7.44

32.38 ± 1.88 60.87 ± 1.74

3.23 ± 0.18 6.08 ± 0.17

113.74 ± 1.70 214.06 ± 5.79

41.11 ± 1.81 74.03 ± 1.50

4.11 ± 0.18 7.40 ± 0.15

144.47 ± 0.35 260.34 ± 6.82

are particle size and zeta potential.53,54 Table 1 shows particle size, polydispersity index, and zeta potential of 5-FU/CTABGNPs prepared at pH 11.5 and different 5-FU/CTAB molar ratios. The 5-FU/CTAB molar ratio had a profound effect on GNPs size where there was a general and significant increase in the particle size with increasing the molar ratio (p < 0.05) (Table 1). This might be attributed to ionic interactions between 5-FU and CTAB-GNPs, which led to drug loading and, in turn, increasing the particle size. Previous reports showed similar effect on particle size.40,55 Polydispersity index of most of the nanoparticles was in the range of 0.1−0.3, indicating their satisfactory monodispersity (Table 1). CTAB-GNPs prepared in the absence of 5-FU had a zeta potential of +49.61 ± 1.32 mV (Table 1). Loading of different concentrations of 5-FU resulted in a general and progressive decrease in the magnitude of the zeta potential, probably due to neutralization of CTAB upon interaction with 5-FU (Table 1). All the differences in the zeta potential of 5-FU/CTAB-GNPs having different 5-FU/CTAB molar ratios were statistically significant (p < 0.05) except those between 0:1 and 0.6:1. This might be taken as an evidence for drug loading through ionic interactions with CTAB. Nanoparticles having a drug/CTAB molar ratios in the range of 0:1 to 1.3:1 had zeta potential values larger than +30 mV. Previous reports showed that nanoparticles having absolute surface charge greater than 30 mV achieved good colloidal stability through electrostatic repulsion between the nanoparticles.56,57 Furthermore, positive surface charge of nanoparticles is an asset for topical drug delivery since it facilitates nanoparticle interaction with the

noticeably better stability of 5-FU/CTAB-GNPs against salinity is possibly attributed to tighter ionic interactions between the permanently charged CTAB and 5-FU. The UV−vis spectra of the nanoparticles were measured to further confirm their resistance to salinity. Figure 3B shows that the characteristic SPR peaks of the nanoparticles were slightly shifted to a higher wavelength and that their intensity was reduced. These characteristic SPR peaks of 5-FU loaded onto glutathioneGNPs and thioglycolic acid-GNPs disappeared at salt concentration as low as 50 mM confirming the better stability of 5-FU/CTAB-GNPs.40 Previous studies showed similar enhanced stability of CTAB-coated GNPs against salinity.51,52 Particle Size and Zeta Potential of the Nanoparticles. Nanoparticle stability and performance as drug delivery systems are affected by several parameters, the most important of which

Figure 7. (A) Growth curves of A431 tumors in C57BL/6 mice. (B) Weights of tumors at the end of the study. (C) Changes in the body weight of A431 tumor-bearing mice. Statistically significant differences were calculated using GraphPad Prism software. ***p < 0.001 compared with untreated control; *p < 0.05 compared with 5-FU GNPs gel. G

DOI: 10.1021/acs.molpharmaceut.8b00047 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 8. Representative images of tumor tissues excised from mice receiving different treatments after H&E staining (A, 10×; B, 40×). The red arrows show the change in epidermis thickness due to the infiltrating tumor cells. There was a marked increase in epidermis thickness in untreated group or groups treated with free 5-FU gel and cream. The groups treated with 5-FU/GNP gel and cream had remarkable regression of epidermal and dermal infiltration by the malignant cells.

ature and at 4 °C, and their properties were studied for up to 4 months. There was no significant change in particle size for the nanoparticles stored at 4 °C and at room temperature during the first month (p > 0.05) (Table 2). However, after one month, there was significant increase in particle size (p < 0.05) for samples stored at both temperatures, but the particle size was still under 20 nm (Table 2). Further, there was no significant change in zeta potential and drug entrapment efficiency for both nanoparticle samples during the whole study period (p > 0.05). No precipitation or aggregation was observed for all the tested samples. This excellent stability was further confirmed by UV−vis studies, which show that all the samples maintained their characteristic SPR peaks at the same λ, albeit with slight decrease in intensity after 4 months

negatively charged skin surface, which might result in enhanced drug efficacy.58 In order to test the nanoparticle stability under physiological salt concentration, size and zeta potential of 5-FU/CTABGNPs (5-FU/CTAB molar ratio: 0.6:1, pH 11.5) were measured in the presence of 150 mM NaCl. Data in Table 1 show a moderate decrease in the nanoparticle zeta potential and small increase in particle size (significant difference, p < 0.05). Sodium chloride might cause dehydration and neutralization of GNP surface charge leading to increased particle size and reduced zeta potential.59 Stability Study of 5-FU-Loaded GNPs. To confirm 5FU/CTAB-GNPs (drug/CTAB molar ratio: 0.6:1, pH 11.5) stability upon storage, aliquots were stored at room temperH

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mechanism is controlled by diffusion (Table 4). The Hixson− Crowell cube root model is applied to systems that dissolve or erode over time and takes account of the change in surface area and diameter over time. By fitting the release data to this model, the correlation coefficient of 0.995 was obtained for 5FU/CTAB-GNPs at pH 7. The Korsmeyer−Peppas release exponent (n) of most formulations was in the range of 0.5 < n < 1, which suggests anomalous (non-Fickian) diffusion controlled by a combination of Fickian diffusion and swelling due to relaxation of polymeric chains.34 Ex Vivo Skin Permeation Studies. In order for a given drug to be successfully delivered through the skin, it must overcome the stratum corneum (SC) barrier.65 SC limits drug permeability through skin to drugs having considerable lipophilicity (log octanol−water partition coefficient of 1−3), molecular weight of less than 500 Da, and melting point of less than 200 °C.65 5-FU has a log octanol−water partition coefficient of −0.89, which results in poor skin permeability.11,66 Figure 6 and Table 5 show the permeation of 5-FU from different preparations through mouse skin. Compared with the drug aqueous solution, 5-FU/CTAB complex and 5FU/CTAB-GNPs had significantly higher drug flux (p < 0.05), presumably due to the drug hydrophilic nature.10,11 Hydrophilic molecules are likely to permeate poorly via the intracellular route.67 Interestingly, 5-FU/CTAB-GNPs incorporated into gel and cream bases had significantly higher drug flux (p < 0.05) when compared with any of the other tested preparations. This might be attributed to neutralization of 5-FU charges upon complexation with CTAB-GNPs. Previous studies showed that ion pairing effectively increased the lipophilicity and transport rate of polar drugs across lipid membranes.68,69 In addition, GNPs were able to achieve substantial enhancement in drug delivery in the epidermis and dermis.23 GNPs penetration through the skin was found to be dependent on their size, shape, charge, and surface functionality.70 Small and positively charged GNPs had high skin permeability.16,71 Thus, Sonavane et al. showed that 15 nm GNPs had better permeation through rat skin and intestine compared with those having a size of 102 or 198 nm.71 GNPs might permeate the skin by shunt routes through appendages, such as hair follicles and sweat ducts. The shape of GNPs also affects their permeation properties through biological membranes. Fernandes et al. found that gold nanorods had better permeation through human and mouse skin compared with gold nanospheres having the same surface charge.70 Data in Figure 6 and Table 5 show that the cream preparations had significantly higher flux compared with their corresponding gel preparations. The opposite effect was expected due to the bioadhesive properties of Pluronic gel used in this study.72 Vanishing cream base used in this study is an oil/water emulsion. Complexation of 5-FU with CTABGNPs might help its partitioning in the oil phase of the cream leading to better skin permeability.73 In addition, glycerin used in the cream base was reported to have permeability enhancing properties.74 In Vivo Antiskin Cancer Studies. We used A431 xenograft-bearing mice to test the anticancer efficacy of 5FU/CTAB-GNP gel and cream formulations in comparison with the same preparations containing the drug alone. As shown in Figure 7A, A431 tumor cells grew aggressively in the negative control group. After 6 days of drug application (day 30), the tumor volume was lower in the groups receiving

(Figure 4). The good stability of 5-FU/CTAB-GNPs might be explained on the basis of strong interactions between 5-FU and CTAB-GNPs. Furthermore, the strong zeta potential of the nanoparticles help maintain their colloidal stability through electrostatic repulsion. In Vitro Drug Release Studies. The effect of pH on 5-FU release from its CTAB-GNPs is shown in Figure 5A. 5-FU aqueous solution, used as a control, had rapid and complete release in about 3 h. 5-FU complexation with CTAB-GNPs significantly slowed down its release at all the studied pH values (p < 0.05). Solution pH had a profound effect on 5-FU release rate. Slowest drug release rate was detected at pH 8, probably due to considerable ionization of 5-FU leading to tight ionic interactions with CTAB-GNPs.45,46 Decreasing the pH to 7 resulted in a significant increase in the drug release rate up to 12 h (p < 0.05), after which it was insignificantly different from that at pH 8. Further decrease in pH to 5 and 3 resulted in even faster drug release rate. After 24 h at pH 3, the drug was completely released from the nanoparticles. Faster 5-FU release in acidic medium might be attributed to reduced ionization of 5-FU leading to weakened ionic interactions with CTAB and, in turn, complex dissociation. Selected 5-FU/CTAB-GNPs (5-FU/CTAB molar ratio of 1:1, pH 11.5) were incorporated into Pluronic F-127 gel and vanishing cream bases, and their release properties were studied at pH 7.4 in comparison with the drug aqueous solution and the free drug gel and cream (Figure 5B). Slower release was observed for the free drug when incorporated into gel and cream (Figure 5B). Likewise, 5-FU/CTAB-GNPs incorporated into gel and cream bases had considerably slower drug release compared with the corresponding free drug gel and cream. After 24 h, the cumulative percent of 5-FU released from the nanoparticle gel and cream was significantly lower than that of the free drug gel and cream (p < 0.05). Furthermore, the cream had a significantly slower drug release compared with the gel for both free 5-FU and 5-FU/CTAB-GNPs (p < 0.05). Drug release rate from nanoparticles incorporated into a gel or cream base is dependent on its release rate from the nanoparticles and its subsequent diffusion through the matrix of gel or cream. In such cases, the viscosity of the gel or cream was reported to have an important effect on the drug release rate.60 Previous studies showed that drug release rate was inversely proportional to the viscosity of the gel or cream.61,62 Viscosity of the prepared cream was almost twice that of the gel (Table 3). This might explain, at least in part, the slower release rate observed for free 5-FU and 5-FU/CTAB-GNP cream compared to the corresponding gel preparation. Kinetic Analysis of Drug Release Data. Drug release data was fitted using various models to determine the exact release mechanism. GNPs are not polymeric systems, yet when they are confined within a dialysis membrane their drug release patterns might be modeled similar to a degradable polymeric system.63 According to the results, drug release from 5-FU/ CTAB-GNP solutions followed the Higuchi model as indicated by the highest correlation coefficients, except 5-FU/CTABGNPs solution at pH 8, which followed the zero-order model (Table 4). Drug release data from all GNP gel and cream formulations were best fit by the zero-order model. The Baker and Lonsdale drug release model is applied to the spherical matrix where the drug release takes place by diffusion.64 By fitting 5-FU release data to this model, high correlation coefficients (0.985−0.998) were obtained for 5-FU/ CTAB-GNPs solution at pH 3 and 5 indicating that the release I

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entrapment efficiency was affected by solution pH and 5-FU/ CTAB molar ratio with maximum entrapment being observed at pH 11.5 and molar ratio of 1:1. 5-FU/CTAB-GNPs had good stability against salinity and after storage for 4 months at room temperature and at 4 °C. 5-FU release was pH dependent where faster release was observed in acidic media. Optimal drug-loaded GNPs formulations (5-FU/CTAB: 1:1, pH 11.5) were incorporated into gel and cream bases. Higher ex vivo drug permeability through mice dorsal skin was observed for 5-FU/ CTAB-GNP cream compared with all other tested preparations. Same cream preparation achieved the highest antiskin cancer effect, in vivo as evidenced by smallest tumor volume and weight. These results are promising, given the possibility of topical application of anticancer drugs with reduced systemic side effects, enhanced therapeutic outcome, and better patient compliance.

different 5-FU formulations compared with the untreated control. For 5-FU/GNPs gel and cream, the tumor volume started to decrease after 8 days (day 32) of treatment. During these initial 8 days, the permeated 5-FU might have not been enough to suppress the multiplication of cancerous cells. The cream or gel containing 5-FU alone very significantly reduced the tumor volume (p < 0.01). Thus, after 29 days of treatment the tumor volume was 1.7- and 2.2-fold lower in the mice treated with 5-FU gel and cream, respectively, compared with the negative control. Furthermore, 5-FU GNPs cream and gel were very significantly more efficacious in inhibiting the tumor growth compared with the negative control or the formulations containing the drug alone (p < 0.01). Thus, the GNPs gel and cream achieved 6.8- and 18.4-fold lower tumor volume compared with the negative control, respectively. The extraordinarily better effect observed for 5-FU/CTAB-GNP gel and cream might be related to the ability of GNPs to enhance drug penetration through skin, as well as CTAB ionic and hydrophobic interactions with the skin.75 Better anticancer efficacy observed for the cream compared with the gel formulations corroborates the results described above for the skin permeability studies. At the end of the study, the mice were euthanized, and tumor tissues were collected and weighed.14 Tumor weight followed the same trend as the tumor volume. Thus, the mice treated with 5-FU/CTAB-GNP cream had very significantly smaller tumor weight compared to all other groups (p < 0.01) except the 5-FU/CTAB-GNP gel group where the difference was significant at p < 0.05 (Figure 7B). The body weights of the mice were also monitored during the treatment period (Figure 7C). Weights of untreated mice or those treated with free 5-FU gel and cream were significantly smaller than those of mice treated with 5-FU/CTAB-GNPs cream or gel (p < 0.05). This confirms the general good health of mice receiving 5-FU GNP cream and gel. The tumor tissues were also examined microscopically (Figure 8). H&E staining revealed increased thickness of the epidermis by the infiltration of tumor cells and marked pleomorphism of the cells with hyperchromatic nuclei and mitoses in the negative control group (untreated mice). Mice treated with either the gel or cream containing the drug alone showed minimal and moderate reduction in the pathological features, respectively. Interestingly, remarkable reduction of the pathological changes and regression of epidermal and dermal infiltration by the malignant cells were observed in the mice treated by 5-FU/CTAB-GNPs gel. More reduction of the pathological changes and near complete regression of epidermal and dermal infiltrations were seen in mice treated by 5-FU/ CTAB-GNP cream. Taken together, these in vivo results confirm the enhanced efficacy of topical 5-FU GNP gel and cream formulations against skin cancer. Topical 5-FU delivery might reduce its systemic adverse effects with subsequent improvement in patient compliance. These interesting results are thought to be due to charge neutralization of 5-FU upon complexation with CTAB-GNPs. Positive charge of these nanoparticles in addition to their small size might have also contributed to their enhanced skin penetration.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +201013427311. E-mail: [email protected]. ORCID

Ghareb M. Soliman: 0000-0002-9674-1254 Notes

The authors declare no competing financial interest.

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REFERENCES

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CONCLUSION GNPs were successfully prepared using NaBH4 as a reducing agent and CTAB as a stabilizing agent. CTAB was also used to load 5-FU at pH ≥ 8.5 through electrostatic interactions. 5-FU J

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