FIELD OF THE INVENTION
[0001] The present disclosure relates generally to anti-microbial formulations. More specifically, the disclosure is directed to a topical nanocomposite gel formulation for anti-microbial activity comprising: (a) silver nanoparticles; (b) zinc oxide nanoparticles; (c) humic acid and (d) a gelling agent. The present disclosure also provides a process of preparing the formulation.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Silver nanoparticles have been manufactured extensively due to its unique properties. The nanoparticles are known to have anti-microbial effects due to its small size and the continuous release of silver ions that effectively bind to cell wall and membrane of the microbes to destroy them. Further, unlike other anti-biotic drugs the microbes do not develop anti-biotic resistance to silver nanoparticles. However, despite its efficacy stabilization of silver nanoparticles and its delivery in a subject is generally challenging.
[0004] Topical formulations provide improved delivery of actives without their intestinal digestion and minimal systemic effects. Conventional compositions for topical delivery of silver nanoparticles suffer from poor delivery of the nanoparticles at the site of action due to low permeability across biological membranes. Possible solutions for anti-microbial nanoparticles’ permeation enhancers include emulsions, solutions, creams, and ointments prepared by conventional mixing and chemical cross-linking methods. Creams, ointments, and solutions have their limitation of applicability, flow stability and uncontrolled drug release which further causes irregularity in drug release and low permeability across the biological membrane. Much like silver nanoparticles, zinc oxide nanoparticles have also shown considerable anti-microbial activity and its compositions face similar defects in topical applications such as low permeability and poor actives delivery.
[0005] The inventors of the present disclosure provide a pharmaceutical formulation for topical application, comprising silver nanoparticles and zinc oxide nanoparticles, that is stable and has high permeability.

OBJECTS OF THE INVENTION
[0006] An object of the present disclosure is to increase the permeability of silver nanoparticles and zinc oxide nanoparticles across the biological membrane in topical compositions.
[0007] An object of the present disclosure is to provide a topical nanocomposite gel formulation for anti-microbial activity comprising silver nanoparticles and zinc-oxide nanoparticles.
[0008] Yet another object of the present disclosure is to provide a process of preparation of a topical nanocomposite gel formulation for anti-microbial activity.

SUMMARY OF THE INVENTION
[0009] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0010] Aspects of the present disclosure provide a formulation for improved permeation of nanoparticles actives, specifically zinc oxide nanoparticles and silver nanoparticles, across the biological membrane.
[0011] In an aspect, the present disclosure provides a topical nanocomposite gel formulation for anti-microbial activity comprising: (a) silver nanoparticles, (b) zinc-oxide nanoparticles, (c) humic acid, and (d) a gelling agent.
[0012] In an embodiment, the silver nanoparticles may be synthesized from silver nitrate employing Sea Buckthorn seed extract as a reducing agent. In a preferred embodiment, the silver nanoparticles may be synthesized from silver nitrate by a process comprising the steps of: (a) mixing equal proportions of Sea Buckthorn extract and a silver salt and incubating the mixture at about 35-55 ºC for about 2-5 hours; and (b) drying followed by centrifuging the silver particles using methanol to remove the debris to give the silver nanoparticles.
[0013] In an embodiment, the formulation comprises silver nanoparticles in a range of about 25% w/w to about 40% w/w of the formulation. In an embodiment, the concentration of silver nanoparticles in a solution may be about 5-15% w/v.
[0014] In an embodiment, the zinc oxide nanoparticles (ZnO NPs) may be synthesized by precipitation of a zinc salt. In an embodiment, the zinc oxide nanoparticles may be synthesized by a process comprising the steps of: (a) preparing a solution of potassium hydroxide in water; (b) preparing a solution of zinc nitrate in water; (c) transferring the zinc nitrate solution into potassium hydroxide solution and stirring for about an hour; and (d) centrifuging the solution of step (c) to give the zinc oxide nanoparticles.
[0015] In an embodiment, the formulation comprises zinc oxide nanoparticles in a range of about 25% w/w to about 40% w/w of the formulation.
[0016] In an embodiment, the concentration of zinc oxide nanoparticles in a solution may be about 5-15% w/v.
[0017] In an embodiment, the formulation comprises humic acid in a range of about 2% w/w to about 10% w/w of the formulation.
[0018] In an embodiment, the gelling agent may be selected from a polyacrylic acid. In an embodiment, the formulation comprises the gelling agent in a range of about 0.5% w/w to about 10% w/w of the formulation.
[0019] In another aspect, the present disclosure provides a process of preparing a topical nanocomposite gel formulation for anti-microbial activity, wherein the process comprises the steps of: (a) reducing a silver salt with Sea Buckthorn extract to give silver nanoparticles; (b) precipitating a zinc salt in potassium hydroxide to give zinc oxide nanoparticles; (c) mixing equal volumes of the silver nanoparticles and zinc oxide nanoparticles with humic acid; (d) adding a gelling agent to the mixture of step (c) with continuous stirring to give a gel; and (e)neutralizing the gel with triethanolamine then keeping the gel undisturbed to expel trapped air to give the topical nanocomposite gel formulation.
[0020] Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0022] Figure 1A provides the Transmission Electron Microscopy (TEM) image showing morphology of silver nanoparticles prepared as per an exemplary embodiment of the present disclosure. Figure 1B provides the Transmission Electron Microscopy (TEM) image showing morphology of zinc oxide nanoparticles prepared as per an exemplary embodiment of the present disclosure. Figure 1C provides an ultraviolet (UV) spectral analysis of silver and zinc nanoparticles after 0(blue line), 3 (yellow line, Ag NPs), 6 (brown line, Ag NPs), 3 (red line, ZnO NPs), and 6 (light blue line, ZnO NPs) months; Figure 1C (inset) provides the variation in colour change after incubation for (a)5 min, (b)10 min, (c) 15 min, (d) 20 min, and (e)25 min due to formation of silver nanoparticles as per an exemplary embodiment of the present disclosure; and Figure 1D provides the Scanning Electron Microscopy (SEM) image showing morphology of the nanocomposite gel prepared as per an embodiment of the present disclosure.
[0023] Figure 2 provides the percentage drug release (%) with time for: (a)silver nanoparticles from silver nanoparticles solution; (b) zinc nanoparticles from zinc oxide nanoparticles solution; (c) silver nanoparticles from nanocomposite gel as per an exemplary embodiment of the present disclosure; and (d) zinc oxide nanoparticles from nanocomposite gel as per an exemplary embodiment of the present disclosure.
[0024] Figure 3 provides the images of zone of inhibition for antimicrobial activity against: (A) Gram negative (E. coli) and (B) Gram positive (S. aureus) bacteria for (A) Ag nanoparticles solution alone, (B) ZnO nanoparticles solution alone, (C) Negative Control i.e., DMSO, (D) nanocomposite gel as per an exemplary embodiment of the present disclosure; and (E) A gel loaded with positive control i.e. ciprofloxacin hydrochloride.

DETAILED DESCRIPTION OF THE INVENTION
[0025] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0026] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0027] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0028] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0029] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0030] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0031] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0032] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0033] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0034] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0035] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0036] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0037] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0038] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0039] As described herein, the term ‘effective amount’ refers to the amount of the formulation required to bring about a change or improvement in a subject without side effects or overdosing.
[0040] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia' , including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.
[0041] The term, ‘management’, or ‘treatment’ as used herein refers to alleviate, slow the progression, attenuation, prophylaxis or as such treat the existing disease or condition. Treatment also includes treating, preventing development of, or alleviating to some extent, one or more of the symptoms of the diseases or condition.
[0042] As used herein, the term ‘nanoparticles’ refers to, small particles that range between 1 to 100 nanometers in size. Undetectable by the human eye, nanoparticles can exhibit significantly different physical and chemical properties compared to their large sized counterparts.
[0043] The terms, ‘formulation’, ‘nanocomposite gel’, ‘gel formulation’ and ‘topical gel’, have been used interchangeably in the present disclosure.
[0044] Aspects of the present disclosure provide a synergistic formulation that is topically administered to a subject to enhance the penetration of silver and zinc oxide nanoparticles; consequently improving the release profile of the nanoparticles and enhancing the anti-microbial activity of the nanoparticles.
[0045] In an embodiment, the present disclosure provides a topical nanocomposite gel formulation for anti-microbial activity comprising: (a) silver nanoparticles, (b) zinc-oxide nanoparticles, (c) humic acid, and (d) a gelling agent.
[0046] In an embodiment, the silver nanoparticles (Ag NPs) may be synthesized by reduction of a silver salt, specifically silver nitrate. In an embodiment, the silver nanoparticles may be synthesized from a silver salt employing Sea Buckthorn seed extract as a reducing agent.
[0047] In a preferred embodiment, the extract of Sea Buckthorn seed may be an aqueous extract.
[0048] In a preferred embodiment, the silver nanoparticles may be synthesized from silver nitrate by a process comprising the steps of: (a) mixing equal proportions of Sea Buckthorn extract and a silver salt and incubating the mixture at about 35-55 ºC for about 2-5 hours; and (b) drying followed by centrifuging the silver particles using methanol to remove the debris to give the silver nanoparticles.
[0049] The progress of the reaction may be monitored by the color change undergone by silver nanoparticles upon formation.
[0050] In an embodiment, the formulation comprises silver nanoparticles in a range of about 25% w/w to about 40% w/w of the formulation. In an embodiment, the formulation comprises silver nanoparticles in a range of about 30% w/w to about 40% w/w of the formulation.
[0051] In an embodiment, the concentration of silver nanoparticles in a solution may be about 5-15% w/v, preferably 5% w/v.
[0052] In an embodiment, the zinc oxide nanoparticles (ZnO NPs) may be synthesized by precipitation of a zinc salt, preferably zinc nitrate.
[0053] In an embodiment, the zinc oxide nanoparticles may be synthesized by a process comprising the steps of: (a) preparing a solution of potassium hydroxide in water; (b) preparing a solution of zinc salt in water; (c) transferring the zinc salt solution into potassium hydroxide solution and stirring for about an hour at about 35-55°C; and (d) centrifuging, washing and drying the solution of step (c) to give the zinc oxide nanoparticles.
[0054] The centrifugation may be performed at about 4000 rpm and the washing may be done with distilled water. The zinc oxide nanoparticles may be dried in an oven or vacuum dried for about 3-5 hours to give the nanoparticles.
[0055] In an embodiment, the formulation comprises zinc oxide nanoparticles in a range of about 25% w/w to about 40% w/w of the formulation. In an embodiment, the formulation comprises zinc oxide nanoparticles in a range of about 30% w/w to about 40% w/w of the formulation.
[0056] In an embodiment, the concentration of zinc oxide nanoparticles in a solution may be about 5-15% w/v.
[0057] Humic acids are macromolecular organic compounds that are found and isolated from soil organic matter. Humic acid increases the permeability of the nanoparticles and provides them stability. Thus, humic acid acts as a permeation enhancer, which works by increasing or enhancing the absorption capacity of actives across the biological membrane.
[0058] In an embodiment, the formulation comprises humic acid in a range of about 2% w/w to about 10% w/w of the formulation. In an embodiment, the formulation comprises humic acid in a range of about 5% w/w to about 10% w/w of the formulation.
[0059] In an embodiment, the gelling agent may be selected from a polyacrylic acid or acrylic acid. In an embodiment, the polyacrylic acid may be selected from Carbopol® 934 or Carbopol® 940.
[0060] In an embodiment, the formulation comprises the gelling agent in a range of about 0.5% w/w to about 10% w/w of the formulation. In an embodiment, the formulation comprises the gelling agent in a range of about 5% w/w to about 10% w/w of the formulation.
[0061] In an embodiment, the formulation may further comprise a solvent. In some embodiments, the solvent may be present in a range of about 70% w/v to about 85%w/v, preferably about 85% w/v of the formulation. The solvent employed may be distilled water.
[0062] The formulation comprising Ag NPs and ZnO NPs having small size and spherical shape are more susceptible to the release of Ag+ and Zn2+ ions owing to their greater surface area. The aggregation of Ag NPs and ZnO NPs reduces the release of Ag+ and Zn2+ ions and without being bound to theory, it is believed that this issue is resolved in the present formulation with the usage of humic acid as capping and stabilizing agent, which effectively modifies the surfaces of Ag NPs and ZnO NPs, and thus improves the dissolution activities of the Ag NPs and ZnO NPs. Addition to these inherent properties of Ag NPs and ZnO NPs, the surrounding media also influences the release of Ag+ and Zn2+ ions. Thus, the formulation is synergistic with improved release characteristics of the ions and enhanced permeability.
[0063] The formulation comprises silver nanoparticles and zinc oxide nanoparticles that contribute to synergistic anti-microbial activity, decrease the disease burden and counter anti-microbial resistance.
[0064] The formulation provides high permeability of actives, controlled release through the skin, uniform dosage, and flow stability for the nanoparticles. The formulation also provides ease of application as opposed to creams, ointments, or solutions. The topical gel has high patient compliance compared to oral dosage and other topical administrative forms.
[0065] In an embodiment, the formulation need not be removed after application owing to its biodegradability. In an embodiment, the formulation is stable and has high permeability. Sustained release of the actives is seen from the formulation for upto 24 hours as compared to silver and zinc nanoparticles released from a non-gel formulation showing drug release for upto 10 hours. Thus, the nanocomposite gel improves delivery of actives for longer periods of time.
[0066] In an embodiment, the present disclosure provides a medicament comprising the formulation as recited above for anti-microbial activity.
[0067] In another embodiment, the present disclosure provides a process of preparing a topical nanocomposite gel formulation for anti-microbial activity, wherein the process comprises the steps of: (a) reducing a silver salt with Sea Buckthorn extract to give silver nanoparticles; (b) precipitating a zinc salt in potassium hydroxide to give zinc oxide nanoparticles; (c) mixing equal volumes of the silver nanoparticles and zinc oxide nanoparticles with humic acid; (d) adding a gelling agent to the mixture of step (c) with continuous stirring to give a gel; and (e)neutralizing the gel with triethanolamine then keeping the gel undisturbed to expel trapped air to give the topical nanocomposite gel formulation.
[0068] In a preferred embodiment, the silver nanoparticles may be synthesized from silver nitrate by a process comprising the steps of: (a) mixing equal proportions of Sea Buckthorn extract and a silver salt and incubating the mixture at about 35-55 ºC for about 2-5 hours; and (b) drying followed by centrifuging the silver particles using methanol to remove the debris to give the silver nanoparticles.
[0069] In an embodiment, the zinc oxide nanoparticles may be synthesized by precipitation of a zinc salt, preferably zinc nitrate. In an embodiment, the zinc oxide nanoparticles may be synthesized by a process comprising the steps of: (a) preparing a solution of potassium hydroxide in water; (b) preparing a solution of zinc salt in water; (c) transferring the zinc salt solution into potassium hydroxide solution and stirring for about an hour at about 35-55°C; and (d) centrifuging, washing and drying the solution of step (c) to give the zinc oxide nanoparticles.
[0070] The process of the present disclosure is simple, economical and industrially scalable.
[0071] In an embodiment, the present process provides a loading efficiency of about 80% to about 85% for silver nanoparticles. In an embodiment, the present process provides a loading efficiency of about 78% to about 88% for zinc oxide nanoparticles.
[0072] In an embodiment, the present disclosure provides use of a topical nanocomposite gel formulation as recited above for the management of anti-microbial infection and diseases.
[0073] In an embodiment, the present disclosure provides a method of treatment, amelioration or prophylaxis of anti-microbial infection in a subject by administering an effective amount of the topical nanocomposite gel formulation as recited above.
[0074] In an embodiment, the anti-microbial infection may be selected from bacterial skin infections, acute ulcers, among others. In an embodiment, the microbe may be selected from a fungi, single-cell eukaryotes, virus, protozoa, or bacteria, including but not limited to, Staphylococcus and Escherichia, specifically Staphylococcus aureus and Escherichia coli. The formulation may be effective against gram positive and gram negative bacteria.
[0075] In an embodiment, the formulation inhibits the growth of the microbe or kills the microbe. Without being bound to theory, it is believed that Ag NPs and ZnO NPs continually release silver and zinc ions, which kill microbes. Owing to electrostatic attraction and affinity to sulfur proteins, silver and zinc ions can adhere to the cell wall and cytoplasmic membrane. The adhered ions have higher permeability through the cytoplasmic membrane and lead to disruption of the bacterial envelope. After the uptake of free silver ions into cells, respiratory enzymes can be deactivated, generating reactive oxygen species but interrupting adenosine triphosphate production. Reactive oxygen species can be a principal agent in the provocation of cell membrane disruption and deoxyribonucleic acid (DNA) modification. As sulfur and phosphorus are important components of DNA, the interaction of silver ions with the sulfur and phosphorus of DNA can cause problems in DNA replication, cell reproduction, or even result in termination of the microorganisms. Moreover, silver ions can inhibit the synthesis of proteins by denaturing ribosomes in the cytoplasm.
[0076] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0077] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
[0078] Materials: Sea Buckthorn seeds were received from Institute of Himalayan Bioresources and Technology, Palampur, H.P, India. All other chemicals were obtained from Himedia Pvt Ltd, India and were of analytical grade. Double distilled water was used throughout the study.
Example 1: Synthesis of Nanoparticles
1.1 Synthesis of silver nanoparticles:
[0079] The Sea Buckthorn seeds were washed thoroughly with distilled water and further soaked for one hour to remove the dust on the periphery of the seed. The soaked seeds were further washed with distilled water and ground with 10 ml of sterile distilled water. The paste was centrifuged at 10000 rpm for 15 min to collect the upper supernatant. The supernatant was transferred into a brown bottle and kept in the dark room for 24 h.
[0080] For the synthesis of silver nanoparticles, green chemistry approach was used using Sea Buckthorn extract. Briefly, an aqueous solution of silver nitrate (1 mM) was prepared by dissolving 17 mg of silver nitrate in 100 ml distilled water. To varying concentrations of Sea Buckthorn seed extract (i.e. 2, 4, 6 and 8 mg/ml), equal volumes of silver nitrate (1 mM) solution was added and the solution was allowed to react at 45 ºC for 3 hours with continuous stirring on magnetic stirrer. The transformation of the color of the solution was observed during the incubation of process within 10–15 min. The Ag nanoparticles solution was dried and centrifuged using methanol at 10,000 rpm for 10 min to remove the debris. Periodic sampling after 5, 10, 15, 20 and 25 min was carried out in order to monitor the color change as well as absorbance for the formation of silver nanoparticles. The color changes are shown in inset image of Figure 1C as indication of the formation of AgNPs from the Sea Buckthorn seed extract (a, b, c, d, and e represents color change of samples after 5, 10, 15, 20 and 25 min of incubation). The silver nanoparticles with 5% w/v were taken for further studies. Figure 1A shows the morphology of the silver nanoparticles using transmission electron microscopy (TEM).
1.2 Synthesis of zinc oxide nanoparticles:
[0081] ZnO nanoparticles were prepared by precipitation method as follows: Briefly, 1.5 M of KOH was prepared by dissolving 8.41g of KOH in 100 mL deionized water in a beaker. Then, 14.87g of ZnNO3 was dissolved in 100 ml distilled water (0.5 M), transferred to the beaker containing the KOH solution, and magnetically stirred for 1 h at 45°C. After 1h, white precipitates of ZnO NPs were obtained, centrifuged at 4000 rpm, and washed thrice with distilled water. Then the precipitates were collected and dried in hot air oven for 4 h. The zinc oxide nanoparticles with 5% w/v were taken for further studies. Figure 1B shows the morphology of the zinc oxide nanoparticles using transmission electron microscopy (TEM)
[0082] Figure 1C shows UV spectroscopy of the synthesized zinc nanoparticles and silver nanoparticles after 0, 3, and 6 months of storage conditions as an indicator of their stability (Temperature 45 °C; and 75% relative humidity conditions).
Example 2: Synthesis of topical nanocomposite gel formulation
[0083] For the preparation of the Carbopol® 934 gel system of varying concentrations, i.e. 0.5% w/w to 3%w/w, aqueous solution of Carbopol®934 was prepared by dissolving the required amount of Carbopol®934 in double distilled water with continuous stirring until completely dissolved. The above gel was then mixed with equal quantities of silver nanoparticles (5%w/v) and zinc oxide nanoparticles (5%w/v) of the formulation with continuous stirring for 24h for complete mixing to give the gel formulation (refer Table 1). The prepared gel was further neutralized using triethanolamine and allowed to stand overnight to remove entrapped air.
Table 1: Composition of nanocomposite gel
S. No. Formulation Code Carbopol®934
(%w/v) Silver Nanoparticles (5%w/v) Zinc oxide Nanoparticles (5%w/v) Humic acid
(%w/v)
1 F0 NA 5.0 5.0 5.0
2 F1 0.5 5.0 5.0 4.5
3 F2 1.0 5.0 5.0 4.0
4 F3 1.5 5.0 5.0 3.5
5 F4 2.0 5.0 5.0 3.0
6 F5 3.0 5.0 5.0 2.0

[0084] Nanocomposite gel of S.No. F6 of Table No. 1 was taken for further studies.
2.1 Scanning Electron Microscopy (SEM):
[0085] Figure 1D provides the scanning electron microscopic images of the nanocomposite gel.
2.2 % Drug Loading Efficiency:
[0086] The nanocomposite gel comprising Ag NPs and ZnO NPs, was suspended in 5 ml of ethanol and the mixture was sonicated for 5 min to allow complete solubilization of particles in ethanol. The resulting solution was then filtered through a 0.22-µm filter and assayed spectrophotometrically at 430 nm. The percentage drug loading efficiency (% DLE) of the gel was determined using eq. 1 and was noted to be 89.2% for silver nanoparticles and 87.6% for zinc oxide nanoparticles.
% drug loading efficiency = eq.1
2.3 Zeta Potential:
[0087] Zeta potential of the nanocomposite gel was obtained using Nanotrac Wave Zetasizer (Microtrac, USA). Samples were diluted with de-ionized water before measurement. Zeta potential of the samples was measured as an indicator of the physical stability within the media. It was found to be +6.2 mV which indicates good stability of the formulation. (A zeta potential in the range of ±10 mV is considered a stable formulation).
2.4 % Drug release studies:
[0088] The release profile of silver ions and zinc ions from the nanocomposite gel was studied at phosphate buffer pH 6.8. Approximately 5g of the formulation was incubated in 5 ml of phosphate buffer pH 6.8 at 37± 0.5ºC at 75 rpm. After predetermined time interval of 0, 1, 2, 4, 6, 10, 12, 14, 16, 20, 24, 28 and 30 h, 3 ml of sample was withdrawn and replaced by 3ml of fresh media. Optical density of each sample was measured using UV spectrophotometer at 435 nm for silver ions and 377 nm for zinc ions. Control experiments to determine the release behavior of the free silver from silver nanoparticles solution and zinc ions from zinc nanoparticles solution was also performed in comparison to the formulation of the present disclosure. Table 2 provides the percentage drug release over time from the three formulations. Figure 2 provides the percentage drug release from the formulation over time. It shows that the drug release from the formulation is a sustained release of drug.
Table 2: Percentage drug release study in phosphate buffer
Time(h) % Drug release from silver nanoparticles % Drug release from zinc oxide nanoparticles % Drug release from the Formulation (ZnO NPs) % Drug release from the formulation (Ag NPs)
0 0 0 0 0
1 16.4 14.8 6.4 4.8
2 29.5 25.9 9.5 5.9
4 32.1 32.6 12.1 12.6
6 53.8 47.9 13.8 17.9
8 68.1 69.6 18.1 19.6
10 85.5 88.1 25.5 28.1
12 40.3 39.6
14 48.5 48.2
16 51.5 56.2
18 66.3 61.2
20 78.9 69.8
24 89.2 84.3

[0089] 2.5 Zone of inhibition:
[0090] The zone of inhibition was determined using modified agar well diffusion assay. Under aseptic condition inside the bio safety chamber, 20 ml of nutrient agar was dispensed into the pre-sterilized glass Petri plates. Once the media solidifies it was then inoculated with micro-organism suspended nutrient broth. The media was punched with 6 mm diameter hole and filled with DMSO (Dimethyl sulfoxide) as negative control (C), silver nanoparticles (AgNPs) as (A), ZnNPs as (B), nanocomposite gels containing AgNPs and ZnNPs as (D), and positive control Ciprofloxacin hydrochloride as (E). Finally, the petriplates were sealed using paraffin films and kept at 37 °C for overnight. The diameter of zone of inhibition was measured by the clear area which was devoid of growth of microbes. The results of the zone of inhibition tests are provided in Table 3.
Table 3: Zone of inhibition Studies
S. No. Sample E. coli (Gram negative; mm) S. aureus (Gram positive; mm)
1 Silver nanoparticles 3.2 2.9
2 Zinc nanoparticles 2.6 2.4
3 Present formulation 7.2 6.8

[0091] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein merely for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention and should not be construed so as to limit the scope of the invention or the appended claims in any way.

ADVANTAGES OF THE PRESENT INVENTION
[0092] The present disclosure provides a formulation that enhances the drug release and permeability of silver and zinc nanoparticles for topical applications.
[0093] The present disclosure provides a formulation that gives synergistic anti-microbial activity and stability to the nanoparticles.

We Claims:

1. A topical nanocomposite gel formulation for anti-microbial activity comprising: (a) silver nanoparticles, (b) zinc-oxide nanoparticles, (c) humic acid, and (d) a gelling agent.

2. The formulation as claimed in claim 1, wherein the silver nanoparticles are synthesized from a silver salt employing Sea Buckthorn seed extract as a reducing agent.

3. The formulation as claimed in claim 1, wherein the formulation comprises silver nanoparticles in a range of 25% w/w to 40% w/w of the formulation.

4. The formulation as claimed in claim 1, wherein the zinc oxide nanoparticles are synthesized by precipitation of a zinc salt.

5. The formulation as claimed in claim 1, wherein the formulation comprises zinc oxide nanoparticles in a range of 25% w/w to 40% w/w of the formulation.

6. The formulation as claimed in claim 1, wherein the formulation comprises humic acid in a range of 2% w/w to 10% w/w of the formulation.

7. The formulation as claimed in claim 1, wherein the gelling agent is selected from a polyacrylic acid or acrylic acid.

8. The formulation as claimed in claim 1, wherein the formulation comprises the gelling agent in a range of 0.5% w/w to 10% w/w of the formulation.

9. A process of preparing a topical nanocomposite gel formulation for anti-microbial activity, wherein the process comprises the steps of: (a) reducing a silver salt with Sea Buckthorn extract to give silver nanoparticles; (b) precipitating a zinc salt in potassium hydroxide to give zinc oxide nanoparticles; (c) mixing equal volumes of the silver nanoparticles and zinc oxide nanoparticles with humic acid; (d) adding a gelling agent to the mixture of step (c) with continuous stirring to give a gel; and (e)neutralizing the gel with triethanolamine then keeping the gel undisturbed to expel trapped air to give the topical nanocomposite gel formulation.