Gold Nanoparticle and Hydrophobic Nanodiamond Based Synergistic

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Gold Nanoparticle and Hydrophobic Nanodiamond Based Synergistic System: A Way to Overcome Skin Barrier Function Thayyath Sreenivasan Anirudhan, and Syam S. Nair Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00417 • Publication Date (Web): 11 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

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Stratum Corneum

Drug

Graphical representation of the (A) untreated skin, skin after the application of (B) AuD0 and (C) AuD3

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Bioconjugate Chemistry 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

Gold Nanoparticle and Hydrophobic Nanodiamond Based Synergistic System: A Way to Overcome Skin Barrier Function T. S. Anirudhan*, Syam S. Nair Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom, Trivandrum-695 581, India

*Corresponding author: Tel: +91 471 2308682 E mail address: [email protected]


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Abstract Gold nanoparticles (AuNP) have attracted ample attention as a transdermal (TND) drug delivery platform for improving the skin permeability of drug molecules. Herein a novel TND device formed from AuNP and oleylamine functionalized nanodiamond (AuD) has been developed successfully for the TND delivery of Ketoprofen (KP), a model drug. Polyvinyl alcohol/Polybuytl methacrylate (PVA/PBMA) film has selected as the matrix of the TND device, as they furnish excellent skin adhesion properties. The PVA/PBMA membranes loaded with different concentrations of AuD have been characterized in terms of surface morphology, thermomechanical properties, water vapor permeability (WVP), optical transmittance, cosmetic attractiveness, skin adhesion behavior and drug encapsulation efficiency (DEE). The matrix loaded with 3.0 % AuD displayed enhanced thermo mechanical and DEE due to the uniform distribution of nanofillers in the membrane. The in vitro skin permeation test proved that a higher amount of KP was delivered by AuD, suggesting improved TND behavior. The synergistic management of AuNP and nanodiamonds (ND) has caused the enhanced skin permeation behavior of the device. The obtained results revealed that AuD may be employed as an effective carrier to substitute NDs for TND delivery. Additionally, while investigating the storage stability of the device we observed that the membrane kept at low temperature presented stability over time. More importantly, the results from cell viability assay and environmental fitness test revealed that AuD based TND system is a high security device as it is non-cyto toxic and microbial resistant. The developed device provides a novel and handy approach to the TND delivery of drug molecules.



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Introduction Skin possessing an approximate surface area of 1.5-2.0 m2 is an attractive portal to deliver drug molecules into systemic circulation.1 The TND pathway of drug delivery provides distinct beneficial features over oral and traditional injection. It prevents first pass liver effects 2 and emotional trauma,1 respectively related to oral and hypodermic injection. Moreover TND route offers self-administration therapy and reduced side effects.3 Nevertheless the potential of TND delivery is substantially limited due to the poor permeability of extremely complex skin layers.4 The principal hindrance to TND drug permeation is the stratum corneum (SC) of 10-15 µm thickness comprising of several layers of corneocytes, embedded in a lipid matrix consisted of long chain fatty acids, ceramides and cholesterol.5 The development of TND device now a day faces significant challenges involving selection of biocompatible materials and the harsh fabricating conditions, which restricts the class of drugs that can be loaded.6 Recently nanoparticles and nanofibres have been explored as an efficient TND drug delivery platform to enhance the skin permeability.7 An ideal skin permeation enhancer must satisfy the following criteria. 1) The skin tissue must immediately return to its usual barrier behavior, 2) the enhancer should display noncytotoxicity, 3) it should not cause skin irritation and 4) the duration of the enhancing effect should be predictable and specific.8 Owing to this combined properties, ND may behave as a perfect

penetration

enhancer.

Moreover

ND

exhibits

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biocompatibility, robust surface structure, large surface area, and enhanced cell tolerance.9 In addition, ND has been predicted to furnish excellent tensile properties necessary for a TND patch.10 In this project ND is hydrophobically modified using oleylamine, as it is expected to enhance skin permeation via mechanism called lipid phase separation and lipid fluidization.11


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On the other hand AuNP received a great deal of attention as penetration enhancers ascribed to their ability to create transient openings on skin surface. Furthermore AuNP demonstrates unique behavior comprising excellent biocompatibility, ease of synthesis and the ability to transport several molecules for example DNA, peptides and small drug molecules.12 Several experimental studies relating the influence of size, shape and surface of AuNP on their TND drug delivery efficacy has been reported.13,14,15 These investigations revealed the potential of AuNP to productively penetrate across the SC layers. Upon compared to other chemical penetration enhancers like azones, ethanol etc, AuNP generates only local disruptions. Hence the defects produced are absolutely reversible and the SC layers regain its original architecture as soon as the particles are removed. As the highly compact lipid packing of skin layers regulate its barrier activity, the SC lipid perturbing capacity of AuNP play a crucial part in enhancing skin transportation. Therefore in this work the potential of AuNP to enhance the skin permeability is further improved in combination with hydrophobic ND. Membrane is the chief component of a TND patch that holds the drug molecules and allows it to effuse steadily towards the skin in the final stage. Polyvinyl alcohol (PVA) is chosen as the membrane due to its biocompatibility, hydrophilicity and physiological inertness.16 The US Food and Drug Administration detailed that the skin adhesion behavior of a TND system is pivotal to the safety, potential and standard of the device. Even a slight lift of the device from skin surface prevents the transport of therapeutically effective concentration of drug molecules across the skin. The situation leads to improper dosing and finally results in the failure of the system. Furthermore if the device does not effectively adhere, it will frequently fall off and the patient thinks that the money he spends for the treatment is not worthy.17 Previous studies have shown that methacrylates which furnish significant flexibility and optical transparency are



peculiarly fit for TND application to improve the skin adhesion properties.18 Herein PBMA is incorporated with PVA with former to enhance the adhesion behavior. KP, a nonsteroidal antiinflammatory drug is selected as the model drug. In the present study we fabricated KP loaded TND patch composed of PVA/PBMA film reinforced with AuNP/oleylamine conjugated ND hybrid nanofiller (AuD). The TND delivery potential of the constructed device was systematically investigated on in vitro rat skin using Franz diffusion cell. Results and Discussion DH loaded TND films reinforced with AuD nanofillers were successfully formulated by solvent casting method and is schematically represented in Figure 1. To enhance the ability of ND to penetrate SC layers, it was hydrophobically modified using oleylamine and is sketched in Figure 2A. To conjugate the amine, ND was first oxidized by strong heating at 420 ℃. The evidence of the surface functionalization of ND is furnished by FTIR spectra presented in Figure 2B. The FTIR bands of pristine ND are well in line as reported by Song et al. 19 The infrared bands centered around 1341 cm–1 is ascribed to the deformation of alkyl group while that pointed at 1659 cm-1 is attributed to the stretching vibration of the aromatic sp2 carbon bonds. A broad signal observed around 3419 cm-1 corresponds to the O-H stretching vibrations. The vibrational peaks located at 1121 and 1727 cm–1 corresponds to the epoxy C-O stretching vibration and C=O stretching vibration in the carboxylic acid and carboxyl groups, respectively. In oxidized ND, the infrared signal of C=O is shifted from 1724 to 1780 cm-1 ascribed to the conversion of oxygen carrying moieties like ester, ketone and alcohol to carboxylic group. 20 Finally HND was synthesized by covalently conjugating oleylamine to oxidized ND surface via EDC-NHS


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coupling. As depicted in FTIR spectrum, the prominent signals appeared in HND at 1561, 1630 and 2888-2900 cm-1 are attributed to N-H bending, C=C and C-H stretching, respectively.21 The appearance of these peaks confirmed the successful attachment of oleylamine onto ND surface.

Figure 1. Schematic representation of the synthesis of AuD incorporated TND film



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A Figure 2. (A) Schematic of the synthesis of HND and (B) FTIR spectra of ND, oxidized ND and HND Next AuD was synthesized by the reduction of HAuCl4 on HND surface. Details regarding the adhesion of AuNPs on the surface of HND were obtained using XRD pattern and TEM image. The XRD profile of HND (Figure 3A) show peaks at 2θ values of approximately 44 and 75o, respectively indexed to the (111) and (220) lattice planes of the ND core.22,23 The formation of AuD was confirmed by the appearance of additional peaks at 38, 64 and 77o indexed to (111), (220) and (311), respectively, characteristics of AuNP.24 The TEM image of AuD displayed in Figure 3B clearly indicated that a number of AuNP of approximate size of 1520 nm were deposited on the surface of HND via in situ chemical reduction. Discrete AuNPs were developed, revealing that most AuNPs were grown on the surface of HND. The morphology of AuD was studied using SEM image (Figure S1, Supporting Information ) which displayed clumped mass of hydrophobic ND particles with approximately 250-300 nm size.


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After the successful synthesis of AuD, TND device was prepared by incorporating predetermined amount of AuD in PVA/PBMA matrix.

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Figure 3. (A) XRD profiles of HND and AuD and (B) TEM image of AuD Characterization of AuD Incorporated PVA/PBMA Films SEM investigation was performed to study the morphological changes happened during the preparation of AuD incorporated films. As presented in Figure 4, in the SEM micrograph of AuD1, few tiny particles of AuD were randomly appeared throughout the matrix of PVA/PBMA. The images revealed that the concentration of AuD in the matrix increased with increasing the loading of nanofillers. The surface morphology of AuD3 definitely displayed that AuD particles disperse uniformly throughout the matrix, which lead to an enhancement in thermomechanical properties. However the homogeneous distribution of AuD was lost with the addition 4.0 % of hybrid fillers. Figure 4D depicted the SEM image of AuD4. Aggregation of AuD was observed. As a result, they do not contribute towards effective reinforcement as will be elucidated below. The



distribution of fillers in AUD3 and AuD4 film was further visualized using AFM images presented in Figure S2, Supporting Information. AuD3 clearly presented homogeneous distribution of nanofiller throughout the matrix indicating favorable matrix-filler interaction. However the fillers possessed irregular size and shape. On the other hand, AuD4 is remarkably different from other membranes whereby displayed agglomerates of nanofillers of irregular morphology.

Figure 4. SEM image of (A) AuD1, (B) AuD2, (C) AuD3 and (D) AuD4 An ideal TND membrane should resist mechanical abrasion. So it is necessary to examine the mechanical properties of the device. As can be seen from Figure 5A, the TS of composite films possessed an upward trend with increase in AuD concentration. The addition of 1.0 and 2.0 % AuD, increased the TS, respectively to 67.0 and 74.0 MPa. The membrane incorporated with


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3.0 % nanofiller presented the highest TS of 85.0 MPa. The enhancement in TS upon the addition of nanofiller is explained as follows.

The significantly small size and uniform

distribution of AuD created a large interfacial area, producing strong interfacial interaction between matrix and AuD.25 In addition, the uniform stress distribution and reduction in stress concentration area occurred due to the efficient load transfer from PVA/PBMA to AuD played an essential role in improving the tensile strength.26 Notably the tensile strength of AuD4 is 76.0 MPa, which was little lower than AuD3 ascribed to the agglomeration of AuD particles. As depicted in Figure 5B, the Eb values of the films were also influenced by AuD content. The Eb values of composite membranes reduced slightly suggesting the retardation of macromolecular motion after AuD addition.27 The effect of storage time on tensile strength was displayed in Figure 5A. The membranes stored for 30 and 60 days are, respectively abbreviated as AuD3A and AuD3B. It can be concluded that the storage of film remarkably improved the tensile strength. The storage of membrane generated an efficient packing of molecular segments which results in better nodular packing density. Therefore for the mobility of segments an appreciable force is required which results in enhanced tensile strength. Similarly storage of membrane has a great impact on Eb values.28 A remarkable reduction in Eb values was obtained after the storage and is ascribed to the changes in the polymeric structure. This change is happened mainly in the amorphous region and is enumerated in terms of free volume. During storage peculiar relaxation occurred in the internodular segments which decreases the free volume and ultimately reduces the Eb values.29



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Figure 5. (A) TS, (B) Eb, (C) WVP and (D) thermogram of AuD0, AuD1, AuD2, AuD3 and AuD4 An improved water vapor barrier property of TND patch may upshot in enhanced risk of microbial deposition and skin occlusion. Skin occlusion ultimately leads to drastic stress in Langerhan’s cells and extreme variation in epidermal lipids.30 Hence the examination of WVP is crucial. Figure 5C presents the WVP values of membranes with and without AuD. The most permeable film was AuD0 with a WVP value of 0.55 gcm224h-1. At a nanofiller load of 1.0 %, the WVP of membrane reduced to 0.49 gcm224h-1. The film gave WVP of 0.46 and 0.40 gcm224h-1 for 2.0 and 3.0 % nanofiller load, respectively. As expected the WVP was reduced


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0.36 gcm224h-1 at the higher nanofiller content (4.0 %). A clear reduction in WVP was observed with AuD loading. The reinforcement of nanofiller blocks the direct diffusion of water molecules through the membrane and a tortuous pathway is created. This winding pathway exceptionally increased the mean free path of piercing water molecules.31 Nevertheless as depicted in Figure 5C, the WVP values of all the prepared patches were much above 0.05 g cm2 24 h-1 and hence fit for skin application.32 TG analysis of the prepared membranes was performed to decipher the impact of AuD on the thermal stability of the composite films. Thermograms displayed in Figure 5D indicated a three stage degradation format in all the test specimens. The first event of weight loss noticed over the temperature range between 70 and 140 ℃ is associated with the evaporation of water molecules absorbed on the film surface. Next a dramatic weight loss was happened between 200 and 350 ℃, affiliated to the degradation of polymeric segments. The final stage degradation began above 380 ℃ corresponds to the decomposition of carbonaceous fragments.33 It can be seen that the residual weight percent of AuD containing films is higher than that of pristine PVA/PBMA films, ascribed to the phenomenal thermal stability of ND. The reinforced ND function as a barrier to retard the volatile decomposition throughout the membrane, thus improving the thermal stability.34 Literature revealed that ND forms nano confined sectors in the matrix bulk around which polymeric strings cannot maintain their consistent coil conformation.35 Furthermore in the reinforced regions, the mobility of polymeric strings is obstructed, producing some variations in the molecular transition dynamics.36 This retarded mobility decelerates the thermal decomposition of membranes and enhanced the thermal stability of the samples. Thus AuD played a remarkable part in improving the thermal stability of the composite films.



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It is worth noticing that the decomposition rate of AuD3 is remarkably slower than that of other composite films. The well dispersion of AuD in the PVA/PBMA matrix and superior interfacial adhesion contributed to the outstanding enhancement in thermal stability.34 On the other hand, the enhancement in thermal stability was only significant for films up to 3.0 % loading. Further addition of AuD reduced the thermal stability, which is mostly connected with the agglomeration of AuD at higher concentration.

a

b

c

d

e

Figure 6. (A) Optical transmittance and (B) photographic images of films AuD0 (a), AuD1 (b), AuD2 (c), AuD3 (d) and AuD4 (e) placed on arbitrary letters


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The optical transparency of the prepared membranes was quantified using spectral scanning and the results have been illustrated in Figure 6A. The AuD0 membrane displayed more than significant transmittance at 550 nm. The light transmittance of AuD1 decreased remarkably at the same wavelength. As expected the optical transmittance of the membranes decreased on increasing the concentration of AuD. The incorporation of AuD generate light scattering while it pass through the membrane and the higher AuD concentration leads to more severe scattering of light.37 Nevertheless all the membranes displayed reasonable optical transmittance necessary for a TND patch. Furthermore the arbitrary letters underneath the composite membrane as displayed in Figure 6B indicated that the membranes were transparent. It can be seen that the clarity of the letter beneath all the films were exceptional. The decent transparency presented by AuD1, AuD2, AuD3 and AuD4 films indicated that test membranes are cosmetically attractive and suitable for topical applications. Drug Encapsulation Efficiency The KP encapsulation efficiency of the prepared patches is shown in Figure 7A. The DEE of AuD0 was found to be 53.0 %. As the concentration of AuD increased, the DEE displayed an increasing trend up to 3.0 % addition of AuD. The presence of NH and CO groups present on the surface on hydrophobic ND furnishes better DEE on the basis of hydrogen bonding interaction with drug molecules. The DEE was beyond 60.0 % for AuD incorporated patches and maximum DEE was exhibited by AuD3. The higher the content of AuD, the greater the functional moieties to accommodate KP molecules. This could have led to an enhancement in DEE. Zhu et al.38 reported the exceptional capacity of ND to encapsulate chemotherapeutic agents doxorubicin and molaridine. Wang and coworkers39 successfully loaded epirubic onto ND surface with high loading efficiency. Tob et al9 reported that the distinct truncated octahedral



morphology empower efficient binding of drug molecules. It can be seen in the encapsulation profile that the addition of 4.0 % AuD in the matrix reduced the DEE. This can be explained by the fact that agglomeration of AuD at higher concentration reduces the possible hydrogen bonding sites for the binding KP molecules. Literature stated that ND is an ideal skin penetration enhancer8 and have remarkable capacity to encapsulate higher content of drug molecules9,38,39. In vitro Skin Permeation test The in vitro skin permeation test began with experiments to study the effect of AuD concentration on the TND delivery of KP. The hydrogen atom present in the OH group of matrix has strong capacity to form a hydrogen bond to an oxygen atom of DMSO. The wetting of TND device with DMSO results in the entry of solvent molecules into the matrix. During the entry of DMSO drug molecules are released in exchange of solvent molecules and eventually reaches the skin surface. As can be seen from permeation profile (Figure 7B), approximately 60.0 % KP was delivered from AuD0 after 24 h, whereas AuD1 delivered 74.0 % during the same period. The improvement in skin permeation is attributed to the synergistic action of AuNP and hydrophobic ND. The skin permeation enhancement of AuNP is due to its ability to induce transient and reversible openings across the SC.40 The lipids present on SC layer positioned themselves in head to head and tail to tail mode. Interestingly, the head to head sector produces fine openings of around 0.3 nm size. Literature contains reports about the availability of around 5.0 × 107 natural openings per cm2 of lipid bilayers. Nevertheless the size of the pores is inconsistent. But the point is that AuNP abundantly perturb the lipid architecture, which subsequently increases the size of SC openings. Furthermore due to the nanometric size and large surface area, AuNP can be wholly in contact with the lipid architecture so that it can productively disturb the lipid barrier.41 Due to the increased porosity of skin produced by the efficient interaction of AuNP, the


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skin permeability enhanced markedly. On the other hand hydrophobic ND acted as a penetration enhancer by discrete mechanism called lipid phase separation and lipid fluidization.11 AuD3 displayed the highest release rate attributed to the uniform distribution of hybrid fillers in the matrix. However AuD4 possessed the lowest permeability rate compared to AuD3, suggesting that above a particular concentration of AuD, the incorporation of nanofillers decrease the KP release. This behavior is ascribed to the agglomeration of AuD at higher concentration which in turn blocks the facile diffusion of KP molecules. The morphological changes happened after the application of optimized film is studied. The SEM images of skin before and after the application of TND device are displayed in Figure S3, Supporting Information. The application of TND device remarkably changed the morphology of skin surface. This variation in skin morphology significantly facilitated the permeation of drug molecules across the skin. After optimizing the concentration of AuD, the highly efficient TND patch (AuD3) was employed to investigate the influence of storage time and temperature. As presented in Figure 7C, compared to patch stored for 0 day, the storage of TND membrane resulted in strong initial blow out of KP. The intensity of initial blow out was more significant in device stored for 60 days. The distribution of KP was uniform before the storage of the TND patch. However considerable number of pores and cracks would be produced in the device kept for 30 and 60 days. As a result KP molecules diffused into the PVA/PBMA film surface from its core and finally the diffused molecules are arranged heterogeneously in the device.42,43 Due to the excessive accumulation, KP molecules are blow out rapidly from the membrane surface when fixed in rat skin. Interestingly a decrease in KP delivery was observed after the premature leakage, attributed to the condensed morphology of the membrane as discussed in literature.44



The AuD3 patches were stored for 60 days at 4.0 ± 1.0 ℃, 30.0 ± 1.0 ℃ and 45.0 ± 1.0 ℃ and were applied to rat skin to investigate the effect of storage temperature on TND delivery (Figure 7D). As discussed earlier the patch kept for 60 days promoted strong premature leakage. This effect is more marked if the patch is kept at 45.0 ± 1.0 ℃. The number and size of cracks developed during the storage becomes remarkable when kept at high temperature. Thus a significant amount of KP molecules diffuses from the core to device surface and blow out swiftly upon skin application. Notably as soon as the KP accumulated on patch surface was blown out, an additional reduction in KP permeation was observed. This is because the film becomes tighter and promotes the entrapment of KP in the condensed patch architecture.44 As a result of this entrapment the leakage of KP molecules diminishes. However the performance of the patch kept at 4.0 ± 1.0 ℃ is excellent as it yield no premature leakage. The test identified that the patches are stable to storage when kept at low temperature. The skin penetration potential of device incorporated with and without AuD was represented diagrammatically in Figure 8

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Figure 7. (A) DEE and (B) skin permeation profile of AuD0, AuD1, AuD2, AuD3 and AuD4, skin permeation profile of (C) device stored for 0, 30 and 60 days at room temperature and (D) device stored for 60 days at 4.0 ± 1.0 ℃ (P), 30.0 ± 1.0 ℃ (Q) and 45.0 ± 1.0 ℃ (R) The surface of oleylamine conjugated ND contains functional moieties such as NH, CO etc. The prepared TND membrane loaded with AuD can efficiently encapsulate and deliver those drug molecules that can form hydrogen bond with the surface functionalities of oleylamine conjugated ND including lidocaine, ibuprofen etc. Herein the developed device enhanced the skin permeability of KP by the synergistic action of AuNP and ND. In addition to KP, the device can efficiently transport other hydrophobic drugs such as lidocaine, ibuprofen etc.

Stratum Corneum

Drug

Figure 8. Graphical representation of the (A) untreated skin, skin after the application of (B) AuD0 and (C) AuD3 In Vitro Skin Adhesion Test In terms of adhesion behavior, an ideal TND patch must easily adhere to skin surface upon the application of slight pressure and produce no residue after its removal. To designate the



adhesion properties of TND patches, research groups commonly utilize a five point scale based on visual examination as suggested by FDA. Howbeit the detailed strategy proposed by FDA is not effectively grounded. Herein peel adhesion force, the force necessary to detach an adhered material from test surface was employed to designate the degree of adhesiveness of the prepared TND patches.45 Routinely stainless steel surface is used to measure the peeling force. As the surface energy of skin and steel surface differ greatly in magnitude our group carried out adhesion test on the surface of rat skin. As presented in Figure S4, Supporting Information, the peel adhesion force of AuD0, AuD1, AuD2, AuD3 and AuD4 was measured. The value of peeling force suggested that the concentration of AuD displayed no significant effect on peeling force. These outcomes are well in line with our previous reports. The addition of AuNP/nanotitania hybrid filler showed no influence on the peeling force of methacrylated β cyclodextrin film.18 The peel adhesion force of guar gum based polyelectrolyte film was not varied significantly when reinforced with AuNP/ nanocellulose.16 Interestingly the peel adhesion force of the developed patches was identified between 0.4 and 2.0 N/cm2, within a range specified for a TND device.46 Cell Viability Test, In vitro Skin Irritation Test and Environmental Fitness Assay In order to guarantee safety, cell viability test of various formulations of AuD3 was carried out using MTT assay. It was advocated that a TND specimen possessing cell viability above 80.0 % is non-cytotoxic.47 As depicted in Figure S5, Supporting Information, the cell viability of all the test formulations was above 80.0 %, within the range recommended for a TND patch. Evidently all the test formulations were displayed to be non-toxic against HaCaT cells. Schrand et al detailed that ND was compatible to a large number of cell types as it did not generate remarkable reactive oxygen species. They also proved that cells can grow on ND coated


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substrate without producing any morphological variations.48 Yu et al also reported that NDs exhibits no sign of cytotoxicity.49 The biocompatibility of ND is ascribed to their chemical stability and biostability.8,50 On the other hand literature contains report about the cytocompatability and non-toxicity of AuNP at low concentration.51 After cell viability assay, Draize test52 was employed to examine the irritation potential on rat skin applied with the prepared TND patches. Figure S6, Supporting Information displayed the photographic images of the rat skin after peeling of the TND patches. All the skin samples displayed no appreciable sign of edema or erythema. The application of TND patches produced no remarkable skin reactions and PII was found to be less than 2. A device with PII of 2 or less than 2 is considered as non-skin irritant. As the test was negative, the developed patches could be deemed as non-skin irritant. The TND devices were engineered to deliver the therapeutic molecules sustainably and so the patches need to wear for 1-3 days. Hence the quality of the device is of first importance. The deposition of microorganisms on the surface of TND film, induce biofilm formation and finally leads to the failure of the device. The microbial colonization and biofilm formation can be hindered only if the devices possess anti-microbial properties. Hence it is necessary to evaluate the ability of the prepared patches to prevent the microbial colonization To investigate the antimicrobial behavior of AuD loaded patches, Escheria coli was employed as model organism. The test result is displayed in Figure S7, Supporting Information. The antibacterial test revealed that the AuD incorporated patches are highly efficacious antimicrobial films that quickly compromise bacterial survival and biofilm formation. The bacterial deposition and succeeding evolution of biofilms were phenomenally defended by the



synergistic effect provided by two discrete mechanisms of ND and AuNP. Turchenuik et al. in a recent article reported that NDs have exceptional inhibitory capacity against Staphylococcus aureus and Escheria coli.53 Medina and coworkers reported that ND remarkably inhibit the growth of Pseudomonas aeruginose and can be used as an antimicrobial coating for medicinal implants.54 The antimicrobial activity of ND surface is ascribed to its non-specific interaction with biomolecules. The reactive group of ND interacts with molecules on cell wall, adjacent proteins and intercellular components. This binding inhibits proteins and vital enzymes, producing a quick failure of bacterial metabolism and ultimately cell death.55 The detailed mechanism prevents the microbial adhesion and subsequent biofilm formation on patch surface. AuNPs on the other hand have the capacity to attach into the bacterial cell membrane, ultimately result in the bursting of cell wall, gathering of intracellular reactive oxygen species and lastly the cell apoptosis.56 Furthermore AuNPs remarkably deactivates the microbial secretions that have phenomenal ability to annihilate patch surface. Antibacterial testing of Rai’s group revealed that AuNPs have exceptional antibacterial activity against Escheria coli and Staphylococcus aureus.57 Zheng et al. reported that AuNPs could remarkably disturb bacterial metabolism that kills bacterial cells ultimately.58 The investigation revealed that apart from skin penetration enhancer, AuD as a kind of antimicrobial agent had a substantial promise in TND applications. More importantly the test revealed that the developed patches are environmentally fit for TND purpose. Conclusion Synergistic TND systems containing AuNP and ND provide a way to successfully overcome the skin barrier properties. AuD reinforced PVA/PBMA could be easily prepared by


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solvent cast method. The fabricated device featured excellent WVP and optical transmittance. The patch loaded with 3.0 % AuD present robust thermomechanical and DEE due to the well dispersion of nanofillers in PVA/BMA matrix. The skin permeability of AuD loaded device is higher than that without AuD revealing the crucial role of AuNP and ND to disrupt compact lipid architecture of skin. Hydrophobic ND enhanced the permeability by discrete mechanism called lipid phase separation and lipid fluidization, while AuNP due to its capacity to create transient and reversible openings across the SC layers. With the application of patch containing 3.0 % AuD, the amount of drug delivered across the skin reached maximum due to uniform distribution of nanofillers. The patch stored at low temperature offered stability over time, hence opens auspicious avenues in storage and TND delivery of drug molecules. Lastly, the fabricated patches are also shown to be non-cyto toxic and strongly defended microbial colonization. The collective results suggested that the described synergistic TND approach disclose fascinating scenarios to pursue strategies for enhanced skin permeation. EXPERIMENTAL SECTION Materials Gold(111) chloride trihydrate (HAuCl4·3H2O), NaBH4, PBMA, KP, and PVA (98–99% hydrolyzed) were supplied by Sigma-Aldrich (USA). Nanodimond (ND), Oleylamine, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Tokyo Chemical Industry (Japan). All solvents were supplied by Merck Specialities Pvt Ltd. (Mumbai). Methods Synthesis of Oleylamine Conjugated ND (HND)



ND was purified according to the procedure reported by Behler et al.59 ND was oxidized by strong heating at 420 ℃ in air atmosphere for 2.0 h. The oxidized ND is further employed to conjugate oleylamine on ND surface. Typically NHS (0.5 mmol) and EDC (O.5 mmol) were added to a solution containing dimethyl formamide and oleylamine (0.3 mmol). The mixture was vigorously stirred for 2.0 h and oxidized ND (0.2 mmol) was added, left stirring for another 24.0 h. The resulting HND was dialyzed, lyophilized and stored. Synthesis of AuNP Deposited HND (AuD) First a solution of 8.0 mL of HNT (23.0 mg/mL) was prepared in cyclohexane. An aqueous solution of HAuCl4.3H2O (0.5 mL, 20.3 mmol) was charged into the solution and stirred vigorously. AuNPs were deposited on HND surface by adding 0.1 M aqueous NaBH4. The mixture was strirred for 10 minutes. The synthesized HND was centrifuged and washed with deionized water until a colorless supernatant was produced.60 Preparation of AuD Reinforced TND Membranes A solution of 4.0 wt % PVA was prepared in DMSO at 70 ℃. PVA/PBMA film was prepared by adding 5.0 wt % of PBMA (of the mass of PVA) into PVA solution maintained at 70 ℃. After thorough mixing the film was cast in a clean petri plate. The AuD reinforced film was prepared by adding definite amount of AuD to PVA/PBMA suspension to obtain film containing 0, 1.0, 2.0, 3.0 and 4.0 wt % (of the mass of PVA/PBMA) AuD. KP loaded TND patches were prepared by adding 5.0 wt % KP in the suspension. The membrane containing 0, 1.0, 2.0, 3.0 and 4.0 wt % AuD is abbreviated as AuD0, AuD1, AuD2, AuD3 and AuD4, respectively. The DEE of the prepared patches was examined spectrophotometrically based on our previous investigation.61


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Characterization and Analysis The surface functionalization of ND was studied using infrared spectrometry Agilent Cary attenuated total reflectance (ATR) spectrometer. The decoration of AuNPs on HND surface was visualized using transmission electron microscope (FEI, TEC-NAIS Twin microscopy) operating at 100 KV. The X-ray diffraction (XRD) pattern of HND and AuD was obtained with Siemen’s D5000 X-ray diffractometer (Germany) equipped with Ni filtered Cu Kα radiation with the Xray tube operating at 40 kV and 30 mA. The FESEM images of test films were obtained using Nova Nano SEM NPEP 252 high resolution FE-scanning electron microscope. The morphology of skin samples was studied using a Carl Zeiss EVO 18 Scanning Electron Microscope (SEM). Atomic Force Microscope (AFM) images were recorded on Bruker DIMENSION Edge with Scan ASYST instrument. The tensile tests were performed on tensile tester (Hounsfield universal testing machine) at room temperature. The impact of storage time on tensile strength and elongation at break (Eb) was also investigated by storing the device for 30 and 60 days at room temperature. To examine the thermal stability of the prepared membranes, thermogravimetric (TG) analysis was performed using TG analyser (TA Q50) with a linear heating rate of 10 ℃/min under the nitrogen atmosphere. Thermogram was recorded over the temperature range of 30-800 °C. Water Vapor Permeability The tests were performed at 37 ℃ using circular membranes with a test area of 1.13 cm2. A glass vial was filled with 8.0 g deionized water and then the test membrane was placed on the top of the vial. Next the vial was kept in a climate chamber for 24.0 h. The WVP was valued by using the following formula. Three measurements were carried out for each membrane and the mean values were reported.61



……………………………………………………………………………. (2)

where W (g) is the weight loss of vial, S (cm2) exposed surface area of membrane and t (24 h) time.

Assessment of Optical Transparency and Cosmetic Appearance

Light transmittance by neat PVA/PBMA film and AuD reinforced films was measured in UV-visible spectrometer (Jasco V-630). The tests were performed in the wavelength range of 400-800 nm. The cosmetic attractiveness of the membranes was portrayed against arbitrary letters.

In vitro Skin Permeation Studies

The skin permeation tests were carried out according to permission approved by animal ethical committee, University of Kerala. The in vitro study of KP delivery on rat skin was performed using modified Franz diffusion cell. Male Wister rats (10-12 weeks old) were used. The animals were culled by the prolonged chloroform inhalation and the excised skin was processed following the procedure reported by our previous project.61 Rat skin was mounted onto Franz apparatus with the SC pointing the donor chamber. The receiver chamber filled with phosphate buffer saline was stirred at 450 rpm speed and maintained at room temperature. The KP loaded TND patch was then casted carefully into the skin specimen at the donor chamber and wetted with two drops of DMSO. The receptor medium was spectrophotometrically analyzed for KP at predetermined time intervals. The patch with highest permeability was further employed to research the impact of storage time and temperature on the KP release profile. Patches stored for 0, 30 and 60 days at room temperature were used to test the influence of storage time. Next the


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highly efficient patches were further kept for 60 days at 4.0 ± 1.0 ℃, 30.0 ± 1.0 ℃ and 45.0 ± 1.0 ℃ to research the impact of storage temperature.

In Vitro Skin Adhesion Test

The in vitro skin adhesion behavior of the Patches was determined in fresh rat skin following the strategy reported elsewhere.45,62 The experiment was performed on INSTRON testing machine (INSTRON, UK). After completely removing the hairs, the full thickness rat skin was mounted cautiously on the moving cartilage of the test machine using cyanoacrylate. The TND patches were wetted and fixed on the wet skin surface. The adhered patches were peeled off at 900 stripping angle and measured the peel adhesion force.

Cell viability and Environmental Fitness Assay

The cell viability of the optimized membrane was investigated in HaCaT cell lines using MTT assay following the procedure detailed in our previous work.16,18 Next the microbial resistance of the film reinforced with AuD was studied against Escheria coli. The test specimen were added to LB broth and mixed thoroughly. The obtained formulations were autoclaved for 1.0 h at 120 ℃ and 15.0 Psi pressure. After autoclaved the formulations were poured into petri dishes and embedded with previously cultured Escheria coli cells. After incubating at 37 ℃ for 24.0 h, the specimens were checked for any visible features of microbial growth. The specimens that resisted the microbial growth were branded as environmental fit and suitable for topical application. All tests were carried out as duplicates, and repeated on two independent days.63



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