Formulation Development, Physicochemical Characterization, and In Vitro Evaluation of A Silver Nanoparticle-Loaded Phytoconstituent-Based Nanogel System for Enhanced Topical Drug Delivery

Topical drug delivery systems have emerged as an integral component of contemporary pharmaceutics because of their capacity to deliver drugs directly to the site of action, thereby achieving localized therapeutic effects while minimizing systemic exposure and associated adverse effects. [1] In addition to improving therapeutic precision, topical systems enhance patient compliance due to their non-invasive nature, ease of administration, and improved acceptability. Among the various topical dosage forms, nanogels—hydrogel-based three-dimensional polymeric networks incorporating nanostructured entities—have gained considerable attention as advanced drug delivery platforms. Their high-water content, soft consistency, biocompatibility, and structural flexibility enable efficient encapsulation of diverse bioactive agents. Furthermore, the integration of nanomaterials within gel matrices significantly enhances drug permeation across the stratum corneum, improves stability, and facilitates controlled or sustained release profiles. [2] Silver nanoparticles (AgNPs) represent one of the most extensively investigated nanomaterials for biomedical applications due to their remarkable broad-spectrum antimicrobial, anti-inflammatory, and wound healing properties. The enhanced biological activity of AgNPs is primarily attributed to their nanoscale dimensions, increased surface-area-to-volume ratio, and high surface reactivity, which enable effective interaction with microbial cell membranes and intracellular components. AgNPs have demonstrated potent activity against a wide range of pathogenic microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and certain viruses. However, the biological performance and safety of silver nanoparticles are strongly influenced by the method of synthesis, as particle size, morphology, surface charge, and stability depend on the preparation technique. In this context, green synthesis approaches utilizing plant extracts have gained prominence as environmentally benign, cost-effective, and non-toxic alternatives to conventional chemical and physical methods of nanoparticle production. [3,4,5]

Fig. 1: Lantana camara leaves

Lantana camara is a well-recognized medicinal plant widely distributed in tropical and subtropical regions. Traditionally, it has been employed in ethnomedicine for its antimicrobial, antioxidant, wound healing, and anti-inflammatory properties. The leaves of this plant are rich in diverse bioactive phytoconstituents, including flavonoids, terpenoids, phenolic compounds, and essential oils. These secondary metabolites not only contribute to its intrinsic therapeutic efficacy but also function as natural reducing and stabilizing agents during the green synthesis of silver nanoparticles. Consequently, the utilization of Lantana camara leaf extract for nanoparticle fabrication offers a synergistic advantage by combining the pharmacological activity of phytoconstituents with the potent antimicrobial effects of nanosilver within a single delivery system. The development of a nanogel incorporating silver nanoparticles synthesized from Lantana camara leaf extract is therefore conceptualized as a strategy to integrate traditional herbal therapeutics with modern nanotechnological advancements. Such a formulation is anticipated to enhance dermal permeation, provide sustained and controlled drug release, and exhibit pronounced antimicrobial activity, making it particularly suitable for the management of skin infections, inflammatory dermatoses, and wound conditions. Additionally, the hydrogel base serves as an appropriate carrier system by ensuring uniform drug distribution, ease of topical application, aesthetic acceptability, and improved patient comfort. [6,7,8] The present study is thus directed toward the systematic formulation development and comprehensive evaluation of a phytoconstituent-loaded silver nanoparticle nanogel. The research encompasses green synthesis of silver nanoparticles using Lantana camara leaf extract, physicochemical and morphological characterization of the synthesized nanoparticles, incorporation into a suitable gel matrix, and detailed evaluation of the final formulation with respect to physicochemical parameters, in vitro drug release behavior, antimicrobial efficacy, antioxidant potential, and stability under specified storage conditions. The successful execution of this work may establish a scientifically validated, natural, and efficacious topical therapeutic system that effectively bridges the principles of traditional herbal medicine with the innovations of nanotechnology.

MATERIALS AND METHODS:

MATERIALS:

The study utilized authenticated Lantana camara leaves, collected and verified by the Department of Pharmacognosy. Analytical-grade solvents including ethanol, methanol, acetone, and chloroform were sourced from Merck, SRL, and HiMedia for extraction. Carbopol 940 (Lubrizol) served as the gelling agent, while triethanolamine (Loba Chemie) was used to adjust pH. Silver nitrate (Merck) was employed for nanoparticle synthesis. Antioxidant assays used DPPH (Sigma-Aldrich) and ascorbic acid (SRL). Antimicrobial testing involved nutrient agar and ciprofloxacin discs (HiMedia), with DMSO as the solvent. Dialysis membranes (MWCO 12,000–14,000) from HiMedia or Sigma-Aldrich were used for diffusion studies. All materials met research-grade specifications to ensure reliability of results.

Collection and authentication of plant materials:

The leaves of Lantana camara were carefully collected from a biodiverse region, ensuring sustainable harvesting at the optimal time for phytochemical content from Pune region of Maharashtra.

Morphological Study

The morphological evaluation of Lantana camara leaves was conducted to ensure accurate identification and authentication of the plant material. Using standard pharmacognostic techniques, the macroscopic and organoleptic characteristics such as color, odor, texture, and shape were carefully observed. These sensory attributes serve as critical indicators in the preliminary assessment and standardization of crude herbal materials. Establishing these morphological features provides a solid basis for consistent quality control in future formulation and phytopharmacological studies. [9,10,11,12]

Physicochemical Parameters of Lantana Camara

The physicochemical profiling of Lantana camara leaves was carried out following protocols from official pharmacopoeias to determine purity, stability, and quality. Parameters such as moisture content, ash values (total, acid-insoluble, and water-soluble), extractive values in different solvents, and pH were assessed to ensure the suitability of the plant for medicinal use. [13,14,15]

Preparation of Lantana Camara Leaf Extracts

Leaf extracts of Lantana camara were prepared using the Soxhlet extraction technique, a reliable method for isolating phytochemicals. Dried, powdered leaves were placed in the thimble of a Soxhlet apparatus, and various solvents including ethanol, methanol, chloroform, acetone, and water were used sequentially for exhaustive extraction. The solvent vapors condensed and repeatedly washed the plant material, allowing efficient extraction of both polar and non-polar bioactive compounds. After completion, the solvent was removed under reduced pressure using a rotary evaporator, yielding concentrated plant extracts. This method ensured thorough recovery of phytoconstituents with minimal thermal degradation, making the extracts ideal for further formulation into nanogels. [16-22]

Fig. 2: Extraction of Lantana camara Leaves powder by Soxhlet Apparatus

Phytochemical Investigation Methods for Lantana camara Extract

The phytochemical investigation of Lantana camara involves a combination of qualitative and quantitative techniques aimed at identifying and quantifying the bioactive compounds in the plant. Each method detects specific phytochemicals, providing a comprehensive profile of the plant's constituents. [23-28]

Quantitative Evaluation of Bioactive Compounds in Lantana camara Extract

Quantitative analysis of Lantana camara extract is vital to identify and correlate its bioactive constituents with therapeutic properties like antimicrobial, anti-inflammatory, and wound healing activity. Various phytochemicals are evaluated using established methods. Total phenolic content (TPC) is measured by the Folin-Ciocalteu method and expressed as mg gallic acid equivalents/g, indicating antioxidant potential. Total flavonoid content (TFC) is determined by aluminum chloride colorimetry and reported as mg quercetin equivalents/g, showing bioactivity. Alkaloids are quantified via the Harborne method using solvent extraction and colorimetry, representing analgesic and antimalarial potential. Tannins are estimated using the Vanillin-HCl method and expressed as catechin equivalents, linked to astringency and wound healing. Saponins are evaluated through froth tests or spectrophotometry, known for antimicrobial and anti-inflammatory roles. Terpenoids are assessed via the Salkowski method using color intensity from chloroform and sulfuric acid, indicating antibacterial and antifungal effects. These evaluations support the pharmacological relevance of Lantana camara. [29-32]

Qualitative Analysis of Inorganic Elements

The ash of Lantana camara leaves was digested using nitric acid and hydrochloric acid in a 3:1 ratio and filtered. The resulting filtrate was tested for inorganic elements. Calcium was identified by the formation of a white calcium oxalate precipitate. Magnesium was detected by a white crystalline precipitate upon reaction with sodium phosphate. Sodium was confirmed by a yellow crystalline precipitate with uranyl magnesium acetate. Potassium showed a yellow precipitate with sodium cobalt nitrite. Iron presence was confirmed by dark blue coloration with potassium ferrocyanide. Sulphate formed a white precipitate with lead acetate, soluble in sodium hydroxide. Phosphate produced a yellow crystalline precipitate with ammonium molybdate upon heating. Chloride was indicated by a white precipitate with lead acetate, soluble in hot water. Nitrate was identified by a brown ring formed at the junction of layers after adding ferrous sulphate and sulfuric acid. [33,34]

Qualitative Analysis of Vitamins

Vitamin A was detected by the formation of a transient blue color after reacting the chloroform extract with antimony trichloride. Vitamin C gave a blue coloration after sequential addition of sodium nitroprusside, sodium hydroxide, and hydrochloric acid. Vitamin D was identified by a pinkish-red color upon reaction with antimony trichloride in chloroform extract. Vitamin E was confirmed by a bright red color developing on a white background, which gradually turned pink when ethanol extract was treated with ferric chloride and dipyridyl reagents. [35,36,37]

UV-Visible Spectroscopic Analysis

The ethanol extract of Lantana camara leaves and the synthesized silver nanoparticles were subjected to UV-Visible spectroscopic analysis using a Perkin Elmer spectrophotometer. The scanning was performed over a wavelength range of 200–1000 nm to detect characteristic peaks confirming nanoparticle formation. [38-42]

FTIR Analysis

Methanol extract of the leaves was analyzed using Fourier Transform Infrared (FTIR) spectroscopy to identify functional groups. Spectra were recorded within 400–4000 cm⁻¹. Repeated runs ensured peak confirmation, highlighting phytochemicals involved in nanoparticle synthesis and stabilization. [43-45]

Synthesis and Characterization of Silver Nanoparticles

For biosynthesis, 5 ml of Lantana camara leaf extract was mixed with 45 ml of 1 mM silver nitrate solution and incubated in the dark for 5 hours. A brown coloration indicated nanoparticle formation. The nanoparticles were purified via centrifugation and freeze-dried for further use. Characterization included UV-Vis and FTIR spectroscopy to confirm reduction and capping, and SEM analysis to examine particle morphology, revealing spherical nanoparticles between 15–36 nm. [46,47]

In-Vitro Antioxidant Activity

The antioxidant potential of both extract and silver nanoparticles was tested through several assays. DPPH radical scavenging activity measured absorbance at 517 nm, indicating free radical neutralization. Total antioxidant capacity was assessed using the molybdate method, showing color intensity at 695 nm. Superoxide scavenging and Fe²⁺ chelation assays measured absorbance at 560 nm and 562 nm respectively, confirming dose-dependent antioxidant behavior of both extract and nanoparticles. [48,49,50]

Nanogel Formulation and Evaluation

The nanogel was prepared by integrating green-synthesized silver nanoparticles into a Carbopol 940-based gel. The ethanolic extract was first obtained via Soxhlet extraction. Nanoparticles were synthesized by mixing this extract with silver nitrate solution, confirmed via UV-Vis. A gel base was prepared by dispersing Carbopol in water, followed by addition of moisturizers (glycerin, propylene glycol) and preservatives (methylparaben, propylparaben). The silver nanoparticles were then incorporated with gentle stirring, and the gel was neutralized using triethanolamine to achieve pH 6.0–6.5. The final formulation was stored in sterile containers for further testing. [51,52,53]

Table 1: Composition of Nanogel Formulations Containing Silver Nanoparticles of Lantana camara Extract

Evaluation Parameters of the Developed Nanogel Formulation

To establish the suitability, performance, and stability of the developed silver nanoparticle-loaded nanogel for topical application, a comprehensive evaluation protocol was implemented. The assessment encompassed physicochemical characterization, rheological behavior, drug release performance, antimicrobial efficacy, stability profiling, and statistical validation to ensure formulation reliability and reproducibility. (53-59)

Physical Appearance:

All prepared nanogel formulations were subjected to visual inspection for organoleptic characteristics, including color, clarity, homogeneity, consistency, presence of lumps, phase separation, and particulate contamination. A smooth, uniform, and lump-free gel with no visible phase separation was considered indicative of appropriate polymer hydration and successful incorporation of silver nanoparticles within the gel matrix.

pH Determination:

The pH of each formulation was measured by dispersing 1 g of nanogel in 10 mL of distilled water, followed by analysis using a calibrated digital pH meter. Maintaining a pH range between 5.5 and 6.5 was considered optimal to ensure compatibility with the physiological pH of the skin, thereby minimizing the risk of irritation and enhancing patient acceptability.

Viscosity Measurement:

Rheological analysis was performed at room temperature using a Brookfield viscometer. Viscosity is a critical parameter influencing spreadability, retention time, and drug release behavior. Appropriate viscosity ensures that the gel remains localized at the site of application without runoff, while still allowing convenient application.

Spreadability Assessment:

Spreadability was evaluated using the slip-and-drag method involving two glass slides. The ease with which the gel spread under a standardized applied weight was recorded. Adequate spreadability is essential for uniform application over the affected area and directly contributes to therapeutic efficiency and patient compliance.

Extrudability Test:

Extrudability was determined by measuring the force required to expel the gel from a collapsible aluminum tube under standardized conditions. Optimal extrudability ensures ease of dispensing, uniform dosing, and user convenience during practical application.

Drug Content Uniformity:

Drug content analysis was carried out by dissolving an accurately weighed quantity of nanogel in a suitable solvent such as ethanol or buffer solution, followed by filtration and spectrophotometric quantification. This test confirmed the uniform distribution and efficient incorporation of the phytoconstituent and silver nanoparticles within the gel matrix.

In Vitro Drug Release Study:

Drug release behavior was evaluated using Franz diffusion cells with either synthetic membranes or excised rat skin as the diffusion barrier. Samples were withdrawn at predetermined intervals and analyzed spectrophotometrically to determine cumulative drug release. The study provided insight into release kinetics, permeation characteristics, and sustained delivery performance of the formulation.

Antibacterial Activity:

The antimicrobial efficacy of the silver nanoparticle-based nanogel was assessed using the agar well diffusion method against Staphylococcus aureus and Escherichia coli. The diameter of the zone of inhibition was measured to evaluate antibacterial potency, reflecting the synergistic effect of silver nanoparticles and phytoconstituents present in the formulation.

Stability Studies:

Optimized formulations were subjected to stability testing under both room temperature conditions and accelerated conditions (40°C ± 2°C/75% ± 5% RH) for a duration of three months. Periodic evaluation of physical appearance, pH, viscosity, and drug content was performed to assess formulation integrity and predict shelf life.

Statistical Analysis:

All experimental studies were conducted in triplicate to ensure reproducibility. Results were expressed as mean ± standard deviation (SD). Statistical comparisons among formulation batches were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, with a significance level set at p < 0.05. Collectively, these evaluation parameters provided a systematic and scientific basis for confirming the quality, safety, efficacy, and stability of the developed nanogel formulation intended for topical therapeutic use.

RESULTS AND DISCUSSION:

Morphological and Organoleptic Evaluation of Lantana camara Leaves (Concise Paragraph)

The leaves of Lantana camara are ovate to oblong in shape with serrated margins, measuring approximately 3–8 cm in length and 2–5 cm in width. They are arranged oppositely on the stem and exhibit pinnate venation. The upper surface is coarse and hairy, while the lower side feels velvety. Fresh leaves are dark green, turning pale green upon drying. They emit a strong, pungent odor, especially when crushed, and possess a bitter, slightly astringent taste. These morphological and organoleptic characteristics are key for the identification and standardization of the plant material prior to further pharmacological studies.

Table 2: Morphological Evaluation of Lantana camera leaves

Table 3:  Organoleptical observation of Lantana camera leaves