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Biosynthesis of AgNPs and their effective wound healing activity in nursing care in children after surgery
Journal of Drug Delivery Science and Technology 55 (2020) 101425
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Biosynthesis of AgNPs and their effective wound healing activity in nursing care in children after surgery
T
Li-Na Xua, Hai-Xia Wangb, Ling Zhaoc,∗ a
Department of Operation, Linyi Central Hospital, Linyi, 276400, Shandong, China Department of Nursing, Linyi Central Hospital, Linyi, 276400, Shandong, China c Complaint Center, Jining NO.1 People's Hospital, Affiliated Jining NO.1 People's Hospital of Jining Medical University, Jining Medical University, Jining, 272111, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: AgNPs Wound healing Polyphenols
The current work illustrated the potency of wound healing activity of biofabricated silver nanoparticles synthesized by using Ocimum Sanctum leaf extract by avoiding chronicity of wound compared to conventional silver treatments. The prepared AgNPs were studied using various characterization techniques such as X-ray diffraction (XRD), Ultra Violet-Visible (UV–Visible) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and Highresolution transmission electron microscopy (HR-TEM). Additionally, the superior wound recovering potential of nanosilver gel compared to conventional formulations was illustrated in vitro in animals utilizing several criteria's including TEWL, visual observations and histological study. In conclusion, utilizing biofabricated AgNPs, the Carbopol-dependent nanosilver gel is a gold class treatment for wound recovery and offering up a modern era for medicinal applications in nursing care in children after surgery.
1. Introduction Any burn therapy's main objective is to accomplish epithelization and wound healing as quickly as feasible to avoid infection without creating any cosmetic and functional side effects [1]. Systematic infection from the injury location called wound sepsis is the major source of death in the patients suffering from burning, which is because of the chronicity. A wound's chronicity can contribute to a high inflammatory load equivalent to that of an ex-aggregated infectious condition [2]. Factors adding to the delayed wound healing can include the development of biofilm, bioburden at the injury location and microbial species, however not restricted to these factors though [3]. The function of primary microorganisms like Pseudomonas spp., Staphylococcus spp., Enterococcus, Streptococcus spp., etc. in chronic injuries has been demonstrated by various microbiological approaches as well as by microbial profiling studies [4,5]. These types of microbial groups have shown growing resistance to conventional antibacterial treatments and are therefore needed to develop modern approaches for eradicating biofilm for chronic wound management [6]. The latest advances utilize noble metal antibacterial agents against such burning diseases and the most common of these is silver [7]. Prior to the introduction of vaccines in the early 20's, silver (Ag) was utilized
for its medicinal and bactericidal properties against multiple inflammatory diseases [8–13]. Revitalizing Ag in the shape of silver nitrate post losing it entirely in the period of IInd World War lasted several years for increasing its interest [14]. Silver is currently regarded as a feasible choice for treating structural diseases induced by microbes [15]. Conventional materials such as silver sulfadiazine and silver nitrate are having the gold standard reputation in the treatment of inflammatory burn diseases among the numerous silver-dependent compounds [13,16]. These compounds, however, suffer from the disadvantages such as delay in the process of wound healing [17], cosmetic abnormality [19] and cytotoxic function on host cells [18]. Recently advanced silver-dependent products like AquacelTM, ActisorbTM, ActicoatTM, ArglaesTM, SilverlonTM, ActisorbTM etc. have a little more sustained and controlled delivery of nanocrystalline Ag into the wounded area [20]. This regulated releasing path enables reducing nosocomial infection, extended duration of function, decreasing price of medication, etc. [13,16,19,20]. The aim of the current study is to develop silver-dependent nanoformulations for topical burn injury healing, thus maintaining an equilibrium between toxicity and therapeutic activity. Silver nanoparticles (AgNPs) of average particle size 20 nm were produced and developed as topical medicine by Ocimum Sanctum. In order to prove
∗ Corresponding author. No. 6 Jiankang Road, Complaint Center, Jining NO.1 People's Hospital, Affiliated Jining NO.1 People's Hospital of Jining Medical University, Jining Medical University, Jining, 272111, Shandong, China E-mail address: [email protected] (L. Zhao).
https://doi.org/10.1016/j.jddst.2019.101425 Received 9 November 2019; Received in revised form 26 November 2019; Accepted 28 November 2019 Available online 13 December 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 55 (2020) 101425
L.-N. Xu, et al.
the wound healing impact of silver nanoformulations on rats’ skin, effective in vitro and in vivo research were conducted. 2. Materials and methods 2.1. Preparation of plant extract During February, freshly cut leaves of Ocimum Sanctum were obtained from the university campus. To eliminate debris as well as other hazardous organic materials, the leaves are surface washed with flowing tap water, accompanied by double distilled water and then dried in air at room temperature. In a flask comprising 200 mL of double distilled water, finely sliced leaves of around 20 gm were placed and then boiled for about 30 min. The temperature of the mixture was brought down and with the help of Whatman no.1 filter paper, the extract was filtered and for further utilization, the mixture was preserved at 4 °C. Fig. 1. UV–Visible spectrum of AgNPs with varying the Ocimum Sanctum extract concentration.
2.2. Green synthesis of silver nanoparticles Erlenmeyer flask was utilized to prepare 100 mL of 1 mM silver nitrate solution. Then Ocimum Sanctum leaf extract of 5, 4, 3, 2 and 1 mL was added with 10 mL solution of silver nitrate separately, maintaining 1 mM concentration. Dark chamber was utilized to incubate this setup at room temperature, in order to mitigate silver nitrate photo-activation. The change in the color of the solution from colorless to brown verified Ag+ reduction to Ag0. 2.3. Preparation of nano-silver gel AgNPs gel matrix was prepared with the help of Carbopol® Ultrez 10 NF and this supplied adequate stability to facilitate dispensation. In brief, 1% (w/v) of Carbopol® Ultrez 10 NF was gently spread in AgNPs subjection with the help of a 1000 rpm stirrer fixed overhead for about 3 h until the gelling agent was fully hydrated using water. 10% (w/v) triethanolamine was utilized to adjust the pH to 6 [21].
Fig. 2. FTIR spectrum of AgNPs prepared using Ocimum Sanctum extract.
2.4. Characterization of synthesized silver nanoparticles Spectrophotometer Shimadzu UV–Vis (UV-1800, Shanghai) was utilized to perform the analysis of UV–Vis spectrum. With 1 nm resolution between 200 nm and 800 nm, the UV–Vis absorption spectrophotometer was used. A diluted nano colloidal solution was used for optical absorption measurements. FTIR spectrophotometer, PerkinElmer 1750, UK was utilized to record the measurements of FT−IR spectrum. With the help of transmission electron microscopy (TEM), JEM-1400, Japan that was functioned at 120 kV accelerated voltage, the surface morphology and particle size were studied. A drop of diluted nanocolloid was placed on the copper grid followed by drying under vacuum, which later was observed under TEM for imaging.
Fig. 3. EDS spectrum of AgNPs.
developed. All sample formulations from the day of burning are added once in 24 h for continuous three weeks.
2.5. Burn healing protocol SD Male rats of weight 160–180 g were collected and separated into 3 groups of 6 rats each for the burn wound healing test. Every animal was placed in an independent cage and supplied with ad libitum water and food. The gel base of Blank Carbopol was provided to group I and was considered as control group. Burn healTM [indicated formulations comprising of 0.5% of chlorhexidine hydrochloride and 1% of silver sulfadiazine] was applied for group II animals (positive control) externally supplying silver ions (Ag0) of 1 mg/cm2 at wounded region. AgNPs gel comprising a similar concentration of 1 mg AgNPs (Ag0) was treated to group III animals per cm2 of wounded area. Animals are anesthetized with a combination of 10 mg/kg of xylazine and 80 mg/kg of ketamine, then depilated before burning on the anterior-dorsal side. Post applying blood hot rod for about 30 s, a 3rd-degree burn was
2.6. Visual observation The percent of wound healed for the tested areas was obtained by following the method demonstrated in the earlier reports, which states that by evaluating the degree of reduction in wound edema and inflammation [22]. Burnt wound healing characteristic was assessed depending on the minimum cessation period for the proportion of injury contraction. 2.7. Measurement of Trans Epidermal Water Loss (TEWL) A vapometer (Shanghai, China) was utilized to measure trans 2
Journal of Drug Delivery Science and Technology 55 (2020) 101425
L.-N. Xu, et al.
Fig. 4. XRD pattern of AgNPs.
Fig. 5. HR-TEM images (A,B) and SAED pattern (C) of AgNPs.
were represented as g/m2 h. 2.8. Histology of skin Histological examination confirmed the microscopic assessment of treated skin's internal structure. Different formulations have been tested to determine possible modifications to skin layers induced by the use of different formulations. The treated skin region was excised in histological assessment, 50% (w/v) formalin was utilized to keep the removed fat and later, dehydrated by graded alcohol sequence. Then, the skin was administrated with xylene followed by embedding into paraffin. For microscopic analysis, 5 μm thickness of skin pieces are taken of every specimen and coated with haematoxyl–eosin. With the help of a light microscope, we examined the stained sections of skin in order to analyze the histological shifts in the skin morphology [23] (see Fig. 1). 3. Results and discussion 3.1. Characterization of AgNPs At specific concentrations of leaf extract such as 1–5 mL, silver nanoparticles are fabricated using silver nitrate of 1 mM. The formed silver nanoparticles were analyzed with the help of Plasmon resonance band UV spectra at 436–446 nm and the results obtained were close to those published in one of the earlier literature [24]. When we raise the concentration of the leaf extract to 4 mL, the wavelength raised correspondingly to 448 nm as shown in Fig. 2a. The small differences in the absorbance parameters suggest that the differences are due to the particle sizes [25]. FT-IR study of AgNPs demonstrated the plant extract's dual function as a capping and reducing agent along with the existence of certain functional groups. Because of the N–H stretching vibrations of OH and NH2 groups, a wide band occurred at 3454 cm−1 and the overlapping of the stretching vibration was ascribed to Ocimum Sanctum leaf extract and water molecules. The bands present at 2083 and 1636 cm−1 are because of the alkyne group existing in the extract phytoconstituents
Fig. 6. Visual images represent burn healing activity with marketed formulation, blank Carbopol gel and AgNPs gel.
epidermal water loss (TEWL) in order to determine skin integrity prior to the treatment as well as after the treatment. Before measuring the TEWL, the vapometer was balanced at chamber temperature and humidity. The probe comprised of a sensor was placed over the treated skin surface in a horizontal position. In order to prevent any manual errors or fluctuations, 3 readings/animals are recorded. The readings 3
Journal of Drug Delivery Science and Technology 55 (2020) 101425
L.-N. Xu, et al.
Fig. 7. Effect of different gel formulations on the reduction of TEWL values with time. Fig. 8. Histological images showing the burn wound healing upon treatment with (A) Carbopol gel, (B) marketed formulation, and (C) AgNPs gel.
and amide C]O stretching [Fig. 2]. The peak found at 1113 cm−1 reflect either the bonds of –C−OC− or –C–O−. The bands found were mostly due to the excessive presence of terpenoids and flavonoids in the plant extract [26,27]. In contrast, a strong and wide band was observed from the prepared sample extract at 553 cm−1. The outcomes of the current study are found to be in accordance with one of the earlier literature results [28]. From the results of FT–IR, it can be inferred that because of few bioorganic compounds of Ocimum Sanctum extract, an intense capping or coating was formed on the AgNPs. However, similar kind of biomolecules capped nanoparticles was observed where plant extracts have been used for synthesis of different nanomaterials [29–33]. Biosynthesized AgNP's EDX spectrum confirms the existence of elemental silver signals (Fig. 3). Due to the bio-molecules bound to the biofabricated AgNPs, a few small weak peaks of Cr and Cl are also noticed in the EDX range apart from the high strong peaks. In the XRD pattern, we observed 5 prominent peaks of diffraction (Fig. 4), at 2θ values of 77.38°, 64.28°, 44.54° and 38.18° that corresponds to Bragg's reflections of (311), (220), (200) and (111) the face-centered cubic (FCC) structure of metallic Ag, respectively. In addition, the peaks detected in the XRD pattern are also noticed to be in strong accordance with the Joint Powder Diffraction Standard Card No-087-0720 and AgNPs SAED (Fig. 2) reference structure. Furthermore, additional three dissimilar peaks of diffraction at 2θ values of 46.53°, 32.52° and 28.10° that corresponds to the lattice planes (220), (200) and (111) of FCC structure related to AgCl NPs. AgCl is a popular step in bio-fabrication of Ag NPs [34]. The signals broadening pattern indicates that the formed nanoparticles are in nanosize. Further, the morphology of prepared AgNPs was studied by using TEM analysis (Fig. 5A and B). It is found that the NPs are spherical in shape with an average particle size of 20 nm. Also, the SAED pattern showed the crystalline nature of the biofabricated AgNPs (Fig. 5 C). Visual examination as shown in Fig. 6 explored the burn wound healing ability of formulations. It is found that the blank gel was unsuccessful in curing the membrane that was burnt and displayed no
inherently related burn healing or antimicrobial characteristic of the blank gel. Marketed treatments cured the wound relatively fast, however, scar persisted on the body, which is known to be an unusual cosmetic. Moreover, the total time taken to accomplish relative healing by the marketed treatments was much greater. When compared, for improved cosmetic results (no agyria or scars), AgNPs gel successfully cured the skin at a much quicker rate. It can be due to AgNPs capacity to modulate cytokine levels that in turn impacts inflammatory result post a burnt wound. Moreover, it is understood that Ag has an antimicrobial effect that performs a bioburden check at the injured area [35]. One of the earlier literatures demonstrated the mechanism of which Ag works to inhibit microbes [36]. Not a single animal model can be evaluated exclusively with visual observation. TEWL calculation was therefore introduced to test the role of skin's water barrier [37,38]. The theory of measuring TEWL is '' the skin protective layer is more ideal, then the water content is higher, thus making the TEWL lower. ‘' The ideal imperforated skin creates a defensive layer and decreases the water content, hence the TEWL quality. Determining the value of TEWL is a significant support for researching the process of burn recovery. Fig. 7 displays the results achieved with the determination of TEWL. In the current work, Carbopol® Ultrez 10 gel (negative control) of 1% (w/w), when treated on the animal skin displayed shift in the value of TEWL to 91.33 from 180 g/m2 h. In the equal period of administration, AgNPs gel decreased the value of TEWL significantly to 8.33 from 179.8 g/m2h (near to the initial value), whereas Burn heal™ lowered to 29.33 from 179.8 g/m2h. This greater decrease in the value of TEWL displayed nanogel's effectiveness in obtaining similar skin integrity to normal skin. Initial accelerated wound healing usually stops systemic infection from developing. In the context of AgNPs gel, the preliminary healing speed was also greater (p < 0.05) when compared with the marketed formulations as shown by the values of TEWL (110 g/m2 h Burn heal™ vs 53 g/ m2 h AgNPs gel, post treating for 10 days), offering enhanced septicemia protection. Studies of burn recovery showed that AgNPs can heal significantly in lower time relative to the marketed formulations 4
Journal of Drug Delivery Science and Technology 55 (2020) 101425
L.-N. Xu, et al.
without any symbol of scar. Throughout the research, healing progressed at a quicker rate relative to the marketed formulations in the context of AgNPs gel (However, it was not possible to entirely recover the injury as well as the scar persisted on administrated skin) (Fig. 7). Eventually, in the scenario of AgNPs gel (the value of TEWL post treating was similar to the original value), full skin consistency was obtained, but this is not achieved in the context of marketed formulations within a defined timeframe. Histology is another testing method for verifying the regenerative efficiency of formulations (curing of hair follicles, vacuoles, sebaceous glands) following treatment on burnt skin. Histological assessments indicate the regeneration of sebaceous glands, vacuoles, and hair follicles at the completion of therapy. Histological studies indicate the regeneration of hair follicles, vacuoles as well as sebaceous glands at the start of the procedure. Fig. 8 demonstrates the histological assessment of various formulations on removed animal skin. Fig. 8A revealed that in the control group, epidermis was not formed well and the dermal area was a deficit of hair vacuoles or follicles suggesting the lack of regeneration. A significant quantity of enucleated cells showed the presence of dense scarring alone in the dermis. Fig. 8B shows the impact of marketed formulations in the process of burn healing. Despite thick layers of keratinocytes displaying thickening, epidermis remained preserved. The existence of sebaceous glands and hair follicles indicates, though limited in quantity, the recovery during curing. Because of the large amounts of enucleated cells, dermis has bruises. Those are less than the control group, however. Fig. 8C shows the impact on the layers of the skin and their constituents by AgNPs gel. Epidermis are natural with the keratin becoming thick on the surface. The appearance of hair follicles was indeed a strong indicator of a feature's recovery that was missing in the group of control and limited in the marketed formulations. The most impressive aspect that distinguishes AgNPs gel from the marketed formulations was the appearance of greater quantity of intact cells in the dermis, which suggests reduced scars.
[7] [8]
[9]
[10]
[11] [12] [13] [14]
[15] [16] [17] [18]
[19] [20] [21]
[22]
[23]
[24] [25]
4. Conclusions
[26]
In conclusion, we showed the wound healing potential of biofabricated AgNPs synthesized by using Ocimum Sanctum leaf extract by avoiding chronicity of wound compared to conventional silver treatments. Also, the superior wound recovering potential of nanosilver gel compared to conventional formulations was illustrated in vitro in animals utilizing several criteria's including TEWL, visual observations and histological study. In conclusion, utilizing biofabricated AgNPs, the Carbopol-dependent nanosilver gel is a gold class treatment for wound recovery and offering up a modern era for medicinal applications.
[27]
[28]
[29]
[30]
CRediT authorship contribution statement
[31]
Li-Na Xu: Conceptualization. Hai-Xia Wang: Data curation, Writing - original draft. Ling Zhao: Conceptualization, Methodology.
[32] [33]
References [34]
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Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Biosynthesis of AgNPs and their effective wound healing activity in nursing care in children after surgery
T
Li-Na Xua, Hai-Xia Wangb, Ling Zhaoc,∗ a
Department of Operation, Linyi Central Hospital, Linyi, 276400, Shandong, China Department of Nursing, Linyi Central Hospital, Linyi, 276400, Shandong, China c Complaint Center, Jining NO.1 People's Hospital, Affiliated Jining NO.1 People's Hospital of Jining Medical University, Jining Medical University, Jining, 272111, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: AgNPs Wound healing Polyphenols
The current work illustrated the potency of wound healing activity of biofabricated silver nanoparticles synthesized by using Ocimum Sanctum leaf extract by avoiding chronicity of wound compared to conventional silver treatments. The prepared AgNPs were studied using various characterization techniques such as X-ray diffraction (XRD), Ultra Violet-Visible (UV–Visible) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and Highresolution transmission electron microscopy (HR-TEM). Additionally, the superior wound recovering potential of nanosilver gel compared to conventional formulations was illustrated in vitro in animals utilizing several criteria's including TEWL, visual observations and histological study. In conclusion, utilizing biofabricated AgNPs, the Carbopol-dependent nanosilver gel is a gold class treatment for wound recovery and offering up a modern era for medicinal applications in nursing care in children after surgery.
1. Introduction Any burn therapy's main objective is to accomplish epithelization and wound healing as quickly as feasible to avoid infection without creating any cosmetic and functional side effects [1]. Systematic infection from the injury location called wound sepsis is the major source of death in the patients suffering from burning, which is because of the chronicity. A wound's chronicity can contribute to a high inflammatory load equivalent to that of an ex-aggregated infectious condition [2]. Factors adding to the delayed wound healing can include the development of biofilm, bioburden at the injury location and microbial species, however not restricted to these factors though [3]. The function of primary microorganisms like Pseudomonas spp., Staphylococcus spp., Enterococcus, Streptococcus spp., etc. in chronic injuries has been demonstrated by various microbiological approaches as well as by microbial profiling studies [4,5]. These types of microbial groups have shown growing resistance to conventional antibacterial treatments and are therefore needed to develop modern approaches for eradicating biofilm for chronic wound management [6]. The latest advances utilize noble metal antibacterial agents against such burning diseases and the most common of these is silver [7]. Prior to the introduction of vaccines in the early 20's, silver (Ag) was utilized
for its medicinal and bactericidal properties against multiple inflammatory diseases [8–13]. Revitalizing Ag in the shape of silver nitrate post losing it entirely in the period of IInd World War lasted several years for increasing its interest [14]. Silver is currently regarded as a feasible choice for treating structural diseases induced by microbes [15]. Conventional materials such as silver sulfadiazine and silver nitrate are having the gold standard reputation in the treatment of inflammatory burn diseases among the numerous silver-dependent compounds [13,16]. These compounds, however, suffer from the disadvantages such as delay in the process of wound healing [17], cosmetic abnormality [19] and cytotoxic function on host cells [18]. Recently advanced silver-dependent products like AquacelTM, ActisorbTM, ActicoatTM, ArglaesTM, SilverlonTM, ActisorbTM etc. have a little more sustained and controlled delivery of nanocrystalline Ag into the wounded area [20]. This regulated releasing path enables reducing nosocomial infection, extended duration of function, decreasing price of medication, etc. [13,16,19,20]. The aim of the current study is to develop silver-dependent nanoformulations for topical burn injury healing, thus maintaining an equilibrium between toxicity and therapeutic activity. Silver nanoparticles (AgNPs) of average particle size 20 nm were produced and developed as topical medicine by Ocimum Sanctum. In order to prove
∗ Corresponding author. No. 6 Jiankang Road, Complaint Center, Jining NO.1 People's Hospital, Affiliated Jining NO.1 People's Hospital of Jining Medical University, Jining Medical University, Jining, 272111, Shandong, China E-mail address: [email protected] (L. Zhao).
https://doi.org/10.1016/j.jddst.2019.101425 Received 9 November 2019; Received in revised form 26 November 2019; Accepted 28 November 2019 Available online 13 December 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 55 (2020) 101425
L.-N. Xu, et al.
the wound healing impact of silver nanoformulations on rats’ skin, effective in vitro and in vivo research were conducted. 2. Materials and methods 2.1. Preparation of plant extract During February, freshly cut leaves of Ocimum Sanctum were obtained from the university campus. To eliminate debris as well as other hazardous organic materials, the leaves are surface washed with flowing tap water, accompanied by double distilled water and then dried in air at room temperature. In a flask comprising 200 mL of double distilled water, finely sliced leaves of around 20 gm were placed and then boiled for about 30 min. The temperature of the mixture was brought down and with the help of Whatman no.1 filter paper, the extract was filtered and for further utilization, the mixture was preserved at 4 °C. Fig. 1. UV–Visible spectrum of AgNPs with varying the Ocimum Sanctum extract concentration.
2.2. Green synthesis of silver nanoparticles Erlenmeyer flask was utilized to prepare 100 mL of 1 mM silver nitrate solution. Then Ocimum Sanctum leaf extract of 5, 4, 3, 2 and 1 mL was added with 10 mL solution of silver nitrate separately, maintaining 1 mM concentration. Dark chamber was utilized to incubate this setup at room temperature, in order to mitigate silver nitrate photo-activation. The change in the color of the solution from colorless to brown verified Ag+ reduction to Ag0. 2.3. Preparation of nano-silver gel AgNPs gel matrix was prepared with the help of Carbopol® Ultrez 10 NF and this supplied adequate stability to facilitate dispensation. In brief, 1% (w/v) of Carbopol® Ultrez 10 NF was gently spread in AgNPs subjection with the help of a 1000 rpm stirrer fixed overhead for about 3 h until the gelling agent was fully hydrated using water. 10% (w/v) triethanolamine was utilized to adjust the pH to 6 [21].
Fig. 2. FTIR spectrum of AgNPs prepared using Ocimum Sanctum extract.
2.4. Characterization of synthesized silver nanoparticles Spectrophotometer Shimadzu UV–Vis (UV-1800, Shanghai) was utilized to perform the analysis of UV–Vis spectrum. With 1 nm resolution between 200 nm and 800 nm, the UV–Vis absorption spectrophotometer was used. A diluted nano colloidal solution was used for optical absorption measurements. FTIR spectrophotometer, PerkinElmer 1750, UK was utilized to record the measurements of FT−IR spectrum. With the help of transmission electron microscopy (TEM), JEM-1400, Japan that was functioned at 120 kV accelerated voltage, the surface morphology and particle size were studied. A drop of diluted nanocolloid was placed on the copper grid followed by drying under vacuum, which later was observed under TEM for imaging.
Fig. 3. EDS spectrum of AgNPs.
developed. All sample formulations from the day of burning are added once in 24 h for continuous three weeks.
2.5. Burn healing protocol SD Male rats of weight 160–180 g were collected and separated into 3 groups of 6 rats each for the burn wound healing test. Every animal was placed in an independent cage and supplied with ad libitum water and food. The gel base of Blank Carbopol was provided to group I and was considered as control group. Burn healTM [indicated formulations comprising of 0.5% of chlorhexidine hydrochloride and 1% of silver sulfadiazine] was applied for group II animals (positive control) externally supplying silver ions (Ag0) of 1 mg/cm2 at wounded region. AgNPs gel comprising a similar concentration of 1 mg AgNPs (Ag0) was treated to group III animals per cm2 of wounded area. Animals are anesthetized with a combination of 10 mg/kg of xylazine and 80 mg/kg of ketamine, then depilated before burning on the anterior-dorsal side. Post applying blood hot rod for about 30 s, a 3rd-degree burn was
2.6. Visual observation The percent of wound healed for the tested areas was obtained by following the method demonstrated in the earlier reports, which states that by evaluating the degree of reduction in wound edema and inflammation [22]. Burnt wound healing characteristic was assessed depending on the minimum cessation period for the proportion of injury contraction. 2.7. Measurement of Trans Epidermal Water Loss (TEWL) A vapometer (Shanghai, China) was utilized to measure trans 2
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Fig. 4. XRD pattern of AgNPs.
Fig. 5. HR-TEM images (A,B) and SAED pattern (C) of AgNPs.
were represented as g/m2 h. 2.8. Histology of skin Histological examination confirmed the microscopic assessment of treated skin's internal structure. Different formulations have been tested to determine possible modifications to skin layers induced by the use of different formulations. The treated skin region was excised in histological assessment, 50% (w/v) formalin was utilized to keep the removed fat and later, dehydrated by graded alcohol sequence. Then, the skin was administrated with xylene followed by embedding into paraffin. For microscopic analysis, 5 μm thickness of skin pieces are taken of every specimen and coated with haematoxyl–eosin. With the help of a light microscope, we examined the stained sections of skin in order to analyze the histological shifts in the skin morphology [23] (see Fig. 1). 3. Results and discussion 3.1. Characterization of AgNPs At specific concentrations of leaf extract such as 1–5 mL, silver nanoparticles are fabricated using silver nitrate of 1 mM. The formed silver nanoparticles were analyzed with the help of Plasmon resonance band UV spectra at 436–446 nm and the results obtained were close to those published in one of the earlier literature [24]. When we raise the concentration of the leaf extract to 4 mL, the wavelength raised correspondingly to 448 nm as shown in Fig. 2a. The small differences in the absorbance parameters suggest that the differences are due to the particle sizes [25]. FT-IR study of AgNPs demonstrated the plant extract's dual function as a capping and reducing agent along with the existence of certain functional groups. Because of the N–H stretching vibrations of OH and NH2 groups, a wide band occurred at 3454 cm−1 and the overlapping of the stretching vibration was ascribed to Ocimum Sanctum leaf extract and water molecules. The bands present at 2083 and 1636 cm−1 are because of the alkyne group existing in the extract phytoconstituents
Fig. 6. Visual images represent burn healing activity with marketed formulation, blank Carbopol gel and AgNPs gel.
epidermal water loss (TEWL) in order to determine skin integrity prior to the treatment as well as after the treatment. Before measuring the TEWL, the vapometer was balanced at chamber temperature and humidity. The probe comprised of a sensor was placed over the treated skin surface in a horizontal position. In order to prevent any manual errors or fluctuations, 3 readings/animals are recorded. The readings 3
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Fig. 7. Effect of different gel formulations on the reduction of TEWL values with time. Fig. 8. Histological images showing the burn wound healing upon treatment with (A) Carbopol gel, (B) marketed formulation, and (C) AgNPs gel.
and amide C]O stretching [Fig. 2]. The peak found at 1113 cm−1 reflect either the bonds of –C−OC− or –C–O−. The bands found were mostly due to the excessive presence of terpenoids and flavonoids in the plant extract [26,27]. In contrast, a strong and wide band was observed from the prepared sample extract at 553 cm−1. The outcomes of the current study are found to be in accordance with one of the earlier literature results [28]. From the results of FT–IR, it can be inferred that because of few bioorganic compounds of Ocimum Sanctum extract, an intense capping or coating was formed on the AgNPs. However, similar kind of biomolecules capped nanoparticles was observed where plant extracts have been used for synthesis of different nanomaterials [29–33]. Biosynthesized AgNP's EDX spectrum confirms the existence of elemental silver signals (Fig. 3). Due to the bio-molecules bound to the biofabricated AgNPs, a few small weak peaks of Cr and Cl are also noticed in the EDX range apart from the high strong peaks. In the XRD pattern, we observed 5 prominent peaks of diffraction (Fig. 4), at 2θ values of 77.38°, 64.28°, 44.54° and 38.18° that corresponds to Bragg's reflections of (311), (220), (200) and (111) the face-centered cubic (FCC) structure of metallic Ag, respectively. In addition, the peaks detected in the XRD pattern are also noticed to be in strong accordance with the Joint Powder Diffraction Standard Card No-087-0720 and AgNPs SAED (Fig. 2) reference structure. Furthermore, additional three dissimilar peaks of diffraction at 2θ values of 46.53°, 32.52° and 28.10° that corresponds to the lattice planes (220), (200) and (111) of FCC structure related to AgCl NPs. AgCl is a popular step in bio-fabrication of Ag NPs [34]. The signals broadening pattern indicates that the formed nanoparticles are in nanosize. Further, the morphology of prepared AgNPs was studied by using TEM analysis (Fig. 5A and B). It is found that the NPs are spherical in shape with an average particle size of 20 nm. Also, the SAED pattern showed the crystalline nature of the biofabricated AgNPs (Fig. 5 C). Visual examination as shown in Fig. 6 explored the burn wound healing ability of formulations. It is found that the blank gel was unsuccessful in curing the membrane that was burnt and displayed no
inherently related burn healing or antimicrobial characteristic of the blank gel. Marketed treatments cured the wound relatively fast, however, scar persisted on the body, which is known to be an unusual cosmetic. Moreover, the total time taken to accomplish relative healing by the marketed treatments was much greater. When compared, for improved cosmetic results (no agyria or scars), AgNPs gel successfully cured the skin at a much quicker rate. It can be due to AgNPs capacity to modulate cytokine levels that in turn impacts inflammatory result post a burnt wound. Moreover, it is understood that Ag has an antimicrobial effect that performs a bioburden check at the injured area [35]. One of the earlier literatures demonstrated the mechanism of which Ag works to inhibit microbes [36]. Not a single animal model can be evaluated exclusively with visual observation. TEWL calculation was therefore introduced to test the role of skin's water barrier [37,38]. The theory of measuring TEWL is '' the skin protective layer is more ideal, then the water content is higher, thus making the TEWL lower. ‘' The ideal imperforated skin creates a defensive layer and decreases the water content, hence the TEWL quality. Determining the value of TEWL is a significant support for researching the process of burn recovery. Fig. 7 displays the results achieved with the determination of TEWL. In the current work, Carbopol® Ultrez 10 gel (negative control) of 1% (w/w), when treated on the animal skin displayed shift in the value of TEWL to 91.33 from 180 g/m2 h. In the equal period of administration, AgNPs gel decreased the value of TEWL significantly to 8.33 from 179.8 g/m2h (near to the initial value), whereas Burn heal™ lowered to 29.33 from 179.8 g/m2h. This greater decrease in the value of TEWL displayed nanogel's effectiveness in obtaining similar skin integrity to normal skin. Initial accelerated wound healing usually stops systemic infection from developing. In the context of AgNPs gel, the preliminary healing speed was also greater (p < 0.05) when compared with the marketed formulations as shown by the values of TEWL (110 g/m2 h Burn heal™ vs 53 g/ m2 h AgNPs gel, post treating for 10 days), offering enhanced septicemia protection. Studies of burn recovery showed that AgNPs can heal significantly in lower time relative to the marketed formulations 4
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without any symbol of scar. Throughout the research, healing progressed at a quicker rate relative to the marketed formulations in the context of AgNPs gel (However, it was not possible to entirely recover the injury as well as the scar persisted on administrated skin) (Fig. 7). Eventually, in the scenario of AgNPs gel (the value of TEWL post treating was similar to the original value), full skin consistency was obtained, but this is not achieved in the context of marketed formulations within a defined timeframe. Histology is another testing method for verifying the regenerative efficiency of formulations (curing of hair follicles, vacuoles, sebaceous glands) following treatment on burnt skin. Histological assessments indicate the regeneration of sebaceous glands, vacuoles, and hair follicles at the completion of therapy. Histological studies indicate the regeneration of hair follicles, vacuoles as well as sebaceous glands at the start of the procedure. Fig. 8 demonstrates the histological assessment of various formulations on removed animal skin. Fig. 8A revealed that in the control group, epidermis was not formed well and the dermal area was a deficit of hair vacuoles or follicles suggesting the lack of regeneration. A significant quantity of enucleated cells showed the presence of dense scarring alone in the dermis. Fig. 8B shows the impact of marketed formulations in the process of burn healing. Despite thick layers of keratinocytes displaying thickening, epidermis remained preserved. The existence of sebaceous glands and hair follicles indicates, though limited in quantity, the recovery during curing. Because of the large amounts of enucleated cells, dermis has bruises. Those are less than the control group, however. Fig. 8C shows the impact on the layers of the skin and their constituents by AgNPs gel. Epidermis are natural with the keratin becoming thick on the surface. The appearance of hair follicles was indeed a strong indicator of a feature's recovery that was missing in the group of control and limited in the marketed formulations. The most impressive aspect that distinguishes AgNPs gel from the marketed formulations was the appearance of greater quantity of intact cells in the dermis, which suggests reduced scars.
[7] [8]
[9]
[10]
[11] [12] [13] [14]
[15] [16] [17] [18]
[19] [20] [21]
[22]
[23]
[24] [25]
4. Conclusions
[26]
In conclusion, we showed the wound healing potential of biofabricated AgNPs synthesized by using Ocimum Sanctum leaf extract by avoiding chronicity of wound compared to conventional silver treatments. Also, the superior wound recovering potential of nanosilver gel compared to conventional formulations was illustrated in vitro in animals utilizing several criteria's including TEWL, visual observations and histological study. In conclusion, utilizing biofabricated AgNPs, the Carbopol-dependent nanosilver gel is a gold class treatment for wound recovery and offering up a modern era for medicinal applications.
[27]
[28]
[29]
[30]
CRediT authorship contribution statement
[31]
Li-Na Xu: Conceptualization. Hai-Xia Wang: Data curation, Writing - original draft. Ling Zhao: Conceptualization, Methodology.
[32] [33]
References [34]
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