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Fabrication of Polymeric Nanostructured Hydrophobic Surfaces and Evaluation of Antimicrobial Activity.

Introduction

Bio contamination is of great concern in numerous applications including biosensors, biomedical devices, surgical equipments, protective apparels of hospital, food packaging, food storage [1] , water purification system, industrial [2] and marine equipment (3,4). It is one of the major causes of failure in medical implantations and increased rate of hospital associated infections.

Although the risk of infection through implanted devices ranges between 1 and 7%, the consequences of associated infection are of great concern. In the US, about 100,000 out of over two million device implants result in some form of postoperative infection. The cost of treating an implant-associated infection ranges from $30,000 to $300,000 and often involves repeated hospitalizations, increased morbidity, mortality, and treatment cost and fixation failure [5]. The increasing prevalence of these devices and the concomitant rise in associated infections have led to widespread dissemination of antibiotics, resulting in the emergence and rapid spread of drug-resistant microbes. The development of device coatings capable of resisting microbial colonization has become a major thrust of research Ideal non-fouling coatings not only resist adhesion of fouling agents (i.e., microorganisms), but also allow easy removal of contamination that may occur. The "self-cleaning" superhydrophobic surfaces existing naturally include lotus leaves exhibiting a static water contact angles >150[degrees].

Organic antibacterial agents had been broadly utilized but due to their low melting and boiling point they volatilize and lose their toxicity. Therefore, inorganic-organic formulations have been used by many researchers and have found to have better chemical durability, thermal resistance and fewer side effects on human body [6]. polymeric molecules such as 5- chloro-8-quinolinyl acrylate [7] methacrylate containing pendent biquaternary ammonium moieties based on (DABCO) [1,4diazabicyclo-[2.2.2]-octane] [8] are examples of commonly studied antibacterial agents that have been used for this purpose [9].

Out of the other polymer based fabrications, recent studies reporting the antibacterial properties of PMMA based composites indicate its prime importance as efficient coating material. It has been reported to posses' antibacterial activity against Staphylococcus aureus and Escherichia coli which varies with the molecular weight of the polymer. The polymer was found to kill bacteria on direct contact, without releasing any active component, which meets the demand of development of antibacterial environmental friendly biomaterials [10]. PMMA has been successfully used both as polymer matrix on plane surface and as nanostructures with other composites [11].

These polycationic polymers are known to affect adversely bacteria by disrupting the net negative charge of the membrane of the bacteria, which causes cell lysis and death. Hydrophobicity seems to weaken bacterial attachment to surface which can be exploited in applications that require bacteria free environment. The use of antimicrobial polymers not only enhances the efficacy of existing antimicrobial agents but also minimizes the environmental problems associated with conventional antimicrobial agents like toxicity of these agents, increasing their efficiency and selectivity, and prolonging the lifetime of the antimicrobial agents [1]. In this regard, the improvements in the field of polymer science have successfully reported the formulation and fabrication of polymer based coatings with hydrophobic properties. In the present study, a PMMA based coating was fabricated to serve in improving the hydrophobicity of the surface and reducing bacterial adhesion and contamination. Organic solvents were used to study their effect on the morphological properties of the coating. Chemical etching was performed onto these coatings to fabricate a nanostructured polymeric surface to improve the hydrophobicity of surface coating.

Such hydrophobic coatings can be applied stably onto glass or plastic surfaces making them resistant to moisture and bacterial adhesion. It can also be hypothesized that such coating may have vital role in regulating the formation of biofilms onto marine surfaces. Such coatings may also have tremendous role in medical equipments such as catheters, prosthetic devices, contact lenses [12] and in immunological assays like enzyme-linked immunosorbent assay (ELISA), in devices for drug delivery, and in materials for patterned cell culture [13,14]. Further, antimicrobial coatings can be used for water treatment and for several non-medical applications such as antimicrobial protection of toilet seats, children's toys, telephone receivers and sports fabrics.

Materials and Method

PMMA with average molecular wt-350,000 in the form of powder were procured from-Aldrich. The stains used in this study namely E.coli MTCC -1610 was procured from Himedia, India and were maintained in nutrient agar slants. All the chemicals such as ethyl acetate, chloroform, and acetone were purchased from CDH Mumbai and are of AR grade.

Preparation of Coating Solution

Various coating materials with different compositions were prepared by dissolving 10% PMMA in different solvent systems. In brief 8 grams of PMMA was mixed in 80 ml of different solvent compositions of chloroform, ethyl alcohol and toluene. For complete dissolution the mixture is stirred on magnetic stirrer for two hrs at 600 rpm at room temperature.

Coating Process

A glass substrate of -X--and having--thickness was used. The substrate was first cleaned with ethanol ultrasonically, rinsed with double distilled water and then dried at 60[degrees]C for 1 hrs. The coating material was applied on glass substrate by simple dip coating with withdrawal speed of 6 cm/ min. and dried at room temperature for 5 min.

Chemical etching of coatings

To create nanostructured morphology and prepare hydrophobic surface the coated surfaces were etched by dipping in different organic solvents which were already used for dissolution of PMMA. By this way the solvents selectively etch the PMMA and create nanostructures.

Scanning Electron Microscopic Studies

Surface morphology of the hydrophobic polymeric coated surface was done by using Zeiss scanning electron microscope (SEM). For SEM, the films were first mounted on stub and then fix with the help of good adhesive (tape, paste) and put in the gold coating unit for 30 minute. This unit give the thickness of 10-15AO to the sample and make conductive for the transmission of electron beam for images and collected the images of polymer with different solvents at variable magnification at different scale as possible.

Contact angle studies

The hydrophobicity of the film was measured by checking contact angle of water dropped on the film surface using contact angle goniometer. This was performed on Easy Drop-standard from KRUSS; Germany with pre-installed Drop Shape Analysis Software DSA-1. The sessile drop method was used to measure the contact angle using an optical subsystem to capture the profile of a pure liquid on a solid substrate. This method involves depositing a liquid drop on a smooth solid surface and measuring the angle between the solid surface and the tangent to the drop profile at the drop edge.

Antimicrobial Activity Studies

Antimicrobial activity of PMMA coated surface was done by dipping the small piece of coated surfaces into liquid bacterial culture of E. coli having a standard density 0.5 McFarland for 24 hrs at 37[degrees]C. After Removal the coated pieces were dried and stained. The stained pieces were visualized under trinoccular microscope to observe bacterial adhesion on coated surface. Same treatment was applied on plane glass surface as positive control to compare the results.

Results and Discussion

Fabrication of Coating

Various coating formulations were prepared and successfully applied on glass surfaces. The nanostructures were created by solvent etching. Compositions of various coating formulations and solvents used for etching were shown in Table 1 with their codes.

Scanning Electron Microscopic Studies of coated surfaces

SEM images of various coatings were shown in figure 1. SEM images of all coating shows micro and nanostructures on surface as compared to plane glass slide (ref.). These micro and nanostructures leads to surface roughness and responsible for hydrophobicity on surface. PCEE coated surface shows aspen leaf like nanostructures which shows hydrophobic nature.

Effect of coating on Contact angle

Water drop contact angle on coated substrates were measured and results are shown in table 1and the pictures are shown in figure 2. The maximum contact angle was found to be 123.76[degrees] for [PCE.sub.E] coating. Contact angle on plane glass is 80.1o (less than 90[degrees]) hence hydrophilic in nature whereas all the coated substrates shows contact angle more than 90o and hence hydrophobic in nature. Further the results are in accordance with SEM images where all the images show surface roughness with micro and nanostructures. Due to this surface roughness the water drops make contact angle more than 90o.

Effect of coating on microbial adhesion

Microbial adhesion on coated surface where evaluated by using e-coli liquid culture. The results are shown in table 1 and pictures are shown in figure 3. Results clearly show less bacterial adhesion on coated surfaces as compared to plane glass surface. It was also noticed that the surface with high contact angle shows minimum adhesion as in case of [PCE.sub.E] coating. Thus hydrophobicity leads to less microbial adhesion.

Conclusion

Various polymeric coatings solutions was prepared by using PMMA in combination with organic solvents. The micro and nanostructures was developed by solvent selective etching. All coated surfaces shows considerable hydrophobicity as evident from contact angle measurement. SEM images clearly shows presence of nano and microstructures on coated surfaces. Hydrophobic surfaces show good antimicrobial activity and most hydrophobic surface shows least bacterial adhesion.

References

[1.] E. R. Kenawy, S. D. Worley, R. Broughton. The Chemistry and Applications of Antimicrobial Polymers: A State-of-the-Art Review. Biomacromolecules, 8 (5), pp 1359-1384, 2007.

[2.] E. Almeida, T. C. Diamantino, O. de Sousa. Marine paints: the particular case of antifouling paints. Prog Org Coat. 59:2-20, 2007

[3.] N. Cole, E. B. H. Hume, A. K. Vijay, P. Sankaridurg, N. Kumar, M. D. P. Willcox. In Vivo Performance of Melimine as an Antimicrobial Coating for Contact Lenses in Models of CLARE and CLPU, Invest. Ophthalmol. Visual Sci. 51 , 390 -395, 2010.

[4.] K. Vasilev, J. Cook, H. J. Griesser, Antibacterial surfaces for biomedical devices. Expert Rev. Med. Devices. 6(5), 553-67, 2009.

[5.] P. Asuri, S. S. Karajanagi, R. S. Kane, J. S. Dordick. Polymer nanotube enzyme composites as active antifouling films, Small, 3, 50-53, 2007.

[6.] J.M. Schierholz, L.J. Lucas, A. Rump, and G. Pulverer, Efficacy of silver-coated medical devices. J. Hosp. Infect. 40, 257, 1998.

[7.] Bankova, M.; Petrova, Ts.; Manolova, N.; Rashkov, I. Eur. Polym. J. Homopolymers of 5-chloro-8-quinolinyl acrylate and 5-chloro-8-quinolinyl methacrylate and their copolymers with acrylic and methacrylic acid., 32, 569-578, 1996.

[8.] Dizman, B.; Elasri, M.O.; Mathias, L.J., Synthesis and Antimicrobial Activities of New Water-soluble Bis-quaternary Ammonium Methacrylate Polymers, J. Appl. Polym. Sci., 94, 635-642, 2004.

[9.] Siedenbiedel, F. and Tiller, J.C. Antimicrobial polymers in solution and on surfaces: overview and functional principles. Polymers, 4, 46-71, 2012.

[10.] Huajiang Zuo, Dingcai Wu, Ruowen Fu. Preparation of antibacterial poly(methyl methacrylate) by solution blending with water insoluble antibacterial agent poly[(tertbutylamino)ethyl methacrylate]. Journal of applied polymer science, 125 (5):3537-3544, 2012.

[11.] Sanches LM, Petri DFS, Carrasco LD and Carmona-Ribeiro AM. The antimicrobial activity of free and immobilized poly (diallyldimethylammonium) chloride in nanoparticles of poly (methylmethacrylate) Journal of Nanobiotechnology, 13:58, 2015.

[12.] Mark D.P. Willcox, Emma B.H. Hume, Ajay K. Vijay and Robert Petcavich. Ability of Silver impregnated contact lenses to control microbial growth and colonization. J Optom. 3(3): 143-148, 2010.

[13.] K. Page, M. Wilson and I. P. Parkin. Antimicrobial surfaces and their potential in reducing the role of the inanimate environment in the incidence of hospital-acquired infections. J. Mater. Chem., Vol 19, 3819-3831, 2009.

[14.] A. Hucknall , S. Rangarajan , A. Chilkoti. In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins, Adv. Mater.21 , 2441-2446, 2009.

Abhilasha Mishra *, Richa Jha

Department of Chemistry, Graphic Era University, Dehradun, Uttarakhand, India

Department of Chemistry and biotechnology Uttaranchal University, Dehradun, Uttarakhand, India

Received 24 December 2017; Accepted 11 January 2017; Published online 31 December 2017

* Coresponding author: Dr. Abhilasha Mishra; E-mail: abhi_virgo1 @rediffmail. com

Caption: Figure 1: SEM Images of plane glass (ref.), coated surfaces (PCTc PCEC PCTt PCEe) and aspen leaf (a)

Caption: Figure 2: Contact angle Images of plane glass (ref.) and coated surfaces (PCT, PCEC, PCTt, PCEe)

Caption: Figure 2: Microbial adhesion Images of plane glass (ref.) and coated surfaces (PCTc PCEC PCTt PCEe)
Table 1: Showing Coating Composition, contact angle and the Effect of
microbial growth on various PMMA coatings

    Coating Code   Composition of Coating             Solvent used
                                                      for etching

1   Ref            Plane Glass Surface
3   [PCT.sub.c]    PMMA+ Chloroform + Toluene         Chloroform
4   [PCT.sub.T]    PMMA+ Chloroform + Toluene         Toluene
5   [PCE.sub.C]    PMMA+ Chloroform+ ethyl acetate    Chloroform
6   [PCE.sub.E]    PMMA+ Chlorofonn+ ethyl acetate    ethyl acetate

    Contact     Microbial growth
    Angle       study bv Pouring
                Method

1   80.10               +++
3   94.93                ++
4   100.49               ++
5   101 03               +
6   123.76               -
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Title Annotation:Original Article
Author:Mishra, Abhilasha; Jha, Richa
Publication:Trends in Biomaterials and Artificial Organs
Date:Jan 1, 2017
Words:2107
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