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

1 Composi e nano ib ous memb anes o PLGA/Aloe e a con aining lipid nanopa icles o wound d essing applica ions I xaso Ga cia-O ue 1,2, Ga azi Gainza3, Pa icia Ga cia-Ga cia4, F ancisco Bo ja Gu ie ez5, Jose Ja ie Agui e3,5, Rosa Ma ia He nandez1,2, A aceli Delgado5, Manoli Iga ua1,2 1 NanoBioCel G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU). 2 Biomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN). Vi o ia-Gas eiz, Spain. 3 Biop axis Resea ch AIE, Miñano, Vi o ia-Gas eiz. 4 Depa men o Chemical Enginee ing and Pha maceu ical Technology, School o Pha macy, Ins i u e o Biomedical Technologies (ITB), Cen e o Biomedical Resea ch o he Cana y Islands (CIBICAN), Uni e si y o La Laguna, Tene i e, Spain 5 Hospi al Uni e si a io de Ála a (HUA) Txago i xu, Vi o ia-Gas eiz, 01009, Spain. M. Iga ua (manoli.iga [email protected]) and A. Delgado ([email protected]) equally sha e c edi o senio au ho ship. *Co esponding au ho : A. Delgado Depa men o Chemical Enginee ing and Pha maceu ical Technology. School o Pha macy. Uni e si y o La Laguna, Tene i e, Spain. Telephone: +34922318507 Fax: +34922318506 E-mail: [email protected] **Co esponding au ho : M. Iga ua Labo a o y o Pha maceu ics, Uni e si y o he Basque Coun y. School o Pha macy, Paseo de la Uni e sidad, 7. 01006 – Vi o ia- Gas eiz, Spain Telephone: +34 945013007 Fax: +34 945013040 E-mail: manoli.iga [email protected] ABSTRACT Elec ospun nano ib ous d essings p esen sui able cha ac e is ics o be used in wound healing, such as high po osi y and high su ace a ea- o- olume a io. In his s udy, a wound d essing based on PLGA and Aloe e a con aining lipid nanopa icles (NLCs) was de eloped. NLCs we e added in o de o add a lipid componen ha could a oid he adhesion o he d essing o he wound and imp o e i s handling. Memb anes wi h and wi hou NLCs we e composed o uni o m ibe s o abou 1 µm in diame e . Thei po osi y was abo e 80 % and hei hickness was abou 160 µm. Bo h d essings showed simila wa e apou ansmission a e 1100 g/m2day. The o mula ion con aining NLCs p esen ed a highe ul ima e ensile s eng h (2.61 ± 0.46 MPa) and a highe wa e up ake. Bo h o mula ions we e biocompa ible in i o. Fu he mo e, he cell adhesion assay demons a ed ha bo h memb anes had a low adhe ence p o ile, al hough i was lowe wi h he d essing con aining NLCs. Finally, hei e icacy was e alua ed in a ull hickness wound healing assay conduc ed in db/db mice, whe e bo h enhanced healing simila ly. Acco dingly, he PLGA-AV-NLC memb ane migh be a p omising s a egy o he ea men o ch onic wounds, since i imp o ed handling in compa ison o he o mula ion wi hou NLCs. KEYWORDS Elec ospinning, wound healing, Aloe e a, PLGA, lipid nanopa icles, db/db mice This is he accep ed manusc ip o he a icle ha appea ed in inal o m in In e na ional Jou nal o Pha maceu ics 556 : 320-329 (2019), which has been published in inal o m a h ps://doi.o g/10.1016/j.ijpha m.2018.12.010. © 2018 Else ie unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/) 2 1. In oduc ion Elec ospinning is a echnique o ob ain memb anes composed o polyme ic nano ibe s, which uses elec ic o ce o elu e nano ibe s om a polyme ic solu ion. Due o he elec os a ic epulsion p oduced by he applica ion o high ol age cha ges o he solu ion, he polyme ic d ople is s e ched and ejec ed o he collec o . The inal nano ibe s a e o med du ing he ejec ion p ocess in which sol en is e apo a ed, allowing he a i al o solid nano ibe s o he collec o (Felguei as and Amo im 2017; Liu, e al. 2017). This p ocess p oduces memb anes composed o nonwo en polyme ic nano ibe s ha mimic he h ee dimensional s uc u e o ex acellula ma ix (Ab igo, e al. 2014). Thei dis inc i e cha ac e is ics a e high po osi y and a high su ace a ea- o- olume a io. Those p ope ies make hem sui able o de elop d essings o wound healing, as hey allow gas pe mea ion and hus cells b ea hing (Ga cia-O ue, e al. 2017). In addi ion, hey help o egula e wound mois u e, enhancing issue egene a ion (Ma hew S. B own, e al. 2018), since hey p omo e he emo al o exuda es om he wound bed, and hey e ain mois u e o p e en wound desicca ion (Pachuau 2015). Fu he mo e, he small size o he po es hinde s he en ance o mic oo ganisms, and hus wound in ec ion (Felguei as and Amo im 2017). Resea ch o de elop no el wound d essings has gained impo ance due o he g ea inc ease in ch onic wound incidence; in ac , only in he US, ch onic wounds annually a ec 5.7 million people (a ound 2% o he popula ion) and cos $20 billions (Jä b ink, e al. 2017). A ac o in ol ed in ha g ow h is he ise o diseases associa ed wi h wound ch onici y, such as, diabe es, enous insu iciency and obesi y (Han and Ceilley 2017; Sen, e al. 2009). Wounds occu ing in pa ien s su e ing hose diseases, usually ail o p og ess h ough he o ganized s eps o physiological healing ha comp ises he ollowing subsequen bu o e lapping phases: hemos asis, in lamma ion, p oli e a ion and emodelling phase (Diegelmann and E ans 2004; Velna , e al. 2009). Ch onic wounds emain s agna ed in o he in lamma o y phase, wi h a cons an in il a ion o mac ophages and neu ophils o he wound bed (Sch eml, e al. 2010). Those cells sec e e a g ea amoun o p oin lamma o y cy okines and p o eases, ha deg ade healing media o s and ex acellula ma ix and hampe he o ma ion o new epi helia, leading o a delay in healing (B iquez, e al. 2015; Velna , e al. 2009). In he cu en s udy, we de eloped a composi e nano ib ous memb ane o PLGA (poly lac ic- co-glycolide acid) and Aloe e a ex ac con aining lipid nanopa icles (nanos uc u ed lipid ca ie s o NLC) (Ga cia-O ue, e al. 2016a). PLGA is a syn he ic polyme which has good biocompa ibili y and biodeg adabili y (Che eddy, e al. 2016). In compa ison o na u al polyme s, i p esen s some ad an ages, among which a e lowe p ice; well-de ined s uc u e 3 and deg ada ion kine ics; eliabili y (Ga g, e al. 2015b); be e mechanical p ope ies, ha make hem mo e easily elec ospinable (Ga g, e al. 2015a); and he p esence o lac a e as a deg ada ion p oduc , which has p o en o accele a e wound healing (Po po a o, e al. 2012). In o de o imp o e he wound healing p ope ies o he PLGA nano ibe s, hey we e also composed o Aloe e a, which has been widely used in wound healing since ancien imes (Da , e al. 2012), because i has shown o p omo e healing in addi ion o i s an i-in lamma o y, an i ungal, an ibac e ial and hypoglycemic p ope ies (Choi and Chung 2003). Due o he ac ion o glucomannan, Aloe e a a ec s ib oblas g ow h ac o (FGF), s imula ing he ac i i y and p oli e a ion o ib oblas , and hus, enhancing hei collagen p oduc ion and sec e ion, as well as he ans e sal connec ion among collagen chains (Hashemi, e al. 2015; Su jushe, e al. 2008). In addi ion, Aloe e a con ains some i amins, amino acids and an h aquinones in ol ed in he enhancemen o wound healing due o hei an ioxidan ac i i y (Boud eau and Beland 2006). Finally, i is no ewo hy o men ion ha due o i s an imic obial ac i i y, Aloe e a can help in he p e en ion o wound in ec ion (Hashemi, e al. 2015). Fo all he abo e easons, Aloe e a has been al eady used o de elop nano ib ous wound d essings using di e en polyme s. In ha ega d, a esea ch g oup de eloped a nano ib ous d essing composed o poly- cap olac one (PCL), silk ib oin, cu cumin and Aloe e a ex ac . The o al con en o Aloe e a was 16.6 % (w/w), and he d essing showed sui able p ope ies o be used as a wound d essing (Ka uppuswamy, e al. 2014). The combina ion o silk ib oin and Aloe e a was used o de elop ano he wo d essings. The i s one was composed o Aloe e a (25% w/w), silk ib oin and poly-lac ic-co-ε-cap olac one (PLLA) and i showed a a ou able e ec in ib oblas p oli e a ion and collagen sec e ion (Suganya, e al. 2014). The second one was composed o Aloe e a (10-20% w/w), silk ib oin and poly( inyl) alcohol nano ibe s which had loaded s a ch nanopa icles con aining i amin E, and i was able o imp o e ib oblas a achmen , p oli e a ion and collagen deposi ion due o Aloe e a and i amin E (Khe ad a , e al. 2018). In a pos e io s udy ca ied ou by ou esea ch g oup, a nano ib ous memb ane composed o PLGA, Aloe e a (50% w/w) and EGF was de eloped and was able o imp o e ib oblas p oli e a ion in i o and eepi helisa ion and wound closu e in a ull hickness wounds in lic ed o db/db mice (Ga cia-O ue, e al. 2016a). Finally, Jouyba e al. de eloped a poly-L-lac ic acid (PLLA) nano ib ous memb ane and hey coa ed i wi h esh Aloe e a gel. In i o, he d essings accele a ed wound healing in a ull hickness skin de ec in lic ed o mice (Jouyba , e al. 2017). T adi ionally, p ima y d essings ha e been imp egna ed in aseline o pa a in o p e en adhesion o he wound su ace. Ne e heless, he lipid componen equen ly was abso bed 4 in o he seconda y d essing o in he wound, d ying he p ima y d essing and inc easing he isk o adhe ence o he wound (Benbow 2002; Da id, e al. 2018). In o de o a oid ha limi a ion, U go ul™ was de eloped, a non-occlusi e hin shee composed o a polyes e ne imp egna ed wi h hyd ocolloid pa icles dispe sed in a pe oleum jelly ma ix. In con ac wi h wound exuda es he hyd ocolloid pa icles hyd a e, and join ly wi h he pe oleum jelly, hey o m a lipido-colloid in e phase ha p e en wound adhe ence (Benbow and Iosson 2004; Tan, e al. 2009). Conside ing his, in he p esen s udy, NLCs we e inco po a ed o he PLGA/Aloe e a o mula ion in o de o add a lipid componen ha could a oid adhesion o he wound. The NLCs we e dis ibu ed in he PLGA nano ib ous s uc u e du ing he elec ospinning p ocess, in o de o a oid i s di usion o he seconda y d essing and hus, dec ease he isk o adhe ence. Fu he mo e, we hypo hesise ha he addi ion o he NLCs could imp o e some ea u es o he d essing, such as, handling, elas ici y and occlusi i y. The ab ica ed nano ib ous PLGA-AV-NLC memb anes we e subjec ed o physical, mechanical and cy ocompa ibili y e alua ion. Thei wound healing e icacy was assessed in i o in a splin ed ull hickness wound model pe o med in diabe ic db/db mice. 2. Ma e ial and me hods 2.1 Nano ibe s p epa a ion PLGA-AV-NLC nano ibe s we e p oduced elec ospinning an emulsion con aining all he componen s. The o ganic phase was composed o 300 mg o PLGA (Resome , LG824; E onik, Ge many) in o 2.5 ml o hexa luo oisop opanol (HFIP, Fluka, Swi ze land), and he aqueous phase was composed o 300 mg o Aloe e a ex ac (Ago a Valencia SL:, Spain) and 30 mg o NLCs in 1.2 ml o a 0.5 % (w/ ) PVA solu ion. To c ea e he emulsion bo h phases we e o exed a le el 10 o 3 min (Vo ex-Genie 2, Scien i ic Indus ies Inc., USA). The esul ing emulsion was loaded in o a Lue -lock sy inge (No m-Jec ) con aining a 14 G needle and a ached o a pump (Ha a d Appa a us, MA) ha p o ided a low a e o 2.7 mL/h. The nano ibe s we e elec ospun ho izon ally on a o a ing collec o (250 pm) loca ed a 8 cm om he needle, unde 10 kV powe supply. Simila ly, PLGA-AV nano ibe s we e p epa ed wi hou adding NLCs o he aqueous phase. The NLCs inco po a ed in o he nano ibe s we e p epa ed ollowing he p ocedu e desc ibed p e iously by ou esea ch g oup (Gainza, e al. 2014; Gainza, e al. 2015; Ga cia-O ue, e al. 2016b). B ie ly, an aqueous phase composed o 40 mg o Tween® 80 (Pan eac, Spain) and 20 mg o Poloxame 188 (Pan eac, Spain) in 3 ml MilliQ wa e and a lipid phase composed o 200 mg o P eci ol® ATO 5 (Ga e ossé Spain, Spain) and 20 mg o Mygliol 812N (Sasol Ge many 5 GmbH), we e hea ed sepa a ely un il he lipid phase mel ed in o a clea solu ion (40 °C). Then, he aqueous phase was added o he lipid phase and he mix u e was sonica ed o 15 s a 50 W (B anson® 250 Soni ie , CT, USA). The esul ing emulsion was s o ed a 4°C o e nigh o allow he e-c ys allisa ion o he lipid. I is no ewo hy o men ion ha be o e he in i o and in i o s udies he memb anes we e s e ilised by keeping hem unde UV ligh o 30 min. 2.2 Nanopa icle and nano ibe s cha ac e isa ion The mean pa icle size (Z-a e age diame e ) and he polydispe si y index (PDI) o he NLCs inco po a ed in o he nano ibe s we e measu ed h ough Dynamic Ligh Sca e ing (DLS) and hei ze a po en ial was de e mined by Lase Dopple mic o-elec opho esis (Mal e n® Ze asize Nano ZS, Model Zen 3600; Mal e n ins umen s L d., UK). The elec opho e ic mobili y was measu ed in wa e (pH 5.6) and i was con e ed in o ze a po en ial h ough he Smoluchowski app oxima ion. The mo phology o he nano ib ous memb anes, namely, ibe diame e and memb ane quali y, was assessed using Scanning Elec on Mic oscopy pho og aphs (SEM, Jeol JSM-6300) and hei hickness was measu ed using s e eo mic oscopy pho og aphs (Leica M205 C, Leica LAS, 3 so wa e, Ge many). Memb ane’s po osi y (P) was calcula ed using he ollowing equa ion (Eq. 1): P (%)=�1 − ρapp ρ eal� x 100 (1) Whe e ρ eal is he eal densi y ha was assessed by means o a helium pycnome e (Mic ome i ics, AccuPyc 1330, USA); and ρapp is he appa en densi y ha was calcula ed di iding he weighed mass o he memb anes by hei olume (leng h × wid h × heigh ). The mono onic ensile es s o bo h d essings (PLGA-AV and PLGA-AV-NLC memb anes) we e pe o med unde displacemen con ol on a ex u e analyse , using a 5 N ull scale load cell (Ins on 5848 mic o es e , Ins on®, UK). The samples we e loaded a a displacemen a e o 0.01 mm/s up o up u e. The load-displacemen cu e ob ained om hose es s was ans o med in o a s ess-s ain cu e and he ul ima e ensile s eng h was ob ained om i . A leas 5 samples we e es ed om each memb ane and he esul s we e shown as mean ± s anda d de ia ion (SD). To de e mine he wa e up ake o he di e en nano ib ous memb anes 1.3x1.3 cm pieces o he memb anes we e cu and weighed. Then, he samples we e imme sed in 1 ml o PBS and 6 incuba ed a 37°C o 72 h. A e incuba ion, he excess o wa e was d ied blo ing hem wi h il e pape and hey we e weighed again o calcula e he wa e up ake using he ollowing equa ion (Eq. 2): Wa e up ake (%) = M−M0 M0×100 (2) Whe e M0 and M a e he mass o he memb anes be o e and a e 72 h incuba ion in PBS, espec i ely. The Wa e Vapou T ansmission Ra e (WVTR) o he memb anes was quan i ied ollowing a modi ied p ocedu e o he me hod desc ibed by Li e al. (Li, e al. 2013). The mou h o a cup illed wi h silica gel desiccan (1 cm in diame e ) was ho oughly sealed wi h a piece o he nano ib ous memb ane, o make he memb ane he only way wa e apou could en e o he cup. The assembly was weighed and placed in a chambe wi h a cons an ela i e humidi y o 75% a 30°C. A e 24 h, he assembly was weighed again o calcula e he WVTR h ough he ollowing equa ion (Eq. 3): WVTR =M1−M0 A × T (3) Whe e M0 is he weigh o he assembly a he beginning o he assay, M1 is i s weigh a e he incuba ion ime, T is he exposu e ime (1 day) and A is he exposu e a ea (0.79 cm2). The he mal beha iou o he nano ib ous memb anes, he physical blend o hei componen s and he componen s hemsel es was analysed using Di e en ial Scanning Calo ime y (DSC-50, Shimadzu, Japan). 1-2 mg o each sample was weighed and sealed in o an aluminium pan. Then, he samples we e hea ed om 25°C o 350°C a a hea ing a e o 10°C pe minu e. 2.3 In i o cell cul u e s udies 2.3.1 Cell cul u e The cell lines used in his s udy we e HaCaT ke a inocy es and BalbC/3T3 A31 ib oblas s (ATCC, Manassas, USA). The i s one was cul u ed on Dulbecco’s modi ied Eagle’s medium (DMEM) (41965-039, Gibco®, Ma, USA) supplemen ed wi h 10% ( / ) oe al bo ine se um (FBS) and 1% ( / ) penicillin-s ep omycin. The ib oblas s we e cul u ed on DMEM (30-2202, ATCC, Manassas, USA) supplemen ed wi h 10% ( / ) oe al cal se um (FCS) and 1% ( / ) penicillin-s ep omycin. Cell lines we e incuba ed in a humidi ied incuba o a 37°C wi h a 5% CO2 a mosphe e and cell passages we e done e e y 2-3 days depending on he cell line. 2.3.2 Cell iabili y s udies 7 The e ec o he nano ib ous memb anes on cell iabili y was assessed incuba ing hei ex ac ed medium wi h ib oblas and ke a inocy es. The cells we e seeded in a 96 well-pla e, ib oblas s a a densi y o 6000 cell/well and ke a inocy es a 12000 cell/well. Cells we e cul u ed o e nigh o allow cell a achmen , and hen, he ollowing samples we e added: (i) s a ing medium as nega i e con ol, (ii) medium incuba ed wi h a 1x1 cm piece o PLGA-AV memb ane o 24 hou s, (iii) medium incuba ed wi h a 1x1 cm piece o PLGA-AV-NLC memb ane o 24 hou s. The s a ing medium o he con ol and samples incuba ed wi h HaCaT cells, was DMEM con aining 0.5% ( / ) o FBS and o he samples incuba ed wi h ib oblas s was DMEM wi h 0.2% ( / ) o FCS. Cells we e incuba ed wi h he samples o 48 h, and a e wa ds he iabili y was assessed using a CCK-8 ki (cell coun ing ki -8, Sigma-Ald ich, Sain Louise, USA). B ie ly, 10 µL o he CCK-8 eagen was added o he cells. A e 4 hou s o incuba ion he abso bance o he mix u e was ead a 450 nm, using 650 nm as e e ence wa eleng h (Pla e Reade In ini e M200, Tecan, Swi ze land). The abso bance and he numbe o li ing cells in each well we e di ec ly p opo ional. 2.3.3 Adhesion assay The abili y o he cells o adhe e o he nano ib ous memb anes was e alua ed by seeding cells on op o hem and measu ing he numbe o adhe ed cells a e an incuba ion ime. PLGA-AV and PLGA-AV-NLC memb anes we e cu in disks o 14 mm in diame e and ixed o he bo om o 24 well-pla es using 10 µl o ib in as adhesi e. Memb anes we e incuba ed o 30 min o allow he o ma ion o ib in clo and hen cells we e seeded on op o hem, ib oblas a a densi y o 20,000 cells/well and ke a inocy es a a densi y o 40,000 cells/well. Con ol wells we e also seeded wi h he same cell densi y. Cells we e incuba ed o e nigh o allow hei a achmen o he memb anes. Then, memb anes we e washed wi h PBS and cells we e de ached incuba ing hem wi h ypsin o 10 minu es. A e ypsin neu aliza ion, cells we e collec ed and cen i uged o 5 minu es a 100 pm. Finally, cells we e coun ed using an au oma ed cell coun e (Au oma ed Cell Coun e TC20™, Bio-Rad, Cali o nia, USA). The adhesion o he memb anes was exp essed as he pe cen age o cells coun ed compa ing o he con ol. Th ee independen s udies we e pe o med. In addi ion, SEM images we e aken o obse e he adhe ed cells and hei mo phology. Fo his assay, memb anes and cells we e incuba ed as in he p e ious s udy bu a a highe 8 densi y, ib oblas a a densi y o 100,000 cells/well and ke a inocy es a 200,000 cells/well. A e o e nigh incuba ion, ins ead o de aching hem, cells we e ixed in a 2.5 % glu a aldehyde solu ion and dehyd a ed in g aded e hanol se ies. Finally, mic opho og aphs o he memb anes we e aken using a SEM mic oscope (Hi achi S4800, Tokyo, Japan). 2.4 In i o wound healing assay 2.4.1 Animals Fo he in i o s udy 24 male db/db mice (BKS.Cg-m+/+Lep db/J) o 6 weeks old we e used (Jan ie labo a o ies, Sain Be he in Cedex, F ance). All he expe imen s we e conduc ed ollowing he p o ocols app o ed by he Ins i u ional E hical Commi ee o Animal Expe imen a ion o he Uni e si y o he Basque Coun y (P ocedu e numbe : M20_2015_155_ HERNÁNDEZ MARTÍN). Each mouse was housed indi idually unde a 12 h ligh -da k cycle, and hey had ad libi um access o s anda d oden chow and wa e . 2.4.2. Wound healing assay This assay was pe o med ollowing he p ocedu e desc ibed by Michaels e al. (Michaels, e al. 2007). Mice we e anes he ised wi h iso lu ane (Iso lo®, Es e e, Spain) and hei do sal hai was emo ed. In o de o a oid healing h ough wound con ac ion, and hus enhance eepi helisa ion, wo silicone ings o 1 cm in diame e we e su u ed on he back o he mice, in each side o he midline using a 3-0 nylon su u e (A agó, Spain). In he middle o each splin , a ull hickness wound ex ending h ough he panniculus ca nosus was c ea ed using an 8 mm in diame e punch biopsy ool (Acu-Punch, Acude m, USA). A e wa ds, ea men s we e applied and inally he wounds we e co e ed wi h pe ola um gauze (Tegade m®, 3M, Minneso a, USA) and adhesi e. On days 4, 8, and 11, he memb anes we e emo ed and new ea men s we e applied. On day 8, hal o he mice we e sac i iced h ough CO2 inhala ion, and he emaining mice we e sac i iced on day 15. Mice we e di ided in 3 g oups o 8 animal each (n=8). Each g oup ecei ed a di e en ea men : (i) un ea ed con ol, (ii) a d essing o 1.5x1.5 cm o PLGA-AV memb ane p e iously hyd a ed in PBS, and (iii) a d essing o 1.5x1.5 cm o PLGA-AV-NLC memb ane p e iously hyd a ed in PBS. 2.4.3 E alua ion o wound healing 9 The e ec i eness o he ea men s was e alua ed assessing he wound closu e pe cen age in each wound. On days 1, 4, 8, 11 and 15 pho og aphs o he wounds we e aken using a digi al came a (Lumix FS16, Panasonic®, Japan) and he a ea o each wound (px2) was measu ed using an image analysis p og amme (ImageJ®, Biopho onics Facili y, Uni e si y o McMas e , Canada). The wound closu e pe cen age was calcula ed using he ollowing equa ion (Eq. 4): 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝑐𝑐𝑐𝑐𝑊𝑊𝑐𝑐𝑊𝑊𝑐𝑐𝑐𝑐 (%)=𝐹𝐹𝐹𝐹𝑊𝑊𝐹𝐹𝑐𝑐 𝑤𝑤𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝐹𝐹𝑐𝑐𝑐𝑐𝐹𝐹 (𝑝𝑝𝑝𝑝2) 𝐼𝐼𝑊𝑊𝐹𝐹𝐼𝐼𝐹𝐹𝐹𝐹𝑐𝑐 𝑤𝑤𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝐹𝐹𝑐𝑐𝑐𝑐𝐹𝐹 (𝑝𝑝𝑝𝑝2)×100 (4) 2.4.4 His ological analysis o wound healing A e mice sac i ice, he wound and su ounding issue (abou 1x1 cm) we e excised and ixed in 3.7% pa a o maldehyde. Tissue was allowed o ix du ing 24 h and hen, he biopsies we e bisec ed, embedded in pa a in and sec ioned in laye s o a hickness o 5 µm. Those slices we e p ocessed by hema oxylin-eosin (H&E) s aining o e alua e hei p og ess h ough wound healing. The eepi helisa ion p ocess was e alua ed in acco dance wi h he scale es ablished by Sinha e al. (Sinha and Gallaghe 2003). Each wound was semi-quan i a i ely a ed wi h a alue wi hin a ange om 0 o 4: 0, eephi elised a ea was con ined o wound ma gins; 1, he new epi helium co e s less han hal o he wound a ea; 2, he new epi helium co e s mo e han hal o he wound a ea; 3, he en i e wound is eepi helised wi h i egula hickness; and 4, he en i e wound is eepi helised and he new epi helium has no mal hickness. The esolu ion o he in lamma o y p ocess and wound ma u i y was assessed ollowing he scale es ablished by Co an e al. (Co an, e al. 2000). Wounds we e sco ed acco ding o he ollowing c i e ia: 0, absence o in lamma ion; 1, acu e in lamma ion, in his phase he ib in clo and he pyogenic memb ane a e o med and he leucocy es and polynuclea neu ophils mig a e o he wound; 2, di use acu e in lamma ion, his phase comp ises he o ma ion o he g anula ion issue, and he disappea ance o he pyogenic memb ane; 3, ch onic in lamma ion, his phase consis s on ib oblas p oli e a ion and 4; esolu ion and healing: his phase consis on he disappea ance o ch onic in lamma ion, al hough occasionally ound cells can be obse ed. 2.4.5 Immunohis ochemical analysis In o de o pe o m immunohis ological s udies, issue slices we e depa a inised and au oma ically p ocesses acco ding o he U Ul a View DAB de ec ion ki (Roche, Swi ze land). Fi s , issue biopsies we e incuba ed wi h he p ima y an ibodies a 37°C. The incuba ion 16 The main easons o include NLCs in o he nano ibe s we e he hypo hesis ha hey could imp o e hei handling and o ease he emo al o he d essing om he wound, a oiding pain and damage o he newly o med issue du ing d essing change. The cha ac e isa ion o he memb anes showed ha NLCs we e able o imp o e he handling and mechanical s eng h o he nano ibe s. The e o e, cell adhesion was analysed and i e ealed a lowe ke a inocy e adhesion o he o mula ion con aining NLCs. Ne e heless, no di e ences we e ound in ib oblas a achmen , since bo h memb anes wi h o wi hou NLCs p esen ed a e y low adhesion. Conside ing hose esul s, mo e s udies should be pe o med o assess d essing a achmen in o wounded issue, analysing he d essing emo al in i o o using a ex u e analyse o e alua e he adhesion s eng h o he wounded issue. Finally, he e icacy o he memb anes was e alua ed in i o in a ull hickness splin ed wound model ca ied ou in db/db mice. Db/db mice we e chosen because hey p esen an impai ed wound healing seconda y o diabe es, and hus, hey esemble be e a ch onic wound model. In addi ion, hey mimic be e human wound healing han o he oden models, since hey ha e impai ed wound con ac ion due o hei obesi y, and he e o e hei healing occu s mainly ia eepi helisa ion, as human healing (Fang and Mus oe 2008; Tkalce ic, e al. 2009). In o de o impai e en mo e con ac ion and enhance eephi elisa ion, silicone splin s we e su u ed a ound he wounds (Michaels, e al. 2007). O e all, bo h memb anes achie ed simila imp o emen in wound healing. Compa able esul s we e ob ained in wound closu e and eepi helisa ion, as bo h we e able o accele a e healing in compa ison o un ea ed con ol. Rega ding he esolu ion o he in lamma o y p ocess, on day 15 only PLGA-AV memb anes p esen ed an imp o ed ou come in compa ison o he con ol g oup. On day 8, no di e ences we e obse ed in he his ological analysis, al hough he de eloped o mula ions, and especially he PLGA-AV-NLC memb anes, we e able o educe he mac ophage in il a ion in he wound bed, which usually is augmen ed in oden wound models wi h diabe es o impai ed healing. (Tsubame Nishikai-Yan Shen, e al. 2017; Yeh, e al. 2010). In addi ion, he mac ophages ound on hose wounds p esen an impai ed abili y o phagocy e apop o ic cells, inc easing he le el o p o-in lamma o y cy okines, and hus, pe pe ua ing a con inuous in lamma o y s a e (Sa i a Khanna, e al. 2010). Acco dingly, bo h o mula ions showed an enhancemen o wound ma u a ion, he PLGA-AV-NLC memb anes on mac ophage in il a ion on he ea ly s age o healing and he PLGA-AV memb anes on he gene al in lamma o y s a e o he la e s age. 17 The e ec o he nano ib ous d essings on wound healing can be pa ially explained by he inco po a ion o Aloe e a, since i has shown o imp o e wound healing, mainly by a ec ing ib oblas g ow h ac o , and hus, imp o ing hei ac i i y and p oli e a ion (Boud eau and Beland 2006). The e ec o Aloe e a in o PLGA-AV memb anes was p o en in he p e ious s udy conduc ed by ou esea ch g oup, whe e PLGA-AV memb anes achie ed an imp o ed wound healing in compa ison o PLGA memb anes (Ga cia-O ue, e al. 2016a). In addi ion, he cha ac e is ics o he nano ib ous s uc u e also con ibu e o he imp o emen o wound healing. In ac , he high su ace o olume a ea and he nanopo osi y c ea e an adequa e en i onmen o cell mig a ion and p oli e a ion o he wound bed. Mo eo e , ha p oli e a ing en i onmen is in ol ed in he imp o emen o he g anula ion issue o ma ion and eepi helisa ion by enhancing he elease o healing media o s, such as g ow h ac o s, angiogenic ac o s o collagen (Ab igo, e al. 2014; Shah e di, e al. 2014). Finally, he chosen polyme is also in ol ed in he enhancemen o eepi helisa ion, since one o i s deg ada ion p oduc s, lac a e, has shown o be able o induce a as e wound healing (Po po a o, e al. 2012). Rega ding o he inclusion o NLCs in o he memb anes, hei e ec on he emo al o he d essing could no be obse ed in i o, since bo h d essings we e humec ed wi h PBS p io o hei elimina ion, in o de o a oid any possible damage on he newly o med issue. Ne e heless, an imp o emen on he handling o he memb anes con aining NLCs was obse ed along he cha ac e isa ion o he o mula ions. Hence, he nano ib ous d essing wi h NLCs showed a bene i conce ning handling, al hough mo e s udies a e needed o assess hei e ec in d essing emo al. 5. Conclusion In he cu en s udy wo composi e elec ospun d essings we e de eloped, he i s one was composed o an emulsion o PLGA and Aloe e a (1:1), and in he second one lipid nanopa icles (NLCs) we e added o he a o emen ioned emulsion. Bo h d essings showed a simila cha ac e isa ion, al hough an enhanced handling was obse ed in he PLGA-AV-NLC o mula ion ega ding o elas ici y and hickness. Finally, hei e ec i i y in wound healing was assessed in a ull hickness wound healing assay pe o med in db/db mice, achie ing simila esul s wi h bo h o mula ions. Acco dingly, he PLGA-AV-NLC nano ib ous memb ane migh be a p omising s a egy o he ea men o ch onic wound, since i imp o ed handling in compa ison o he o mula ion wi hou NLCs. 6. Acknowledgmen s 18 I. Ga cía-O ue hanks he Basque Go e nmen o he ellowship g an . The au ho s hank o echnical and human suppo p o ided by SGIke o UPV/EHU and Eu opean unding (ERDF and ESF). 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The wound healing p ocess: an o e iew o he cellula and molecula mechanisms. J. In . Med. 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. Impai ed skin wound healing in lumican-null mice. B . J. De ma ol., 163, 1174. doi: 10.1111/j.1365-2133.2010.10008.x. 22 Fig 1. SEM images o he PLGA-AV and PLGA-AV-NLC memb anes. The scale ba o each image indica es 100 µm. Fig 2. Cell iabili y s udy. (A) CCK-8 esul s a e cul u ing he memb anes´ ex ac ed medium wi h ib oblas s. *** p<0.001 compa ing all g oups. (B) CCK-8 esul s a e cul u ing he memb anes´ ex ac ed medium wi h ke a inocy es. *** p<0.001 compa ing PLGA-AV memb anes wi h he con ol; and ** p<0.01 compa ing PLGA-AV-NLC nano ibe s wi h he con ol. Resul s a e gi en as he mean % o li ing cells ega ding o he con ol ± SD. 23 Fig. 3. In i o adhesion assay. (A) SEM images o memb anes wi h cells seeded on op: 1, PLGA-AV-NLC memb anes incuba ed wi h ke a inocy es; 2, PLGA-AV-NLC memb anes incuba ed wi h ib oblas s; 3, PLGA-AV memb anes incuba ed wi h ke a inocy es; and 4, PLGA- AV memb anes incuba ed wi h ib oblas s. The scale ba in each image indica es 25 µm. (B) Ke a inocy es adhesion pe cen age. *** p<0.001 compa ing PLGA-AV-NLC memb anes wi h con ol g oup; ** p<0.01 compa ing PLGA-AV memb anes wi h con ol g oup; * p<0.05 compa ing bo h memb anes. (C) Fib oblas s adhesion pe cen age. *** p<0.001 compa ing PLGA-AV-NLC and PLGA-AV memb anes wi h con ol g oup. Fig 4. In i o wound closu e. (A) Wounds pho og aphs o each g oup on days 1, 4, 8, 11 and 15. (B) Wound closu e ep esen ed as he pe cen age o educ ion o he ini ial a ea on days 4, 8, 11 and 15 pos inju y. The scale ba indica es 5 mm. * p<0.05 compa ing PLGA-AV-NLC memb anes g oups wi h he un ea ed g oups, *** p>0.001 compa ing wi h he un ea ed g oup. 24 Fig. 5. His ological e alua ion o he wounds. (A) His ological images o issue sec ions o each g oup on days 8 and 15, p ocessed wi h H&E. (B) Reepi helisa ion g ade on days 8 and 15. *** p<0.001 compa ing g oups ea ed wi h he d essings and un ea ed g oup. (C) G ade o esolu ion o he in lamma o y p ocess. ** p0.01 compa ing he g oup ea ed wi h PLGA-AV memb anes and he un ea ed g oup. Fig 6. Immunohis ological analysis. (A) Numbe o lymphocy es (CD4+ and CD8+ cells) on day 8. (B) Ra io be ween CD8+ and CD4+ cells on day 8. (C) Numbe o CD68+ cells on day 8. ** p<0.01 compa ing he g oup ea ed wi h PLGA-AV memb anes wi h he un ea ed g oup. *** p<0.001 compa ing he g oup ea ed wi h PLGA-AV-NLC memb anes wi h he un ea ed g oup. 25 Figu e S1. DSC he mog ams. (A) The mog ams o he aw ma e ials used o he p oduc ion o nano ibe s, PLGA, AV and NLCs. (B) he mog ams o he PLGA-AV-NLC and PLGA-AV nano ibe s and he physical blend o he componen s, i.e., PLGA, AV and NLC. I is no ewo hy o men ion ha he da a in able 2, e e s o he mean alue o he endo he mic peaks o h ee independen expe imen s, while he he mog ams o his igu e a e om one o hose expe imen s, hus he alues o he peaks a ies sligh ly.