Formulation and evaluation of a nanocomposite plant stem cell spray for wound healing

Abstract

A novel nanocomposite spray formulation developed for advanced wound healing by combining the therapeutic potential of plant stem cell extract with innovative polymeric film-forming technology, the novel nanocomposite spray formulation comprising the following: at least one plant stem cell extract, selected from Centella asiatica stem cell extract and Curcuma longa stem cell extract; and a polymeric film-forming matrix comprising ethylcellulose and Eudragit, wherein the formulation, when applied to a wound surface, forms a thin, transparent and adhesive film, thereby promoting accelerated wound healing.

Description

[0001] Maintaining a moist environment, preventing microbial infections, reducing inflammation, and promoting rapid tissue regeneration are key objectives. Conventional wound care methods include topical ointments, creams, gels, dressings, and bandages containing antibiotics, antiseptics, or growth promoters. While widely used, these methods often have limitations, such as poor drug penetration, frequent reapplication, uneven drug distribution, delayed healing, and patient discomfort.

[0002] Spray-based wound care formulations have attracted attention due to their touchless application, ease of use, uniform coverage of irregular wound surfaces, and reduced risk of secondary infection. Existing wound sprays typically contain antiseptics, polymers, or film-forming agents; however, many lack the ability for sustained drug release and sufficient bioactivity to actively accelerate tissue regeneration.

[0003] Nanotechnology is increasingly being explored for wound healing applications due to the unique physicochemical properties of nanoparticles, such as high specific surface area, improved bioavailability, controlled drug release, and enhanced interaction with biological tissue. Nanoparticles, including metallic nanoparticles (such as silver, zinc oxide, and copper), polymeric nanoparticles, lipid-based nanoparticles, and biopolymer-based nanomaterials, have been incorporated into wound dressings, hydrogels, and topical formulations. These nanomaterials exhibit antimicrobial, anti-inflammatory, and antioxidant properties that are beneficial for wound healing. However, nanoparticle aggregation, stability issues, and limited retention at the wound site remain challenges.

[0004] Nanocomposites, which combine nanoparticles with polymeric or biopolymeric matrices, have been developed to overcome the limitations of individual nanomaterials. Nanocomposite systems offer improved mechanical stability, controlled drug release, enhanced antimicrobial activity, and better biocompatibility. Previous research has described nanocomposite films, scaffolds, hydrogels, and membranes for wound care applications. However, many of these systems require direct contact with the wound surface, which can cause discomfort during application or removal and may not be suitable for large or sensitive wounds.

[0005] Existing spray formulations with nanomaterials are limited in their ability to maintain long-term adhesion, sustained release, and optimal therapeutic efficacy at the wound site. Furthermore, challenges such as nozzle clogging, uneven spray distribution, nanoparticle sedimentation, and formulation stability have restricted the widespread adoption of nanocomposite-based spray systems.

[0006] Accordingly, there remains a need in the field of technology for an improved wound healing formulation that combines the advantages of nanocomposite materials with a spray-based delivery system. Such a system should offer uniform coverage, improved antimicrobial protection, controlled release of active ingredients, enhanced wound healing efficacy, and user-friendly application, while ensuring the stability and biocompatibility of the formulation. SUMMARY

[0007] In light of the foregoing, one embodiment herein provides a novel nanocomposite spray formulation designed for advanced wound healing by combining the therapeutic potential of plant stem cell extract with innovative polymeric film-forming technology. The novel nanocomposite spray formulation comprises (i) at least one plant stem cell extract selected from Centella asiatica stem cell extract and Curcuma longa stem cell extract; and (ii) a polymeric film-forming matrix comprising ethylcellulose and Eudragit, wherein the formulation, when applied to a wound surface, forms a thin, transparent, and adhesive film, thereby promoting accelerated wound healing.

[0008] In some respects, the stem cell extract from Centella asiatica stimulates the proliferation of fibroblasts, angiogenesis and collagen remodeling at the wound site.

[0009] In some aspects, Curcuma longa stem cell extract exhibits anti-inflammatory, antioxidant and regenerative properties.

[0010] In some respects, the combination of Centella asiatica stem cell extract and Curcuma longa stem cell extract offers a synergistic therapeutic effect for improved tissue repair and a reduction in oxidative stress.

[0011] In some aspects, the stem cell extracts are incorporated into a nanocomposite matrix to improve stability, skin penetration, and the controlled release of bioactive ingredients.

[0012] In some respects, the nanocomposite matrix prevents the aggregation and degradation of the bioactive components during storage and use.

[0013] In some respects, ethylcellulose gives the formed film integrity, hydrophobic balance and mechanical strength.

[0014] In some respects, Eudragit improves adhesion to the wound surface, modulates permeability and ensures a sustained release of the bioactive components.

[0015] In some respects, ethylcellulose and Eudragit act as complementary film-forming agents to create a semi-open and breathable wound dressing.

[0016] In some respects, the sprayed formulation forms a semi-open, breathable and microbially resistant film over the wound surface.

[0017] In some aspects, the formed film adapts to irregular wound geometries without tearing or peeling off.

[0018] In a certain sense, the film immobilizes the active ingredients immediately after application to the wound site, thus reducing the risk of contamination.

[0019] In some aspects, the formulation improves wound closure rate, re-epithelialization and collagen deposition compared to conventional wound dressings.

[0020] In some aspects, the formulation is non-irritating and suitable for topical application on human skin.

[0021] In some respects, the formulation makes conventional bandages or secondary dressings unnecessary.

[0022] In some respects, the formulation is suitable for the treatment of acute wounds, chronic wounds, surgical wounds, burns, ulcers and diabetic wounds.

[0023] These and other aspects of the embodiments described herein can be better understood and comprehended when considered in conjunction with the following description and the accompanying drawings. It is understood, however, that the following descriptions, although they specify preferred embodiments and numerous details thereof, serve only for illustration and not as limitations. Many changes and modifications can be made to the embodiments described herein without departing from their spirit, and the embodiments described herein encompass all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The embodiments described herein will be better understood with reference to the following detailed description and the drawings, in which: Fig. shows a formulation composition and a formula for preparation according to an embodiment of the present invention; and Fig. illustrates an active ingredient release mechanism of the formulation according to an embodiment of the present invention; and Fig. illustrates a comparative anti-inflammatory effect of the formulation according to an embodiment of the present invention. DETAILED DESCRIPTION OF PREFERRED EXECUTION FORMS

[0025] The embodiments described herein, along with their various features and advantageous details, are further explained with reference to the non-limiting embodiments illustrated in the accompanying drawings and described in detail in the following description. Descriptions of known components and processing techniques are omitted to avoid unnecessarily obscuring the embodiments described herein. The examples used herein serve only to facilitate understanding of the possibilities for implementing the embodiments described herein and to enable those skilled in the art to implement them. Accordingly, the examples should not be interpreted as limiting the scope of the embodiments described herein.

[0026] Fig. Figure 1 shows a formulation composition and a formula for preparation according to an embodiment of the present invention. A preparation of a stem cell extract-loaded polymeric spray formulation comprises (i) the preparation of a polymeric solution comprising Eudragit and ethylcellulose, which were precisely weighed and dissolved in ethyl acetate under continuous stirring. The process was continued until a clear, transparent polymeric solution was obtained, indicating complete dissolution of the polymers. This ensured a stable base matrix for further formulation steps.

[0027] (ii) Incorporation of plasticizer and stabilizer: Citric acid (as a stabilizer) and PEG 6000 (as a plasticizer) were gradually added to the polymer solution. The components were thoroughly mixed until completely dissolved to ensure improved flexibility of the polymer film and stabilization of the dispersion system. The presence of PEG 6000 also contributed to improved solubility and biocompatibility of the final formulation.

[0028] (iii) Homogenization of the polymer mixture: The prepared solution was subjected to high-speed homogenization for 15 minutes at 15,000 rpm using a homogenizer. This step facilitated the uniform distribution of the excipients within the polymer matrix, reduced the particle size, and ensured the consistency of the formulation. The intensive homogenization process also contributed to the formation of a nanoformulation, thereby improving the stability and bioavailability of the final product. As a result of this step, which is crucial prior to biological incorporation, a homogeneous and stable dispersion was achieved.

[0029] (iv) Incorporation of stem cell extracts Centella asiatica and Curcuma longa: The stem cell extracts were carefully incorporated into the homogenized polymer dispersion. This step required controlled handling to prevent degradation of the bioactive substances while ensuring maximum incorporation efficiency.

[0030] (v) Gentle mixing and uniform distribution: The mixture was gently stirred to distribute the stem cell extract evenly throughout the polymer dispersion. In contrast to homogenization, gentle mixing was used in this phase to minimize shear stress and avoid compromising cell viability, while simultaneously achieving uniform cell distribution throughout the formulation.

[0031] The evaluation of nanoparticles includes various characterization parameters such as particle size, PDI (polydispersity index) and zeta potential, which were determined for the production of the nanoparticles using Malvern instruments.

[0032] Droplet size and polydispersity index: One drop of stabilized nanoemulsion is diluted with 10 ml of water in this Litesizer 500. Using a dilute dynamic light scattering technique at 250 °C and a measurement angle of 1750 °C, the particle size and polydispersity index are measured from this 3 / 4 volume in a polystyrene cuvette.

[0033] Zeta potential: The zeta potential for investigating the velocity of particles in an electric field was analyzed using the Litesizer 500 with electrophoretic light scattering at 250 °C. Dilute samples were stored in the transparent zeta cuvette (omega cuvette) with closed tips, which is used to determine the zeta potential.

[0034] The evaluation of the formulation and the patch includes the following: (i) Digital pH meters were used to measure the pH of the nanocomposite spray formulation at 25 °C. 100 ml of distilled water was added, the mixture was set aside for 2 hours, and then approximately one gram of the sample was taken. The pH of the formulation was measured three times, and the average value was calculated. (ii) The viscosity of the sample was determined using an Ostwald viscometer, which is based on the principle of measuring the time it takes for a liquid to flow under gravity through a capillary tube. The viscometer was first thoroughly cleaned and then placed vertically in a water bath at a constant temperature of 25 ± 0.1 °C to ensure a consistent measurement. Distilled water was used as the reference liquid and was added to the viscometer up to the mark above the upper flask.Using a suction device, the fluid was drawn over the upper time mark and then allowed to flow freely downwards. The time it took for the meniscus to flow between the two marked points was measured with a stopwatch. This process was repeated three times, and the average flow time (tref) was calculated. The same procedure was applied to the test sample after the viscometer had been rinsed with a small amount of it. The average flow time (tsample) was recorded.

[0035] The viscosity of the sample was calculated relative to water using the following formula: η2=η1×(t1t2)×(ρ1ρ2)

[0036] where η2 is the viscosity of the sample, η1 is the viscosity of water, t1 is the flow time of water, t2 is the flow time of the sample, ρ1 is the density of water and ρ2 is the density of the sample.

[0037] (iii) The thickness of the prepared patch was measured at three different locations using a digital micrometer. The average thickness was calculated to assess the uniformity of the patch.

[0038] (iv) The spray pattern of the formulation was evaluated by spraying it onto a clean white paper placed at a fixed distance from the spray nozzle. The resulting pattern was visually examined for shape, spread, and uniformity.

[0039] (v) The spray angle was determined by measuring the diameter of the spray zone on the paper at a fixed distance (10-15 cm) from the nozzle. The spray angle (θ) was calculated using the following formula: θ=2×tan−1(D / 2L) where θ is the spray angle, D is the spray width or spray diameter, and L is the distance between the nozzle and the paper.

[0040] The surface morphology of the patch was examined using scanning electron microscopy (SEM). The samples were cut into small sections, mounted on an aluminum stub, sputter-coated with gold to improve conductivity, and analyzed under the SEM at suitable magnifications.

[0041] Fig. Figure 1 illustrates a drug release mechanism of the formulation according to an embodiment of the present invention. In vitro permeation studies include in vitro drug permeation studies performed using a cellophane membrane mounted on a Franz diffusion cell. The membrane, tailored to the size and shape of the diffusion cell, separated the donor and acceptor compartments. The receptor compartment was filled with a phosphate buffer solution at a pH of 6.8 to simulate physiological conditions. The method involved placing a 2.4 cm diameter cellophane membrane between the donor and receptor compartments. A volume of 0.1 ml of the stem cell extract was introduced into the receptor compartment in gel form.To maintain a constant temperature similar to that of the skin, 32 °C water was circulated through the outer shell of the diffusion cell using a water pump. A stirrer was set to 100 rpm to ensure uniform distribution. Samples were taken from the receptor chamber at regular intervals over a period of up to one hour. Each sample was diluted to 10 ml in a volumetric flask, and the absorbance was measured using a UV spectrophotometer at 228 nm and 232 nm to identify two compounds. The drug concentration was determined based on the absorbance values ​​and the calibration curve equation. Furthermore, the release kinetics of the optimized formulation were evaluated using various mathematical models, including zero-order, first-order, Higuchi, and Korsmeyer-Peppas models.

[0042] Fig.Figure 1 illustrates a comparative anti-inflammatory effect of the formulation according to an embodiment of the present invention. The anti-inflammatory test comprises HaCaT keratinocyte cell lines: The anti-inflammatory effect of the optimized formulation was compared with the commercially available preparation using HaCaT keratinocyte cell lines. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin, under standard culture conditions (37°C, 5% CO2, 95% relative humidity).

[0043] In some embodiments, HaCaT cells were experimentally placed in 24-well plates with a density of 2 × 10 5Cells were seeded into a well and allowed to adhere for 24 hours. The inflammatory response was induced by treatment with lipopolysaccharide (LPS, 1 µg / ml) for 6 hours. After induction, the medium was replaced with fresh DMEM containing different concentrations (25, 50, 75, and 100 µg / ml) of the optimized formulation and the commercially available sample. LPS-stimulated cells without further treatment served as positive controls.

[0044] In some embodiments, the percentage inhibition of inflammatory markers was calculated in comparison to LPS-treated controls to determine the comparative anti-inflammatory efficacy of the optimized formulation and the marketed product.

[0045] In some embodiments using RAW 264.7 macrophage cell lines: RAW 264.7 macrophage cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics at 37 °C and 5% CO2. For cytotoxicity screening, the cells were saturated at a density of 2.5 × 10 4Cells / wells were seeded and treated with different concentrations (25, 50, 75, and 100 µg / ml) of the optimized formulation and the commercially available sample. Cell viability was assessed using an MTT assay, with absorbance measured at 520 nm. Anti-inflammatory activity was evaluated by measuring nitric oxide (NO) release after stimulation with 1 µg / ml LPS. Nitrite concentrations in the culture supernatant were quantified at 540 nm using Griess reagent with sodium nitrite as the standard. The percentage inhibition of inflammatory markers compared to LPS-treated controls was calculated to determine the comparative anti-inflammatory efficacy of the optimized formulation and the commercially available sample.

[0046] In some embodiments, the keratinocyte scratch test includes evaluating the wound-healing potential of the film-forming spray formulation using a keratinocyte scratch test. Keratinocyte cells were cultured under standard conditions until they formed a uniform monolayer suitable for migration studies. Subsequently, a linear gap was introduced into the monolayer to simulate a wound area, after which loose cell debris was removed to allow a clear view of the scratched area. The test formulation was applied such that the resulting film gently came into contact with the cell layer without disturbing the scratched area, while maintaining appropriate controls, including untreated cells and samples containing only the vehicle.After treatment, fresh culture medium was replenished, and the scratched areas were documented under a microscope at baseline and at predetermined intervals to monitor the extent of cell migration. Wound healing progress was quantified by measuring changes in the scratched area over time using image analysis software, and the results were compared between the treatment groups to evaluate the effect of the film-forming spray on keratinocyte migration.

[0047] In some embodiments, the forced degradation studies included an active ingredient content stability study conducted to evaluate the effects of temperature and humidity on formulation integrity over a 24-hour period. Samples were stored at 25°C / 60% RH, 30°C / 65% RH, 40°C / 75% RH, and 60°C (thermal stress). Aliquots were taken at predetermined intervals (0, 2, 4, 6, and 24 hours) and analyzed by UV spectrophotometry to determine the percentage of remaining active ingredient. All samples were diluted accordingly and scanned at the λmax of the active ingredient. The measured absorbance values ​​were used to calculate the percentage of active ingredient, which was then compared to the initial value of 100% to evaluate degradation under the respective conditions.

[0048] In some embodiments, the stability studies included an accelerated stability study conducted for 45 days at 25 °C ± 2 °C / 60% RH ± 5%. Samples were evaluated on days 0, 15, 30, and 45 for pH, viscosity, organoleptic properties, and drug content. pH was measured using a calibrated digital pH meter, and viscosity was determined using a Brookfield viscometer. Organoleptic properties were assessed visually, while drug content was estimated by UV-Vis spectrophotometry. The percentage change for pH and drug content relative to baseline values ​​was calculated to monitor changes during storage.

[0049] Development of a stem cell extract-loaded nanocomposite spray formulation for wound healing, combining stem cell extracts from Centella asiatica and Curcuma longa with a polymer matrix of Eudragit and ethylcellulose. Preformulation studies, including FTIR, solubility, and compatibility assessments, confirmed the stability and suitability of the bioactive substances and polymers. The formulation exhibited a suitable nanoparticle size (~419 nm), moderate polydispersity, and an acceptable zeta potential (-19.2 mV), ensuring sufficient physical stability. Physicochemical evaluations revealed a skin-friendly pH (4.7), optimal viscosity (3.83 cP), a uniform spray pattern and angle, and a suitable patch thickness (0.004 cm). 2 ). The SEM analysis revealed a smooth, porous surface that promotes cell adhesion and tissue regeneration.

[0050] In vitro studies demonstrated controlled drug release, efficient permeation, and significant anti-inflammatory activity (up to 86.45% inhibition). Overall, the developed spray forms a thin, semi-occlusive film that accelerates wound healing, promotes tissue regeneration, and provides a safe, effective, and dressing-free wound care system suitable for further preclinical and clinical development.

Claims

[1] A novel nanocomposite spray formulation developed for advanced wound healing by combining the therapeutic potential of plant stem cell extract with innovative polymeric film-forming technology, wherein the novel nanocomposite spray formulation comprises: at least one plant stem cell extract, selected from Centella asiatica stem cell extract and Curcuma longa stem cell extract; and a polymeric film-forming matrix comprising ethylcellulose and Eudragit, wherein the formulation, when applied to a wound surface, forms a thin, transparent and adhesive film, thereby promoting accelerated wound healing. [2] Nanocomposite spray formulation according to claim 1, wherein the Centella asiatica stem cell extract stimulates fibroblast proliferation, angiogenesis and collagen remodeling at the wound site. [3] Nanocomposite spray formulation according to claim 1, wherein the Curcuma longa stem cell extract has anti-inflammatory, antioxidant and regenerative properties. [4] Nanocomposite spray formulation according to claim 1, wherein the combination of Centella asiatica stem cell extract and Curcuma longa stem cell extract provides a synergistic therapeutic effect for improved tissue repair and a reduction of oxidative stress. [5] Nanocomposite spray formulation according to claim 1, wherein the stem cell extracts are incorporated into a nanocomposite matrix to improve stability, skin penetration and controlled release of bioactive components. [6] Nanocomposite spray formulation according to claim 1, wherein the nanocomposite matrix prevents the aggregation and degradation of the bioactive components during storage and application. [7] Nanocomposite spray formulation according to claim 1, wherein ethylcellulose imparts film integrity, hydrophobic balance and mechanical strength to the formed layer. [8] Nanocomposite spray formulation according to claim 1, wherein Eudragit improves adhesion to the wound surface, modulates permeability and maintains the release of the bioactive components. [9] Nanocomposite spray formulation according to claim 1, wherein the ethylcellulose and Eudragit act as complementary film-forming agents to create a semi-occlusive and breathable wound covering. [10] Nanocomposite spray formulation according to claim 1, wherein the sprayed formulation forms a semi-permeable, breathable and microbially resistant film over the wound surface. [11] Nanocomposite spray formulation according to claim 1, wherein the formed film adapts to irregular wound geometries without cracking or detachment. [12] Nanocomposite spray formulation according to claim 1, wherein the film immobilizes the active ingredients immediately after application to the wound site, thereby reducing the risk of contamination. [13] Nanocomposite spray formulation according to claim 1, wherein the formulation improves the wound closure rate, re-epithelialization and collagen deposition compared to conventional wound dressings. [14] Nanocomposite spray formulation according to claim 1, wherein the formulation is non-irritating and suitable for topical application on human skin. [15] Nanocomposite spray formulation according to claim 1, wherein the formulation eliminates the need for conventional bandages or secondary dressings. [16] Nanocomposite spray formulation according to claim 1, wherein the formulation is suitable for the treatment of acute wounds, chronic wounds, surgical wounds, burns, ulcers and diabetic wounds.

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