Integrated poly-D,L-lactide-co-glycolide/silver nanocomposite: synthesis, characterization and wound healing potential in Wistar Albino rats

Abstract

Over the past few centuries, interest in novel metal nanoparticles has expanded quickly with the inclusion of new nanocomposites into a variety of products and technologies. Recently, nanocomposites prepared by the distribution of inorganic nanoparticles in polymeric matrices have found a prominent role in biomedical applications. In this report, we attempt to formulate a biodegradable poly-D,L-lactide-co-glycolide (PLGA) polymer based biogenic silver nanocomposite using a modified solvent casting method. The silver nanoparticles mediated by Origanum vulgare aqueous leaf extract (Ag NPs) and formulated silver polymeric nanocomposite (PLGA/Ag NC) show absorbance spectra at 420 nm under UV-visible spectroscopy. Fourier transform infrared spectroscopy confirms that Ag NPs utilized the carbonyl functional group present in the PLGA polymer for the successful formation of PLGA/Ag NC. Scanning electron microscopy analysis notably showed that PLGA/Ag NC was found to be spherical and irregular in shape, distributed in the polymer matrix. A dynamic light scattering measurement showed that PLGA/Ag NC has an average particle size distribution of 115 nm, and the zeta potential was −33 mV. The PLGA/Ag NC crystalline nature was confirmed using X-ray diffraction analysis. Furthermore, PLGA/Ag NC improves wound healing in the excision wound which was established using wound closure, histopathology, protein profiling, and matrix metalloproteinases 2 and 9 expression. The analysis results clearly show that PLGA/Ag NC enhances the wound healing activity by the sustained release of Ag NPs, upregulates protein expression and inhibits pathogenic bacterial growth in the wound area. In conclusion, PLGA/Ag NC could be a novel therapeutic agent for wound treatment.

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Introduction

The most important and largest organ in our body system is the skin. In most cases skin damage occurs due to physical force, chemical injury and bacterial pathogenic infections, which result in the loss of normal cellular activity and the functional continuity of living tissues.^1,2 Impaired wounds, such as acute and chronic wounds, fail to progress through the normal stages of the healing process.³ Such wounds regularly enter a state of pathologic inflammation owing to a postponed, incomplete and uncoordinated healing process.⁴ Impaired wounds and their complications are generally associated with diseases like diabetes, obesity and vascular disorders.⁵ Delayed wound healing is mainly due to the presence of predominantly pathogenic bacterial populations in the injured site.⁶ With a modern lifestyle, wounds and their related complications are unavoidable and a principal burden.⁷ Some wound healing agents are available in the market, but their effects on severe wounds are still not yet proven. Hence, we have a need for novel and environmentally friendly agents to treat wounds.

In recent times the usage of metal nanoparticles in the biomedical field has extremely increased due to their unique size, shape and large surface to volume ratio.⁸ Size, surface charge and shape are major factors for the action of nanoparticles in biological systems.⁹ A suitable size for nanoparticles for therapeutic purposes might be in the range of 2 to 200 nm.¹⁰ The surface charge of the nanoparticles mostly determines the cellular uptake, biodistribution and interaction with other biological environments.¹¹ Changes in the desired shape of the nanoparticles might make them useful as a specific therapeutic agent by themselves via changes in ligand targeting, cellular uptake, transport, and degradation.¹² Among the abundant metal nanoparticles, silver nanoparticles (Ag NPs) are largely used for various biological applications, and their potent anticancer, antimicrobial, and wound healing activity are well established.^13,14 The ingenuity of Ag NPs is that one can easily engineer the size, shape and surface characteristics depending on the requirements. Ag NPs synthesised using environmentally friendly biological agents as mediators showed an improved activity as compared with other methods of mediating Ag NP synthesis.¹⁵ What’s more, traditional medicinal plant leaf extract mediated synthesis of Ag NPs displayed a wide range of upregulated biomedical activity.¹⁶ Moreover, we have recently proven that Origanum vulgare plant leaf extract mediated Ag NPs display antibacterial and anticancer activities.¹⁷

Recently, polymer nanocomposites have gained much attention for modern nanotechnologies due to their design flexibility, unique applications and lower life cycle costs.¹⁸ The biodegradable poly-D,L-lactide-co-glycolide (PLGA) polymer is a highly preferable material for nanocomposite preparation because of its extended applications in imaging, tissue regeneration and targeted drug delivery.^19,20 Nanocomposites prepared by the distribution of inorganic nanoparticles in a polymeric matrix have improved their physicochemical and biological properties.²¹ Additionally, biodegradable polymer based metal nanocomposites have been used for various disease diagnoses and treatments. It has been recently reported that PLGA polymer based metal nanocomposites strongly inhibit pathogenic bacterial growth through surface modification.²² No effort has been made to prepare a green chemistry based polymeric nanocomposite for beneficial biological applications. Therefore, in the present study we make an attempt to formulate biodegradable PLGA polymer based biogenic synthesized silver nanocomposites, and tried to explore its role in the excision wound model.

Experimental section

Biogenic synthesis of silver nanoparticles

An Origanum vulgare aqueous leaf extract preparation was accomplished using the established method.¹⁷ For the synthesis of biogenic Ag NPs, 90 mL of 5 mM silver nitrate (Sigma-Aldrich, India) solution was mixed with 10 mL of aqueous leaf extract and allowed to stay at room temperature for 24 h. The formed reddish brown color indicates the synthesis of Ag NPs, which were centrifuged at 12 000 rpm for 20 minutes and washed three to four times with deionized water.

Formulation of silver polymeric nanocomposite

The PLGA/Ag NC was formulated using a modified solvent casting method.²² Briefly, PLGA polymer (PLGA, L/G = 50 : 50, a gift sample from Prof. Dragan Uskokovic, Institute of Technical Sciences of SASA, Serbia) (1% w/v) was dissolved in chloroform and sonicated in an ultrasonic bath for 30 minutes. Similarly, biogenic Ag NPs (0.5% w/v) were dissolved in chloroform, and sonicated in an ultrasonic bath for 5 h to improve their dispersion in the solvent and promote the interaction with the polymer matrix. For the formulation of the nanocomposite, Ag NPs were added drop by drop to the PLGA polymer solution with uniform stirring at room temperature. After overnight stirring, the formed blackish brown colored PLGA/Ag NC was used for further studies.

Characterization of silver polymeric nanocomposite

UV-visible spectroscopy analysis of the Ag NPs and PLGA/Ag NC was performed using a UV-visible double beam spectrophotometer (UV-1601, Shimadzu, Japan) in the range of 200–800 nm. Fourier transform infrared (FT-IR) spectra were obtained using a spectrum RX-1 instrument in diffuse reflectance mode functioning at a resolution of 4 cm⁻¹ in the range of 4000–400 cm⁻¹. The topographical information of the formulated PLGA/Ag NC was attained using a scanning electron microscope (SEM) with an EDAX (VEGA3 TESCAN, 30.0 KV) instrument. A Malvern Zetazier (Nano ZS90, UK) instrument was used to find out the size distribution and stability of the formulated PLGA/Ag NC. An X-ray diffraction (XRD) pattern of PLGA/Ag NC was obtained using a powder X-ray diffractometer (Philips X'Pert Pro X-ray diffractometer) in the 2θ range from 10° to 80°.

Experimental animals

Male Wistar Albino rats of 2 to 3 months old, weighing 150–200 g, were used for the wound healing experiment. The animals were housed in sterile polypropylene cages and maintained in standard laboratory conditions (12 hour light–dark cycle; 25 ± 3 °C; 35–60% humidity), and feed and water were provided ad libitum. The experimental protocols were approved by the Institutional Animal Ethics Committee, Bharathidasan University, Tiruchirappalli, India.

Animal grouping, wound creation and drug administration

A total of 9 animals were separated into three groups, named group-I (control), group-II (treatment with Ag NPs) and group-III (treatment with PLGA/Ag NC). For the creation of the excision wound, the hair on the rats’ back side was shaved using a sterile surgical blade and an approximately 2 cm² full thickness patch of skin was removed under standard anesthesia conditions.⁶ Group-I animals were treated with 2 mL of normal physiological saline, group-II animals were treated with Ag NPs (10 mg kg⁻¹ body weight) and group-III animals were treated with PLGA/Ag NC (10 mg kg⁻¹ body weight), applied topically (PLGA/Ag NC in chloroform solution; the treatment’s organic solvent evaporates and the nanocomposite forms a film like structure on the wound area), once daily, for a period of 12 days.

Wound contraction

The wound contraction of all of the animals was observed planimetrically by tracking the wound margin on a graph sheet (0, 4, 8 and 12th day). The animals’ wound images were taken on scheduled days for visual comparison. The percentage of wound contraction was calculated using the following formula.¹

Histopathological analysis

At the end of the experiment, the wound granulation tissue was removed and separately fixed with 10% formalin solution, dehydrated, cleaned with xylene and embedded in paraffin wax.¹ The 3 μm of paraffin wax embedded tissue was cut and stained with Hematoxylin and Eosin. The stained tissue sections were inspected under a phase contrast light microscope and photographs were taken at 40× objectives.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

The collected animals’ wound granulation tissue was washed with normal physiological saline followed by pH 7.4 phosphate-buffered saline. 2 mg of all of the group animals’ wound granulation tissue was separately sliced into small pieces and collected in 1 mL of Tris buffer (50 mM, pH 7.5) containing 10 mM potassium chloride, 1 mM magnesium chloride, 1 mM ethidium diamide tetra-acetic acid and 1 mM protease inhibitors. The tissue was homogenized in a polytron homogenizer with three strokes of 12 seconds each in an ice bath. Then, the homogenized tissue samples were centrifuged at 8000 rpm for 10 minutes and the supernatant containing the total protein was analyzed using the Bradford method. 25 μg of the total protein samples was treated with loading dye containing β-mercaptoethanol, denatured at 95 °C for 5 minutes before loading. Separation of the proteins was achieved using SDS-PAGE analysis using 5% (v/v) stacking and 10% (v/v) separation gels, followed by staining with Coomassie brilliant blue R-250 (Hi Media Laboratories, India) for 2 h. The stained gel was destained, and the protein molecular weight was determined using standard prestained dual protein markers (250–10 kDa, Bio-Rad, USA).

Western blot analysis

The SDS-PAGE analyzed proteins were transferred from the gel to a nitrocellulose membrane for 1 h at 60 V using a transfer buffer (glycine: 39 mmol L⁻¹, tris: 48 mmol L⁻¹ and 20% methanol). The membrane was blocked with blocking solution (5% milk powder) for 1 h. Then the membrane was incubated at 4 °C overnight with specific primary antibodies (matrix metalloproteinase-2, 9, and β-actin in 1 : 1000 dilutions). The blots were washed with TBST and TBS solutions, and incubated with secondary antibodies (horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies in 1 : 1000 dilutions) for 1 h. The blots were washed with TBST and TBS solutions, and developed using BCIP/NBT substrates (GeNei-Merck, India).

Statistical analysis

The statistical analysis was done using SPSS software Version 16 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to express the significance. Statistical significance was accepted at a level of p < 0.05.

Results and discussion

Formulation of silver nanoparticles and silver polymeric nanocomposite

Initially, biogenic Ag NPs were synthesized from Origanum vulgare aqueous leaf extract with 5 mM silver nitrate solution. A 24 h time period was required for the complete synthesis of Ag NPs. Here, the Origanum vulgare aqueous leaf extract was used as a reducing, capping and stabilizing agent for the successful formation of Ag NPs.¹⁷ The synthesized biogenic Ag NPs were used for PLGA polymer based nanocomposite preparation. Optimized magnetic stirring at room temperature is mandatory for the formation of PLGA/Ag NC. The Ag NPs utilized carbonyl functional groups present in the PLGA polymer for the successful formation of the nanocomposite. The development of a dark brown and blackish brown color was primarily used to confirm the formation of Ag NP and PLGA/Ag NC (Fig. 1), respectively. Surface plasmon resonance (SPR) of the Ag NPs and PLGA/Ag NC showed an absorbance spectra at 420 nm using UV-visible spectroscopy (Fig. 1(b and c)), and there was no detectable absorption peak in the PLGA polymer (Fig. 1(a)).

Fig. 1 Color intensity pattern and UV-vis spectroscopy of (a) PLGA polymer; (b) Ag NPs; (c) PLGA/Ag NC. The development of a dark brown and blackish brown color confirmed the formation of Ag NPs and PLGA/Ag NC, respectively (b & c). UV-visible spectroscopy analysis of Ag NPs and PLGA/Ag NC which displays an intense SPR peak at 420 nm (b & c).

Characterization of silver polymeric nanocomposite

The Origanum vulgare aqueous leaf extract is reported to possess bioactive molecules, which play a huge role in the Ag NP and PLGA/Ag NC formation.¹⁷ This was confirmed using FT-IR spectroscopy analysis. The FTIR peaks at 3434 cm⁻¹, 1607 cm⁻¹, and 1071 cm⁻¹ show amide N–H stretching, carbonyl C=O stretching and amine C–N stretching, respectively (Fig. 2(a–c)). The PLGA polymer has a functional peak at 1607 cm⁻¹ (carbonyl C=O stretching)²³ which has shifted in the PLGA/Ag NC, suggesting that the Ag NPs utilized the carbonyl functional group present in the PLGA polymer for the successful formation of the nanocomposite (Fig. 2(a and c)). The Ag NP strong functional peak at 1223 cm⁻¹ disappeared in PLGA/Ag NC, also confirming the nanocomposite formation. The appearance of specific peaks in the Ag NPs and PLGA/Ag NC at 565 cm⁻¹ established the silver vibration (Fig. 2(b and c)). Similarly, Sathishkumar et al., 2015, reported that the reducing agent chrysin has a carbonyl functional group used for stable metal nanoparticle formation.²⁴ The SEM topographical image shows that synthesized Ag NPs are spherical and irregular in shape with surface roughness, and are embedded in the PLGA polymer (Fig. 3(a)). The highest percentage of the elemental silver peak present in the PLGA/Ag NC was confirmed using EDAX analysis (Fig. 3(b)). The SEM analysis results visibly showed the successful formation of PLGA/Ag NC. Armentano et al., 2010, demonstrated that Ag NPs induce surface morphological changes in the PLGA polymer and affect the nanocomposite surface wettability and roughness, and their use in enhancing bacterial adhesion on the nanocomposite surface.²⁵

Fig. 2 FTIR spectra of (a) PLGA polymer; (b) Ag NPs; and (c) PLGA/Ag NC. The PLGA polymer has a functional peak shifted in the PLGA/Ag NC, suggesting that Ag NPs utilized the C=O functional group for the formation of the nanocomposite.

Fig. 3 Scanning electron microscopy analysis of (a) PLGA/Ag NC taken at 5 and 10 μm; and (b) EDAX spectrum. The SEM analysis revealed that the Ag NPs are spherical and irregular in shape, and are embedded in the polymeric matrix.

The DLS analysis result of the Ag NP average particle size distribution was 115 nm (Fig. 4(a)), and the zeta potential was −33 mV (Fig. 4(b)). Using our synthesis procedure, we were able to reproduce similarly sized Ag NPs. Duan and Y. Li, 2013, showed that nanoparticles with sizes in between 2 and 200 nm were highly preferred for biological therapeutic purposes.¹⁰ It is well accepted that a zeta potential value of about −25 mV ensures a high energy barrier for the stabilization of nanosuspension.¹⁷ The PLGA/Ag NC crystalline nature was confirmed using XRD analysis. The XRD pattern of the PLGA/Ag NC matches the face centered cubic (FCC) structure of the bulk silver with broad peaks as shown in ESI Fig. S1.† The distinct peaks at 38.60°, 64.58°, and 77.39° are those of the indexed planes (111), (220), and (311) of the FCC structure of silver nanoparticles.

Fig. 4 DLS measurements of Ag NPs (a) particle size distribution; (b) zeta potential measurement. The analysis results showed that the Ag NPs’ average particle size was 115 nm and the zeta potential was −33 mV.

Wound healing activity of silver polymeric nanocomposite

For many centuries, silver sulfadiazine has been used as a well known agent for open wounds. But as a metal, silver may lose some of the benefits and recent nanotechnology knowledge has been used to develop highly stable Ag NPs. The wound healing activity of the biogenic Ag NPs and PLGA/Ag NC against the excision wound is shown in Fig. 5. The study results clearly show that the PLGA/Ag NC treated wound showed enhanced healing activity without any complications as compared with the control and Ag NP-alone treated animals. The PLGA polymer sustainedly releases Ag NPs into the wound area, thereby for a long time arresting bacterial growth, leading to enhanced wound healing activity. The engineered Ag NPs loaded in the biodegradable polymer showed potent wound healing activity through the inhibition of pathogenic bacterial growth and also modulated inflammatory cytokines, which are involved in the delayed wound healing process.²⁶ Xu et al., 2010, demonstrated that a continued antibacterial effect was achieved in the biodegradable fibres containing finely dispersed Ag NPs.²⁷ Fortunati et al., 2011, reported that Ag NP release was controlled by the polymer degradation process and demonstrated a prolonged antibacterial effect.²²

Fig. 5 Photographic representation of the animal wounds on different days after treatment with the control, Ag NPs, and PLGA/Ag NC (scale bar = 5 mm). The images were compared with the control and treatment.

The PLGA/Ag NC treated wound shows significant wound contraction during the treatment days as compared with the Ag NPs and control, and maximum wound closure was noticed in the final day of the experiment (Fig. 6). Likewise, the reduced wound size and significant wound closure of wounds treated with Ag NPs has been reported by Bidgoli et al., 2013.²⁸ The Ag NP release in the wound area is actively controlled by the polymer degradation process, supporting a promising sustained antibacterial effect.²² The nanocomposite has a longer release time of the silver loading, since the Ag NPs are reduced in the degradation process by the inhibition of the autocatalytic action. Additionally, the Origanum vulgare plant leaf extract having bioactive compounds capped in the Ag NPs also helps to enhance the wound healing activity.¹ Moreover, we had recently proven that Origanum vulgare mediated Ag NPs strongly arrest the growth of pathogenic bacteria, which might be involved in the delayed wound healing process.^6,17 Ag NPs inhibit the pathogenic bacterial growth due to structural changes in the bacterial cell membrane, the generation of reactive oxygen species and signal transduction inhibition.²⁹

Fig. 6 Percentage of wound closure on different days of treatment with the control, Ag NPs, and PLGA/Ag NC. All the data are expressed as the mean ± SD.

The PLGA/Ag NC wound healing activity was further confirmed using histopathological analysis. On the final day of the experiment, wound granulation tissue was subjected to histopathological analysis. The results showed the aggressive formation of collagen, enhanced epithelialization and neovascularization, and mature macrophages and fibroblast formation in the Ag NPs and PLGA/Ag NC treated wound tissue (Fig. 7(b and c)), whereas the control wound has limited collagen, immature macrophages and fibroblasts (Fig. 7(a)). The formation of collagen gives the skin its tensile strength, and plays a major role in each phase of the wound healing process. The collagen also provides substances for the new tissue growth in the injured site. The PLGA polymer supports the sustained release of Ag NPs which controls the inflammatory cytokines by decreasing the lymphocyte and mast cell infiltration.³⁰

Fig. 7 Histopathological analysis of (a) control; (b) Ag NPs; (c) PLGA/Ag NC. The images were taken at 40× objective under a phase contrast light microscope (scale bar = 100 μm). Wound granulation tissue was used for this study on the final day of the experiment.

The PLGA/Ag NC regulates various protein expression levels in the wound tissue for the enhancement of the healing activity. As compared with the control and Ag NPs the PLGA/Ag NC upregulates various proteins (Fig. 8). At this junction, PLGA/Ag NC enhances proteins around 86 kDa and 79 kDa, which are a heterotrimeric form of collagen.³¹ The enhanced collagen level regulates various key factors which are actively involved in the healing process. The matrix metalloproteinase (MMP) family are remodeling enzymes, which are part of the collagenases that are known to reduce extracellular matrix components, which enables the migration of cells and the remodeling of the wound. Balanced MMP expression is needed for normal wound healing, and they have a positive and important role in healing and the response to injury.³² MMP-2 and 9 are known as a subclass of the MMPs owing to their gelatinolytic activity, and have been shown to contribute to the wound healing response. Based on the available reports, we hypothesise that the PLGA/Ag NC enhanced wound healing activity is also due to the regulation of MMP expression. To confirm the MMP role in wound tissue, we carried out the western blotting technique. The results notably show that PLGA/Ag NC upregulates MMP-2 and 9 expression as compared with the Ag NPs and control (Fig. 9). In the wound healing process, MMP-2 and 9 are significantly involved in keratinocyte migration and granulation tissue remodeling.³³ MMP-9 is involved in inflammation, matrix remodeling, epithelialization and is highly essential for the normal progression of wound closure.³² Moreover, Chereddy et al., 2013, described how PLGA polymeric metabolite lactate activates the procollagen and angiogenic factors in the wound tissue, thereby helping in the wound healing process.³⁴ Overall our study results conclude that PLGA/Ag NC enhanced the wound healing activity via the inhibition of bacterial growth, the sustained release of Ag NPs from PLGA polymer and activated MMP expression in the wound.

Fig. 8 SDS-PAGE analysis. Lane 1 shows the standard protein molecular weight markers with molecular weights given in kilodaltons; Lane 2 shows the control; Lane 3 shows the Ag NP treatment; and Lane 4 shows the PLGA/Ag NC treatment.

Fig. 9 Western blotting analyses of MMP-2 and 9. Wound granulation tissue was used for this study on the final day of the experiment. The substrate developed blot image was taken using a Biorad gel documentation system. β-Actin was used as an internal control.

Conclusion

In the present work, we conclude that we have developed a modified solvent casting method for the successful formulation of a biodegradable polymer based biogenic synthesized silver nanocomposite. The PLGA/Ag NC showed enhanced wound healing activity as compared with Ag NPs, due to the sustained release of Ag NPs from the PLGA polymer. The PLGA/Ag NC strongly arrests the growth of pathogenic bacterial populations and also enhances MMP expression. The activated MMPs are involved in inhibiting inflammatory signals and promoting re-epithelialization processes in the injured site, leading to an upregulated wound healing activity. Overall, our preliminary study results showed that the PLGA/Ag NC maybe a suitable candidate for wound treatment.

Acknowledgements

We are grateful to the Department of Science and Technology (DST) for providing the financial assistance to Mr Renu Sankar through the INSPIRE Fellowship scheme (DST/INSPIRE Fellowship/2010/229C). We extend our acknowledgement to the Department of Science and Technology – Fund for Improvement of S & T Infrastructure in Universities and Higher Educational Institutions (DST-FIST) for their infrastructure support to our department.

References

1. R. Sankar, R. Dhivya, K. S. Shivashangari and V. Ravikumar, J. Mater. Sci.: Mater. Med., 2014, 25, 1701–1708.
2. S. Guo and L. A. Dipietro, J. Dent. Res., 2010, 89, 219–229.
3. T. N. Demidova-Rice, M. R. Hamblin and I. M. Herman, Adv. Skin. Wound Care, 2012, 25, 304–314.
4. N. B. Menke, K. R. Ward, T. M. Witten, D. G. Bonchev and R. F. Diegelmann, Clin. Dermatol., 2007, 25, 19–25.
5. R. Nunan, K. G. Harding and P. Martin, Dis. Models & Mech., 2014, 7, 1205–1213.
6. R. Sankar, A. Baskaran, K. S. Shivashangari and V. Ravikumar, J. Mater. Sci.: Mater. Med., 2015, 26, 214.
7. C. K. Sen, G. M. Gordillo, S. Roy, R. Kirsner, L. Lambert, T. K. Hunt, F. Gottrup, G. C. Gurtner and M. T. Longaker, Wound Repair Regen., 2009, 17, 763–771.
8. V. V. Mody, R. Siwale, A. Singh and H. R. Mody, J. Pharm. BioAllied Sci., 2010, 2, 282–289.
9. D. H. Jo, J. H. Kim, T. G. Lee and J. H. Kim, Nanomedicine, 2015, 11, 1603–1611.
10. X. Duan and Y. Li, Small, 2013, 9, 1521–1532.
11. M. L. Schipper, G. Iyer, A. L. Koh, Z. Cheng, Y. Ebenstein, A. Aharoni, S. Keren, L. A. Bentolila, J. Li, J. Rao, X. Chen, U. Banin, A. M. Wu, R. Sinclair, S. Weiss and S. S. Gambhir, Small, 2009, 5, 126–134.
12. J. A. Champion, Y. K. Katare and S. Mitragotri, J. Controlled Release, 2007, 121, 3–9.
13. S. Gurunathan, J. K. Jeong, J. W. Han, X. F. Zhang, J. H. Park and J. H. Kim, Nanoscale Res. Lett., 2015, 10, 35.
14. G. Thirumurugan, V. S. Veni, S. Ramachandran, J. V. Rao and M. D. Dhanaraju, J. Biomed. Nanotechnol., 2011, 7, 659–666.
15. R. Sankar, R. Maheswari, S. Karthik, K. S. Shivashangari and V. Ravikumar, Mater. Sci. Eng., C, 2014, 44, 234–239.
16. D. Qu, W. Sun, Y. Chen, J. Zhou and C. Liu, Int. J. Nanomed., 2014, 9, 1871–1882.
17. R. Sankar, A. Karthik, A. Prabu, S. Karthik, K. S. Shivashangari and V. Ravikumar, Colloids Surf., B, 2013, 108, 80–84.
18. I. Armentano, C. R. Arciola, E. Fortunati, D. Ferrari, S. Mattioli, C. F. Amoroso, J. Rizzo, J. M. Kenny, M. Imbriani and L. Visai, Sci. World J., 2014, 2014, 410423.
19. W. Gu, C. Wu, J. Chen and Y. Xiao, Int. J. Nanomed., 2013, 8, 2305–2317.
20. R. Sankar, S. Karthik, N. Subramanian, V. Krishnaswami, J. Sonnemann and V. Ravikumar, Mater. Sci. Eng., C, 2015, 51, 362–368.
21. I.-Y. Jeon and J.-B. Baek, Materials, 2010, 3, 3654–3674.
22. E. Fortunati, L. Latterini, S. Rinaldi, J. M. Kenny and I. Armentano, J. Mater. Sci.: Mater. Med., 2011, 22, 2735–2744.
23. S. S. Zhao, M. A. Bichelberger, D. Y. Colin, R. Robitaille, J. N. Pelletier and J. F. Masson, Analyst, 2012, 137, 4742–4750.
24. G. Sathishkumar, R. Bharti, P. K. Jha, M. Selvakumar, G. Dey, R. Jha, M. Jeyaraj, M. Mandal and S. Sivaramakrishnan, RSC Adv., 2015, 5, 89869–89878.
25. I. Armentano, M. Dottori, E. Fortunati, S. Mattioli and J. M. Kenny, Polym. Degrad. Stab., 2010, 95, 2126–2146.
26. J. Tian, K. K. Wong, C. M. Ho, C. N. Lok, W. Y. Yu, C. M. Che, J. F. Chiu and P. K. Tam, ChemMedChem, 2007, 2, 29–36.
27. X. Xu, Q. Yang, Y. Wang, H. Yu, X. Chen and X. Jing, Eur. Polym. J., 2006, 42, 2081–2087.
28. S. A. Bidgoli, M. Mahdavi, S. M. Rezayat, M. Korani, A. Amani and P. Ziarati, Acta Med. Iran., 2013, 7, 203–208.
29. S. Prabhu and E. K. Poulose, Int. Nano Lett., 2012, 2, 32.
30. T. Gunasekaran, T. Nigusse and M. D. Dhanaraju, J. Am. Coll. Clin. Wound Spec., 2012, 3, 82–96.
31. P. Ramasamy and A. Shanmugam, Int. J. Biol. Macromol., 2015, 74, 93–102.
32. K. L. Derrick and M. C. Lessing, Eplasty, 2014, 14, e43.
33. T. Salo, M. Mäkelä, M. Kylmäniemi, H. Autio-Harmainen and H. Larjava, Lab. Invest., 1994, 70, 176–182.
34. K. K. Chereddy, R. Coco, P. B. Memvanga, B. Ucakar, A. des Rieux, G. Vandermeulen and V. Préat, J. Controlled Release, 2013, 171, 208–215.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra23212k

‡ Current address: Global Research Laboratory for RNAi Medicine, Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Republic of Korea.

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