LL37 loaded nanostructured lipid carriers (NLC): A new strategy for the topical treatment of chronic wounds

1 LL37 LOADED NANOSTRUCTURED LIPID CARRIERS (NLC): A NEW STRATEGY FOR THE TOPICAL TREATMENT OF CHRONIC WOUNDS Itxaso Garcia-Oruea, Garazi Gainzaa,c, Cecilia Girbaud, Rodrigo Alonsod, José Javier Aguirree, José Luis Pedraza,b, Manoli Igartuaa,b and Rosa Maria Hernandeza,b,* a NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, Vitoria-Gasteiz 01006, Spain. b Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Vitoria-Gasteiz, Spain. c Biopraxis Research AIE, Miñano, Vitoria-Gasteiz d Department of Immunology, Microbiology and Parasitology, School of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, Vitoria-Gasteiz 01006, Spain e Hospital Universitario de Álava (HUA) Txagorritxu, Vitoria-Gasteiz, 01009, Spain *Corresponding author: Prof. Rosa Maria Hernandez Laboratory of Pharmaceutics, University of the Basque Country School of Pharmacy, Paseo de la Universidad, 7 01006 – Vitoria-Gasteiz, Spain Telephone: +34 945013095 Fax: +34 945013040 E-mail: [email protected] This is the accepted manuscript of the article that appeared in final form in European Journal of Pharmaceutics and Biopharmaceutics 108 : 310-316 (2016), which has been published in final form at https://doi.org/10.1016/j.ejpb.2016.04.006. © 2016 Elsevier under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 ABSTRACT The LL37 is a human antimicrobial peptide which not only has a broad spectrum of antimicrobial activity, but it has also been proved to modulate wound healing by participating in angiogenesis, epithelial cell migration and proliferation, and immune response. In this work, LL37 has been encapsulated in nanostructured lipid carriers (NLCs), produced by the melt-emulsification method, in order to improve its effectiveness. The characterization of the NLC-LL37 showed a mean size of 270 nm, a zeta potential of -26 mV and an encapsulation efficiency of 96.4%. The cytotoxicity assay performed in Human Foreskin Fibroblasts demonstrated that the NLC-LL37 did not affect cell viability. Moreover, the in vitro bioactivity assay evidenced that the peptide remained active after the encapsulation, since the NLC-LL37 reversed the activation of the macrophages induced by LPS in the same way as the LL37 in solution. In addition, the in vitro antimicrobial assay revealed the NLC-LL37 activity against E. coli. The effectiveness of the nanoparticles was assessed in a full thickness wound model in db/db mice. The data demonstrated that NLC-LL37 significantly improved healing compared to the same concentration of the LL37 solution in terms of wound closure, reepithelisation grade and restoration of the inflammatory process. Overall, these findings suggest a promising potential of the NLC-LL37 formulation for chronic wound healing. KEYWORDS LL37, antimicrobial peptide, lipid nanoparticles, NLC (nanostructured lipid carriers), chronic wounds 3 1. INTRODUCTION The incidence of suffering non-healing chronic wounds has exponentially increased with aging of population together with the resultant comorbidities of diabetes, venous insufficiency and associated chronic diseases. It has been estimated that 1-2% of the population in the developed countries would experience a chronic wound in their lifetime, representing 2% of the European health budget according to the United Nations [1,2]. Cutaneous wound healing is an orderly and timely reparative process, designed to restore the skin barrier function and homeostasis. It is accomplished by regulated processes that overlap in space and time, including an initial inflammatory response, a proliferative phase and a final remodelling phase [3,4]. Chronic wounds fail to proceed in this subsequent phases, stagnating in a permanent inflammatory state in which the wound do not cure. This inflammation maintains a constant infiltration of macrophages and neutrophils, which produce an excessive amount of collagenases, proteases and reactive oxygen species that degrade healing mediators and hamper the formation of the extracellular matrix and new epithelia. In addition, these lesions are usually infected and remain open for extended periods of time, occasionally endangering the life of the patient [1,5]. Current advances in wound care have focused on finding new treatments for wound healing, such as the administration of healing mediators for wound repair. In this regard, one of the strategies that is gaining a notorious interest is the administration of the human antimicrobial peptide LL37. It should be noted that the downregulation of LL37 has been associated with an increased risk in suffering chronic ulcers [6]. The LL37 has been detected in an inactive preform (hCAP18) in several immune and dermal-epithelial cells. After a skin injury, hCAP18 is released to the extracellular environment due to cell degranulation, leading to its activation into the LL37 peptide [4,7]. It has been proven that this molecule modulates wound healing by activating angiogenesis [8,9] and epithelial cell migration and proliferation [10,11]. Furthermore, it has demonstrated a broad spectrum of antimicrobial, antiviral and antifungal activity [12-15]. In addition, the LL37 presents an immunomodulatory effect, since it is chemotactic to monocytes, neutrophils and dendritic cells [13]. The LL37 also shows the ability to 4 neutralise the proinflamatory responses exerted by macrophages (very important in chronic wounds) by its union to lipopolysaccharide (LPS) [13,16]. The in vivo wound healing studies conducted to date, have required high doses and dosing frequencies of free LL37 because of the rapid degradation of the peptide [15,17] or gene therapy [18] to achieve improved healing. With the aim of overcoming these limitations, Chereddy and cols. encapsulated the LL37 peptide into PLGA nanoparticles obtaining a significantly improvement in the wound healing activity after intradermal administration [19]. In line with this approach, the encapsulation of LL37 into nanostructured lipid carriers (NLC) is presented as a promising alternative to optimise the administration of the LL37 in terms of dose, delivery pattern, and safety. The NLCs, besides of protecting the encapsulated peptide against the degradation of the proteases, can be administered through the topical route, thereby reducing the systemic effects due to a lower systemic bioavailability of the drug. Furthermore, the NLCs allow a sustained release of the drugs at the site of action and present an excellent biocompatibility, making them a suitable option for the topical treatment of chronic wounds [20-22]. Thus, the aim of our study is the development of a topical formulation based on LL37 loaded-NLC (NLC-LL37) for the treatment of chronic wounds. In vitro studies were carried out to determine the biocompatibility of the formulation and the activity against LPS of the encapsulated LL37. In addition, antimicrobial tests were undertaken in E. coli to analyse the efficacy of NLC-LL37 against bacteria. Finally, the wound healing activity of the NLC-LL37 was evaluated in vivo in a full thickness wound model in db/db mice. 2. MATERIALS AND METHODS 2.1 NLC-LL37 preparation The NLC-LL37 were prepared trough the melt emulsification method and following the procedure previously described by our group [23,24]. Briefly, a warm aqueous phase (heated at 40 °C, 1 min) containing 3 ml of water, 20 mg of Poloxamer 188 (Panreac, Spain) and 40 mg of Tween® 80 (Panreac, Spain) was added to a melted lipid phase containing 200 mg of the solid lipid Precirol® ATO 5 (Gattefossé Spain, Spain) and 20 mg of the liquid lipid Miglyol® 812N (Sasol Germany GmbH). Then, 50 µL of an 80 mg/mL LL37 (95.0% pure, Caslo ApS, DK) aqueous solution was added and 5 immediately after, the mixture was emulsified for 15 s at 50 W (Branson® 250 sonifier, CT, USA). The resulting emulsion was stored at 4°C to allow the re-crystallisation of the lipid for the NLC formation. On the following day, the particles were collected using a 100-kDa molecular weight cut-off centrifugal filter unit (Amicon, “Ultracel100k”, Millipore, Spain) at 2,500 rmp for 10 minutes and washed three times with MillliQ water. Finally, the NLC suspension was freeze dried with the cryoprotectant trehalose (Sigma-Aldrich, Spain) in a final concentration of 15% (w/w) of the weighed lipid. The target loading of LL-37 in NLC-LL37 was 2% (w/w). 2.2 Nanoparticle characterisation The mean particle size (Z-average diameter) and the polydispersity index (PDI) were measured by Dynamic Light Scattering (DLS), and the zeta potential was determined through Laser Doppler micro-electrophoresis (Malvern® Zetasizer Nano ZS, Model Zen 3600; Malvern instruments Ltd., UK). The measurement medium for zeta potential was water (pH 5.6), and the measured electrophoretic mobililty was converted into zeta potential through Smoluchowski approximation. Each assay was performed in triplicate after nanoparticles lyophilisation. Nanoparticle morphology was examined by transmission electron microscopy (TEM, Philips EM208S). The encapsulation efficiency (EE) of the NLC-LL37 was assessed indirectly by measuring the free LL-37 (non-encapsulated) in the supernatant obtained after the filtration/centrifugation process described in Section 2.1. The amount of free LL-37 was quantified using a commercially available ELISA kit for LL-37 (human LL-37 ELISA kit, Hycult® biotech, Netherlands). The EE (%) was determined using the following equation (1): 𝐸𝐸 󰇛%󰇜𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝐿37 𝐹𝑟𝑒𝑒 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝐿37 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝐿37 100 󰇛1󰇜 2.3 In vitro cell culture studies 2.3.1 Cell culture Human Foreskin Fibroblasts (HFF) (ATCC, Manassas, USA) were cultured on Dulbeccos’s modified Eagle’s medium (DMEM) (ATCC, Manassas, USA) supplemented with 15% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine and 1% (v/v) penicillin-streptomycin. 6 The RAW 264.7 cell line (Murine Macrophages; ATCC, Manassas, USA) was cultured on a specific growth medium DMEM/F-12, GlutaMAX™ Supplement (Gibco®/Life technologies, Spain) supplemented with 10% (v/v) FBS and 1% (v/v) penicillinstreptomycin. The cell lines were maintained at 37°C in a humidified incubator with a 5% CO2 atmosphere. The cell passages were performed every 2-3 days depending on the cell line. 2.3.2 Cell viability studies The effect of the NLC-LL37 on cell viability was assayed using HFF cells. 1000 cells/well were seeded into 96-well culture plates and incubated 24 h to allow cell attachment. Then, the medium was replaced by serial concentrations of NLC-LL37 (corresponding to 5000-50 ng of LL37/mL) and empty NLC resuspended in 1% serumsupplemented-DMEM. After 48 h of incubation, the cell viability was measured by adding 10 µL of CCK-8 reagent (Sigma-Aldrich, Saint Louise, USA) to the wells. The mixture was incubated for 4 h and the absorbance was then read at 450 nm and at 650 nm as the reference wavelength. The absorbance was directly proportional to the number of living cells in the culture. 2.3.3 Inhibition of macrophages activation induced by LPS This assay was conducted in the RAW 264.7 cell line. 105 cells/well were seeded into a 96-well culture plate and incubated for 24 h to allow cell attachment. Then, the medium was replaced by the following samples (all of the samples were resuspended in 1 % serum-supplemented DMEM containing 20 ng/ml of LPS prior to their incorporation into the cell culture): (i) 5000 ng/ml of free LL37, (ii) the concentration of NLC-LL37 equivalent to 5000 ng/ml of LL37 and (iii) the same concentration of empty NLC. As negative control 1% serum-supplemented medium was used and as positive control 1% serum-supplemented medium with 20 ng/ml of LPS. After 6 h of incubation, the supernatant of the wells was collected to quantify the TNF-α released from the macrophages using a commercially available ELISA kit (Murine TNFα Elisa Development kit, PeproTech). The results were displayed as the percentage of absorbance value of each group compared to those obtained in the negative control. 7 2.4 Antimicrobial assay In order to test the antimicrobial activity of the formulations, Escherichia coli ATCC 25922 was grown overnight at 37°C in Mueller Hinton broth (Conda, Pronadisa, Spain). Then, the bacterial suspension was diluted to 105 CFU/ml. Free LL37 (20 µg/ml), NLCLL37 (the concentration corresponding to 20 µg/ml LL37) and empty NLC (the same nanoparticle concentrations as the NLC-LL37 group) were incubated with 1 ml of this bacterial suspension at 37°C with gentle agitation. In addition, 1 ml of the bacterial suspension alone was also incubated, to use it as control. At 4 h of incubation, aliquots were taken from each suspension, diluted in PBS and 100 µl were inoculated in Mueller Hinton agar plates (Conda/Pronadisa, Spain). The plates were incubated for 24 h at 37°C and subsequently, the colony forming units were determined as the total number of colonies grown on each plate. Three independent experiments were performed. The antibacterial effect was assessed as the percentage of death cells compared to the control. 2.5 In vivo wound healing study 2.5.1. Animals For the in vivo experiment, 16 eight week-old male db/db (BKS.Cg-m+/+Leprdb/J) mice were used (Janvier laboratories, Sain Berthevin Cedex, France). Every experiment was conducted according to the protocols approved by the Institutional Ethical Committee for Animal Experimentation of the University of the Basque Country (Procedure number: CEBA/243/2012/HERNANDEZ MARTIN). Animals were housed in individual cages that were maintained on a 12 h light-dark cycle, and were given standard rodent chow and water ad libitum. 2.5.2 Wound healing assay The wound healing assay was performed adapting the procedure described by Michaels et al. [25]. Human main healing mechanism is through granulation tissue formation and re-epithelisation, while mice’s one is through wound contraction. In order to mimic human healing process and avoid mice’s main process, two silicone rings of 1 cm in diameter were sutured on each side of the midline using a 3-0 nylon suture (Aragó, Spain), after anesthetising the mice and removing their dorsal hair. Then, two full thickness wounds of 8 mm in diameter and extending through the panniculus carnosus were created using a punch biopsy tool (Acu-Punch, Acuderm, USA). After the 8 administration of the treatments, the wounds were covered with one layer of petrolatum gauze (Tegaderm® 3M) and two layers of adhesive. The mice were divided in 4 groups (n=4): (i) untreated control, (ii) 6 µg of free LL37, (iii) 6 µg of LL37 encapsulated into NLC-LL37 and (iv) 2 µg of LL37 encapsulated into NLC-LL37. Treatments, previously resuspended in 10 µL of MilliQ water, were administered topically allowing them to spread over the wound bed, on day 1 and 4 after the wound induction. On day 8, the mice were sacrificed through CO2 inhalation. 2.5.3 Evaluation of wound healing The effectiveness of the treatments was evaluated by measuring the wound area (px2) on days 1, 4 and 8 after the surgery. Those days, photographs of the wounds were taken using a digital camera (Lumix FS16, Panasonic®, Spain), and the wound area was assessed with an image analysis programme (ImageJ®, Biophotonics Facility, University of McMaster, Canada). The wound closure was expressed as the percentage of the initial wound area. 2.5.4 Histological analysis of wound healing After the sacrifice of the mice, the wound and surrounding tissue (~1 cm) were excised and fixed in 3.7% paraformaldehyde for 24 h. Then the tissues were bisected, embedded in paraffin, and sectioned in 5 µm thick layers. The samples were processed by H&E staining for wound healing evaluation and by Masson trichrome staining for collagen deposition evaluation. The re-epithelisation process was assessed following the scale established by Sinha et al., 2003 [26]. The score of each wound was determined semi-quantitatively giving to each wound a value within a range from 0 to 4: 0, re-epithelisation at the margin of the wound; 1, re-epithelisation covering less than half of the wound; 2, re-epithelisation covering more than half of the wound; 3, re-epithelisation covering the entire wound with irregular thickness, and 4, re-epithelisation covering the entire wound with normal thickness. The resolution of the inflammatory process and wound maturity was determined according to the scale described by Cotran et al., 2000 [27]. 1, acute inflammation, with the formation of fibrin clot and migration of leucocytes and polynuclear neutrophils; 2, diffuse acute inflammation, with the predominance of granulation tissue formation and angiogenesis and with barely presence of pyogenic membrane; 3, chronic inflammation, 9 with the presence of granulation tissue and with fibroblast proliferation and 4, resolution and healing, disappearance of chronic inflammation, although occasionally round cells can be found. The deposition of collagen was determined following the scale described by Gal et al, 2006. [28]: 0, absent of collagen; 1, mild content of collagen; 2, moderate content of collagen and 3, marked content of collagen. 2.5.5 Immunohistochemistry In order to assess neoangiogenesis in the wound bed, immunohistochemical studies were performed using a specific monoclonal anti-CD31 antibody (JC70 clon, 760-4378, Ventana-Roche). 5 µm thick tissue slides were incubated with the primary antibody for 36 min at 37°C, and subsequently the layers were treated with Ultraview Universal DAB detection kit (760-500, Ventana-Roche) to visualize the antigen. Afterwards, the number of blood vessels was counted per field. 2.6 Statistical analyses All of the data are expressed as the mean ± standard deviation. Based on the result of the normality test and the Levene test for the homogeneity of variances, the means were compared through one-way ANOVA, with the subsequent application of StudentNewman-Keuls post-hoc; or through Mann-Whitney U test. All the statistical calculations were performed using SPSS 22.0.01 (SPSS®, INC., Chicago, IL, USA). 3. RESULTS AND DISCUSSION 3.1 Nanoparticle characterisation As illustrated in Table 1 the NLC-LL37 and empty NLC showed similar mean size, however, the NLC-LL37 exhibited a slightly higher size: 273.6± 27.64 nm and 220.6 ± 5.48 nm, respectively. The polydispersity index (PDI) was below 0.4 in both formulations, indicating a stable polydisperse system. The analysis of the zeta potential revealed that both formulations presented a similar surface charge of about -31 mV in the case of NLC-LL37 and about -26 mV in the case of empty NLC. In addition, the NLC-LL37 presented high encapsulation efficiency (96.40 % ± 0.41) and a peptide loading of 16.76±0.07 µg LL37/mg nanoparticle. According to the TEM photographs of the nanoparticles, they showed a similar size to that obtained with DLS (Fig. 1). 16 [13] Y. Kai-Larsen, B. 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TEM photographs of NLC-LL37 and NLC-B. The scale bar indicates 200 nm. Fig. 2. Cell viability after NLC treatment. The results are given as the mean % of living cells relative to the control ± SD. Controls are C (cells without any addition) and DMSO (cells after addition of DMSO). 19 Fig. 3. Inhibition of the activation of the macrophages. The results are given as the mean % of TNF-α production relative to the control ± SD. ** significantly greater than empty NLC and C+ (p<0.01). Controls are C- (cells without any addition) and C+ (cells after the addition of LPS). Fig. 4. Antimicrobial assay. The results are given as the mean percentage of death bacteria relative to the control ± SD. *** p<0.001 between the three groups. Fig 5. In vivo wound closure. (A) Wound closure represented as the percentage of the reduction of initial area. * Significantly greater than untreated group (p<0.05); ○ significantly greater than untreated group (p>0.05), + significantly greater than the rest of the groups (p<0.05). (B) Wound images. 20 Fig 6. Histological analysis. (A) Reepithelisation grade. ** Significantly greater than the rest of the groups (p>0.01). (B) Grade of resolution of inflammation. ** Significantly greater than NLC-LL37 low dose (p<0.05); ● significantly greater than untreated group (p<0.05). (C) Collagen deposition. (D) Number of new vessels in immunohistochemically stained tissue slides. All the results are shown as mean ±SD. Table 1.Characterisation of NLCs: Mean size, PDI, zeta potential, EE and peptide loading. Data are shown as the means ± SD. Size (nm) PDI Zeta Potential(mV) EE % Peptide loading (µg LL37/mg NLC) NLC-LL37 273.6 ± 27.64 0.31 ± 0.06 -31.63 ± 1.94 96.40 ± 0.41 16.76±0.07 Empty NLC 220.6 ± 5.48 0.27 ± 0.03 -26.10 ± 0.53