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Synthesize of Titanium dioxide nanoparticles using Moringa oleifera leaves and evaluation of wound healing activity
Accepted Manuscript Title: Synthesize of Titanium dioxide nanoparticles using Moringa oleifera leaves and evaluation of wound healing activity Author: V. Sivaranjani P. Philominathan PII: DOI: Reference:
S2213-9095(15)30005-7 http://dx.doi.org/doi:10.1016/j.wndm.2015.11.002 WNDM 65
To appear in: Received date: Revised date: Accepted date:
28-7-2015 19-10-2015 6-11-2015
Please cite this article as: V. Sivaranjani, P. Philominathan, Synthesize of Titanium dioxide nanoparticles using Moringa oleifera leaves and evaluation of wound healing activity, Wound Medicine (2015), http://dx.doi.org/10.1016/j.wndm.2015.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Synthesize of Titanium dioxide nanoparticles using Moringa oleifera leaves and evaluation of wound healing activity
an
V. Sivaranjania and P. Philominathanb , * a,b PG and Research Department of Physics AVVM Sri Pushpam College, Poondi, Thanjavur, India.
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October 19, 2015
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Abstract
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1
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Titanium dioxide nanoparticles(TiNPs) were emerged as wound healing enhancer as well as an antibacterial agent against Gram-positive and Gramnegative bacteria due to its enhanced mechanical properties. Moreover, the green synthesized Titanium dioxide exhibited significant wound healing activity in Albino rats which was confirmed by measuring wound closure. Titanium dioxide nanoparticles were effectively synthesized using aqueous leaf extract of Moringa oleifera under pH and temperature dependent condition. 5 mM Titanium dioxide solution worked as a primary source for the synthesize of titanium dioxide nanoparticles. The green synthesized TiNPs excitation was confirmed using UV-Vis spectrophotometer at 428 nm. The high-resolution scanning electron microscope results showed the spherical shape of TiNPs with a mean particle size around 100 nm. Furthermore, the wound healing activity of nano-sized particles(Control Group) when compared with micro-sized(Positive Control Group) was examined through the 1
* corresponding author E-mail id:[email protected] Mobile No: 9443170304
1
Page 1 of 13
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excision wound model by measuring wound closure and the control group revealed significant wound healing activity in Albino rats.
Keywords
Introduction
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Titanium dioxide, Titanium nanoparticles, Moringa oleifera,Wound healing.
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In recent times, wounds are among major world-wide clinical problems because of morbidity associated with prolonged periods required for repair and regeneration of the injured tissue, bleeding, risk for infections and septicemias, keloids and scar formation. These risk factors are further exacerbated in patients with impaired wound healing and the usage of external wound healing medications can minimize these risk factors. Moreover, due to the increment in the proportion of aged people’s population in the coming decades, the wound healing process cost in clinical aspect may likely to increase [1]. Generally, the wound infections are commonly caused by plenty of bacteria or fungi; Gram-positive (staphylococcus) and Gram-negative (pseudomonas aeruginosa) are the most common bacteria responsible for the majority of wound infections. These bacteria can easily contaminate the surface of wounds and access the underlying tissue, thereby delaying the healing process. According to the increase in health care costs and increasing antibiotic resistances, the economic burden for the treatment of chronic wounds is rapidly growing. Considering that resistance against newly approved antibiotics developed within two years, there is an urgent need for newer generation of antibiotics to fight infections [2]. In spite of so many medical advancements, wound healing still remains as an inefficiently managed area. The process of wound healing involves four types of stages: Haemostasis, Inflammation, Proliferation and remodeling ultimately leading to scar formation. The ultimate goal of wound healing is to have a speedy recovery with minimal scaring. Nowadays, nanotechnology is emerging rapidly with the development of nanoscale materials which have potential biomedical applications, especially in fighting and preventing diseases. The new age drugs include the nanoparticles of polymers, metals,
2
Page 2 of 13
an
us
cr
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ceramics which can fight against human pathogens like bacteria and even cancer. The importance of nanoparticles having potent bactericidal activity is inevitable because of their effect against resistant strains of pathogens. Moreover, nanoparticles increase the chemical activity due to their large surface volume ratio. In recent times in fact, the metallic nanoparticles have proven that they are the most suited candidate among all the other nano particles. In particular, metal oxide nanoparticles (NPs) are known to possess strong antimicrobial activity. However, Titanium nanoparticles are one of the most important metal oxide nanoparticles compared to others and are used in glass ceramics, electrical ceramics, catalyst, solar cell sensors, electric conductors and chemical intermediates but still a limited evidence available in biological related aspects. TiO2 have good mechanical properties, antibacterial effect against Gram-postive and Gram-negative bacteria, cell growth and high corrosion resistance.
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This investigation was aimed to study the wound healing activity of green synthesized TiO2 nanoparticles. Although, the main phytochemicals responsible for the synthesize of nanoparticles are terpenoids, flavones, ketones, aldehydes, amides etc., here we wish to report a facile, green and one pot synthesize method using the leaf extract of Moringa oleifera for synthesizing titanium dioxide nanoparticles. As the biosynthetic route for nanoparticles has not yet been extended for the synthesize of titanium dioxide nanoparticles and its evaluation of wound healing activity, the present study has been employed with utmost care and reported here.
2
2.1
Materials and Methods Preparation of Titanium nanostructures
For the preparation of aqueous leaf extract (ALE) solution of Moringa oleifera, 10 g of Moringa oleifera powder dissolved in 100 ml of deionized water, boiled at 600 C for 10 min to kill the pathogens in ALE solution. After cooling, ALE solution was filtered using Whatman No.1 filter paper. Titanium dioxide nanoparticles were synthesized by adding 10 ml of filtered ALE solution to 90 ml of 5 mM titanium dioxide solution (pH 1.5) in an Erlenmeyer flask under stirring at 500 C. After 5 h, the developed dark brown colour confirmed the formation of titanium dioxide nanoparticles (TiNPs)[3]. Finally, 3
Page 3 of 13
3.1
Characterization techniques
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3
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well formed titanium dioxide nanoparticles were acquired by centrifugation at 10000 rpm for 15 minutes and thus separated titanium dioxide nanoparticles (TiNPs) were dried and used for further analytical techniques.
UV-Vis spectral analysis
SEM analysis of Titanium nanoparticles
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3.2
an
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Colour change in the reaction mixtures is the evidence for TiNPs formation. The reduction of pure titanium ions was monitored by measuring the UVVis spectrum of the reaction medium after 5 hours. UV-Vis spectral analysis was carried out by UV-Vis spectrophotometer (Perkin Elmer Lamda 35)in the range 200-800 nm.
Excision and treatment on Animals
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3.3
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Scanning Electron Microscopic (SEM) analysis was done using VEGA3 TESCAN equipment. Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid. Extra solution was removed using a blotting paper and then the films on the SEM grid were allowed to dry under a mercury lamp for 5 min.
The wound healing activity was examined upon the Male albino rats of Wistar strain with weight of 160 - 1100 g and these animals were purchased from Indian Institute of Science, Bangalore. The rats were housed in spacious polypropylene cages bedded with rice husk and the room was well ventilated maintained under standard experimental conditions (Temperature 27 ±20 C and 12 hour light/dark cycle) throughout the experimental periods. All the animals were fed with standard pellet diet and water ad libitum. These animals were acclimatized to the environment for one weak prior to experimental use. The food composition to animal was taken by the following ratios, crude protein (22.3 %), crude oil (4.01 %), crude fibre (4.02 %) and sand silical (1.02 %). The wound site of the animals was produced by excision wound model [4]. First, the animals were anesthetized with diethyl ether and the hairs on the
4
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skin of the back, shaved with sterilized razor blades. Then the dorsal fur was shaved and full thickness of the excision wound of 1.5 cm in width and 0.2 cm depth was created in animals. After completion of wounding process, the wound part was well washed with normal saline solution. Animals bearing partial thickness wound were distributed into various groups such as Group I, II and III where Group I is treated with gel alone to serve as self immunity (Negative control group), Group II animals are treated with gel based ointment(TiNPs+gel)(control group). The standard drug sulfadiazine purchased from medical store was treated on Group III (sulfadiazine+gel)(positive control) animals. The very purpose of selecting a standard drug is to ascertain the size of particles which play a crucial role in wound healing activity, rather than other medicinal and ethical aspects. In this directions, sulfadiazine was chosen as standard drug which contains micro-sized particles. It is found to be a good antibiotic for the treatment and prevention of local infections particularly, in animals. Each group has six animals.
Preparation of gel based ointment
Wound measurement
Ac ce p
3.5
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The gel based ointment was prepared using titanium nanoparticles (TiNPs) by the following procedure: 0.5 g of TiNPs was dispersed in gel with mild stirring and is allowed to swell for 5 minutes to obtain 0.5 % of medicated gel.
The measurement of the wound area was carried out from the day of the excision of the wound and at every three (3) days interval until the epithelisation of the wound was completed. The area of the wound contraction was measured in different treated and control groups on 3rd , 6th 9th and 12th day. Wound contraction which contributes to wound closure was studied by tracing of the site. A meter ruler was placed over the wound and that estimate provides scale calculation. The percentage of wound closure was calculated as follows using the initial and final area drawn on glass slides during the experiments [5]. % of WH =
W A0 ×W An W A0
×100
5
Page 5 of 13
where,
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WH - Wound Healing
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WA0 - Wound area on day 0
and n = 3, 6, 9 and 12th day.
Titanium nanoparticles
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4.1
Results and discussion
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WAn - Wound area on day n
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The colour change due to the reaction of T iO2 with Moringa oleifera is displayed in Fig. 1 and the colour changes observed in the vessels is due to the reduction of titanium ions. This same type of reaction (i.e colour formation) was found in earlier reported article shankar et al., (2014)[3]. It is well known that the titanium nanoparticles exhibit brown colour in aqueous solution due to the excitation of surface plasmon vibrations. The appearances of brown colour in the reaction vessels suggest the formation of titanium nanoparticles.
Figure 1: TiNPs formation after the addition of leaf extract to TiO2 as indicated by the colour change.
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4.2
UV-Vis spectral analysis
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Titanium nanoparticles were characterized using UV-Vis spectroscopy to find the structural properties of TiNPs as depicted in Fig. 2. It can be seen that the spectra was recorded as a function of reaction time (5 hrs) of TiNPs. From this analysis it is observed that the maximum absorbance of TiNPs was attained at 428 nm as evident from absorbance spectrum and this peak was assigned due to the presence of surface plasmons.
SEM analysis
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Figure 2: UV-Vis absorption spectrum of titanium nanoparticles after 5 hrs.
Ac ce p
SEM (Scanning Electron Microscope) analysis was carried out to understand the topology and the size of the TiNPs, which showed the formation of higher density polydispersed spherical TiNPs of various sizes. The SEM image showing the high density titanium nanoparticles synthesized by the leaf extract further confirmed the development of titanium nanostructures. Most of the nanoparticles were scattered, as observed under SEM (Fig. 3). The SEM analysis showed the spherical titanium nanoparticles of particle size around 100 nm as well.
4.4
Wound healing treatment
The wound healing activity was carried out for the rate of wound contraction by excision wound model. The percentage of wound contraction was recorded by observing the change in the wound area of Group I(negative control group), Group II(control group), Group III(positive control group) animals at fixed intervals of time viz., 3rd , 6th , 9th and 12th day. However, on 7
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Figure 3: Scanning Electron Microscopic (SEM) image of polydispersed titanium nanoparticles (TiNPs) of approximately 100 nm.
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the post wound day(12th ), that is after healing, the Group I animals exhibited 78.07 % healing which is due to the self immunity of animals, whereas the control group (Group II) animals showed 92.36% of healing and finally standard treated group (Group III) displays 83.55% of healing which is tabulated in Table.1 and the histogram representation of the table is shown in Fig.4.
Figure 4: Percentage of wound healing activity of Group I(control group),Group II(TiNPs treated), Group III(standard treated) Wound healing comprises of different phases such as contraction, epithelization, granulation and collagenation. It normally involves an initial in8
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12th DAY (in %) 75.23± 0.58 92.36± 0.5∗] 83.55± 0.57
in
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Table 1: Percentage of wound healing activity of TiNPs. DAYS GROUPS 0 DAY 3rd DAY (in 6th DAY (in 9th DAY (in (in %) %) %) %) Negative control 22.28± 0.17 35.87± 0.24 57.02± 0.35 group Control 28.23± 0.25 52.00± 0.37 55.36± 0.58 group Positive control 20.35± 0.14 34.53± 0.22 60.23± 0.42 group Values were expressed as mean ± Standard Deviation(SD) for six rats each group. ∗ Significantly different from control (p<0.05); ] Significantly different from standard (p<0.05).
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flammatory phase followed by fibroblast proliferation, formation of collagen fibres and shrinking, occurring concurrently but independent of one another. Many synthetic and natural compounds are known to promote wound healing process mainly by their astringent and antimicrobial property [6-7]. The present investigation demonstrated that TiNPs were capable of promoting wound healing activity. Enhanced wound contractions suggest that TiNPs have the potential in the management of wound healing when compared to any standard. The potential wound healing activity of TiNPs may be responsible for its antimicrobial property. This study confirms such agreement with earlier studies [8,3]. The investigators here, wish to record just the role of medicinal particle’s size and their related induced metabolic changes for the enhancement in the wound healing property. Further, the effectiveness and validity of either sulfadiazine or TiNPs, in terms of medical consideration were not considered in this investigation and undoubtedly beyond our limitations. However, the experiments were carried out well within the guidelines of the committee for the purpose of control and super vision of experiments on animals(CPCSEA). This present study showed the wound healing efficacy of titanium nanoparticles by preventing the wound chronicity in comparison to conventional standard treatments. It also describes the synthesize of titanium nanoparticles 9
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Figure 5: Comparision of Wound healing activity of Group I(control group), Group II(TiNPs) and Group III(Standard drug).
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Conclusion
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using Moringa oleifera. Excellent wound healing efficacy of nano-titanium in comparison to standard was demonstrated in vitro in rats using various criteria including visual observations and measurement of wound size as shown in Fig. 5. As a result, synthesize and characterization of titanium nanostructures with regard to novel morphology are of great interest in the wound healing material.
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The rapid synthesize of stable titanium dioxide nanoparticles using Moringa oleifera leaf extract solution has been demonstrated. The prepared nanoparticles exhibit spherical morphology and the particle size is in the range of 100 nm as evident from SEM. The present work deals with the usage of low cost precursor, that is, titanium dioxide and the use of Moringa oleifera leaf extract for the biosynthesize of titanium dioxide nanoparticles. This simple, cost effective, time saving and environmental friendly synthetic method gives a potential avenue for various applications. The eco-friendly green chemistry approach using leaf extract for the synthesize of nanoparticles will increase the economic viability and sustainable management. So the exploration of the plant systems as the potential nanofactories has heightened interest in the biological synthesize of nanoparticles. Conclusively, preparation of nanotitanium gel using bio synthesized titanium nanoparticles is a highly effective technique towards the wound healing and opens a new paradigm of medical research. Hence this novel TiNPs investigated in this work could be promising approach for enhanced wound healing and various skin infection treatments.
Acknowledgment One of the authors(VS) wish to thank the University Grants Commission, New Delhi, for the financial support in the form of Rajiv Gandhi National Fellowship (F1-17.1/2011-2012/RGNF-SC-TAM-438/dt.06.06.2012).
Disclosure The authors report no conflicts of interest in this work.
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References
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[1] Sen CK, Gordillo GM, Roy S. Human skin wounds: A major and snowballing threat to public health and the economy. Journal of Wound Repair and Regeneration. 2009; 17:763-771.
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[2] Hasan Nurhasni, Jiafu Cao, Moonjeong Cho, Kim, Bol Luel Lee, Yunjin Jung, Jin-wook Yoo.Nitric oxide-releasing poly(lactic-co-glycolic acid)polyethylenimine nanoparticles for prolonged nitric oxide release, antibacterial efficacy, and in vivo wound healing activity. International Journal of Nanomedicine. 2015; 10:3065-3080.
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[3] Shankar R, Dhivya R, Subramanian K, Ravikumar VJ. Wound healing activity of Origanum vulgare engineered titanium dioxide nanoparticles in Wistar albino rats. Journal of Material science: material medicine. 2014; 25:17011708.
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[4] Vishal Gupta, Yadav SK, Dinesh singh, Naveen Gupta, Evaluation of wound healing activity of herbal drug combination of Rubia cordifolia, Centella asiatica,Terminalia belerica,Plumbago Zeylanica and Withania somnifera. International Journal of Pharm. and Life Sci. 2011; 2:952-954.
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[5] Wilkinson LJ, White RJ, Chipman JK. Silver and nanoparticles of silver in wound dressings: A review of efficacy and safety. Wound Care. 2011; 20:543549. [6] Vinothapooshan G, Sundar K. Wound healing effect of various extracts of adhatoda vasica. International Journal of Pharma and Bio sciences. 2010; 4:230-37. [7] Venkatanarayana D, Saravana kumar A, Mohana lakshmi S. Review on natural wound healing agents. International Journal of Phytopharmacy research. 2010; 1:1-4 [8] Sundrarajan M, Gowri s. Green synthesis of titanium dioxide nanoparticles by Nyctanthes arbor-tristis leaf extract. Chalcogenide letters. 2011; 8:447-451. [9] Winkler, Jochen. Titanium Dioxide. Hannover: Vincentz Network 2003; 5:148-9. 12
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[10] Kurtoglu ME, Longenbach T, Gogotsi Y. Preventing Sodium Poisoning of Photocatalytic TiO2 Films on Glass by Metal Doping. International Journal of Applied Glass Science. 2011; 2:108116.
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[11] Jump up, Koleske JV. Paint and Coating Testing Manual. ASTM International. 1995; 232.
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S2213-9095(15)30005-7 http://dx.doi.org/doi:10.1016/j.wndm.2015.11.002 WNDM 65
To appear in: Received date: Revised date: Accepted date:
28-7-2015 19-10-2015 6-11-2015
Please cite this article as: V. Sivaranjani, P. Philominathan, Synthesize of Titanium dioxide nanoparticles using Moringa oleifera leaves and evaluation of wound healing activity, Wound Medicine (2015), http://dx.doi.org/10.1016/j.wndm.2015.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ip t
us
cr
Synthesize of Titanium dioxide nanoparticles using Moringa oleifera leaves and evaluation of wound healing activity
an
V. Sivaranjania and P. Philominathanb , * a,b PG and Research Department of Physics AVVM Sri Pushpam College, Poondi, Thanjavur, India.
M
October 19, 2015
te
Abstract
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1
Ac ce p
Titanium dioxide nanoparticles(TiNPs) were emerged as wound healing enhancer as well as an antibacterial agent against Gram-positive and Gramnegative bacteria due to its enhanced mechanical properties. Moreover, the green synthesized Titanium dioxide exhibited significant wound healing activity in Albino rats which was confirmed by measuring wound closure. Titanium dioxide nanoparticles were effectively synthesized using aqueous leaf extract of Moringa oleifera under pH and temperature dependent condition. 5 mM Titanium dioxide solution worked as a primary source for the synthesize of titanium dioxide nanoparticles. The green synthesized TiNPs excitation was confirmed using UV-Vis spectrophotometer at 428 nm. The high-resolution scanning electron microscope results showed the spherical shape of TiNPs with a mean particle size around 100 nm. Furthermore, the wound healing activity of nano-sized particles(Control Group) when compared with micro-sized(Positive Control Group) was examined through the 1
* corresponding author E-mail id:[email protected] Mobile No: 9443170304
1
Page 1 of 13
ip t
excision wound model by measuring wound closure and the control group revealed significant wound healing activity in Albino rats.
Keywords
Introduction
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Titanium dioxide, Titanium nanoparticles, Moringa oleifera,Wound healing.
Ac ce p
te
d
M
an
In recent times, wounds are among major world-wide clinical problems because of morbidity associated with prolonged periods required for repair and regeneration of the injured tissue, bleeding, risk for infections and septicemias, keloids and scar formation. These risk factors are further exacerbated in patients with impaired wound healing and the usage of external wound healing medications can minimize these risk factors. Moreover, due to the increment in the proportion of aged people’s population in the coming decades, the wound healing process cost in clinical aspect may likely to increase [1]. Generally, the wound infections are commonly caused by plenty of bacteria or fungi; Gram-positive (staphylococcus) and Gram-negative (pseudomonas aeruginosa) are the most common bacteria responsible for the majority of wound infections. These bacteria can easily contaminate the surface of wounds and access the underlying tissue, thereby delaying the healing process. According to the increase in health care costs and increasing antibiotic resistances, the economic burden for the treatment of chronic wounds is rapidly growing. Considering that resistance against newly approved antibiotics developed within two years, there is an urgent need for newer generation of antibiotics to fight infections [2]. In spite of so many medical advancements, wound healing still remains as an inefficiently managed area. The process of wound healing involves four types of stages: Haemostasis, Inflammation, Proliferation and remodeling ultimately leading to scar formation. The ultimate goal of wound healing is to have a speedy recovery with minimal scaring. Nowadays, nanotechnology is emerging rapidly with the development of nanoscale materials which have potential biomedical applications, especially in fighting and preventing diseases. The new age drugs include the nanoparticles of polymers, metals,
2
Page 2 of 13
an
us
cr
ip t
ceramics which can fight against human pathogens like bacteria and even cancer. The importance of nanoparticles having potent bactericidal activity is inevitable because of their effect against resistant strains of pathogens. Moreover, nanoparticles increase the chemical activity due to their large surface volume ratio. In recent times in fact, the metallic nanoparticles have proven that they are the most suited candidate among all the other nano particles. In particular, metal oxide nanoparticles (NPs) are known to possess strong antimicrobial activity. However, Titanium nanoparticles are one of the most important metal oxide nanoparticles compared to others and are used in glass ceramics, electrical ceramics, catalyst, solar cell sensors, electric conductors and chemical intermediates but still a limited evidence available in biological related aspects. TiO2 have good mechanical properties, antibacterial effect against Gram-postive and Gram-negative bacteria, cell growth and high corrosion resistance.
Ac ce p
te
d
M
This investigation was aimed to study the wound healing activity of green synthesized TiO2 nanoparticles. Although, the main phytochemicals responsible for the synthesize of nanoparticles are terpenoids, flavones, ketones, aldehydes, amides etc., here we wish to report a facile, green and one pot synthesize method using the leaf extract of Moringa oleifera for synthesizing titanium dioxide nanoparticles. As the biosynthetic route for nanoparticles has not yet been extended for the synthesize of titanium dioxide nanoparticles and its evaluation of wound healing activity, the present study has been employed with utmost care and reported here.
2
2.1
Materials and Methods Preparation of Titanium nanostructures
For the preparation of aqueous leaf extract (ALE) solution of Moringa oleifera, 10 g of Moringa oleifera powder dissolved in 100 ml of deionized water, boiled at 600 C for 10 min to kill the pathogens in ALE solution. After cooling, ALE solution was filtered using Whatman No.1 filter paper. Titanium dioxide nanoparticles were synthesized by adding 10 ml of filtered ALE solution to 90 ml of 5 mM titanium dioxide solution (pH 1.5) in an Erlenmeyer flask under stirring at 500 C. After 5 h, the developed dark brown colour confirmed the formation of titanium dioxide nanoparticles (TiNPs)[3]. Finally, 3
Page 3 of 13
3.1
Characterization techniques
cr
3
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well formed titanium dioxide nanoparticles were acquired by centrifugation at 10000 rpm for 15 minutes and thus separated titanium dioxide nanoparticles (TiNPs) were dried and used for further analytical techniques.
UV-Vis spectral analysis
SEM analysis of Titanium nanoparticles
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3.2
an
us
Colour change in the reaction mixtures is the evidence for TiNPs formation. The reduction of pure titanium ions was monitored by measuring the UVVis spectrum of the reaction medium after 5 hours. UV-Vis spectral analysis was carried out by UV-Vis spectrophotometer (Perkin Elmer Lamda 35)in the range 200-800 nm.
Excision and treatment on Animals
Ac ce p
3.3
te
d
Scanning Electron Microscopic (SEM) analysis was done using VEGA3 TESCAN equipment. Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid. Extra solution was removed using a blotting paper and then the films on the SEM grid were allowed to dry under a mercury lamp for 5 min.
The wound healing activity was examined upon the Male albino rats of Wistar strain with weight of 160 - 1100 g and these animals were purchased from Indian Institute of Science, Bangalore. The rats were housed in spacious polypropylene cages bedded with rice husk and the room was well ventilated maintained under standard experimental conditions (Temperature 27 ±20 C and 12 hour light/dark cycle) throughout the experimental periods. All the animals were fed with standard pellet diet and water ad libitum. These animals were acclimatized to the environment for one weak prior to experimental use. The food composition to animal was taken by the following ratios, crude protein (22.3 %), crude oil (4.01 %), crude fibre (4.02 %) and sand silical (1.02 %). The wound site of the animals was produced by excision wound model [4]. First, the animals were anesthetized with diethyl ether and the hairs on the
4
Page 4 of 13
3.4
M
an
us
cr
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skin of the back, shaved with sterilized razor blades. Then the dorsal fur was shaved and full thickness of the excision wound of 1.5 cm in width and 0.2 cm depth was created in animals. After completion of wounding process, the wound part was well washed with normal saline solution. Animals bearing partial thickness wound were distributed into various groups such as Group I, II and III where Group I is treated with gel alone to serve as self immunity (Negative control group), Group II animals are treated with gel based ointment(TiNPs+gel)(control group). The standard drug sulfadiazine purchased from medical store was treated on Group III (sulfadiazine+gel)(positive control) animals. The very purpose of selecting a standard drug is to ascertain the size of particles which play a crucial role in wound healing activity, rather than other medicinal and ethical aspects. In this directions, sulfadiazine was chosen as standard drug which contains micro-sized particles. It is found to be a good antibiotic for the treatment and prevention of local infections particularly, in animals. Each group has six animals.
Preparation of gel based ointment
Wound measurement
Ac ce p
3.5
te
d
The gel based ointment was prepared using titanium nanoparticles (TiNPs) by the following procedure: 0.5 g of TiNPs was dispersed in gel with mild stirring and is allowed to swell for 5 minutes to obtain 0.5 % of medicated gel.
The measurement of the wound area was carried out from the day of the excision of the wound and at every three (3) days interval until the epithelisation of the wound was completed. The area of the wound contraction was measured in different treated and control groups on 3rd , 6th 9th and 12th day. Wound contraction which contributes to wound closure was studied by tracing of the site. A meter ruler was placed over the wound and that estimate provides scale calculation. The percentage of wound closure was calculated as follows using the initial and final area drawn on glass slides during the experiments [5]. % of WH =
W A0 ×W An W A0
×100
5
Page 5 of 13
where,
ip t
WH - Wound Healing
cr
WA0 - Wound area on day 0
and n = 3, 6, 9 and 12th day.
Titanium nanoparticles
M
4.1
Results and discussion
an
4
us
WAn - Wound area on day n
Ac ce p
te
d
The colour change due to the reaction of T iO2 with Moringa oleifera is displayed in Fig. 1 and the colour changes observed in the vessels is due to the reduction of titanium ions. This same type of reaction (i.e colour formation) was found in earlier reported article shankar et al., (2014)[3]. It is well known that the titanium nanoparticles exhibit brown colour in aqueous solution due to the excitation of surface plasmon vibrations. The appearances of brown colour in the reaction vessels suggest the formation of titanium nanoparticles.
Figure 1: TiNPs formation after the addition of leaf extract to TiO2 as indicated by the colour change.
6
Page 6 of 13
4.2
UV-Vis spectral analysis
M
an
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cr
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Titanium nanoparticles were characterized using UV-Vis spectroscopy to find the structural properties of TiNPs as depicted in Fig. 2. It can be seen that the spectra was recorded as a function of reaction time (5 hrs) of TiNPs. From this analysis it is observed that the maximum absorbance of TiNPs was attained at 428 nm as evident from absorbance spectrum and this peak was assigned due to the presence of surface plasmons.
SEM analysis
te
4.3
d
Figure 2: UV-Vis absorption spectrum of titanium nanoparticles after 5 hrs.
Ac ce p
SEM (Scanning Electron Microscope) analysis was carried out to understand the topology and the size of the TiNPs, which showed the formation of higher density polydispersed spherical TiNPs of various sizes. The SEM image showing the high density titanium nanoparticles synthesized by the leaf extract further confirmed the development of titanium nanostructures. Most of the nanoparticles were scattered, as observed under SEM (Fig. 3). The SEM analysis showed the spherical titanium nanoparticles of particle size around 100 nm as well.
4.4
Wound healing treatment
The wound healing activity was carried out for the rate of wound contraction by excision wound model. The percentage of wound contraction was recorded by observing the change in the wound area of Group I(negative control group), Group II(control group), Group III(positive control group) animals at fixed intervals of time viz., 3rd , 6th , 9th and 12th day. However, on 7
Page 7 of 13
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Figure 3: Scanning Electron Microscopic (SEM) image of polydispersed titanium nanoparticles (TiNPs) of approximately 100 nm.
Ac ce p
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the post wound day(12th ), that is after healing, the Group I animals exhibited 78.07 % healing which is due to the self immunity of animals, whereas the control group (Group II) animals showed 92.36% of healing and finally standard treated group (Group III) displays 83.55% of healing which is tabulated in Table.1 and the histogram representation of the table is shown in Fig.4.
Figure 4: Percentage of wound healing activity of Group I(control group),Group II(TiNPs treated), Group III(standard treated) Wound healing comprises of different phases such as contraction, epithelization, granulation and collagenation. It normally involves an initial in8
Page 8 of 13
12th DAY (in %) 75.23± 0.58 92.36± 0.5∗] 83.55± 0.57
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Table 1: Percentage of wound healing activity of TiNPs. DAYS GROUPS 0 DAY 3rd DAY (in 6th DAY (in 9th DAY (in (in %) %) %) %) Negative control 22.28± 0.17 35.87± 0.24 57.02± 0.35 group Control 28.23± 0.25 52.00± 0.37 55.36± 0.58 group Positive control 20.35± 0.14 34.53± 0.22 60.23± 0.42 group Values were expressed as mean ± Standard Deviation(SD) for six rats each group. ∗ Significantly different from control (p<0.05); ] Significantly different from standard (p<0.05).
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flammatory phase followed by fibroblast proliferation, formation of collagen fibres and shrinking, occurring concurrently but independent of one another. Many synthetic and natural compounds are known to promote wound healing process mainly by their astringent and antimicrobial property [6-7]. The present investigation demonstrated that TiNPs were capable of promoting wound healing activity. Enhanced wound contractions suggest that TiNPs have the potential in the management of wound healing when compared to any standard. The potential wound healing activity of TiNPs may be responsible for its antimicrobial property. This study confirms such agreement with earlier studies [8,3]. The investigators here, wish to record just the role of medicinal particle’s size and their related induced metabolic changes for the enhancement in the wound healing property. Further, the effectiveness and validity of either sulfadiazine or TiNPs, in terms of medical consideration were not considered in this investigation and undoubtedly beyond our limitations. However, the experiments were carried out well within the guidelines of the committee for the purpose of control and super vision of experiments on animals(CPCSEA). This present study showed the wound healing efficacy of titanium nanoparticles by preventing the wound chronicity in comparison to conventional standard treatments. It also describes the synthesize of titanium nanoparticles 9
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Figure 5: Comparision of Wound healing activity of Group I(control group), Group II(TiNPs) and Group III(Standard drug).
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Conclusion
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using Moringa oleifera. Excellent wound healing efficacy of nano-titanium in comparison to standard was demonstrated in vitro in rats using various criteria including visual observations and measurement of wound size as shown in Fig. 5. As a result, synthesize and characterization of titanium nanostructures with regard to novel morphology are of great interest in the wound healing material.
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The rapid synthesize of stable titanium dioxide nanoparticles using Moringa oleifera leaf extract solution has been demonstrated. The prepared nanoparticles exhibit spherical morphology and the particle size is in the range of 100 nm as evident from SEM. The present work deals with the usage of low cost precursor, that is, titanium dioxide and the use of Moringa oleifera leaf extract for the biosynthesize of titanium dioxide nanoparticles. This simple, cost effective, time saving and environmental friendly synthetic method gives a potential avenue for various applications. The eco-friendly green chemistry approach using leaf extract for the synthesize of nanoparticles will increase the economic viability and sustainable management. So the exploration of the plant systems as the potential nanofactories has heightened interest in the biological synthesize of nanoparticles. Conclusively, preparation of nanotitanium gel using bio synthesized titanium nanoparticles is a highly effective technique towards the wound healing and opens a new paradigm of medical research. Hence this novel TiNPs investigated in this work could be promising approach for enhanced wound healing and various skin infection treatments.
Acknowledgment One of the authors(VS) wish to thank the University Grants Commission, New Delhi, for the financial support in the form of Rajiv Gandhi National Fellowship (F1-17.1/2011-2012/RGNF-SC-TAM-438/dt.06.06.2012).
Disclosure The authors report no conflicts of interest in this work.
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