Effect of curcumin caged silver nanoparticle on collagen stabilization for biomedical applications

Accepted Manuscript Title: Effect of curcumin caged silver nanoparticle on collagen stabilization for biomedical applications Author: Srivatsan Kunnavakkam Vinjimur Duraipandy N Shajitha Begum Rachita Lakra Usha Ramamurthy Purna Sai K Manikantan Syamala Kiran PII: DOI: Reference:

S0141-8130(15)00061-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.01.050 BIOMAC 4865

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

22-7-2014 19-12-2014 16-1-2015

Please cite this article as: S.K. Vinjimur, D. N, S. Begum, R. Lakra, U. Ramamurthy, P.S. K, M.S. Kiran, Effect of curcumin caged silver nanoparticle on collagen stabilization for biomedical applications, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.01.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights: Curcumin caged silver nanoparticles are ecologically safe and biocompatible alternatives for collagen crosslinking

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Curcumin caged silver nanoparticle provide multisite interaction for enhanced cross-linking of collagen

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Ideal as biomaterial scaffolds with enhanced antimicrobial activity and cytocompatibilty

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Effect of curcumin caged silver nanoparticle on collagen stabilization for biomedical applications

Srivatsan Kunnavakkam Vinjimur1, Duraipandy N1, 2, Shajitha Begum3, Rachita Lakra1, Usha

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Ramamurthy1, Purna Sai K1, 2 and Manikantan Syamala Kiran1, 2*

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1. Council of Scientific and Industrial Research, Central Leather Research Institute, Adyar,

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Chennai 600020, India

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3. University of Madras, Chennai, India

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2. Academy of Scientific and Innovative Research, New Delhi, India

Corresponding author: Dr. M. S. Kiran, CSIR-CLRI,

Adyar, Chennai- 600020, India.

E-mail:[email protected] [email protected]

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Abstract The current study aims at understanding the influence of curcumin caged silver nanoparticle

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(CCSNP) on stability of collagen. The results indicated that curcumin caged silver nanoparticles efficiently stabilize collagen, indicated by enhanced tensile strength, fibril formation and viscosity.

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The tensile strength of curcumin caged silver nanoparticle cross-linked collagen and elongation at break was also found to be higher than glutaraldehyde cross-linked collagen. The physicochemical

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characteristics of curcumin caged nanoparticle cross-linked collagen exhibited enhanced strength. The thermal properties were also good with both thermal degradation temperature and hydrothermal

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stability higher than native collagen. CD analysis showed no structural disparity in spite of superior physicochemical properties suggesting the significance of curcumin caged nanoparticle mediated cross-linking. The additional enhancement in the stabilization of collagen could be attributed to

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multiple sites for interaction with collagen molecule provided by curcumin caged silver nanoparticles. The results of cell proliferation and anti-microbial activity assays indicated that

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curcumin caged silver nanoparticles promoted cell proliferation and inhibited microbial growth making it an excellent biomaterial for wound dressing application. The study opens scope for nano-

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biotechnological strategies for the development of alternate non-toxic cross-linking agents

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facilitating multiple site interaction thereby improving therapeutic values to the collagen for biomedical application.

Keywords: Curcumin, Silver Nanoparticles, Collagen, Stabilization, Anti-microbial, Biocompatible

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1.

Introduction

Collagen is one of the most commonly used biomaterial due to its structural stability,

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biocompatibility and ability to support cell adhesion and proliferation[1–4]. The major reason for

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utility of collagen in biomedical applications relies in its ability to form fibers with extra strength and stability through self-aggregation and cross-linking properties [5–8]. However, collagen after

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isolation and reconstitution has poor mechanical strength, thermal stability and susceptibility to

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proteolytic degradation [9–11]. Modifying collagen by inter-helical and intra-helical cross-links significantly enhances the thermal stability and renders enzymatic resistance to the tri-peptide. Low

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antigenicity of the cross-linked peptide is an additional advantage [12–15]. A variety of chemical, physical and enzymatic cross-linking methods have been used for collagen stabilization [16,17].

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Glutaraldehyde (GA) is the most frequently used chemical cross-linking agents, but the presence of

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un-reacted agents or functional groups and the release of these functional groups during enzymatic

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degradation makes it less biocompatible owing to in vivo cytotoxicity [18,19]. Furthermore, unreacted GA also facilitates the formation of heterogeneous cross-linking structure that leads to local incompatibility, inflammation, encapsulation and calcification, along with limited cell growth. Aldehydic derivatives, ether derivatives, plant polyphenols, cyclodextrins, epoxy residues and physical methods like dehydrothermal cross-linking, γ- irradiation methods have also been extensively used for cross-linking collagen but were found to have several drawbacks [10,20–28]. Thus there is a pressing need for identifying ecologically and biologically safe molecules for crosslinking collagen. Nutraceuticals are gaining attention in this direction. Nutraceuticals provides several health benefits by preventing various diseases [29]. One such nutraceutical is curcumin that has wide range of biological activities like anti-tumourigenic, anti4 Page 4 of 42

oxidant, anti-proliferative activities [30–33]. Curcumin is a diarylheptanoid compound and has been reported to enhance the physiochemical properties of collagen [34]. However, curcumin as such has not been widely accepted as a therapeutic agent due to its poor bioavailability, low solubility and

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staining ability [35]. The use of functional analogs or conjugation with therapeutic compounds could solve such problems. Nano-biotechnological intervention has proven to be effective in

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increasing the physicochemical properties of molecules [36]. Nano-silver has been reported to

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exhibit different and enhanced physicochemical properties compared to bulk silver. Silver nanoparticles have been used in wide range of applications such as drug delivery, molecular imaging of

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cells and has been reported to possess anti-inflammatory, anti-microbial and wound healing activities [37–42]. The present study deals with understanding the role of curcumin caged silver

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nanoparticle (CCSNP) on stabilization of collagen. The study essentially involves a combination of

Experimental

2.1.

Materials

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2.

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nanoparticle.

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robust anti-microbial, wound healing and cross-linking properties of curcumin caged silver

All the chemicals were procured from Ms. Sigma Aldrich, unless and otherwise mentioned. HaCaT cells (immortalized human keratinocytes) were purchased from NCCS Pune, India. All the tissue culture wares were from Ms Nunc, Denmark. 2.2.

Isolation of collagen from Rat Tail Tendon (RTT)

Tendons from rat tails were washed 4 times with 1:1 diethyl ether and chloroform, followed by two methanol washes. The tendons were then washed with 1% sodium chloride (4 times) and ground in 0.05 M acetic acid. Pellets were removed after centrifugation and the supernatant was precipitated 5 Page 5 of 42

with 5% sodium chloride. The collagen thus obtained was dialyzed against 0.05M acetic acid and lyophilized for further use [43,44].

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2.3.1. Synthesis of curcumin caged Silver nanoparticles (CCSNP) To 1 ml of 100 mM silver nitrate, 200 µl of KOH containing different concentrations of curcumin

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(20µM to 100 µM) was added and shaken for 10 minutes. The nanoparticles were collected by

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spinning at 7500 rpm for 10 minutes. The particles were then washed twice with double distilled water to remove unbound curcumin, frozen overnight at -80 ºC and lyophilized to obtain a fine

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powder [45,46].

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2.3.2. Caging efficiency of curcumin

1 mg of curcumin caged silver nanoparticle (CCSNP) was solubilized in DMSO, centrifuged and

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supernatant was collected. The optical density of supernatant (solubilized curcumin caged silver

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nanoparticle) were read at 450nm in BIORAD ELISA plate reader and compared with various

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concentrations of standards (25µM to 1 mM).

2.3.3. Fourier Transform Infrared Spectroscopy (FTIR) FTIR was performed using a Spectrum two - Perkin-Elmer Co., USA model spectrophotometer. Curcumin caged silver nanoparticles, solubilized curcumin from curcumin caged silver nanoparticles (CCSNP) and pure curcumin were made into a pellet using potassium bromide (KBr). A scan was run from 450 to 4000 cm -1 with 8 scans per sample and a resolution of 1 cm -1. 2.4.

Characterization of Nanoparticles

2.4.1. Particle Size Analysis

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The particle size and the zeta potential of the curcumin caged silver nanoparticle (CCSNP) was determined using a Photo Correlation spectrophotometer (PCS) at 90º from Malvern Instruments,

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Zetasizer 3000 HSA equipped with a digital auto-correlator. 2.4.2. SEM with EDAX

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The shape and morphology of the curcumin caged silver nanoparticle (CCSNP) was determined

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using Quanta 200 FEG Scanning Electron Microscope equipped with Energy dispersive X-Ray (EDAX) Spectrometer. The nanoparticles were spread on the EM stubs, sputtered for 2-3 minutes

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with gold for conductivity and analyzed in high vaccum mode. The shape, morphology and

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conjugation of curcumin on silver nanoparticles were further confirmed by TEM analysis. 2.4.3. Powder XRD

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X-ray diffraction pattern of the curcumin caged silver nanoparticle (CCSNP) was measured using

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Bruker D8 advance diffractometer instrument with Cu κα 1.54Å radiation and detected using a

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Brukerlyric eye detector. Measurement temperature and slit size were set at 25ºC and 0.6 as default for all measurements respectively. The X-Ray diffraction spectra were recorded in the range 2θ from 10.0 to 60.0 with a step increment of 0.02 and count time of 5s. 2.5.

Collagen Stability

2.5.1. Fibrillation Assay

Fibrillation assay was done by initializing fibril formation by mixing 500 µg/mL final concentration of collagen with 0.2 M phosphate buffer and 2 M sodium chloride. The pH was adjusted to 7.2 using 1.25 N sodium hydroxide. The turbidity was measured at 313 nm using Perkin Elmer UV–Vis

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spectrophotometer. The rate of fibril formation was determined by measuring the time taken to reach half the value of final turbidity (t1/2). Tensile Strength

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2.5.2.

The tensile strength of the collagen scaffolds were measured using Instron Universal Testing

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machine until the rupture of the sample. Samples with uniform thickness, length (5 cm) and width (4

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cm) were cut from the scaffolds. A load of 10 N was applied and measuring speed was 5 mm/min with relative humidity of 60% at 25°C. Viscosity

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2.5.3.

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Shear viscosity of collagen molecules in presence and absence of curcumin caged nanoparticle was measured by Brookefield R/S+ Rheometer. 0.1 % collagen solution was used throughout the study.

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The solutions were kept overnight with continuous stirring at 4º C before performing the

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measurements with and without CCSNP for cross-linking [47].

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2.5.4. Differential Scanning Calorimetry (DSC) Thermal stability of hermetically sealed collagen scaffold was determined by DSC analysis using DSC Q200 (V23.10 Build 79) Differential Scanning Calorimeter from 25ºC to 250º C in nitrogen atmosphere at a flow rate of 50 ± 5 ml/min. The temperature was standardized using iridium as standard. The heating rate was set at 5º C /min. 2.5.5. Thermo Gravimetric Analysis Thermo gravimetric analysis (TGA) measures the amount and rate of change in the mass of a sample as a function of temperature in a controlled atmosphere. Collagen films cross-linked with glutaraldehyde, curcumin caged silver nanoparticle and native collagen were subjected to TG 8 Page 8 of 42

analysis using TGA Q50 (V20.6 Build 31) from 20 ºC to 800 ºC at a constant heating rate of 20ºC/min under nitrogen atmosphere.

2.5.6.1.

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2.5.6. Conformational studies Circular Dichroism (CD) measurement

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The influence of the curcumin caged nanoparticle on conformation of collagen was studied at 20 ºC

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using Circular Dichroic spectropolarimeter in N2 atmosphere. 0.5 ml of collagen solution containing 0.1% collagen was used for recording the spectra. Two scans per sample and a scan speed of 50nm

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per min were set for recording the spectra. A reference spectrum was also recorded with collagen

Biological Activity

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2.6.1. Antimicrobial Activity

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2.6.

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and 0.05 M acetic acid as control and blank respectively.

Anti-microbial activity of the curcumin caged nanoparticle was tested using Escherichia coli and

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Bacillus subtilis by broth dilution method using LB broth and Kirby Bauer agar diffusion method [48,49]. In broth dilution assay, collagen scaffolds cross-linked with 10 and 20 µM curcumin caged nanoparticle were added in a total volume of 2 ml culture media along with culture inoculums and incubated overnight. After incubation, 10 µl of medium was diluted 25 times and was read at 630nm in Bio-Rad ELISA plate reader. The antimicrobial activity of cross-linked collagen sheets on agar plates was also determined by measuring the diameter of zones of inhibition. 2.6.2. MTT assay The influence of stabilized collagen on cell attachment and proliferation was studied by performing MTT assay. Collagen cross-linked with curcumin, glutaraldehyde (50µM) (positive control) and 9 Page 9 of 42

curcumin caged silver nanoparticle and native collagen (control) were coated on culture plates to perform the assay. HaCaT cells were used to carry out the assay. Approximately 12,000 cells were treated with three different concentrations of curcumin caged silver nanoparticles (from 10µM to

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20µM). At different time intervals (Day 1, Day 3 and Day 5) the culture medium was removed and the cells were incubated with 3- (4, 5 dimethylthiazolyl – 2) - 2, 5- diphenyl tetrazolium bromide

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(MTT) salt containing 0.5 mg/ml in PBS at 37º C. After 4 hours of incubation, the blue/ purple

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formazan crystals formed were solubilized using DMSO (Dimethyl Sulphoxide) and the absorbance

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was read 570 nm in Bio-Rad ELISA plate reader[50]. 2.6.3. Statistical Analysis

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The data given are mean ± standard deviation (S.D). Statistical analysis was performed using SPSS

Synthesis of curcumin caged silver nanoparticle

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3.1.

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3. Results and Discussion

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Statistics 22 software. A value of P < 0.05 was considered significant.

Curcumin caged nanoparticle was synthesized by simple oxido-reduction method using potassium hydroxide and silver as starting material. The ratio of 1:1 curcumin and silver nitrate was found to be the ideal concentration for the synthesis of nanoparticle. The unstable silver hydroxide was formed as an intermediate, which disintegrates to form Ag2O but in presence of curcumin, it gets caged on to silver during breakdown of silver hydroxide to silver oxide, which resulted in a dark colored colloidal solution. We observed significant aggregation in control nanoparticles as indicated by higher sedimentation of nanoparticles to the bottom of the reaction vessel compared to curcumin caged silver nanoparticles where they remained in suspension. The results are provided in supplementary Fig. 1. We assume that caging of curcumin on silver nanoparticle prevents 10 Page 10 of 42

intermolecular interactions between silver nanoparticles and induce repulsion preventing their aggregation and sedimentation when compared with control nanoparticles. Estimation of curcumin incorporated in the Silver nanoparticle complex

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3.1.1.

The amount of curcumin that has been incorporated on the nanoparticle was determined by

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solubilizing curcumin from CCSNPs in 0.5 M alcoholic KOH. The supernatant containing

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solubilized curcumin was collected after centrifugation and was read along with curcumin standards (25-1000µM) at 450nm colorimetrically. Curcumin loaded on to the nanoparticle was calculated

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using the following formula - Amount of drug (curcumin) loading = [(O.D of test)*(conc. standard)]/ [(O.D of standard)*(vol of test)]. The caging efficiency of curcumin onto nanoparticle

FT-IR

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3.1.2.

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was found to be 100µM per mg of nanoparticle.

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FTIR analysis was done to further confirm the caging of curcumin onto silver nanoparticles.

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Curcumin, solubilized curcumin from CCSNPs in alcoholic potassium hydroxide (KOH) and curcumin caged silver nanoparticle (CCSNP) was made into pellets using potassium bromide (KBr) and these pellets were analyzed in IR spectrophotometer. Fig. 1 shows the FTIR spectrum of curcumin caged silver nanoparticles, curcumin solubilized from curcumin caged silver nanoparticles and curcumin control (standard). The results indicated that the broad peaks at 3419, 3422 and 3427 cm-1corresponded to (OH- H) of alcoholic or phenolic group, peaks at 2920 and 2908 cm-1represents the O-H stretching vibration of carboxylic acid, the narrow peaks at 1629, 1630, 1637 cm1

corresponded to (C=O) stretching vibrations of carboxylic group or its derivatives, low intensity

peaks at 1423, 1446, 1407 cm-1denote (OH bending) and CH2 bending vibrations of aldehyde or

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ketone group respectively [51,52]. Occurrence of identical peaks in all the three spectra indicates the caging of curcumin on silver nanoparticle in CCSNPs.

Characterization of Nanoparticle

3.2.1.

Particle size analysis

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3.2.

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Preferred location for Fig. 1

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Particle size, zeta potential and dipersity of the nanoparticle were determined by Dynamic Light

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Scattering (DLS) analysis in aqueous solution. The results indicated that the average size of control silver nanoparticles was 800 nm and that of curcumin caged silver nanoparticle was 170 nm (Fig. 2

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a and b). The particle size of the control nanoparticle was found to be higher due to aggregation of Ag2O that occurred during the disintegration of Ag(OH)2, whereas in the case of curcumin caged

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silver nanoparticle, curcumin bonds with silver during the breakdown of Ag(OH) 2, thereby caging

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the silver nanoparticle, preventing clumping and reducing the size of the caged nanoparticle. The

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results are in accordance with the earlier observation indicating aggregation and sedimentation of control nanoparticle. The DLS analysis gives the hydrodynamic diameter since a covering of water over nanoparticle is formed. The polydispersity and zeta potential of silver nanoparticle and curcumin caged nanoparticle is listed in Table 1. The zeta potential of control silver nanoparticle and curcumin caged nanoparticle was -31.0 mV and -35.4 mV respectively. The results suggested that curcumin caged silver nanoparticle have higher stability with a greater zeta potential value compared to control silver nanoparticle since it has been reported that nanoparticles with smaller size having high zeta potential value which will confer stability by resisting aggregation. The stability of nanoparticle dispersion is directly proportional to zeta potential value. The results are consistent with the sedimentation profile of curcumin caged silver nanoparticles as shown in 12 Page 12 of 42

supplementary Fig. 1 where CCSNPs are finely dispersed in solution but control nanoparticles gets sediment to the bottom of reaction vessel.

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3.2.2.

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Preferred location for Table 1 Scanning Electron Microscopy

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The morphology and size are one of the important determinants in the activity of any nanoparticle in

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biological systems. The synthesized nanoparticle was subjected to SEM analysis to determine its shape and size and EDAX analysis to understand its elemental composition. Fig. 3 a, b shows SEM

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image and c, d) shows EDAX image for control silver nanoparticle and curcumin caged silver nanoparticle respectively. The particles were uniform in size. In order to reveal the presence of

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curcumin on the silver nanoparticle, elemental analysis was performed along with SEM. The

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reduction in oxygen content in the curcumin caged silver nanoparticles when compared with the

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silver nanoparticles (unconjugated) clearly indicated the conjugation of curcumin on silver nanoparticle. The SEM EDAX analysis confirmed the conjugation of curcumin on silver nanoparticle. The morphology and size of control and curcumin caged silver nanoparticles were further analyzed by TEM analysis. The results are provided in Supplementary Fig. 2. The TEM image shows that control silver nanoparticles are found as aggregates whereas negligible amount of aggregation was found in curcumin caged nanoparticles when compared with control nanoparticles (without curcumin). The results are consistent with supplementary Fig. 1 where we observe that in control nanoparticles they aggregate and sediment whereas due to lower aggregation and size the curcumin caged silver nanoparticles are in colloidal suspension. The TEM images revealed that the average size of the curcumin caged silver nanoparticles and control nanoparticles was around 28.76 13 Page 13 of 42

nm and 41.28 nm respectively. The shape of these nanoparticles was found to be spherical. The appearance of a translucent covering over silver nanoparticles in TEM images of curcumin caged

curcumin on silver nanoparticles when compared with control nanoparticles.

Powder XRD

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3.2.3.

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Preferred location for Fig. 3

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silver nanoparticles and absence of it in control nanoparticles indicated a uniform caging of

XRD pattern obtained for silver nanoparticles with and without curcumin are provided in Fig. 4.

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The diffraction pattern of silver nanoparticle showed well-defined diffraction peak, corresponding to the crystallographic plane centered at 38.4=2θ, which indicated a relatively high degree of

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crystallinity. The intensity of the peaks reflected high degree of crystallinity for silver nanoparticles.

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The XRD pattern of powdered silver nanoparticles had two peaks at 2θ values of 33.12, 38.4 mdeg

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corresponding to (110), (111) planes. All the reflections corresponded to pure silver metal with face centered cubic symmetry. No spurious diffraction peaks indicated the purity of the synthesized

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curcumin caged silver nanoparticle. Preferred location for Fig. 4 3.3.

Stabilization of collagen

3.3.1. Fibrillation

Fibrillogenesis occurs in two steps, formation of nuclei by aggregation of individual helix molecule and growth of nuclei into fibrils [53]. It is important to study the self-assembly process to prepare a biomaterial using collagen. We tried to study the effects of curcumin caged silver nanoparticle on fibril formation with collagen glutaraldehyde as positive control. The extent of cross-linking at 14 Page 14 of 42

lower concentrations of collagen is hard to determine hence change in absorbance at 313nm is recorded to approximate the relative amount of fibrils formed. Fig. 5 shows the turbidity curve of native and curcumin caged silver nanoparticle cross-linked collagen measured at 32ºC from which

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t1/2 and total turbidity change (Δh) were calculated for all the samples. The t1/2 and Δh for native collagen was 47 min and 0.5219, 40 min and 0.5271 for curcumin caged silver nanoparticle cross-

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linked collagen and 39 min and 0.5578 for glutaraldehyde cross-linked collagen respectively.

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Hydrophobic interactions play a major role in fibril formation, which is greatly influenced by temperature. The thermal stability improves due to the incorporation of cross-links, which in turn

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increases the intermolecular interaction. The rate of fibril formation of collagen self-assembly of curcumin caged silver nanoparticle cross-linked collagen was observed to be similar to

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glutaraldehyde cross-linked collagen scaffolds (positive control) whereas it was significantly greater

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when compared with collagen native control. The kinetics of fibrillogenesis is denoted by sigmoid

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curve where aggregation and alignment of collagen monomers is controlled by electrostatic interaction [54]. The figure also shows that the general shape of the turbidity curves was not

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affected by glutaraldehyde and CCSNPs. The results showed that the rate at which cross-links are formed was significantly high for curcumin caged silver nanoparticles compared to control collagen. The result suggests that lower concentration of curcumin caged silver nanoparticles stimulate fibril formation and promote stabilization of fibrillar forms of collagen. Preferred location for Fig. 5 3.3.2. Tensile strength: Tensile strength and elongation at break denotes the strength and flexibility of collagen scaffolds [55]. It is the spatial arrangement of fibre bundles and interweaving of fibres within the bundles that

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determined the tensile strength of any collagen based materials [56]. The tensile strength of the collagen was determined by breakage point of the collagen sheet. During stress, the sheets start rupturing locally before complete rupture at breakage point. The curcumin caged silver nanoparticle

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cross-linked collagen showed better strength at break of 13.92% compared to 6.25% and 5.92% for glutaraldehyde cross-linked collagen and control native collagen respectively. Table 2 indicates that

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there is an increase in tensile strength as well as increase in elongation at break. The tensile strength

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of the collagen cross-linked with curcumin caged silver nanoparticles was (4.52 N/mm2) higher than the control native collagen (3.48 N/mm2) and collagen cross-linked with glutaraldehyde (2.04

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N/mm2). The results indicated that curcumin caged silver nanoparticles provides better cross-linking

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and strength to collagen when compared to glutaraldehyde.

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3.3.3. Rheology

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Cross-linking greatly influences the stress–relaxation mechanism of collagen based materials [57].

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The viscosity of collagen samples were determined based on the flow rate of collagen with curcumin caged silver nanoparticle and glutaraldehyde into the capillary of the viscometer probe. Fig. 6 (a, b) shows the rheograms indicating the shear viscosity and shear stress vs. shear rate of collagen scaffolds cross-linked with glutaraldehyde, curcumin caged silver nanoparticles and native collagen scaffolds at room temperature. The results (Fig. 6 a) indicated that the shear viscosity increased in curcumin caged silver nanoparticles, which could be due to increased cross-linking of collagen by curcumin caged silver nanoparticles. The pattern of viscoelastic behavior of collagen molecules cross-linked with curcumin caged silver nanoparticles were similar to that observed with glutaraldehyde cross-linked collagen (positive control) but was significantly higher when compared to native collagen. Fig. 6 b indicated that the shear stress increased with shear rate in native 16 Page 16 of 42

collagen, collagen cross-linked with curcumin caged silver nanoparticles and glutaraldehyde crosslinked collagen but the shear stress varied among these groups depending on the cross-linking efficiency of these molecules. The resistance to shear stress was observed to be greater in curcumin

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caged silver nanoparticle cross-linked collagen and glutaraldehyde cross-linked collagen when compared with native collagen. We assume that increased shear stress and shear viscosity in

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curcumin caged silver nanoparticle cross-linked collagen may be due to induction of more cross-

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links by curcumin caged silver nanoparticles that leads to more complex intra-helical cross-links that increased viscosity of collagen when compared to native collagen. The results are found to be

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consistent with the fibrillation efficiency of curcumin caged silver nanoparticles (CCSNP). The results indicated that curcumin caged silver nanoparticles effectively cross-links collagen scaffolds

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similar to glutaraldehyde and can be used as an alternative to cross-link collagen fibrils for

Differential scanning calorimetry

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3.3.4.

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biomaterial applications.

The hydrothermal stability of the films was determined using a differential scanning calorimetry (DSC) which is a widely used method to study the thermal behaviour of materials as they undergo physical and chemical changes upon heating. DSC analysis provides details regarding the heat flow necessary for heating of sample with constant temperature and gives an idea about cross-linking density. It gives a better understanding of unfolding of protein under the influence of temperature. The improved stability of triple helical structure due to cross-linking leads to a low denaturation rate of collagen and higher melting temperature [10,58,59].

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Fig. 7 a. displays thermogram of collagen cross-linked with CCSNP, glutaraldehyde (positive control) and native collagen scaffolds. We observed several minor peaks appearing in the DSC thermogram which implies that different levels of cross-linking occurred during stabilization of

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collagen. We assume that this ambiguity during formation of cross-links may be due to different ways the cross-links have formed or the ways in which triple helices have been packed. The

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thermogram exhibited an endothermic peak centered at 51.27°C, associated to the helical coil

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transition. It is at this temperature collagen loses its ordered triple helical structure to form a random coil. The denaturation temperature (Td) can be considered as a measure of thermal stability of

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collagen, as it gives the temperature for unfolding on heat[60].

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The figure also shows that the presence of curcumin silver nanoparticles increased the denaturation temperature of the scaffolds with broader peaks centered at 94.91°C compared to native collagen

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scaffolds. Formation of broad peaks in the thermograms of CCSNPs and glutaraldehyde cross-

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linked collagen may be due to overlapping of peaks that are closely spaced. Hence this multiple

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transition phases in the collagen represents the features of intra and inter chain bonds in collagen structure [61]. The results indicated that higher denaturation temperature observed in collagen scaffolds cross-linked with curcumin caged silver nanoparticles may have more hydrogen bonds induced by curcumin caged silver nanoparticles that results in bringing collagen fibril closer to each other thereby resulting in lesser hydrophobic interactions which may be the reason for a higher denaturation temperature when compared to native collagen. The peaks at 121.60 ͦC and 131.98 ͦC are water loss peaks of curcumin caged silver nanoparticle and glutaraldehyde cross-linked collagen scaffolds. The lower denaturation temperature in native collagen compared to curcumin caged silver nanoparticle cross-linked collagen is attributed to water holding capacity of collagen. The water content levels in collagen fibers are influenced by cross-linkers, since they occupy the water binding 18 Page 18 of 42

sites in the triple helix, hence more heat is required to break these bonds leading to an increase in denaturation

temperature.

The

denaturation

temperature of

collagen cross-linked

with

glutaraldehyde was 92.86 ºC which was only 2 ºC higher than that observed for collagen scaffolds

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cross-linked with curcumin caged silver nanoparticles which indicated that the denaturation pattern of collagen scaffold cross-linked with curcumin caged silver nanoparticles was almost similar to

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that of glutaraldehyde crosslinked collagen scaffolds. The higher temperature for water loss in

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collagen scaffolds cross-linked with glutaraldehyde and curcumin caged silver nanoparticles in comparison with native collagen results from complexity of cross-links indicated a better stability to

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helical structures in presence of glutaraldehyde and curcumin caged silver nanoparticles.

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Preferred location for Fig. 7

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3.3.5. Thermo gravimetric Analysis

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The effect on collagen thermal properties brought about by curcumin caged silver nanoparticles on collagen was further investigated by TGA. The thermal degradation of collagen shows a four step

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transition [62]. The first step is removal of absorbed water molecule. The second step is the removal of structural water. The third transition is partial breaking down of intermolecular bonding and fourth is the complete thermal decomposition of collagen molecules. As shown in Fig. 7 b. the degradation of the polypeptide chain is initiated from 104.13 °C with weight percentage of 84.55 which is the first transition step of native collagen. In comparison, for the curcumin caged silver nanoparticle cross-linked collagen sample there was a shift to higher transition temperature and weight percentage to 113.72°C and 86.08 respectively. The second transition of collagen crosslinked with curcumin caged nanoparticle starts at 236.79°C with increase in temperature transition than the native collagen. The weights of collagen film are 82.61 and 84.59 for native collagen and curcumin caged silver nanoparticle (CCSNP) cross-linked collagen respectively. Partial 19 Page 19 of 42

denaturation of collagen begins at third transition where the weight percentage of native collagen is 68.95 and 71.63 for curcumin caged silver nanoparticle -cross-linked film. Complete degradation of collagen occurs at fourth temperature transition, which also shows increase in cross-linked collagen

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than the native collagen. The final weight percentage of collagen films were 47.25 and 54.85 respectively for native collagen and curcumin caged silver nanoparticle (CCSNP) cross-linked

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collagen scaffolds. The results indicated that collagen is covalently cross-linked, and exhibits a

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relatively higher thermal stability with a significant improvement brought about by cross-links

3.4.

Conformational studies

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Circular Dichroism (CD) measurements

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induced by curcumin caged silver nanoparticles.

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The influence of nanoparticles induced cross-links on the conformational change of collagen was

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studied using CD. In the far UV region, there is a minimum negative peak obtained at 197nm and maximum positive peak at 220nm with a cross over at 210nm which is characteristic of the triple

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helical structure of collagen in CD spectrum[57,63]. The CD spectra of native collagen and collagen with curcumin caged silver nanoparticles were carried out and are shown in the Fig. 8. The results showed that there was no conformational change in native collagen and collagen cross-linked with CCSNPs which indicated no changes in triple helical structure of collagen after cross-linking. Preferred location for Fig. 8 3.5.

Biological Activity

3.5.1. Cytotoxicity Assay of nanoparticles with collagen

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Collagen has an ability to facilitate cell attachment and proliferation, this ability is vital for cell differentiation and tissue regeneration. The effect of curcumin stabilized and curcumin caged silver nanoparticle stabilized collagen on cell proliferation and viability was studied using MTT assay.

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Approximately 12000 HaCaT cells were seeded into each well coated with collagen scaffolds crosslinked with various concentration of curcumin and respective concentrations of curcumin caged

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silver nanoparticles. Native collagen scaffold was used as control and collagen scaffolds cross-

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linked with glutaraldehyde was used as positive control. We observed that cells attach and spread on the surface of cross-linked collagen matrices and it has been reported that successful cell attachment

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is beneficial for cell proliferation. The results showed that cells undergo proliferation on culturing them on various concentrations of curcumin caged silver nanoparticle cross-linked collagen

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scaffolds. We observed a concentration dependent increase in the cell proliferation rate on collagen

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scaffolds cross-linked with various concentrations of curcumin caged silver nanoparticles. The

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results indicated that the cell also underwent proliferation in collagen scaffolds cross-linked with various concentrations of curcumin, collagen controls and glutaraldehyde cross-linked collagen

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scaffolds but the rate of cell proliferation was observed to be different in each group depending of the efficiency of these scaffolds for promoting cell growth. The results showed that the rate of cell proliferation was significantly higher in cells treated with collagen scaffolds cross-linked with various concentrations of curcumin caged silver nanoparticles when compared to uncross-linked collagen matrices and collagen matrices cross-linked with curcumin as shown in Fig. 9. The rate of cell proliferation was observed to be more or less similar in all groups on day 1, however significant changes in the proliferation rate was observed on day 3 and day 5 among various groups. The pattern of cell proliferation rate in 10 µM and 15 µM curcumin caged silver nanoparticles crosslinked collagen was observed to be similar, however we observed that the cell proliferation rate was significantly higher in cells treated with 20 µM curcumin caged silver nanoparticles cross-linked 21 Page 21 of 42

collagen scaffolds when compared to native collagen and glutaraldehyde cross-linked collagen scaffolds. A representative microphotograph of cell attachment and proliferation on day 3 is given in supplementary Fig. 3. The antimicrobial properties of both silver and curcumin will negate

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chances of any contamination that could occur at the wound site. The results indicated that collagen scaffolds cross-linked with curcumin caged silver nanoparticles are superior to glutaraldehyde

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cross-linked collagen scaffolds indicating that curcumin caged silver nanoparticle can be used as an

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alternative for crosslinking agent of collagen. The “adding-in” of therapeutic values of curcumin and silver on to the collagen scaffolds when used as a cross-linker of collagen makes this an ideal

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material for wound healing applications.

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Preferred location for Fig. 9

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3.5.2. Antimicrobial activity

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The anti-microbial ability of collagen scaffold cross-linked with curcumin caged silver nanoparticle (CCSNP) was investigated by both agar diffusion method and broth macro dilution method. Broth

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macro dilution assay was carried out in Escherichia coli (gram negative bacteria) and Bacillus subtilis (gram positive bacteria) by culturing them in Luria Bertani broth. Fig. 10 shows the bactericidal kinetics testing results of curcumin caged silver nanoparticle cross-linked collagen scaffolds. Collagen sheets cross-linked with 10 and 20 µM curcumin caged nanoparticle were first tested for anti-microbial activity by agar diffusion assay Fig. 10 a and c. The results showed that collagen cross-linked with curcumin caged silver nanoparticle showed a concentration dependent bactericidal activity in both E. coli and B. subtilis cultures respectively. Collagen scaffolds crosslinked with 20 µM curcumin caged silver nanoparticles had higher inhibition in both E. coli and B. subtilis cells as reflected by a greater zone of inhibition when compared to collagen scaffolds cross-linked with 10 µM curcumin caged silver nanoparticles. Fig. 10 b shows Blank cultures. Fig. 22 Page 22 of 42

10 d and e shows the growth inhibition of 10 and 20 µM curcumin caged silver nanoparticle crosslinked collagen scaffolds on E coli and B subtilis by broth macro dilution assay. The optical density of the growth medium directly correlates to proliferation and viability of the bacterial cells. The

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change in optical density of conditioned media can be associated with the ability of nanoparticle to inhibit the growth of these bacteria. The results showed that at highest concentration tested (20µM)

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there was maximum growth inhibition in both the bacteria (95% and 80 % growth inhibition in E.

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coli and B. subtilis respectively). A concentration dependent increase in inhibition of microbial cell growth was observed in curcumin caged silver nanoparticles treated cultures in comparison to

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control. A growth inhibition of 90% and 65% was observed in gram positive B. subtilis and gram negative E. coli respectively in microbial cultures treated with 10 µM curcumin caged silver

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nanoparticle cross-linked collagen scaffolds. For biomedical applications, use of biomaterials with

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anti-microbial property will be an added advantage. Collagen sheets incorporated with curcumin

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caged nanoparticle having such antimicrobial activity will prevent infection, sepsis and can be a

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good material for preparing wound dressing. Preferred location for Fig. 10 4. Conclusion

The interaction between curcumin, curcumin caged silver nanoparticle and collagen was studied by analyzing the physicochemical properties of collagen and collagen cross-linked with curcumin caged silver nanoparticles. Curcumin caged silver nanoparticle enhanced the viscosity, selfassembly process of collagen and improved its mechanical and thermal properties. The three dimensional conformation of collagen was also retained after cross-linking with curcumin caged silver nanoparticle. The curcumin caged silver nanoparticle also had an enhancing effect on cell viability, in comparison to collagen films cross-linked with or without curcumin. The antimicrobial 23 Page 23 of 42

property of silver and curcumin were retained even after conjugation and with both cell proliferative and anti-microbial property, curcumin caged silver stabilized collagen can be ideal for biomedical applications. In conclusion, curcumin caged silver nanoparticle can be considered as a cross-linking

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agent for collagen and provide a scope for alternate biocompatible interventions in the development of wound dressings.

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Acknowledgements

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We would like to thank The Director, CSIR–CLRI, Chennai, India for providing the facilities to

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carry out the experiments. Financial assistance from CSIR, Govt. of India for the supra-institutional project-STARIT (CSC0201) and NanoSHE (BSC0112) under XII five year plan is greatly

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acknowledged.

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Legends to Figures Fig. 1: FT-IR spectra showing peaks of i) Curcumin nanoparticle (CUR NP), ii) Curcumin

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solubilized (CUR SOL) from silver nanoparticle iii) Curcumin control (CUR CONTROL) Fig. 2: Size distribution by Dynamic Light Scattering analysis a) Control silver nanoparticles b)

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curcumin caged silver nanoparticles.

Fig. 3: Scanning Electron Microscopy analysis (SEM) and Energy dispersive X-Ray Spectroscopy

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(EDAX) a) Control nanoparticle b) curcumin caged silver nanoparticle c) EDAX of Control silver nanoparticles d) EDAX of curcumin caged silver nanoparticle.

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Fig. 4: X-ray diffraction (XRD) pattern of curcumin caged silver nanoparticle.

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Fig. 5: Kinetics of fibril formation in native collagen (Col), curcumin caged silver nanoparticles

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(ColCCSNP) and glutaraldehyde cross-linked collagen (ColGlu). * Statistically significant

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compared to native control collagen (p < 0.05).

Fig. 6: Rheological analysis a) viscosity of collagen cross-linked with curcumin caged silver nanoparticles (ColCCSNP), glutaraldehyde (ColGlu) and native collagen (Col) b) Shear stress vs. shear rate of collagen cross-linked with curcumin caged silver nanoparticles (ColCCSNP), glutaraldehyde (ColGlu) and native collagen (Col). * Statistically significant compared to native control collagen (p < 0.05).

Fig. 7: a) The DSC thermograms of native collagen scaffold (Collagen), collagen scaffolds crosslinked with curcumin caged silver nanoparticle (Collagen Cur) and glutaraldehyde (Collagen Glu). b) TGA curves of native collagen scaffolds (Collagen), collagen scaffolds cross-linked with curcumin caged silver nanoparticle (Col-Cur) and glutaraldehyde (Col-Glu) 28 Page 28 of 42

Fig. 8: Circular dichroic spectra a) native collagen b) collagen cross-linked with glutaraldehyde c) collagen cross-linked with curcumin caged silver nanoparticles.

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Fig. 9: Cytotoxicity and Cell proliferation of collagen scaffold cross-linked with different concentrations of curcumin (C10, C15 and C20), curcumin caged silver- nanoparticle (CNP10,

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CNP15 and CNP20), glutaraldehyde (50µM) (CG) and native collagen control (CC) for day 1, day 3

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and day 5. * - Statistically significant compared to native control collagen on respective days (p < 0.05). # - Statistically significant compared to glutaraldehyde cross-linked collagen on respective

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days (p < 0.05). $ Statistically significant compared to respective concentrations and days of curcumin cross-linked collagen (p < 0.05).

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Fig. 10: Anti-microbial activity of collagen scaffolds cross-linked with 10 and 20 µM curcumin

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caged silver nanoparticle using agar diffusion method. a) and c) shows zone of inhibition in E. coli

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and B. subtilis respectively b) represents blank. Anti-microbial activity of collagen scaffolds crosslinked with curcumin caged silver nanoparticle using broth macro dilution method d) and e)

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represents percentage growth of microbes on treatment with curcumin caged silver nanoparticle cross-linked collagen scaffolds (10 and 20 µM) for E. coli and B. subtilis respectively. * Statistically significant compared to positive control (p < 0.05).

Supplementary Figures

Supplementary Fig. 1: a) Control silver nanoparticle b) Curcumin caged silver nanoparticles c) Supernatant containing curcumin solubilized from curcumin caged silver nanoparticle. Supplementary Fig. 2: TEM microphotograph of control silver nanoparticle and curcumin caged silver nanoparticle. 29 Page 29 of 42

Supplementary Fig. 3: Representative microphotographs showing proliferation of HaCaT cells on collagen scaffold cross-linked with native collagen, positive control, different concentrations of

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curcumin (A) and curcumin caged silver nanoparticles (B). The scale bar represents 10µm

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Curcumin caged Ag NPs

Z- Average (d.nm)

800

170

Poly dispersity Index (Pdi)

0.548

0.241

Zeta potential (mV)

-31.0

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Control Ag NPs

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-35.4

Table1.Comparative Dynamic light scattering analysis of size dispersity and Zeta potential of

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control silver nanoparticles and curcumin caged silver nanoparticles

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Tensile st (N/mm2)

Elongation at break (%)

CC

6.62 ± 0.02

3.48 ± 0.04

6.25 ± 0.02

C C NP

8.58 ± 0.01*

4.52 ± 0.03*

13.92 ±0.01*

Col Glu

3.67 ± 0.02*

2.04 ± 0.01*

5.92 ±0.03

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Max Load(N)

Table 2.Mechanical properties of curcumin caged silver nanoparticles(tensile strength and elongation at break) CC denotes the Control native collagen, C C NP denotes the collagen cross-

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linked with curcumin caged silver nanoparticles whereas Col glu denotes the collagen cross-linked

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with glutaraldehyde. * Statistically significant when compared to control native collagen

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