Composite nanofibrous membranes of PLGA/Aloe vera containing lipid nanoparticles for wound dressing applications

1 Composite nanofibrous membranes of PLGA/Aloe vera containing lipid nanoparticles for wound dressing applications Itxaso Garcia-Orue 1,2, Garazi Gainza3, Patricia Garcia-Garcia4, Francisco Borja Gutierrez5, Jose Javier Aguirre3,5, Rosa Maria Hernandez1,2, Araceli Delgado5, Manoli Igartua1,2 1 NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU). 2 Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Vitoria-Gasteiz, Spain. 3 Biopraxis Research AIE, Miñano, Vitoria-Gasteiz. 4 Department of Chemical Engineering and Pharmaceutical Technology, School of Pharmacy, Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, Tenerife, Spain 5 Hospital Universitario de Álava (HUA) Txagorritxu, Vitoria-Gasteiz, 01009, Spain. M. Igartua ([email protected]) and A. Delgado ([email protected]) equally share credit for senior authorship. *Corresponding author: A. Delgado Department of Chemical Engineering and Pharmaceutical Technology. School of Pharmacy. University of La Laguna, Tenerife, Spain. Telephone: +34922318507 Fax: +34922318506 E-mail: [email protected] **Corresponding author: M. Igartua Laboratory of Pharmaceutics, University of the Basque Country. School of Pharmacy, Paseo de la Universidad, 7. 01006 – VitoriaGasteiz, Spain Telephone: +34 945013007 Fax: +34 945013040 E-mail: [email protected] ABSTRACT Electrospun nanofibrous dressings present suitable characteristics to be used in wound healing, such as high porosity and high surface area-to-volume ratio. In this study, a wound dressing based on PLGA and Aloe vera containing lipid nanoparticles (NLCs) was developed. NLCs were added in order to add a lipid component that could avoid the adhesion of the dressing to the wound and improve its handling. Membranes with and without NLCs were composed of uniform fibers of about 1 µm in diameter. Their porosity was above 80 % and their thickness was about 160 µm. Both dressings showed similar water vapour transmission rate 1100 g/m2day. The formulation containing NLCs presented a higher ultimate tensile strength (2.61 ± 0.46 MPa) and a higher water uptake. Both formulations were biocompatible in vitro. Furthermore, the cell adhesion assay demonstrated that both membranes had a low adherence profile, although it was lower with the dressing containing NLCs. Finally, their efficacy was evaluated in a full thickness wound healing assay conducted in db/db mice, where both enhanced healing similarly. Accordingly, the PLGA-AV-NLC membrane might be a promising strategy for the treatment of chronic wounds, since it improved handling in comparison to the formulation without NLCs. KEYWORDS Electrospinning, wound healing, Aloe vera, PLGA, lipid nanoparticles, db/db mice This is the accepted manuscript of the article that appeared in final form in International Journal of Pharmaceutics 556 : 320-329 (2019), which has been published in final form at https://doi.org/10.1016/j.ijpharm.2018.12.010. © 2018 Elsevier under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 1. Introduction Electrospinning is a technique to obtain membranes composed of polymeric nanofibers, which uses electric force to elute nanofibers from a polymeric solution. Due to the electrostatic repulsion produced by the application of high voltage charges to the solution, the polymeric droplet is stretched and ejected to the collector. The final nanofibers are formed during the ejection process in which solvent is evaporated, allowing the arrival of solid nanofibers to the collector (Felgueiras and Amorim 2017; Liu, et al. 2017). This process produces membranes composed of nonwoven polymeric nanofibers that mimic the three dimensional structure of extracellular matrix (Abrigo, et al. 2014). Their distinctive characteristics are high porosity and a high surface area-to-volume ratio. Those properties make them suitable to develop dressings for wound healing, as they allow gas permeation and thus cells breathing (Garcia-Orue, et al. 2017). In addition, they help to regulate wound moisture, enhancing tissue regeneration (Matthew S. Brown, et al. 2018), since they promote the removal of exudates from the wound bed, and they retain moisture to prevent wound desiccation (Pachuau 2015). Furthermore, the small size of the pores hinders the entrance of microorganisms, and thus wound infection (Felgueiras and Amorim 2017). Research to develop novel wound dressings has gained importance due to the great increase in chronic wound incidence; in fact, only in the US, chronic wounds annually affect 5.7 million people (around 2% of the population) and cost $20 billions (Järbrink, et al. 2017). A factor involved in that growth is the rise of diseases associated with wound chronicity, such as, diabetes, venous insufficiency and obesity (Han and Ceilley 2017; Sen, et al. 2009). Wounds occurring in patients suffering those diseases, usually fail to progress through the organized steps of physiological healing that comprises the following subsequent but overlapping phases: hemostasis, inflammation, proliferation and remodelling phase (Diegelmann and Evans 2004; Velnar, et al. 2009). Chronic wounds remain stagnated into the inflammatory phase, with a constant infiltration of macrophages and neutrophils to the wound bed (Schreml, et al. 2010). Those cells secrete a great amount of proinflammatory cytokines and proteases, that degrade healing mediators and extracellular matrix and hamper the formation of new epithelia, leading to a delay in healing (Briquez, et al. 2015; Velnar, et al. 2009). In the current study, we developed a composite nanofibrous membrane of PLGA (poly lacticco-glycolide acid) and Aloe vera extract containing lipid nanoparticles (nanostructured lipid carriers or NLC) (Garcia-Orue, et al. 2016a). PLGA is a synthetic polymer which has good biocompatibility and biodegradability (Chereddy, et al. 2016). In comparison to natural polymers, it presents some advantages, among which are lower price; well-defined structure 3 and degradation kinetics; reliability (Garg, et al. 2015b); better mechanical properties, that make them more easily electrospinable (Garg, et al. 2015a); and the presence of lactate as a degradation product, which has proven to accelerate wound healing (Porporato, et al. 2012). In order to improve the wound healing properties of the PLGA nanofibers, they were also composed of Aloe vera, which has been widely used in wound healing since ancient times (Dat, et al. 2012), because it has shown to promote healing in addition to its anti-inflammatory, antifungal, antibacterial and hypoglycemic properties (Choi and Chung 2003). Due to the action of glucomannan, Aloe vera affects fibroblast growth factor (FGF), stimulating the activity and proliferation of fibroblast, and thus, enhancing their collagen production and secretion, as well as the transversal connection among collagen chains (Hashemi, et al. 2015; Surjushe, et al. 2008). In addition, Aloe vera contains some vitamins, amino acids and anthraquinones involved in the enhancement of wound healing due to their antioxidant activity (Boudreau and Beland 2006). Finally, it is noteworthy to mention that due to its antimicrobial activity, Aloe vera can help in the prevention of wound infection (Hashemi, et al. 2015). For all the above reasons, Aloe vera has been already used to develop nanofibrous wound dressings using different polymers. In that regard, a research group developed a nanofibrous dressing composed of polycaprolactone (PCL), silk fibroin, curcumin and Aloe vera extract. The total content of Aloe vera was 16.6 % (w/w), and the dressing showed suitable properties to be used as a wound dressing (Karuppuswamy, et al. 2014). The combination of silk fibroin and Aloe vera was used to develop another two dressings. The first one was composed of Aloe vera (25% w/w), silk fibroin and poly-lactic-co-ε-caprolactone (PLLA) and it showed a favourable effect in fibroblast proliferation and collagen secretion (Suganya, et al. 2014). The second one was composed of Aloe vera (10-20% w/w), silk fibroin and poly(vinyl) alcohol nanofibers which had loaded starch nanoparticles containing vitamin E, and it was able to improve fibroblast attachment, proliferation and collagen deposition due to Aloe vera and vitamin E (Kheradvar, et al. 2018). In a posterior study carried out by our research group, a nanofibrous membrane composed of PLGA, Aloe vera (50% w/w) and EGF was developed and was able to improve fibroblast proliferation in vivo and reepithelisation and wound closure in a full thickness wounds inflicted to db/db mice (Garcia-Orue, et al. 2016a). Finally, Jouybar et al. developed a poly-L-lactic acid (PLLA) nanofibrous membrane and they coated it with fresh Aloe vera gel. In vivo, the dressings accelerated wound healing in a full thickness skin defect inflicted to mice (Jouybar, et al. 2017). Traditionally, primary dressings have been impregnated in vaseline or paraffin to prevent adhesion to the wound surface. Nevertheless, the lipid component frequently was absorbed 4 into the secondary dressing or in the wound, drying the primary dressing and increasing the risk of adherence to the wound (Benbow 2002; David, et al. 2018). In order to avoid that limitation, Urgotul™ was developed, a non-occlusive thin sheet composed of a polyester net impregnated with hydrocolloid particles dispersed in a petroleum jelly matrix. In contact with wound exudates the hydrocolloid particles hydrate, and jointly with the petroleum jelly, they form a lipido-colloid interphase that prevent wound adherence (Benbow and Iosson 2004; Tan, et al. 2009). Considering this, in the present study, NLCs were incorporated to the PLGA/Aloe vera formulation in order to add a lipid component that could avoid adhesion to the wound. The NLCs were distributed in the PLGA nanofibrous structure during the electrospinning process, in order to avoid its diffusion to the secondary dressing and thus, decrease the risk of adherence. Furthermore, we hypothesise that the addition of the NLCs could improve some features of the dressing, such as, handling, elasticity and occlusivity. The fabricated nanofibrous PLGA-AV-NLC membranes were subjected to physical, mechanical and cytocompatibility evaluation. Their wound healing efficacy was assessed in vivo in a splinted full thickness wound model performed in diabetic db/db mice. 2. Material and methods 2.1 Nanofibers preparation PLGA-AV-NLC nanofibers were produced electrospinning an emulsion containing all the components. The organic phase was composed of 300 mg of PLGA (Resomer, LG824; Evonik, Germany) into 2.5 ml of hexafluoroisopropanol (HFIP, Fluka, Switzerland), and the aqueous phase was composed of 300 mg of Aloe vera extract (Agora Valencia SL:, Spain) and 30 mg of NLCs in 1.2 ml of a 0.5 % (w/v) PVA solution. To create the emulsion both phases were vortexed at level 10 for 3 min (Vortex-Genie 2, Scientific Industries Inc., USA). The resulting emulsion was loaded into a Luer-lock syringe (Norm-Ject) containing a 14 G needle and attached to a pump (Harvard Apparatus, MA) that provided a flow rate of 2.7 mL/h. The nanofibers were electrospun horizontally on a rotating collector (250 rpm) located at 8 cm from the needle, under 10 kV power supply. Similarly, PLGA-AV nanofibers were prepared without adding NLCs to the aqueous phase. The NLCs incorporated into the nanofibers were prepared following the procedure described previously by our research group (Gainza, et al. 2014; Gainza, et al. 2015; Garcia-Orue, et al. 2016b). Briefly, an aqueous phase composed of 40 mg of Tween® 80 (Panreac, Spain) and 20 mg of Poloxamer 188 (Panreac, Spain) in 3 ml MilliQ water and a lipid phase composed of 200 mg of Precirol® ATO 5 (Gattefossé Spain, Spain) and 20 mg of Mygliol 812N (Sasol Germany 5 GmbH), were heated separately until the lipid phase melted into a clear solution (40 °C). Then, the aqueous phase was added to the lipid phase and the mixture was sonicated for 15 s at 50 W (Branson® 250 Sonifier, CT, USA). The resulting emulsion was stored at 4°C overnight to allow the re-crystallisation of the lipid. It is noteworthy to mention that before the in vitro and in vivo studies the membranes were sterilised by keeping them under UV light for 30 min. 2.2 Nanoparticle and nanofibers characterisation The mean particle size (Z-average diameter) and the polydispersity index (PDI) of the NLCs incorporated into the nanofibers were measured through Dynamic Light Scattering (DLS) and their zeta potential was determined by Laser Doppler micro-electrophoresis (Malvern® Zetasizer Nano ZS, Model Zen 3600; Malvern instruments Ltd., UK). The electrophoretic mobility was measured in water (pH 5.6) and it was converted into zeta potential through the Smoluchowski approximation. The morphology of the nanofibrous membranes, namely, fiber diameter and membrane quality, was assessed using Scanning Electron Microscopy photographs (SEM, Jeol JSM-6300) and their thickness was measured using stereo microscopy photographs (Leica M205 C, Leica LAS, v3 software, Germany). Membrane’s porosity (P) was calculated using the following equation (Eq. 1): P (%)=�1 − ρapp ρreal� x 100 (1) Where ρreal is the real density that was assessed by means of a helium pycnometer (Micromeritics, AccuPyc 1330, USA); and ρapp is the apparent density that was calculated dividing the weighed mass of the membranes by their volume (length × width × height). The monotonic tensile tests of both dressings (PLGA-AV and PLGA-AV-NLC membranes) were performed under displacement control on a texture analyser, using a 5 N full scale load cell (Instron 5848 microtester, Instron®, UK). The samples were loaded at a displacement rate of 0.01 mm/s up to rupture. The load-displacement curve obtained from those tests was transformed into a stress-strain curve and the ultimate tensile strength was obtained from it. At least 5 samples were tested from each membrane and the results were shown as mean ± standard deviation (SD). To determine the water uptake of the different nanofibrous membranes 1.3x1.3 cm pieces of the membranes were cut and weighed. Then, the samples were immersed in 1 ml of PBS and 6 incubated at 37°C for 72 h. After incubation, the excess of water was dried blotting them with filter paper and they were weighed again to calculate the water uptake using the following equation (Eq. 2): Water uptake (%) = M−M0 M0×100 (2) Where M0 and M are the mass of the membranes before and after 72 h incubation in PBS, respectively. The Water Vapour Transmission Rate (WVTR) of the membranes was quantified following a modified procedure of the method described by Li et al. (Li, et al. 2013). The mouth of a cup filled with silica gel desiccant (1 cm in diameter) was thoroughly sealed with a piece of the nanofibrous membrane, to make the membrane the only way water vapour could enter to the cup. The assembly was weighed and placed in a chamber with a constant relative humidity of 75% at 30°C. After 24 h, the assembly was weighed again to calculate the WVTR through the following equation (Eq. 3): WVTR =M1−M0 A × T (3) Where M0 is the weight of the assembly at the beginning of the assay, M1 is its weight after the incubation time, T is the exposure time (1 day) and A is the exposure area (0.79 cm2). The thermal behaviour of the nanofibrous membranes, the physical blend of their components and the components themselves was analysed using Differential Scanning Calorimetry (DSC-50, Shimadzu, Japan). 1-2 mg of each sample was weighed and sealed into an aluminium pan. Then, the samples were heated from 25°C to 350°C at a heating rate of 10°C per minute. 2.3 In vitro cell culture studies 2.3.1 Cell culture The cell lines used in this study were HaCaT keratinocytes and BalbC/3T3 A31 fibroblasts (ATCC, Manassas, USA). The first one was cultured on Dulbecco’s modified Eagle’s medium (DMEM) (41965-039, Gibco®, Ma, USA) supplemented with 10% (v/v) foetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin. The fibroblasts were cultured on DMEM (30-2202, ATCC, Manassas, USA) supplemented with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin-streptomycin. Cell lines were incubated in a humidified incubator at 37°C with a 5% CO2 atmosphere and cell passages were done every 2-3 days depending on the cell line. 2.3.2 Cell viability studies 7 The effect of the nanofibrous membranes on cell viability was assessed incubating their extracted medium with fibroblast and keratinocytes. The cells were seeded in a 96 well-plate, fibroblasts at a density of 6000 cell/well and keratinocytes at 12000 cell/well. Cells were cultured overnight to allow cell attachment, and then, the following samples were added: (i) starving medium as negative control, (ii) medium incubated with a 1x1 cm piece of PLGA-AV membrane for 24 hours, (iii) medium incubated with a 1x1 cm piece of PLGA-AV-NLC membrane for 24 hours. The starving medium for the control and samples incubated with HaCaT cells, was DMEM containing 0.5% (v/v) of FBS and for the samples incubated with fibroblasts was DMEM with 0.2% (v/v) of FCS. Cells were incubated with the samples for 48 h, and afterwards the viability was assessed using a CCK-8 kit (cell counting kit-8, Sigma-Aldrich, Saint Louise, USA). Briefly, 10 µL of the CCK-8 reagent was added to the cells. After 4 hours of incubation the absorbance of the mixture was read at 450 nm, using 650 nm as reference wavelength (Plate Reader Infinite M200, Tecan, Switzerland). The absorbance and the number of living cells in each well were directly proportional. 2.3.3 Adhesion assay The ability of the cells to adhere to the nanofibrous membranes was evaluated by seeding cells on top of them and measuring the number of adhered cells after an incubation time. PLGA-AV and PLGA-AV-NLC membranes were cut in disks of 14 mm in diameter and fixed to the bottom of 24 well-plates using 10 µl of fibrin as adhesive. Membranes were incubated for 30 min to allow the formation of fibrin clot and then cells were seeded on top of them, fibroblast at a density of 20,000 cells/well and keratinocytes at a density of 40,000 cells/well. Control wells were also seeded with the same cell density. Cells were incubated overnight to allow their attachment to the membranes. Then, membranes were washed with PBS and cells were detached incubating them with trypsin for 10 minutes. After trypsin neutralization, cells were collected and centrifuged for 5 minutes at 100 rpm. Finally, cells were counted using an automated cell counter (Automated Cell Counter TC20™, Bio-Rad, California, USA). The adhesion to the membranes was expressed as the percentage of cells counted comparing to the control. Three independent studies were performed. In addition, SEM images were taken to observe the adhered cells and their morphology. For this assay, membranes and cells were incubated as in the previous study but at a higher 8 density, fibroblast at a density of 100,000 cells/well and keratinocytes at 200,000 cells/well. After overnight incubation, instead of detaching them, cells were fixed in a 2.5 % glutaraldehyde solution and dehydrated in graded ethanol series. Finally, microphotographs of the membranes were taken using a SEM microscope (Hitachi S4800, Tokyo, Japan). 2.4 In vivo wound healing assay 2.4.1 Animals For the in vivo study 24 male db/db mice (BKS.Cg-m+/+Leprdb/J) of 6 weeks old were used (Janvier laboratories, Sain Berthevin Cedex, France). All the experiments were conducted following the protocols approved by the Institutional Ethical Committee for Animal Experimentation of the University of the Basque Country (Procedure number: M20_2015_155_ HERNÁNDEZ MARTÍN). Each mouse was housed individually under a 12 h light-dark cycle, and they had ad libitum access to standard rodent chow and water. 2.4.2. Wound healing assay This assay was performed following the procedure described by Michaels et al. (Michaels, et al. 2007). Mice were anesthetised with isoflurane (Isoflo®, Esteve, Spain) and their dorsal hair was removed. In order to avoid healing through wound contraction, and thus enhance reepithelisation, two silicone rings of 1 cm in diameter were sutured on the back of the mice, in each side of the midline using a 3-0 nylon suture (Aragó, Spain). In the middle of each splint, a full thickness wound extending through the panniculus carnosus was created using an 8 mm in diameter punch biopsy tool (Acu-Punch, Acuderm, USA). Afterwards, treatments were applied and finally the wounds were covered with petrolatum gauze (Tegaderm®, 3M, Minnesota, USA) and adhesive. On days 4, 8, and 11, the membranes were removed and new treatments were applied. On day 8, half of the mice were sacrificed through CO2 inhalation, and the remaining mice were sacrificed on day 15. Mice were divided in 3 groups of 8 animal each (n=8). Each group received a different treatment: (i) untreated control, (ii) a dressing of 1.5x1.5 cm of PLGA-AV membrane previously hydrated in PBS, and (iii) a dressing of 1.5x1.5 cm of PLGA-AV-NLC membrane previously hydrated in PBS. 2.4.3 Evaluation of wound healing 9 The effectiveness of the treatments was evaluated assessing the wound closure percentage in each wound. On days 1, 4, 8, 11 and 15 photographs of the wounds were taken using a digital camera (Lumix FS16, Panasonic®, Japan) and the area of each wound (px2) was measured using an image analysis programme (ImageJ®, Biophotonics Facility, University of McMaster, Canada). The wound closure percentage was calculated using the following equation (Eq. 4): 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝑐𝑐𝑐𝑐𝑊𝑊𝑐𝑐𝑊𝑊𝑐𝑐𝑐𝑐 (%)=𝐹𝐹𝐹𝐹𝑊𝑊𝐹𝐹𝑐𝑐 𝑤𝑤𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝐹𝐹𝑐𝑐𝑐𝑐𝐹𝐹 (𝑝𝑝𝑝𝑝2) 𝐼𝐼𝑊𝑊𝐹𝐹𝐼𝐼𝐹𝐹𝐹𝐹𝑐𝑐 𝑤𝑤𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝐹𝐹𝑐𝑐𝑐𝑐𝐹𝐹 (𝑝𝑝𝑝𝑝2)×100 (4) 2.4.4 Histological analysis of wound healing After mice sacrifice, the wound and surrounding tissue (about 1x1 cm) were excised and fixed in 3.7% paraformaldehyde. Tissue was allowed to fix during 24 h and then, the biopsies were bisected, embedded in paraffin and sectioned in layers of a thickness of 5 µm. Those slices were processed by hematoxylin-eosin (H&E) staining to evaluate their progress through wound healing. The reepithelisation process was evaluated in accordance with the scale established by Sinha et al. (Sinha and Gallagher 2003). Each wound was semi-quantitatively rated with a value within a range from 0 to 4: 0, reephitelised area was confined to wound margins; 1, the new epithelium covers less than half of the wound area; 2, the new epithelium covers more than half of the wound area; 3, the entire wound is reepithelised with irregular thickness; and 4, the entire wound is reepithelised and the new epithelium has normal thickness. The resolution of the inflammatory process and wound maturity was assessed following the scale established by Cotran et al. (Cotran, et al. 2000). Wounds were scored according to the following criteria: 0, absence of inflammation; 1, acute inflammation, in this phase the fibrin clot and the pyogenic membrane are formed and the leucocytes and polynuclear neutrophils migrate to the wound; 2, diffuse acute inflammation, this phase comprises the formation of the granulation tissue, and the disappearance of the pyogenic membrane; 3, chronic inflammation, this phase consists on fibroblast proliferation and 4; resolution and healing: this phase consist on the disappearance of chronic inflammation, although occasionally round cells can be observed. 2.4.5 Immunohistochemical analysis In order to perform immunohistological studies, tissue slices were deparaffinised and automatically processes according to the U Ultra View DAB detection kit (Roche, Switzerland). First, tissue biopsies were incubated with the primary antibodies at 37°C. The incubation 16 The main reasons to include NLCs into the nanofibers were the hypothesis that they could improve their handling and to ease the removal of the dressing from the wound, avoiding pain and damage of the newly formed tissue during dressing change. The characterisation of the membranes showed that NLCs were able to improve the handling and mechanical strength of the nanofibers. Therefore, cell adhesion was analysed and it revealed a lower keratinocyte adhesion to the formulation containing NLCs. Nevertheless, no differences were found in fibroblast attachment, since both membranes with or without NLCs presented a very low adhesion. Considering those results, more studies should be performed to assess dressing attachment into wounded tissue, analysing the dressing removal in vivo or using a texture analyser to evaluate the adhesion strength to the wounded tissue. Finally, the efficacy of the membranes was evaluated in vivo in a full thickness splinted wound model carried out in db/db mice. Db/db mice were chosen because they present an impaired wound healing secondary to diabetes, and thus, they resemble better a chronic wound model. In addition, they mimic better human wound healing than other rodent models, since they have impaired wound contraction due to their obesity, and therefore their healing occurs mainly via reepithelisation, as human healing (Fang and Mustoe 2008; Tkalcevic, et al. 2009). In order to impair even more contraction and enhance reephitelisation, silicone splints were sutured around the wounds (Michaels, et al. 2007). Overall, both membranes achieved similar improvement in wound healing. Comparable results were obtained in wound closure and reepithelisation, as both were able to accelerate healing in comparison to untreated control. Regarding the resolution of the inflammatory process, on day 15 only PLGA-AV membranes presented an improved outcome in comparison to the control group. On day 8, no differences were observed in the histological analysis, although the developed formulations, and especially the PLGA-AV-NLC membranes, were able to reduce the macrophage infiltration in the wound bed, which usually is augmented in rodent wound models with diabetes or impaired healing. (Tsubame Nishikai-Yan Shen, et al. 2017; Yeh, et al. 2010). In addition, the macrophages found on those wounds present an impaired ability to phagocyte apoptotic cells, increasing the level of pro-inflammatory cytokines, and thus, perpetuating a continuous inflammatory state (Savita Khanna, et al. 2010). Accordingly, both formulations showed an enhancement of wound maturation, the PLGA-AV-NLC membranes on macrophage infiltration on the early stage of healing and the PLGA-AV membranes on the general inflammatory state of the later stage. 17 The effect of the nanofibrous dressings on wound healing can be partially explained by the incorporation of Aloe vera, since it has shown to improve wound healing, mainly by affecting fibroblast growth factor, and thus, improving their activity and proliferation (Boudreau and Beland 2006). The effect of Aloe vera into PLGA-AV membranes was proven in the previous study conducted by our research group, where PLGA-AV membranes achieved an improved wound healing in comparison to PLGA membranes (Garcia-Orue, et al. 2016a). In addition, the characteristics of the nanofibrous structure also contribute to the improvement of wound healing. In fact, the high surface to volume area and the nanoporosity create an adequate environment for cell migration and proliferation to the wound bed. Moreover, that proliferating environment is involved in the improvement of the granulation tissue formation and reepithelisation by enhancing the release of healing mediators, such as growth factors, angiogenic factors or collagen (Abrigo, et al. 2014; Shahverdi, et al. 2014). Finally, the chosen polymer is also involved in the enhancement of reepithelisation, since one of its degradation products, lactate, has shown to be able to induce a faster wound healing (Porporato, et al. 2012). Regarding to the inclusion of NLCs into the membranes, their effect on the removal of the dressing could not be observed in vivo, since both dressings were humected with PBS prior to their elimination, in order to avoid any possible damage on the newly formed tissue. Nevertheless, an improvement on the handling of the membranes containing NLCs was observed along the characterisation of the formulations. Hence, the nanofibrous dressing with NLCs showed a benefit concerning handling, although more studies are needed to assess their effect in dressing removal. 5. Conclusion In the current study two composite electrospun dressings were developed, the first one was composed of an emulsion of PLGA and Aloe vera (1:1), and in the second one lipid nanoparticles (NLCs) were added to the aforementioned emulsion. Both dressings showed a similar characterisation, although an enhanced handling was observed in the PLGA-AV-NLC formulation regarding to elasticity and thickness. Finally, their effectivity in wound healing was assessed in a full thickness wound healing assay performed in db/db mice, achieving similar results with both formulations. Accordingly, the PLGA-AV-NLC nanofibrous membrane might be a promising strategy for the treatment of chronic wound, since it improved handling in comparison to the formulation without NLCs. 6. Acknowledgments 18 I. García-Orue thanks the Basque Government for the fellowship grant. The authors thank for technical and human support provided by SGIker of UPV/EHU and European funding (ERDF and ESF). This project has been funded by the Basque Government (ELKARTEK 2015, Nanoplatform, KK-2015/0000036 and Consolidated Groups, IT-428-10 and IT-528-10). 7. References Abrigo, M., McArthur, S.L., Kingshott, P., 2014. Electrospun nanofibers as dressings for chronic wound care: advances, challenges, and future prospects. Macromol. Biosci., 14, 772-792. doi: 10.1002/mabi.201300561. Benbow, M., 2002. Urgotul™: alternative to conventional non-adherence dressings. Br. J. Nurs. , 11, 135-138. doi: 10.12968/bjon.2002.11.2.9315. Benbow, M., Iosson, G., 2004. 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Res., 37, 1528-1542. doi: 10.1177/147323000903700531. Yeh, J., Yeh, L., Jung, S., Chang, T., Wu, H., Shiu, T., Liu, C., Kao, W.W., Chu, P., 2010. Impaired skin wound healing in lumican-null mice. Br. J. Dermatol., 163, 1174. doi: 10.1111/j.1365-2133.2010.10008.x. 22 Fig 1. SEM images of the PLGA-AV and PLGA-AV-NLC membranes. The scale bar of each image indicates 100 µm. Fig 2. Cell viability study. (A) CCK-8 results after culturing the membranes´ extracted medium with fibroblasts. *** p<0.001 comparing all groups. (B) CCK-8 results after culturing the membranes´ extracted medium with keratinocytes. *** p<0.001 comparing PLGA-AV membranes with the control; and ** p<0.01 comparing PLGA-AV-NLC nanofibers with the control. Results are given as the mean % of living cells regarding to the control ± SD. 23 Fig. 3. In vitro adhesion assay. (A) SEM images of membranes with cells seeded on top: 1, PLGA-AV-NLC membranes incubated with keratinocytes; 2, PLGA-AV-NLC membranes incubated with fibroblasts; 3, PLGA-AV membranes incubated with keratinocytes; and 4, PLGAAV membranes incubated with fibroblasts. The scale bar in each image indicates 25 µm. (B) Keratinocytes adhesion percentage. *** p<0.001 comparing PLGA-AV-NLC membranes with control group; ** p<0.01 comparing PLGA-AV membranes with control group; * p<0.05 comparing both membranes. (C) Fibroblasts adhesion percentage. *** p<0.001 comparing PLGA-AV-NLC and PLGA-AV membranes with control group. Fig 4. In vivo wound closure. (A) Wounds photographs of each group on days 1, 4, 8, 11 and 15. (B) Wound closure represented as the percentage of reduction of the initial area on days 4, 8, 11 and 15 postinjury. The scale bar indicates 5 mm. * p<0.05 comparing PLGA-AV-NLC membranes groups with the untreated groups, *** p>0.001 comparing with the untreated group. 24 Fig. 5. Histological evaluation of the wounds. (A) Histological images of tissue sections of each group on days 8 and 15, processed with H&E. (B) Reepithelisation grade on days 8 and 15. *** p<0.001 comparing groups treated with the dressings and untreated group. (C) Grade of resolution of the inflammatory process. ** p0.01 comparing the group treated with PLGA-AV membranes and the untreated group. Fig 6. Immunohistological analysis. (A) Number of lymphocytes (CD4+ and CD8+ cells) on day 8. (B) Ratio between CD8+ and CD4+ cells on day 8. (C) Number of CD68+ cells on day 8. ** p<0.01 comparing the group treated with PLGA-AV membranes with the untreated group. *** p<0.001 comparing the group treated with PLGA-AV-NLC membranes with the untreated group. 25 Figure S1. DSC thermograms. (A) Thermograms of the raw materials used for the production of nanofibers, PLGA, AV and NLCs. (B) thermograms of the PLGA-AV-NLC and PLGA-AV nanofibers and the physical blend of the components, i.e., PLGA, AV and NLC. It is noteworthy to mention that the data in table 2, refers to the mean value of the endothermic peaks of three independent experiments, while the thermograms of this figure are from one of those experiments, thus the values of the peaks varies slightly.