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Phomopsidione nanoparticles coated contact lenses reduce microbial keratitis causing pathogens
Experimental Eye Research 178 (2019) 10–14
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Phomopsidione nanoparticles coated contact lenses reduce microbial keratitis causing pathogens
T
Muhammad Yusoff Bin Sahadana, Woei Yenn Tonga,∗, Wen Nee Tanb, Chean Ring Leonga, Mohamad Najib Bin Misric, Murphy Chand,e, See Yuan Chengf, Shahrulzaman Shaharuddina a Universiti Kuala Lumpur, Malaysian Institute of Chemical and Engineering Technology, Lot 1988 Kawasan Perindustrian Bandar Vendor, Taboh Naning, 78000, Alor Gajah, Melaka, Malaysia b School of Distance Education, Universiti Sains Malaysia, 11800, Gelugor, Pulau Pinang, Malaysia c Massey University, Palmerston North, Auckland, Wellington, New Zealand d Management Science University, University Drive, Off Persiaran Olahraga, 40100, Shah Alam, Selangor, Malaysia e Eyecon Optometri, G10 Bangunan Kings Hotel, Lebuh Ayer Keroh, 75450, Melaka, Malaysia f Faculty of Mechanical Engineering, Universiti Teknikal Malaysia Melaka, Hang Tuah Jaya, 76100, Durian Tunggal, Melaka, Malaysia
A R T I C LE I N FO
A B S T R A C T
Keywords: Contact lens Microbial keratitis Phomopsidione Nanoparticles
Microbial keratitis is the infection caused by pathogenic microorganisms that commonly occurs among the contact lens users. Various antimicrobial compounds were coated on contact lenses to kill keratitis causing microorganisms, however these compounds caused several adverse side effects. Hence, the aim of this study is to develop a silicone hydrogel contact lens coated with phomopsidione nanoparticle that inhibit keratitis causing clinical isolates. Phomopsidione nanoparticles were synthesized using polyvinyl alcohol as encapsulant. The nanoparticles showed an average size of 77.45 nm, with neutral surface charge. Two drug release patterns were observed in the drug release profile, which are the initial slow release phase with extended drug release (release rate 46.65 μg/h), and the burst release phase observed on Day 2 (release rate 2224.49 μg/h). This well-regulated drug delivery system enables the control of drug release to meet the therapeutic requirements. On agar diffusion assay, 3 out of 5 test microorganisms were inhibited by phomopsidione nanoparticle coated contact lenses, including two Gram negative bacteria. Besides, all test microorganisms showed at least 99% of growth reduction, with the treatment of the contact lens model. The drug loaded onto the nanoparticles is sufficient to prevent the bacterial growth. In conclusion, this study provides an effective alternative to combat keratitis-causing microorganisms among contact wearers.
1. Introduction Microbial keratitis is referring to microbial infection on cornea tissue (Jeng, 2003). Approximately 52% of microbial keratitis incidences in 2012 were related to contact lens wear (Dyavaiah et al., 2015). These infections may result in permanent visual loss due to corneal scarring or perforation (Cheng et al., 1999). The corneal ulcers remain as the major cause for visual loss globally. The infections are mainly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens (Kilvington et al., 2013). These bacteria are predominantly present on the surface of the contact lens or in the storage cases. Furthermore, the colonization of bacteria in the form of biofilm showed an increase resistance to disinfectants due to the poor antimicrobial diffusion, induction of phenotypic variability, and adaptive stress responses (Bassyouni et al., 2016). ∗
Microbial keratitis is common among contact lenses users. Contact lenses are effective mode of vision correction used by more than 125 million people worldwide (Eltis, 2011). Silicone hydrogel is the most commonly used contact lens material, it makes up 64% of contact lens production worldwide (Subbaraman and Jones, 2013). Advances in contact lens manufacturing technologies and lens care in the past few decades have further improved the safety and efficacy of contact lens wear. However, some adverse responses to microbial contamination on contact lenses still occur (Miller et al., 2007). Many approaches were attempted to reduce the risk of microbial contamination on contact lenses, including the lens care solution that provide effective disinfection and frequent lens cases replacement (Bazzaz et al., 2014). However, these approaches are less effective due to the emergence of multidrug-resistant pathogens and the formation of biofilm by the microbes on the surface of the contact lens.
Corresponding author. E-mail address: [email protected] (W.Y. Tong).
https://doi.org/10.1016/j.exer.2018.09.011 Received 14 June 2018; Received in revised form 2 August 2018; Accepted 19 September 2018 Available online 20 September 2018 0014-4835/ © 2018 Elsevier Ltd. All rights reserved.
Experimental Eye Research 178 (2019) 10–14
M.Y. Bin Sahadan et al.
Series 200) with C18 Column (250 mm × 4.6 mm, 5 μ particle size) at the wavelength of 264 nm using UV–Vis detector. The mobile phase used was phosphate buffer and methanol at the ratio of 7:3 (v/v) with flow rate 0.5 ml/min. The amount of phomopsidione in the sample was determined based on the calibration curve. Encapsulation efficiency and nanoparticle yield were calculated based on the equation below:
Coating of antimicrobial compounds onto contact lenses is one of the effective measurements to prevent microbial keratitis. Silver ions are commonly used as antimicrobial coating for contact lenses. However, the application of silver ions on ocular surface induced argyrosis, the toxic-blackening of the mucus membrane (Lansdown, 2006). The antimicrobial efficiency of nanosilver was reported by Huang et al. (2016) and Shayani et al. (2016) on microbial keratitis causing microorganisms. Even so, high concentration of nanosilver caused cytotoxicity to the mitochondrial activity and it is not suitable for ophthalmic application (Rai et al., 2009). Thus, seeking a natural alternative is crucial to replace silver ions. In this study, we aimed to develop an antimicrobial contact lens model impregnated with phomopsidione nanoparticles. The antimicrobial efficiency of the contact lens model was tested on bacterial keratitis clinical strains. Phomopsidione (C7H10O4) is a novel ketone derivative isolated from Diaporthe flaxinii ED2. It is worth noting that previous toxicity study experiments in animal models have demonstrated that phomopsidione has low toxicity and therefore represents a chemotherapeutic effect worthwhile of further investigations (Yenn et al., 2017). Nanostructured biomaterial or nanoparticle in particular, are widely used to enhance the chemical and physical properties of pharmaceutical compounds. The technology has been applied to facilitate the administration of antimicrobial drugs, thus overcoming some of the limitation in traditional antimicrobial chemotherapies (Zhang et al., 2010). Besides improving the bioavailability of the drugs, nanoparticles are also used to facilitate antimicrobial drug delivery to the infectious site. However, only very few drug delivery systems were approved for ocular use. In this study, we developed a nano-scale drug delivery system for silicone contact lenses, by using polyvinyl alcohol (PVA) as functional polymer matrix.
Encapsulation efficiency (%) =
Concentration drug in nanoparticle Concentration of drug fed initially
× 100% Nanoparticle Yield (%) =
(1)
Weight of nanoparticle Weight of drug and polymer fed initially × 100%
(2)
2.3.3. Zeta potential The phomopsidione nanoparticle was dissolved in distilled water and the surface charge and zeta potential of the nanoparticles were analysed by dynamic light scattering (DLS) on a zeta sizer (Malvern Instrument) software Version 7.11. Each parameter was measured thrice. 2.4. Phomopsidione nanoparticles coating onto silicone hydrogel contact lens This coating was performed according to Jung et al. (2013) with modifications. The phomopsidione nanoparticle were coated onto silicone hydrogel contact lens by soaking the lenses (ACUVUE® TrueEye™14.2 mm diameter) into 2% nanoparticle solution for 24 h at 4 °C. The resulting contact lenses were then utilized for the tests. A control was included by immersing the lenses in 2% nanoparticle solution synthesized previously without phomopsidione.
2. Materials and method 2.1. Test compound
2.5. Scanning electron microscope (SEM) observation of contact lens model
The test compound, phomopsidione was generously donated by Professor Darah Ibrahim, Universiti Sains Malaysia. The compound was first discovered by her research team (Yenn et al., 2017).
The surface morphology of contact lens coated with Phomopsidione nanoparticle were observed under SEM (Jeol 6010Plus).
2.2. Synthesis of phomopsidione nanoparticle
2.6. Drug release behaviour
Phomopsidione nanoparticles were prepared using a solvent and anti-solvent precipitation method (Lee et al., 2015). In this process, 0.02 g of phomopsidione was firstly dissolved in 60 ml of ethanol at room temperature. After that, 50 ml of Pluronic F127 (1% w/v) (Sigma) was prepared at 4 °C in an ice bath. Next, both solutions were mixed and homogenized (Heidolph) at 10,000 rpm for 2 min. Next, 1% of PVA (R&M) solution was added. The mixture was homogenized until a colorless transparent solution was obtained. The solidified solution was then freeze dried (Labconco) for 48 h. A control was included by synthesizing the nanoparticle without phomopsidione.
Two contact lenses coated with phomopsidione nanoparticle were soaked in 0.1 M (pH 7.4) phosphate buffer solution at room temperature. In every 24 h, 1 ml sample was withdrawn from the solution. The amount of the phomopsidione released were then determined as per protocols described in Section 2.3.2. 2.7. Test microorganisms The test microorganisms used in this study were Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, Candida utilis and Candida albicans. The microbial strains were previously isolated from patients in Hospital Seberang Jaya, Penang, Malaysia, with a cohort of microbial keratitis. The inoculum suspension was prepared by transferring several bacterial colonies into sterile physiological saline. The turbidity of the inoculum suspension was adjusted to match the 0.5 McFarland standard which equivalent to the density of microbial suspension 1.5 × 108 colony forming unit (CFU).
2.3. Characterization of phomopsidione nanoparticle 2.3.1. Transmission electron microscope (TEM) observation The morphology and particle size of phomopsidione nanoparticle were observed under a TEM (Phillips CM12), operating at 120 kV. The samples were stained using uranyl acetate and placed on copper grid to be examined using TEM. 2.3.2. Encapsulation efficiency and nanoparticle yield The phomopsidione nanoparticle was dispersed and dissolved in 0.1 M (pH 7.4) phosphate buffer solution. The solution was then filtered using PTFE syringe filter hydrophobic (Bioflow) with 0.22 μm pore size. Then, 20 μl of the resulting solution was then injected into the HighPerformance Liquid Chromatography (HPLC) system (Perkin Elmer
2.8. Agar diffusion assay This assay was performed to screen the antimicrobial activity of phomopsidione nanoparticles coated contact lens on clinical isolates. Mueller Hinton agar (Merck) plate was prepared by pouring 20 ml of sterile molten nutrient agar onto 9 cm diameter Petri dish. The 11
Experimental Eye Research 178 (2019) 10–14
M.Y. Bin Sahadan et al.
Besides, PVA also used to encapsulate ketone derivative as reported by She et al. (2005). Encapsulation is defined as a process of enclosing a core material (Goran and Richard, 2016). The encapsulation efficiency was determined by the concentration of the incorporated material detected in the formulation over the initial concentration used to make the formulation (Goran and Richard, 2016). Based on the results, the nanoparticle yield and encapsulation efficiency obtained were 28.7% and 33.75% respectively. The encapsulation efficiency was significantly low compared to studies reported by Papadimitriou and Bikiaris (2009) and Lee et al. (2015), which also use PVA as encapsulant. The low encapsulation efficiency is possibly caused by low stirring speed during nanoparticle preparation. The encapsulation efficiency can be increased by creating high shear stress which facilitates the dispersion of viscous droplets (Li et al., 2017). The surface chemistry of nanoparticles affects the efficiency in ocular drug deliver. Zeta potential is the electrostatic potential at the electrical double layer surrounding the phomopsidione nanoparticle. The magnitude of the zeta potential gives an indication of the potential stability of the nanoparticles (Bhattacharjee, 2016). The zeta potential of phomopsidione nanoparticle was measured at −5.03 mV, with a conductivity of 0.656 mS/cm. The zeta potential confirmed that the nanoparticles are neutral in charge. The result is in agreement with Mura et al. (2011) and Ai et al. (2011). This characteristic allows even dispersion of nanoparticles in the silicone material, which further improves the drug delivery to the ocular system. Fig. 2 shows the SEM micrograph of phomopsidione nanoparticle coated contact lens. The hydrogel matrix showed a mesh-like structure. This contact lens is made up of siloxane and hydrogel material with the surface treatment using galafylcon A. This resulted the formation of ultrastructure network which consist of several layer of microstructure made up from different material, which can be clearly seen on the micrograph (González et al., 2006). Ethanol was used to create microscopes on the ultrastructure on the lens, which allows the coating of nanoparticles onto the surface (Jung et al., 2013). One of the challenge in ocular drug delivery is to control the release of the drug. This is particularly important to provide long hour protection against microorganisms among contact lens wearers. In this study, 2 drug release patterns were observed in the drug release profile of phomopsidione nanoparticles (Fig. 3), the initial slow release phase (extended release) and the burst release on Day 2. The typical release profile of PVA particulate delivery system was observed. The initial release was slow but gradual (46.65 μg/h) on Day 1–2, as PVA forms a barrier which contribute to the slower release of drug (Gupta et al.,
microbial suspension was spread uniformly on the agar plate by using sterile cotton swab. Next, the phomopsidione nanoparticles coated contact lens and control were gently placed on the surface of agar medium. The contact lens with blank nanoparticle acts as negative control. All plates were incubated at 37 °C for 24 h. The average diameter of clear zones surrounding the contact lens were measured and recorded after the incubation period. 2.9. Growth reduction assay The bacteria that showed inhibitory activity on agar diffusion assay were selected. The contact lenses were placed into 900 μl of Mueller Hinton broth (Merck), containing 100 μl of bacterial inoculum. The cultures were then incubated at 37 °C for 48 h. After incubation period, viable cell count was performed using spread plate method to quantify the living bacterial cells in the sample. The number of living bacterial cells were expressed in number of colony forming units (CFU) per ml of sample. 3. Result and discussion In the field of ophthalmic drug delivery, many approaches were explored to increase the efficiency and residence time of antimicrobial drugs on contact lenses. One of the most common method is by encapsulating the drug in the form of nanoparticles (Kim et al., 2008). In this study, phomopsidione nanoparticles were synthesized via solvent and anti-solvent precipitation method. A FDA-approved polymer, PVA was used as non-ionic stabilizer to encapsulate phomopsidione. Besides, pluronic F-127 was also added to prevent the coalescence between nanoparticles, while maintaining their interfacial energy. This is because nanoparticles tend to agglomerate in order to minimize their surface energy (Lee et al., 2015). Fig. 1 shows the TEM micrograph of phomopsidione nanoparticles. The nanoparticles are of sphere like morphology, with an average size of 77.45 ± 11.7 nm. The particle sizes were obtained by analyzing the recorded images, and the findings indicated the small mean particle size, which is less than 100 nm. PVA is a water soluble synthetic polymer which belongs to the class of nonionic polymers containing a vinyl group (Moukwa et al., 1993). The polymer is an ideal choice for ophthalmic applications, as it is nontoxicity, non-carcinogenic, and low immunogenicity (DeMerlis and Schoneker, 2003). Besides, the polymer also stabilizes the nanoparticles during freeze-drying process through steric hindrance (Lee et al., 2015).
Fig. 1. TEM micrograph of phomopsidione nanoparticles. The average size of the nanoparticles is 77.45 nm ± 11.7 nm.
Fig. 2. SEM micrograph of contact lens coated with phomopsidione nanoparticles. The mesh-like matrix of silicone hydrogel was observed. 12
Experimental Eye Research 178 (2019) 10–14
M.Y. Bin Sahadan et al.
Fig. 3. The drug release behaviour of phomopsidione from the PVA-encapsulated nanoparticles. Two drug release patterns were observed.
bacteria in causing contact lens related keratitis (Fleiszig and Evans, 2010). The contact lens also showed good inhibitory activity on S. marcescens, which frequently associated with keratitis and ulcerations among contact lens wearers. However, in this study, no anti-yeast activity was reported. A quantitative antibacterial assessment of the contact lens models was tested in a broth medium. The contact lens coated with phomopsidione nanoparticle showed a significant growth reduction on all test microorganisms, with the percentage of growth reduction up to 99%. The results are in agreement with agar diffusion assay. The drug loaded onto the nanoparticles is sufficient to prevent the bacterial growth, for a period of 48 h.
Table 1 Antimicrobial activity of the contact lenses coated with phomopsidione nanoparticles. The contact lenses inhibited the growth of 3 test microorganisms, with at least 99% of growth reduction. Test microorganisms
S. marcescens P. aeruginosa MRSA P. mirabilis C. utilis
Average diameter of clear zone (mm) Control
Contact lenses with phomopsidione nanoparticles
– – – – –
41.6 ± 3.2 51.3 ± 2.9 24.0 ± 4.0 – –
Percentage of growth reduction (%)
99.9 99.9 99.34 – –
4. Conclusion In conclusion, phomopsdione nanoparticles coated contact lens showed sustainable release pattern of drug for 48 h. The contact lens models exhibited inhibited keratitis causing microorganisms, particularly on Gram negative bacteria. The contact lens models also caused a significant growth reduction on the test microorganisms, with the percentage of growth reduction up to 99%. The study could be extended to in vivo keratitis models to evaluate the antimicrobial potential on animal models.
*The average diameter of clear zone was expressed in average diameter ± standard deviation. The experiments were conducted in triplicates in separate occasions.
2010). A total of 17% of drug was released in 48 h of test period. Such a well-regulated drug delivery system enables the control of drug amount released to meet the therapeutic requirements. A burst release state was observed on Day 2 with release rate 224.49 μg/h. The drug release at later phase was rapid and high concentration of phomopsidione was detected in the test medium. This is partially due to the complete swelling of the silicone hydrogel in the test medium, which increase the total area exposed (Diebold and Calonge, 2010). The release was complete on Day 4, following the first order of kinetic. In contrast, Kim et al. (2008) reported that the release of the ocular drug from the silicone hydrogel contact lenses can be prolonged up to 150 days. The drug release profile of nanoparticles is influenced by structure of the silicone hydrogel matrix, and also the physiochemical characteristics of the drug. The antimicrobial efficiency of the contact lens models was tested on several clinical pathogens. Drug delivery of therapeutic contact lenses is a challenge as the complex silicone hydrogel matrix hinders the release of drug (Paradiso et al., 2016). On agar diffusion assay, 3 out of 5 test microorganisms were inhibited by phomopsidione nanoparticles coated contact lenses, including 2 Gram negative bacteria. No clear zones were observed surrounding the controls. This indicates that the antimicrobial activity was solely due to phomopsidione present in the nanoparticles. Most of the severe cases of microbial keratitis were caused by Gram-negative bacteria (Jeng, 2003). They are hardly killed by the conventional disinfectants due to their ability to form biofilm, which reduce the penetrating capacity of disinfectant. In this study, P. aeruginosa showed the largest clear zone, with a diameter of 51.3 mm (Table 1). P. aeruginosa is considered as the most virulent pathogenic
Funding This study is supported by Fundamental Research Grant Scheme (FRGS/1/2017/STG05/UNIKL/02/5). Acknowledgement The test compound, phomopsidione was generously donated by Professor Darah Ibrahim, Universiti Sains Malaysia. References Ai, J., Biazar, E., Jafarpour, M., Montazeri, M., Majdi, A., Aminifard, S., Zafari, M., Akbari, H.R., Rad, H.G., 2011. Nanotoxicology and nanoparticle safety in biomedical designs. Int. J. Nanomed. 6, 1117. Bassyouni, R.H., Kamel, Z., Abdelfattah, M.M., Mostafa, E., 2016. Cinnamon oil: a possible alternative for contact lens disinfection. Contact Lens Anterior Eye 39 (4), 277–283. Bazzaz, B.S.F., Khameneh, B., Jalili-Behabadi, M.M., Malaekeh-Nikouei, B., Mohajeri, S.A., 2014. Preparation, characterization and antimicrobial study of a hydrogel (soft contact lens) material impregnated with silver nanoparticles. Contact Lens Anterior Eye 37 (3), 149–152. Bhattacharjee, S., 2016. DLS and zeta potential–What they are and what they are not? J. Contr. Release 235, 337–351. Cheng, K.H., Leung, S.L., Hoekman, H.W., Beekhuis, W.H., Mulder, P.G., Geerards, A.J., Kijlstra, A., 1999. Incidence of contact-lens-associated microbial keratitis and its
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Lee, W.H., Bebawy, M., Loo, C.Y., Luk, F., Mason, R.S., Rohanizadeh, R., 2015. Fabrication of curcumin micellar nanoparticles with enhanced anti-cancer activity. J. Biomed. Nanotechnol. 11 (6), 1093–1105. Li, X., Wang, L., Wang, B., 2017. Optimization of encapsulation efficiency and average particle size of Hohenbuehelia serotina polysaccharides nano-emulsions using response surface methodology. Food Chem. 229, 479–486. Miller, M., Chalmers, R., Winterton, L., 2007. Creating antimicrobial surfaces and materials for contact lenses and lens cases. Eye Cont. Lens. 33 (6), 426–429. Moukwa, M., Youn, D., Hassanali, M., 1993. Effects of degree of polymerization of water soluble polymers on concrete properties. Cement Concr. Res. 23 (1), 122–130. Mura, S., Hillaireau, H., Nicolas, J., Le Droumaguet, B., Gueutin, C., Zanna, S., Tsapis, N., Fattal, E., 2011. Influence of surface charge on the potential toxicity of PLGA nanoparticles towards Calu-3 cells. Int. J. Nanomed. 6, 2591. Papadimitriou, S., Bikiaris, D., 2009. Novel self-assembled core–shell nanoparticles based on crystalline amorphous moieties of aliphatic copolyesters for efficient controlled drug release. J. Contr. Release 138 (2), 177–184. Paradiso, P., Serro, A.P., Saramago, B., Colaço, R., Chauhan, A., 2016. Controlled release of antibiotics from vitamin E–loaded silicone-hydrogel contact lenses. J. Pharmacol. Sci. 105 (3), 1164–1172. Rai, M., Yadav, A., Gade, A., 2009. Silver nanoparticles as a new generation of antimicrobials. Biotechnol. Adv. 27 (1), 76–83. She, S., Zhou, Y., Zhang, L., Wang, L., Wang, L., 2005. Preparation of fluorescent polyvinyl alcohol keto-derivatives nanoparticles and selective determination of chromium (VI). Spectrochem. Acta A. 62 (1–3), 711–715. Shayani, R.M., Khameneh, B., Sabeti, Z., Mohajeri, S.A., Fazly Bazzaz, B.S., 2016. Antibacterial activity of silver nanoparticle-loaded soft contact lens materials: the effect of monomer composition. Curr. Eye Res. 41 (10), 1286–1293. Subbaraman, L., Jones, L., 2013. Measuring friction and lubricity of soft contact lenses: a review. Contact Lens Spectr. 28, 28–33. Yenn, T.W., Ring, L.C., Nee, T.W., Khairuddean, M., Zakaria, L., Ibrahim, D., 2017. Endophytic Diaporthe sp. ED2 produces a novel anti-candidal ketone derivative. J. Microb. Biotechnol. 27 (6), 1065–1070. Zhang, L., Pornpattananangkul, D., Hu, C.M., Huang, C.M., 2010. Development of nanoparticles for antimicrobial drug delivery. Curr. Med. Chem. 17 (6), 585–594.
related morbidity. Lancet 354 (9174), 181–185. DeMerlis, C.C., Schoneker, D.R., 2003. Review of the oral toxicity of polyvinyl alcohol (PVA). Food Chem. Toxicol. 41 (3), 319–326. Diebold, Y., Calonge, M., 2010. Application of nanoparticles in ophthalmology. Prog. Retin. Eye Res. 29 (6), 596–609. Dyavaiah, M., Phaniendra, A., Sudharshan, S.J., 2015. Microbial keratitis in contact lens wearers. JSM Ophthalmol. 3 (3), 1036. Eltis, M., 2011. Contact-lens-related microbial keratitis: case report and review. J. Optom. 4 (4), 122–127. Fleiszig, S.M., Evans, D.J., 2010. The pathogenesis of contact lens-associated microbial keratitis. Optom. Vis. Sci. 87 (4), 225. González, M.,J.M., López, A.,A., Almeida, J.B., Parafita, M.A., Refojo, M.F., 2006. Microscopic observations of superficial ultrastructure of unworn siloxane‐hydrogel contact lenses by cryo‐scanning electron microscopy. J. Biomed. Mater. Res. B Appl. Biomater. 76 (2), 419–423. Goran, T.V., Richard, G.H., 2016. Encapsulation efficiency. In: Encyclopedia of Membranes. Springer, Berlin Heidelberg, pp. 703–707. Gupta, H., Aqil, M., Khar, R.K., Ali, A., Bhatnagar, A., Mittal, G., 2010. Sparfloxacinloaded PLGA nanoparticles for sustained ocular drug delivery. Nanomed. Nanotechnol. Biol. Med. 6 (2), 324–333. Huang, J.F., Zhong, J., Chen, G.P., Lin, Z.T., Deng, Y., Liu, Y.L., Yuan, J., 2016. A hydrogel-based hybrid theranostic contact lens for fungal keratitis. ACS Nano 10 (7), 6464–6473. Jeng, B.H., 2003. Microbial keratitis. Br. J. Ophthalmol. 87 (7), 805–806. Jung, H.J., Abou-Jaoude, M., Carbia, B.E., Plummer, C., Chauhan, A., 2013. Glaucoma therapy by extended release of timolol from nanoparticle loaded silicone-hydrogel contact lenses. J. Contr. Release 165 (1), 82–89. Kilvington, S., Shovlin, J., Nikolic, M., 2013. Identification and susceptibility to multipurpose disinfectant solutions of bacteria isolated from contact lens storage cases of patients with corneal infiltrative events. Contact Lens Anterior Eye 36 (6), 294–298. Kim, J., Conway, A., Chauhan, A., 2008. Extended delivery of ophthalmic drugs by silicone hydrogel contact lenses. Biomaterials 29 (14), 2259–2269. Lansdown, A., 2006. Silver in healthcare: antimicrobial effects and safety in use. Biofunctional Text. Ski 33, 17–34.
14
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Phomopsidione nanoparticles coated contact lenses reduce microbial keratitis causing pathogens
T
Muhammad Yusoff Bin Sahadana, Woei Yenn Tonga,∗, Wen Nee Tanb, Chean Ring Leonga, Mohamad Najib Bin Misric, Murphy Chand,e, See Yuan Chengf, Shahrulzaman Shaharuddina a Universiti Kuala Lumpur, Malaysian Institute of Chemical and Engineering Technology, Lot 1988 Kawasan Perindustrian Bandar Vendor, Taboh Naning, 78000, Alor Gajah, Melaka, Malaysia b School of Distance Education, Universiti Sains Malaysia, 11800, Gelugor, Pulau Pinang, Malaysia c Massey University, Palmerston North, Auckland, Wellington, New Zealand d Management Science University, University Drive, Off Persiaran Olahraga, 40100, Shah Alam, Selangor, Malaysia e Eyecon Optometri, G10 Bangunan Kings Hotel, Lebuh Ayer Keroh, 75450, Melaka, Malaysia f Faculty of Mechanical Engineering, Universiti Teknikal Malaysia Melaka, Hang Tuah Jaya, 76100, Durian Tunggal, Melaka, Malaysia
A R T I C LE I N FO
A B S T R A C T
Keywords: Contact lens Microbial keratitis Phomopsidione Nanoparticles
Microbial keratitis is the infection caused by pathogenic microorganisms that commonly occurs among the contact lens users. Various antimicrobial compounds were coated on contact lenses to kill keratitis causing microorganisms, however these compounds caused several adverse side effects. Hence, the aim of this study is to develop a silicone hydrogel contact lens coated with phomopsidione nanoparticle that inhibit keratitis causing clinical isolates. Phomopsidione nanoparticles were synthesized using polyvinyl alcohol as encapsulant. The nanoparticles showed an average size of 77.45 nm, with neutral surface charge. Two drug release patterns were observed in the drug release profile, which are the initial slow release phase with extended drug release (release rate 46.65 μg/h), and the burst release phase observed on Day 2 (release rate 2224.49 μg/h). This well-regulated drug delivery system enables the control of drug release to meet the therapeutic requirements. On agar diffusion assay, 3 out of 5 test microorganisms were inhibited by phomopsidione nanoparticle coated contact lenses, including two Gram negative bacteria. Besides, all test microorganisms showed at least 99% of growth reduction, with the treatment of the contact lens model. The drug loaded onto the nanoparticles is sufficient to prevent the bacterial growth. In conclusion, this study provides an effective alternative to combat keratitis-causing microorganisms among contact wearers.
1. Introduction Microbial keratitis is referring to microbial infection on cornea tissue (Jeng, 2003). Approximately 52% of microbial keratitis incidences in 2012 were related to contact lens wear (Dyavaiah et al., 2015). These infections may result in permanent visual loss due to corneal scarring or perforation (Cheng et al., 1999). The corneal ulcers remain as the major cause for visual loss globally. The infections are mainly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens (Kilvington et al., 2013). These bacteria are predominantly present on the surface of the contact lens or in the storage cases. Furthermore, the colonization of bacteria in the form of biofilm showed an increase resistance to disinfectants due to the poor antimicrobial diffusion, induction of phenotypic variability, and adaptive stress responses (Bassyouni et al., 2016). ∗
Microbial keratitis is common among contact lenses users. Contact lenses are effective mode of vision correction used by more than 125 million people worldwide (Eltis, 2011). Silicone hydrogel is the most commonly used contact lens material, it makes up 64% of contact lens production worldwide (Subbaraman and Jones, 2013). Advances in contact lens manufacturing technologies and lens care in the past few decades have further improved the safety and efficacy of contact lens wear. However, some adverse responses to microbial contamination on contact lenses still occur (Miller et al., 2007). Many approaches were attempted to reduce the risk of microbial contamination on contact lenses, including the lens care solution that provide effective disinfection and frequent lens cases replacement (Bazzaz et al., 2014). However, these approaches are less effective due to the emergence of multidrug-resistant pathogens and the formation of biofilm by the microbes on the surface of the contact lens.
Corresponding author. E-mail address: [email protected] (W.Y. Tong).
https://doi.org/10.1016/j.exer.2018.09.011 Received 14 June 2018; Received in revised form 2 August 2018; Accepted 19 September 2018 Available online 20 September 2018 0014-4835/ © 2018 Elsevier Ltd. All rights reserved.
Experimental Eye Research 178 (2019) 10–14
M.Y. Bin Sahadan et al.
Series 200) with C18 Column (250 mm × 4.6 mm, 5 μ particle size) at the wavelength of 264 nm using UV–Vis detector. The mobile phase used was phosphate buffer and methanol at the ratio of 7:3 (v/v) with flow rate 0.5 ml/min. The amount of phomopsidione in the sample was determined based on the calibration curve. Encapsulation efficiency and nanoparticle yield were calculated based on the equation below:
Coating of antimicrobial compounds onto contact lenses is one of the effective measurements to prevent microbial keratitis. Silver ions are commonly used as antimicrobial coating for contact lenses. However, the application of silver ions on ocular surface induced argyrosis, the toxic-blackening of the mucus membrane (Lansdown, 2006). The antimicrobial efficiency of nanosilver was reported by Huang et al. (2016) and Shayani et al. (2016) on microbial keratitis causing microorganisms. Even so, high concentration of nanosilver caused cytotoxicity to the mitochondrial activity and it is not suitable for ophthalmic application (Rai et al., 2009). Thus, seeking a natural alternative is crucial to replace silver ions. In this study, we aimed to develop an antimicrobial contact lens model impregnated with phomopsidione nanoparticles. The antimicrobial efficiency of the contact lens model was tested on bacterial keratitis clinical strains. Phomopsidione (C7H10O4) is a novel ketone derivative isolated from Diaporthe flaxinii ED2. It is worth noting that previous toxicity study experiments in animal models have demonstrated that phomopsidione has low toxicity and therefore represents a chemotherapeutic effect worthwhile of further investigations (Yenn et al., 2017). Nanostructured biomaterial or nanoparticle in particular, are widely used to enhance the chemical and physical properties of pharmaceutical compounds. The technology has been applied to facilitate the administration of antimicrobial drugs, thus overcoming some of the limitation in traditional antimicrobial chemotherapies (Zhang et al., 2010). Besides improving the bioavailability of the drugs, nanoparticles are also used to facilitate antimicrobial drug delivery to the infectious site. However, only very few drug delivery systems were approved for ocular use. In this study, we developed a nano-scale drug delivery system for silicone contact lenses, by using polyvinyl alcohol (PVA) as functional polymer matrix.
Encapsulation efficiency (%) =
Concentration drug in nanoparticle Concentration of drug fed initially
× 100% Nanoparticle Yield (%) =
(1)
Weight of nanoparticle Weight of drug and polymer fed initially × 100%
(2)
2.3.3. Zeta potential The phomopsidione nanoparticle was dissolved in distilled water and the surface charge and zeta potential of the nanoparticles were analysed by dynamic light scattering (DLS) on a zeta sizer (Malvern Instrument) software Version 7.11. Each parameter was measured thrice. 2.4. Phomopsidione nanoparticles coating onto silicone hydrogel contact lens This coating was performed according to Jung et al. (2013) with modifications. The phomopsidione nanoparticle were coated onto silicone hydrogel contact lens by soaking the lenses (ACUVUE® TrueEye™14.2 mm diameter) into 2% nanoparticle solution for 24 h at 4 °C. The resulting contact lenses were then utilized for the tests. A control was included by immersing the lenses in 2% nanoparticle solution synthesized previously without phomopsidione.
2. Materials and method 2.1. Test compound
2.5. Scanning electron microscope (SEM) observation of contact lens model
The test compound, phomopsidione was generously donated by Professor Darah Ibrahim, Universiti Sains Malaysia. The compound was first discovered by her research team (Yenn et al., 2017).
The surface morphology of contact lens coated with Phomopsidione nanoparticle were observed under SEM (Jeol 6010Plus).
2.2. Synthesis of phomopsidione nanoparticle
2.6. Drug release behaviour
Phomopsidione nanoparticles were prepared using a solvent and anti-solvent precipitation method (Lee et al., 2015). In this process, 0.02 g of phomopsidione was firstly dissolved in 60 ml of ethanol at room temperature. After that, 50 ml of Pluronic F127 (1% w/v) (Sigma) was prepared at 4 °C in an ice bath. Next, both solutions were mixed and homogenized (Heidolph) at 10,000 rpm for 2 min. Next, 1% of PVA (R&M) solution was added. The mixture was homogenized until a colorless transparent solution was obtained. The solidified solution was then freeze dried (Labconco) for 48 h. A control was included by synthesizing the nanoparticle without phomopsidione.
Two contact lenses coated with phomopsidione nanoparticle were soaked in 0.1 M (pH 7.4) phosphate buffer solution at room temperature. In every 24 h, 1 ml sample was withdrawn from the solution. The amount of the phomopsidione released were then determined as per protocols described in Section 2.3.2. 2.7. Test microorganisms The test microorganisms used in this study were Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, Candida utilis and Candida albicans. The microbial strains were previously isolated from patients in Hospital Seberang Jaya, Penang, Malaysia, with a cohort of microbial keratitis. The inoculum suspension was prepared by transferring several bacterial colonies into sterile physiological saline. The turbidity of the inoculum suspension was adjusted to match the 0.5 McFarland standard which equivalent to the density of microbial suspension 1.5 × 108 colony forming unit (CFU).
2.3. Characterization of phomopsidione nanoparticle 2.3.1. Transmission electron microscope (TEM) observation The morphology and particle size of phomopsidione nanoparticle were observed under a TEM (Phillips CM12), operating at 120 kV. The samples were stained using uranyl acetate and placed on copper grid to be examined using TEM. 2.3.2. Encapsulation efficiency and nanoparticle yield The phomopsidione nanoparticle was dispersed and dissolved in 0.1 M (pH 7.4) phosphate buffer solution. The solution was then filtered using PTFE syringe filter hydrophobic (Bioflow) with 0.22 μm pore size. Then, 20 μl of the resulting solution was then injected into the HighPerformance Liquid Chromatography (HPLC) system (Perkin Elmer
2.8. Agar diffusion assay This assay was performed to screen the antimicrobial activity of phomopsidione nanoparticles coated contact lens on clinical isolates. Mueller Hinton agar (Merck) plate was prepared by pouring 20 ml of sterile molten nutrient agar onto 9 cm diameter Petri dish. The 11
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Besides, PVA also used to encapsulate ketone derivative as reported by She et al. (2005). Encapsulation is defined as a process of enclosing a core material (Goran and Richard, 2016). The encapsulation efficiency was determined by the concentration of the incorporated material detected in the formulation over the initial concentration used to make the formulation (Goran and Richard, 2016). Based on the results, the nanoparticle yield and encapsulation efficiency obtained were 28.7% and 33.75% respectively. The encapsulation efficiency was significantly low compared to studies reported by Papadimitriou and Bikiaris (2009) and Lee et al. (2015), which also use PVA as encapsulant. The low encapsulation efficiency is possibly caused by low stirring speed during nanoparticle preparation. The encapsulation efficiency can be increased by creating high shear stress which facilitates the dispersion of viscous droplets (Li et al., 2017). The surface chemistry of nanoparticles affects the efficiency in ocular drug deliver. Zeta potential is the electrostatic potential at the electrical double layer surrounding the phomopsidione nanoparticle. The magnitude of the zeta potential gives an indication of the potential stability of the nanoparticles (Bhattacharjee, 2016). The zeta potential of phomopsidione nanoparticle was measured at −5.03 mV, with a conductivity of 0.656 mS/cm. The zeta potential confirmed that the nanoparticles are neutral in charge. The result is in agreement with Mura et al. (2011) and Ai et al. (2011). This characteristic allows even dispersion of nanoparticles in the silicone material, which further improves the drug delivery to the ocular system. Fig. 2 shows the SEM micrograph of phomopsidione nanoparticle coated contact lens. The hydrogel matrix showed a mesh-like structure. This contact lens is made up of siloxane and hydrogel material with the surface treatment using galafylcon A. This resulted the formation of ultrastructure network which consist of several layer of microstructure made up from different material, which can be clearly seen on the micrograph (González et al., 2006). Ethanol was used to create microscopes on the ultrastructure on the lens, which allows the coating of nanoparticles onto the surface (Jung et al., 2013). One of the challenge in ocular drug delivery is to control the release of the drug. This is particularly important to provide long hour protection against microorganisms among contact lens wearers. In this study, 2 drug release patterns were observed in the drug release profile of phomopsidione nanoparticles (Fig. 3), the initial slow release phase (extended release) and the burst release on Day 2. The typical release profile of PVA particulate delivery system was observed. The initial release was slow but gradual (46.65 μg/h) on Day 1–2, as PVA forms a barrier which contribute to the slower release of drug (Gupta et al.,
microbial suspension was spread uniformly on the agar plate by using sterile cotton swab. Next, the phomopsidione nanoparticles coated contact lens and control were gently placed on the surface of agar medium. The contact lens with blank nanoparticle acts as negative control. All plates were incubated at 37 °C for 24 h. The average diameter of clear zones surrounding the contact lens were measured and recorded after the incubation period. 2.9. Growth reduction assay The bacteria that showed inhibitory activity on agar diffusion assay were selected. The contact lenses were placed into 900 μl of Mueller Hinton broth (Merck), containing 100 μl of bacterial inoculum. The cultures were then incubated at 37 °C for 48 h. After incubation period, viable cell count was performed using spread plate method to quantify the living bacterial cells in the sample. The number of living bacterial cells were expressed in number of colony forming units (CFU) per ml of sample. 3. Result and discussion In the field of ophthalmic drug delivery, many approaches were explored to increase the efficiency and residence time of antimicrobial drugs on contact lenses. One of the most common method is by encapsulating the drug in the form of nanoparticles (Kim et al., 2008). In this study, phomopsidione nanoparticles were synthesized via solvent and anti-solvent precipitation method. A FDA-approved polymer, PVA was used as non-ionic stabilizer to encapsulate phomopsidione. Besides, pluronic F-127 was also added to prevent the coalescence between nanoparticles, while maintaining their interfacial energy. This is because nanoparticles tend to agglomerate in order to minimize their surface energy (Lee et al., 2015). Fig. 1 shows the TEM micrograph of phomopsidione nanoparticles. The nanoparticles are of sphere like morphology, with an average size of 77.45 ± 11.7 nm. The particle sizes were obtained by analyzing the recorded images, and the findings indicated the small mean particle size, which is less than 100 nm. PVA is a water soluble synthetic polymer which belongs to the class of nonionic polymers containing a vinyl group (Moukwa et al., 1993). The polymer is an ideal choice for ophthalmic applications, as it is nontoxicity, non-carcinogenic, and low immunogenicity (DeMerlis and Schoneker, 2003). Besides, the polymer also stabilizes the nanoparticles during freeze-drying process through steric hindrance (Lee et al., 2015).
Fig. 1. TEM micrograph of phomopsidione nanoparticles. The average size of the nanoparticles is 77.45 nm ± 11.7 nm.
Fig. 2. SEM micrograph of contact lens coated with phomopsidione nanoparticles. The mesh-like matrix of silicone hydrogel was observed. 12
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Fig. 3. The drug release behaviour of phomopsidione from the PVA-encapsulated nanoparticles. Two drug release patterns were observed.
bacteria in causing contact lens related keratitis (Fleiszig and Evans, 2010). The contact lens also showed good inhibitory activity on S. marcescens, which frequently associated with keratitis and ulcerations among contact lens wearers. However, in this study, no anti-yeast activity was reported. A quantitative antibacterial assessment of the contact lens models was tested in a broth medium. The contact lens coated with phomopsidione nanoparticle showed a significant growth reduction on all test microorganisms, with the percentage of growth reduction up to 99%. The results are in agreement with agar diffusion assay. The drug loaded onto the nanoparticles is sufficient to prevent the bacterial growth, for a period of 48 h.
Table 1 Antimicrobial activity of the contact lenses coated with phomopsidione nanoparticles. The contact lenses inhibited the growth of 3 test microorganisms, with at least 99% of growth reduction. Test microorganisms
S. marcescens P. aeruginosa MRSA P. mirabilis C. utilis
Average diameter of clear zone (mm) Control
Contact lenses with phomopsidione nanoparticles
– – – – –
41.6 ± 3.2 51.3 ± 2.9 24.0 ± 4.0 – –
Percentage of growth reduction (%)
99.9 99.9 99.34 – –
4. Conclusion In conclusion, phomopsdione nanoparticles coated contact lens showed sustainable release pattern of drug for 48 h. The contact lens models exhibited inhibited keratitis causing microorganisms, particularly on Gram negative bacteria. The contact lens models also caused a significant growth reduction on the test microorganisms, with the percentage of growth reduction up to 99%. The study could be extended to in vivo keratitis models to evaluate the antimicrobial potential on animal models.
*The average diameter of clear zone was expressed in average diameter ± standard deviation. The experiments were conducted in triplicates in separate occasions.
2010). A total of 17% of drug was released in 48 h of test period. Such a well-regulated drug delivery system enables the control of drug amount released to meet the therapeutic requirements. A burst release state was observed on Day 2 with release rate 224.49 μg/h. The drug release at later phase was rapid and high concentration of phomopsidione was detected in the test medium. This is partially due to the complete swelling of the silicone hydrogel in the test medium, which increase the total area exposed (Diebold and Calonge, 2010). The release was complete on Day 4, following the first order of kinetic. In contrast, Kim et al. (2008) reported that the release of the ocular drug from the silicone hydrogel contact lenses can be prolonged up to 150 days. The drug release profile of nanoparticles is influenced by structure of the silicone hydrogel matrix, and also the physiochemical characteristics of the drug. The antimicrobial efficiency of the contact lens models was tested on several clinical pathogens. Drug delivery of therapeutic contact lenses is a challenge as the complex silicone hydrogel matrix hinders the release of drug (Paradiso et al., 2016). On agar diffusion assay, 3 out of 5 test microorganisms were inhibited by phomopsidione nanoparticles coated contact lenses, including 2 Gram negative bacteria. No clear zones were observed surrounding the controls. This indicates that the antimicrobial activity was solely due to phomopsidione present in the nanoparticles. Most of the severe cases of microbial keratitis were caused by Gram-negative bacteria (Jeng, 2003). They are hardly killed by the conventional disinfectants due to their ability to form biofilm, which reduce the penetrating capacity of disinfectant. In this study, P. aeruginosa showed the largest clear zone, with a diameter of 51.3 mm (Table 1). P. aeruginosa is considered as the most virulent pathogenic
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