Bioadhesive, Hemostatic, and Antibacterial in Situ ChitinâFibrin

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Bioadhesive, Hemostatic and Antibacterial in situ Chitin-Fibrin Nanocomposite gel for Controlling Bleeding & Preventing Infections at Mediastinum M. Nivedhitha Sundaram, Vignesh Krishnamoorthi Kaliannagounder, Vignesh Selvaprithiviraj, Maneesha K. Suresh, Raja Biswas, Anil Kumar Vasudevan, Praveen Kerala Varma, and Rangasamy Jayakumar ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00915 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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ACS Sustainable Chemistry & Engineering

Bioadhesive, Hemostatic and Antibacterial in situ Chitin-Fibrin Nanocomposite gel for Controlling Bleeding & Preventing Infections at Mediastinum M. Nivedhitha Sundaram1#, Vignesh Krishnamoorthi Kaliannagounder1#, Vignesh Selvaprithiviraj1, Maneesha K. Suresh1, Raja Biswas1, Anil Kumar Vasudevan2, Praveen Kerala Varma3 and R. Jayakumar1* 1

Center for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences

and Research Centre, Amrita Vishwa Vidyapeetham, Kochi-682041, India 2

Department of Microbiology, Amrita Institute of Medical Sciences and Research

Centre, Amrita Vishwa Vidyapeetham, Kochi-682041, India 3

Department of Cardio Vascular and Thoracic Surgery, Amrita Institute of Medical

Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi-682041, India

---------------------------------------------------------------------------#

These authors contributed equally.

*Corresponding Author. Dr. R. Jayakumar E-mail: [email protected] & [email protected]

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ABSTRACT: Mediastinitis occurs after cardiac surgery and is a major threat to patient’s life due to postoperative bleeding and deep sternal wound infection. Major challenge in treating this condition is that it demands a material that should adhere to the applied site and act both as a hemostatic and an antibacterial agent. Based on this we have developed an in situ forming tissue adhesive chitin-fibrin (CH-FB) gel with tigecycline loaded gelatin nanoparticles (tGNPs) for controlling bleeding and preventing bacterial infection. Spherical shaped tGNPs (231 ± 20 nm) were prepared and characterized. In situ forming tGNPsCH-FB gel was formed using a dual syringe applicator in which one syringe was loaded with a mixer of fibrinogen solution, chitin gel and tGNPs; the other syringe was loaded with a mixture of thrombin solution, chitin gel and tGNPs, both these mixtures were injected together. In situ gel formed within a minute and also exhibited excellent tissue adhesive property. tGNPsCH-FB gel was found to be cyto-compatible against Human umbilical vein endothelial cells (HUVECs). Sustained release of tigecycline from tGNPsCH-FB gel was found to be for 21 days. In vitro antibacterial activity of tGNPsCH-FB gel was tested against Staphylococcus aureus, Methicillinresistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli) and their clinical isolates. Further, in vivo hemostatic potential of tGNPsCH-FB gel was evaluated in deep organ injuries created in Sprague Dawley rats. Developed gel exhibited rapid blood clotting potential by achieving hemostasis within 154 s and 84 s during femoral artery (pressured) and liver (oozing) bleeding condition. Hence these findings exhibit the potential application of the developed tGNPsCH-FB gel to adhere at surgical site for controlling bleeding and prevent bacterial infection after cardiac surgery.

KEYWORDS: Mediastinitis, in situ forming gel, tigecycline containing gelatin nanoparticles, fibrin, chitin, hemostasis, infection, bioadhesive. 2 ACS Paragon Plus Environment

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INTRODUCTION Mediastinitis is a deep sternal wound infection (DSWI) that mostly occurs after median sternotomy, which is a common procedure during cardiac surgery. Mediastinitis leads to morbidity, prolonged hospitalization and also has a risk of 10 to 40% mortality.1–3 Recent clinical studies report the incidence of DSWI to be 1 to 5%.4 Patients who go through coronary artery bypass graft (CABG) surgery are mostly prone to postoperative bleeding and deep sternal wound infection.5,6 So, to control postoperative bleeding from sternal edges after sternotomy, bone wax is the most commonly used hemostatic agent but there are reports that bone wax causes foreign body reaction and also enhances infection at the surgical site.7 Most common pathogen isolated from infected sternal wound is Staphylococcus aureus (Gram positive organism) (70-80%) followed by polymicrobial infection and Gram negative bacilli (15-20%).8 Different strategies have been developed for the treatment of DSWI such as intense course of antibiotic therapy along with series of debriments, vacuum-assisted closure therapy and sternal closure with muscle flap.9,10 Vancomycin possess bactericidal properties towards Methicillinresistant Staphylococcus aureus (MRSA) strain (major infectious agent) due to which its systemic administration11,12 and paste form13,14 is the commonly employed method for treating infections like mediastinitis. But it has been reported that frequent dosage of vancomycin systemically leads to renal failure and hearing loss12 and frequent dosage of vancomycin paste will increase the risk of developing vancomycin resistant S. aureus pathogen.15 Vancomycin paste has also been reported to not reduce the incidence of polymicrobial infections of Gram negative bacilli in DSWI as its activity is limited to Gram positive bacteria.13,16 Many methods have been developed to control bleeding and infection in mediasternitis condition however there are very limited treatments in clinics for



prevention of infection after cardiac surgery.17,18 This is due to the lack of a suitable delivery system that would adhere at the surgical site and release antibiotics locally in a sustained and effective manner. In situ forming gels can adhere and retain at the site of application and deliver antibiotics locally in a sustained manner.19,20 Tissue adhesive hydrogel system has been potentially used for hemostasis application. It has also been reported that nanoparticles incorporated into hydrogel systems posses the ability to bring polymer chains together and hence enhance its tissue adhesive property.21 Fibrin sealant is a FDA approved in situ forming material that is widely used in clinics as hemostatic agent/tissue adhesive.22,23 Main drawback of using fibrin gel as a drug delivery carrier is that it degrades fast from the site of application due to the action of plasmin in our body.24 Chitin has been in use for promoting hemostasis25,26 and it has also shown great potential as a drug delivery system.27,28 Incorporation of chitin into fibrin system would aid in the formation of an interconnected chitin-fibrin system that would form an adhesive gel and at the same time slower the rate of fibrin degradation and deliver drug in a sustained manner. Osteoblast cell adhesion property of chitin29 is also an added advantage of using it to form an in situ gel for application at the sternal edge as a treatment for mediastinitis. Tigecycline is a broad spectrum antibiotic (glycylcycline class of antibiotic) which effectively inhibits both Gram positive; Gram negative and even many other antibiotic resistant organisms.30 It hinders the protein synthesis of bacteria by binding to H34 region on 30S subunit of ribosome thereby blocking the entry of aminoacyl transfer RNA to its ribosomal binding site.31,32 Controlled and sustained release of drug for a long period is desirable for preventing mediasternitis.33 Nanoparticles have been in use as carrier system for drug delivery applications.34,35 Gelatin nanoparticles have been widely used for encapsulating


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many bioactive molecules.36 Based on this we developed an in situ forming tigecycline loaded gelatin nanoparticles incorporated chitin-fibrin gel system which can effectively control bleeding and prevent bacterial infections at surgical site.

EXPERIMENTAL SECTION Materials. α-Chitin in powder form (degree of acetylation>90% with average molecular weight of ~100kDa) was purchased from Koyo Chemical, Japan. Fibrinogen and Gelatin B were purchased from Himedia and Nitta Gelatin, India respectively. Thrombin (bovine plasma), potassium dihydrogen phosphate, calcium chloride, sodium chloride and methanol were purchased from Merck Chemicals, U.S.A. Isopropanol, disodium phosphate and triton-X 100 was purchased from Spectrochem Pvt .Ltd., India. Potassium

chloride

was

purchased

from

Sdfine-Chem

Ltd.,

India.

Tris(hydroxymethyl)aminomethane buffer (Tris), lysozyme, and sodium hydroxide were purchased from Qualigens fine chemicals, India. 25% glutaraldehyde solution (w/v) was purchased from Nice Chemicals (p) Ltd., India. Tigecycline was purchased from BDR Pharmaceuticals International Pvt. Ltd., India. Antibiotic-Antimycotic (AA), Iscove’s Modified Dulbecco’s Medium (IMDM), Alamar Blue Reagent, Fetal Bovine Serum (FBS), 0.25% trypsin-EDTA and Large Vessel Endothelial Supplement (LVES) were obtained from Invitrogen, India. 0.9% sodium chloride (saline) was purchased from Infutec Healthcare Ltd., India. 3.2% sodium citrate coated vacutainer was obtained from BD, USA. Bacterial growth mediums like Luria Bertani (LB) and Muller Hinton (MH) Broth; agar-agar were obtained from Himedia, India.



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Methods. Preparation

of

Tigecycline

Loaded

Gelatin

Nanoparticles

(tGNPs).

tGNPs were prepared by nanoprecipitation technique.37,38 Here, gelatin B (200 mg) was added to double distilled water (10 mL). Gelatin was completely dissolved by increasing the solution temperature to 40°C. 0.25 N sodium hydroxide was then added to bring the pH of the solution to 6. After complete dissolution of gelation, the temperature of the solution was brought back to room temperature (25°C) and 500 µL of 1% tigecycline was added under stirring condition. 15 mL of isopropanol was then added to generate gelatin nanoprecipiates. Dropwise addition of 50 µL of 25% glutaraldehyde solution (w/v) was used as a cross-linker to form gelatin nanoparticles. Similarly bare gelatin nanoparticles (GNPs) were prepared by the above mentioned procedure without the addition of tigecycline. Upon centrifugation of solution containing the prepared nanoparticles at 13,500 rpm for 15 min (HERMLE Z 326 K centrifuge using 220.78 rotor) nanoparticle pellet was obtained. The obtained nanoparticles pellet was washed thrice using double distilled water to remove impurities. Nanoparticles pellet was lyophilized and stored at room temperature for further use (Figure 1A).


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Figure 1. Schematic diagram illustrates the synthesis of in situ forming tGNPsCH-FB gel. Size Analysis and Characterization of prepared tGNPs. Hydrodynamic diameter, polydispersity index (PDI) and zeta potential (ZP) of tGNPs was evaluated using Dynamic light scattering (DLS-Malvern Zeta Sizer 3000, Nano series). Particle size of tGNPs was further confirmed using Scanning Electron Microscope (SEM; JEOL Analytical SEM JSM-6490LA). For SEM analysis, prepared nanoparticle pellet was diluted (700 times with double distilled water) and dropped on aluminium stub, after which it was air dried. These samples were then coated at 10 mA using gold sputter coater (JEOL JFC- 1600) for 90 s and imaged at an acceleration voltage of 15 kV. 7 ACS Paragon Plus Environment


Stability analysis. Approximately 5 mg of tGNPs was dispersed in 1 mL of phosphate buffer saline (PBS). These suspensions were maintained at 4°C, 25°C and 37°C. Stability study was carried out for a month during which particle size, PDI and zeta potential of tGNPs was noted at different time points using DLS. Encapsulation and Loading Efficiency. Two different methods were used to evaluate the encapsulation efficiency of the developed tGNPs. In the first method, the supernatant obtained after centrifugation of tGNPs suspension (13,500 rpm for 15 min in HERMLE Z 326 K centrifuge using 220.78 rotor) was collected and its absorbance (245 nm) was obtained using UV spectrophotometer (UV-1700, Shimadzu). The free drug present in the supernatant was calculated using the formula obtained from standard tigecycline absorbance curve at 245 nm. The encapsulation efficiency (EE) and loading efficiency (LE) were further determined using formulas as given below39:

% =

− × % =

× !" "

In the second method, the amount of drug encapsulated within the tGNPs was evaluated using lyophilized tGNPs after digesting it with trypsin.38 0.25% trypsin-EDTA (0.5 mL) was used to digest tGNPs (2 mg) in PBS (1.5 mL) and the digestion was allowed to happen for 5 hrs. Digested tGNPs suspension was then centrifuged (13,500 rpm for 15 min in HERMLE Z 326 K centrifuge using 220.78 rotor) and the absorbance (245nm) of the obtained solution was measured. Then the amount of tigecycline encapsulated within tGNPs was calculated. Preparation of in situ forming Chitin-Fibrin (CH-FB) Gel. α-chitin (100 mg) was dissolved in saturated calcium chloride-methanol solvent (10 mL) and regenerated by adding equal volume of methanol. Regenerated chitin gel was obtained upon 8 ACS Paragon Plus Environment

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centrifugation (9000 rpm for 15 min in HERMLE Z 326 K centrifuge using 220.78 rotor). Chitin gel so obtained was washed thrice by centrifugation (9000 rpm for 15 min in HERMLE Z 326 K centrifuge using 220.78 rotor) using double distilled water to remove excess methanol and calcium chloride.40 Fibrinogen solution was prepared by dissolving 50 mg of fibrinogen powder in 1 mL PBS and 0.1 M Tris buffer (pH 8.8)41 in the ratio (1:1). 20U thrombin was dissolved in 1 mL of 35 mM calcium chloride solution. In the prepared fibrinogen and thrombin solutions, 400 mg of chitin gel was dispersed and maintained at 37ºC for 10-15 min before injection. Fibrinogen solution containing chitin gel was taken in one syringe and in another syringe, thrombin solution containing chitin gel was taken. Both the solutions were taken in equal volume (1 mL). These solutions were injected simultaneously using a dual syringe applicator to form the in situ CH-FB gel (Figure 1B). Fibrinogen, thrombin and chitin concentrations were varied. We found that only when 50 mg fibrinogen in 1 mL of PBS:Tris buffer, 20U thrombin in 1 mL calcium chloride and 400 mg chitin gel were taken, uniform gel was formed. Other concentrations didn’t form uniform gel. Preparation of in situ tGNPs loaded Chitin-Fibrin (tGNPsCH-FB) gel. 20 mg tGNPs was dispersed separately in both fibrinogen and thrombin solutions containing chitin gel (400 mg). Equal volume (1 mL) of these solutions were then loaded into a dual syringe and injected simultaneously to form in situ tGNPsCH-FB gel (Figure 1C). tGNPsCH-FB gel represents 1g CH-FB gel with 40 mg tGNPs. Characterization of Developed Gel Systems. Gelation time of the developed gel was calculated by vial tilting method.42 200 µL of developed gel were directly injected into vials using dual syringe applicator. Vials were then tilled and the time at which there was no flow of solution upon inversion was noted as the gelation time of the developed gel. The surface morphology of the developed chitin, fibrin, CH-FB and tGNPsCH-FB



gels were examined using SEM. Gels were air dried, placed in aluminium stub; coated at 10 mA using gold sputter coater (JEOL JFC- 1600) for 90 s and imaged at an acceleration voltage of 15 kV. Fourier transformed infrared spectroscopy (FTIRShimadzu IRAffinity-1S) of the lyophilized samples (tigecycline, gelatin, tGNPs, chitin, fibrin and tGNPsCH-FB gel) was recorded in the frequency range of 4000-500 cm-1. Rheological Studies of Developed Gel Systems. Malvern Kinexus pro rheometer (Malvern Instruments, UK) was used for all rheological measurements with parallel plate geometry of 20 mm diameter and 0.5 mm gap between the upper and lower plates.43 To mimic the physiological temperature, all tests were carried out at 37°C using a peltier heating unit with an environmental enclosure. Viscoelastic Study. Amplitude sweep analysis, frequency sweep analysis and yield stress analysis was evaluated for the gel systems as per the previously established protocol.43 This was done to determine the nature, strength, and stability of developed gel systems. Modulus and yield stress of chitin, fibrin and CH-FB gels were compared with tGNPsCH-FB gel. Temperature Sweep Analysis. Elastic (G’) and viscous modulus (G’’); phase angle (δ) of in situ forming fibrin, CH-FB, tGNPsCH-FB gel were measured by varying temperature from 25°C to 40°C with a fixed frequency and shear rate. This study was carried out to understand the stability and transitional property of the in situ gel from room temperature to human physiological temperature.


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Tack Test (Inverted Probe Test). In tack test an inverted probe is brought in contact with the developed gels and the adhesive force was measured.44 This is the maximum force (negative tension force) required to separate two parallel plates having a defined volume of material (gel) between them. The contact time and gapping speed employed in the test were 90 s and 1 mm/s respectively.

In Vitro Tissue Adhesion Test. Lap Shear Test. The tissue adhesive nature of chitin, fibrin, CH-FB and tGNPsCH-FB gel systems were determined by lap shear test by tension loading according to ASTM standard protocol (ASTM F2255-05).45 This test was carried out using universal tensile machine (Tinius Olsen, H5KL) with load cell of 150 N. Briefly, two pieces of fresh porcine skin (5 cm × 2 cm) size and 3 mm width was taken and soaked in water. These soaked skin pieces were then taken and 200 mg of developed gel (chitin, fibrin, CH-FB and tGNPsCH-FB) was applied between the pieces. After 15 min of curing, the skin pieces were clamped to tensile machine and stretched at a rate of 1 mm/min. Maximum adhesion strength was noted as the point at which the two skin pieces detach. Burst Pressure Test. Burst pressure test was carried out as per ASTM standard procedure with slight modification (ASTM F 2392-04).46 Porcine small intestine of length 10 cm was used in this experiment. Pressure sensor instrument (pressure volume controller,GDS, UK) with a reservoir containing PBS is connected to one end of the porcine intestine and another end was fixed with artery clamp to ensure it is leak proof. In this experiment porcine intestine was used as positive control and gel applied on 3 mm hole created in the middle of the intestine was the sample tested. 200 mg of gel (chitin, fibrin, CH-FB and tGNPsCH-FB) was taken for this study. Samples were placed at 37°C for 15 min as curing time. Maximum pressure of 250 mmHg which is significantly greater than normal arterial pressure was applied to the connected porcine



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intestines. Upon gradual increase in pressure, the pressure at which there is leak from the intestine is noted as the maximum pressure that the applied gel system can withstand. Degradation Study. Degradation study of the developed gels was carried out to understand their degradation pattern as this influences the drug release from the gels. Briefly, 1g of fibrin, CH-FB, tGNPsCH-FB gels was injected into storage vial and the initial weight of the gels was noted. To carry out this enzymatic degradation study, PBS solution (5 mL containing 10,000U of lysozyme/mL) was then added to the storage vial and maintained at 37°C shaking incubator (50 rpm). For every 24 hrs, solution was completely removed and the wet weight of the gels was noted. After noting the wet weight of gels same volume of fresh solution was added to the storage vial. The percentage degradation of gel was calculated using the formula shown below:47 % =

In

Vitro

# !" " − $ !" " × # !" "

Cytocompatiblity

Study

of

the

Developed

Gel

Systems.

The

cytocompatibility of the developed chitin, fibrin, CH-FB, and tGNPsCH-FB gel systems were analysed against Human umbilical cord derived endothelial cells (HUVEC) by Alamar Blue assay.48 HUVEC from umbilical cord were isolated by following previously established protocol.49 HUVEC (passage 4, 50,000 cells/well) was seeded in a 24 well plate containing equal amounts (180 mg) of sterile chitin, fibrin, CH-FB and tGNPsCHFB. IMDM complete medium with LVES was used as culturing medium for HUVEC. Cells were then incubated in cell culture incubator at 37°C, 5% CO2 for 24 and 48 hrs. After each time point, 500 µL of 10 % Alamar Blue® reagent in basal IMDM media was added to each well after complete removal of culture media and gels. Well plate was again incubated in the cell culture incubator for 6 hrs after which the optical density (OD) value of samples were measured at 570 and 600 nm using a microplate 12 ACS Paragon Plus Environment

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spectrophotometer (Biotek Power Wave XS, USA).50 HUVEC alone in culture medium was used as a control. Tigecycline release from tGNPs and tGNPsCH-FB gel. 40 mg of tGNPs alone, 1 g tGNPsCH-FB gel system, and an equivalent amount of bare tigecycline in 1 g CH-FB gel were taken in centrifuge tubes containing PBS (8 mL) and was kept in a 37°C shaking incubator (50 rpm) for 21 days. Each day the tubes were centrifuged at 9000 rpm for 10 min (HERMLE Z 326 K centrifuge using 220.78 rotor) and the presence of tigecycline in the supernatant was quantified at 245 nm using UV spectrophotometer. Percentage release of tigecycline was calculated using the formula given below:39

% % = × ( &

/ In Vitro Antibacterial Study. Preparation of Bacterial Culture. All bacterial strains were cultured overnight in Mueller Hinton Broth (MHB) or Luria-Bertani (LB) broth at 37°C with 120 rpm shaking. E. coli strain DH5α, S. aureus strain SA113 (ATCC 35556), MRSA (ATCC 43300) and clinical strains of E. coli; MRSA were used to evaluate the antibacterial activity of the developed tGNPsCH-FB gel system. Luria-Bertani (LB agar) plates were used for disc diffusion assays. Broth Microdilution Method. Broth microdilution method51 was performed to determine MIC of tigecycline and tGNPs against S. aureus, E. coli and MRSA. 100 µL of serially diluted tigecycline (32 µg/mL to 0.015 µg/mL) and tGNPs (3 mg/mL to 1.46 µg/mL) were added separately to wells of a 96 well plate to which 1 µL of overnight bacterial culture was then added and incubated at 37°C. After 24 hrs of incubation, OD value of bacterial cultures were measured (at 578 nm) using micro plate spectrophotometer (Biotek Power Wave XS, USA).52



Agar Well Diffusion Method. This method53 was performed to evaluate the antibacterial activity of tGNPsCH-FB gel against S. aureus, E. coli, and MRSA.100 µL of overnight bacterial cultures adjusted to turbidity of 0.5 McFarland (1-2 ×108 CFU/mL) was spread on LB agar in a petri plate (90 mm diameter). Wells (7 mm diameter) was punched in the agar plate using agar puncher.54 100 µL suspension of bare drug (tigecycline-200 µg/mL), 200 mg of CH-FB gel and tGNPsCH-FB gel containing (2 mg, 4 mg, 8 mg of tGNPs) were added to the wells of the well plates and then incubated at 37°C for 24 hrs in upright position. After the incubation period, zone of inhibition formed by the samples was measured and photographed (Bio-Rad Gel Doc, USA).

In Vitro Antibacterial Activity of tGNPsCH-FB gel against Clinical Isolates (E .coli and MRSA). Antibacterial activity of developed tGNPsCH-FB gel was evaluated against ten different clinical isolates of E. coli and MRSA. 100 µL of overnight culture of clinical isolates were spread plated on LB agar medium containing petri plate. After which 7 mm diameter wells were created in the agar. 200 mg of CH-FB gel containing 8 mg tGNPs was added into the wells and the plates were incubated at 37°C for 24 hrs in upright position. After the incubation period, zone of inhibition formed by the samples was measured and photographed (Bio-Rad Gel Doc, USA).

In Vitro Blood Compatibility Study. Blood Clotting Study. Whole blood was collected from healthy human volunteers in sodium citrated vacutainer tubes. 500 µL of citrated blood was taken in vials maintained at 37ºC. To this 100 µL of 0.25 M calcium chloride was added to recalcified the blood. Blood was allowed to clot. This served as control. In the test samples; 180 mg of chitin, fibrin, CH-FB, and tGNPsCH-FB gels were taken in vials to which recalcified whole blood was added. Added blood was allowed to clot. Time at which there is no flow of blood upon inversion is noted as the blood clotting time.


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Hemolysis Assay.1 mL of anti-coagulated blood was diluted by addition of 9 mL saline. 100 µL of 0.1% Triton-X (positive control), 100 µL saline (negative control), 180 mg of gels (chitin, fibrin, CH-FB and tGNPsCH-FB) were taken in vials to which 500 µL of diluted blood was added and incubated at 37ºC for 1 hr. After an hour, samples were centrifuged (3500 rpm) for 10 min (HERMLE Z 326 K centrifuge using 220.78 rotor) to obtain plasma. 100 µL of the plasma (supernatant) was collected from each sample and absorbance (OD value) of the samples was measured using (at 540 nm, Biotek, power wave XS, US) a plate reader. Then hemolysis (%) was calculated using formula given below:55

* − * ) % = × ( * − * In Vivo Hemostasis Study. In vivo hemostasis studies were performed in rat model (Male, Sprague Dawley rats of weight 200-300 g, 7-9 weeks). The study protocols were approved by the Animal Ethical Committee of Amrita Institute of Medical Science, Kochi, India (Approval No: IAEC/2017/1/7). Animals inbred at animal house of Amrtia Institute of Medical Science, Kochi, India were obtained for the study. All animals were sacrificed at the end of the experiment. Hemostasis Study on Rat Femoral Artery Injury Model. For this experiment, animals were anesthetized (0.6 mL of ketamine and xylazine in the ratio 2:3) and a midline skin incision approximately 1-1.5 cm long was made adjacent to the femoral vessels. Femoral artery was then exposed and a back ground was placed beneath the femoral artery for its better visualization. Artery was then wiped with gauze to remove any fluid present. Femoral artery injury was creating with a 24-gauge needle56 (~0.3 mm depth) and 200 mg gel was immediately applied on the bleeding site. Time taken to achieve complete hemostasis was noted to evaluate the hemostatic effect. Pre weighted gauze pieces were used to collect blood from the injury site. The difference of pre and post 15 ACS Paragon Plus Environment


weight of the gauze pieces gives the mass of blood loss due to the injury. The groups taken for this study are sham control (n=4), fibrin gel (n=6), chitin gel (n=6), CH-FB gel (n=6) and tGNPsCH-FB gel (n=6). Hemostasis Study on Rat Liver Injury Model. Animals were anesthetized (0.6 mL of ketamine and xylazine in the ratio 2:3) and a middle line abdominal incision was made to expose liver. Liver was then wiped with gauze to remove any fluid present. Liver injury was created using a 5 mm biopsy punch and 200 mg gel was immediately applied on the bleeding site. Time taken to achieve complete hemostasis was noted to evaluate the hemostatic effect. Pre weighted gauze pieces were used to collect the blood from the injury site. The difference of pre and post weight of the gauze pieces gives the mass of blood loss due to the injury. The groups taken for this study are sham control (n=4), fibrin gel (n=6), chitin gel (n=6), CH-FB gel (n=6) and tGNPsCH-FB gel (n=6). Statistical Analysis. Results of experiments in this study were represented as average ± standard deviation, n>3. Statistical analysis was done using ANOVA with Tukey's multiple comparisons test. Probability level (P values) less than 0.05, 0.01, and 0.001 were represented as *, **, and *** in figures.

RESULTS AND DISCUSSION Preparation and Characterization of tGNPs. The tGNPs and bare GNPs were synthesised by nanoprecipitation37 method and were then characterized. Tigecycline is reported to undergo oxidation at pH greater than 7 and non-enzymatic epimerization at pH less than 5. So, pH 6 was chosen for particle preparation31. DLS results of bare GNPs showed an average particle size of 172 ± 30nm with zeta potential and PDI of 30.2 mV and 0.090. tGNPs showed an average particle size of 231 ± 20 nm with zeta potential and PDI of -28.16 mV and 0.093 respectively (Figure 2B & 2C). The increase


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in the particle size of the tGNPs compared to GNPs might be due to the entrapment of drug inside the gelatin nanoparticles. Higher zeta potential of tGNPs shows strong anion to anion electrostatic repulsive force between the nanoparticles, which would prevent its aggregation.56 PDI value less than 0.5 indicates the monodispersion of tGNPs in suspension. SEM image of the particles show a spherical morphology with average diameter of 220 ± 13 nm (Figure 2A) and the particle size of developed tGNPs are in correlation with its DLS data. Further, the nanoparticle stability was studied for 1 month at 4°C, 24°C and 37°C. Particle size and PDI of tGNPs at 4°C, 24°C and 37°C is 233 ± 2 nm, 229 ± 4 nm, 227 ± 3 nm and 0.128, 0.128, 0.125 respectively (Figure 2D).The zeta potential of tGNPs at 4°C, 24°C, and 37°C is -27.03 mV, -25.43 mV and -24.23 mV respectively (Figure 2E).These values were found to be statistically insignificant, although slight variation in these parameters might be due to the interaction of the particles with the ions in PBS solution.57 The yield of tGNPs was found to be 55 ± 5% and its EE and LE were found to be 70 ± 5% and 3.5 ± 0.2% respectively.



Figure 2:(A) SEM image of tGNPs, (B) DLS data of tGNPs, (C) zeta Potential of tGNPs, (D&E) stability of tGNPs at 4°C, 24°C and 37°C, (F) gelation time of fibrin, CH-FB and tGNPsCH-FB gel, (G) SEM images of chitin, fibrin, CH-FB, and tGNPsCH-FB gel and (H) FTIR spectra of tigecycline, gelatin, tGNPs, fibrin gel, chitin gel and tGNPsCH-FB gel. Preparation and Characterization of in situ CH-FB and tGNPsCH-FB Gels. Chitin gel was prepared by regeneration chemistry after addition of excess methanol into chitin dissolved in saturated calcium chloride-methanol solvent. Gel formation is mainly due to the regeneration of destructured α-crystalline structure of chitin from random coiled structure due to the addition of excess methanol and repeated washing with double 18 ACS Paragon Plus Environment

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distilled water.40 An in situ forming fibrin gel was obtained by injecting fibrinogen solution and thrombin solution simultaneously using a dual syringe applicator. Thrombin cleaves fibrinopeptide of fibrinogen to form fibrin network which then assembles to form fibrin gel.57 Similarly, in situ forming tGNPsCH-FB gel was prepared by taking a mixer of fibrinogen solution, chitin gel, and tGNPs in one syringe and a mixture of thrombin solution, chitin gel, and tGNPs in an another syringe, these mixtures in equal volume is taken and injected simultaneously using a dual syringe applicator. SEM images revealed the smooth morphology of chitin gel and fibrous morphology of the network formed by fibrin gel. Both smooth morphology of chitin and fibrous structure of fibrin network are observed in the SEM image of the developed CH-FB gel, which depicted the formation of interconnected fibrin network within chitin gel. Further, a homogenous distribution of tGNPs in the chitin-fibrin network was also observed in the SEM image of tGNPsCH-FB composite gel system (Figure 2G). The functional groups present in tigecycline, gelatin, tGNPs, fibrin gel, chitin gel and tGNPsCH-FB gel were analysed using FTIR. The characteristic FTIR peaks of chitin are 1650 and 1550 (amide I, II), 1374 and 1070 cm−1 (asymmetrical deformation and β-1,4 glycosidic bond).58 Fibrin characteristic peaks are 1650, 1550 and 1230 cm−1 for amide I, II and III.59 The characteristic peaks of chitin and fibrin were present in the developed CH-FB gel. Gelatin being a protein has characteristic peaks at 1640-1690, 1550 and 1240 cm−1 for its amide I, II and III.60 The peak at 1521 cm−1 corresponds to the most intense tigecycline peak61 (Figure 2H). The specific peaks of gelatin and tigecycline were found in synthesised tGNPs. Similarly all the characteristic peaks of individual components of the final system were present in tGNPsCH-FB gel. In order to act as a physiological barrier and stop bleeding rapidly, a quick stable network has to be formed by in situ forming gel system at the surgical site.62 So it is



important to know the gelation time of the developed in situ forming gels (fibrin, CH-FB, and tGNPsCH-FB). When fibrinogen and thrombin solutions were mixed in situ in a vial, fibrin network was formed and the gelation time was noted to be 40 ± 1 s. Similarly, CHFB and tGNPsCH-FB gels also showed a gelation time of 40 ± 2 s (Figure 2F). These results indicate that even after incorportion of tGNPs and chitin gel into fibrinogen and thrombin solutions the tGNPsCH-FB gel system forms a stable gel within minutes. Rheological Study. Amplitude sweep was carried out at different shear strain (%) to determine the LVER and gel strength. The values of G’, G”, phase angle, and yield stress analysis (complex shear strain and sigma prime) are tabulated in Table I. Storage modulus (G’) of fibrin and tGNPsCH-FB gel systems was found to be 272 ± 7.8 Pa and 5460 ± 102 Pa respectively (Figure 3A). This significant increase in the storage modulus of tGNPsCH-FB gel would be due to the addition of chitin gel to fibrin system. But incorporation of tGNPs in to CH-FB gel didn’t affect the G’ value of the final system. This increase in G’ of tGNPsCH-FB gel might also be attributed to the formation of dense network between fibrin and chitin gel systems. Frequency sweep analysis was studied to understand the structural relaxation of the developed (fibrin, chitin, CH-FB, and tGNPsCH-FB) gel systems over a period of time (0.1 to 10 f (Hz)). Their G’ values remained stable over the frequency range. These results indicated that all the gel systems showed very less structural relaxation once a complete network is formed.63 The phase angle analysis revealed that the developed gel systems had a phase angle below 10° indicating that it is solid dominant64, which stipulates that the developed gel systems won’t flow unless a force is applied. Further, the yield stress analysis provides the peak stress (stress required to break the gel network and induce flow) and strain value (brittleness or ductility property of material) of the gel systems. CH-FB and


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tGNPsCH-FB gels exhibited high yield strength compared to chitin and fibrin gels. This indicates that more stress has to be applied on CH-FB and tGNPsCH-FB gel systems to initiate the flow which means, once tGNPsCH-FB gel formed after injection it could stay at the surgical site. In case of yield strain, highest strain was observed for fibrin gel followed by CH-FB, tGNPsCH-FB and chitin gel systems. This result revealed the brittle property of chitin gel and ductile property of the other gel systems. Table I. Viscoelastic properties of developed gels.

Figure 3. Rheological characterization of gel. (A) Amplitude sweep analysis with the shear strain ranges from 0.1% to 100%, (B) temperature sweep analysis from 25°C to 21 ACS Paragon Plus Environment


40°C, (C) tack test indicating the adhesive force of developed gel systems and (D) photographic images showing the adhesive nature of fibrin gel and tGNPsCH-FB gel during tack test experiment. Further, temperature sweep analysis was evaluated to understand the transitional behaviour of in situ forming gel at different temperature points from 25°C to 37°C. Elastic and viscous modulus, phase angle, complex viscosity and complex modulus for fibrin, CH-FB, and tGNPsCH-FB gel systems at 25°C and 37°C are tabulated in Table II. It was noted that fibrin had less elastic modulus; less complex viscosity and greater phase angle at 25°C, which might be due to low cross-linking density of the fibrin network. As the temperature was increased to 37°C there was reduction in phase angle (28.89° to 5.64°), significant increase in strength (19.83 to 539 Pa) and viscosity (3.605 to 85.95 Pa s). This implied that a strong fibrin network was formed only at 37°C as a result of high cross-linking density. At 25°C, both CH-FB and tGNPsCH-FB gel systems compared to fibrin gel exhibited high elastic modulus, high complex viscosity and less phase angle (Figure 3B). This might be due to the incorporation of chitin gel into fibrin system that gave solid dominance property to tGNPsCH-FB gel system at 25°C itself. As temperature increases to 37°C, tGNPsCHFB gel attained its maximum strength which was also higher than the strength obtained by fibrin gel system. Table II. Properties of developed gel systems at 25 and 37°C.


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The adhesive strength of developed gels was studied using tack test. The tGNPsCH-FB gel had a peak normal force value of -19.95 N which was similar to the value obtained by fibrin gel (Figure 3C). This result clearly points out that addition of chitin and tGNPs into fibrin network did not affect the basic adhesive property of fibrin gel (Figure 3D). The gap between parallel plates is equivalent to peak force which is related to critical strain upto which the material can deform. Similarly, area under curve (AUC) obtained in this study is directly proportional to adhesive strength of the material. The tGNPsCH-FB gel reached a maximum gap of 0.199 mm and highest AUC of 45.93 mm2 while chitin gel reached a gap of 0.049 mm and AUC of 1.650 mm2, fibrin gel reached a gap of 0.164 mm and AUC of 26.87mm2; CH-FB gel reached a gap of 0.187 mm and AUC of 28.72 mm2. These results collectively imply that the developed tGNPsCH-FB gel system exhibited a strong adhesive property.



In Vitro Tissue Adhesion test. For effective control of bleeding from surgical site, gel applied at site should adhere to the surrounding tissue and should also withstand high blood pressure. So it is necessary to evaluate the adhesion and burst pressure strength of the developed gel systems. For this lap shear test and burst pressure analysis were carried out as per ASTM standards. In the lap shear test the gel systems were formed in situ between the surface of wet porcine skin pieces and the adhesion strength of these gels were evaluated (Figure 4A). Fibrin and tGNPsCH-FB gel showed adhesion strength of 14.4 ± 0.5 kPa and 12.45 ± 0.35 kPa respectively (Figure 4B). Fibrin gel has been in use in clinics as tissue sealant.65,66 This result illustrated that the adhesion strength of tGNPsCH-FB gel is comparable to fibrin gel. From this result it is clear that incorporation of tGNPs and chitin gel into fibrin system has not altered the basic adhesion strength of fibrin gel. Burst pressure test was carried out with porcine small intestine (Figure 4C). The pressure holding capacity of the porcine intestine without hole was found to be 225 mmHg and porcine intestine with 3 mm hole is 10.12 ± 8.4 mmHg. The burst pressure strength of fibrin and tGNPsCH-FB gels applied to porcine intestine with hole was evaluated to be 24.75 ± 8.4 mmHg and 170.25 ± 12.73 mmHg (Figure 4D) respectively. From this result it was evident that fibrin has less burst pressure strength compared to CH-FB and tGNPsCH-FB gels. This might be because of the formation of a thin film of fibrin gel at the site of application due to which the fibrin gel was not able to withstand high pressure. There are also reports that show the inefficacy of fibrin gel to form a firm clot at high pressure bleeding conditions.46 CH-FB and tGNPsCH-FB gel showed burst pressure strength even higher than normal arterial blood pressure (120 mmHg). This might be mainly due to the addition of chitin gel into fibrinogen and thrombin solutions,


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there by the spreading out of these solutions at the punctured site was overcome and a firm in situ gel was formed at the site of application.

Figure 4. (A) Experimental steps of lap shear test using porcine skin,(B) graphical representation of adhesion strength obtained for chitin, fibrin, CH-FB and tGNPsCH-FB gel systems (**p

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