PROCESS OF PREPARING ERI COCOON SILK PROTEIN WOUND DRESSING INCORPORATED WITH GREEN SYNTHESIZED SILVER NANOPARTICLES

Abstract

Process of preparing Eri cocoon silk protein wound dressing incorporated with green synthesized silver nanoparticles comprising; Preparation of aloe vera gel, Green synthesize of silver nanoparticles, Pre-processing of Eri cocoon, Preparation of Eri cocoon silk protein wound dressing, and Blending of Eri cocoon silk protein wound dressing with green synthesized silver nano particles. The present invention of Eri silk protein wound dressing escalates antimicrobial resistance crisis and improve wound healing outcomes.

Specification

Description:FIELD OF INVENTION
The present invention relates to field of sericulture. Specifically, the present invention relates to synthesis of green silver nano particles. More specifically, the present invention relates to process of preparing Eri cocoon silk protein wound dressing incorporated with green synthesized silver nanoparticles. The green synthesized nano particles of the present invention are produced from Aloe vera extract.
BACKGROUND
The novel combination of Eri silk protein, green-synthesized silver nanoparticles, and Aloe vera presents a synergistic approach to wound care. The wound dressing demonstrates a broader spectrum of antimicrobial coverage against common selected human pathogens like Escherichia coli, Staphylococcus aureus and Candida albicans. The Eri silk protein component effectively retains moisture, promoting optimal wound healing and minimizing the risk of desiccation compared to commercially available wound dressings. The incorporation of silver nanoparticles enhances the water absorption capacity and porosity of the wound dressing, potentially improving material permeability and biocompatibility.
The global rise of Antimicrobial Resistance (AMR) presents a significant threat to public health, ranking among the top 10 global health challenges, according to the WHO. Antibiotic overuse and misuse are fueling this crisis, leading to drug-resistant pathogens. Effective wound care solutions require innovative materials. Eri Silk Protein Wound Dressing, a natural silk protein, offers a promising alternative. The present invention provides a facile pathway for compelling evidence for the potential of silk fibroin-green-synthesized silver nanoparticles wound dressings as a novel and effective approach to combating selected human pathogens.
AgNPs are incorporated into various wound dressings, topical creams, and medical devices to provide effective protection against a wide range of pathogens. The green synthesis of silver nanoparticles (AgNPs) using A. vera as a reducing agent is growing rapidly due to their eco-friendly and cost-effective nature.
These dressings can be tailored to specific patient needs, providing a sustainable and innovative approach to wound healing. Their potent antimicrobial properties, coupled with their ability to promote wound healing, make them an attractive option for preventing infections and accelerating tissue regeneration.
There are some known prior arts worked on green synthesis of nano particles and they are provided below:
One known scholarly article of Tiara Egga Agustina et al discloses the UV-VIS Spectrum Analysis From Silver Nanoparticles Synthesized Using Diospyros maritima Blume. Leaves Extract from "Advances in Biological Sciences Research", volume 14 Proceedings of the 3rd KOBI Congress, International and National Conferences (KOBICINC 2020).
Another known scholarly article of Kun Yu et al discloses in situ assembly of Ag nanoparticles (AgNPs) on porous silkworm cocoon-based wound film: enhanced antimicrobial and wound healing activity from Scientific reports | (2017) 7:2107 | DOI:10.1038/s41598-017-02270-6
The present invention comes with a unique solution comprising properties of the wound dressing that is customized by adjusting the concentrations of Eri silk protein, silver nanoparticles, and A. vera, concerning the type of wound application, allowing for optimal performance.
OBJECT OF THE INVENTION
The main object of the present invention is to provide process of preparing Eri cocoon silk protein wound dressing incorporated with green synthesized silver nanoparticles.
It is one object of the present invention, wherein the said green synthesized silver nanoparticles is produced from Aloe vera gel extract.
It is one object of the present invention, wherein Eri cocoon preparation comprises pre-preparing of cocoon by harvesting and cutting cocoon edges at equal aera.
It is one object of the present invention, wherein preparing Eri cocoon silk protein wound dressing comprising immersing the prepared cocoon in Ajisawa's solution.
It is one object of the present invention, wherein Eri silk protein wound dressing incorporated with green-synthesized silver nanoparticles wound dressings offers a promising solution for wound care and improved antimicrobial properties
It is one object of the present invention, wherein Eri silk protein wound dressing escalates antimicrobial resistance crisis and improve wound healing outcomes.

SUMMARY
The main aspect of the present invention is to provide, a process of preparing Eri cocoon silk protein wound dressing incorporated with green synthesized silver nanoparticles comprising, Preparing aloe vera gel: Harvesting matured leaves of aloe vera for fresh parenchyma, Centrifuging and homogenizing is done to eliminate fibre and Collecting supernatant and storing.
It is one aspect of the present invention, wherein the said green synthesis of silver nanoparticles comprises adding 10 ml of different concentration ranging from 5, 10, 50, 100, 200 and 300 mm of AgNO3 to 20 ml of the supernatant, and identifying the formation of silver nanoparticles by color change from transparent to brown solution.
It is another aspect of the present invention, wherein pre-processing of Eri cocoon comprising harvesting Eri cocoon from mountage, cutting of ends of cocoon and removing pupae, and procuring equal area of cocoon.
It is one another aspect of the present invention, wherein preparing Eri cocoon silk protein wound dressing comprises immersing prepared cocoons in Ajisawa's solution (Cacl2 - C2H5 OH - H2O) with molar ratio of 1:2:8, respectively, incubating the said cocoon in water bath at 58℃ for 20 minutes, washing the obtained transparent cocoons several times with deionized water and sterilizing the cocoons with UV radiation for fabrication
It is yet another aspect of the present invention, wherein blending of Eri cocoon silk protein wound dressing with green synthesized silver nano particles comprises lyophilizing the prepared aloe vera gel green synthesized silver nano particles, dissolving the lyophilized nano particle in lactic acid (20ml ) using magnetic stirrer, adjusting the pH to 4.0 ± 0.2 at 47℃, impregnating the said solution with the fabricated Eri cocoon silk protein wound dressing in individual petri dishes for each concentration, sterilizing the obtained film with UV radiation and storing the final product store at room temperature
Brief description of drawings
Figure 1 illustrates UV-vis spectroscopic determination of AgNP concentrations, according to the present invention.
Figure 2 illustrates FTIR analysis of green-synthesis silver nanoparticles, according to the present invention.

Figure 3 illustrates evaluation of chemical stability of non-blended and blended Eri SPD through FTIR, according to the present invention.
Figure 4 illustrates surface morphology and EDX analysis of non-blended Eri SPD, according to the present invention.
Figure 5 illustrates surface morphology and EDX analysis of blended Eri SPD, according to the present invention.
DETAILED DESCRIPTION OF INVENTION WITH RESPECT TO ACCOMPANING DRAWINGS
The present invention relates to Process of preparing Eri cocoon silk protein wound dressing incorporated with green synthesized silver nanoparticles comprising; a. Preparation of aloe vera gel, b. Green synthesize of silver nanoparticles, c. Pre-processing of Eri cocoon, d. Preparation of Eri cocoon silk protein wound dressing, and e. Blending of Eri cocoon silk protein wound dressing with green synthesized silver nano particles.
The first step of the said process comprises preparation of alae vera gel wherein matured leaves of aloe vera are harvested and fresh parenchyma is taken, centrifugation and homogenization are undergone to eliminate the fibre and Supernatant is collected and used for further use.
The next step of the said process comprises green synthesize of silver nanoparticles, wherein 10 ml of various concentration (5, 10, 50, 100, 200 and 300 mm) of AgNo3 are added to 20ml of aloe gel extract. The color change from transparent to brown solution indicates the formation of silver nanoparticles.
Another step of the said process comprises pre-processing of Eri cocoon, wherein Eri cocoon were harvested from the mountage, ends of cocoon were first cutoff and the pupae was removed and equal area of cocoon were procured for further experimentation.
The further step of the said process comprises preparation of Eri cocoon silk protein wound dressing, prepared cocoons were immersed in Ajiraw's solution (Cacl2 - C2H5 OH - H2O) with molar ration of 1:2:8, respectively and incubated in water bath at 58C at 20 minutes. Now the cocoons become transparent and are washed several times with deionized water. The obtained cocoons were sterilized with UV radiation for fabrication
The final step of the said process comprises blending of Eri cocoon silk protein wound dressing with green synthesized silver nano particles, wherein prepared aloe vera gel green synthesized silver nano particles were lyophilized (freeze dried), and the lyophilized nano particles are were dissolved in lactic acid (20ml) using magnetic stirrer, pH adjusted to 4.0+-0.2 at 47C. The said solution is impregnated with fabricated Eri cocoon silk protein wound dressing in individual petri dishes for each concentration and the obtained film was sterilized with UV radiation. The obtained final products are stored at room temperature until further use.
With reference to figure 1, The green-synthesized silver nanoparticles were characterized using UV-Vis spectroscopy and Fourier transform infrared spectroscopy (FTIR). FTIR spectroscopy evaluated the chemical stability and interactions between silver nanoparticles and silk fibroin. Additionally, the disc diffusion method was performed to compare the antimicrobial activity with commercially available wound dressing (Surgicom®).
With reference to figure 2 Fourier transform infrared spectroscopy evaluated the chemical stability and interactions between silver nanoparticles and silk fibroin. FTIR analysis of A. vera synthesized silver nanoparticles revealed characteristic peaks indicating the presence of hydroxyl groups (O-H stretching), aromatic double bonds (C=C stretching), and other functional groups. The spectra of AVAg1 and AVAg showed slight variations in peak positions, suggesting subtle differences in the interactions between the A. vera extract and silver ions during nanoparticle formation. The presence of a new peak in the AVAg spectrum suggests a unique interaction or structural change associated with the higher concentration of A. vera extract.

FTIR analysis of Eri SPD-AVAg blends revealed spectral changes indicating chemical interactions between the components. These interactions, particularly in the O-H, C-H, C=C, and C-O functional groups, suggest potential enhancements in the material's stability and functional properties. The presence of a prominent peak at 1515 cm⁻¹ confirmed the integrity of the silk protein structure. Overall, the results suggest that AVAg incorporation affects the secondary structure of the silk proteins, potentially improving the material's properties.
The Eri SPD films with different concentrations of silver nanoparticles were evaluated for physical parameters such as water absorbability, WVTR and porosity.
Porosity analysis indicated that Eri SPD films treated with silver ions exhibited higher porosity compared to the control (Table 1.). The control and positive control exhibited WVTRs of 525 and 2916.67 g/m²/day, respectively. AVAg1 to AVAg6 demonstrated a progressive increase in WVTR values, with AVAg6 exhibiting the highest at 2500 g/m²/day. These results indicate that the incorporation of silver ions substantially enhances the water vapor permeability of EriSPD-AVAg blends.
Water absorption studies revealed that Blended EriSPD samples (AVAg1 to AVAg6) exhibited enhanced water uptake compared to the control (Table 2.). The initial absorption rates and final values were significantly higher in the silver-treated samples, suggesting that silver ion incorporation improves the hydrophilic properties of the material. Table 3. shows the water vapor permeability (WVTR) of EriSPD-AVAg blends. The control and positive control exhibited WVTRs of 525 and 2916.67 g/m²/day, respectively. AVAg1 to AVAg6 showed increasing WVTR values, with AVAg6 having the highest at 2500 g/m²/day.
Table 1. Evaluation of the Porosity (%)
Sample Porosity (%)
Eri SPD AVAg1 78.38 (62.29)f
Eri SPD AVAg2 79.65 (63.19)d
Eri SPD AVAg3 78.98 (62.71)e
Eri SPD AVAg4 83.13 (65.75)b
Eri SPD AVAg5 82.35 (65.16)c
Eri SPD AVAg6 88.46 (70.14)a
Control 77.17 (61.46)g
Positive control 88.46 (70.14)a
S.Ed 0.29
CD (P=0.05) 0.8
Values in parentheses are arc sine transformed values
Values with different alphabets within a column differ significantly

Table 2. Evaluation of Water absorbability (%)

% water absorbability
Time
(in hours) Eri SPD AVAg1 Eri SPD AVAg2 Eri SPD AVAg3 Eri SPD AVAg4 Eri SPD AVAg5 Eri SPD AVAg6 Control Positive control
12h 44.4 (41.78)e 73.6 (59.08)d 52.6 (46.49)e 55 (47.87)e 58.6 (49.95)e 58.8 (50.07)e 62.2 (52.06)e 44.4 (41.78)e
24h 55.5 (48.16)d 80 (63.43)c 63.1 (52.59)d 70 (56.79)d 75.8 (60.53)d 79.4 (63.01)d 82.2 (65.05)d 55.5 (48.16)d
48h 61.1 (51.41)c 88.4 (70.09)b 68.4 (55.8)c 72 (58.05)c 78.6 (62.44)c 80.8 (64.01)c 83.5 (66.03)c 61.1 (51.41)c
72h 65.5 (54.03)b 89.5 (71.09)ab 78.9 (62.65)b 79 (62.73)b 83.1 (65.73)b 87.3 (69.12)b 88.2 (69.91)b 65.5 (54.03)b
96h 68.8 (56.04)a 90.2 (71.76)a 81.5 (64.53)a 83.5 (66.03)a 86.5 (68.44)a 88.8 (70.45)a 90.2 (71.76)a 68.8 (56.04)a
S.Ed 0.23 0.29 0.31 0.20 0.26 0.27 0.27 0.34
CD (P=0.05) 0.70 0.90 0.94 0.62 0.81 0.84 0.83 1.04

Values in parentheses are arc sine transformed values
Values with different alphabets within a column differ significantly
Table 3. Evaluation of Water vapor permeability of AgNPs coated Eri SPD using water vapor transmission rate (WVTR) test

Sample WVTR (g/m²/day)
Control (Eri SPD) 525 (22.91)h
Positive Control 2916.67 (54.01)a
Eri SPD AVAg1 833.33 (28.87)g
Eri SPD AVAg2 1041.67 (32.27)f
Eri SPD AVAg3 1458.33 (38.19)e
Eri SPD AVAg4 1666.67 (40.82)d
Eri SPD AVAg5 2340 (48.37)c
Eri SPD AVAg6 2500 (50)b
S. Ed 0.31
CD (P=0.05) 0.95













Values in parentheses are square root transformed values
Values with different alphabets within a column differ significantly
Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray (EDX) analysis used to examine the surface morphology, elemental composition, and nanoparticle size distribution of blended and non- blended Eri SPD. SEM-EDX analysis of Eri SPD-AVAg blends revealed significant morphological changes compared to the untreated control. The control exhibited a smooth, fibrous structure without silver nanoparticles. In contrast, the blended samples showed a distinct presence of silver nanoparticles, with higher densities observed in AVAg5 and AVAg6. EDX analysis confirmed the presence of silver in these samples, with AVAg6 exhibiting the highest concentration of silver (78.66% Ag atom%).
Additionally, the disc diffusion method was performed to compare the antimicrobial activity with commercially available wound dressing (Surgicom®). The study revealed significant differences in antimicrobial activity among Eri SPD-AVAg formulations (Table 4.). Eri SPD AVAg2 consistently demonstrated the highest antimicrobial efficacy against E. coli, S. aureus, and C. albicans, while Eri SPD AVAg3 displayed the lowest.
The pooled mean values further confirmed these trends. All Eri SPD-AVAg formulations exhibited superior antimicrobial activity compared to the negative control, and most outperformed the positive control. These findings suggest that the formulation of Eri SPD-AVAg plays a crucial role in determining its antimicrobial effectiveness, with AVAg2 being
particularly effective against all three pathogens.
Table 4. Antimicrobial assay using blended Eri SPD-AVAg

Treatments E. coli S. aureus
C. albicans
Eri SPD AVAg1 13.43 (3.66)c 13.02 (3.61)d 13.39 (3.66)d
Eri SPD AVAg2 13 (3.61)d 13.69 (3.7)c 17.55 (4.19)a
Eri SPD AVAg3 12.05 (3.47)e 9.85 (3.14)e 8.37 (2.89)g
Eri SPD AVAg4 10.89 (3.3)f 15.43 (3.93)b 13.21 (3.63)e
Eri SPD AVAg5 13.53 (3.68)b 17.05 (4.13)a 14.2 (3.77)c
Eri SPD AVAg6 14.42 (3.8)a 9.68 (3.11)f 15.39 (3.92)b
Negative control 0 (0.0)h 0 (0.00)h 0 (0.00)h
Positive control 8.22 (2.87)g 8.84 (2.97)g 9.16 (3.03)f
S. Ed 0.3 0.24 0.31
CD (P= 0.05) 0.89 0.75 0.91


Advantages:
• The present invention offers a wound dressing material with superior antimicrobial properties, moisture-retention capabilities, and enhanced biocompatibility. These dressings exhibit broad-spectrum antimicrobial activity, effectively targeting bacteria, fungi, and viruses, thereby reducing the risk of infections in wounds.
• They maintain an optimal moisture balance, crucial for faster wound healing and preventing the wound from drying out by utilizing natural reducing agents like A. vera in the synthesis of AgNPs ensures biocompatibility and minimizes adverse reactions.
• The green synthesis methods employed are environmentally friendly and cost-effective, making the production of these dressings sustainable. The properties of these dressings can be tailored to meet specific patient needs, enhancing their effectiveness in various wound care scenarios. AgNPs promote tissue regeneration and reduce inflammation, leading to accelerated wound healing.
• Additional advantages include reduced antibiotic resistance by providing an alternative antimicrobial mechanism, long-lasting antimicrobial properties that reduce the need for frequent dressing changes, and non-toxicity to human cells when synthesized using green methods, ensuring safety for prolonged use. Their potent antimicrobial properties, coupled with their ability to promote wound healing, make them an attractive option for preventing infections and accelerating tissue regeneration.
• Green synthesized AgNPs are utilized in the pharmaceutical industry for advanced wound care products, including bandages and dressings with enhanced antimicrobial properties.

, Claims:WE CLAIM
1. Process of preparing Eri cocoon silk protein wound dressing incorporated with green synthesized silver nanoparticles comprising;
a. Preparing aloe vera gel:
i. Harvesting matured leaves of aloe vera for fresh parenchyma;
ii. Centrifuging and homogenizing is done to eliminate fibre; and
iii. Collecting supernatant and storing;

b. Green synthesis of silver nanoparticles:
i. Adding 10 ml of different concentration ranging from 5, 10, 50, 100, 200 and 300 mm of AgNO3 to 20 ml of the supernatant; and
ii. Identifying the formation of silver nanoparticles by color change from transparent to brown solution;
c. Pre-processing of Eri cocoon:
i. Harvesting Eri cocoon from mountage;
ii. Cutting of ends of cocoon and removing pupae; and
iii. Procuring equal area of cocoon;
d. Preparing Eri cocoon silk protein wound dressing:
i. Immersing prepared cocoons in Ajisawa's solution (Cacl2 - C2H5 OH - H2O) with molar ratio of 1:2:8, respectively;
ii. Incubating the said cocoon in water bath at 58℃ for 20 minutes;
iii. Washing the obtained transparent cocoons several times with deionized water; and
iv. Sterilizing the cocoons with UV radiation for fabrication.
e. Blending of Eri cocoon silk protein wound dressing with green synthesized silver nano particles:
i. Lyophilizing the prepared aloe vera gel green synthesized silver nano particles;
ii. Dissolving the lyophilized nano particle in lactic acid (20ml) using magnetic stirrer; and
iii. Adjusting the pH to 4.0 ± 0.2 at 47℃;
iv. Impregnating the said solution with the fabricated Eri cocoon silk protein wound dressing in individual petridishes for each concentration;
v. Sterilizing the obtained film with UV radiation; and
vi. Storing the final product store at room temperature.

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