Synthesis of poly(3-hydroxybutyrate- co ...

Synthesis of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/Chitosan/Silver Nanocomposite Material

with Enhanced Antimicrobial Activity

M. Rennukka

School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

C.S. Sipaut

School of Engineering and Information Technology, Universiti Malaysia Sabah, P.O. Box 2073, 88999, Kota

Kinabalu, Sabah, Malaysia

A.A. Amirul

School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

Malaysian Institute of Pharmaceuticals and Nutraceuticals, MOSTI, 11900, Bayan Lepas, Penang, Malaysia

Corresponding author: e-mail: [email protected], phone: +604-6533888 ext 4013

This work aims to shed light in the fabrication of poly(3-hydroxybutyrate-co-44%-4-

hydroxybutyrate)[P(3HB-co-44%4HB)]/chitosan-based silver nanocomposite material using different contents

of silver nanoparticle (SNP); 1- 9 wt%. Two approaches were applied in the fabrication; namely solvent casting

and chemical cross-linking via glutaraldehyde (GA). A detailed characterization was conducted in order to

yield information regarding the nanocomposite material. XRD analysis exhibited the nature of the three

components that exist in the nanocomposite films: P(3HB-co-4HB), chitosan and SNP. In term of mechanical

properties, tensile strength and elongation at break were significantly improved up to 125% and 22%,

respectively with the impregnation of the SNP. The melting temperature of the nanocomposite materials was

increased whereas their thermal stability was slightly changed. SEM images revealed that incorporation of 9

wt% of SNP caused agglomeration but the surface roughness of the material was significantly improved with

the loading. Staphylococcus aureus and Escherichia coli were completely inhibited by the nanocomposite films

with 7 and 9 wt% of SNP, respectively. On the other hand, degradation of the nanocomposite materials

outweighed the degradation of the pure copolymer. These bioactive and biodegradable materials stand a good

chance to serve the vast need of biomedical applications namely management and care of wound as wound

dressing.

Keywords: poly(3-hydroxybutyrate-co-4-hydroxybutyrate), chitosan, SNP, nanocomposite material,

antimicrobial, biodegradability

Formulation and Engineering of Biomaterials Biotechnology ProgressDOI 10.1002/btpr.1986

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article asdoi: 10.1002/btpr.1986© 2014 American Institute of Chemical Engineers Biotechnol ProgReceived: Sep 01, 2013; Revised: Aug 21, 2014; Accepted: Aug 22, 2014

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Introduction

Many natural polymers have potential as biotechnological and biomedical materials due to their

attractive properties such as non-toxicity, biodegradability and biocompatibility. However, a single

homopolymer is unable to meet the vast criteria of a biomaterial, thus blending and composite formations are

selected to upgrade their performance.1

Polyhydroxyalkanoate (PHA) is synthesized by microbes under the

stress condition such as limitation of phosphorus or nitrogen, but in the presence of excess carbon.2 Poly(3-

hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the PHA copolymer that can be tailored

into different monomer compositions by the utilization of two different carbon sources.3 P(3HB-co-4HB) has a

good potential in the biomedical applications as the 4-hydroxybutyrate (4HB) is a natural human metabolite.4

In

spite of this, P(3HB-co-4HB) has gained much attraction in medical field due to its ability to be hydrolyzed by

eukaryotic lipases into natural metabolites similar to those present in human body.5 All these indicate that

P(3HB-co-4HB) copolymer has a great future in medical and pharmaceutical applications due to its attractive

features.6 In addition, P(3HB-co-4HB) can be modified in many ways to overcome or minimize the chief

drawbacks in their properties such as absence of hydrophilicity and bioactivity, so that a new low cost product

can be obtained.7

Therefore, blending with natural polymer such as chitosan (CS) is an excellent way to develop a novel

biomaterial because it has active groups such as amine, amide and hydroxyl, which will enable the interaction

with the copolymer.8 CS consists of 2-acetamido-2-deoxy-β-ᴅ-glucopyranose and 2-amino-2-deoxy-β-ᴅ-

glucopyranose residues and it is found by Professor C. Rouget in 1859.9 CS is a good functional material

because it has many desirable attributes such as biodegradability, biocompatibility, non-toxicity and adsorption

properties.10

Moreover, its antimicrobial activity arises from the cationic charge and enables it to aggressively

bind onto the microbial cell surface causing membrane shrinkage and finally death of the cell.11

Both of the

polymers namely P(3HB-co-4HB) and chitosan have a good potential in wound dressing field due to their

features.

Based on our previous study, the mechanical properties of the blend material were not significantly

improved with the blending. Moreover, antimicrobial performance of the blend material was not sufficient to

inhibit and completely kill the microorganisms.12

Therefore, it intrigues us to incorporate another material that is

able to enhance both the mechanical and antimicrobial properties. Decades ago, the benefit of SNP is undeniable

as it is greatly known as an anti-infection agent but it is pushed aside by the presence of antibiotics and other

disinfectants. Recently, incorporating SNP has regained popularity because of the improved physicochemical

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and biological properties it offers when compared with the bulk parent materials 13

SNP is non-toxic and

environmentally friendly antibacterial material but it has poor binding affinity to the surface. Therefore, SNP is

normally cross-linked with the surface of the polymer matrix. So, researchers are focusing in developing the

polymer silver nanocomposite as it has promising future in biomedical applications.14

Chemical cross-linking is a direct method to produce a permanent network by the formation of covalent

bonding between the polymeric chains.18

Chemical cross-linking agent that is commonly utilized is GA and

formation of acetyl bridges will occur when this cross-linking agent is used together with sulphuric acid.19

The

mechanism takes place with nucleophilic attack by nitrogen of the amino group on the carbon group of GA,

which replaces the oxygen of the aldehyde forming imine bond (C=N).20

Herein, it is noteworthy that chemical

cross-linking agent can be used to fulfil the demand of skin tissue engineering as the physical cross-linking

cannot yield high cross-linking degree.21

Our previous study suggested that blend films of P(3HB-co-4HB) with 20 wt% of chitosan were more

hydrophilic with optimal pore size and biocidal activity.12

In this present investigation, nanocomposite material

was designed through the fabrication of P(3HB-co-44%4HB)/chitosan/silver whereby the content of the SNP

was varied from 1 to 9 wt%. In addition, the nanocomposite material was subjected to chemical cross-linking

using GA to form permanent network in the polymeric matrix. The pivotal goal of this work is to enhance the

mechanical and antimicrobial activity of the nanocomposite material. In depth characterization was also

performed in order to determine the effect of SNP loading towards the physical and thermal properties.

Furthermore, biodegradability of the nanocomposite materials was also explored as it could be one of the added

advantages of the nanocomposite material.

There are number of published works about the silver nanocomposites based on chitosan. According to

Rhim and co-workers, nanocomposite of chitosan and SNP has higher tensile strength and it is bacteriostatic and

bactericidal against both Gram negative and Gram positive bacteria compared with neat chitosan.15

Another

finding is done by Vimala and co-workers whereby polyethylene glycol (PEG)/chitosan/SNP is cross-linked

with GA and this nanocomposite has improved mechanical properties in terms of mechanical strength and has

exhibited good antimicrobial property.16

It is obvious that incorporation of SNP into the polymer matrix is

favourable for the improvement of mechanical strength and antimicrobial property. Shameli and co-workers

have prepared silver/poly(lactic acid) nanocomposite and they have achieved strong antibacterial activity with

high content of SNP (32 wt%) which have been detected by qualitative method (zone of inhibition) alone.17

To

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the best of our knowledge, there is no research that has been reported regarding the silver nanocomposite of

P(3HB-co-4HB)/chitosan.

Experimental

Bacterial strains, chemicals and reagents

P(3HB-co-44%4HB) copolymer was produced by fermentation using Cupriavidus sp. USM1020 (DSM

19416), isolated from sludge samples at Lake Kulim, Malaysia which was reported previously.22

Chitosan

powder from crab shells with high degree of deacetylation (90%) and molecular weight (600-800 kDa) was

purchased from Acros Organics Company, Belgium. SNP (327085) and lysozyme from chicken egg white

(lyophilized powder, protein ≥ 90% and ≥ 40 000 units/mg protein) [L6876] were purchased from Sigma-

Aldrich. Chloroform (Technical Grade AR), glacial acetic acid, sodium hydroxide, hydrochloric acid and GA

(cross-linking agent) were purchased from R&M Chemicals, United Kingdom. Two Gram negative bacteria

such as Escherichia coli (ATCC 11303); Pseudomonas aeruginosa (USM-AR2) and two Gram positive bacteria

Staphylococcus aureus (ATCC 12600); Bacillus subtilis (ATCC 6633) were used in this work.

Fabrication of P(3HB-co-4HB)/chitosan/silver nanocomposite films

The nanocomposite films of P(3HB-co-44%4HB)/20 wt% of chitosan were fabricated by varying the

content of SNP; 1, 3, 5, 7 and 9 wt%. First of all, chitosan (20 wt%) was dissolved in 30 mL of glacial acetic

acid solution by stirring at 90 °C for 20 minutes and cooled at room temperature. The pH of the solution was

adjusted to 5.5 using 0.1 M NaOH and the solution was filtered through 0.20 µm syringe filters to remove the

impurities.15

About 1.5 g of P(3HB-co-44%4HB) was dissolved in 30 mL of chloroform and was mixed with the

chitosan solution. The mixture was stirred to obtain a homogenous solution. Later, SNP was added into the

mixture and dispersed via ultra sonification using sonicator (IKA®

T25 digital ULTRA-TURRAX®, China) for

15 minutes and sonicator (Virsonic cell disrupter model 16-850, Germany) for 1 hour. The mixture was casted

on a glass Petri dish with a diameter of 11 cm. The solvents were evaporated and the nanocomposites films were

peeled of the Petri dish. Later, the samples were dried in constant weight in vacuum.

Cross-linking of the nanocomposite films

The nanocomposite films were cross-linked by using 5% (w/v) of GA up to 120 minutes. The cross-

linking mixture was consisted of isopropanol/water (90/10, v/v), 1% (v/v) of HCl as a catalyst and GA as the

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cross-linker. After the cross-linking reaction, the films were washed with distilled water and dried at room

temperature.23

X-ray diffraction (XRD) analysis

The structures of the SNP, chitosan and P(3HB-co-4HB) in the polymer matrix were evaluated using a

Simens D5000 X-Ray Powder Diffractrometer, Germany (40kV, 30 mA) with Cu Kα radiation with a wave

length λ= 0.154 nm. The scanning range was carried out between 2θ = 0-100° at a rate of 0.5°/minute.16

Tensile test

A standard test piece (75 mm gauge length and 4 mm width) in the shape of dumbbell was gripped at

either end by the suitable apparatus in the tensile machine and cut using the steel ASTM regulation punches

(GOTECH A1-3000 with U60 software, Taiwan), which slowly exerted an axial pull at the rate of 10

mm/minute so that the polymer was stretched until it broke. The thickness of the films was measured before the

testing. Tensile strength, Young’s modulus and elongation at break were deduced from the stress/strain curves.

The average value of five replicates was taken for each sample.24

Differential scanning calorimeter (DSC)

Thermal properties such as glass transition temperature (Tg), crystallization temperature (Tc) and

melting temperature (Tm) were determined using differential scanning calorimeter (Pyris 1 DSC, Perkin Elmer,

Germany) equipped with liquid nitrogen cooling accessory. Approximately, 5-6 mg of sample was encapsulated

in aluminium pans. First heating was carried at a rate of 10°C/minute from -50°C to 200°C. The samples were

cooled to -50°C at a rate of 20°C/minute before it was heated again to 200°C at 10°C/minute in the second

heating. The Tm and Tc were determined from the DSC endotherms whereas Tg was taken as the midpoint of the

heating capacity change.25

Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was conducted using Mettler-Toledo TGA/SDTA 851 (Mettler-

Toledo Corporation, Switzerland) thermobalance to determine the thermal stability. About 10 mg of the sample

was heated under nitrogen and air flows to 800 °C at a rate of 10 °C/minute. The thermal decomposition

temperature (Td) was determined from the thermogram.16

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Scanning electron microscopy (SEM)

Surface morphology of the P(3HB-co-4HB), chitosan, blend, cross-linked films and nanocomposite

material was observed using scanning electron microscopy (SEM, Leo Supra 50 VP Field Mission SEM; Carl-

Ziess SMT, Oberkochan, Germany). The films were mounted on aluminium stumps and coated with gold.

Quadrant back scattering detector (QBSD) was used to observe the SNP distribution in the polymeric matrix of

the nanocomposite material. Elemental mapping was also done by energy dispersive X-ray (EDX) facility of

SEM.26

Atomic force microscopy (AFM)

Atomic force microscopy (AFM) (Model Dimension Edge Bruker, Germany) was used to observe the

surface topography and to determine the surface roughness of the films. The images were recorded under

ambient conditions using a multimode nanoscope (Version 1.2) operating in the tapping mode regime.

Meanwhile, the sampling areas were standardized at 10 µm × 10 µm. Five different spots per film were

analyzed. Microfabricated silicon cantilever tips (MPP-11100-10) with a resonance frequency of 299 kHz and a

spring constant of 20–80 Nm were used.27

Disc diffusion assay

In the disc diffusion method, the films were cut into disc shape (5 mm in diameter) and sterilized under UV light

for 30 minutes. The Gram-positive and Gram-negative bacteria were cultured separately in nutrient rich (NR)

medium [5 g of peptone, 2 g of yeast extract, 1 g of beef extract, and 5 g of NaCl in 1 L distilled water] for 16-

18 hours at room temperature. After that, the bacterial cultures were diluted using the sterile distilled water until

107

colony forming units per millilitre (CFU/mL) was obtained. The diluted suspension was spread on the NA

plate before the discs were placed onto the plates which were incubated at 37 °C for 24 hours and the zone of

inhibition was measured using a ruler.15

Viable cell count method

For the viable cell count method, all the bacterial strains were grown and diluted according to the

previous method. Film samples were cut into square pieces (1 x 1 cm) and sterilized under UV light for 30

minutes. The films were placed into the universal bottles aseptically. Later, the diluted bacterial suspension was

transferred into universal bottles containing 5 mL of NR medium. All the bottles were incubated for 5 hours

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and the bottle without the blend film was the control. Finally, 50 µL of the suspension from the universal bottle

was spread on the NA plate which was incubated at 37°C for 24 hours. After the incubation, the number of the

colonies formed (CFU/mL) was counted and the reduction of the colonies due to the antibacterial activity was

determined in percentage.15

In vitro biodegradation test

The biodegradation studies of the P(3HB-co-44%4HB), P(3HB-co-44%4HB)/20 wt% chitosan cross-

linked with 5% (w/v) of GA and its nanocomposite films which were incorporated with 1 and 9 wt% of SNP

were conducted at 37 °C in 0.1 M phosphate buffer (PBS, pH 7.2–7.4) containing 2 mg/mL of lysozyme from

chicken egg white (Sigma-Aldrich). The UV sterilized films were cut into squares (1 x 1 cm) and incubated in

the reaction solution with gentle shaking (150 rpm). The samples were removed at various time points (every 7

days), washed in distilled water and allowed to dry in air until a constant weight was obtained. Three replicates

were used and the degradation rate (%) was determined by the ratio of the weight loss to the initial weight of

samples as shown below:

Degradation rate �%�= Wb-Wa

Wb ×100 (1)

Wa is the weight of the film after the degradation whereas Wb is the weight of the film before the degradation.28

Statistical analysis

The statistical analysis was performed by using SPSS 15.0 for Windows Software. The data for the

different parameters were statistically analysed using one way ANOVA with Tukey comparison test to

determine the presence of significant differences. A p value < 0.05 was considered as significant.

RESULTS AND DISCUSSION

XRD analysis of the nanocomposite films

Generally, X-ray diffraction (XRD) analysis was carried out to confirm the formation of SNP and to

reveal the crystal structure of the nanoparticles.29

Figure 1 illustrates the XRD diffractograms of the P(3HB-co-

44%4HB)/ 20 wt% CS cross-linked blend film, SNP and the nanocomposite films which were incorporated with

1, 5 and 9 wt% of SNP. P(3HB-co-44%4HB)/ 20 wt% CS blend film cross-linked with 5% (w/v) of GA

exhibited 5 intensity peaks at 2θ with the value of 13, 17, 25, 27 and 28°. The four peaks which were detected at

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13, 17, 25 and 27° were attributed to the (0 2 0), (1 1 0), (1 2 1) and (0 4 0) of the orthorhombic lattice structure

of the P(3HB) unit cells, respectively.30, 31

According to the reported data, the crystalline peak of chitosan was

detected around 22°.32

Therefore, the peak at 28° in the diffractogram might be the crystalline peak of the

chitosan. In the case of SNP, four prominent peaks at 2θ values about 38, 44, 64, 77 and 81° were observed and

they represented the 111, 200, 220, 311 and 222 Bragg’s reflections of the face-centered cubic (fcc) phase of the

silver.33

The XRD patterns of the nanocomposite films revealed the nature of the three components that exist in

the nanocomposite films: P(3HB-co-4HB), chitosan and SNP. Only one of the crystal lattice of the P(3HB) was

present in all the nanocomposite films around 13° (0 2 0). Besides, the nanocomposite films that were

impregnated with 1, 5 and 9 wt% had the chitosan’s crystalline peak at 32 and 36°, respectively. The

nanocomposite films all had the silver’s fcc phase, which was verified by the strong reflections around 38, 44,

64, 77 and 81°. The d-spacing values of the nanocomposites were similar with the blend and SNP. Therefore,

this gave clear evidence that the SNP was present in the nanocomposites.

Mechanical properties of the nanocomposite films

It is true that only antimicrobial materials with good mechanical strength will able to serve in the

wound dressing applications.16

Therefore, serious attention was paid in order to develop the nanocomposites

with enhanced mechanical properties than the cross-linked blend films. The mechanical properties of the

nanocomposite films are displayed in the Table 1. Based on the result, the nanocomposite’s mechanical strength

and elongation at break were significantly improved up to 125% and 22%, respectively in parallel with the

increasing content of the SNP. On the other hand, the Young’s modulus of the nanocomposite films was

decreased about 44% as the content of SNP was increased. There was a strong influence of the SNP

reinforcement towards the mechanical strength of the nanocomposites. By increasing the content of SNP, the

nanofillers restricted the polymer chain mobility due to their large surface area and their van der Waals

attraction to the polymer matrix contributed to the strengthening of the mechanical strength.34

It was imperative

to note that elongation at break value of the nanocomposite was relied on its molecular weight. Therefore, the

enhancement of the elongation at break with the higher SNP loading was due to the increase in the molecular

weight of the nanocomposite. This was also attributed by the formation of complexes with the silver ions.35

Moreover, the decrease in the Young’s modulus values of the nanocomposite films could be explained by the

strong bonding of the SNP with the polymeric matrix. It was because increase in Young’s modulus followed

with decrease in the tensile strength and elongation at break when the silver content was raised could be

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attributed by poor bonding of SNP with the polymeric matrix.36

These findings proved that mechanical

properties of the nanocomposites will give the wound better elasticity and good strength to assist the use of

wound dressing in high stress.37

In addition, flexibility of the nanocomposite material also ensures the material

can be easily peeled off from the wound surface without any secondary injury or pain and enables better

conformation to the wound surface.38

Thermal properties of the nanocomposite films

The thermal properties, namely melting temperature and thermal stability (thermal decomposition

temperature), are shown in Table 2. According to the findings, only the melting point (Tm) of the nanocomposite

could be detected in the thermogram and within the measurement sensitivity not much difference was seen for

crystallization temperature. In contrast with the uncross-linked and cross-linked blend films, different pattern of

melting temperature was observed for the nanocomposite films when the content of SNP was increased from 1

to 9 wt%. The melting point of the nanocomposite films was significantly increased with the increasing content

of SNP. Moreover, the increment was up to 167°C which was 6.4% higher than the melting temperature of the

P(3HB-co-44%4HB)/20 wt% chitosan cross-linked with 5% (w/v) of GA. Such phenomenon was usual for the

polymer nanocomposites because nanoparticle could act as a nucleating agent for the crystallisable polymers

which leaded to a more perfect crystalline structure.39

In addition, this was also due to SNP which act as filler

did not reduced the mobility of the polymeric chain when attached to the surface of the polymeric matrix.40

The

thermal stability of the nanocomposite films was not much affected by the addition of the different contents of

SNP. Moreover, the thermal decomposition temperatures of the nanocomposite film were only reduced about

10% from 300°C to 270°C when compared with the P(3HB-co-44%4HB)/20 wt% chitosan cross-linked blend

film. This result demonstrated that presence of SNP promotes the degradation of the nanocomposite film.41

Surface morphology of the nanocomposite films

SEM-EDX via quadrant back scattering detector (QBSD) and AFM were employed to observe the

morphology of the nanocomposite films in term of dispersion of the SNP in the polymeric matrix and effect of

the filler towards the roughness of the nanocomposite films. EDX was used to investigate the elemental

distribution or chemical composition of the nanocomposite films in order to verify the presence and the

distribution of fillers in the polymer matrix.42

The SEM images and the elemental mapping including the

analysis of the nanocomposite film are represented in Figure 2. The SEM images indicated the presence and

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even distribution of the SNP throughout the surface of the polymeric matrix. It was evident that the dispersion of

SNP was homogenous in all the nanocomposite films except the one filled with 9 wt% of SNP. The image of the

latter nanocomposite clearly showed the existence of agglomeration on the surface of the polymer. Therefore,

there was a limit in the incorporation of the SNP because it is susceptible to agglomeration when the content of

the SNP exceeded 7 wt%. It was indicated that high concentration of SNP tends to change the shape of the

particle from spherical to asymmetrical that caused the clusters to coagulate into large particles.43

Besides that,

the elemental mapping via EDX exhibited the presence of element namely carbon, nitrogen and silver which

stands for P(3HB-co-4HB), chitosan and SNP, respectively.

The AFM topographic images (Figure 3) demonstrated that all the nanocomposite film’s surface was

rougher with grain like morphology. Furthermore, 3D images of the nanocomposite films enabled more precise

view of the surface compared with the 2D images. The experimental results indicated that impregnation of SNP

had significantly increased the surface roughness of the nanocomposite films. Based on the comparison among

the nanocomposite films, it was revealed that increase in SNP content was accompanied with high degree of

surface roughness (Table 3). The SNP tend to increase the Ra from 0.79 to 1.01 µm whereas RMS from 314 to

708 nm of the nanocomposite film. A dramatic increase in the RMS value showed that the heights of the peaks

were drastically increased with the addition of the SNP compared to the mean surface roughness of the entire

volume of the film (Ra). The surface roughness was generally caused by the solvent evaporation. In addition,

formation of aggregates was due to the small silver particles that clustered when the solvent was evaporating.44

The changes in the surface roughness might be the reflection of the rearrangement of the macromolecules due to

the presence of SNP.45

Basically, the surface properties of the material namely topography and/or surface energy

could influence the cell adhesion and proliferation which would determine the acceptance or rejection of the

antibacterial dressing material.46

Based on the findings, higher surface roughness is crucial for the cell

attachment and proliferation.

Assessment of antimicrobial activity of the nanocomposite films

The diameter of the zone of inhibition against the bacteria was found to increase when the content of

the SNP was raised from 1 to 9 wt% (Table 4). The formation of zone of inhibition is due to the diffusion of the

silver ion from the nanocomposite material into the agar which prevent the growth of the bacteria in that

particular area. Silver metal is known to slowly change to silver ions and interact with the bacterial cells.

Besides, the concentration of the silver ions is not high enough to cause normal human cells damage.47

It was

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interesting that the nanocomposite films could inhibit the bacteria whether it is a Gram-negative or Gram-

positive bacterium. It was well known that the inhibition against Gram-negative bacteria was greater than Gram-

positive due to the presence of chitosan. After the addition of this filler, the efficiency of the nanocomposite

films was equally improved than the cross-linked blend films. In addition, antimicrobial efficacy of these

nanocomposite films were further clarified by the reduction in the number of colonies of the bacteria when they

were cultured together with the nanocomposite films (Table 5). The bactericidal effect of the nanocomposite

films was excellent because 100% of the E. coli was killed after 24 hours of incubation when the content of the

filler was 7 wt%. Meanwhile, nanocomposite film incorporated with 9 wt% of SNP was required for total

reduction of P. aeruginosa, S. aureus and B. subtilis. It should be noted that agglomeration on the surface of the

nanocomposite with 9 wt% of SNP (SEM image) did not interfere with its antimicrobial performance. The

antibacterial property of the nanocomposite films was significantly promoted by impregnating SNP in different

contents. As noted earlier, SNP was selected based on its strong bactericidal effects against broad spectrum of

microorganisms. It was expected that particle size would influence the bactericidal action because change in

particle size would change the surface area which would be in contact with the bacterial species. Moreover, in

particular filler geometry, the total surface area of the filler particles was significantly increased with the

growing filler content.48

Therefore, this implied that silver in nano size had a great tendency inhibiting the

growth of the bacteria.49

In addition, SNP could constitute sophisticated biomaterials that could serve in a

number of promising applications.50

As a wound dressing material, silver ions were constantly inactivated by

chloride or organic matter would require the maintenance of silver concentration by frequently changing the

dressing. The nano-silver was less affected by the wound environment compared to the ionic silver whereby the

same concentration is enough to induce the bactericidal activity.51

Furthermore, the capability of the blend films

to bind to the negatively charged bacteria would reduce the primary wound contamination and protects the

wound from secondary bacterial infection.52

Besides biotic control, silver ions could be also detected in the

abiotic control such as water and soil. The concentrations of the SNP in different environmental compartments

are much lower than other types of nanomaterials. However, transformation of the SNP by environment is the

one which will determine its chemical composition, bioavailability, eco-toxicity and its direct effects on

humans.53

In vitro biodegradation

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In vitro biodegradability test was performed in order to investigate the effect of composite formation

towards the biodegradability of the material (Figure 4). The P(3HB-co-44%4HB) copolymer film was only

degraded up to 5% at 8th

week of degradation whereby the percentage of degradation became constant during

the 6th

week of degradation. The enzymatic degradation of the cross-linked blend and nanocomposite films with

1 and 9 wt% of SNP was much faster than the pure P(3HB-co-4HB) film. The blend film exhibited 22% of

degradation whereas nanocomposite with 1 and 9 wt% of SNP exhibited 19 and 11% of degradation,

respectively. In addition, the degradation was 2-3 folds higher than the pure film. Besides, surface erosion was

also observed on the surface of the films that were degraded for 56 weeks (Fig. 5). Biodegradation is the

degradation that takes place in a biological environment whereas in vitro degradation test was carried out to

inspect the performance of the biodegradable polymer in the clinical situation. It was expected that enzyme

would play an important role in the enzymatic degradation of biomaterial whereby it would catalyze the

hydrolysis.54

Lysozyme was chosen for this biodegradation because it had the capability to degrade chitosan.

Moreover, chitosan is biodegradable in humans because lysozyme can be found in numerous body fluids and

tissues, serum, tears and mucosa.55

There are two possible contributions to the improved biodegradability of the

cross-linked nanocomposite; namely, the reduced crystallinity of P(3HB-co-4HB) and the hydrophilicity of the

chitosan which made the it easier for the enzyme to attack. The synergistic effect of lower crystallinity and good

hydrophilicity of chitosan accelerated the degradation of the nanocomposite material.56

However, it was clear

that high content of SNP somehow hindered the degradation of the nanocomposite film. The retardation was due

to the decreased polymer chain’s mobility caused by the SNP and consequent suppression of the degradation

reactions of the nanocomposite. Besides, the increasing content of silver embedded in the polymeric matrix also

slowed down the degradation of the nanocomposite.57

Conclusions

P(3HB-co-44%4HB)/20 wt% of CS cross-linked with 5% (w/v) of GA was selected for the preparation

of the silver nanocomposite based on the improved mechanical strength, high percentage of cross-linking, good

hydrophilicity, larger pore size, excellent surface roughness and enhanced antimicrobial activity. The presence

of the SNP in the nanocomposite film was confirmed by the XRD analysis. The mechanical strength was

increased up to 18 MPa whereas the melting temperature was also higher than the cross-linked blend films. In

addition, SEM images revealed that the surface of the nanocomposite films was homogenous except the film

with 9 wt% of SNP. However, the surface roughness tends to significantly increase with the content of the filler.

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Last but not least, the antimicrobial performance of the nanocomposite films was excellent because 100% of

inhibition against two of the bacteria was achieved. Finally, the biodegradability test showed that the cross-

linked nanocomposite films were degraded faster than the pure copolymer.

Acknowledgements

The author acknowledged the short term research grant (304/PBIOLOGI/6313019) provided by

Universiti Sains Malaysia, MyMaster (MyBrain15) and USM Science Fellowship

(RU:1001/441/CIPS/AUPE001) awarded to Rennukka that have resulted in this article.

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Table 1. Mechanical properties of nanocomposite films of the P(3HB-co-44%4HB)/20 wt% CS

cross-linked with 5% (w/v) of GA incorporated with different contents of SNP

Concentrations of SNP Tensile strength Elongation at break Young's Modulus

(wt%) (MPa)a

(%)a

(MPa)a

0 6 ± 1D

196 ± 2F

5 ± 1A

1 8 ± 1CD 205 ± 3E 5 ± 1A

3 10 ± 1C

215 ± 2D

5 ± 1A

5 13 ± 1B

226 ± 3C

4 ± 1AB

7 15 ± 2B 236 ± 2B 3 ± 1B

9 18 ± 1A

250 ± 3A

3 ± 1B

aDetermined using tensile machine. Values are means ± SD of three replications

Mean in the same column that was labelled with different letters are significantly different at p = 0.05 and

confidence interval for the difference between means is 95% Tukey test (HSD)

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Table 2. Thermal properties of nanocomposite films of the P(3HB-co-44%4HB)/20 wt% CS cross-

linked with 5% (w/v) of GA incorporated with different contents of SNP

Concentrations of SNP Thermal properties (°C)

(wt%) Tga Tc

a Tma Td

b

0 ND ND 157.3 ± 0.4E 300

1 ND ND 159.5 ± 1.1D 290

3 ND ND 163.3 ± 0.8C 290

5 ND ND 165.3 ± 0.7B 290

7 ND ND 166.4 ± 0.5AB

280

9 ND ND 167.3 ± 0.5A 270

a Glass transition and melting temperature determined using DSC a Values are means ± SD of three replications

ND means not detected b

Decomposition temperature determined using TGA b Values are means of two replications

Mean in the same column that was labelled with different letters are significantly different at p = 0.05

and confidence interval for the difference between means is 95% Tukey test (HSD)

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Table 3. Surface roughness of nanocomposite films of the P(3HB-co-44%4HB)/20 wt% CS cross-

linked with 5% (w/v) of GA incorporated with different contents of SNP

Concentrations of SNP Ra (µm)a Rms (nm)

a

(wt%)

0 0.70 ± 0.03E 242 ± 4F

1 0.79 ± 0.02D

314 ± 4E

3 0.84 ± 0.03CD

436 ± 3D

5 0.90 ± 0.04BC 554 ± 2C

7 0.95 ± 0.03AB

670 ± 4B

9 1.01 ± 0.03A

708 ± 5A

a Calculated based on AFM images. Values are means ± SD of three replications


and confidence interval for the difference between means is 95% Tukey test (HSD).

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Table 4. Qualitative assessment of nanocomposite films of the P(3HB-co-44%4HB)/20 wt%

chitosan cross-linked with 5% (w/v) of GA incorporated with different contents of SNP

Contents of Diameter of zone of inhibition (mm)

SNP Escherichia Pseudomonas Staphylococcus Bacillus

(wt%) coli aeruginosa aureus subtilis

1 15 ± 1d 11 ± 1

d 9 ± 1

d 8 ± 1

c

3 16 ± 1cd

13 ± 1c

11 ± 1c

9 ± 1c

5 17 ± 1bc

15 ± 1b

12 ± 1bc

11 ± 1b

7 18 ± 1ab 17 ± 1a 13 ± 1ab 12 ± 1ab

9 19 ± 1a

18 ± 1a

14 ± 1a

13 ± 1a

Values are means ± SD of three replications


and confidence interval for the difference between means is 95% Tukey test (HSD)

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Table 5. Antimicrobial activity via colony count method of nanocomposite films of the P(3HB-co-44%4HB)/20 wt% chitosan cross-linked with 5% (w/v) of

GA incorporated with different contents of SNP

Bacteria E. coli P. aeruginosa S. aureus B. subtilis

Contents of SNP (CFU/mL) Reduction (CFU/mL) Reduction (CFU/mL) Reduction (CFU/mL) Reduction

(wt%) (107) (%) (10

7) (%) (10

7) (%) (10

7) (%)

Control 4.0 ± 1.0 0 4.2 ± 1.2 0 4.2 ± 1.1 0 4.2 ± 1.0 0

1 2.4 ± 0.4 40 ± 4d 2.4 ± 0.5 43 ± 4

d 3.0 ± 0.3 29 ± 4

c 3.3 ± 0.6 21 ± 6

d

3 1.7 ± 0.3 58 ± 6c 1.4 ± 0.4 67 ± 8

c 2.7 ± 0.2 36 ± 3

c 2.6 ± 0.5 38 ± 4

c

5 0.8 ± 0.2 80 ± 2b 0.7 ± 0.1 83 ± 6

b 1.4 ± 0.5 67 ± 6

b 1.6 ± 0.3 62 ± 5

b

7 0 100 ± 0a 0.1 ± 0 98 ± 0a 0.3 ± 0.1 93 ± 5

a 0.4 ± 0.1 90 ± 3

a

9 0 100 ± 0a 0 100 ± 0

a 0 100 ± 0

a 0 100 ± 0

a

Values are means ± SD of three replications

Mean in the same column that was labelled with different letters are significantly different at p = 0.05 and confidence interval for the difference between means is 95%

Tukey test (HSD)

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Figure 1. XRD patterns of the cross-linked P(3HB-co-44%4HB)/20 wt% CS (a), SNP (b) and its

nanocomposite films incorporated with 1 (c), 5 (d) and 9 (e) wt% of SNP.

a

b

c

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Figure 1. Continued

d

e

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Figure 2. Surface morphology and elemental mapping of nanocomposite films of the P(3HB-co-

44%4HB)/20 wt% CS cross-linked with 5% w/v of GA incorporated with 1 (a), 3 (b), 5

(c), 7 (d) and 9 (e) wt% of SNP under 100x magnification using quadrant back

scattering detector (QBSD).

a

b

c

d

e

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Figure 3. AFM topographic images of nanocomposite films of the P(3HB-co-44%4HB)/20 wt% CS

cross-linked with 5% w/v of GA incorporated with 1(a), 3 (b), 5 (c), 7 (d) and 9 (e) wt%

of SNP and their 3 dimensional images (f, g, h, i & j).

a

b

c

d

e

f

g

h

i

j

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Figure 4. The degradation rates of the P(3HB-co-44%4HB)/20 wt% CS blend film (A), P(3HB-co-

44%4HB)/20 wt% incorporated with 1 (B), 9 (C) wt% of SNP and P(3HB-co-44%4HB)

(D). Values are means ± SD of three replications. Point that was labelled with different

letters are significantly different at p = 0.05 and confidence interval for the difference

between means is 95% Tukey test (HSD).

0

5

10

15

20

25

0 7 14 21 28 35 42 49 56

Deg

radat

ion (

%)

Time (days)

A

B

C

D

bcc

d

de

eff

a

abbc

cd

de

ef

f

g

ab

bcbc

cdde

dee

aaab

bccd

dd

b

a

a

a

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Figure 5. SEM images of the P(3HB-co-44%4HB) (a), cross-linked P(3HB-co-44%4HB)/20 wt%

chitosan (b), nanocomposite films incorporated with 1 (c) and 9 (d) wt% of SNP before

degradation and its images after degradation (e, f, g & h).

Legend: 1cm = 100 µm

a e

b

c

d

f

g

h

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