Fluorouracil-Loaded Gold Nanoparticles for The Treatment of Skin

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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 Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00047 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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Molecular Pharmaceutics

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§,ǂ, Ghareb M. Soliman§,*, Douaa Sayedǁ, Mohamed A. Attia§

§

ǂ

Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt

Department of Pharmaceutics, Faculty of Pharmacy, South Valley University, Qena, Egypt

ǁ

Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University, Assiut,

Egypt

*Corresponding author. Tel. +201013427311, E-mail: [email protected]

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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 is was loaded into a gold nanoparticles (GNPs)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. GNPs 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 5-FU 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 GNPs 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 GNPs 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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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,

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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 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 dermatological origin due to off-target cytotoxic effects of 5-FU.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,

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Several delivery systems having 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 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 ACS Paragon Plus Environment

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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 disulphide 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. Optimized 5-FU/CTAB-GNPs formulation was incorporated into a cream and gel bases and their ex vivo drug permeability through mice skin was tested. Anticancer efficacy of 5-FU/CTABGNPs cream and gel was tested, in vivo in a mouse skin cancer model. Experimental section Materials 5-Flourouracil 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


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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 CTAB-coated GNPs were separated by centrifugation at 14,000 RPM using a Microlitre centrifuge, Micro 200R, Germany for 1 h at 25 °C. 5-FU was loaded through electrostatic interactions between CTAB cationic charges and 5-FU anionic ones. Aliquots of 5-FU 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) 5FU in the supernatant was assayed spectrophotometrically at 266 nm. 5-FU entrapment efficiency (EE%) was calculated from equation (1). UV-Vis spectra of samples were recorded on a UV-Vis Spectrophotometer (Shimadzu-50-02, Kyoto, Japan) in the range of 500-700 nm. 5-FU EE (%) =

× 100

(1)

The effect of pH on 5-FU entrapment efficiency was evaluated by adjusting the pH of 5FU/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


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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 GNPs 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 (NanoZS, 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/CTAB-GNPs (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. ACS Paragon Plus Environment

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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 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 non-boiling 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 water bath and mixed with CTAB-GNPs loaded with 100 mg 5FU 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 5-FU released was plotted ACS Paragon Plus Environment

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vs time. For 5-FU/CTAB-GNPs gel and cream, a one-gram sample containing 10 mg 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 5-FU 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 5-FU/CTABGNPs gel or cream (1 g equivalent to 10 mg of 5-FU) 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 ACS Paragon Plus Environment

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(Jss) was calculated from the slope of the linear regression line.35 The curves had good linearity as indicated by their high correlation coefficient (0.995-0.968). The apparent permeability coefficient (Papp) was calculated using the following equation: Papp =

(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 (%) =

(") # $ (%)

(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 mice was injected subcutaneously with 400,000 cells in 400 µL phosphate buffer in the hair-trimmed area on day 0. On day 24 when palpable tumors were developed, mice were randomly divided into 5 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 5-FU/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:


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Tumor volume (mm

.

)=

/ ×1 ×1 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 results are shown as mean ± SD. Data analysis was carried out using GraphPad Prism software version 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 post-hoc test. Results and discussion Synthesis of CTAB-capped GNPs GNPs were prepared using NaBH4 as a reducing agent due to its ability to produce nanoparticles of small size and good stability.37 CTAB was a 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 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 CTAB-GNPs 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. ACS Paragon Plus Environment

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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 facilitate 5-FU loading through ionic interactions between 5-FU anionic charges and cationic ones of CTAB. CTAB is 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 5-FU 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.


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CTAB-coated GNPs

5-FU-loaded GNPs

1.8 1.6

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


B

525 nm

1.4 1.2

CTAB coated GNPs 5-FU/CTAB coated GNPs GNPs without CTAB

C

527 nm

1.0 0.8 0.6 0.4 0.2 500

550

600

650

700

Wavelength (nm)

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).


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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 5-FU/CTAB molar ratios. 5-FU pKa values are 8 and 13, which results 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 Next, the nanoparticles were prepared at different 5FU/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 5FU/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.


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80 5-FU entrapment efficiency (%)

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pH 5 pH 7 pH 8.5 pH 9.5 pH 10.5 pH 11.5

60

40

20

0 0.6:1

1:1 1.3:1 2:1 5-FU/CTAB molar ratio

4:1

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

Ionic strength studies Figure 3 shows that the drug entrapment efficiency decreased insignificantly upon adding 50 mM NaCl (p>0.05). Salts concentrations above 50 mM resulted in significant decrease in drug entrapment efficiency (p

Fluorouracil-Loaded Gold Nanoparticles for The Treatment of Skin

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