Antimicrobial activity of poly( l -lactide acid)/silver nanocomposite fibers

Abstract

Silver (Ag) nanoparticles (NP) and poly(lactide acid) (PLA) granules were microcompounded to form a nanocomposite. A series of PLA nanocomposite fibers containing, respectively, 0, 0.5, 1, 3 or 5 wt% Ag were produced and their antimicrobial activity against Gram-negative and Gram-positive bacteria evaluated. It was found that the PLA/Ag nanocomposite fibers exhibited increased antimicrobial activity, depending on the filler content. On the other hand, mechanical and thermal characterization tests, including thermogravimetric analysis, differential scanning calorimetry and tensile testing, showed that increasing concentrations of Ag hindered the mechanical properties of Nanocomposites due to partial agglomeration, leading to the generation of flaws. The crystallinity of the fibers was found to decrease by about 23% if the Ag content was increased to 5%. This could be attributed to a more rapid cooling rate resulting from the high thermal conductivity of the Ag particles.

Keywords

Antimicrobial nanocomposite poly(lactide acid)silver nanoparticles

Research on biodegradable polymers has increased in importance in recent years due to their wide range of applications in biomedicine, packaging and agriculture. Aliphatic–aromatic polyesters, such as poly(lactide acid) (PLA), polycaprolactone, poly(butylene adipate terephthalate) and polyhydroxybutyrate are examples of popular biodegradable polymers. Poly(lactide acid) [PLA; (–CHCH₃–CO–O–) _n ] is a biodegradable biomedical polyester. It can reduce wastage by decaying to nontoxic products. PLA is biocompatible and in the body undergoes scission to monomeric units of lactic acid, which is a natural intermediate in carbohydrate metabolism.¹ These properties make PLA suitable for use in absorbable sutures, as carriers for the controlled release of drugs, and in implants in orthopedic surgery and blood vessels.² PLA is synthesized from l- and d-lactic acid produced by the fermentation of polysaccharides such as sugar feedstocks, corn, wheat and other starch sources. PLA generally implies poly-l-lactide acid (PLLA), which contains a greater proportion of l-lactic acid and is the form employed commercially, since PLLA gives polymers of higher crystallinity.³

Silver nanoparticles (AgNPs) have the novelty of combining long-lasting antimicrobial properties with high temperature stability and low volatility.⁴ The inherent heat resistance of silver makes AgNPs acceptable for high temperature processing of thermoplastics. The antimicrobial activity of metallic silver is still not well understood, although that of silver ions has been well established. In previous studies it has been confirmed that the activity of AgNPs is similar to that of silver cations, Ag⁺.⁵ The latter bind strongly to electron donor groups in biological molecules containing sulphur, oxygen or nitrogen. In order to be effective silver-containing antimicrobial polymers have to release Ag⁺ into a pathogenic environment. Oxidation of metallic silver to the active species, Ag⁺, is possible through interaction of the silver with water molecules. A steady and prolonged release of the Ag⁺ at a concentration level of 0.1 ppb is required for antimicrobial efficacy.^4–7 The Ag⁺ ions bind to the cell membrane and can also penetrate inside the bacteria. When the ions penetrate the bacterial cell, they interact with phosphorus-containing compounds such as DNA. Also within the bacterial cell, they can attack its respiratory chain and inhibit cell division, leading to death of the bacterium.^8,9

Four methods are available for synthesis of nanocomposites – solution intercalation, in situ polymerization, melt intercalation and template synthesis. Melt intercalation is the most suitable method, due to its versatility and its compatibility with polymer processing equipment. In this method, nano fillers and polymer are added together above the melting temperature of the polymer. They may be held at this temperature and shear applied to give intercalation.^3,7 In the present study antimicrobial PLA/Ag nanocomposite fibers were prepared by the intercalation method and their antimicrobial activities against Gram-negative and Gram-positive bacteria evaluated. The fibers themselves were characterized by differential scanning calorimetry (DSC), thermo-gravimetric analysis (TGA), scanning electron microscopy (SEM), water uptake and tensile testing. As a result of these tests the optimum concentration could be determined for preserving antibacterial activity while maintaining fiber characteristics. The applicability of these fibers to the prevention of infection in medical applications, such as sutures and wound dressings, has been investigated.

Experimental details

Materials

The PLA granules were fiber grade 6201D, purchased from Naturework, USA. The melting temperature of the granules was 170℃, and the melt-spinning temperature was 190℃. The AgNPs were purchased from Sigma–Aldrich, USA. The particles were below 100 nm in size; they had a surface area of 5.0 m² g⁻¹ and a melting point of 960℃.

Methods

Nanocomposite fibers were extruded in a DSM Xplore micro-compounder by feeding 10 g PLA granules and, depending on filler percentages, 0.05, 0.10, 0.31, or 0.53 g AgNPs. Before melt spinning, the polymer granules were dried at 80℃ for 24 h to give a moisture content below 5 wt%. Polymer granules and AgNPs were fed into the compounder at a temperature of 190℃ and a screw speed of 100 rpm, and mixed for 15 min. These conditions were used in all cases. The melt was extruded through a spinneret (D = 0.8 mm) and carried over the godets at a constant draw ratio (λ = 3). Neat PLA fibers were prepared similarly.

A JEOL JSM 5310, Scanning Electron Microscope (SEM) was used to assess the dispersion state of the AgNPs in the PLA matrix. The fiber surface and cross-section were examined at 10 kV acceleration voltage to obtain approximately 200× and 1000× magnification. The fibers for SEM examination were initially fractured in liquid nitrogen and silver coated before observation.

TGA and DSC were used to determine the thermal properties of the fibers. TGA was performed using a TGA Q 500 (TA Instruments) according to ASTM E1131–08 in order to evaluate the thermal stability of the fibers. The TGA scans were performed in a nitrogen atmosphere and during the scans the temperature was increased from room temperature to 700℃ at a rate of 10℃ min⁻¹. The DSC Q 1000 (TA instruments) was used according to ASTM D 7426–08 to determine the thermal properties of the fibers. Each sample was heated from −20℃ to 220℃ at a scanning rate of 10℃ min⁻¹, and subsequently the samples were cooled to −20℃ at a scanning rate of 20℃ min⁻¹. In the thermal tests three replicates were carried out in each case. During the DSC scans, the glass transition temperatur (T_g), crystallization temperature (T_c), melting temperature (T_m), crystallization enthalpy (ΔH_c) and melting enthalpy (ΔH_m) were determined. The percentage of the crystalline fraction was calculated from the enthalpies according to^10,11
$χ = \frac{1}{(1 - w_{f})} [\frac{Δ H_{m} - Δ H_{c}}{Δ H_{m}^{0}}] \cdot 100$
(1)
where w_m is the weight ratio, $Δ H_{m}$ is the melting enthalphy, $Δ H_{c}$ is the crystallization enthalphy, and $Δ H_{m}^{0}$ is the melting enthalpy for 100% crystalline PLA (93 J g⁻¹).

An Instron 5567 tensile tester was used to determine the mechanical properties of the fibers according to ASTM D3822–07. For the tensile tests the fibers were cut into 25 mm lengths and placed between the clips of the tensile tester. The samples were drawn at a rate of 100 mm min⁻¹ until they broke. The stress and strain curves were obtained and the initial modulus calculated.¹²

Water uptake was determined according to ASTM D 570–98 (immersion method). The fibers were cut into 25.4 mm lengths and dried in an oven at 105–110℃ for 1 h. The dry weight of the fibers was measured, and the fibers were then immersed in distilled water at a temperature of 23 ± 1℃ for 24 h. Following immersion their weight was again determined and the water uptake of the fiber was calculated.¹³

Antimicrobial activity was assessed against Staphylococcus aureus (ATCC 6538) as a Gram-positive bacterium and Klebsiella pneumoniae (ATCC 4352) as a Gram-negative bacterium, according to ASTM E2149–01. In accordance with this standard, an ASTM buffer solution was prepared by dissolving 34 g potassium dihydrogen phosphate in 1000 ml distilled water. The bacteria cultures were diluted with the buffer solution to give a final concentration of 1.5 × 10⁵ colony-forming unit (CFU) ml⁻¹. Buffer solution (50 ± 0.1 ml) was placed in flasks and 50 ± 0.1 µl of bacteria solution added. 1 ± 0.1 g of samples cut into small pieces and sterilized with ethylene oxide were added to the flasks, which were then shaken at maximum speed for 1 ± 0.5 s. 1 ± 0.01 ml of the solutions were immediately transferred to test tubes, diluted serially (10⁰, 10¹, 10²) and then inoculated on agar, Chapman agar for S. aureus and Endo agar for K. pneumoniae. After inoculation, the samples were incubated in Petri dishes at 37℃ for 48 h. As soon as the 0th contact time sub-samples were prepared, the flasks were returned to the shaker incubator at maximum speed for 24 h, and the procedure was repeated up to the 24th contact time. After incubation of the Petri dishes, the number of bacterial colonies in each dish were counted and the percentage reduction in the bacteria calculated according to
$R (%) = \frac{A - B}{A} \cdot 100$
(2)
where B is the number of bacteria from the flask containing the treated substrate after 24 h contact and A is the number of bacteria for the addition of treated substrate at zero contact time. A and B are expressed in CFU ml⁻¹. R(%) is the percentage reduction ratio.¹⁴

Results and discussion

The surface morphology and particle distribution in the cross-section of the fibers were examined under SEM (Figures 1 and 2). Analysis indicated a reasonable distribution of the particles, with occasional agglomerates seen both within and near the surface of the fibers, particularly at higher concentrations of AgNP.
Figure 1.
Cross-section micrograph of the nanocomposite fibers: (a) at 5% filler content, (b) at 1% filler content.

Figure 2.
Surface micrograph of the nanocomposite fibers: (a) at 5% filler content, (b) at 1% filler content.

The thermal stability of neat PLA fibers was determined and compared with that of the PLA/Ag nanocomposite fibers. TGA scans for all fibers were carried out, under a nitrogen atmosphere, to determine the thermal stability of the samples and the percentage of AgNP in the matrix. From the TGA results the addition of AgNP was shown to increase the thermal stability of the PLA matrix. With filler added the decomposition temperature (Td_0.05) of the PLA nanocomposite fıbers increased progressively from 332.0℃ to 341.5℃ (Table 1). The percentage of AgNP was confirmed to be 0.5, 1, 3, and 5 wt%, respectively.
Table 1.
TGA results for PLA neat and nanocomposite fibers

Sample Td_0.5 (℃) Td_0.05 (℃) Filler content (%)

M2D1K0 366.1 332.0 0.0

M2D1K1 374.5 337.0 0.5

M2D1K2 373.9 339.6 0.9

M2D1K3 375.4 339.8 3.0

M2D1K4 378.8 341.5 5.0

DSC tests were used to measure the thermal properties and the degree of crystallinity of the fibers. As shown in Table 2, the addition of AgNPs caused a decrease in crystallinity from 17% to 13%. The AgNPs appear to hinder the formation of crystals and hence to cause the reduction in crystallinity, possibly due to the presence of aggregated NPs, which prevent crystal growth.
Table 2.
Thermal characteristics of PLA neat and nanocomposite fibers

Sample T_g (℃) T_m (℃) T_c (℃) ΔH_m (J g⁻¹) ΔH_c (J g⁻¹) χ (%)

M2D1K0 60.9 167.9 107.8 45.3 29.5 17.0

M2D1K1 60.8 168.1 109.4 47.9 32.6 16.6

M2D1K2 61.5 168.4 110.4 41.5 27.8 15.9

M2D1K3 61.5 168.7 109.6 42.2 29.8 13.3

M2D1K4 61.4 168.5 110.9 45.9 33.0 13.0

The tensile properties of PLA neat and nanocomposite fibers are summarized in Table 3. The addition of AgNP sharply decreased the tensile properties of the fibers (statistical significance (p value) <0.0001, coefficient of determination (R²) = 0.84). The initial modulus of the fibers was significantly decreased (p value <0.0001, R²= 0.93).
Table 3.
Tensile properties of the neat PLA and nanocomposite fibers

Sample Stress at break (MPa) Strain at break (%) Initial modulus (GPa)

M2D1K0 75.5 42.2 2.12

M2D1K1 72.2 19.7 2.44

M2D1K2 64.7 23.2 2.00

M2D1K3 50.4 15.9 1.82

M2D1K4 38.1 16.7 1.70

Similar results have been reported in the literature. Fortunati et al.¹⁵ produced PLA/Ag nanocomposite fibers and compared their thermal and mechanical properties with those of unmodified PLA fibers. They observed that there was no change in thermal properties, but noted a decline in tensile properties. The latter may be due to the agglomeration of NPs at high concentrations, the agglomerates acting as stress concentration centers during tensile testing.

Since the addition of Ag leads to a decrease in crystallinity, this is reflected in the modulus and other mechanical properties. Water uptake is also a feature of the crystallinity of the polymer matrix (Table 4). Since the crystallinity of the fibers was decreased by the addition of NPs, the degree of water uptake increased. With increasing water uptake, the diffusion rate of the nanoparticles also increased, leading to greater antibacterial activity.
Table 4.
Water uptake of neat PLA and nanocomposite fibers

Sample Water uptake (%)

M2D1K0 2.2

M2D1K1 2.8

M2D1K2 2.8

M2D1K3 4.6

M2D1K4 4.9

The antibacterial activities of PLA/Ag nanocomposite fibers were evaluated after a specific contact time and calculated from the percentage reduction in S. aureus and K. pneumoniae colonies. As shown in Figure 3, the PLA sample provided good conditions for growth of Gram-positive and Gram-negative bacteria over 24 h and confirmed that samples had been attacked by bacteria during the antibacterial test.
Figure 3.
Antibacterial efficiency of neat PLA fibers against (a) Klebsiella pneumoniae and (b) Staphylococcus aureus.

Figure 4 demonstrates the variation in the effectiveness of fibers against Gram-positive and Gram-negative bacteria. AgNPs were more effective against Gram-positive bacteria, confirming similar results in the literature. Parikh et al.¹⁶ produced Ag-filled wound dressings and evaluated their antimicrobial activity against S. aureus and K. pneumoniae, and found that Ag was more effective against S. aureus. Kvitek et al. researched the antimicrobial activity of AgNPs against both Gram-positive and Gram-negative bacteria and found that AgNPs were more active against Gram-positive bacteria.¹⁷ As seen in Table 5, the antibacterial efficiency of the samples against K. pneumoniae was determined only at 3 and 5 (wt%), and this sample exhibited 82% and 86% efficiency, respectively.
Figure 4.
Antibacterial efficiency of PLA/Ag fibers against Klebsiella pneumoniae and Staphylococcus aureus; (a) 0th contact time (b) 24th contact time.

Table 5.
Antibacterial efficiency of samples

Antibacterial activity

Sample (Klebsiella pneumoniae) (%) (Staphylococcus aureus) (%)

M2D1K0 0 0

M2D1K1 0 55

M2D1K2 0 72

M2D1K3 82 96

M2D1K4 86 99.9

The antibacterial efficiency of the samples against S. aureus was observed at 1, 3 and 5 (wt%), confirming that AgNPs have greater antimicrobial efficiency against Gram-positive bacteria, since they can more readily penetrate the Gram-positive bacteria cell (Figure 5).
Figure 5.
Antimicrobial efficacy of nanocomposite fibers.

Conclusions

In this study the preparation of antimicrobial PLA/Ag nanocomposite fibers by melt-spinning was investigated. Fibers containing AgNPs of particle size below 100 nm were produced at four different percentages and their thermal, mechanical and water uptake properties determined. Finally, the antimicrobial activity of the fibers against K. pneumoniae and S.aureus was evaluated according to ASTM E2149–01. The test results showed that the nanocomposite fibers were effective against both Gram-positive and Gram-negative bacteria, but the effect was greater against Gram-positive bacteria, optimal behavior being found at 5 wt% filler. Furthermore, the addition of NP had a noticeable effect on the properties of the fibers. The NPs increased water uptake, and caused a sharp decrease in initial modulus, breaking stress and crystallinity. These properties may be useful in wound dressings, particularly those used in the treatment of burns.

Sample	Td_0.5 (℃)	Td_0.05 (℃)	Filler content (%)
M2D1K0	366.1	332.0	0.0
M2D1K1	374.5	337.0	0.5
M2D1K2	373.9	339.6	0.9
M2D1K3	375.4	339.8	3.0
M2D1K4	378.8	341.5	5.0

Sample	T_g (℃)	T_m (℃)	T_c (℃)	ΔH_m (J g⁻¹)	ΔH_c (J g⁻¹)	χ (%)
M2D1K0	60.9	167.9	107.8	45.3	29.5	17.0
M2D1K1	60.8	168.1	109.4	47.9	32.6	16.6
M2D1K2	61.5	168.4	110.4	41.5	27.8	15.9
M2D1K3	61.5	168.7	109.6	42.2	29.8	13.3
M2D1K4	61.4	168.5	110.9	45.9	33.0	13.0

Sample	Stress at break (MPa)	Strain at break (%)	Initial modulus (GPa)
M2D1K0	75.5	42.2	2.12
M2D1K1	72.2	19.7	2.44
M2D1K2	64.7	23.2	2.00
M2D1K3	50.4	15.9	1.82
M2D1K4	38.1	16.7	1.70

Sample	Water uptake (%)
M2D1K0	2.2
M2D1K1	2.8
M2D1K2	2.8
M2D1K3	4.6
M2D1K4	4.9

Antibacterial activity
M2D1K0	0	0
M2D1K1	0	55
M2D1K2	0	72
M2D1K3	82	96
M2D1K4	86	99.9

Footnotes

Acknowledgements

The authors wish to thank Distinguished Prof. Dr. Mukerrem Cakmak, University of Akron, for support with the SEM and tensile tests and analysis of the thermal results. The authors are grateful to Ekoteks Lab Isl. Ltd. for allowing use of their laboratory facilities for the antibacterial tests, and particularly to Mrs Sevim Atmaca Razaq, Technical Manager of Ekoteks Lab Isl. Ltd. for her support. The authors also thank Mrs Haike Hilke of the University of Borås for technical support in the compounding and thermal tests, and Miss Emel Duran, biologist at Ekoteks Lab Isl. Ltd. for technical support and experimental assistance with the antibacterial tests.

Funding

This work was supported by Istanbul Technical University Scientific Research Project Fund (grant number BAP–32856) and the Scientific and Technological Research Council of Turkey (International Research Fellowship Program TUBITAK BIDEB–2214).

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Antibacterial activity
Sample	(Klebsiella pneumoniae) (%)	(Staphylococcus aureus) (%)
M2D1K0	0	0
M2D1K1	0	55
M2D1K2	0	72
M2D1K3	82	96
M2D1K4	86	99.9