Cyclodextrin-Based Nanosponges as a

Article Copyright © 2014 American Scientific Publishers All rights reserved Printed in the United States of America

Journal of Nanopharmaceutics and Drug Delivery Vol. 2, 311–324, 2014 www.aspbs.com/jnd

Cyclodextrin-Based Nanosponges as a Nanotechnology Strategy for Imiquimod Delivery in Pathological Scarring Prevention and Treatment Chiara Bastiancich1 † , Sara Scutera2 , Daniela Alotto3 , Irene Cambieri3 , Mara Fumagalli3 , Stefania Casarin3 , Silvia Rossi2 , Francesco Trotta4, Maurizio Stella3 , Roberta Cavalli1 ∗ , Tiziana Musso2 , and Carlotta Castagnoli3 1

Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, 10125, Italy Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Università degli Studi di Torino, 10126, Italy 3 Dipartimento di Chirurgia Generale e Specialistiche, Banca della Cute, AOU Città della Salute e della Scienza di Torino, 10126, Italy 4 Dipartimento di Chimica, Università degli Studi di Torino, 10125, Italy 2

Although hypertrophic scars are a common complication in a high percentage of burn injuries, to date their optimal treatment still remains to be established. Herein, the antiproliferative effect of Imiquimod on human fibroblasts is evaluated and an innovative nanotechnological approach for Imiquimod delivery by pyromellitic cyclodextrin-based nanosponges is proposed. We observed that Imiquimodby inhibits cell proliferation andto: induces apoptosis of normal skin and hypertrophic Delivered Publishing Technology Chiara Bastiancich scar fibroblasts. When incorporated cyclodextrin-based nanosponges, Imiquimod shows a high encapsulation effiIP:into 81.245.241.98 On: Wed, 09 Mar 2016 20:20:29 Copyright: American Scientific ciency and a slow and prolonged in vitro kinetic release. These featuresPublishers give Imiquimod-loaded nanosponges a greater antiproliferative effect on human fibroblasts and on an immortalized non-tumorigenic keratinocyte cell line, compared to free Imiquimod. In conclusion, we suggest that this innovative nanomedicine formulation could provide an important contribution to the prevention and therapy of post-burn hypertrophic scars by combining the antiproliferative capacity of Imiquimod with the ability of the nanosponges to enhance the drug activity.

KEYWORDS: Skin Wound Healing, Hypertrophic Scars, Imiquimod, Nanosponges, Drug Nanodelivery System.

INTRODUCTION Hypertrophic scar (HS) formation is an aberrant wound healing response due to excessive fibrosis and deposition of extracellular matrix. HSs are observed as a common complication in a high percentage of burn injuries. Excessive deposition of collagen, tissue hyperplasia and increase in cellular turnover cause pain, pruritus, rigidity and aesthetic problems that limit the working and social life of those affected.1 2 HS pathogenesis involves several immunocompetent and structural cells, such as lymphocytes, monocytes and fibroblasts.3 Dermal fibroblasts are key contributors of the wound healing process, and ∗

Author to whom correspondence should be addressed. Email: [email protected] † Present address: Université Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Belgium. Received: 19 January 2015 Revised/Accepted: 6 May 2015 J. Nanopharmaceutics Drug Delivery 2014, Vol. 2, No. 4

an imbalance between their proliferation and apoptosis leads to impaired wound healing.4 Since the pharmacological therapy has not provided satisfactory results yet, the current therapeutic approach to HSs is based on surgical excision or on the application of pressure garments and silicone sheets. However, to date these techniques leave much to be desired in terms of patient compliance and of therapeutic, functional and good cosmetic outcome. The key role of fibroblasts and the involvement of the immune system in HS generation has been fully documented, prompting numerous researchers to evaluate the use of Imiquimod as one of the emerging options for HS prevention and therapy.5 Imiquimod (1-[2methylpropyl]-1H-imidazo[4,5-c]quinolin-4-amine) is an immune response modifier which enhances both the innate and cellular immune responses by acting on a number of cell types. Imiquimod stimulates the maturation of pDC and Langerhans cell migration.6 7 Furthermore, Imiquimod 2167-9312/2014/2/311/014

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CDNS as a Nanotechnology Strategy for Imiquimod Delivery in Pathological Scarring Prevention and Treatment

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induces profound Th1 responses and activates NK cells, units and polar free carboxylic acid groups that allow macrophages and B-lymphocytes.8 Cells activated by nanosponges to host both apolar organic molecules and Imiquimod via TLR-7 secrete cytokines such as IFN-, positively charged substances.21 8 IL-6 and TNF-. Imiquimod also induces membranePyromellitic cyclodextrin-based nanosponges or pyrodeath receptor-independent apoptosis as well as autophagic nanosponges (CDNS) can be incorporated into creams or cell death in various cell types.9–11 gels for topical slow and sustained drug delivery.22 The ™ Five percent of Imiquimod cream (Aldara was firstly prolonged release kinetics reduces irritancy by decreasing approved by the FDA in 1997 for the topical treatment the local concentration of free drug in contact with the of external genital and perianal warts and in 2004 for skin. the treatment of actinic keratosis and superficial basal The aim of our study was to evaluate the antiprolifcell carcinoma in adults. Since then, the unlicensed use erative action of Imiquimod on HS fibroblasts and to of Imiquimod was extended to the treatment of various develop and characterize a new nano-delivery system condermatologic viral and tumoral pathologies, such as moltaining Imiquimod incorporated in cyclodextrin-based luscum contagiosum, xeroderma pigmentosum, Bowen’s nanosponges cross-linked with pyromellitic dianhydride disease.12–14 Moreover, it was shown that is effective (IMQ-CDNS) for use in the prevention and treatment of against keloids and HSs through local release of IFNs post-burn HS. which degrade excess collagen and block the formation of new collagen.10 15 MATERIALS AND METHODS The use of a topical formulation of Imiquimod for the Materials treatment of dermatological disorders offers several advan-cyclodextrins were kindly donated by Roquette (France), tages over surgical procedures: patients may apply the drug pyromellitic dianhydride was purchased from Sigmaby themselves and the treatment, which is painless and Aldrich (USA). Imiquimod was obtained from In VivoGen 16 economical, reduces the risk of permanent scarring. (USA). Human Factor X was from Haematologic Tech™ However, there are limitations in the use of Aldara : it nologies Inc (USA). necessitates long treatment times with numerous applicaAcetic acid was purchased from Riedel-de Haen tions that may lead to local adverse reactions (e.g., irri(Germany) and Triethylamine from Fluka (Switzerland). tation and hyper-pigmentation), and only small skin areas Delivered by Publishing Technology to: Chiara Bastiancich All the other reagents were supplied by Sigma-Aldrich. 17 can be treated. Psoriasis-like lesions occur in susceptible IP: 81.245.241.98 On: Wed, 09 Mar 2016 20:20:29 All reagents were used without further purification. patients with basal cell carcinoma and actinic keratosis. Copyright: American Scientific Publishers Milli-Q (Merck-Millipore, Germany) water was used Repeated application of Aldara™ on murine skin results throughout the studies. in inflammation and hyperplasia of epidermis resembling human psoriasis.18 Adverse skin reactions seem due to Synthesis of Pyromellitic Cyclodextrin-Based the vehicle cream (isostearic acid) rather than Imiquimod Nanosponges per se.19 The CDNS were prepared using pyromellitic dianhyAs post-burn HSs are normally extended to large skin dride for the cross-linking and a molar ratio between areas, the use of a drug carrier capable of protecting the -cyclodextrin and cross-linker of 1:4, as previously skin from Imiquimod adverse effects by releasing it slowly reported.23 Briefly, an amount of anhydrous -cyclodextrin over time could be an interesting approach for the prevenand pyromellitic dianhydride was put to react with tion and treatment of HSs in post-burn patients. This led dimethyl sulfoxide (DMSO) in presence of triethylamine us to test a new type of cyclodextrin-based nanosponges. and stirred at room temperature for 24 hr. Once reacNanosponges are a class of biocompatible nanostruction was complete, the solid was ground in a mortar tured polymers that can be obtained with , and and washed with deionized water to remove unreacted cyclodextrins cross-linked with a suitable cross-linking pyromellitic acid and Soxlet-extracted with acetone to agent. They are solid particles with a spherical morphology remove DMSO residues and impurities. After purification, and are used in nanomedicine as multifunctional nanoscale CDNS were stored at 25 C until further use. drug delivery systems for their capacity to incorporate different molecules in their structure by forming inclusion Preparation of Imiquimod-Loaded Pyromellitic or non-inclusion complexes.20 For instance, this allows an Cyclodextrin-Based Nanosponges Suspensions increase of the solubility and stability of a drug, to protect An amount of CDNS was weighted and suspended in active molecules from degradation or to obtain the control Milli-Q water to obtain a nanosuspension with a final of the drug release in time.21 concentration of 10 mg/ml. The suspension was sonicyclodextrin-based nanosponges produced by using cated for 5 min in a Soltec Sonica ultrasound bath and pyromellitic dianhydride as cross-linker provide a highly stirred for 30 min. It was then homogenized in an Ultraswellable white powder that forms hydrogels on conturrax Ika T18 high-shear homogenizer at 24,000 rpm tact with water. The compound contains -cyclodextrin 312

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for 5 min, stirred at 25 C for 24 hr and subsequently homogenized with an Avestin Emulsiflex C5 High Pressure Homogenizer for one hr with a back-pressure of 30,000–50,000 kPa to reduce the size of the nanosponges. The suspension was then dialyzed using a dialysis bag with a membrane Spectra/Por® (cut off 12,000–14,000 Da) and left in Milli-Q water under stirring for two hr replacing with new medium every 30 min to remove any impurities of the synthesis. The pH of the suspension was then adjusted to the desired value with a solution of Sodium Hydroxide 0.1 M and stirred for 30 min. The suspension was then stored at 4 C until further use. To prepare the IMQ-CDNS, a solution of Imiquimod (1 mg/ml) was dispersed in the 10 mg/ml CDNS nanosuspension in a ratio of 1:1 (v/v) and was stirred for 24 hr at room temperature. The pH was adjusted to 6.5 and the system was purified by dialysis to eliminate any not encapsulated drug and then stored at −20 C until further use. The IMQ-CDNS nanosuspension was defrosted and stirred for 30 min before use. An amount of the IMQCDNS nanosuspension was freeze-dried using a Modulyo freeze-drier (Edwards, UK) to obtain the drug-loaded nanosponges as a powder.

solution and placed in the electrophoretic cell, where an electric field of about 15 V/m was applied. The nanosponge morphology was evaluated by a Philips CM10 transmission electron microscope (TEM). The suspension of CDNS was sprayed onto a copper grid and dried before observation.

Quantitative Determinations of Imiquimod The quantitative determinations of Imiquimod in the suspensions of IMQ-CDNS were measured by HPLC analysis using a Shimadzu model no. LC-10AD, equipped with a spectrophotometric UV-Vis detector Shimadzu SPD10AV and a Shimadzu C-R6A Chromatopac integrator, under isocratic conditions. The separation was carried out using an Agilent TC C18 column with a pore size of 5 m with a mobile phase containing acetonitrile, acetate buffer (100 mM, pH 4) and triethylamine in a ratio of 30:69.85:0.15 (v/v) as previously reported.24 The detection wavelength was set at 242 nm and the flow rate was maintained at 1 ml/min. To evaluate the total drug content in IMQ-CDNS, an amount of nanosuspension was diluted in the HPLC mobile phase and sonicated in an ultrasound bath at 25 C for 5 min to extract the drug from the nanocarrier. The suspension was then stirred for 30 min and 1 ml was centrifuged at 10,000 rpm for 10 min. The Physicochemical Characterization of Pyromellitic supernatant was injected into the HPLC and the average Cyclodextrin-Based Nanosponges value of three injections was inserted in the calibration Fourier Transform Infrared Spectroscopy Delivered by (FTIR) Publishing Technology to: Chiara Bastiancich curve. IP: 81.245.241.98 On: Wed, 09 Mar 2016 20:20:29 FTIR was carried out by the usage of a Perkin-Elmer Copyright: American Scientific Publishers system 2000 spectrophotometer to investigate any interacDetermination of the Encapsulation Efficiency of tion between the drug and the CDNS. The spectra were Imiquimod Loaded in Pyromellitic Cyclodextrin-Based −1 obtained on KBr pellets in the region of 4,000 cm to Nanosponges −1 650 cm . The encapsulation efficiency (EE) was determined using a filtration technique. One hundred l of IMQ-CDNS Differential Scanning Calorimetry (DSC) nanosuspension that had not been purified by the last dialDSC analysis were carried out using a Perkin Elmer DSC ysis step were placed into an Amicon® Ultra-0.5 cen7 system apparatus equipped with a TAC 7/DX instrument trifugal filter device (Merck-Millipore) consisting of two controller. Samples of 1–3 mg of solid CDNS, freezemicrocentrifuge tubes: one tube is used to collect the fildried IMQ-CDNS and free Imiquimod were weighed in trate, while the other, containing the ultrafiltration device, aluminium sample pans and heated at 10 C/min in the is used to recover the nanosponges. The system was cenrange of 20–200 C under nitrogen purge. trifuged at 15,000 rpm for 30 min in a Beckman Coulter Allegra 64R Centrifuge. Then 20 l of the filtrate at Characterization of Suspensions of Blank the bottom of the external tube were diluted in the HPLC and Imiquimod-Loaded Pyromellitic mobile phase in a ratio of 1:50 (v/v). The solution was then Cyclodextrin-Based Nanosponges agitated in a Velp vortex mixer for one min. The sample CDNS and IMQ-CDNS sizes and polidispersity indexes was then injected into the HPLC to obtain the concentra(PDIs) were measured by dynamic light scattering using tion of unecapsulated Imiquimod in the nanosuspension. a 90 Plus particle sizer (Brookhaven Instrument CorporaThe encapsulation efficiency was calculated as follows tion, USA) equipped with MAS OPTION particle sizing EE% = 1 − free drug/total drug content ∗ 100 software. The measurements were made at a fixed angle of 90 and 25 C for all samples. For the measurement In Vitro Release of Imiquimod from Pyromellitic each sample was suitably diluted with Milli-Q water. Zeta Cyclodextrin-Based Nanosponge Formulations potential measurements were also made using an additional electrode in the same instrument. For zeta potential The in vitro release of Imiquimod from IMQ-CDNS determination the samples were diluted with 0.1 mM KCl was obtained using multi-compartment rotating cells with J. Nanopharmaceutics Drug Delivery 2, 311–324, 2014

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a dialysis membrane (Spectra/Por®, cut off 12,000– 14,000 Da) for a period of 24 hr. The donor phase consisted of suspensions containing IMQ-CDNS at pH 6.5 to mime the chronic wound pH value.25 The receiving phase, which was phosphate buffer (0.05 M pH 7.4) as previously reported for drug release studies for wound healing applications,26 27 was completely withdrawn and replaced with fresh medium at fixed time intervals, suitably diluted and analyzed using the HPLC method described previously. The amount of Imiquimod released was expressed considering 100% the total amount of encapsulated drug.

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Immunohistochemistry for Ki67 Immunostaining for Ki67 was performed on NS fibroblasts untreated or treated with Imiquimod (25 g/ml). The cells were seeded at a density of 2 × 104 and after 72 hr the slides were fixed for 10 min at 4 C in fresh pure acetone and then incubated with monoclonal anti human Ki-67 (clone B56, BD Pharmingen, USA). Endogenous peroxidase activity was inhibited by the addition of methyl alcohol and 0.03% hydrogen peroxide. The immunoperoxidase method with biotinylated secondary antibody and an avidin-biotin-horseradish peroxidase complex LSAB + (DAKO, Denmark) was used for the immunohistochemical staining. The slides were examinated in double blind and microphotographs were taken using a DMLA Leica microscope equipped with a digital camera (Leica DFC 425C). Images were acquired using LAS software (Leica Application Suite). The positive cells were counted on 3 different samples in an epithelial area of 0.05 mm2 .

Biological Studies Cell Cultures Fibroblasts were extracted from cutaneous normal skin (NS) biopsies (N = 3) and HS biopsies (N = 3) taken from patients after having received individual informed consent (approved by the Ethical Committee of the AOU “Città della Salute e della Scienza di Torino”—CTO Hospital, Annexin V Staining Assay protocol n. 0041451) during reconstructive plastic proceThe quantification of cell death was determined by flow dures at the Turin Burn Centre. cytometry using the Annexin V-FITC apoptosis detecBriefly, the tissue was minced and incubated overnight tion kit (Biolegend, USA) according to the manufacturer’s in Dulbecco’s modified Eagle’s medium DMEM (Gibco, instructions. Briefly, fibroblasts were synchronized by starInvitrogen, USA) supplemented with 100 U/ml penicillin vation for 24 hr in DMEM containing 0.5% FBS and then and 100 mg/ml streptomycin. Afterwards, the biopsies treated with Imiquimod or H2 O2 for 48 hr. The cells were were incubated in 2 mg/ml Dispase II (Roche Manheim, stained with Annexin V-FITC and propidium iodide (PI) CO2 for 2byhr.Publishing The dermis, Germany) at 37 C and 5%Delivered Technology to: Chiara Bastiancich and analyzed within 1 hr by FACS Calibur Flow CytomeIP: 81.245.241.98 obtained after epithelial layer removal, was incubatedOn: for Wed, 09 Mar 2016 20:20:29 Copyright: American Scientific Publishers ter (BD Biosciences, USA). 1 week in 24 well plates at 37 C and 5% CO2 in DMEM supplemented with 10% FBS (Sigma) and antibiotics. Real-Time PCR Once an adequate growth of fibroblasts had been detected, Total RNA isolated with the Qiagen RNeasy mini kit was the cells were trypsinized for subcultures in 75 cm2 tistreated with DNase I (Qiagen, Germany) and retrotransue culture flasks. For stimulation experiments, fibroblasts scribed into cDNA by the iScript cDNA Synthesis Kit were used at 4–6 passage of culture. (Bio-Rad Laboratories Inc., USA). Gene specific primers Human immortalized non-tumorigenic keratinocyte cell were: line (HaCaT) was cultured in DMEM (high glucose) sup-actin (sense, 5 -CGCCGCCACCTCACCATG-3 ; plemented with 2 mM L-glutamine solution and 10% FBS. antisense, 5 -CACGATGGAGGGGAAGACGG -3 ); Bax (sense, 5 -AGAGGATGATTGCCGCCGT-3; MTT Colorimetric Assay antisense, 5 -CAACCACCCTGGTCTTGGATC-3 ); Fibroblasts and HaCaT cell viability was measured by the Bcl-xL (sense, 5 -GTAAACTGGGGTCGCATTGT-3 ; MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium antisense, 5 - TGCTGCATTGTTCCCATAGA-3). bromide assay (Roche). Fibroblasts were seeded at a density of 1 × 104 cells/well in a 24-well plates; HaCat were seeded at 2 × 104 cells/well in a 24-well plates. After 2 hr, the cells were either incubated with the indicated compounds or left untreated. Each experimental condition was performed in triplicate. Cell proliferation analysis was performed after 24, 48, 72 and 96 hr. Spectrophotometric readings at 570 nm were performed with an Eppendorf BioPhotometer photometer. Data are shown as optical density or as percentage of average proliferation rate compared to the control unstimulated cells or CDNS. 314

Quantitative real-time PCR was done by the iQ™ SYBR Green Supermix (Bio-Rad Laboratories Inc.), according to manufacturer’s instructions. Reactions were run in duplicate on an iCycler Chromo4™ (Bio-Rad Laboratories Inc.) and the generated products were analyzed by the Opticon Monitor™ 3.0 Software (Bio-Rad Laboratories Inc.). Gene expression was normalized based on the -actin mRNA content. In Vitro Scratch-Wound-Closure Assay The fibroblasts were seeded in 4-well chamber slide (Thermo SC NUNC) at a concentration of 1 × 104 cells. J. Nanopharmaceutics Drug Delivery 2, 311–324, 2014

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Confluent monolayers were synchronized for 2 hr in DMEM containing 0.5% FBS, and wounded by removing a 500–800 m wide strip of cells across the well with a standard 200 l pipette tip. The wounded culture wells were washed with PBS to remove any non-adherent cells, and treated with free Imiquimod (5 g/ml) or IMQ-CDNS (5 g/ml) or CDNS. Wound-closure effects were observed and photographed by an optical microscope (Axiovert, ZEISS) at 0, 24, 48 and 72 hr.

suppresses fibroblast proliferation under basal and profibrogenic conditions.

Evaluation of Apoptosis in Imiquimod-Treated Hypertrophic Scars and Normal Skin Fibroblasts Fibroblast apoptosis is crucial for the resolution of fibrosis and reduced fibroblast apoptosis is associated with pathological scar formation.28 To assess the effect of Imiquimod on apoptotic cell death, HS and NS fibroblasts were treated for 48 hr with Imiquimod (25 g/ml) or H2 O2 (1.5 mM) Statistical Analysis as positive control, and stained with Annexin V-FITC and Statistical significance between the experimental groups propidium iodide (PI). The cytogram of the four quadwas determined by the Mann-Whitney U test or one-way rants in Figure 2(A) was used to distinguish the live cells analysis of variance (ANOVA) with Tukey’s post hoc test (Annexin−/PI−), early apoptotic cells (Annexin+/PI−), (GraphPad Prism version 4.00 for Windows, GraphPad late apoptotic cells (Annexin+/PI+, and necrotic cells Software). (Annexin−/PI+. There was a higher proportion of early apoptotic rates in Imiquimod-treated NS and HS fibroblasts than in the untreated control. An increase of the perRESULTS centage of cells, both in the early and late apoptotic stages, Effect of Imiquimod on the Proliferation of was observed when the fibroblasts were incubated with Hypertrophic Scars and Normal Skin Fibroblasts H2 O2 (Fig. 2(B)). The relative amounts of anti-apoptotic To explore the potential of Imiquimod as a HS treatment (Bcl-xL, Bcl-2, Bcl-w and others) and pro-apoptotic (Bax, agent, we determined its effects on the proliferation of BAD, Bid and others) proteins belonging to the Bcl-2 famfibroblasts derived from NS and HS biopsies. The cells ily control apoptosis. Therefore, so as to investigate into were treated for 72 hr with different concentrations of the mechanism underlying Imiquimod-induced apoptosis Imiquimod (from 5 to 50 g/ml) and MTT assay was in fibroblasts, we examined Bax and Bcl-xL gene expresperformed. A reduction in the proliferation of both NS sion in cells derived from HSs and NS. The expression and HS derived fibroblasts was observed at Imiquimod Delivered by Publishing Technology to: Chiara Bastiancich of Bax mRNA was significantly increased in HS and NS IP: 81.245.241.98 doses of 10–50 g/ml, whilst treatment with 5 g/mlOn: of Wed, 09 Mar 2016 20:20:29 fibroblastsPublishers treated with Imiquimod for 24 hr, whilst no sigAmerican Scientific Imiquimod did not significantly affectCopyright: cell proliferation. nificant differences were detected in the expression levels The inhibitory effect reached significance from a concenof Bcl-xL (Fig. 2(C), left panel). This demonstrated that tration of 25 g/ml for NS and 10 g/ml for HS. The the ratio between Bax/Bcl-xL increased in HS and NS cells proliferation rate of HS fibroblasts treated with Imiquimod after the Imiquimod treatment (Fig. 2(C), right panel). at 10, 25 or 50 g/ml decreased by about 37, 42 and These results indicate that Imiquimod inhibits HS and 56% respectively compared to untreated HS cells. The NS fibroblasts by the induction of apoptosis and highlights proliferation rate decreased by about 37% in NS fibrobthe contribution of Bcl-2 family members in Imiquimodlasts at an Imiquimod concentration of 25 g/ml and 48% induced apoptosis. at a concentration of 50 g/ml (Fig. 1(A)). Time dependent proliferation responses showed a statistically signifiPreparation and Characterization of Blank cant inhibition between 72 and 96 hours when fibroblasts and Imiquimod-Loaded Pyromellitic were treated with Imiquimod (25 g/ml) in comparison to Cyclodextrin-Based Nanosponges untreated cells. No significant differences were observed A new nanocarrier, made up of cyclodextrin-based between 24 and 72 hours (Fig. 1(B)). nanosponges cross-linked with pyromellitic dianhydride The effect of Imiquimod on the proliferation of acti(CDNS), was prepared and characterized as an innovavated fibroblasts was then assessed. As factor Xa (FXa) tive nanodelivery system for the delivery of Imiquimod. is a potent stimulator of profibrotic response, the effect of CDNS were used for Imiquimod formulation at a molar Imiquimod in combination with 1 U/ml FXa was evaluratio of 1:4 between the cyclodextrins and the cross-linker. ated. As shown in Figure 1(C), FXa enhanced HS cell proA schematic representation of the molecular dispersion liferation, whilst this effect was inhibited by co-treatment of Imiquimod in the nanosponge structure is shown in with Imiquimod. Figure 3. The morphology of the nanosponges was deterThe reduction of fibroblast proliferation mediated by mined by TEM analyses, which showed them to be spherImiquimod was also demonstrated using the proliferation ical and with a smooth surface (Fig. 4). marker Ki67 (Fig. 1(D)). The percentage of Ki67 posiThe CDNS suspensions were prepared using an tive nuclei was significantly lower in fibroblasts treated easily reproducible solvent-free method, which is with Imiquimod (25 g/ml) than in controls (13.1 ± cost-effective and applicable to large scale systems. 2.9% vs. 65.4 ± 5.2%). Our data indicate that Imiquimod J. Nanopharmaceutics Drug Delivery 2, 311–324, 2014

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Figure 1. Imiquimod inhibits proliferation of HS- and NS-derived fibroblasts. (A) Fibroblasts derived from NS and HS were treated for 72 hr with increasing doses of Imiquimod. Data of cell proliferation assessed using MTT assay are presented as percentage of cell viability (untreated cells assumed as 100%) (means ± SEM; n = 4). (B) Fibroblasts were treated without (CTRL) or with 25 g/ml Imiquimod (IMQ) and cultured for the indicated times. Cell proliferation is presented as means ± SEM of 3 independent experiments. ∗ p < 005 versus untreated cells. (C) HS fibroblasts were cultured for 72 hr with 25 g/ml IMQ, 1 U/ml factor Xa (FXa), alone or in combination. MTT assay was performed and expressed as % of cell viability (means ± SEM; n = 4). ∗ p < 005 versus untreated cells and # versus FXa treated cells. (D) Representative example of staining with Ki-67 antibody and percentage of Ki67-positive cells per field in dermal HS fibroblasts untreated or treated with Imiquimod (25 g/ml). 5 fields from 3 different experiments were examined. ∗ p < 005.

The physicochemical properties of CDNS and IMQ-CDNS are presented in Table I. The particle size and polydispersity index results demonstrated small dimensions with a narrow size distribution. The zeta potential values showed a marked negative surface charge on the nanosponges of about −28 mV, meaning that the particles have little tendency to aggregate. The concentration of Imiquimod obtained in the CDNS-IMQ nanosuspension was of 0.5 mg/ml and the drug encapsulation efficiency 316

was superior to 97%. The interaction between Imiquimod and the nanosponges was also confirmed by DSC and FTIR analyses, as shown in Figure 5. The FTIR spectra of the blank nanosponges showed the presence of carbonate bond peak at around 1,720 cm−1 , C–O absorption band at 1,000–1,250 cm−1 and O–H absorption band around 3,300 cm−1 . The main characteristic peaks of IMQ were at about 3,100 cm−1 , 2,500 cm−1 , 1,670 cm−1 , 650–900 cm−1 and disappeared in the IMQ-CDNS spectra. In particular, J. Nanopharmaceutics Drug Delivery 2, 311–324, 2014

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Figure 2. Imiquimod inhibits cell proliferation through apoptosis induction. NS and HS fibroblasts were incubated for 48 hr with 25 g/ml Imiquimod (IMQ) or with 1.5 mM H2 O2 , used as positive control. (A) Flow cytometry diagram of FITC-Annexin V-PI double staining in HS fibroblasts. The four quadrants represent different cellular conditions: lower left panel, viable cells; lower right panel, early apoptotic cells; upper right panel, late apoptotic or secondary necrotic cells; upper left panel, primary necrotic cells. (B) Histogram representation of the quantitative percentage of NS and HS fibroblasts at different conditions. Results are expressed as means ± SEM of 5 independent experiments. ∗ p < 005 versus untreated cells (C) Fibroblasts were treated with Imiquimod (25 g/ml) for 24 hr. After incubation total RNA was isolated and real-time RT-PCR was performed using primers specific for Bax and Bcl-xL mRNA and normalized on -actin. Results are presented as fold increase of Bax or Bcl-xL relative to the mRNA levels in untreated cells (assumed as the 1.0 value) in the left panel. Relative expression of the Bax/Bcl-xL ratio at the mRNA level of Imiquimod-treated fibroblasts is summarized in the right panel (means ± SEM; n = 3). ∗ p < 005 versus untreated fibroblasts. J. Nanopharmaceutics Drug Delivery 2, 311–324, 2014

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Figure 3. Synthesis protocol of CDNS with molar ratio of 1:4 between cyclodextrin and cross-linker (1) and schematic representation of the preparation of IMQ-CDNS (2).

In Vitro Release of Imiquimod from the Imiquimod-Loaded Pyromellitic Cyclodextrin-Based Nanosponges There was a gradual, sustained Imiquimod in vitro release from IMQ-CDNS nanosuspensions, i.e., below 45% at 24 hr, confirming a strong interaction between Imiquimod and the nanocarrier at a pH of 6.5. The release profile during the first 8 hr is shown in Figure 6. The absence of burst effect shows that Imiquimod is not absorbed on the surfaceto:ofChiara the nanosponges but it is incorporated within Delivered by Publishing Technology Bastiancich IP: 81.245.241.98 On: Wed, Mar 2016 the09 CDNS matrix20:20:29 from which diffuses slowly. Almost 42% Copyright: American Scientific Publishers of Imiquimod had been released after 8 hr, after which there was only a slight increase in the 24 hr. These results suggest that CDNS could be an effective reservoir for Imiquimod as they strongly interact with the drug and Figure 4. Trasmission electron microscopic (TEM) image of release it slowly over time. CDNS (9000×). Bar = 1 m.

the region corresponding to the aromatic C–H and C C bending differed greatly in the free drug or loaded drug spectra, suggesting that the small and lipophilic molecule of Imiquimod may be hosted inside the nanosponge structure. Although the CDNS and IMQ-CDNS spectra are very similar, they do present some differences between 1,026– 623 cm−1 , 1,340–1,079 cm−1 and 1,721–1,582 cm−1 , confirming definite interaction between the drug and the nanocarrier. The DSC IMQ-CDNS thermogram shows the disappearance of the endothermic peak at around 300 C, which corresponds to the melting point of the drug, confirming the molecular interaction between Imiquimod and the nanocarrier. Table I.

Physicochemical properties of CDNS and IMQ-CDNS at pH 6.5 (means ± SD, n = 3).

CDNS IMQ-CDNS

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Assessment of the Effect of Imiquimod-Loaded Cyclodextrin-Based Nanosponges on Fibroblasts and HaCat Proliferation and Wound Healing Process The antiproliferative effect of IMQ-CDNS was evaluated in comparison with CDNS and free Imiquimod after 72 hr of treatment. According to the dose response graph shown in Figure 7 (upper and middle panels), free Imiquimod and IMQ-CDNS have a similar antiproliferative activity on NS and HS fibroblasts at higher doses, while at a concentration of 5 g/ml only the nanoencapsulated drug still maintains a significant antiproliferative effect on the cells. The effect of IMQ-CDNS was also evaluated on HaCaT cells (Fig. 7, lower panel), that have previously been

Particle size (nm)

Polydispersity index

Zeta potential (mV)

Imiquimod efficiency (%)

258.0 ± 9.18 258.5 ± 11.4

0.236 ± 0.07 0.239 ± 0.02

−28.12 ± 1.06 −28.24 ± 1.96

– 97.78 ± 0.29%


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Figure 5. FTIR spectra (A) and DSC thermogram (B) of solid CDNS, freeze-dried IMQ-CDNS and Imiquimod (IMQ). FTIR spectra (upper panel) and DSC thermogram (lower panel) of solid CDNS, freeze-dried IMQ-CDNS and Imiquimod (IMQ).

Figure 6. In vitro release profile of Imiquimod from IMQCDNS suspension at pH 6.5 for the first 8 hr (means ± SD; n = 3). J. Nanopharmaceutics Drug Delivery 2, 311–324, 2014

shown to be sensitive to Imiquimod.29 The antiproliferative effect of free Imiquimod was observed at 10 and 25 g/ml whilst IMQ-CDNS showed a remarkable inhibitory effect even at 5 g/ml, as observed in the fibroblasts. The fibroblasts migration was evaluated by a scratchwound-closure assay in fibroblasts untreated or treated with CDNS or free Imiquimod or IMQ-CDNS. The results in Figure 8 show that the samples left untreated or treated with CDNS or with free Imiquimod (5 g/ml) completed the wound healing process within 24 hr. Conversely, fibroblasts treated with low doses of IMQCDNS (5 g/ml) showed a delayed healing process as demonstrated by incomplete closure of the scratch after 72 hr. These results indicate that Imiquimod loaded into 319


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HS and NS fibroblasts treated with Imiquimod show a statistically significant inhibition of the cell proliferation. Imiquimod also had a suppressive effect in the presence of Factor Xa, which stimulates the fibroproliferative response of fibroblasts.30 Consistent by our observation, an antiproliferative effect of Imiquimod has been reported in various cancer cell lines including skin cancer cells.9 11 Imiquimod treatment of HS and NS fibroblasts showed a significant increase in the percentage of annexin V-positive cells indicating that apoptosis is involved in the antiproliferative effect. Furthermore, we demonstrated that this increase in apoptosis is mediated by changes in the levels of the Bcl-2 family of proteins. Indeed, although Imiquimod did not affect the Bcl-xL expression, it did markedly increase the expression of the proapoptotic gene Bax, leading to an increase in the Bax/Bcl-xL ratio. It is known that Imiquimod exerts its effects through Toll-like receptor 7 (TLR7) and/or TLR8.31 32 Dermal fibroblasts constitutively express mRNA from TLR1 through TLR9 and an increased TLR7 and TLR8 expression was observed in the fibroblasts from hypertrophic scars and keloids.33 34 Therefore, it is conceivable that TLR7 and/or TLR8 are involved in the antiproliferative effect we observed. However, TLRindependent pathways cannot be excluded as Imiquimod also exerts some effects in TLR7/8-negative cells, such as the HaCaT cell line, through interference with the adenosine receptor signalling mediated by adenylyl cyclase.31 32 Delivered by Publishing Technology to: Chiara Bastiancich Imiquimod 5%20:20:29 cream is indicated for the topical treatIP: 81.245.241.98 On: Wed, 09 Mar 2016 ment of actinic keratosis, superficial basal cell carcinoma Copyright: American Scientific Publishers and genital warts. Recently, various clinical trials have been carried out on Imiquimod as an adjunctive treatment to prevent keloid recurrence after surgical excision and has been reported to reduce the recurrence rate.10 35 However, there is some result variability, but this may well be due to the small sample size, short follow-up period, and poor patient compliance.36 37 Further investigations on the effect of Imiquimod on the prevention and treatment of keloids and HSs is required so as to improve its efficacy through innovative nanomedicine formulations. Figure 7. NS, HS fibroblasts and HaCaT were treated for 72 hr Currently, nanotechnologies represent one of the main with increasing doses of free Imiquimod or CDNS or IMQCDNS (5, 10 or 25 g/ml). Data of cell proliferation assessed fields of interest for pharmaceutical researchers and indususing MTT assay are presented as percentage of cell viability tries, as they could change the future of therapeutics and (untreated cells or CDNS assumed as 100%) (means ± SEM; diagnostics, allowing the formulation of drugs that exploit ∗ n = 3). p < 005 versus untreated or CDNS treated fibroblasts. biocompatible nanocarriers. Nanoscale particles, as drug carriers, have the potential to protect the encapsulated nanosponges is more efficacious on fibroblast proliferation molecules from degradation, to reduce side effects and and migration compared to free Imiquimod. improve the efficacy of a drug by releasing it slowly over time. Moreover, these nanocarriers allow for a targeted DISCUSSION delivery and a controlled release, providing more effective and convenient routes of administration and lower The abnormal wound healing process resulting in hyperdrug toxicity, as well as extending the product life cycle.38 trophic scarring is characterized by an exaggerated In the field of dermatology micro and nano-sized partiimmune and inflammatory response which leads to the cles have been investigated to improve transdermal and fibroblast proliferation and fibrosis. Despite the variety of topical drug delivery.39 For example, nanoparticles can therapeutic strategies, to date no drugs are available for carry resveratrol and coenzyme Q10 efficiently into the a satisfactory treatment of this complication. Our data on 320


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Delivered by Publishing Technology to: Chiara Figure 8. Representative micrographs of scratch-wound-closure assay following freeBastiancich Imiquimod or CDNS or IMQ-CDNS treat81.245.241.98 On: Wed, 09 Mar 20:20:29 ment in HS fibroblasts. Fibroblasts IP: were synchronized for 2 hr, wounded and2016 treated as indicated for 72 hr before being phoCopyright: American Scientific tographed. Each experiment was performed in triplicate. Arrows indicate the Publishers means ± SEM of three replicates of the wound size of each sample. Bars = 100 m. skin whilst maintaining their antioxidant properties.40 41 Several studies concerning nanomedicine and its applications as antibacterial wound dressings and wound healing enhancers have been previously published.27 42–48 Recent studies have reported the use of ethosomes and liposomes for the encapsulation of 5-fluorouracil for their use in the topical treatment of HSs.49 As Imiquimod might be able to act more selectively against the physiopathogenetic mechanisms involved in the formation of HSs we decided to focus our attention on this drug formulation for our research instead of other emerging therapeutic molecules or strategies. A new nanocarrier, made up of cyclodextrin-based nanosponges cross-linked with pyromellitic dianhydride, was purposely prepared and characterized as an innovative nanocargo for the topical delivery of Imiquimod, in an attempt to overcome the limitations of the 5% Imiquimod cream. Imiquimod has been previously incorporated into different nanodelivery systems for the treatment of skin cancers, as a possible aid for chemo-immunotherapy and transcutaneous immunization and as a vaccine adjuvant.50–54 We chose to use nanosponges as they have -cyclodextrin building blocks linked by ester bonds to the pyromellitic J. Nanopharmaceutics Drug Delivery 2, 311–324, 2014

acid to form a hyper cross-linked network capable of hosting molecules, such as Imiquimod, inside their lipophilic cyclodextrin cavities. Further interactions with cross-linker network can cooperate to stabilize imiquimod by ionic bonds. This types of CDNS are soft, swellable and form stable nanohydrogel structure on contact with water, making them very suitable for topical applications.21 55 A recent study by Conte et al. describes the potential of CDNS as a multifunctional ingredient in semisolid formulations for drug delivery to the skin.56 Moreover, they have recently proven to be safe, biodegradable and biocompatible.57 Their use as drug delivery systems has been tested with a wide range of molecules and, thanks to their structure, they might be able to incorporate different molecules at the same time.21 58 The percentage of water uptake increases with the pH of the external medium and reaches the best swelling properties at pHs over 6, due to the presence of dissociated carboxylic groups of the pyromellitic acid network (data not shown). Therefore, we decided to work on CDNS and IMQCDNS nanosuspensions with a pH of 6.5 to mimic the pH of the skin in HS and to obtain a higher electrostatic interaction capacity between the drug and the carrier matrix. 321


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The IMQ-CDNS preparation was tuned by using a optimal rheological and bioadhesive properties, compatisolvent-free method and reducing by homogenization the ble with HS skin and able to have a slow, prolonged drug sizes of the nanosponges so as to obtain homogeneous release. We assume that the great swelling and mucoadnanosuspensions with sizes within the 240–270 nm range hesion properties of the CDNS (data not shown) could and low polydispersity index. The high Imiquimod encapallow to incorporate the nanosuspension in a semisolid forsulation efficiency value (∼97%) proved the affinity of mulation for topical use avoiding the usage of patches or the drug for the NS nanostructure, which was confirmed garments on top of the HS. We will carry on some permeby DSC and FTIR analysis. This behaviour could be ability studies using Franz cells to evaluate the diffusion explained by the evaluation of the chemical structure of the of the drug in normal and HS skin biopsies. We are also drug: Imiquimod is a lipophilic molecule and we hypothcurrently evaluating some animal models to test the bioesize that it might form inclusion complexes with the compatibility, tolerability and efficacy of IMQ-CDNS. cyclodextrin hydrophobic cavities present in the CDNS structure. Futhermore, at a pH value of 6.5 it can form Acknowledgments: This work was supported by funds additional electrostatic interactions with the dissociated from the Compagnia di San Paolo, Turin, Italy, Fondazione carboxylic groups of the cross-linker network, thanks to CRT, Turin Italy, and from Piedmont Foundation of Studthe presence of an ammine group in the drug structure. The ies and Research on Burns Simone Teich Alasia. Irene carrier incorporates Imiquimod strongly and then releases Cambieri, Mara Fumagalli, were supported by a fellowship it slowly in vitro (about 43% in 24 h). As previously from the Compagnia di San Paolo. Stefania Casarin, Silvia reported by Stella and Rajewski and by Loftsson and MasRossi were supported by a fellowship from the Piedmont son we assume that this prolonged release kinetics can Foundation of Studies and Research on Burns. Chiara Basbe correlated to the presence of cyclodextrins.22 59 We tiancich, Roberta Cavalli were supported by University of suppose that the lipophilic part of Imiquimod interacts Turin research founds (ex 60%). The authors also thank with the hydrophobic central cavity of cyclodextrin in the Barbara Wade for her linguistic contribution. nanosponge structure, while the protonated ammine group of the drug forms additional electrostatic bonds with the REFERENCES dissociated carboxylic acids of the cross linker at a pH 1. M. Stella, C. Castagnoli, and E. N. Gangemi, Postburn scars: An of 6.5. update. Int. J. Low. Extrem. Wounds 7, 176 (2008). 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