A Method For Synthesizing Silver Nanoparticles Using Anisomeles Malabarica Plant Extract

Abstract

This invention presents a green synthesis method for preparing silver nanoparticles using Anisomeles malabarica leaf extract and their incorporation into liposomes for advanced drug delivery systems. The process involves collecting and authenticating Anisomeles malabarica, preparing a 1mM silver nitrate (AgNO₃) solution, and synthesizing silver nanoparticles through a bio-reduction reaction with the plant extract. Liposomes are prepared using the thin-film hydration method, utilizing soy phosphatidylcholine, cholesterol, and span 80 as lipid components, and the therapeutic agent Lapatinib as the drug. The silver nanoparticles are incorporated into the liposomes during the hydration phase at elevated temperatures, forming multilamellar vesicles (MLVs).This approach combines the antimicrobial and anticancer potential of silver nanoparticles with the targeted delivery capabilities of liposomes. The resulting nanocomposite system is highly suitable for biomedical applications, such as infection control and cancer therapy. The invention provides a sustainable and efficient platform for next-generation pharmaceutical formulations.

Specification

Description:The invention is related to a method for synthesizing silver nanoparticles using Anisomeles malabarica plant extract. Fig 1 illustrates the method for synthesizing silver nanoparticles. This invention centers on an eco-friendly approach to synthesizing silver nanoparticles (AgNPs) by utilizing Anisomeles malabarica leaf extract, which serves as a natural reducing and stabilizing agent. The process begins with the careful selection and authentication of the plant material to ensure scientific accuracy and reproducibility. The leaves are washed, dried,
and extracted using distilled water to prepare a bioactive-rich solution. This aqueous extract contains secondary metabolites like flavonoids, tannins, terpenoids, and phenolic compounds, which facilitate the reduction of silver ions (Ag⁺) from a silver nitrate (AgNO₃) solution into nanoparticles.
The reaction is initiated by mixing the extract with a 1mM AgNO₃ solution under controlled conditions, such as ambient temperature and pH. A visible color change in the solution, typically to a yellowish-brown hue, indicates the formation of silver nanoparticles. Analytical techniques such as UV-Vis spectroscopy (confirming the surface plasmon resonance peak), Transmission Electron Microscopy (TEM) (revealing nanoparticle morphology), and X-ray Diffraction (XRD) (confirming crystalline structure) validate the synthesis. The method is environmentally benign, eliminating hazardous chemicals and fostering a sustainable approach to nanomaterial production.
Fig 2 illustrates a process for preparing multilamellar vesicle (MLV) liposomes. This invention provides a robust and scalable method for preparing multilamellar vesicles (MLVs), commonly referred to as liposomes, via the thin-film hydration technique. The process begins with dissolving lipid components-such as soy phosphatidylcholine, cholesterol, and Span 80-in an organic solvent like chloroform or methanol. This lipid mixture is subjected to rotary evaporation under controlled conditions (typically 55°C) to remove the solvent, leaving behind a thin lipid film on the inner walls of the flask.
The lipid film is then hydrated with an aqueous solution at an elevated temperature (60°C), resulting in the self-assembly of liposomes. This process creates MLVs with distinct layers, which can encapsulate both hydrophilic and lipophilic therapeutic agents. The size and stability of the liposomes can be fine-tuned by modifying the lipid composition, hydration time, and temperature. The thin-film hydration technique is advantageous due to its simplicity, reproducibility, and ability to scale up for industrial applications. This method ensures efficient encapsulation, prolonged stability, and controlled release of encapsulated drugs, making it ideal for pharmaceutical applications.
Components of formulation prepared by using the emulsification method.

Fig 3 illustrates a method for incorporating silver nanoparticles into liposomes. The present invention allows a novel integration of silver nanoparticles into the lipid bilayers of liposomes to form a composite system with enhanced functionality. During the hydration phase of the thin-film preparation, 2M silver nanoparticles are added to the aqueous phase used to hydrate the lipid film. The nanoparticles interact with the lipids, embedding themselves within the bilayer or being encapsulated within the aqueous core, depending on their size and surface properties.
The resulting nanocomposite benefits from the synergistic properties of liposomes and silver nanoparticles. Liposomes act as protective carriers, preventing premature degradation of the nanoparticles and ensuring their targeted delivery. Silver nanoparticles, known for their broad-spectrum antimicrobial and anticancer activities, enhance the therapeutic potential of the formulation. The incorporation process ensures uniform distribution of nanoparticles and
lipids, achieving high encapsulation efficiency and stability. This innovative method creates a multi-functional system suitable for treating infections, cancer, and other medical conditions requiring targeted and sustained release of active agents.
Fig 4 illustrates a drug delivery system combining liposomes and silver nanoparticles.The invention introduces a dual-action drug delivery platform combining liposomal carriers and silver nanoparticles. The system leverages the drug-carrying capacity of liposomes and the antimicrobial and cytotoxic properties of silver nanoparticles. The liposomes encapsulate the chemotherapeutic agent Lapatinib, which is primarily used for treating cancers. Meanwhile, the incorporated silver nanoparticles contribute to the system's efficacy by providing additional antimicrobial and anticancer effects.
This delivery system addresses challenges such as drug resistance, low bioavailability, and systemic toxicity. Liposomes ensure targeted delivery, protecting the drug from degradation in the bloodstream and releasing it at the desired site of action. The presence of silver nanoparticles augments the therapeutic impact by inducing oxidative stress in cancer cells or microbial pathogens, enhancing treatment outcomes. This system is particularly valuable in cases where traditional therapies have limited efficacy, such as multidrug-resistant infections and aggressive cancers.
The invention also describes a comprehensive, nanotechnology-driven process for creating advanced liposome-silver nanoparticle composite formulations. The process begins with the precise calibration of lipid and nanoparticle ratios to ensure compatibility and optimize encapsulation efficiency. The method employs advanced techniques, such as dynamic light scattering (DLS) and zeta potential analysis, to monitor particle size, charge, and stability during formulation.
The resulting composite system is versatile, allowing for customization based on therapeutic needs. By adjusting lipid composition, nanoparticle size, and drug concentration, the formulation can be tailored for various applications, from antimicrobial treatments to targeted cancer therapies. This nanotechnology approach ensures scalability, reproducibility, and regulatory compliance, making it suitable for industrial-scale production. Furthermore, the process enables the incorporation of other therapeutic agents, broadening its application scope in regenerative medicine, infection control, and advanced drug delivery systems.
The comprehensive evaluation of liposomal formulations ensures the quality, stability, and effectiveness of the system in drug delivery. Various analytical techniques were employed to characterize the liposomes and their interaction with Lapatinib, providing critical insights into their physicochemical and biological properties.
The melting point of the active pharmaceutical ingredient (API), Lapatinib, was determined using a digital melting point apparatus (Digi Melt). A small quantity of the drug was loaded into a capillary tube and gradually heated until the transition from solid to liquid occurred. This procedure confirmed the drug's thermal stability and its suitability for inclusion in liposomal formulations. The melting point was observed in the range of 136-140°C, aligning with its expected stability parameters.
Solubility studies revealed the dissolution behavior of Lapatinib in various media. The drug was found to be poorly soluble in water and phosphate-buffered saline (PBS), but it demonstrated good solubility in ortho phosphoric acid, where it dissolved clearly. These results guided the selection of ortho phosphoric acid as the preferred solvent for formulation processes, ensuring adequate drug loading.
To establish a reliable method for drug quantification, a standard calibration curve was developed. Lapatinib was serially diluted with a co-solvent mixture of ortho phosphoric acid and Milli-Q water (0.1:1 ratio) to achieve concentrations of 10, 20, 30, 40, and 50 µg/mL. UV spectrophotometry was performed to scan the solutions between 200-400 nm, identifying 309 nm as the wavelength of maximum absorbance. The calibration curve, plotted with concentration on the X-axis and absorbance on the Y-axis, exhibited a linear relationship, confirming the drug's consistent spectrophotometric response.
Microscopic analysis was performed to observe the morphology and surface characteristics of the liposomes. Using an Olympus BX-51 optical microscope, images were captured with 40X magnification. The study confirmed the presence of liposomes, which were uniformly distributed with consistent shapes and surface characteristics.
FTIR analysis was conducted to investigate the chemical interactions between Lapatinib and the liposomal components. A Perkin Elmer Spectrum 400 spectrometer was used to record spectra in the 4000-400 cm⁻¹ range. The analysis confirmed the integrity of functional groups and the absence of undesired chemical reactions, ensuring the formulation's stability.
Thermal properties of the liposomes were studied using DSC. Samples weighing 5 mg were sealed in aluminum pans and scanned from 0°C to 200°C at a rate of 10°C per minute. The resulting thermograms provided insights into the phase transitions, crystallinity, and stability of the liposomal formulations.
The particle size and distribution of the liposomes were measured using Photon Correlation Spectroscopy (PCS) on a Zetasizer Nano ZS. Liposomes were prepared at a concentration of 1 mg/mL in ultra-clean water and filtered through a 0.45 µm syringe filter to remove insoluble particles. This analysis ensured uniformity in size, a crucial factor influencing drug release and bioavailability.
Zeta potential analysis assessed the colloidal stability of the liposomal system. A high absolute zeta potential value, whether positive or negative, indicated robust repulsion between particles, preventing aggregation and maintaining dispersion stability. Liposomes with low zeta potential were found to be prone to coagulation, emphasizing the importance of this parameter for sustained stability.
TEM was employed to investigate the structural details of the nanoparticles. Thin samples were prepared in a vacuum chamber, and images were captured with spatial resolutions below 1 nm. TEM provided high-contrast images, revealing uniform structures and the surface modifications of the liposomes, thus confirming successful formulation.
Entrapment efficiency, a critical measure of drug encapsulation, was determined by separating the free drug from the liposomal formulation. The liposomes were centrifuged at 4000 RPM for 1 hour, and the supernatant containing unencapsulated drug was collected. Spectrophotometric analysis at 309 nm quantified the drug concentration in the supernatant.
In-Vitro Dissolution Studies: The release profile of Lapatinib-loaded liposomes was investigated using in-vitro dissolution studies. Liposomes were placed in dialysis membrane bags, which were immersed in phosphate buffer (pH 7.4) at 37 ± 5°C. Samples of the release medium were collected at intervals (2, 4, 6, 8, 10, and 24 hours) and replaced with fresh buffer to maintain sink conditions. The drug concentration in the collected samples was analyzed using UV spectrophotometry at 309 nm.
The dissolution data indicated a controlled and sustained drug release profile, essential for prolonged therapeutic efficacy. Curve-fitting analysis revealed the release mechanism, aligning with models such as zero-order or Higuchi kinetics, based on the optimized formulation.
Preparation and Characterization of Lapatinib Liposomes: The formulation of Lapatinib liposomes involved modifying the composition of soy phosphatidylcholine, cholesterol, and Span 80 to optimize the drug delivery system. The study evaluated the effects of varying lipid compositions on encapsulation efficiency and in vitro release profiles. Results indicated that increasing soy phosphatidylcholine content significantly improved encapsulation efficiency and provided stable in vitro drug release. Conversely, higher cholesterol concentrations showed minimal impact on entrapment efficiency, primarily stabilizing the lipid membrane by filling gaps between phospholipids and reinforcing structural integrity.
The morphological characteristics of liposomes were analyzed using optical microscopy. Observations revealed that the F7 formulation produced small, spherical liposomes with consistent shapes. These spherical structures reflect the effective self-assembly of lipid components in the formulation.
Particle size analysis of the Lapatinib-loaded liposomes showed an average size of 268.6 nm, placing the optimized formulation (F7) within the nano-size range. Zeta potential measurements for the F7 formulation indicated a value of -5.9 mV, suggesting that the negatively charged surface prevented vesicle aggregation and enhanced colloidal stability.
The drug entrapment efficiency across various liposomal formulations ranged from 23.5% to 59.3%. Formulation F7 demonstrated the highest entrapment efficiency due to its optimal lipid-to-cholesterol ratio. Higher lipid concentrations in F7 enhanced the encapsulation capacity, while lower cholesterol content reduced competition for space within the lipid bilayer, resulting in improved drug loading.
The in vitro drug release profiles revealed significant differences among the formulations, largely influenced by their compositions. Formulation F7 exhibited sustained drug release due to its higher soy phosphatidylcholine content and optimized lipid concentration. This formulation maintained controlled release over an extended period, fitting well with Higuchi and Peppas release models, as indicated by the R² values.
Drug-excipient compatibility was evaluated using Fourier Transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). FTIR spectra confirmed the stability of functional groups in the physical mixture of Lapatinib and lipids, while DSC analysis provided evidence of thermal compatibility. These studies verified that the selected excipients were suitable for formulating stable liposomes.
The invention successfully developed and characterized Lapatinib-loaded liposomes using soy phosphatidylcholine derived from plant sources. Various lipid and surfactant concentrations were evaluated, with the F7 formulation demonstrating superior performance in terms of drug entrapment, morphology, and release behavior. Optical microscopy confirmed that the liposomes were spherical with stable, thick bilayers.
Drug entrapment efficiency ranged between 23.5% and 59.3%, with F7 achieving the highest efficiency due to optimized lipid-to-cholesterol ratios. In vitro drug release studies highlighted that F7 exhibited sustained release, suitable for prolonged therapeutic applications. The nano-sized particles with a zeta potential of -5.9 mV ensured stability and prevented aggregation.
Drug-excipient compatibility studies via FTIR and DSC confirmed the stability and compatibility of the components. The findings underscore the potential of soy phosphatidylcholine as an economical and effective phospholipid for anticancer drug delivery systems, emphasizing its utility in the production of liposomal formulations for enhanced therapeutic outcomes.
, Claims:We claim
1. A method for synthesizing silver nanoparticles using Anisomeles malabarica plant extract, comprising;
a) collecting and authenticating Anisomeles malabarica leaves;
b) preparing a plant extract by processing the collected leaves;
c) preparing a 1mM silver nitrate (AgNO₃) solution by dissolving 0.017 g of silver nitrate in 100 ml of distilled water; and
d) reacting the plant extract with the 1mM AgNO₃ solution to produce silver nanoparticles.
2. A process for preparing multilamellar vesicle (MLV) liposomes, comprising:
a) adopting the thin-film hydration method to prepare liposomes;
b) utilizing lipid components including soy phosphatidylcholine, cholesterol, and span 80, and incorporating a drug into the formulation;
c) forming a thin lipid film by rotating the mixture at 55°C to evaporate solvents; and
d) hydrating the thin film using an aqueous phase at 60°C to produce MLV liposomes.
3. A method for incorporating silver nanoparticles into liposomes, comprising
a) introducing 2M silver nanoparticles into the organic phase containing lipid components;
b) hydrating the lipid film with an aqueous phase at 60°C to facilitate the formation of MLV liposomes; and
c) rotating the mixture at 55°C until the organic solvents evaporate completely, forming a thin film on the inner walls of the flask.
4. A formulation for drug-loaded liposomes, wherein the formulation comprises:
a) soy phosphatidylcholine, cholesterol, and span 80 as lipid components;
b) silver nanoparticles synthesized from Anisomeles malabarica incorporated into the lipid bilayer;
c) Lapatinib as a drug payload; and
d) an aqueous phase to hydrate the lipid film during liposome preparation.
5. A pharmaceutical composition comprising silver nanoparticles synthesized from Anisomeles malabarica plant extract and incorporated into liposomes, wherein the composition is produced by the thin-film hydration method and exhibits enhanced therapeutic efficacy.
6. A process for the synthesis of drug-loaded, silver-nanoparticle-incorporated liposomes, wherein the process combines biologically synthesized silver nanoparticles with lipid components to achieve a therapeutic delivery system for anticancer or antimicrobial applications.
7. A method for preparing silver-nanoparticle-laden liposomes, comprising:
a) synthesizing silver nanoparticles using Anisomeles malabarica extract and 1mM silver nitrate solution;
b) preparing a lipid film using soy phosphatidylcholine, cholesterol, and span 80 by evaporating solvents under controlled conditions; and
c) hydrating the film with a silver nanoparticle-containing aqueous phase to form stable liposomal structures.
8. A process for the synthesis of drug-loaded, silver-nanoparticle-incorporated liposomes, wherein the process combines biologically synthesized silver nanoparticles with lipid components to achieve a therapeutic delivery system for anticancer or antimicrobial applications.

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