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    PCL Gelatine

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    International Journal of Pharmaceutics 567 (2019) 118480 Contents lists available a ScienceDinsct International Journal of Pharmaceutics ELSEVIER journal homepage: www olsovier.comMlocate/lipharm Biomimetic PCL-gelatin based nanofibers loaded with ciprofloxacin hydrochloride and quercetin: A potential antibacterial and anti-oxidant dressing material for accelerated healing of a full thickness wound Gufran Ajmal’, Gunjan Vasant Bonde®, Pooja Mittal’, Gayasuddin Khan’ Bharati V. Bakade‘, Brahmeshwar Mishra’ {Peps of Pharnaseal rmerig& Teck, Infos ie of Tc (RU, Vora ni “capron of Chm Engine & Tc stats of Tv (BHU). Vranas la “Pada! Wana’ Cleo Pamary, Yamal Mahrara a ', Vivek Kumar Pandey”, omen Aopen wound highly sere microbial ineton alin leva eve of amity rns Cinch fer prompt rling. won reires a omic drengmaeal with esl hyopicty and ene oer stg, possesing anno and ntostans propery. Atough PCL nanotes have sfc eo tensile strength and biocompatibility, it lacks in terms of optimum hydrophilicity and biodegradation. Therefore, — we fabricated PC-gltin sed stem nanofbem nied with qartn and Gprfixain hye Saryrole Chord (CH. The merge dametr of devloed nanos wot 7259 = 201. 969nm, a devoid of ‘hema! interaction between two drugs and polymers. CH and quercetin exhibited biphasic oto release in phosphate buffer (pit 7.4). The instr anibcteril and antioxidant property of scaffolds were evaluated by Film ifsion aginst Staphylococcus aureus and DPPH axa, respectively. The ation of gelatin along with CH tnd quercetin enhanced the hydrophilicity (contact angle = 48.8 + 295") and biodegradation rate of the na ofbes nye bioompaubly of scaffald was examined by henocompatbiliy and fibroblast ably using MIT assay. The results cone the det application of seaadin the wounded area Further, complete closure of full-hickess wound within 16 days, and regulation of hydroxypotine, SOD and catalase eve! in granulation tissues following treatment with safe, confirmed it applestion fr aceerated wound healing 1. Introduction Wound healing is a complex natural, dynamic, interactive process that consists the participation of various components such as exta- cellular mates, soluble mediators, blood and parenchymal cells erjani et a, 2016). Depending onthe scale of injury, healing proceeds Uhrough three sequential phases of varying and overlapping duration, namely the hemostasis and inflammatory, proliferative and tissue re- ‘modeling (El-Fejani eta, 2016; Park ets, 2011). The inflammatory phase, which starts withthe hemostasis, is associated with the release ff numerous pro-inflammatary cytokines, proteolytic enzymes like protease, reactive oxygen species (ROS), along with several growth factors (Park etal, 2011; Pereira and Bartolo, 2016). These ROS are necessary for phagocytic activity and forms a part of body's defense system, and a homeostatic state is established by endogenous anti- ‘oxidants such as catalase, superoxide dismutase (SOD) and ghtathione peroxidase (Park et al, 2011). However, in severe trauma such as full thickness wounds, microbial infection inereases the inflammatory re- sponse, which results in the excessive generation of reactive species. ‘These excessive ROS entises oxidative damage to the cellular compe nent including flbroblasts, the primary eell responsible for collagen secretion and wound healing (Gomathi et al., 2003; Seivara) and Fathima, 2017). Another problem associated with a full thickness ‘wound is that wounded skin does not regenerate spontaneously. It re 4uires skin regeneration product for prompt closure of the wound ‘Therefore, a tissue engineered scaffold enriched with an antimicrobial land antioxidant woul be beneficial for the healing ofa fll thickness ‘wound. Recently, electrospinning is a widely employed technique for the production of a tissue engineered seaffold. Various properties of an lectrospun nanofibers such 2s it (offer high surface area and highly Porous structure which activate cel signaling and attract fibroblast for ‘Coresponding author at: Department of Pharmaccutical Engineering & Technology, IT (BUD, Varanasi 221006, UP, Indl, ‘Einall adress: bnlshvabhuetrediinall com (8 Misha). htyss//dok.or/10.10167).ipbaem.2019.118480 Received 10 May 2015; Received in revised form 25 June 2019; Accepted 27 June 2019 ‘Available online 28 kane 2019 (03785173/ © 2019 Eeevier RV. All rights ceserve, 6 Aa a extracellular matrix components seeretion, and structural similarity of mative extracellular matrix (ECM) that support cell attachment and its proliferation, (i) proveet the wound from microbial infiltration due to extremely interconnected pores, and (iv) act as drug delivery device with controlled and sustained release profile, and with high drug loading, make it potential candidate for ‘wound dressing (Chong eta, 2007; Kim et al, 2009; Liu etal, 2010s; Lin et al, 201085; Xue eta, 2014). Numerous polymers, ranging from synthetic polymers like poly acticco-lycolic acid) (PLGA), polyethylene oxide, poly (c-capro- lactone) (PCL) and poly (lactic acid) (PLA), to natural polymers such a5, chitosan, gelatin, collagen, sik fibroin and hyaluronic aeid, have been utilized (0 fabricate nanofibers with required properties for specific drug:delivery and tissue regeneration applications (ve et a, 201; Zamani etal, 2013). Although natural polymers possess excellent hy- Arophilicity and biocompatibility, it lacks stficlent “mechanical strength and predictable biodegradation rate. Similarly, PLGA and PLA- based nanofibers have adequate mechanical strength, Dut its rapid de- gradation and resultant monomer Le, lactic acid causes inflammatory response at healing site (Bergsma et al, 1995; Xue et al, 20146), “Therefore, PCL, which is a widely studied synthetic polymer for tissue engineered matrix, was chosen for electraspinning. It offers a wide range of biocompatibility, chemical compatibility and mechanical strength Cue et al, 2014), However, hydrophobic nature, low bio- degradation rate and absence of cell recognition ste in PCL. chain re- sults into inefficient attachment of scaffold at wound site, patil de- gradation and assimilation of nanofibers during the couse of healing, tnd incomplete closure of wound, as observed in our previous study (Ajmal et al, 2019). Further, slow degradation of PCL scaffold in phosphate bulfer (pH 7.4) took six days to release 98.98 + 2.619% ci profloxacin HCl and 85.09 = 2.89% quercetin, as observed in our previous finding (Ajmal etal, 2019), whieh was inadequate to heal a cthranie wound with severe microbial infiltration, Therefore, the blending of PCL with gelatin was proposed to obtain a scaffold with optimum hydrophilicity, biodegradation rate, and mechanical strength, as done by other researchers (Chong etal, 2007; Ghasemi-Mobarakeh Cal 2008; Xue etal, 2014a), Gelatin (GE) isa hydrophilic biopolymer obtained from partial hydrolysis of collagen, the principal extracellular ‘matrix building protein (Dunk et al, 2016; Ghasemi-Mobarakch etal, 2008). Gelatin also contains cell-ecognition site (Arg-GIy-Asp amino acid sequences), which is recognized by integrins and thus aids in cell attachment and is spreading, Ciprottoxacin hydrochloride (CH) is a Muoroquinolone antibiotic, Owing to low minimal inhibitory concentration and lesser incidence of microbial resistance, itis widely used for treating wound Infection, caused by Staphylococeus, a Gram-positive and Pseudomonas, a Gram- rogative bacteria (Jannesari et al., 2011; Keva 2014; Unnithan et al, 2012), Unnithan etal, and Toncheva etal. fabricated the electrospun nanofibers loaded with ciprofloxacin hydrochloride ae Wwound-dressing material, but could not examine the in-vivo wound healing efficacies ofthe nanofibers. Kataria eta, also developed PVA/ sodium alginate-based nanofibers loaded with’ eiprofioxacin hydro- chloride and found it effective for complete wound healing (Kataria eal, 2014; Toncheva et al, 2012; Unnithan et al, 2012). Quercetin (Que) is Havonoid, commonly found in vegetables and fruits. Ie pos: sesses potent antioxidant and strong antiinflammatory activity, which advocates for its possible appliation in wound healing (Arvand et al, 2015; Hatahet etal, 2016; Li etal, 2016). However, poor water s0- Iubilty, low bioavailability, short half-life, physiochemical instability limits its potential benefits. These shortcomings may be overcome by using the drug through an alternative route. Although many potential researchers had fabricated quercetin loaded nanofibers for diferent application (Arvand et al, 2015; da Silva Uebel etal, 2016; Li etal 2014; Vashisth eta, 2016), to the best of our knowledge, we couldn’ find clectrospun nanofibers loaded with quercetin for full thickness wound. Therefore, PCl-gelatin based nanofibers loaded with mena oun of Paracas S67 (2018) 11880 ciprofloxacin hydrochloride and quercetin might be unique for full thickness wound healing application In the present study, we developed PCLGE based nanofibers in ‘corporated with quercetin and ciprofloxacin HCl, and hypothesized that fabricated scaffold would () decrease bacterial growth and hence check infection at the wounded area, (if) scavenge ree radical and thus di ‘minish the inflammatary response, (i) degrade and get assimilated in the granulation tissue, and (i) facilitate the complete wound closure in fan accelerated manner. Further, we evaluated the proposed hypothesis in a rat model after excising fll thickness wounds. 2. Materials and experimental section 2.1. Material and reagents Ciprofloxacin HCL was generously provided as gift sample by Cipla, ‘Mumbai. Poly (ccaprolactone) (average My = 80 kDa) was purchased from Sigma-Aldrich, USA, Quercetin was procured from Cayman, (Chemical, USA. Gelatin from poreine kin, Type A was purchased from imedia, 376-Swiss albino fibroblast cell lines wore obtained from [National Centre for Cell Science, Pune. 1,1,1,3,3,3-hexafluoro-tso peo ‘anol (HFIP) was purchased from Spectrochem Pvt Ltd. Mumbai, Inia, All other chemicals were obtained from eectified distributors. 2.2. Preparation of electrospinning solution and fabrication of nanoftbers ‘A polymeric blend of PCI/Gelatin/HFIP was prepared by mixing 12% w/v of PCL/HEIP and 12% w/v of Gelatin/1EIP in the equal vo: ume ratio (1:1) A small quantity (0.2% v/v of HFIP) of glacial acetic acid was added to the mixture to aid the complete solubilization of gelatin and PCL and avoid phase separation during electrospinning (njam et al, 2017). DMSO was used as a cosolvent for quercetin solubilization. Electrospinning solutions of various compositions had ‘heen prepared by the scheme given in Table I. The electraspinning was carried out with an in-house assembled electospinning equipment fitted with a 5ce syringe and a blunt-end 24G metallic needle, a high voltage (14-16) DC power supply to generate sufficient charge inthe solution, a syringe pump to regulate the low rate of the eleetrospinning solution and a flat metallic collector covered with aluminum foil t0 collect the nanofibers. The nanofiber membrane was careflly zemoved from the collector and stored in a vacuum desiccator for further use 2.3. Characterization of nanofiber membrane 2.3.1. Morphology of elecrospun nanofibers ‘Morphology ofthe fabricated eleetrospun nanofibers were observed, ‘by high-osolution scanning electron micrascopy (HRSEM) (FE, ‘Quanta 2008, Japan) operated at an acceleration voltage of 1OKV. [Before SEM scanning, samples were sputtered with gold for 2min to increase their conductivity. ImageJ software was used to measure fiber diameters and porosity, as reported previously (Liu et al, 2014). At Teast 100 filaments of each sample from random locations were ana lyzed fo obtain the average fiber diameter. The data were further pro cessed by GraphPad Prism 5 to ge the histogram of fiber diameter with respect to its distribution frequency. 2.3.2, Solit-state charactertzations ‘Any possible changes in the chemical structure of drugs (Que and, CCH) due ta interaction with other formulation excipients and effect of clectrospinning on functional groups was determined by fourier-tans form infrared (FTIR) spectrophotometer (SHIMADZU 84005). The FTIR spectra of individual drugs, polymers and nanofber film were obtained by scanning KBr pellet inthe range of 400-4000 em” with a resolution of dem". ‘An X-ray Powder Diffraction (XRPD) study was conducted £0 6x plore the changes inthe drug's crystalline property while encapsulating 6 Aa a mena oun of Paracas S67 (2018) 11880 in the nanofibers. The diffraction pattern of ciprofloxacin HCL, (Quercetin, PCL, Gelatin and PCL-GE-CH-Que nanofibers were studied by an X-ray diffractometer (Mini Flex 600, Rigaku, Japan) equipped with a D/tex Ultra detector and an X-ray source (Cuka. radiation; 2. = 1.54 A) operating at 40KV, 15mA. A rotating holder was em ployed to scan the samples at a scanning rate of 4° min over a dif: Fraction angle (28) range from 5" to 60° i é 3 a : 5 24. Contact angle of nanofiber membrane ‘The hydrophilicity of the nanofibers surface was evaluated by ‘measuring the contact angle between a sessile drop of water and ‘membrane surface. For contact angle measurement, a 2ul drop of ‘water was dropped on the nanofibers surface with the help of a mi- crosyringe and statle image was taken by Drop Shape Analyzer (DSA255, KRUSS, Germany). An average value of three measurements at different postion on the scaffold surface represented asthe contact angle value ofthat scaffold ‘Mbrane Poway C3 gee 25. Entrapment efficiency and in-vitro cumulative drug release profile ‘Mean Dinter (any The entrapment efficiency of PCL-GE-CH-Que nanofibers was de- termined by simultaneous equation method using UV-Vis Spectrophotometer (UV-1800, Shimadzu). Briefly, a known weight of ‘membrane was dissolved in HFIP using 0.2% v/v acetic acid to obtain a clear solution, Il. of the obtained soliton was added dap wise in ‘mL. methanol, which was an antisolvent for polymers. After cen ‘wifugation of resulting solution, polymers got precipitated and drugs remained dissolved in supematant which was determined by UV-Vis Spectrophotometer, and concentration was calculated using Eqs. (1) and (2), Amount of entrapped drug was quantified by the Ea. (3). Aum X ayn = Ay X dp Ban X do = Bam Gin a (Que concentration Aw X ban — Aun X bine mn X as = Bow X Or @ CCH concentration = Smo enters ith ever fle and form ste 725988 = 201965 ‘bbton ‘Fie Morphology Sach anoles i : i Entrapment Efficiency (3) ‘Amount of drug inthe sample (mg) ) an Theoretical amount of drug loading inthe samiple Om), @ here, Arr and Asyp denote the absorbance of the supernatant at 271 and 370 nm. aa and asp i the absorptivity of CH at 271 and 370 nm, ar and Dayo isthe absorptivity of Que at 271 and 370 nm. ‘The release of encapsulated drugs in PCL-GE-CH-Que nanofibers, ‘was carried out using waterbath shaker in phosphate buffer (pH 7.4, Briefly, a known weight of membrane of approximate 1 x Lem? di mension was incubated into 2ml. buffer in a closed val, which was further shaken ina water bath shaker at 80 s per minute at 37 = °C ‘The entire portion of buffer was removed atthe definite time and re- placed with another 2m of fresh buffer to maintain sink condition. The ‘cumulative amount of the drug released was calculated based on the {intial amount of drug encapsulated into the nanofiber membrane ue 6 wv of| Prisms) 26, Freeradical scavenging efficiency of nanofibers porosity and entrapment elelency of nanofibers wit diferent composition. ‘Antioxidant activity ofthe nanafiber membrane was performed by 2.2-diphenyl-1-pierylhydrazyl (DPPH) scavenging ability of fabricated scaffold (Selvara} and Fathima, 2017). Smg of nanofiber membrane ‘was immersed in a 3 ml. methanolie solution of DPPH (100 uM) and the reaction mixture was kept in the dark condition at room temperature. Aiter ineubating for 0.5h, the absorbance ofthe reaction mixture was ‘measured at Asizam by UV-Vis Spectrophotometer. DPPH scavenging cfficacies was determined by Ea. (1) POLO w/ Galan OWA) CH Cow of > Paccecrow 12 “The valves are expresed a means = SD, n= 100; %n=3. Morphology, mean dame sample ‘Table 1 6 Aa a DPPH scavenging efleacis(%) (42) x10 a @ where, Ay and A, cepresents the absorbance of pure methanolic DPPE solution and nanofiber incubated DPPH solution respectively 27, Invitro antibacterial assessment Anubacterial activity of fabricated nanofiber membrane against Staphylococcus aureus (S. aureus) (MTCC1303), a commonly found Gram positive bacteria in open wounds infection, was determined by film difusion method (Unnithan et al, 2012). Briefly, a 100 aliquot of standard suspension (10*cells/mL) ofS. aureus pre-cultured and r= constituted in Luria-Bertani mem was spread onto the surface of a solidified agar plate by using swab sticks. The circular sections (~ mm diameter) of nanofibrous membranes, e, PCL-GE, PCL-GE-CH and PCL CGE-CH-Que were placed on agar plates and symbolized as , F> and F, respectively. The plates were incubated for 24h at 37 + 1°C. After that, the nanofibers were moved to other agar plates seeded with mi- croorganism. The microbial growth on the petr-plate was observed directly and bacterial growth inhibition zone was measured, 28, Biocompatibilty evaluation of nanofiber membrane 28.1. Invitro hemacompaiilty assessment of nanofiber membrane [As the scaffolds were intended for open wound application, the assessment of hemolytic property became essential to evaluate whether the formulation was able to preserve the integrity of RBCS in newly developed blood vessels. Hemocompatibility ofthe nanofibers was as sessed as described by Vijayakumar etal, (2016). RBCs were collected from 2m human blood by centrifugation at 2200rpm for 1S min and rinsed thrice with normal saline (0,936) to separate traces of plasma Finally, erythrocytes pellet were re-suspended gently up to LOmL. in ‘normal saline to obtain standard erythrocytes suspension. 5 mg of each sample ie. PCL-GE, PCL-GE-CH and PCL-GE-CH-Que nanofibers were mixed with 2m of a standard suspension of RBCS and kept at room, temperature for 0.5. An equal volume of standard erythrocytes sus- Pension was mixed with 19 Triton X-100 and normal saline, separately and utilized as postive (completely lysed RBCs) and negative control, respectively. After incubation, the samples were centrifuged and the supernatant was separated and absorbance of oxy-hemoglobin mea- sured at Asam by UV=Vis spectrophotometer. The percentage of RBCS lysed was ealeulated using Eq. (5): Ava Anmart Hrmolysis (6) = a0 o \Where38 As oven enim Nd Api ial FeBEESEN the absorbance of supernatant incubated with nanofibers, normal saline and Triton X- 100, respectively. 28.2. Inviro cel viability assessment ‘The viability of 3 T6:Swiss albino fbroblast on nanofbers was ex- amined by MIT (3-[4,5-dimethylthiazol-2-y1-2,5-diphenyitetrazolinm bromide) assay. Briefly, the fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) enriched with 1036 fetal calf serum, 10% antibiotics in a humidified CO, incubator (85% air/5% CO,) at 37°C. Sterilized nanofiber membrane was atiached on a coverslip ad placed into 24-well microplate. Subsequently the cultured fibroblasts were seeded onto fixed-seaffold at a density of 2 = 10* cells/well, and further incubated for 24h, 48h, 72h in triplicates. A similar cell sus- pension seeded into a well without any seaffod was considered as the Positive control. At the end of the incubation period, 10yL. MIT (1Omg/ml. in PBS solution) was added in cach well and ineubated for another 4h, Afterward, MT solution with culture medium was pis petted put completely and 150 DMSO was added in each well to mena oun of Paracas S67 (2018) 11880 solubilize the internalized insoluble formazan that were proportional (0 Viable cell number. After agitating the plate for 10 min, formazan dis solved and the purple color solution was measured At Ron: Cell viability was calculated by Eq, (0) Aras = Ana Apinonint = ADn0 call rab) <1 © here Aree Apstive sone and Apso Tepresent the optical density of culture mediuon incubated with a nanofiber membrane, without nano fiber membrane, and of DMSO only, respectively. 2.9. Open-wound healing experiment Developed nanofibers were evaluated for wound healing in the ‘animal by excising circular wound, The experiments were carried ou after the approval from the Central Animal Ethical Committe (CAEC) of Banaras Hindu University, Varanasi (No. Dean/2018/CAEC/638), ‘Twenty four healthy adult’ Winstar rats of either sex, weighing 220-250 mg were used as experimental animals. The rats were an ‘sthetized by injecting a combination of ketamine (85 mg/kg) and xy lazine (S mg/kg) intraperitoneally. The back side of rats were shaved off and decontaminated with 70% ethanol and skin wat excised to create a full thickness cireular wound with an approximate diameter of 25cm. The animals were randomly divided into four experimental groups (n= 6), namely, Group 1 (gauze treated), Group 2 (PCL-GE ‘ucated), Group 3 (PCL-GE-CH treated) and Group 4 (PCL-GE-CH-Que treated). The wounds were snapped on a regular interval and wound boundary was traced on a tracing paper which was further re-traced on ‘8 2mm? graph paper to obtain the wound area. The Eq, (7) was utilized to determine the progression of the wound healing area: (4549) co Aa ” ‘where, Ap and A, denotes the wound area on day 0, and on day 4, 8, 12 and 16 post-wounding, respectively. On the 8th and 16th days after surgery, granulation tissue was harvested from the healed area and divided into three parts and preserved for further stues. Woune closure (%) 2.9.1. Histological exaination In order to evaluate the histological changes, formalin-preserved ‘granulation issues were fxed in parafin, dissected into 4 ym thin slices and stained using a routine hematoxylin ~ eosin (H&E) procedure. ‘Microscopie changes at the histological level were visualized under the ‘optical microscope, 2.9.2, Antioxidant enzyme activity in granulation tissues Harvested granulation tissues were washed with ice-cold saline, homogenized at a concentration of 10% w/v in ice-cold potassium phosphate buffer (7.4, 50 mM), centrifuged at 5000 pm for 0.51 and supernatants were separated ott to analyze the free radical scavenging activity of endogenous antioxidants like Superoxide Dismutase (SOD) and Catalase (El-erjani eta, 2016). The activity of SOD and catalase ‘was evaluated as per the procedure used by Beauchamp and Fridovieh (1971) and Aebi (1984), respectively. 2.9.3, Hydroxyproline content in granulation eset Hydroxyproline forms a major constituent of collagen and its dis: Uwibution is highly restricted to collagen, Therefore, its quantitative cstimation can be utilized as an indicator of collagen synthesis. The amount of hydroxyproline was determined in acid-hydrolyzed granu lated tissue according to earlier reported methods (Neuman and Logan, 1950; Reddy and Enwemeks, 1996). 6 Aa a 2.10, Statistical analysis All the results were presented as mean = standard deviation (SD) and statistical comparisons were carried out by GraphPad Prizm soft- ware, version 5.01. The obtained data were elther subjected to one-way ANOVA followed by Tukey's post-test or two-way ANOVA followed by Bonferroni posttest. The experimental result with 95% confidence level (p < 0.05) was considered as statistically significant 3. Results and discussion 3. Characterization of nanofiber membrane 3.1.1. Morphology of elecraspun nanofibers ‘Due to the sol-gel transition of gelatinous protein and the im- miscibility of gelatin and PCL. atthe iselectic point, the mixed solu tion of gelatin/HHIP and PCL/HEIP became cloudy and slowly sepa- rated into «wo layers during electrospinning, which results into defective fiber morphology. When a litle amount of glacial acti acid was added to this opaque solution, the pH ofthe solution reduce fom the isoelectric point of gelatin, which resulted into compete miscibility of gelatin and PCL, and homogenous electrospinning (Anjum ef al 2017; Xue et al, 2014a), ‘The FE-SEM micrographs and corresponding histogram of nanofi- bers diameter are shown in Fg. 1, "The micrograph images demonstrate a randomly oriented, beadless and uniform structure. The mean dia meter of the PCL nanofiber membrane wns found ta be 86.184 = 20.984nm. After blending the gelatin with PCL, the dia meter of nanofibers increased significantly (234.172 = 98.234 nm) and was attibuted to an increase in visesity of the solution (Anjan ct al, 2017; Norouzi et al, 2015). Further incorporation of drugs in PCI-Gelatin matrix resulted in nanofibers with increased average di meter, smoath surface and more uniform size distribution, ‘The porosity of a tissucengineered scaffold becomes a vital para- eter especially when its application is anticipated for skin regenera tion. The optimum porosity ofthe scaffold would be useful for cellular ingrowth and proliferation on the scaffold, infiltration of nutrients, gases and exudates across the nanofiber membrane. Generally, the ideal Porosity of a scaffold for skin regeneration should be within 60-90% range (Chong eta, 2007). The porosities of fabricated nanofibers were found to be within this range (Table 1), ensuring sufficient exchange of nutrient and gas. 3.1.2, Solid-state characterizations FEAR spectroscopy was conducted to investigate any probable drugs and polymer interactions, drug stability and the effect of eleetrospin- ring on the functional groups of drugs in the formulation (Gaonkar Cal, 2017; Khan et al, 2017), FEIR spectra of eiprfloxacin HCl and (Quercetin are shown in Fig. 2(a). Ciprofloxacin HCI showed its char- acteristic peak at 1624.11 cm for ketone C=0 stretching vibration, 43708.99cm~' for carboxylic acid C=O stretching vibration, 804.34 m~" for CF stretching vibration and a broad shoulder for NH and O-H stretching vibration (between 3350 and 3550.¢m™'. Simi- larly, the major characteristic peak of Quercetin appeared at 1383em"' for O-H bending vibration of phenol functional group, 1664,62em- for aryl ketone C=O stretching vibration and a broad peak around 3400 em eorrespond to stretching vibration offve OH phenolic groups. FTIR spectra of manufactured scaffold shoved typical Ciprofloxacin and quercetin peaks with minor wavenumber variations, Which showed thatthe eletrospinning process did not adversely affect the ciprofloxacin and quercetin functional groupe. It can therefore be concluded that drugs and polymers are chemically compatible and ean be electrospun into a scaffold, XRD patterns of ciprofloxacin HCl, Quercetin, PCL, Gelatin and fabricated nanofibers are shown in Fig. 206). Ciprofloxacin HC spectra exhibited sharp diffraction peak at 8.2", 9.04", 19.2", 24.72" and 26.48" mena oun of Paracas S67 (2018) 11880 {in 20 seale (Katria et al, 2014), which signifies the erystalline nature lof the drug. Likewise, few distinct sharp peaks at 10.74", 12.42", 27.34" established the crystalline nature of quercetin (Qi et al, 2015). Two dlifraction peaks situated at 23.9° and 21.6" attributed to the semi crystalline nature of PCL (Xue et al, 201 4a). Absence of any sharp peak gelatin spectra denoted its amorphous nature. In contrast, absence of ‘characteristic peaks of drugs in PCL.GE-CH-Que spectra denotes that a portion of drugs had been entrapped in the nanofibers and rest of the amount got aggregated on nanofibers surface in amorphous frm, since rapid in-situ solidification didn't provide sufficient time for recrystal- lization of drugs molecules on the surface. This finding was further confirmed by drug entrapment and drug release studies of the seaffod 3.2. Contact angle of nanofiber membrane ‘Water contact angle represents the hydrophilicity of the surface of the scaffold which in turn affects the fibroblast adhesion and its pro- liferation on nanofibers. Despite having desirable biocompatible prop: erty, PCL nanofibers suffer from severe hydrophobicity. Therefore, the effet of geatin on hydrophobicity was evaluated and shown in Fig. 3 1 was found that gelatin redaced the contact angle effectively, whieh ‘was attributed to the hydrophilic nature of the protein. Further, the Addition of ciprofloxacin hydrochloride also reduced the contact angle. ‘The average contact angle values for PCL, PCL-GE, and PCL-GE-CH-Que nanofibers were 100.1 = 3.16", 55.5 = 2.10%, and 48.8 = 2.95" re. spectively. The contact angle of PCL.GE and PCI-GE-CH-Que nanofiber ‘membrane reached to O° within the 30, while that of PCL nanofiber ‘membrane did not change significantly til 2 min, 3.3. Entrapment efficiency and in-vitro cumulative drag release profile ‘The entrapment efficiency of fabricated nanofiber membranes Fs shown in Table 1. The high entrapment efficiency of nanofibers might, be due to better miscibility of drugs with polymers which results in good interaction of drugs with the polymer matrix and enhanced dis- persion of drugs in nanofibers, Further, the non-volatile nature of drugs and mixing of optimum concentration of drugs resulted in partial loss of drugs as aggregate on the nanofibers surface during an insty solidi cation of the drugs-polymers mixture. Therefore, fabricated scafold entrapped most of the drugs with a fraction amount appeared on the surface, which also confirmed the assumption of XRD outeome. ‘The release profile of ciprofloxacin HCL and quercetin from PCL-GE- (CH-Que nanofibers in phosphate bulTe (pH 7.4 is displayed in Pig 4. It thas been observed thatthe release of entrapped drugs from nanofibers ‘was biphasic in nature, with intial rapid release followed by slow and a steady release. The probable reason for the inital burst release was {quick solubilization of sucface-aggregate in amorphous form and also due to leaching of drugs which was entrapped near the surface and was in direct contact of dissolution media, The reason forthe extended re Tease could be longer diffusion path from thick nanofibers, slow bio- degradation of gelatin due to entanglement with PCL chains, and the crystallinity of PCL (Xue et ol, 20142). Since the underlying objective ‘was to minimize the microbial infiltration and thas reduced the gen ration of ROS during the inital healing phase, therefore, the rapid release of drugs was deemed necessary, and this had been observed in PCL-GE-CH-Que nanofibers release profile. Further, the addition of gelatin increased the degradation of scaffold which resulted into 99.18 + 4.98 (ciprofloxacin HC) and 88.09 + 6.16 (quercetin) re lease in Adays as against 98.98 + 261 (ciprofloxacin HCI) and 85.09 + 2.89% (quercetin) release in 6 days for PCL-based nanofibers as reported by Ajmal et al. (2019). 3.4, Freeradical scavenging efficiency of nanofibers ‘The Free-radical scavenging efficacies of the nanofibers were es tablished by DPPH reduction method and shown in ig. S. The assay f= 6. Aa a rmenaonal oul of Parmacues 5572018) 18480 Mea amet m= 28.372 ry Pisin “ Peace Eis g RIA i w z =” eo a % @ mw 9 or "Tio 250 300 400 sho 600 700 Diameter (am) Diameter (am) i Hea . 0 300 400 Sho 600 700 Hi” 900 Diameter (1 ‘lo Seo lo Te 0 980 100017001300 Diameter (am) Fig. 1. The FE-SEM micrograph of nanofibers and histogram of fiber diameter with respect to its distbution Gequency: (a €) PCL nanofibers, (bf) PCLAGE ranafbers (1:1), (6g) POL-GE-CH nanofibers, and (,b) PCL GE-CH Que nanclibers based on the principle that DPPH is stable free-adieal and it gives purple color solution in methanol. After accepting an electron of by: drogen from an antioxidant, it reduces to DPPH, and solution color changes to yellow. This change in color is measured at Asiran and utilized for estimation of relative antioxidant efficacies of diferent scaffolds (Selvara) and Fathima, 2017). Tt was found that PCL-GE na- nofibers scavenged the 8.699% DPPH, which might be due to gelatin derived radical-scavenging peptide sequence (His-Gly-Pro-Leu-Gly-Pro Lew), as reported by Mendis etal. (2005). Further, quereetin loaded nanofibers quenched the 55.14% DPPH, which proved the antioxidant activity of phenolic groups present in the quercetin. The enhanced antioxidant property of PCL-GE-GH-Que scaffold in compared to PCL (CH-Que nanofiber membrane (40.13 + 3.01%) as observed in our previous study was attributed to fast degradation gelatin added scaffold (Ajmal eta, 2019), 6. Aa a oe ytd ae wr’ ‘Transmittance (%) rmenaonal oul of Parmacues 5572018) 18480 rnc Intensity wl Wavenumber (em) Dittsetion Angle (20) Fig. 2 Chemical characterization of ciprofloxacin HCL, quercetin, PCL, gelatin, and developed nanofibers via (a) FT-IR ana (b) XRD. 100: age (°) 80: 60: 40: 20: Water Contact 0. PCL PCL-GE — PCL-GE-CH Fig. . Water droplet profile and quancatve value of contact angle for PCL PCL-GE, and PCL-GE-CH-Que nanotber membrane The values are expressed means = 50,0 100: 75 > Ciprofloxacin HCI 50 = Quercetin 28: ‘Cumulative Drug Release (%) 0 20 40 60 80 100 ‘Time (hrs) Fig. 4 The iro clease profile of quercetin and ciprofloxacin HC fom PL: telat nanofibers in phosphate buffer (pH 7.4. The values ae expressed as 120 140 160 3:5, Invitro antibacterial actiity Inhibition of bacterial growth zone was used to determine the ant bacterial property of scafolds. The growth ofS. aureus can be observed ireely from the petridish to evaluate the antibacterial property (ig 6€) and (b). Fis. 6 presents the diameter of inhibition zone for diferent incubation time, It can be observed that placebo nanofibers (&) did not show any antibacterial activity throughout the study period, In contrast, PCI-GE-CH (F) and PCLGE-CH.-Que (Fy) aanofi= bers exhibited wide inhibition zone intially which might be due to the rapid release of antimicrobial Although PCL-GE-CH-Que had a litle broad inhibition zone in comparison to PCL-GE-CH, except on day ‘three, no significant difference had been observed between their values. Farther, it was observed that initially, PCL-GE-CH-Que nanofibers was more aetive thon PCL-CH-Que seaffold as reported by Ajmal eal. which ‘was due to mote burst release ofantimierobial from the scaffold (Ajo ct al, 2019), Hence, it could be established that elecrospun scaffold ‘was sufficiently active to check the bacterial grow during the ex: ‘perimental period and eleetrospinning did not change the antimicrobial property of ciproflaxacin HCI while encapsulating in the nanofibers. 3.6. Blocompatiity assays of nanofber membrane 3.6.1. Invitro Remocompatibity assesment of nanofber membrane Percentage hemolysis represents the degree of exythrocytes lysed ‘when they are expased tothe scaffolds inthe solution. A higher value of hemolysis indicates the poor hemocompatibility of the scaffold in tended for application. The influence of different scaffolds on the in- tegrity of erythrocytes is shoven in Fig. 7(a) The degree of erythrocytes Drokensdown in the presence of PCL-GE-CH-Que nanofibers was 1.072%, which was significantly (p < 0.05) much lower in comparison 1 other scaffolds. Although other seaffolds rendered higher hemolysis {n comparison to PCL-GE-CH-Que, hemolysis caused by all the seafolds ‘wore under the acceptable range of S% (Haghjooy Javanmard et a, 2016). The plausible reason forthe low value of hemolysis caused by PCL-GE-CH-Que nanofibers was protective action of quercetin, a fl vonoid, which diminished the peroxidation of unsaturated fatty acid and oxidative damage of glutathione and thiol group (SH) in the ery throeytes membrane. 36.2. Invitro cell vibilty assessment ‘The viability of 316-Swiss albino fibroblasts on the scaffold was, evaluated by MIT assay, and the results are displayed in Fig, 70). It ‘was observed thatthe cells proliferated well on all the membranes in dicating that the scaffolds were cyto-compatible and nontoxic. All scaffolds showed more than 100% cell viability even after 72h in feubation, which might be due to cell recagniion site (Arg-Gly-Asp Am (a) 18% 1500}. DPPH solation PCLGE t PCL-GE-CH Zoom PoL-ce-ch.ove 2 < 0.500 0.000 400.00 500.00 60000 70000 $00.0 Wavelength (nm) (Gi hisogram representing DPPH attenuation eficences of diferent Sato ig. 5 Pree radical eavenging efficacies ofthe PCL-GE, PCL-GE-CH, and PLC-GE-CH.Que nanofibers after 0.5 inexbaton wi mena oun of Paracas S67 (2018) 11880 (b) DPPH Scavenging (%) PCL-GE PCL-GE-CH PCL-GE-CH-Que DPPH soation: (0) UV-Vis spectra: 3 “p = 005, "p = O00, The values are expressed ax means SD, 0 “# PCL-GE-CH-Que > PCL-GECH e PCL-GE Fig. 6. Antimicrobial activity of nanofber membranes against S. ures (inhibition ze on day 1, ¢) inhibition zone on day 3, graphical stration showing the relationship between diameters of inhibition zone ram) vs Incubation time (4a). F, >, abd Fy represent the PCL-GE, PCL-GE-CH, and PCL-GE-CH Que tnnofber,expectively, The vals are expreied ae reans + 8D, 0 amino acid sequence) provided by gelatin protein and thus high cell adhesion and proliferation on gelatin seaffold. The plausible reason for significanly (p < 0.05) higher cell viability on PCL-GE-CH-Que na- nofiber surface was additional protective nature offered by quercetin, a Aavonoid, which was found to inerease the Rbroblast proliferation and collagenesis as reported by Selvara) and Fathima (2017) PCLGE PCLGE-CH PCL-GECH-Que 24h, aa and 72h incubation. The vals ate expresed as means + SD, 0 p< O05 vs PCLGE CH nanofibers 47. Open-wound healing experiment ‘Throughout the sisteen days of the treatment period, none of the ‘animals showed any post-operative adverse reaction suchas bleeding of granulation tissue, Nuid retention, infection, sepsis, et. All the animals, stayed alive till the end of the experiment and by the end of the 3rd ‘week al the groups showed complete healing. Fig. a) showed the representative images of rats from all the four groups (gauze, PCL-GE, PCLGE-CH, and PCL-GE-CH.Que treated) on the Oday, 8th and 16th BB Control Ee PCL-GE & Pc-ce-cu Ml PCL.GE-CH-Qve athe abe The ig-7- Instr biocompaibiity of develope! nanofibers (a) hemacompatibility of saffol, (b)vabiity of 3T6 Swiss albino fibroblast on diferent eatflds ater ‘3 = 005 vs contol, Ala a (a) PCL-GE Treated Gauze Treated mena oun of Paracas S67 (2018) 11880 PCL-GE-CH-Que Treated PCL-GE-CH Treated Wound Area Closed (%) 4th Day 8th Day 12th Day EB Gauze Treated FER PCL-GE Treated i PCL-GE-CH Treated HID PCL-GE-CH-Que Treated 16th Day Fig. 8. Eet of nanofibers in the healing of fll hess wound: (a) epresentative image of wound healing on day 8 and 16 (b) percentage of wound area closed following teatment with gauze, PCL-GE, PCL-GE-CH and PCL-GE-CH-Que nanofibers on day 4,8, 12 and 16. The values ate expresed as means + SD, = 6, "p< (108 vs Gauze Treat, day, and Pig, s(t) showed wound area closed during the experimental period. Among the four groups, all the scaffold treated groups showed. excellent adherence and complete degradation and assimilation of na- rnofibers in the granulation tissue in contrast to earlier finding (Ajmal etal, 2019), which was due to hydrophilicity provided by the gelatin and they also exhibited significant (p < 0.05) wound healing in eom- parison to control group. Native ECM mimicking structure provided by nanofibers and cel-ecognition site (Arg-Gly-Asp amino acid sequence) ‘on gelatin might have inereased the ell attachment and its spreading On day 16, PCL-GE-CH-Que treated group provided 100% wound clo- sure, whereas wound closed by PCL-GE:CH, PCL-GE and gauze treated group was 89.09%, 78.45 and 71.22, respectively 3.7.1. Histolegial examination ‘The histological changes of gauze and nanofibers treated groups were evaluated by 1 & E staining of granulation tissue on Sth and 16th days and results are displayed in Fig 9. On day 8, the surface layer of granulation tissue of gauze treated group showed high ulceration and the underlying layer was severely infiltrated with inflammatory cells. “The wound in PCI-GE and PCI-GE-CH treated group displayed gran lation tissue with moderate ulceration and infiltration with in flammatory response and with fewer fibroblasts. In contrast, PCL-GE- CCH-Que nanofibers treated group demonstrated moderate epitheliali- zation. The amount of inflammatory response at ths stage inthe PCL GE-CH-Que nanofibers treated group was quite low in comparison to ‘other groups, confirming the anti-oxidant activity of scaffold. A mod crate amount of beoblast in PCL-GE-CH-Que nanofibers treated gran ‘lation tissue established its accelerated healing property On day 16, PCL-GE-CH-Que nanofibers treated group exhibited complete reepithelialization. The epidermal layer was thick and in flrated with keratinocyte while dermal layer had good collagen de position and spars inflammatory cells, demonstrating complete wound Ala a Gaure Treated 0x. 0x mena oun of Paracas S67 (2018) 11880 PCL-GE-CH Treated 10x PCL-GE-CH-Que Treated 10x = ig. 9. Histological images of granulation sue of Gauze, PCLGE, PC-GE:CH and PCL-GE-CH:-Que nanofibers teatment on day 8 and 16, Seale bar a 10% and 40X resoltion are shove in respective figures. healing. Although wound treated with PCL-GE-CH scaffold. also ex hibited good re-epithelislization, it showed more infiltration with in flammatory response and comparatively mare white space indicating low collagen deposition in compasison to PCL-GE:-CH-Que nanofibers treated group. Gauze treated wound showed moderate re-epithelili- zation with inflammatory infiltration, necrotic ibrinofd debris, poor collagen deposition, and capillary hyperproliferation, signifying that inflammation still present in this group. 37.2. Antioxidant enzyme activity in granulation tissues Open full thickness wound is prone to microbial infection which results in excessive production of ROS and thus oxidative damage of Sibroblast and collagen metabolism. During the inflammatory phase of wound healing (1-4 days), even quercetin, an exogenous antioxidant, was not able to enhance the activity of SOD and catalase signifieantly (ig. 10). Although, PCL-GE-CH-Que treated group shown significant Improvement (p ~ 0.05) in SOD and catalase level in contrast to other treated group, sill significant difference (p < 0.05) was found when compared with the sham group. PCL-GE-CH treated group maintained a significant (p < 0.05) improvement in enzymes level throughout the experiment period when compared to gauze and PCL-GE treated group, Which might be due to attenuation of bacterial invasion and thus low ROS generation. On day 16, only PCL-GE-CH-Que treated group (a) SOD Activity (Uniti protein) 16 Day enhanced SOD level up to sham groups (p > 0.05), however, a sig nificant difference (p < 0.05) still found in catalase level. Therefore, the antimicrobial and antioxidant property of a scaffold would be helpful for attenuating ROS and ataining the endogenous anti-oxidant level up to the homeostatic state. 3.7.3. Hydroxyproline content in granulation tissues Due to the highly restricted distribution of hydroxyproline into collagen, its estimation in granulation tissue gives a direct correlation with collagen deposition. On day 8, although all nanofibers treated groups began to achieve statistically signifieant (p < 0.05) inerease in hydroxyproline contents as compared to gauze treated, but no one could reach to an insignificant difference level in comparison to the sham group (Fig. 11). On day 16, hydroxyproline content peaked in PCLGECH-Que nanofibers weated group, which was, 2531 + 0.239 ug/mg of granulation tissue and no significant difer fence was found when compared with the sham group. Although, hy droxyproline content in PCL-GE.CH nanofibers treated group was quite significant (p < 0.05) in comparison with Gauze treated and PCL-GE nanofibers treated group, but still a significant difference exist between PCL-GE-CH nanofibers treated group and sham group. These outcomes revealed that the addition of quercetin had potentiated the healing property of scaffold by attenuating the oxidation of fbroblast and © Sham Group EB Gauze Treated > PCL-GE treated OD PCL-GE-CH Treated Ed PCL-GE-CH-Que Treated 16hDay ig. 10. fect of treatment with iferent nanaibers on endogenous enzymes vie (2) SOD and ()eatalase in granulation ses on day 8 and 16. The values are expressed as means + SD, n= 6, p = 0.05 vs Sham Group; “p< 0.05 vs PCL-GE-CH.Que nanoflbers weated group, and "p > 0.05 vs Sham Group. Ala a 8th Day 16th Day Hydroxyprotine Content (jg/mg wet tissue) mena oun of Paracas S67 (2018) 11880 EB Sham Group FEA Gauze Treated i PCL-GE Treated CD PCL-GE-CH Treated PCL-GE-CH-Que Treated Fig. 11, Effect of diferent scffold on hydroxyproline content in granulation tsa of rats on day and 16 post-woundng The values are expressed as means = SD, = 6, "p< 0.05 veSham Group, “p = 0.05 v= PCLGE-CH nanofibers treated group, and "p > 0.05 vs Sham Group. promoting the collagen synthesis and accelerating the formation of granulation tissue. 4. Conclusion We developed a PCL-gelatin based nanofibers functionalized with ‘quercetin and ciprofloxacin HCL. The addition of gelatin had enhanced, the hydrophilicity (contact angle = 48.8 + 2.95°) and biodegradation rate, The resultant high rate of biodegradation had reduced the release duration of nanofibers, and 67.98% CH and 44.78% quercetin vas r- leased within &h, which was desirable for reducing bacterial infection tnd ROS attenuation during the carly healing phase. The developed. nanofibers had effectively reduced the S. aureus growth on the agar plate and scavenged > 50% DPPH (100 uM) in methanol, which con- firms the potential anti-microbial and anti-oxidant property of nanofi ition site provided by gelatin improved the fibroblast viability on the seafold. Further, the protective action of quereetin against RBCS Iysis confirmed the bio- compatible nature ofthe scaffold and proved as a promising aplication for wound healing. Subsequently, accelerated healing and 100% closure of wounded skin within 16 days endorses the quercetin and cipro- foxacin HCl incorporated PCl-getatin scaffold as a potential dressing rater for full thickness wound, Acknowledgements ‘The authors are highly thankful to Prof, PAK. Mishra, Head, Department of Chemical Engineering, IT-BHU, Varanasi, for permitting ta use his lab facilities. We are also obliged to Central Instrument Facility Center (C1EC), IT (BHU), Varanasi for providing SEM and XRD facilities. The financial support for this rescarch provided by the Ministry of Human Resources and Development, New Deli (India) is thankfully acknowledged. Declaration of Competing Interest “The authors state no conflicting interest. References ‘ei, 1988 (5 Catan pro, Heir Math Enea 121-128, ‘jms. Sond, GX Tokai Pn. Sh, 3 andy. VK. Misa 1, 219, Cowen HCl and quercetin fein’ okewosun analer !neabran:faveaon ands evahaoa nul ks wound bedi A als ‘jum Agabalan A. 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