Chitosan

Aug 10, 2016 - healing. In the present work tigecycline nanoparticles were loaded into chitosan−platelet-rich plasma (PRP) hydrogel. The tigecycline...
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Anti-staphylococcal Activity of Injectable Nano Tigecycline/ChitosanPRP Composite Hydrogel Using Drosophila melanogaster Model for Infectious Wounds T. R. Nimal,† Gaurav Baranwal,† M. C. Bavya, Raja Biswas,* and R. Jayakumar* Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Amrita University, Kochi 682041, India S Supporting Information *

ABSTRACT: Compared to the current treatment modalities, the use of an injectable hydrogel system, loaded with antibiotic encapsulated nanoparticles for the purpose of treating Staphylococcus aureus (S. aureus) chronic wound infections have several advantages. These include adhesiveness to infection site, reduced frequency of dressings, sustained drug release, inhibition of bacterial growth, and increased healing. In the present work tigecycline nanoparticles were loaded into chitosan−platelet-rich plasma (PRP) hydrogel. The tigecycline nanoparticles (95 ± 13 nm) were synthesized through ionic cross-linking method using chitosan, tripolyphosphate, and tigecycline and characterized by dynamic light scattering (DLS), scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR). The synthesized nanoparticles and activated PRP powder were mixed with chitosan hydrogel to form a homogeneous gel. Rheology studies have confirmed the shear thinning property, thermal stability, and injectability of the prepared gel systems. The gel system was further assessed for its drug release property and found that it was released in a sustained manner. Hemolysis and blood-clotting assays demonstrated that the gel system was neither a hemolysin nor a hamper to the clotting cascade. Cell viability results showed that these nanoparticles were cyto-compatible. The bioactivity of PRP loaded chitosan gel toward fibroblast cell line was studied using cell proliferation and migration assay. In vitro antibacterial studies revealed that the gel system inhibited bacterial growth to a great extent. The antibacterial activity was further analyzed using ex vivo porcine skin assay. In vivo anti-Staphylococcal activity of the prepared hydrogels was studied using a Drosophila melanogaster infection model. The tigecycline and tigecycline nanoparticle incorporated chitosan gel showed a significant antibacterial activity against S. aureus. Thus, the gel system is an effective medium for antibiotic delivery and can be applied on the infection sites to effectively forestall various skin infections caused by S. aureus. KEYWORDS: injectable hydrogel, antibacterial, infection, Staphylococcus aureus, chitosan, platelet-rich plasma, tigecycline, Drosophila melanogaster nectin, and thrombospondin-1.6,7 These growth factors are important in modulating mesenchymal cell recruitment, proliferation, and extracellular matrix synthesis that occurs during the healing process.8 Additionally, PRP also suppresses cytokine release and limits inflammation thereby improving tissue healing and regeneration, promoting new capillary growth and acceleration of epithelialization in chronic wounds.9,10 Bacterial infections are considered to be a major local factor that influences wound healing. Bacterial toxins can stimulate inflammatory cells to secrete pro-inflammatory cytokines such as interleukin-1 and tumor necrosis factors-α at the wound site. As a result of this inflammatory process, wound healing gets

1. INTRODUCTION Chronic wounds that are associated with ischemia, diabetes mellitus, and venous stasis disease are often difficult to heal because they lack the necessary growth factors required for the healing process and are frequently infected by bacteria.1,2 Conventional therapies such as dressings, surgical debridement, skin grafting, and systemic antibiotics do not always provide satisfactory healing of chronic wounds.3 Such chronic infectious wounds often require simultaneous treatments with tissue growth factors and antimicrobial agents. Recent research reports have shown that platelet-rich plasma (PRP) treatment can enhance the rate of wound healing.4,5 PRP acts as a natural reservoir for many growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), osteocalcin, osteonectin, fibrinogen, vitronectin, fibro© XXXX American Chemical Society

Received: June 20, 2016 Accepted: August 10, 2016

A

DOI: 10.1021/acsami.6b07463 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic representation of tg-ChNPs-ChPRP gel preparation.

delayed and the wound may become chronic.11 Microbiological investigations have shown that Staphylococcus aureus (S. aureus) plays a major role in delaying the healing of nearly 25−30% of chronic wounds.12 Many chronic ulcers caused by S. aureus do not heal because of the inherent ability of this organism to form biofilms at the wound surface. S. aureus also expresses several virulence factors (e.g., proteases, nucleases, lipases, collagenase, and hyaluronidase), surface proteins (e.g., protein A, fibronectin and collagen-binding proteins, and clumping factors), and toxins (e.g., toxic shock syndrome toxins and enterotoxins) which promote its adhesion to the wound tissue and decreases the polymorphonuclear neutrophils (PMN) function and immune response of the host.13,14 Considering the above criteria, a successful wound healing formulation should contain components for tissue regeneration and antimicrobial agents to prevent bacterial growth. In order to achieve this, we aimed at developing a chitosan hydrogel formulation containing PRP and antibiotic tigecycline. Such an injectable hydrogel will be an ideal solution for chronic wound healing applications. Chitosan gel has good biodegradability and high adsorption capacity and is biocompatible and nontoxic.15 Moreover, chitosan degradation product Nacetylglucosamine mimics the extracellular matrix composition and therefore enhances tissue regeneration.16 Tigecycline is a glycylcycline antibiotic that acts via inhibition of protein synthesis by binding to the 30S ribosomal subunit, thereby blocking aminoacyl tRNA synthesis.17 The presence of bacteria at the wound site can lead to a prolonged inflammation phase.18 A sustained release of antibiotics from hydrogel is important to reduce this inflammation phase. Addition of PRP into the hydrogel system can enhance fibroblast proliferation and migration and promote a granulation phase.9,10 The overall objective of this research work is to develop a novel injectable hydrogel system for infectious wound treatment which can reduce the inflammatory phase (by inhibiting the bacterial growth using tigecycline) and enhance the granulation phase (by the addition of PRP) and simultaneously promote efficient wound healing.

2. EXPERIMENTAL SECTION 2.1. Materials. The polymer chitosan (MW, 100−150 kDa; degree of deacetylation, 85%) was acquired from Koyo Chemical Industry Ltd., Itami, Japan. Tripolyphosphate (TPP) was purchased from Sigma-Aldrich. Platelet-rich plasma (PRP) was obtained from the blood bank of Amrita Institute of Medical Sciences and Research Centre, Kochi, Bengaluru, India. Tigecycline was purchased from Glenmark Pharmaceuticals Ltd. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotic−antimycotic solution, Alamar blue, and Trypsin-EDTA were obtained from Gibco, India. Luria−Bertani (LB) Broth and agar−agar were purchased from HiMedia, Mumbai, India. All chemicals were used without further alteration and purification. 2.2. Methods. 2.2.1. Preparation of Tigecycline Loaded Chitosan Nanoparticles (tg-ChNPs). tg-ChNPs were prepared by the following method. Briefly, 1 mL of 0.5% (w/v) TPP was added dropwise to 10 mL of chitosan solution (0.15% (w/v)) containing 1 mg of tigecycline (tg) at a stirring speed of 1200 rpm.19 Turbidity formation after stirring confirmed the presence of NPs. The NPs suspension was allowed to stand for 30 min to detect any precipitations. The NPs were harvested by centrifugation at 14,000 rpm for 15 min (Beckman Coulter Avanti J-26XP centrifuge using JA-17 rotor). The pellet obtained was resuspended in double distilled water and used for further studies. 2.2.2. Nanoparticle Size Analysis and Characterization. The hydrodynamic diameter, polydispersity index (PDI), and surface ζ potential (ZP) of the nanoparticles was determined using DLS (Malvern Zeta Sizer 3000, Nano series). The particle sizes of the dehydrated nanoparticles were analyzed using scanning electron microscope (SEM; JEOL JSM-6490LA Analytical SEM). For this analysis, tg-ChNPs solution (1 mg/mL) was diluted 50 times using double distilled water and dropped on an aluminum stub. The airdried stub was placed in an automatic fine gold coater (JEOL JFC1600) at 10 mA for 90 s. The nanoparticles were observed at 15 kV acceleration voltages and imaged. 2.2.3. Encapsulation Efficiency and Loading Efficiency. The reaction mixture after nanoparticle synthesis was centrifuged at 14,000 rpm for 15 min (Beckman Coulter Avanti J-26XP centrifuge using JA17 rotor), and the supernatant was collected. The absorbance of the supernatant at 245 nm was measured using a UV spectrophotometer (UV-1700 Pharma spec, Shimadzu). The concentration corresponding to the obtained absorbance was determined using the standard absorbance curve of tigecycline at 245 nm. Based on the amount of B

DOI: 10.1021/acsami.6b07463 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

2.2.7.5. Inversion Test. The flow of gels under the influence of gravity was observed using inversion test. The inversion tests confirm the property of the gels to stay on the infection site without falling. This test was performed by inserting an equal amount of gels in a flat bottomed cylindrical tube and incubating in an inverted position without any disturbance. The flow of gels was observed visually at 0, 1, 2, and 4 days time intervals. 2.2.8. In Vitro Tigecycline Release Study. The in vitro quantification of tigecycline released from tg-ChNPs (1 μg/mg tg), ChPRP gel (10 μg/mg tg-ChNPs and 5 mg/g PRP), and 1 μg/mg tg containing ChPRP gel (tg-ChPRP gel) was studied in 100 mM Tris buffer (pH 8.0) which mimics infectious wound pH. Briefly, 20 mg of tg-ChNPs and 5 g of gel systems were added to seven tubes containing 10 mL of Tris buffer (pH 8.0) and incubated in a 37 °C shaking incubator for 7 days. Each day the supernatants from one tube were collected after separating the gel and nanoparticles by centrifugation at 14,000 rpm for 15 min (Beckman Coulter Avanti J-26XP centrifuge using JA-17 rotor). The absorbance of the supernatant at 245 nm was measured using a UV spectrophotometer and converted into concentration using the standard curve.20,25 The percentage of tigecycline release was calculated using the following formula:

tigecycline present in the supernatant, encapsulation efficiency (EE) and loading efficiency (LE) were determined using the following formulas.20,21

EE/% =

Wi − Ws × 100 Wi

LE/% =

Wi − Ws × 100 Y

where Wi = total amount of tigecycline added for tg-ChNPs preparation, Ws = amount of tigecycline in the supernatant, and Y = total dry weight of tg-ChNPs prepared. 2.2.4. Preparation of Chitosan-PRP Gel. The chitosan hydrogel was prepared aseptically by previously reported method.22 A 2 g amount of chitosan powder was dissolved in 100 mL of 1% (v/v) acetic acid by stirring overnight. The obtained viscous chitosan solution was neutralized to pH 7.4 by dropwise addition of 1% (w/v) sodium hydroxide (NaOH). The hydrogel formed after neutralization was harvested by centrifugation at 10,000 rpm for 10 min (Beckman Coulter Avanti J-26XP centrifuge using JA-17 rotor). The chitosan gel thus obtained was washed thrice with phosphate-buffered saline (PBS) to remove the traces of acetic acid and stored at 4 °C. The human PRP obtained from a blood bank was activated by the addition of 10% (w/v) calcium chloride (CaCl2). The activated PRP gel was freeze-dried (Labconco freezone 2.5 plus) and crushed into fine powder for further use. The chitosan−PRP (hereafter mentioned as ChPRP) gel was prepared by thorough mixing of the chitosan gel with powdered PRP in different ratios (0−5 mg/g) for ensuring the formation of homogeneous gel. 2.2.5. Incorporation of tg-ChNPs into ChPRP Gel. The prepared tg-ChNPs were resuspended in double distilled water at a concentration of 2 mg/mL. The nanosuspesions were added dropwise during the mixing stage of chitosan−PRP (ChPRP) gel preparation. The obtained tg-ChNPs containing ChPRP gel (hereafter mentioned as tg-ChNPs-ChPRPgel) was stored at −20 °C for further use. The preparation of tg-ChNPs-ChPRP gel is shown in Figure 1. 2.2.6. Chemical Characterizations of Gel Using FT-IR. Infrared spectra of chitosan, PRP, and tigecycline and tg-ChNPs-ChPRP gel (10 μg/mg tg-ChNPs and 5 mg/g PRP) were determined between 4000 and 400 cm−1 using FT-IR (Shimadzu IRAffinity-1S). The obtained peak values were correlated using graphical representation and compared with the individual compounds. 2.2.7. Rheological Studies of the Composite Gel. 2.2.7.1. Viscoelastic Study. Rheological studies of Ch gel, ChPRP (5 mg/g PRP), and tg-ChNPs-ChPRP gel (10 μg/mg tg-ChNPs and 5 mg/g PRP) were performed using Malvern Kinexus pro rheometer with a stainless steel cone plate with 20 mm diameter and 4° cone angle. A constant gap of 0.5 mm was maintained between the upper and lower plates at 25 °C. An amplitude sweep was carried out to find the linear viscoelastic region (LVER). The elastic modulus (G′), viscous modulus (G″), and phase angle (δ) at LVER were measured at different strain percentages. The amplitude sweep was started from a strain percentage of 10−1, and the end point was determined automatically by the rheometer. The gel strength and solid/liquid dominating behavior of the gels were determined by studying the frequency sweep between 101 and 10−1 Hz in the LVER region of the gels.23,24 2.2.7.2. Temperature Stability Tests. The complex modulus and complex viscosity of the gels were measured from 25 to 38 °C at constant frequency and shear. This study was done to test the stability of the gels from room temperature to human body temperature. 2.2.7.3. Flow Curve Analysis. The flow behavior of the gels was studied using flow curve analysis. The viscosity of the gels at 25 °C was plotted against different shear rates (10−1 to 10−2 s−1) to obtain the flow curve. 2.2.7.4. Injectability. The flow of gels from a 1 mL syringe with and without a needle was observed visually where the syringe with a 21 gauge needle creates a high shear condition, while the syringe without a needle makes a low shear condition.

release/% = amount of tigecycline released at definite time × 100 total amount of tigecycline entrapped within the nanoparticle/gel

2.2.9. Cell Migration Study−Scratch Assay. L929 cells were seeded in 24-well plates (104 cells/well) and incubated overnight in serum containing culture medium to create a confluent monolayer. The confluent layer was wounded with a 200 μL pipet tip to make a straight scratch. The cell debris around the scratch was removed by washing three times with PBS. The scratched monolayer was supplemented with serum free medium. The tg-ChNPs-ChPRP gels (10 μg/mg tg-ChNPs) with different concentrations of PRP (0.5, 1.5, and 5 mg/g) were added to a corner of the wells and incubated in culture conditions. The scratch was monitored by taking the images at 0, 24, and 48 h using a phase contrast microscope (Nikon ECLIPSE TE2000-U, Gotemba, Japan).26 The areas of wounds at different time points were measured using ImageJ software, and the wound closure rate was calculated using the following equation.

scratch wound closure/% =

SA 0 − SA t × 100 SA 0

where SA0= scratch area at time = 0 and SAt= scratch area at time = t. 2.2.10. Bacterial Growth on Gel Surface and SEM Imaging. A 100 μL aliquot of PBS containing 106 CFU of S. aureus was added on 1 g of aseptically prepared ChPRP (5 mg/g PRP), tg-ChPRP (1 μg/mg tg and 5 mg/g PRP), and tg-ChNPs-ChPRP (10 μg/mg tg-ChNPs and 5 mg/g PRP) gels and incubated for 1 h. Later, 500 μL of LB broth was added to the gels and incubated at 37 °C. After 12 h of incubation, samples were fixed by immersing in 2% (v/v) glutaraldehyde for 30 min. The samples were further dehydrated with graded ethanol series, starting with 25, 50, 75, 90, and 100% ethanol for 15 min each prior to sputter coating and imaging using SEM.27,28 2.2.11. Long-Term in Vitro Antibacterial Activity. The antibacterial activity of the releasate from tg-ChPRP and tg-ChNPs-ChPRP in 14 days was studied. A 500 μg amount of tigecycline and 5 mg of PRP were incorporated in 1 g of tg-ChPRP and tg-ChNPs-ChPRP gels. The gels were added to 3 mL of LB broth and incubated at 37 °C in a shaking incubator. After 24 h of incubation, the tubes were centrifuged and the releasate was harvested. The releasate was replaced with 3 mL of fresh LB broth and incubated further. This replacement of releasate with fresh LB broth was repeated every day until day 14. A 3 × 106 CFU amount of S. aureus was inoculated into the 3 mL of releasate and incubated further to study the influence of releasate on microbial growth. After 24 h incubation the turbidity of the LB broth was measured at 600 nm using a micotiter plate reader. 2.2.12. In Vivo Antibacterial Activity. 2.2.12.1. Drosophila melanogaster Strains and Maintenance. Stocks of wild-type Drosophila melanogaster (D. melanogaster; Flybase Stock 2: C

DOI: 10.1021/acsami.6b07463 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces FBst0000002) were raised on classical banana fly culture medium at room temperature (25 °C) with 55−60% humidity.29 Banana fly culture medium was prepared by boiling 5 g of agar in 250 mL of water for 10 min; to which 250 g wet weight of banana paste (prepared from fresh ripe bananas using a mixer) was added; the volume was adjusted to 500 mL with water (if required). The complete medium was mixed well using a spatula and boiled further for 5 min, before pouring into the glass tubes. A few granules of yeast extract were added to the top of the medium before transferring the flies into new vials. 2.2.12.2. D. melanogaster Infection Model. Female flies 2−5 days old (10 flies per batch in triplicates; n = 30) were selected for infection. Flies were anaesthetized on ice and infected via pricking in the dorsal thorax with a 25 gauge needle dipped halfway into a culture of SApCgf p (optical density (OD) at 600 nm = 2.0).30−32 Flies were returned to standard fly culture vials and fed on different hydrogel− NPs systems, and survivability was observed for the next 24 h. Following infection the number of surviving flies was recorded at 0, 3, 6, 12, and 24 h intervals. Results are expressed as the percentage of flies alive at different time points after infection. 2.2.12.3. Growth of S. aureus in Vivo. Flies were infected in batches of 10 with S. aureus strain. At 16 h after infection, flies were crushed in 0.5 mL of PBS, using a mortar and pestle, and the homogenate was serially diluted in PBS. The number of colonyforming units per fly was determined through overnight growth on LB agar.31,32 2.2.12.4. S. aureus Infections and Fluorescent Imaging of the Flies. To assess the growth and distribution of the bacteria, we have infected the flies with S. aureus strain SApCgf p.27 The flies infected with SApCgf p were observed and imaged under fluorescent stereomicroscope (Leica DFC 295) at different time points starting from 0 to 24 h postinfection. For comparison of the fluorescent intensity, flies with no infection were used as controls. 2.2.13. Statistical Analysis. All the experiments were performed in triplicate, and the results were represented as average ± standard deviation. The significance between samples was analyzed using Student’s t-test. The probability level less than 0.05 were considered significant. p values less than 0.05, 0.01, and 0.001 were represented as *, **, and *** in figures.

Figure 2. (A) SEM images of tg-ChNPs, tg-ChPRP gel, and tgChNPs-ChPRP gel; (B) FT-IR spectra of tg-ChNPs-ChPRP gel, tigecycline, PRP, and chitosan.

Tigecycline is a broad-spectrum antibiotic which can effectively treat bacterial infections in soft tissues and wounds.34,35 But to date, no tigecycline based topical treatments such as gels and bandages are available for infectious wounds.36 PRP has been used in wound care since the past few decades.37,38 Lawlor et al. studied the role of PRP in postoperative wounds of vascular surgery patients.39 Researchers have concluded that the incorporation of PRP did not control wound complications such as infections.39 A topical delivery system with PRP alone cannot help in the wound healing because it is incapable of controlling the microbial growth on the wound. A combination of tigecycline and a PRP loaded topical system could be a novel effective treatment modality for infectious wounds such as venous and arterial ulcers, diabetic ulcers, and pressure ulcers. 3.3. Chemical Characterizations of Gel-FT-IR. The functional groups present in each sample were studied using FT-IR and represented in Figure 2B. The interactions of chitosan with PRP and tg-ChNPs caused a slight shift in chitosan peaks. The characteristic peaks of C−O−C and CO stretching of pure chitosan shifted from 1062 and 1649 cm−1 to 1078 and 1639 cm−1, respectively.29 The tg-ChNPs-ChPRP gel also showed characteristic peaks of amide A and amide III peaks of PRP at 3270 and 1400 cm−1, respectively.30 The presence of tigecycline in the final gel system was confirmed by the peak at 1541 cm−1.31 These results confirmed the integration of tigecycline and PRP in the prepared tg-ChNPsChPRP gel. 3.4. Rheological Studies of the Composite Gel. 3.4.1. Viscoelastic Study. The solid or liquid dominating behavior of the material was confirmed by measuring the elastic (storage) modulus (G′), viscous (loss) modulus (G″), and phase angle (δ) between applied and measured sinusoidal signals (Figure 3A(a)). The frequency sweep of the gels showed that δ is not changing with the frequency like viscoelastic solids and viscoelastic liquids. The independency of δ toward frequency confirmed that the test material is a gel. It was found that G′ of all of the gel systems were greater than G″, which indicated that the gels have a more elastic component. The solid-like behavior of the test material was

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of tg-ChNPs. The nanoparticles were prepared using ionic cross-linking method. The presence of NPs in the opalescent turbid solution formed after cross-linking was confirmed by DLS and SEM. The prepared tg-ChNPs were analyzed for their hydrodynamic diameter, ZP, and PDI by DLS; the particle size of tigecycline loaded was found to be 95 ± 13 nm. The PDI of tg-ChNPs was 0.18 ± 0.07. PDI < 0.3 indicates that the prepared NPs were monodispersed in the suspension. The entrapment of tg into the nanoparticle did not alter the ZP significantly, and it was found to be +37 ± 15 mV. The magnitude of ZP can be used to study the stability of the nanosuspensions, since nanosuspensions are considered to be stable if the magnitude of ZP is higher than 30 mV.33 The prepared tg-ChNPs was stable in water. The SEM imaging of tg-ChNPs confirmed that the particles are spherical in shape with an average diameter of 97 ± 18 nm (Figure 2A). The EE and LE of the tg-ChNPs was found to be about 25 ± 3% and 10 ± 2%, respectively. 3.2. Preparation of ChPRP and tg-ChNPs-ChPRP Gel. The chitosan gel formed after the addition of NaOH was mixed with PRP and tg-ChNPs to get a homogeneous gel. The mixing speed and water content of chitosan gel was optimized to minimize the stirring time. Excess stirring of tg-ChNPs-ChPRP gel may cause a rapid tigecycline release from the tg-ChNPs entrapped in the gel. The SEM images showed that tg-ChPRP gel and tg-ChNPs-ChPRP gel have a smooth surface morphology (Figure 2A). D

DOI: 10.1021/acsami.6b07463 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 3. (A) Rheological characterizations of gels: frequency Sweep (a), viscosity (η*) vs temperature (b), and flow curves (c). (B) Injectability of tg-ChNPs-ChPRP gels through a syringe without needle (a) and with needle (b). (C) Inversion test depicting gel stability of Ch gel (a, d), ChPRP gel (b, e), and tg-ChNPs-ChPRP gel (c, f) under the influence of gravity after 0 h (a, b, c) and 100 h (d, e, f).

determined by measuring the δ. If the δ = 0° stress and strain are exactly in phase, then the material is purely elastic, and if the δ = 90° stress and strain are one-quarter of a cycle out of phase, then the material is purely viscous. The prepared hydrogel showed a δ of around 8°, which confirms that the gel is solidlike and will not flow unless otherwise an external force is applied.40 The addition of PRP and tg-ChNPs did not alter viscoelastic properties of the chitosan gel. 3.4.2. Temperature Stability Tests. The complex modulus (G*) was monitored as a function of temperature to determine the thermal stability in the range of 25−45 °C (Figure 3A(b)). The developed gel systems showed a similar cycling behavior, and less than 5% change at each point across the data set confirmed that the microstructure of the gel system has not changed in the test temperature range. Thus, the gel systems can be stored and transported without any deterioration. In addition, the viscoelastic properties of the gels are unaffected by human body temperature (37 °C). 3.4.3. Flow Curve Analysis and Injectablilty. The viscosity of developed gels was measured from 0.01 to 100 s−1 of shear rate. The correlation between viscosity and shear rate was graphically represented in a flow curve (Figure 3A(c)). The obtained flow curve pattern was used to predict the flow behavior of the gel.40 The viscosity of all the prepared gels decreased with increase in shear rate. The inverse proportionality of viscosity and shear rate confirmed the shear thinning property of the prepared gels.41 The predicted flow behavior was tested by observing the gel flow from a syringe under shear stress (Figure 3B). The smooth and continuous flow of the prepared gels from the syringe confirmed the injectablility. The flow behavior studies confirmed that the developed hydrogels can be injected on the infection site and can stay localized at the region of injection. 3.4.4. Inversion Test. The inversion test confirmed the flow behavior of gel under gravitational force. The result in Figure 3C shows that all of the developed gel system did not displace even after 100 h. The gravitational force acting on the gel

during inversion was negated by the shear stress of the gel, and it remained in its position.33 The results of the inversion test confirmed that the applied gel on the wound can remain on the defect for a long duration without spreading to neighboring tissue or falling under gravity, unless a shear was applied. 3.5. In Vitro Tigecycline Release Study. The in vitro tigecycline release from tg-ChNPs showed a burst release of 37 ± 2.3% after 24 h, with almost 58% release of the tigecycline within a week. The nanoparticle system exhibited a tigecycline release of 2.75 ± 0.56% per day after the second day of the release study. The release profile demonstrated a higher tigecycline release over 48 h (47−52%) followed by a release at a decreasing rate. The tigecycline release from tg-ChNPs-ChPRP gel was compared with ChPRP gel containing a free form of tigecycline (tg-ChPRP gel; Figure 4A). The concentrations of tigecycline in both gel systems were kept constant for comparison. The tigecycline release from tg-ChPRP gel showed a burst release of 50−55% after 1 day. The ChPRP gel containing tigecycline in

Figure 4. (A) In vitro tigecycline release profiles of from tg-ChNPs, tgChPRP, and tg-ChNPs-ChPRP gel and (B) absorbances of microbial flora in the presence of releasate from tg-ChPRP and tg-ChNPsChPRP gels up to 14 days. E

DOI: 10.1021/acsami.6b07463 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

PRP loaded gels showed dose dependent increase in proliferation of L929 cells. As shown in Figure S3D, the tgChNPs-Ch gel did not show any significant increase in cell population. These results confirmed that addition of activated PRP into the chitosan gel matrix stimulated cell division. There are more than 30 bioactive proteins including growth factors present in the PRP that are known to regulate cell proliferation, attachment, differentiation, migration and collagen gene expression, and extracellular matrix synthesis by binding to specific cell receptors.8 The growth factors such as fibroblastic growth factor (FGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) are strongly responsive to fibroblast cells.7,8 Our in vitro results demonstrated that tgChNPs-ChPRP gels can induce the proliferation of the fibroblast cells at the wound site and can fasten the proliferative phase of wound healing. 3.9. Cell Migration Study. The influence of the PRP loaded gel system on fibroblast migration was studied using scratch assay (Figure 5A). The photographs of the scratch and

encapsulated form (tg-ChNPs-ChPRP gel) showed a significantly lower burst release of 12 ± 1.2% and yielded more controlled release of tigecycline over a week. Compared to tgChNPs and tg-ChNPs-ChPRP gel, the gel system exhibited more controlled and sustained release. The release of tg from tg-ChPRP gel by diffusion resulted in a faster release whereas the release of tg from tg-ChNPs-ChPRP gel was delayed because the release happens only after the swelling of chitosan nanoparticle, followed by diffusion. The slow and sustained delivery of a novel broad-spectrum antibiotic such as tigecycline on the wound bed can improve the therapeutic index by eliminating latent bacterial infections and preventing further invasion of bacteria efficiently.12 The tg-ChNPs-ChPRP gel can be used as an extended release delivery system to reduce the induction of antibiotic resistance.42 Additionally, it can improve patient compliance and reduce the frequency of dressing.43 The tigecycline release data were fitted with five kinetic models, and the R2 and n values are shown in Supporting Information Table S2. The R2 values of zero order and first order kinetics were compared, and it was found that all three systems showed a higher R2 for first order kinetics. The first order kinetics describe that the tigecycline release was proportional to the concentration of tigecycline present in the carrier. The Korsmeyer−Peppas model showed better linearity than the Higuchi model for all systems. This indicated that the drug release is based on diffusion. The slopes of the Korsmeyer−Peppas model for the three samples were less than 0.45, which confirmed the Fickian diffusion of drug from the delivery systems.44 3.6. Whole Blood Clotting Study. The hemostatic potential of the prepared tg-ChNPs-ChPRP hydrogel systems were evaluated by calculating blood-clotting indexes (BCI; Figure S3A). All of the gel systems showed a significant decrease in BCI compared to control. The lower BCI represents faster blood clotting. The protonated amino groups of the chitosan polymer chains in the gel systems can accelerate the hemagglutination by attracting the negatively charged plasma membrane proteins of RBC.45 The enhanced recruitment of RBC will help to make a stable clot that stops the bleeding in the wound site. 3.7. Hemolysis Assay. The hemocompatibility of the tgChNPs-ChPRP gels was assessed by hemolysis assay. The supernatant of a Triton X-100 treated sample showed a dark red color, and the remaining samples were translucent. The presence of hemoglobin in the supernatant was quantified and plotted as shown in Figure S3B. All of the test samples showed a hemolysis of less than 5%. The low percent hemolysis indicates that the tg-ChNPs-ChPRP gels are not affecting the integrity of erythrocyte membrane and were not causing any hemoglobin release. 20,46,47 These results confirmed the hemocompatibility of the prepared hydrogels. 3.8. Cell Viability and Proliferation Studies. The cytocompatibility of the prepared tg-ChNPs-ChPRP gels were evaluated using Alamar blue assay.20,46 The viability of L929 cell lines treated with tg-ChNPs-ChPRP gels was measured and plotted (Figure S3C). The hydrogel system did not show any significant cytotoxicity even in the presence of 50 and 250 mg of tg-ChNPs-ChPRP gel. The viability study indicated that the prepared nanocomposite hydrogel does not affect the proliferation of fibroblast cells which plays a major role in wound healing. The tg-ChNPs-Ch gel and tg-ChNPs-ChPRP gel were compared for cell proliferation assay, and it was observed that

Figure 5. (A) Schematic diagram representing the in vitro wound closure protocol. (B) L929 cell migration in the presence of tgChNPs-ChPRP gel with different concentrations of PRP. Photographs of cell migration at different time points. (C) Graphical representation of % scratch wound closure.

the percentage of wound closure at different time points are shown in Figure 5B,C. The results show that the tg-ChNPs-Ch gel did not show any significant scratch wound closure compared to nontreated cells. It was observed that PRP containing tg-ChNPs-Ch gel (tg-ChNPs-ChPRP gel) showed more effectiveness in the migration of fibroblasts compared to tg-ChNPs-Ch gel and nontreated cells at 24 and 48 h. The tgChNPs-ChPRP gel with different PRP concentrations showed noticeable differences in scratch wound closure at 48 h. The growth factors such as PDGF present in the tg-ChNPs-ChPRP gel stimulated fibroblast cell migration.8,48 The results indicated that the tg-ChNPs-ChPRP gel can enhance the wound closure by stimulating the migration of fibroblast cells into the granulating tissue and expedite the wound healing. 3.10. In Vitro and ex Vivo Antibacterial Activity. 3.10.1. In Vitro Antibacterial Activity. The antibacterial activity of the prepared gel systems were confirmed by using agar well diffusion assay. The result showed that the wells containing tg, F

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sustained release of the tigecycline from the tg-ChNPs-ChPRP gel is responsible for the prolonged antibacterial activity. This study confirms that the nanoformulation of drug incorporated hydrogel can offer more sustained release than free drug incorporated gel. Thus, the developed system can be used on an infectious wound site for more duration, and the dressing frequency can be reduced. 3.10.3. Ex Vivo Antibacterial Activity. The antibacterial activity of the prepared gel systems was studied using an ex vivo porcine skin infection model,46 and the results were shown in Figure S5B. The ChPRP gel without tigecycline did not show any antibacterial activity. Similar to in vitro results, tigecycline and tg-ChNPs loaded ChPRP gels showed significant antibacterial activity toward S. aureus. The tigecycline released from tg-ChPRP gel and tg-ChNPs-ChPRP gel inhibited the growth of S. aureus on the skin surface. Our results indicated that the prepared gel systems can be used to treat S. aureus skin infections effectively. 3.11. In Vivo Antibacterial Activity in D. melanogaster Model. 3.11.1. S. aureus Infections and Fly Survival Assay. Figure 7B shows that intrathoracic infection with SApCgf p caused approximately 100% mortality of the D. melanogaster flies within 24 h. Survivability of the flies improved when infected flies were fed on food containing tigecycline. We have observed maximum survival rate (>90%) with tg and tg-ChPRP gels when compared with any other groups possibly due to better release and availability of tigecycline at higher concentration within the flies. The survivability of tg-ChNPsChPRP gel fed flies (∼80%) was lower than tg-ChPRP fed flies (∼90%) and this is very likely due to the slow release of tigecycline from the tg-ChNPs-ChPRP gel (Figure 7B). Further to assess the quantitative growth of the bacterial cells inside the flies, we have crushed the flies and serially diluted and plated them on LB agar plates. The percentage of live bacterial cells per group was plotted. Bacterial survival decreased when flies were treated with tigecycline alone or tigecycline containg hydrogels. No bacterial colonies were observed when flies were fed with tigecycline alone. Bacterial growth was reduced up to 95% when flies were fed with tgChPRP hydrogel and by 80% when fed with tg-ChNPs-ChPRP hydrogel (Figure 7C). Time based fluorescent images of the flies were captured starting from the 0 h until 24th h. We observed infected and untreated flies emitted the brightest green fluorescence intensity while the control noninfected flies emitted no fluorescence. When infected flies were fed on tigecycline, it was observed that the intensity of fluorescence had decreased as the antibiotic inhibited the growth of the SApCgfp (Figure 7D). The better therapeutic effect of tg-ChPRP gel compared to tg-ChNPs-ChPRP gel is possibly due to the enhanced release of tigecycline from the tg-ChPRP gel system. This result also indicated that tg-ChNPs-ChPRP gel could be used as a slow releasing gel formulation to treat a variety of S. aureus infected wounds. The experiments with D. melanogaster flies indicate that all tigecycline formulations are effective in controling S. aureus infections and therefore enhanced the survivability of these flies. Our results using D. melanogaster not only demonstrated the antistaphylococcal activity of tg-ChPRP gel and tg-ChNPsChPRP gel but also provided evidence that the prepared hydrogels were safe for in vivo applications.

tg-ChPRP gel, and tg-ChNPs-ChPRP gel showed a significant zone of inhibition against S. aureus (Figure S4). ChPRP gel alone, failed to demonstrate any antibacterial activity. The Figure S4 depicts that the incorporation of PRP in to the tg-Ch and tg-ChNPs-Ch gel did not enhance or inhibit the antibacterial activity. The study confirms the antibacterial activity of the prepared gel systems in solid medium. The antibacterial activities of different amounts of ChPRP, tgChPRP, and tg-ChNPs-ChPRP gels were studied by broth dilution method, and the results were represented graphically in Figure S5A. The ChPRP gel alone did not show any antibacterial activity (data not shown).49 The tg-ChNPsChPRPgel showed an enhanced antibacterial effect compared to ChNPs-ChPRP gel. The ChPRP gel with the lowest concentration of tg-ChNPs (1 μg/mg) also showed a significant reduction in bacterial growth. As the amount of tg or tg-ChNPs in the ChPRP gel increased, the efficacy to prevent S. aureus growth also increased. The results indicated that when similar concentrations of tg-ChPRP and tg-ChNPs-ChPRP gels were used for the antimicrobial activity assays, tg-ChPRP gel showed better antistaphylococcal activity than tg-ChNPs-ChPRP gel. This is very likely due to the rapid release of tigecycline from the tg-ChPRP gel. The smaller growth inhibition zone produced by tg-ChNPs-ChPRP gel against S. aureus could be due to the slow release of tigecycline from the NPs. The sustained release of tigecycline from the tg-ChNPs-ChPRP gel indicates that the gel system can be used as a slow release antimicrobial gel to treat S. aureus infected wounds. In order to visualize the growth inhibition of S. aureus by the prepared gels, we have added the bacteria at the top of the gel and incubated for defined time intervals. Bacterial growth was observed using SEM (Figure 6). The SEM images corroborate

Figure 6. SEM images demonstrating antistaphylococcal activity of ChPRP, tg-ChPRP, and tg-ChNPs-ChPRP gel.

our earlier findings. The ChPRP gel without tigecycline failed to inhibit the S. aureus growth whereas the growth of S. aureus was inhibited by tg-ChPRP and tg-ChNPs-ChPRP gels. 3.10.2. Long-Term in Vitro Antibacterial Activity. The antibacterial activity of releasate from tg-ChPRP, and tgChNPs-ChPRP gels was studied and the results were represented graphically in Figure 4B. The releasate up to the 7th day from both gel systems showed a significant antibacterial activity against S. aureus, whereas the releasate from tg-ChNPs gel did not inhibit bacterial growth from the 8th day onward. At the same time, releasate from tg-ChNPs-ChPRP gel showed a significant bacterial growth inhibition up to 14 days. The G

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Figure 7. Kaplan−Meier survival curves of D. melanogaster and microbial quantification. (A) Flies infected with S. aureus and fed with ChPRP, tgChPRP, and tg-ChNPs-ChPRP gels; (B) survivability of the flies monitored and plotted; (C) bacterial load within the flies after treatment with different hydrogels. Data were analyzed using the log-rank test to derive the p values (*, p < 0.05; **, p < 0.01; ***, p ≤ 0.001); (D) fluorescence microscopic image demonstrating the antimicrobial activity of different hydrogels. The infected flies fed on different hydrogel systems. Images at 24 h postinfection of uninfected flies, infected flies, and infected flies fed with tg, ChPRP gel, tg-ChPRP gel ,and tg-ChNPs-ChPRP gel.

4. CONCLUSION The tg-ChNPs loaded ChPRP gel was prepared for the treatment of S. aureus infected wounds. The desired properties of therapeutic gel such as injectability, thermal stability, and flow behavior were confirmed using rheology tests. The tigecycline release study confirmed that tigecycline release from tg-ChNPs-ChPRP gel is slower and more sustained, which is ideal for effective infection control. The sustained antibiotic release can decrease the chance of invasion of bacteria into the wound and thereby decrease the frequency of wound dressing. The PRP loaded gels significantly increased the proliferation and migration of fibroblasts. Similar to in vitro studies the PRP

containing gels can accelerate granulation phase of wound healing by stimulating fibroblast cells. The in vitro, ex vivo and in vivo antibacterial activity of the gel against S. aureus was tested to optimize the tg-ChNPs concentration in the ChPRP gel. The long-term antibacterial activity of tg-ChNPs-ChPRP gels further confirmed the sustained release of tigecycline. Thus, taking the above results into consideration, the prepared gel system could be a promising therapeutic gel for infectious wound healing. Although the work was carried out using Staphylococcus aureus as a model organism, the gel can be used for a variety of other microbial wound infections caused by Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, H

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(9) El-Sharkawy, H.; Kantarci, A.; Deady, J.; Hasturk, H.; Liu, H.; Alshahat, M.; Van Dyke, T. E. Platelet-Rich Plasma: Growth Factors and Pro- and Anti-Inflammatory Properties. J. Periodontol. 2007, 78, 661−669. (10) Alsousou, J.; Ali, A.; Willett, K.; Harrison, P. The Role of Platelet-Rich Plasma in Tissue Regeneration. Platelets 2013, 24, 173− 182. (11) Guo, S.; Dipietro, L. A. Factors Affecting Wound Healing. J. Dent. Res. 2010, 89, 219−229. (12) Bowler, P. G.; Duerden, B. I.; Armstrong, D. G. Wound Microbiology and Associated Approaches to Wound Management. Clin Microbiol Rev. 2001, 14, 244−269. (13) Oogai, Y.; Matsuo, M.; Hashimoto, M.; Kato, F.; Sugai, M.; Komatsuzawa, H. Expression of Virulence Factors by Staphylococcus aureus Grown in Serum. Appl. Environ. Microbiol. 2011, 77, 8097− 8105. (14) Liu, G. Y. Molecular Pathogenesis of Staphylococcus aureus Infection. Pediatr. Res. 2009, 65, 71R−77R. (15) Rodriguez-Vazquez, M.; Vega-Ruiz, B.; Ramos-Zuniga, R.; Saldana-Koppel, D. A.; Quinones-Olvera, L. F. Chitosan and Its Potential Use as A Scaffold for Tissue Engineering in Regenerative Medicine. BioMed Res. Int. 2015, 2015, 821279. (16) Suh, J.-K. F.; Matthew, H. W. T. Application of Chitosan-Based Polysaccharide Biomaterials in Cartilage Tissue Engineering: A Review. Biomaterials 2000, 21, 2589−2598. (17) Bauer, G.; Berens, C.; Projan, S. J.; Hillen, W. Comparison of Tetracycline and Tigecycline Binding To Ribosomes Mapped By Dimethylsulphate and Drug-Directed Fe2+ Cleavage of 16S rRNA. J. Antimicrob. Chemother. 2004, 53, 592−599. (18) Guo, S.; DiPietro, L. A. Factors Affecting Wound Healing. J. Dent. Res. 2010, 89, 219−229. (19) Thakur, G. A.; Shaikh, M. M. Synthesis, Characterization, Antibacterial and Cytotoxicity Studies on Some Mixed Ligand Th(IV) Complexes. Acta Pol. Pharm. 2006, 63, 95−100. (20) Kiruthika, V.; Maya, S.; Suresh, M. K.; Kumar, V. A.; Jayakumar, R.; Biswas, R. Comparative Efficacy of Chloramphenicol Loaded Chondroitin Sulfate and Dextran Sulfate Nanoparticles to Treat Intracellular Salmonella Infections. Colloids Surf., B 2015, 127, 33−40. (21) Dhanalakshmi, V.; Nimal, T. R.; Sabitha, M.; Biswas, R.; Jayakumar, R. Skin and Muscle Permeating Antibacterial Nanoparticles for Treating Staphylococcus aureus Infected Wounds. J. Biomed. Mater. Res., Part B 2016, 104, 797−807. (22) Kutlu, B.; Tigli Aydin, R. S.; Akman, A. C.; Gumusderelioglu, M.; Nohutcu, R. M. Platelet-Rich Plasma-Loaded Chitosan Scaffolds: Preparation and Growth Factor Release Kinetics. J. Biomed. Mater. Res., Part B 2013, 101, 28−35. (23) Benz, K.; Stippich, C.; Osswald, C.; Gaissmaier, C.; Lembert, N.; Badke, A.; Steck, E.; Aicher, W. K.; Mollenhauer, J. A. Rheological and Biological Properties of a Hydrogel Support for Cells Intended for Intervertebral Disc Repair. BMC Musculoskeletal Disord. 2012, 13, 54. (24) Priya, M. V.; Kumar, R. A.; Sivashanmugam, A.; Nair, S. V.; Jayakumar, R. Injectable Amorphous Chitin-Agarose Composite Hydrogels for Biomedical Applications. J. Funct. Biomater. 2015, 6, 849−862. (25) Maya, S.; Indulekha, S.; Sukhithasri, V.; Smitha, K. T.; Nair, S. V.; Jayakumar, R.; Biswas, R. Efficacy of Tetracycline Encapsulated OCarboxymethyl Chitosan Nanoparticles Against Intracellular Infections of Staphylococcus aureus. Int. J. Biol. Macromol. 2012, 51, 392−399. (26) Liang, C. C.; Park, A. Y.; Guan, J. L. In Vitro Scratch Assay: A Convenient and Inexpensive Method for Analysis of Cell Migration In Vitro. Nat. Protoc. 2007, 2, 329−333. (27) Biswas, R.; Voggu, L.; Simon, U. K.; Hentschel, P.; Thumm, G.; Gotz, F. Activity of The Major Staphylococcal Autolysin Atl. FEMS Microbiol. Lett. 2006, 259, 260−268. (28) Lboutounne, H.; Chaulet, J. F.; Ploton, C.; Falson, F.; Pirot, F. Sustained Ex Vivo Skin Antiseptic Activity of Chlorhexidine in Poly(εCaprolactone) Nanocapsule Encapsulated Form and as A Digluconate. J. Controlled Release 2002, 82, 319−334.

Methicillin-resistant Staphylococcus aureus (MRSA), and Clostridium perf ringens.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b07463. Procedure, results of kinetic modeling of in vitro tigecycline release, whole blood clotting study, hemolysis assay, cell viability and proliferation studies, and in vitro and ex vivo antibacterial activity (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; jayakumar77@yahoo. com. Tel.: +91 484 2801234. Fax: +91 484 2802020. *E-mail: [email protected]; raja.biswas100@gmail. com. Tel.: +91-484-2801234. Fax: 91-484-2802030. Author Contributions †

N.T.R. and G.B. contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We are thankful to the Department of Biotechnology (DBT), Government of India, for the financial support for this work (Grant No. BT/PR6758/NNT/28/620/2012). The author B.M.C. is grateful to Nanomission, Department of Science and Technology, Government of India, for providing an M. Tech PG Programs research grant to Amrita Centre for Nanosciences and Molecular Medicine (Reference No. SR/NM/PG-01/ 2015). We gratefully acknowledge Dr. Gopi Krishnan Anjaneyan, Mr. Vivek Vinod, and Mr. Sajin. P. Ravi for their suggestions and technical assistance.



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