Electrospun Polymeric Coreâsheath Yarns as Drug Eluting Surgical

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Electrospun Polymeric Core-sheath Yarns as Drug Eluting Surgical Sutures Smrithi Padmakumar, John Joseph, Madhuri Harsha Neppalli, Sumi Elizabeth Mathew, Shantikumar V Nair, Sahadev A Shankarappa, and Deepthy Menon ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00874 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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ACS Applied Materials & Interfaces

Electrospun Polymeric Core-sheath Yarns as Drug Eluting Surgical Sutures Smrithi Padmakumar, John Joseph, Madhuri Harsha Neppalli, Sumi Elizabeth Mathew, Shantikumar V Nair, Sahadev A Shankarappa *and Deepthy Menon* *Sahadev A Shankarappa and Deepthy Menon are co-corresponding authors

Amrita Centre for Nanosciences & Molecular Medicine, Amrita Institute of Medical Sciences & Research Centre, Amrita Vishwa Vidyapeetham, Kochi 682041, Kerala, India

ACS Paragon Plus Environment

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KEYWORDS Yarns, Core-sheath, Suture, Electrospinning, Aceclofenac, Insulin ABSTRACT Drug-coated sutures are widely used as delivery depots for antibiotics and anti-inflammatory drugs at surgical wound sites. Although drug-laden coating provides good localized drug concentration, variable loading efficiency and release kinetics limits its use. Alternatively, drug incorporation within suture matrices is hampered by the harsh fabrication conditions required for suture-strength enhancement. To circumvent these limitations, we fabricated mechanically robust electrospun core-sheath yarns as sutures,with a central Poly-L-Lactic acid core, and a drugeluting Poly-Lactic-co-Glycolic acid sheath. The electrospun-sheath was incorporated with aceclofenac or insulin to demonstrate versatility of the suture in loading both chemical and biological class of drugs. Aceclofenac and insulin incorporated sutures exhibited 15 % and 4 % loading, and release for 10 and 7 days respectively. Aceclofenac sutures demonstrated reduced epidermal hyperplasia and cellularity in skin-inflammation animal model, while insulin loaded sutures showed enhanced cellular migration in wound healing assay. In conclusion, we demonstrate an innovative strategy of producing mechanically-strong, prolonged drug-release sutures loaded with different classes of drugs.


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1. INTRODUCTION Drug-eluting surgical sutures have been long examined for their potential use in local drug delivery. The concept of delivering pre-loaded local anesthetics1, analgesics, anti-inflammatory agentsor antibiotics2 from the suture directly into the wound vicinity provides a direct and efficient route for drug delivery. Drug-coated sutures are available commercially that offer highmechanical strength, biodegradability, and demonstrate low tissue responsiveness. However, their major drawback is the difficulty in attaining efficient drug loading and poorcontrol over its subsequent release kinetics.3–7 To overcome this shortcoming of surface coating, the alternative logical option is to incorporate drugs within the suture polymeric matrix. This however, poses a technical challenge. Melt spinning, the most common technique used to produce commercial sutures utilizes high temperature to process polymers.8 However, incorporating heat-sensitive compounds especially drugs, into the polymeric blend during high temperature processing steps, would render them inactive. Alternatively, electrospinning is anothercommon technique adopted to produce micro or nanosized fibers that can be used to produce drug-loaded sutures.1,9–11Although electrospun polymeric sutures demonstrate stable drugloading and controlled release, the inherent low mechanical property of electrospun fibers limits their use as strong surgical sutures.1,9,12 In addition, it would be difficult to incorporate heat-sensitive drugs (e.g. peptides) within electrospun matrices, since fiber strength enhancement methods require harsh post-processing conditions.13,14 Also, incorporation of higher molecular weight compounds such as serum albumin within the polymeric matrix is known to markedly decrease the mechanical strength of electrospun fibers.15 In the current study, we address the challenge of producing multifunctional sutures possessing both high mechanical strength and favorable drug-loading / release using a single-step


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electrospinning technique that yields continuous yarns.16 The idea is to develop a mechanically strong small-diameter electrospun ‘core-yarn’, with an outer drug-loaded electrospun ‘sheath’, for suture application. Maintaining a small core diameter is crucial for fabricating sutures with optimal radial size. The strength-enhancing post-processing of core-yarn prior to the deposition of the drug-laden sheath allows for the fabrication of thin, yet strong sutures. Since the burden of mechanical strength is borne by the core yarn, the sheath offers flexibility in drug loading, multiple drug loading, and controlled drug release, without compromising the overall mechanical strength of the suture, or without any concerns of drug degradation by high temperature processing steps. Here, we have used Poly-L-Lactic Acid (PLLA) for fabricating the mechanically strong core, while Poly Lactic-co-Glycolic Acid (PLGA 50:50) was used as the drug loadable sheath. Aceclofenac and insulin were chosen as model drugs. Anti-inflammatory properties of aceclofenac-loaded sutures were tested in a cutaneous inflammatory animal model, while wound-healing ability of insulin-loaded sutures was tested in an in vitro cell migration assay.


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2.

MATERIALS AND METHODS

2.1 Materials PLLA (molecular weight 45,000 - 50,000 gmol-1, density 1.5 gcm-3 at 25 °C) was purchased from Goodfellow, UK. PLGA 50:50 (molecular weight 50 kDa, density 1.34 gcm-3) was purchased from Polysciences Inc., USA. Chloroform and acetone (HPLC Grade) were purchased from Merck, India. Aceclofenac, insulin (recombinanthuman insulin peptide, Mw ~ 5800 Da), indocyaninegreen (ICG), dichloromethane, 12-O-Tetradecanoylphorbol 13-acetate (TPA) and paraformaldehyde (PFA) were procured from Sigma Aldrich, USA. Acetonitrile (HPLC Grade) for HPLC study was procured from Spectrochem, India. Mouse dermal fibroblasts (L929) were procured from National Centre for Cell Science (NCCS, India), Dulbecco’s modified Eagle’s medium (DMEM) with glucose and L-glutamine (4.5 gL-1) was purchased from Lonza Chemicals, USA. 2.2 Fabrication of fibrous core yarns Fibrous core in the form of continuous yarns was fabricated using a modified electrospinning method developed by our group.16 Briefly, PLLA was dissolved in a mixture of chloroform and acetone in the ratio 2:1 (v/v) at 14 % w/v to obtain a clear, homogenous solution. Similarly, PLGA was dissolved in a mixture of chloroform and acetone in the ratio 4:1 (v/v) at 12.5 % w/v to obtain a clear and viscous solution. Aceclofenac was dissolved in 50 % (w/w) in both of the blended solutions. Electrospinning was carried out using a modified electrospinning assembly consisting of a rotating collector having a plurality of point electrodes, and an infusion pump (KD Scientific, USA) fitted with a syringe (BD Emerald, India) loaded with polymeric solution. A high DC voltage of +10 kV (Gamma High Voltage, USA) was applied to the blunted end of


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the polymer ejection needle. Continuous fibrous yarns were twisted and drawn using a guide wirefrom the rotating collector and wound over a rotating bobbin, whose speed (100 rpm)was synchronous with that of the collector. To develop mechanically strong fibrous PLLA core yarns, the as-spun yarns were heat-stretched to 6 times the original length at an optimum temperature of 120 °C. 2.3 Fabrication of core-sheath yarns PLGA (9 % w/v) was dissolved in a mixture of chloroform and acetone in the ratio 4:1 and electrospun over the pre-fabricated hot stretched PLLA core yarn which was wound over a bobbin fitted within the modified collector as a uniform coating to yield yarns with core-sheath architecture. Drug-loaded core-sheath yarns were developed by dissolving the desired drug in the PLGA solution and electrospun at similar conditions as above. Aceclofenac and insulin were dissolved in 50 % and 3 % (w/w) respectively in the PLGA polymer solution. To fabricate mechanically strong sutures, drug-loaded core-sheath yarns were double plied using a custommade plying setup consisting of a set of winding and unwinding motors. For in vivo studies, sutures with USP dimension of 3-0 were developed. 2.4 Morphological Analysis Fiber morphology of all samples was examined using the scanning electron microscope (JEOL, JSM-6490LA, Japan). Samples were sputter coated with gold before imaging at an accelerating voltage of 15 kV.


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2.5 Mechanical testing Tensile strength and maximum break force of fabricated yarns were determined by straight pull test using a mechanical testing machine (H5KL, Tinius Olsen, USA), equipped with pneumatic grips and a load cell of 10 N. Sample gauge was set at 40 mm and pulled at a test speed of 15 mm/min until failure. The grips were set at anapproach speed of 5 mm/minute. Stress, extension and preload were 10 MPa, 100 % and 0.01 N respectively. Tests were repeated thrice on separate samples. 2.6 Suture characterization 2.6.1 Fourier Transform Infrared Spectroscopy: FTIR transmission spectra were recorded using an FTIR spectrometer (IR Affinity-1s ATR-FTIR, Shimadzu, Japan) in a spectral range from 500 to 4000 cm-1. All yarns were fragmented prior to FTIR analysis. 2.6.2 X-Ray Diffraction Analysis: Sample yarns were fabricated and wound over a glass slide and diffraction spectra was recorded using an X-ray diffractometer (XpertPro, PANalytical, Germany) with Cu Kα radiation (40 kV, 30 mA). The diffraction scans were recorded at 2θ values in the angular range of 5-60 ° at a step size of 0.03 °. 2.6.3 Differential Scanning Calorimetry Analysis: Thermal profile of yarn samples was gathered by differential scanning calorimetry (DSC) (822e, Metler Toledo, Columbia). 4-6 mg of each sample was placed in a hermetically sealed aluminium pan and heated at a rate of 10 °C/min from 20 to 250 °C in a nitrogen atmosphere.


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2.7 In vitro degradation study Core-sheath yarns were cut into 5 cm segments and weighed. They were then immersed in PBS (37 °C) for in vitro degradation study. At time points of days 0, 7, 14, 21, 28 and one year, triplicate samples were taken out and dried. The yarns were then weighed and subjected to morphological analysis by SEM and mechanical evaluation. 2.8 In vitro drug release Three sets of aceclofenac-loaded PLGA core yarns, PLLA core yarns and core-sheath yarns (5 cm) were immersed in PBS (pH 7.4) at 37 °C under stirring condition (80 rpm). PBS (500 µl) was withdrawn at different time points (0, 10, 20, 40 minutes, 24, 48, 72 hours, up to 25 days) and replaced with the same volume of fresh PBS. Aliquots were stored at -20 °C until further use. Drug analysis and drug content quantification experiments were performed using reverse phase high performance liquid chromatography (RP-HPLC, 20AD, Shimadzu, Japan). The sample (20 µL) was injected into a 4.6 х 250 mm Qualisil gold 5 µm C-18 column maintained at 30 °C. The column was eluted with 35:65 acetonitrile: NaH2PO4 / triethanolamine, (pH 5.2 with H3PO4) at 1 mLmin-1. Aceclofenac was detected by UV absorbance at 273 nm for a total analysis time of 15 minutes. The drug release profile of insulin-loaded core-sheath yarns was investigated by determining the concentration of eluted insulin over a period of 7 days. Three 10 cm insulin-loaded core-sheath yarn segments were submerged separately in PBS (2mL, pH 7.4) and incubated at 37 °C under shaking conditions. PBS (100 µL) was withdrawn at different time points (0, 2, 4, 8, 12, 24, 48, 72 hours, up to 7 days) and replaced with the same volume of fresh PBS. The withdrawn samples were stored at 4 °C until analysis. Insulin concentration was determined using CBQCA (3-(4-


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carboxylbenzoyl) quinolone-2-carboxaldehyde) Protein Quantitation Kit (Molecular Probes ® Invitrogen ™). The assay was performed according to the manufacturer's protocol and sample fluorescence emission was read at 550 nm, with excitation at 465 nm. To determine the total insulin load, yarn segments were dissolved in a mixture of 1:1 volume ratio of dichloromethane (DCM) and 0.1 N hydrochloric acid (HCl) and centrifuged at 2000 rpm for 3 minutes. The resulting supernatant was used to determine total insulin loaded using the CBQCA kit. 2.9 Cell migration assay L929 mouse fibroblasts were seeded in a 6 well plate and incubated at 37 °C with 5 % CO2. Upon formation of a confluent monolayer, cells were exposed to serum-freemedia for 48 hours to minimize proliferation. At the end of 48 hours, a scratch defect was applied on the monolayer using a pipette tip (200 µl). Cells in the naïve group continued to receive serum-free media, while other groups received free-insulin (5 µgml-1) or were exposed to yarn segments (10 cm, insulin-loaded or drug-free) placed within transwell inserts (Corning Incorporated, New York). The cell defects were imaged using an Olympus PEN-E-PL1 digital camera at different time points (0, 2, 4,8, 12, 16 and 24 hours) and the defect area analyzed using ImageJ software (National Institute of Health, USA). Each experiment was done in triplicates and repeated three times to ensure reproducibility. 2.10 Animal studies This study was conducted using protocols approved by the Institutional Animal Ethical Committee (IAEC) of AIMS, Kochi, India in accordance with the principles of laboratory animal care.17 All animals were housed in pairs, allowed standard rat diet and water ad libitum, and


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maintained on a 10 h / 14 h light / dark cycle. Adult male Sprague-Dawley (SD) rats (initial body weight 250 g) were randomly divided into various groups as described below. 2.10.1 In vivo dye release: Under ketamine / xylazine anaesthesia, rats were shaved and aseptically prepped and draped. A single 2 cm long, full skin thickness incision was made on a shaved portion of the lower back area. The incision was closed using ICG containing electrospun core-sheath suture. ICG was chosen as a representative tracer since the molecular weight of ICG (0.7 kDa), comes close to that of aceclofenac (0.3 kDa). Fluorescent images of localized dye distribution within the sutured rat skin were captured at 0, 1, 4, 24, 48, 72, 120 and 144 hours using an in vivo imaging system (Kodak Multispectral Imaging System, Carestream USA). At the end of study, animals were euthanized by CO2 inhalation. 2.10.2 Skin inflammation model: To analyze the functionality of aceclofenac released from sutures, an inflammatory model was established in SD rats by a single topical application of a phorbol ester (TPA).18,19 TPA (1 µg in 200 µL acetone) was applied on a shaved area of the dorsal flank of rats (dimensions: 2 x 2 cm2). Suturing was done on an incision made over the TPA applied area, one hour after TPA application. Animals were classified into six groups (n=5) with each group receiving either baresuture, Vicryl®, topical TPA, topical TPA with bare or aceclofenac suture. Naïve animals were included as controls. All animals were euthanized after 48 hours and skin harvested from suturesite. Skin samples were then fixed with 4 % PFA, paraffin embedded and sectioned for histological analysis using haematoxylin and eosin staining. Histo-morphometric evaluation was performed on images acquired using a Leica compound microscope (DM500, Germany)


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connected with a Leica microscope camera (ICC50 HD, Germany). Epidermal thickness and epidermal cell count were quantified from captured images using the ImageJ software. Epidermal thickness was measured at ten different sites per field (4 random fields / section). Similarly, epidermal cells in six ROI's (100 µm x 100 µm) per field in a total of 4 fields per section were counted and quantified. A total number of 5 randomly selected sections were analyzed per animal. 2.11 Statistical analysis All data are shown as mean ± standard deviation (SD). Difference in mean values between various groups was tested for statistical significance using one-way ANOVA with either HolmSidak's (in vitro analysis) or Tukey (in vivo analysis) post-hoc multiple comparison tests. Statistical significance was set at p

Electrospun Polymeric Coreâsheath Yarns as Drug Eluting Surgical

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