Methacrylate Stitched Î²-Cyclodextrin Embedded with Nanogold

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Methacrylate Stitched #-Cyclodextrin Embedded with Nanogold/NanotitaniaA Skin Adhesive Device for Enhanced Transdermal Drug Delivery Thayyath Sreenivasan Anirudhan, Syam S. Nair, and Athira V Sasidharan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16686 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

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ACS Applied Materials & Interfaces

Methacrylate Stitched β-Cyclodextrin Embedded with Nanogold/NanotitaniaA Skin Adhesive Device for Enhanced Transdermal Drug Delivery T. S. Anirudhan*, Syam S. Nair, Athira V. Sasidharan Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom, Trivandrum-695 581, India

Keywords Gold nanoparticle, Titanium nanotube, β Cyclodextrin, Methacrylate, Transdermal delivery *Corresponding author: Tel: +91 471 2308682 E mail address: [email protected]

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Abstract Transdermal (TD) drug delivery is an attractive technique for drug delivery compared to oral and intravenous injection. However the permeation of drug molecules across the skin is difficult due to the presence of highly ordered lipid barrier. This investigation details the development of a novel TD system, which has the potential to simultaneously enhance the skin permeability and adhesion behavior. Ibuprofen (IP) was selected as model drug. The ability of gold nanoparticle (AuNP) and hydrophobic titanium nanotube (TNT) to enhance the skin permeability was explored. Additionally, β-cyclodextrin (βCD) which can exceptionally encapsulate poorly water soluble drugs is grafted with methacrylates to improve the skin adhesion property. Finally AuTNT nanocomposite was deposited onto methacrylate grafted βCD matrix. The developed material was characterized through NMR, FTIR, SEM, TEM, XRD, and Raman spectra. The characteristics of the film including water vapor permeability (WVP), thermo mechanical properties, etc. were examined in terms of Au-TNT content. The TD delivery of IP with different concentrations of Au-TNT was evaluated via an in vitro skin permeation study through rat skin. It is revealed that the prepared TD film exhibited an improved drug delivery performance due the synergistic action of AuNP and hydrophobic TNT. The cumulative percent of IP delivered across the skin is extremely depending on nanofiller content, lipophilicity and thickness of the membrane and the device incorporated with 4.0 % Au-TNT displayed the best performance. In addition a study on storage stability was performed by storing the films for two months at different temperatures. The study revealed that the device possessed excellent storage stability when stored at low temperature. The developed film offers excellent WVP, drug encapsulation efficiency (DEE), thermomechanical properties and skin adhesion behavior. Moreover the device was cosmetically attractive, non-cytotoxic and resistant to microbial growth, hence extremely 2 ACS Paragon Plus Environment

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reliable for skin application. The developed skin permeation strategy may open new avenues in TD drug delivery.



Introduction Among numerous viable routes of administration, the therapeutic molecules are predominantly delivered either orally or by hypodermic injection.1 Even if oral route is the most convenient and carries the lowest cost, the first pass hepatic metabolism significantly reduce the bioavailability of several orally administered drugs.2 Parenteral injection is more agreeable and can remarkably overcome the above mentioned drawbacks by administering molecules straight into the blood stream.1 Nonetheless injection induces pain, infection, phlebitis, hypothermia etc.3 In this scenario TD route of drug delivery is explored. TD drug delivery involves the administration of drug molecules across the skin for systemic distribution. TD route appears more attractive as it is non-invasive, avoids first pass hepatic metabolism, provides the possibility of self administration, easy termination of therapy etc.4 Despite its exceptional possibilities, the development of a TD device is challenging, ascribed to the extremely ordered structure of outermost layer of skin, stratum corneum (SC).5 From a global point of view, TD drug delivery system has been evolved in three generations. The first generation device contains drug molecules which can easily penetrate across the skin with little or no enhancement strategies. The candidates of second generation include conventional chemical enhancers, non-cavitational ultrasound and iontophoresis. The systems of third generation target their effects to SC and enable the skin transportation of drug molecules using electroporation, thermal ablation and microneedles.3,6,7 However, these systems failed in attaining enhanced permeation, while protecting deeper skin layers from injury. In this context, we exploit AuNP as a safe and potent penetration enhancer to overcome the skin barrier functions. Previous studies revealed that the interaction of AuNP with lipid blocks of SC


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creates transient and reversible pores which stimulate the effortless permeation of drugs into deeper skin layers.8 Recently, Chen et al.9 enhanced the transdermal delivery of vascular endothelial growth factor using surface modified gold nanoparticles. Lee et al.10 utilized surface engineered AuNPs for the transdermal treatment of surgical wounds. In the present investigation the capacity of AuNP to induce lipid modulation was used to deliver IP, a model lipophilic drug. Among the polysaccharides, βCD plays a crucial part in drug delivery applications and could be remarkably preferable in the fabrication of TD film.11 These biocompatible, non-toxic, cyclic polysaccharides have exceptional potential to solubilize lipophilic drugs via molecular encapsulation. The superior solubilization capacity of this biopolymer permits a substantial amount of the drug molecules to be encapsulated and is hypothesized to enhance the skin permeability.12 Literatures revealed that silicones and polyisobutylenes are commonly employed adhesives to fabricate TD devices.13 We display in this investigation that methacrylates which offer excellent flexibility and transparency are exceptionally suited for the development of TD film with improved adhesion behavior. In contrast to silicones and polyisobutylenes whose pretty limited properties prevent their application in vivo, methacrylates seems to be comparatively suitable. Herein we grafted βCD with glycidyl methacrylate (GMA) and butyl methacrylate (BMA) and research the possibility of using them as adhesion enhancing agents. Uekama et al.14 in an article specified the potential utilization of βCD in drug delivery systems thanks to the excellent physicochemical behavior and encapsulation capacity. However its poor tensile strength significantly restricts their in vivo topical applications. The chemically modified derivatives of polysaccharides using synthetic monomers yet not produce any encouraging enhancement in mechanical properties. The ultimate beneficial section of composites research comprises the mechanical improvement of films using nanometric particles as reinforcing 5 ACS Paragon Plus Environment


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phase.15 TNTs have attracted considerable recognition as excellent building blocks to enhance the tensile properties and drug encapsulation behavior. This behavior ascribed to their improved Young’s modulus and hollow cylindrical nano architecture.16,17 Nevertheless the as prepared TNTs precipitates briskly in organic solvents since it do not produce stable suspensions. Hence it is necessary to functionalize TNT surface with hydrophobic moieties such as oleic acid (OA). OA is a biocompatible fatty acid commonly used to fabricate devices for the delivery of lipophilic molecules.18 Such chemical modification is critical not only to improve the solubility of suspension in organic solvents but also enhance the encapsulation of poorly water soluble drugs. Presently, our attention of is lying on the development of methacrylate grafted βCD film reinforced with Au-TNT nanocomposite. The novelty of the investigation is that although AuNP have been displayed to bypass SC barrier, the present work aimed to fabricate a TD device employing the synergistic effect of AuNP and hydrophobic TiO2 nanoparticles. To the best of our knowledge, the skin penetration enhancement using the synergistic effect of AuNP and hydrophobic TNT has not been reported earlier. In addition the device has the potential to simultaneously enhance both skin permeation and adhesion behavior. The possibility of TD delivery of IP in vitro was evaluated by application of the device in rat skin. In addition the physicochemical behavior and the stability of patch over time and temperature were examined. EXPERIMENTAL SECTION Materials Gold(III) chloride trihydrate (HAuCl4.3H2O), sodium citrate dihydrate (Na3Cit), (3Aminopropyl)triethoxysilane

(APTEOS),

1-ethyl-3-(3-dimethylaminopropyl) 6

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carbodiimide

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(EDC), N-hydroxysuccinimide (NHS), and 2,21-azobis(2-methylpropionitrile) (AIBN) were purchased from Sigma-Aldrich (USA). GMA, BMA, and IP were supplied by Tokyo Chemical Industry (Japan). Titanium dioxide, OA, NaOH and all organic solvents were obtained from Merck specialties Pvt Ltd (Mumbai). All aqueous solutions were prepared using deionized water. Methods Synthesis of TNT TNT was prepared by alkaline hydrothermal method.19 Briefly 10.0 g TiO2 (anatase) was added to 100.0 mL 10.0 M NaOH solution and stirred for 1 h. The obtained suspension was placed in a poly(tetrafluoroethylene) lined autoclave maintained at 135 0C for 72 h. The product was washed thrice with water and 0.1 M HNO3 and dried at 100 0C. Finally it was calcined at 400 0C for 2 h, grinded and sieved in a 200 mesh. Synthesis of Aminated TNT (APTEOS-TNT) Briefly, 0.05 g TNT and 40.0 mL ethanol was mixed thoroughly for 2 h. Next 200.0 µL of APTEOS was added drop wise into the mixed solution, followed by the addition of 2.0 mL each of water and 5.0 % ammonia. The obtained solution was further stirred for 12 h at room temperature. Finally the precipitate was collected by centrifugation and washed with ethanol and water thrice to eliminate the unreacted APTEOS.20 Synthesis of Oleic Acid Functionalized TNT (OA- APTEOS-TNT) OA modified TNT was prepared by carbodiimide mediated coupling of APTEOS-TNT with OA. Typically, a solution of OA (0.3 mmol) in DMF was prepared followed by adding NHS (0.5 mmol) and EDC (0.5 mmol). The solution was stirred for 1 h at ambient temperature. After stirring, 0.2 mmol APTEOS-TNT was introduced drop wise into the mixture and allowed to react 7 ACS Paragon Plus Environment


for 24 h under nitrogen gas. Finally, the product was dialyzed to remove unreacted species, lyophilized and stored.21 Following the same procedure, OA- APTEOS-TNT samples with the addition of 0.1 and 0.2 mmol OA was also synthesized to investigate the effect of oleyl functionalization on drug release profile. Synthesis of AuNP/OA- APTEOS-TNT (Au-TNT) A solution of 8.0 mL OA coated TNT (23.0 mg/mL) in hexane and an aqueous solution of HAuCl4. 3H2O (0.5 mL, 20.0mM) was mixed thoroughly.22 The mixture was then reduced by adding 1.00 mL 0.1 M aqueous NaBH4 in a drop wise manner to complete the reduction of Au (III) ions. The suspension was maintained for 2 h at 60 0C. The developed Au-TNT was centrifuged and then washed with deionized water until a colorless supernatant was obtained. Synthesis of GMA Grafted βCD (CDG) CDG was synthesized through the reaction of βCD and GMA in the presence of NaOH.23 Briefly a 5.0 wt % solution of βCD was prepared in 100.0 mL dimethyl formamide containing 0.5 M NaCl and alkalinized with 5.0 mL 10.0 % NaOH. The reaction mixture was magnetically stirred for 1 h. After thorough mixing 3.5 wt % GMA was added drop wise. The reaction was allowed to carry out for 24 h maintained at 70-80 0C. The product was washed with 0.9 % NaCl solution at 70-80 0C and finally dried at room temperature. Synthesis of BMA Grafted CDG (CDGB) and Preparation of Au-TNT Reinforced TD film BMA was introduced into CDG using AIBN as initiator. The polymerization was performed in a 3-necked RB flask fitted with nitrogen inlet containing 3:7 THF and water. To the solvent medium, CDG and BMA were added at 1:1 Molar ratio and 1.0 wt % AIBN and stirred at 70 0C


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for 3 h. The product was purified by several precipitation operations into acetonitrile after dissolving in THF.24 To 0.02 g CDGB in 10.0 mL DMSO, predetermined amount of Au-TNT was added to obtain film with 1.0, 4.0, 7.0 and 10.0 % nanofillers abbreviated as CDGB1, CDGB4, CDGB7 and CDGB10, respectively followed by the addition of IP (5.0 wt %). The resulting mixture was ultrasonicated for 15 minutes to obtain sufficient drug-matrix interaction. The entire mixture was then transferred into a petri dish and finally solvent casted to obtain TD film. The percentage grafting of GMA on the backbone of βCD was determined using the following equation

………………………………….............................. (1)

where m1 and m2 are the weight of initial substrate and grafted copolymer, respectively. Next the influence of βCD concentration on the grafting percentage was investigated. Details of experiment were given in the supporting information. The drug encapsulation efficiency (DEE) of each device was investigated spectrophotometrically as described by our previous study at 221 nm.25

* 100 …………………………………... (2)

DEE (%) =

Characterization and Analysis 1

H NMR spectrum was recorded on a Bruker Avance 500 MHz spectrometer at room

temperature in CD3OD solvent. The success of the chemical reactions was also confirmed by infrared analysis recorded using Agilent Cary ATR spectrometer in the range 4000-650 cm-1. The mass of CDG was characterized using matrix-assisted laser desorption/ionization time-of9 ACS Paragon Plus Environment


flight mass spectrometry (MALDI-TOF-MS, UltrafleXtreme, Bruker Daltonic) with 2,5dihydroxybenzoic acid as matrix. The molecular weight of CDGB was investigated by Gel permeation chromatography (Agilent Technologies-1260 instrument equipped with a PL-gel 20 µm MIXED-B column and RI detector) using tetrahydrofuran as eluent. The formation of AuTNT was visualized through TEM image recorded using FEI, TEC-NAIS Twin microscopy operating at 100 KV. The test sample was subjected to ultrasonication, then drop casted onto formvar coated copper grid and dried before analysis. The energy dispersive X-ray (EDX) elemental mapping was performed using Nova Nano SEM NPEP 252 equipped with EDX spectrometer. X-ray diffraction (XRD) profiles were recorded with Siemen’s D5000 X-ray diffractometer (Germany) with Ni filtered Cu Kα radiation with the X-ray tube operating at 40 kV and 30 mA. Raman spectra were recorded with a micro-Raman spectrometer Lab Ram UV HR, Jobin-Yvon. The surface morphology of the prepared samples was viewed using Carl Zeiss EVO 18 Scanning Electron Microscope (SEM) at an acceleration voltage of 15 KV. The FESEM images of test films were obtained using Nova Nano SEM NPEP 252 high resolution FEscanning electron microscope. The thickness of the prepared membranes was measured using thickness gauge (S. C. Dey & CO, India). Tensile measurements were performed on rectangular membrane samples using Hounsfield universal testing machine at room temperature. To investigate the influence of storage time on mechanical properties the measurements were also conducted for membranes stored for 30 and 60 days at room temperature. The influence of nanofiller loading on the thermal stability of the prepared membranes was studied using thermograms. The thermogravimetric analysis (TGA) was conducted using TG analyser (TA Q50) under nitrogen purge and 10 0C heating rate. The measurements were recorded in the temperature range 30-800 0C. Next the WVP, optical transmittance, in vitro skin permeation,


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skin adhesion properties, cell viability and environmental fitness of the developed films were evaluated. The details of these experiments were provided in supporting information. RESULTS AND DISCUSSION The fabrication of TD film consists of three main episodes. First TNTs were synthesized using the method reported by Kolen'ko et al.19 To improve its compatibility with CDGB matrix and enhance the solubility of poorly water soluble IP; TNTs were hydrophobically coated using OA. To conjugate OA, TNTs were first amine functionalized by reacting with APTEOS. Hydrophobic TNT was then prepared by covalently conjugating OA to amine functionalized TNT using EDC-NHS coupling. The schematic representation of the synthesis of OA-APTEOSTNT is presented in Figure 1A. The evidence supporting the successful synthesis of hydrophobic TNT and Au-TNT was collected from the ATR spectra presented in Figure 1C. The infrared signals of synthesized TNT are well in line as reported earlier.26 In ATR spectrum of APTEOSTNT, the development of overlapped bands that elongated from 1000 to 900 cm-1 confirmed the occurrence of an organic-inorganic connection. Moreover a sharp peak ascribed to O-Si asymmetric vibration was also visible at 1150 cm-1.27 As presented in the spectrum of OAAPTEOS-TNT, the characteristic stretching vibration band of -CO- appeared at 1717 cm-1 and CH2 anti-symmetric and symmetric modes of OA at 2920 and 2850 cm-1 respectively evidenced the successful synthesis of OA-APTEOS-TNT.



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APTEOS-TNT

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Figure 1 Schematics of the synthesis of (A) OA-APTEOS-TNT and (B) Au-TNT, (C) ATR spectra of TNT, APTEOS-TNT, OA-APTEOS-TNT and Au-TNT. Next Au-TNT was synthesized following previous report with some modifications.22 The advance in Au (III) reduction was clearly visualized by the remarkable color change of the mixture from yellow to wine-red. The schematic representation of the synthesis of Au-TNT is displayed in Figure 1B. The attachment of AuNP to TNT surface was confirmed using TEM, XRD, EDX elemental mapping, Raman, ATR and UV-visible spectroscopy.


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Figure 2 (A) TEM image of Au-TNT, (B) High resolution TEM image of Au-TNT (C) Raman spectra of (a) Hydrophobic TNT and (b) Au-TNT and (D) XRD profiles of (a) AuNP (b) Hydrophobic TNT and (c) Au-TNT. TEM image of Au-TNT shown in Figure 2A demonstrated the presence of AuNP of 10-12 nm size along the tubular particle of TNT of length approximately 200 nm. The titania tubes were significantly covered with AuNPs, which enhanced the skin permeability and anti-microbial properties of the film as elucidated later. The Raman spectra of pure TNT and Au-TNT are 13 ACS Paragon Plus Environment


presented in figure 2B. Pure TNT displayed characteristic peaks centered at 140, 398, 514 and 640 cm-1 assigned to the anatase phase of TNT.28 Au-TNT showed a remarkably enhanced Raman band intensity than that observed in TNT. Besides, the characteristic Raman features of TNT, Au-TNT clearly showed slight wave number shift upon decoration with AuNP. The interfacial interaction between AuNP and TNT is accountable for the significant rise in intensity and wave number shift.29 XRD profile presented in Figure 2D further confirmed the successful synthesis of Au-TNT. Pure AuNP displayed characteristics peaks at 38, 43, 64 and 770 indexed to (111), (200), (220) and (311), respectively.30 Hydrophobic TNT displayed characteristics signals at 2θ values 25, 38, 48 and 540 indexed to (101), (004), (200) and (105), respectively.28 The Incorporation of AuNP also produced a near XRD pattern to that of hydrophobic TNT. However the appearance of additional peak at 430 (marked with asterisk) ascribed to the fcc of Au, confirmed the successful preparation of Au-TNT.30 In the ATR spectra of Au-TNT, a slight blue shift in the position of –CO- band from 1650 t0 1624 cm-1 is observed in addition to the reduction in peak intensity. This is ascribed to the favorable interaction between oleyl functionalized TNT and AuNP.31 The energy dispersive X-ray (EDX) spectrum (Figure S1, Supporting information) clearly revealed the existence of AuNP on the surface of TNT. The characteristic Au peak appeared at 2.12 keV indicated that AuNP was successfully deposited on TNT surface. In addition, other peaks appeared at 0.27, 0.39, 0.51 and 1.74 keV respectively attributed to the presence of C, N, O and Si. The peaks corresponding to Ti was observed at 4.52 and 4.94 keV. The elemental composition obtained by EDX mapping was presented in Table S1, Supporting information. The EDX data revealed that approximately 3.1 % of AuNP was deposited on TNT surface. The UV-visible spectra of pure AuNP and Au-TNT were presented in Figure S2, Supporting information. Pure AuNP showed an intense absorption signal at 520 nm


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attributed to the surface plasmon excitation of smaller Au particles.30 The red shift and broadening of surface plasmon resonance band of Au particles appeared in the spectrum of AuTNT supported the successful formation of composite. The second step involves the modification of βCD with GMA and BMA to enhance its skin adhesion behavior (Figure 3A). GMA has two polymerizable functionalities including epoxide and methacrylate. In the presence of NaOH, the hydroxyl group of βCD reacted with epoxide part of GMA generating stable ether bond with a percentage grafting of 93.4 %. The synthesis of CDG was confirmed by ATR, NMR and MALDI mass spectrum presented in Figure 4 (A-C). Besides the characteristic signals of βCD, the appearance of peaks centered around 1579 and 1117 cm-1 corresponds to C=C stretching vibrations and carbonyl group, respectively, furnished the evidence of successful grafting of GMA onto βCD.25 The MALDI-TOF mass spectrum displayed in Figure 4B revealed that the molecular weight of CDG is 2129.288 g/mol. The 1H NMR spectrum of CDG is presented in Figure 4C. The characteristic NMR signals for the ring protons of βCD are appeared between 3.1 to 4.8 ppm.32 GMA grafting was achieved, as evidenced by signals appeared at 5.8 and 6.0 ppm ascribed to the protons on the vinyl carbon and a signal pointed at 2.4 ppm ascribed to the methyl group on the methacylate end.33 Moreover the absence of resonance peak of epoxide normally appeared between 2.4 and 2.9 ppm substantiated the generation of ether bond between βCD and GMA by breaking the epoxide moiety of GMA.34 The as prepared CDG carried polymerizable methacrylate termination which provides direct polymerization sites for BMA. The polymerization initiated by AIBN produced bioadhesive CDGB film. The molecular weight of CDGB was investigated by gel permeation chromatography and found to be 12.4 kDa (Figure S3, Supporting information). The combination of ATR (Figure 4A) and 1H NMR (Figure 4D) data furnished strong evidence for 15 ACS Paragon Plus Environment


the successful synthesis of CDGB. The disappearance of ATR signal pointed around 1579 cm-1, characteristics of C=C bond confirmed the successful synthesis of CDGB.24 The characteristic resonance of –O-CH2 observed at 4.10 ppm substantiated the grafting of GMA and BMA onto βCD backbone. In addition appearance of peak at 1.27 ppm corresponds to the CH3 protons of BMA and that at 0.90 ppm represents the methyl group linked near to carbonyl carbon of BMA and GMA. The retention of signals of the ring protons of βCD and emergence of the characteristic peaks of BMA and GMA supported the claim that GMA and BMA was effectively incorporated into βCD backbone. Finally IP encapsulated TD devices based on as synthesized CDGB reinforced with Au-TNT with average thickness of about 0.082 ± 0.01 nm were obtained on petri plates through solution casting method.


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Figure 3 Schematics of the (A) synthesis of CDGB, (B) In vitro skin permeation test using Franz diffusion cell and (C) Transdermal release of drug.



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50

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Figure 4 (A) ATR spectra of βCD, CDG and CDGB, (B) MALDI-TOF mass spectrum of CDG, (C) 1H NMR spectrum of CDG and (D) CDGB and (E) Effect of βCD concentration on grafting percentage.


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Next the influence of βCD concentration on the grafting percentage was investigated. In the presence of NaOH, βCD at different weight ratios were grafted to GMA-BMA backbone at 70 0C at fixed monomer concentration, total reaction volume and time. The results were summarized in Figure 4E. The grafting percent showed an increasing trend on increasing the βCD concentration to a certain value and is ascribed to the increase in the availability of primary hydroxyl group that can react with epoxide moiety of GMA-BMA backbone. The grafting percent was maximum (56.0 %) when the weight ratio of βCD increased to 1:4. The decrease in grafting percent at higher feed of βCD may be due to the increase in viscosity of the reaction medium, which reduce the rate of diffusion of molecules to the active sites.35 Characterization of Au-TNT Reinforced Films SEM micrograph of βCD (Figure 5A) clearly presented particles with irregular size. In CDG, the particles closely clumped together into clusters with rough surface. On further reaction with BMA, the clumped mass turned to a sheet like morphology. To clearly visualize the surface morphological changes during the preparation of composite films, FESEM studies were performed. The dispersed Au-TNTs were observed as bright spot in the relatively coarse film matrix. The film surface of CDGB1 contained only limited bigger particles whereas tiny particles appeared as bright dots arranged randomly in the film matrix. The dark phase of the polymer matrix was plainly noticed as the concentration of Au-TNT was minimum. Figure 5E distinctly displayed that a uniform distribution of nanofillers was accomplished throughout the film matrix at 4.0 % loading of Au-TNT. Beyond this nanofiller loading, some aggregation of Au-TNTs was observed. Finally, an appreciable fragment of Au-TNT aggregate absolutely and cohere after the incorporation of 10.0 % Au-TNT (Figure 5G).



Figure 5 SEM images of (A) βCD, (B) CDG, and (C) CDGB, FESEM images of (D) CDGB1, (E) CDGB4, (F) CDGB7 and (G) CDGB10. With the intention to investigate the mechanical and reinforcing effect of Au-TNT, we compared the tensile properties of Au-TNT loaded films with that of its neat counterpart. The mechanical test results were plotted in Figure 6(A&B). Among the test samples neat CDGB possessed 20 ACS Paragon Plus Environment

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poorest mechanical properties as its tensile strength (TS) was only around 46.0 MPa. With the incorporation of 1.0 and 4.0 % Au-TNT, the TS increased remarkably and are ascribed to several reasons. First homogeneous dispersion of Au-TNT produced favorable matrix-filler interaction.36 Second, the productive load transfer from matrix to Au-TNT, developing homogeneous stress distribution and lowering of stress concentration area.37 Furthermore it is interesting to observe that loading of 7.0 and 10.0 % Au-TNT did not enhance the TS. The agglomeration of Au-TNT at higher concentration produced lower rate of load transfer which reduced the TS to 62.0 and 58.0 MPa respectively for CDGB7 and CDGB10. These tensile results are comparable with TD films reported earlier.38,39 Figure 6B depicted that the Eb values decreased slightly with the increase in loading level of Au-TNT. The reduction is ascribed to the limited motion of polymer chain arose due to matrix-filler interaction.40 Even though the incorporation of Au-TNT reduced the Eb, the values were not too much less than the neat counterpart. The impact of storage time on the mechanical behavior of the developed films was also investigated and results are depicted in Figures 6(A&B). As the storage time increased, the Eb values were reduced marginally and that was described as follows. The storage of films for few months significantly changes the polymeric structure predominantly in the amorphous region and is



Figure 6 (A) Tensile strength, (B) Elongation at break, (C) Water vapor permeability and (D) Thermogram of CDGB, CDGB1, CDGB4, CDGB7 and CDGB10. specified in terms of free volume. Upon storage, the exceptional relaxation occurred on the internodular segments reduces the free volume and hence decreases the Eb values.41 The TS values for membranes kept for 30 and 60 days were 81.0 and 85.0 MPa, respectively. The remarkable increase in the TS upon storage was due to efficient packing of segments leading to improved nodular packing density. Hence an appreciable force is required for the mobility of molecular segments, resulted in increased TS.42


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A perfect TD device should sustain evaporative moisture loss from skin surface at an optimal rate. British Pharmacopoeia recommended that a membrane possessing a WVP above 0.05 g cm2 24 h-1 could be scrutinized as moisture permeable without risking skin occlusion and microbial growth.43 The calculated values of WVP of various TD films are summarized in Figure 6C. The addition of 1.0 % Au-TNT resulted in no substantial lowering of WVP of the device. But higher concentration of Au-TNT significantly reduced the WVP. This effect is predicted as hydrophobically modified TNT generated a physical barrier to the diffusion of water vapor. Moreover the homogeneous distribution of Au-TNT created a winding track for piercing water molecules. This phenomenon produced a significant enhancement in mean free path for the diffusing water molecules.44 Figure 6C indicated that the WVP was minimum for CDGB10 and is attributed to the extensive agglomeration of Au-TNT. All the prepared films displayed WVP values within the range suggested by British Pharmacopoeia and are ideal as TD film in terms of WVP. The thermogravimetric profiles of CDGB and Au-TNT incorporated films are presented in Figure 6D. All the samples displayed three main weight loss episodes. The first event happened over the temperature ranging between 80 and 140 0C, attributed to the evaporation of adsorbed moisture. The most significant event appeared between 200 and 400 0C, accounted for about 55.0 % mass loss and is ascribed to the degradation of polymer chains. The final transition was occurred around a temperature above 500 0C accredited to the degradation of carbonaceous fragments.45 Figure 6D, depicted that including three transitions, a sum of around 85.0 % mass was lost from the materials. Thermograms evidently signalized the variation in thermal properties of the film upon the incorporation of Au-TNT. The thermal degradation temperature and residue yield of the test 23 ACS Paragon Plus Environment


samples was increased with the addition of nanofillers. The increasing effect was more pronounced in the case of CDGB4, accredited to the uniform distribution of Au-TNT in the matrix and notable matrix-filler interaction, which significantly reduced the thermal mobility of polymer strings.46 Thermograms also signalized that the agglomeration of Au-TNT at higher concentration decreased the thermal stability of CDGB7 and CDGB10. In wind up, Au-TNT played an exceptional part in decelerating the thermal decomposition of test samples with CDGB4 possessed maximum thermal stability.

90

100 90

85

80

80

70 60

DEE (%)

Transmittance (%)

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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CDGB CDGB1 CDGB4 CDGB7 CDGB10

50 40 30 20

75 70 65 60

10 0 400

55 500

600

Wavelength (nm)

700

800

A

CDGB

CDGB1

CDGB4

CDGB7 CDGB10

Samples

C

B Figure 7 (A) Optical transmittance curve, (B) Photographic images on arbitrary letters of CDGB, CDGB1, CDGB4, CDGB7 and CDGB10 and (C) Drug encapsulation efficiency. 24 ACS Paragon Plus Environment

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The optical transmittance is an effective criterion to evaluate film transparency and can related to cosmetic attractiveness of the device. As displayed in Figure 7A, the optical transmittance of films was found to be depending on the Au-TNT content. The TD film with 1.0 % Au-TNT showed improved percentage of optical transmittance as that of CDGB, attributed to negligible size and excellent distribution of Au-TNT in the matrix. In addition absence of agglomeration and entanglement of nanofillers at lower concentration also favored better light transmittance. Noteworthy the reduction of optical transmittance in CDGB10 was caused by the agglomeration of nanofillers which scatter the light severely.47 Furthermore the cosmetic attractiveness of the device was evaluated by placing the film in arbitrary letters (Figure 7B). The prepared membranes such as CDGB, CDGB1, CDGB4 and CDGB7 were exceptionally transparent as specified by the clarity and sharpness of the letters beneath each film. In contrast CDGB10 was identified as more opaque as the letter got blurry with the raise in Au-TNT concentration. Finally it can be concluded that the cosmetic attractiveness of the films were proved by transparency test. Drug Encapsulation Efficiency and In vitro Skin Permeation Study The encapsulation behavior of IP loaded membranes is presented in Figure 7C. As depicted in encapsulation profile, pure CDGB membrane showed 59.0 % DEE while CDGB4 provided the highest encapsulation of 86.0 % followed by CDGB1 of 81.0 %. The encapsulation profile revealed that the developed membranes displayed improved DEE right from the 1.0 % loading of Au-TNT. Previous researches detailed that TNT have exceptional capacity to encapsulate different payloads of drugs due to its hollow cylindrical nano architecture.48 In the present investigation the structural modification of TNT using OA coating remarkably enhanced the encapsulation of hydrophobic IP. This is due to the hydrogen bonding interaction of carbonyl 25 ACS Paragon Plus Environment


group of IP with –NH- group of OA-APTEOS-TNT, in addition to the hydroxyl group present in CDGB matrix. Thus the incorporation of Au-TNT as nanofiller raised the encapsulation of IP, ascribed to its interaction with uniformly distributed nanofillers. However CDGB7 and CDGB10 gave a depreciated DEE of 78.0 and 75.0 %, respectively. This may due to the agglomeration of Au-TNT at higher nanofiller content. The in vitro skin permeation study was carried out using Franz diffusion cell. The weight of the film used in diffusion experiment was 0.018 g. The hydrogen atom present in the OH group of βCD has strong capacity to form a hydrogen bond to an oxygen atom of DMSO.49 The wetting of TD device with DMSO results in the entry of solvent molecules into the matrix. As a result the hydrogen bonds between the IP molecules and matrix collapses and new hydrogen bonds were formed between hydrogen atom of OH group of βCD matrix and oxygen atom of DMSO. During the entry of DMSO both AuNP and drug molecules are released in exchange of solvent molecules and eventually reaches the skin surface.50 First control experiments were performed to investigate the effect of Au and TNT on IP release profile. The cumulative release profiles of films incorporated with 1.0 % Au, 1.0 % TNT and without nanofillers were displayed in Figure S4, supporting information. The membrane without any nanofiller presented extremely slow release pattern with almost 52.0 % IP was released in 20 h, whereas the devices incorporated with 1.0 wt % AuNP and OA coated TNT released 59.0 and 56.0 % respectively. The permeation profile revealed that IP permeation was enhanced by the addition of AuNP and TNT. The improvement in skin permeation in the presence of AuNP is accredited to its capacity to produce reversible and transient openings on the SC.8 In the SC, the lipids organize themselves in a head to head and tail to tail configuration, with the head to head sector probably forming small openings of about 0.3 nm size. Literature stated that there were about 5.0 × 107 natural pores 26 ACS Paragon Plus Environment

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were available per cm2 of the SC.51 AuNP efficiently disturb the lipid bilayers and this disturbance lead to an increase in skin porosity and size of extracellular gaps. As a result of this enhanced skin porosity produced by AuNP, the permeation increased. In addition smaller AuNP can achieve distinct physicochemical properties including large surface energy, excellent interfacial attraction and biological behavior due to its quantum size effect. Hence they can efficiently disturb lipid layers due to its favorable size which resulting in enhanced permeability.8 In addition OA coated TNT also penetrates the SC layer by mechanisms called lipid fluidization and lipid phase separation.52 Next the effect of Au-TNT on TD delivery was studied. The permeation profile of IP across rat skin from TD patches with and without Au-TNT is displayed in Figure 8A. CDGB presented extremely slow release pattern with almost 52.0 % of IP was released in first 20 h, whereas CDGB1 and CDGB4 released 73.0 and 82.0 % respectively. The Au-TNT embedded patches followed a quick release profile as compared to neat CDGB. Au-TNT enhanced the skin permeation by the synergistic action of Au and hydrophobic TNT. The disruption of lipid bilayers in presence of Au-TNT and enhanced permeation of drug molecules across the skin is visualized in Figure 3C. However higher Au-TNT content did not further improve IP delivery where about 73.0 and 66.0 % of the loaded IP was delivered respectively from CDGB7 and CDGB10. This observation is attributed to the agglomeration of Au-TNT which in turn intercepts the easy diffusion of IP molecules. The most effective TD device (CDGB4) was stored for 30 and 60 days to explore the impact of storage time on the IP release profile. As can be seen from Figure 8B, unstored CDGB4 exhibited weak initial blow out profile whereby approximately 46.0 % of the loaded IP has been delivered within the first 5 h. However the patches stored for 30 and 60 days demonstrated 27 ACS Paragon Plus Environment


remarkable premature leakage. The strongest initial blow out was observed from the film stored for 60 days, which indicated the premature leakage intensified with increase in storage time and is ascribed to the following reasons. The concentration of IP was homogeneous inside the device before storage. But the storage of patches for many days creates cracks and pores in the membrane.53,54 This results in the slow diffusion of IP from core to the device surface, which ultimately produce non-uniform drug distribution throughout the matrix. This movement generated excessive IP concentration on the membrane surface. A fraction of IP that has moved to the device surface blow out rapidly during the TD application. As storage time increased the rate of diffusion of IP into the surface increased and finally produced higher IP concentration at the membrane surface. Furthermore, after the premature leakage approximately 81.0 and 80.0 % of IP was released from the device stored for 30 and 60 days, respectively after 24 h. The reduction in the IP delivery after the initial blow out was mainly due to the condensed structure of the membrane as reported previously.55


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Figure 8 (A) Cumulative drug release profile of CDGB, CDGB1, CDGB4, CDGB7 and CDGB10, (B) films stored for 0 (TDO), 30 (TD 30) and 60 days (TD 60), (C) film stored for 60 days at 4 ± 1 0C (TD60A), 30 ± 1 0C (TD60) and 45 ± 1 0C (TD60B), (D) film prepared from 0.3 mmol (TD1), 0.2 mmol (TD2), 0.1 mmol OA (TD3) and without OA (TD4), (E) Peeling force and (F) Cell viability of CDGB4 at concentration (A-1.5), (B-3.0), (C-6.25), (D-12.5) and (E25.0 mg/ml). The impact of storage temperature on IP delivery was also studied. The permeation profile of CDGB4 stored for 60 days at three different temperatures 4 ± 1 0C, 30 ± 1 0C and 45 ± 1 0C is presented in Figure 8C. The profile indicated no significant variation in IP release for the films stored at 4 ± 1 0C and 30 ± 1 0C. However the premature leakage was remarkable for film kept at 45 ± 1 0C. Moreover once the IP concentrated on the surface was delivered in a burst an additional decrease in IP delivery was identified. This reduction in IP release is explained as follows. The storage of device at high temperature made the membrane tighter which induced IP entrapment in the remarkably condensed membrane architecture. This entrapment made the IP release difficult.55 Finally the in vitro test inferred that the membrane kept at low temperature produced no significant premature leakage and hence exhibited storage stability. Next the effect of oleyl functionalization on IP release profile was investigated. The drug release profile of device fabricated from 0.1, 0.2, and 0.3 mmol OA and without OA is displayed in Figure 8D. The device without OA coating exhibited a slow release profile and approximately 72.0 % of IP was delivered within 20 h. At the same time, the film prepared from 0.1, 0.2 and 0.3 mmol OA delivered nearly 76.0, 80.0 and 82.0 %, IP respectively. The reduced permeability of device without OA coating is ascribed to its inefficiency in disturbing lipid structure of SC, compared to highly lipophilic films. As depicted in figure 8D, IP release rate was remarkably 30 ACS Paragon Plus Environment

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improved with increase in lipophilic character of the film. This is because OA moiety can efficiently interact and disturb the ordered lipid bilayers by mechanisms called lipid fluidization and lipid phase separation.52 The obtained results were aligned with previous literatures. Yang et al.56 reported that poly(amidoamine) dendrimers with long chain fatty acid exhibited increased skin permeation compared to its pure counter parts. The incorporation of highly lipophilic octyl methoxycinnamate in poly(ε-caprolactum) remarkably increased its capacity to enhance skin permeability.57 In addition to lipohilicity and storage conditions, TD delivery also depends on the size of AuNP. Literature revealed that the skin penetration of AuNP increases with decrease in particle size.58 Sonavane et al.59 investigated the skin penetration of 15, 62 and 108 nm AuNP and reported that 15 nm AuNP displayed enhanced skin penetration compared to bigger particles. However, previous studies revealed that AuNP of size less than 10 nm are markedly cyto-toxic.60 Xia et al.61 studied the effect of AuNP size on genotoxicity and showed that 5 nm AuNP induced obvious DNA damage and promoted the production of reactive oxygen species. It is interesting that the size of AuNP prepared in this work was about 10-12 nm. Hence they are acceptable in terms of skin permeability and cyto-compatibility. Finally the influence of membrane thickness on TD delivery was studied. The thickness of the membrane can be controlled by dropping different amount of CDGB solution onto the petri plates. The drug release profile was presented in Figure S5, Supporting information. After 20 h, approximately 82.0, 80.0 and 77.0 % of IP were delivered from the films of thickness 0.082, 0.087 and 0.094 nm, respectively. The results clearly indicated that TD delivery decreased with increase in thickness. As membrane thickness increases, the effective path length for the migration of drug molecules towards the surface of the membrane increases. This prevents the easy migration of drug molecules to the film surface and hence the percentage of drug release 31 ACS Paragon Plus Environment


decreased.62 This result is consistent with previous reports that drug release decreases with increase in thickness of the TD films.63,64 In vitro Skin Adhesion Behavior and Cell Viability Assay One of the vital requirements for a TD device is the superior adhesion behavior. In this project we utilized peeling force to specify the skin adhesion behavior of the patch. Peel adhesion force is a measure of force necessary to remove an adhered membrane from the test surface.65 Since it is not possible to duplicate skin moisture and elasticity in steel panels, we selected fresh rat skin as test surface. As depicted in Figure 8E, the mean 900 peel adhesion force of CDGB, CDGB1, CDGB4, CDGB7 and CDGB10 was found to be 0.873, 0.867, 0.864, 0.859 and 0.855 N/cm2, respectively. The test outcomes revealed that the peel adhesion force of the prepared membranes was independent of Au-TNT concentration. The results also suggested that all the test samples exhibited peeling force between 0.4 and 2.0 N/cm2 which was necessary for a TD membrane.66 After the successful preparation of TD patches, their cell viability was studied in details to examine the biological safety. The cell viability of HaCaT cells treated with CDGB4 at different concentrations in the growth medium was calculated by MTT assay. As presented in Figure 8F, the cell viability of CDGB4 was 92.8, 91.6, 89.9, 88.8 and 85.2 % respectively, for 1.5, 3.0, 6.25, 12.5 and 25.0 mg/ml of CDGB4. These results suggested the dose dependent behavior in cell viability with in the investigated concentration range. Literature stated that the samples possessing cell viability more than 80.0 % were considered as non-toxic and cyto-compatible.67 The outcomes of the cytotoxic assay are consistent with reported works where AuNP in low concentration produced no damaging effect on cell viability.68 Furthermore, Yang et al.69 in their recent article reported that TNT did not induce cell toxicity and avoids the risk of infection. The


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assay concluded that the test sample exhibited cell viability with in an acceptable range, indicating that the developed device is non-skin irritant. Finally the environmental stability of the films was also evaluated and the results were discussed in supporting information. Conclusions In this investigation a novel TD device was introduced that significantly enhances the skin permeability. For this Au-TNT nanocomposite was prepared for the synergistic enhancement of skin permeability. Further methacrylate grafted βCD was synthesized to improve skin adhesion behavior and finally these polymers were reinforced with AU-TNT. The device loaded with 4.0 % Au-TNT possessed exceptional enhancement in thermomechanical properties and DEE attributed to the uniform dispersion of Au-TNT in the polymer matrix. The films were cosmetically attractive, permeable to water vapor and exhibited peeling force values suggested for TD device. Using rat skin as model, the cumulative percent of IP delivered across the skin was determined and found to be heavily depending on Au-TNT content. At 4.0 % Au-TNT, TD IP permeation was maximum and about 82.0 % was delivered within 20 h. The incorporation of Au-TNT above 4.0 % remarkably reduced the permeability due to the agglomeration of AuTNT. It was further noticed that the drug release profile depend on lipophilicity, thickness, storage time and temperature of the patch. Finally cyto-toxic and environmental fitness assay have furnished the evidence that the developed devices possessed excellent cell viability and strongly defended microbial colonization. These results displaying the potential of AuNP based TD device are encouraging and may disclose new avenues in TD drug delivery.



Acknowledgments The authors thank Head of the Department, Department of Chemistry, University of Kerala, India for support and usage of the characterization equipments. One of the authors would like to acknowledge University of Kerala, for providing assistance in the form of research fellowship (Grant no: Part INon Plan-MH 64(b)-scholarships-Sub Head 9-E Code 3330). References (1)

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TOC Graphics

Schematic of the transdermal penetration of drug molecules

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Methacrylate Stitched Î²-Cyclodextrin Embedded with Nanogold

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