GELATIN/CELLULOSE/CATECHIN/COPPER OXIDE NANOCOMPOSITES AS TOPICAL AGENT PREPARATION AND ITS USES THEREOF

Abstract

The invention is focused on an innovative nanocomposite topical agent composed of cellulose, gelatin, catechin and copper oxide nanoparticles in the form of nanocomposites. The nanocomposite is uniform, has very high entrapment efficacy, and can penetrate the skin and release copper in sustained manner. It has DPPH scavenging property (62.54%), mitigates oxidative stress, and degradation efficiency of bromophenol blue at 180 min is 99.63%. The nanocomposite exhibits a good spreadability, biocompatible and can be used for topical applications.

Specification

Description:The following examples are presented merely for the purpose of illustrative purposes and should not be interpreted as restricting the scope or applicability of the current invention. The invention is illustrated through these examples.
Example1: Preparation of Copper oxide (CuONp): To synthesize CuO nanoparticles, 1.25 g of copper chloride is dissolved in 50 mL of distilled water in a round-bottom flask. Simultaneously, 150 mg of polyethylene glycol is dissolved in 1 mL of distilled water, after which the solution is incorporated into the copper chloride solution within the round-bottom flask. This is agitated continually using a magnetic stirrer. A gradual addition of 5 mL of a 30% ammonia solution is added while stirring and the color changes from light blue to deep blue, indicating complex development, while stirring persists for 5 minutes. The solution was subsequently heated to 100°C for one hour, resulting in the formation of a blackish-brown precipitate. The precipitate is thereafter removed as supernatant and rinsed three times with distilled water to eliminate residual contaminants. The purified precipitate is black CuO nanoparticles (CuONp). 2 mg CuO np was weighed and dissolved in the 3 mL of distilled water using magnetic stirring and makeup the volume for 15 mL of distilled water. Kept stirring for 2 hours at 500 RPM.
Preparation of Catechin (C): In 50 mL beaker, weighed 60 - 120 mg of C was added and dissolved in 2 mL of distilled water under the stirring condition and then makeup the volume for 15 mL of distilled water. Kept stirring for 2 hours at 500 RPM.
Preparation of Gelatin (G): weighed 300- 600 mg of G was added and dissolved in 5 mL of distilled water under the stirring condition and makeup the volume for 15 mL of distilled water. Kept stirring for 2 hours at 500 RPM.
Preparation of Cellulose (CEL): 300 mg CEL (sodium carboxymethyl cellulose) was dissolved in 5 mL of distilled water in the 50 mL beaker under the stirring condition. Once its dissolved makeup the volume for 15 mL of distilled water. Kept stirring for 2 hours at 500 RPM.
Preparation of Gelatin (G) + catechin (C) : G+C
In 2 mL effendrof tube, weighed 120 mg C was dissolved in 2 mL of distilled water and vortexed for 5 mins. And kept aside. In 50 mL beaker, weighed 600 mg of G was added and dissolved in 5 mL of distilled water under the stirring condition. Once it dissolves completely added drop wise catechin solution in the beaker. After added makeup the volume for 15 mL distilled water. Kept stirring for 2 hours at 500 RPM.
Preparation of Cellulose (CEL) + Gelatin (G) + catechin (C) : CEL+G+C
In 2 mL effendrof tube, weighed 120 mg C was dissolved in 2 mL of distilled water and vortexed for 5 mins. And kept aside. 300 mg CEL was dissolved in 5 mL of distilled water in another beaker. In 50 mL beaker, weighed 600 mg of G was added and dissolved in 5 mL of distilled water under the stirring condition. Then added CEL solution in drop wise once it dissolved completely and add C solution also drop wise in the 50 mL beaker. After added makeup the volume for 15 mL of distilled water. Kept stirring for 2 hours at 500 RPM
Preparation of Cellulose (CEL) + Gelatin (G) + catechin (C) + copper oxide (CuONp): CEL+G+C+CuONp
300 - 600 mg of G was weighed and dissolved in 5 mL of distilled water in 50 mL beaker under the stirring condition until the solution dissolves. 300 mg of CEL was dissolved in 5 mL of distilled water in a separate 50 mL beaker under the stirring condition. Once it is dissolved this solution was added to the solution containing G. In 2 mL eppendorf tube, weighed 60 - 120 mg of C was added (for cross linking) and dissolved in 2 mL of distilled water and vortexed. This solution was added to the solution containing G and CEL under the stirring condition. 2 mg CuONp was weighed and dissolved in the 3 mL of distilled water and added to the solution containing CEL+G+C crosslinked nanocomposites. This was kept for stirring for 2 hours at 500 RPM. After 2 hours, solution was centrifuged for 15 minutes at 2000 RPM. Then the pellets (CEL+G+C+CuONp ) were separated and made up to 10 mL using distilled water. To understand the shift in the peak of the formulated nanocomposite, UV-Vis spectrophotometric analysis was carried out and the spectra were recorded. The hydrodynamic diameter was done to understand the size distribution and zeta potential measurements were carried out to understand the degree of stability and the charge of the molecules that are bound on the surface. FT-IR studies on nanocomposites and drugs alone was done
Example-2: DPPH+TRISHCl+G+C, DPPH+TRISHCl+CEL+G+C, DPPH+TRIS HCl+CEL+G+C+CuONp, DPPH+TRIS HCl+CuONp, also DPPH+TRIS HCl+G, DPPH+TRIS HCl+C, DPPH+TRIS HCl+CEL and DPPH+TRIS HCl was added to 2 mL tubes and incubated for 1h. OD was taken at 517 nm and % of radical scavenging was calculated based on the OD of DPPH+TRIS HCl. Hemolysis assay was performed for G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, also G, C, CEL using chicken blood erythrocytes and compared with negative control - NC with PBS and positive control - PC with SDS. For this 10 µL of G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, also G, C, CEL was taken and incubated with erythrocytes. After 1 h of incubation and centrifugation at 8000 rpm for 5 mins, the OD was taken at 545 nm and % of hemolysis was calculated with respect to NC and PC. Pollutant degradation study was done by using1 mg bromophenol blue was weighed and added in 10 mL distilled water pH adjusted to 12 with NaOH. For this, 1 mL Bromophenol blue+ 1 mL distilled water; 1 mL Bromophenol blue + 1 mL CuONp; 1 mL Bromophenol blue + 1mL CEL+G+C+CuONp was taken and the samples were kept in dark for 3 h. OD at 590 nm was taken and the degradation % was calculated with respect to the OD obtained from 1 mL Bromophenol blue + 1 mL distilled water. For skin penetration studies, Chicken skin was cleaned with PBS and was placed in the Franz cell between the donor and receptor compartment. The donor compartment was filled with PBS and CEL+G+C+CuONp or CuONp was placed on the skin placed on the receptor compartment. The study was carried for 2 h. the sample was withdrawn from the donor compartment and replaced with fresh PBS every time. After 1 and 2 h, and ICP- OES analysis of copper present was calculated as drug release and penetration was calculated as percentage based on amount of CuONp added. To understand the spreadability of CEL+G+C+CuONp,100 µL of CEL+G+C+CuONp was added to a Petri plate and was spread. The spreadability of the nanocomposite was measured at initial and final stage. For water Retention Study: The samples (CuONp, CEL+G+C+CuONp) were dried completely without water content and were placed in 2 petri plates with same weight. The initial weight was noted. Following this, 20 L of distilled water was added to the 2 plates and its weight was noted at 0th h. The plates were placed in a desiccator and the weight was taken and noted for every 1 h interval till 3 h. % water retention = final weight/ initial weight *100
Example-3: The antibacterial activity was assessed by a growth inhibition assay conducted on two bacterial strains: S. aureus and E. coli. One hundred microliters of samples, G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, also G, C, CEL were cultured for three hours with the bacterial strains S. aureus or E. coli. Following incubation for 3 h, the optical density at 600 nm was measured, and the percentage of growth inhibition was determined. Also, optical images of bacteria S. aureus and E. coli and in the presence of samples G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, also G, C, CEL was taken using Gram Staining technique. For this, the samples cultured with bacterial strains were heat-fixed on a clean glass slide and stained with crystal violet and safranin O. The slides were delicately cleansed and examined under a microscope at 100X magnification.
.ADVANTAGES OF THE INVENTION
A simple method to entrap copper oxide nanoparticles in cellulose, gelatin crosslinked nanocomposite and the ability to form materials that can be used as topical applicants.
The main advantage of such nanocomposite is that it can penetrate skin and release copper oxide nanoparticles in a sustained manner
The presence of gelatin in nanocomposite aids in excellent antioxidant activity.
The presence of cellulose in nanocomposite exerts spreadability of nanocomposite which is an important criterion of topical applicants to be applied on skin.
The presence of catechin provides antioxidant property as well as antibacterial activity to the nanocomposite.
The nanocomposites cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) are biocompatible in nature.
The nanocomposites cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) size is in the nanometer region.
EXPLANATION OF FIGURES
Figure 1 - Fig 1 A indicates the schematic representation of the nanocomposites of CEL, G, C and CuONp which was prepared with the help of crosslinking G+CEL with C and then entrapment of CuONp into the nanocomposite of CEL, G, C. Fig 1 B indicates the hydrodynamic diameter (d. nm) of G+C, CEL+G+C, CEL+G+C+CuONp, CuONp. Fig 1 C indicates the UV-Vis spectra of G+C, CEL+G+C, CEL+G+C+CuONp. Fig 1 D indicates the zeta potential values of G+C, CEL+G+C, CEL+G+C+CuONp, CuONp. Fig 1 E indicates the IR spectra of CEL, G, C, G+C, CEL+G+C, CEL+G+C+CuONp. The circles in the IR spectra indicate the changes in the molecular vibrations with respect to the formation of nanocomposites using catechin as a crosslinker. (Example 1).
Figure 2. A represents the antioxidant assay as determined by the DPPH scavenging activity of G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, C, G, CEL. Fig 2 B indicates the hemolysis study of PC treated with SDS and negative control treated with PBS also the erythrocytes treated with 10 µL of G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, C, G, CEL. Fig 2 C indicates the experimental setup of penetration studies done for CuONp and CEL+G+C+CuONp using chicken skin ex vivo sample. Here the graph indicates the penetration of CuONp/ CEL+G+C+CuONp at the end of 1st h and 2nd h. Fig 2 D indicates the control bromophenol blue dye, bromophenol blue dye treated with CuONp and bromophenol blue dye treated with CEL+G+C+CuONp. As seen from the figure, the nanocomposites CEL+G+C+CuONp could degrade bromophenol blue at 99.63%. Fig 2 E indicates the water retention study done for CuONp and CEL+G+C+CuONp. The photograph indicates the dried weight and wet weight of CuONp and CEL+G+C+CuONp. The table indicates the % of water retention by CuONp and CEL+G+C+CuONp. The spreadability of CEL+G+C+CuONp can be seen from fig 2 F and from the figure it can be seen initially it was around 1.1 cm and after spreading it was around 3.9 cm. (Example 2).
Figure 3. Fig 3A indicates the antibacterial activity of G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, G, CEL, C when compared to control which was done for both gram positive (S. aureus) and gram negative (E. coli) bacteria. Fig 3 B indicates the representative optical micrographs of control as well as S. aureus/ E. coli treated with G+C, CEL+G+C, CEL+G+C+CuONp, CuONp, G, CEL, C indicating the highest inhibition of bacteria by CEL+G+C+CuONp nanocomposite. (Example 3).
, Claims:I/We Claim:
1. The cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) nanocomposites as topical agents comprises following characteristics:-
i) nanocomposite made of cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) was found to be in an homogeneous material with zetapotential measurement of -4.62 mV.
ii) nanocomposite made of cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) was found to be in nanometer range and the hydrodynamic diameter as determined by the DLS is 133.9 d.nm.
iii) nanocomposite made of cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) had entrapment efficiency of 100 % for CuONp and 100 % for catechin.
iv) nanocomposite made of cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) could penetrate the skin and release CuONp in a sustained manner and was hemobiocompatible with no hemolysis monitored.
v) nanocomposite made of cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) had DPPH scavenging activity (62.54%) thereby reduced oxidative stress and had the ability to degrade pollutant at 99.63 %.
vi) nanocomposite made of cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) had spreadability at 3.9 cm.
2. The nanocomposite made of cellulose (CEL), gelatin (G) crosslinked with catechin (C) and nanocomposites entrapped with polyethylene glycol coated copper oxide nanoparticles (CuONp) (CEL+G+C+CuONp) as claimed in claim 1, method of preparation comprising of steps:-
i) A process involving copper chloride solution that is prepared by dissolving 1.25g in 50mL of distilled water and 150 mg of polyethylene glycol in 1mL of distilled water and the solution is stirred continuously, and ammonia solution is added. The solution is heated to 100°C for an hour, forming a blackish-brown precipitate which is then collected and rinsed three times with distilled water and purified precipitate is black CuO nanoparticles (CuONp).
ii) 300 - 600 mg of G was weighed and dissolved in 5 mL of distilled water in 50 mL beaker under the stirring condition until the solution dissolves. 300 mg of CEL was dissolved in 5 mL of distilled water in a separate 50 mL beaker under the stirring condition. Once it is dissolved this solution was added to the solution containing G.
iii) In 2 mL eppendorf tube, weighed 60 - 120 mg of C was added (for crosslinking) and dissolved in 2 mL of distilled water and vortexed. This solution was added to the solution containing G and CEL under the stirring condition. 2 mg CuONp was weighed and dissolved in the 3 mL of distilled water and this was added to the solution containing CEL+G+C crosslinked nanocomposites. This was kept for stirring for 2 hours at 500 RPM.
iv) After 2 hours, solution was centrifuged for 15 minutes at 2000 RPM. Then the pellets (CEL+G+C+CuONp ) were separated and made up to 10 mL using distilled water.

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