System for the phytosynthesis of anisotropic silver and gold nanoparticles with anti-cancer effects on ovarian cancer cells

Abstract

A system for the phytosynthetic synthesis of anisotropic metal nanoparticles, comprising: a) a leaf extraction unit for the production of Bombax Ceiba leaf extract; b) a metal precursor manufacturing unit configured to produce metal salt solutions; c) a nanoparticle synthesis unit configured to combine the leaf extract with metal salt solutions at room temperature and neutral pH; d) a characterization unit comprising several analytical instruments for analyzing the synthesized nanoparticles; and e) a unit for evaluating anticancer activity, configured to test the synthesized nanoparticles on cancer cell lines.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for the phytosynthetic synthesis of anisotropic metal nanoparticles, in particular silver and gold nanoparticles. BACKGROUND OF THE INVENTION

[0002] Cancer remains a critical global health challenge. Millions of people die from cancer each year, and the number of new cases is expected to rise. Conventional cancer treatments such as radiation and chemotherapy are limited due to their lack of specificity, resulting in significant damage to normal cells and numerous side effects. These limitations have prompted researchers to explore more targeted and innovative approaches to cancer treatment.

[0003] Metal nanoparticles, particularly anisotropic silver (AgNPs) and gold (AuNPs), have emerged as a promising alternative in cancer therapy. These nanoparticles offer unique advantages, including lower toxicity, chemical stability, and the ability to penetrate deep into biological tissue. Their shape and size can be engineered to enhance certain properties, such as surface plasmon resonance (SPR) in the near-infrared range, which enables more effective interaction with cancer cells. The development of environmentally friendly synthesis methods using plant extracts such as those from Bombax Ceiba represents a significant advance in the creation of biocompatible and environmentally friendly nanoparticles for medical applications.

[0004] In view of the foregoing discussion, the present invention provides a system for the phytosynthesis of anisotropic AgNPs and AuNPs using Bombax ceiba leaf extract, harnessing the plant's rich medicinal properties. By employing advanced characterization techniques and carefully controlled synthesis conditions, the invention aims to produce nanoparticles with precise morphology and potential anticancer activity. SUMMARY OF THE INVENTION

[0005] The present disclosure relates to a system for the phytosynthetic synthesis of anisotropic metal nanoparticles, particularly silver and gold nanoparticles. This invention provides a system for producing anisotropic silver and gold nanoparticles (AgNPs and AuNPs) using Bombax ceiba leaf extract. The system comprises components for the synthesis, characterization, and evaluation of nanoparticles and focuses on the creation of environmentally friendly, biocompatible nanoparticles with potential anti-cancer applications. The system is designed for the preparation of a leaf extract, its combination with metal salt solutions at room temperature and neutral pH, and the production of anisotropic nanoparticles within minutes.The synthesized nanoparticles are thoroughly characterized using advanced analytical techniques, including UV-Vis spectroscopy, X-ray diffraction, transmission electron microscopy, dynamic light scattering, and zeta potential analysis. The novelty lies in its green synthesis approach, in which the shape and size of the nanoparticles are controlled by interactions between plant extract proteins and metal ions, ultimately resulting in nanoparticles with potential therapeutic applications, particularly in the targeted treatment of cancer cells.

[0006] The present disclosure is intended to provide a system for the phytosynthetic synthesis of anisotropic metal nanoparticles. The system comprises: a) a leaf extraction unit for producing Bombax ceiba leaf extract; b) a metal precursor production unit for producing metal salt solutions; c) a nanoparticle synthesis unit for combining the leaf extract with metal salt solutions at room temperature and neutral pH; d) a characterization unit with multiple analytical instruments for analyzing the synthesized nanoparticles; and e) an anticancer activity evaluation unit for testing the synthesized nanoparticles against cancer cell lines.

[0007] An object of the present disclosure is to provide a system for the phytosynthetic synthesis of anisotropic metal nanoparticles, in particular silver and gold nanoparticles.

[0008] Another objective of the present disclosure is to provide a system for environmentally friendly and sustainable synthesis of anisotropic silver and gold nanoparticles using plant extracts.

[0009] Another objective of the present disclosure is to develop a systematic approach to control the shape, size and morphology of metal nanoparticles through biomolecular interactions in plant extracts.

[0010] Another objective of the present disclosure is to create a comprehensive characterization system that enables precise analysis of synthesized nanoparticles using multiple advanced analytical techniques.

[0011] Another objective of the present disclosure is to evaluate the potential anticancer activity of biogenically synthesized anisotropic nanoparticles against certain cancer cell lines.

[0012] Another objective of the present disclosure is to reduce the environmental and biotoxicity associated with conventional chemical methods of nanoparticle synthesis by implementing an environmentally friendly, plant extract-based approach.

[0013] To further clarify the advantages and features of the present disclosure, a more detailed description of the invention will be given with reference to specific embodiments thereof, which are illustrated in the accompanying drawings. It should be noted that these drawings represent only typical embodiments of the invention and are therefore not to be considered limiting its scope. The invention will be described and explained in additional detail and in greater detail with reference to the accompanying drawings. SHORT DESCRIPTION OF THE FIGURE

[0014] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Where: Fig. 1 shows a block diagram of a system for phytosynthetic synthesis of anisotropic metal nanoparticles according to an embodiment of the present disclosure.

[0015] Furthermore, those skilled in the art will appreciate that elements in the drawings are shown for convenience and may not necessarily be drawn to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, with respect to the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawing may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art who would benefit from the description herein. DETAILED DESCRIPTION:

[0016] To facilitate an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and described in specific language. It is to be understood, however, that no limitation on the scope of the invention is intended thereby, since such changes and further modifications of the illustrated system, and such further applications of the principles of the invention as illustrated therein, are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0017] It will be understood by one skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0018] References in this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, occurrences of the phrase "in one embodiment," "in another embodiment," and similar language throughout this specification may or may not all refer to the same embodiment.

[0019] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only includes those steps, but may also include other steps not expressly listed or inherent in such process or method. Likewise, one or more devices or subsystems or elements or structures or components preceded by "comprises...a" does not preclude, without further limitation, the existence of other devices or other subsystems or other elements or other structures or other components or additional devices or additional subsystems or additional elements or additional structures or additional components.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0021] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0022] Fig. 1 shows a block diagram of a system (100) for phytosynthetic synthesis of anisotropic metal nanoparticles according to an embodiment of the present disclosure.

[0023] With reference to Fig.the system (100) comprises: a) a leaf extraction unit (102) for producing a Bombax Ceiba leaf extract; b) a metal precursor production unit (104) configured to produce metal salt solutions; c) a nanoparticle synthesis unit (106) configured to combine the leaf extract with metal salt solutions at room temperature and neutral pH; d) a characterization unit (108) comprising a plurality of analytical instruments for analyzing the synthesized nanoparticles; and e) an anticancer activity evaluation unit (110) configured to test the synthesized nanoparticles on cancer cell lines.

[0024] In one embodiment, the leaf extraction unit (102) comprises: a) a leaf washing subsystem for cleaning Bombax Ceiba leaves; b) a leaf drying subsystem for drying the leaves in the shade; c) a grinding mechanism for converting the dried leaves into powder; d) an extraction vessel for boiling leaf powder in distilled water; e) a filtering mechanism for separating the leaf extract; and f) a centrifuge device operated at 2000 rpm for a specified period of time, the device centrifuging the filtered leaf extract to separate heavy biomass.

[0025] In one embodiment, the metal precursor production unit (104) is configured to produce 0.001 M solutions of AgNO3 and HAuCl4.

[0026] In one embodiment, the nanoparticle synthesis unit (106) comprises: a) a mixing vessel with a constant stirring mechanism, wherein the stirring mechanism comprises a magnetic stirrer; b) a dropwise addition system for introducing leaf extract into the mixing vessel containing prepared solutions of AgNO3 and HAuCl4; c) a temperature control mechanism for maintaining room temperature; and d) a pH monitoring system ensuring a neutral medium.

[0027] In one embodiment, the nanoparticle synthesis unit (106) is configured to produce anisotropic silver and gold nanoparticles within 5 minutes of addition of the leaf extract.

[0028] In one embodiment, the characterization unit (108) comprises: a) a UV-Vis-NIR spectrophotometer for optical characterization; b) an X-ray diffractometer for crystallinity analysis; c) a transmission electron microscope for shape and size determination; d) a dynamic light scattering instrument for particle size distribution; e) a zeta potential analyzer for evaluating nanoparticle stability; f) a Fourier transform infrared spectrometer for composition analysis.

[0029] In one embodiment, the anticancer activity evaluation unit (110) is configured to test nanoparticles on the cell lines HIO180 and HeyA8 and determine IC50 values.

[0030] In one embodiment, the characterization unit (108) further comprises a high-resolution liquid chromatography-mass spectrometry system (HR-LCMS) for analyzing the chemical composition of the leaf extract.

[0031] The present invention relates to a system for synthesizing anisotropic AgNPs and AuNPs using BC leaf extract for anticancer applications. The synthesized nanoparticles were subjected to characterization using chromatographic techniques (HR-LCMS, HPLC) to determine the extract composition in the leaf extract responsible for the formation of anisotropic AgNPs and AuNPs. The anisotropic AgNPs and AuNPs nanoparticles were thoroughly characterized using various characterization techniques. Finally, the anisotropic AgNPs and AuNPs were tested for their anticancer activity against HIO180 and HeyA8 cells.

[0032] To produce nanoparticles, BC leaves were collected in Kolhapur, India. The collected leaves were thoroughly cleaned, first with tap water and then with distilled water. After washing, the leaves were allowed to dry in the shade for one week. Once dry, they were ground into a fine powder. To prepare the extract, 5 grams of these powdered leaves were mixed with 200 milliliters of distilled water and boiled for 15 minutes. The mixture was then cooled to room temperature and filtered with Whatman No. 1 filter paper. The resulting filtrate was centrifuged at 2000 rpm for 5 minutes to remove heavy biomass, leaving a purified extract for further use.

[0033] The synthesis of the nanoparticles is carried out at room temperature and natural pH. The slow formation and aggregation rate of metal nanoparticles (MNPs) under acidic conditions can be attributed to the electrostatic repulsion of anions in the solution. On the other hand, a basic pH environment can lead to the precipitation of silver hydroxide (AgOH). Based on these observations, neutral pH conditions were considered optimal for the synthesis of MNPs using BC leaf extract. For the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs), 100 ml of 0.001 M AgNO3 and 0.001 M HAuCl4 were used. - A solution was prepared in 250-ml beakers. 3 ml of BC leaf extract was added dropwise to these solutions with continuous stirring. The formation of AgNPs and AuNPs was observed within five minutes, and the nanoparticles were subsequently used for further analysis.

[0034] The phytosynthesis mechanism of MNPs comprises three main phases. The first phase, the activation phase, involves the reduction of metal ions and the formation of reduced metal atoms. The second phase, the growth phase, involves the spontaneous fusion of small nanoparticles into larger particles. Finally, the termination phase determines the final shape of the nanoparticles. Prolonging the growth phase can lead to the aggregation of nanoparticles and the formation of various types of MNPs. Studies have shown that the amino acid sequence of proteins significantly influences the size, shape, and yield of nanoparticles. For example, replacing the N- and C-terminal amino acid residues in peptides can slow down the reduction process, resulting in shape-controlled MNPs. Peptides and proteins in plant extracts are thought to play a crucial role in the shaping of nanoparticles and influence their overall yield.

[0035] The prepared nanoparticles were characterized using various instruments. The formation of silver and gold nanoparticles (AgNPs and AuNPs) was monitored using a UV-Vis-NIR spectrophotometer (Shimadzu, UV-3600). Crystallinity and average size of the synthesized materials were analyzed using X-ray diffraction (XRD). Shape and size of the materials were determined using transmission electron microscopy (TEM). Images were acquired using a JEOL JEM 2100 TEM equipped with a high-resolution Gatan CCD camera. Particle size distribution and nanoparticle stability were determined using dynamic light scattering (DLS) and zeta potential measurements, which were performed on a Malvern Zetasizer (Nano ZS-90) with a 4 mW, 633 nm He-Ne laser at 25 °C and a fixed angle of 90° in disposable polystyrene cuvettes.Fourier transform infrared spectra (FT-IR) were recorded using a Perkin-Elmer infrared spectrometer. The chemical composition of the BC leaf extract was investigated using high-resolution liquid chromatography-mass spectrometry (HR-LCMS) on an Agilent Technologies 1290 Infinity UltraHPLC system coupled with a 1260 Infinity Nano HPLC and Chipcube, as well as a 6550 iFunnel Q-TOF mass spectrometer.

[0036] The anticancer potential of the prepared nanoparticles was tested by performing the cell proliferation assay after treatment with AgNPs and AuNPs. Normal ovarian epithelial cells (HIO180) and high-grade ovarian serous adenocarcinoma cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco). After reaching confluence, the cells were detached using 0.25% trypsin-EDTA (Gibco) and counted using a Cellometer mini (Nexcelom). For the experiment, 5,000 cells were seeded per well in a 96-well plate and incubated overnight to facilitate attachment. The cells were then exposed to increasing concentrations of gold and silver nanoparticles for 72 hours. After the incubation period, 10 µl of CCK-8 reagent (Dodjindo) was added to each well and the plates were incubated for another three hours at 37 °C.Absorbance was measured at 450 nm to assess cell viability. The percentage survival rate was calculated relative to an untreated control group, which was assumed to have 100% survival. The IC50 value, or the concentration causing 50% cell death, was determined from the generated survival curve. All tests were performed in triplicate.

[0037] FT-IR analysis confirmed the presence of various phytochemicals in the BC leaf extract, which play a dual role in reducing metal ions to nanoparticles and stabilizing their structures. The spectrum highlighted functional groups such as alcohols, phenols, flavonoids, and terpenoids. Shifts in the OH, C≡C, and CO bending bands after nanoparticle synthesis indicate binding between the phytochemicals and the nanoparticle surfaces. The reduced intensity in the AuNP spectra indicates lower functionality and confirms the role of phytochemicals in regulating the size and shape of nanoparticles.

[0038] HR-LCMS identified a variety of compounds in BC leaf extract, including alcohols, phenols, amino acids, and carboxylic acids. These biomolecules were involved in the stabilization of the nanoparticles by resonance-stabilized quinones, which prevent agglomeration. This mechanism confers stability to the nanoparticles and enhances their anticancer properties.

[0039] UV-Vis analysis confirmed the successful synthesis of AgNPs and AuNPs with distinct surface plasmon resonance (SPR) bands at 434 nm and 547 nm, respectively. The difference in SPR peak positions reflects variations in the size and shape of the nanoparticles, which is confirmed by the anisotropic shapes observed for AgNPs.

[0040] XRD patterns confirmed the face-centered cubic (FCC) crystal structure for both AgNPs and AuNPs. The Scherrer equation yielded average crystallite sizes of 7 nm for AgNPs and 10 nm for AuNPs, demonstrating the effectiveness of BC leaf extract in controlling nanoparticle size.

[0041] XPS analysis revealed the chemical states of Ag and Au in the synthesized nanoparticles. Binding energies confirmed the presence of metallic Ag (Ag 3d) and Au (Au 4f), further supporting successful nanoparticle synthesis.

[0042] DLS and zeta potential measurements showed nanoparticle sizes of approximately 15 nm and zeta potential values ​​of -14.5 mV for AgNPs and -10.4 mV for AuNPs. The negative zeta potentials indicate good colloidal stability due to surface charge repulsion.

[0043] TEM revealed the morphology and size distribution of the nanoparticles. AgNPs exhibited various shapes ranging from 2 to 10 nm, while AuNPs exhibited triangular shapes ranging from 5 to 10 nm. Selected area electron diffraction (SAED) patterns confirmed the crystalline nature of the nanoparticles.

[0044] Cell proliferation assays on HIO180 and HeyA8 cells demonstrated significant cytotoxic effects of AgNPs and AuNPs. The nanoparticles exhibited dose-dependent activity, with maximum efficacy at 60 µg / ml. The IC50 values ​​underscored the superior anticancer potential of the biosynthesized nanoparticles compared to previous studies. It has been suggested that phytoconstituents attached to the surface of the nanoparticles synergistically enhance the anticancer activity.

[0045] The above results clearly demonstrate the significant potential of biosynthesized AgNPs and AuNPs against HIO180 and HeyA8 cancer cells. The anticancer activity increases due to the synergistic effect of phytochemicals from BC leaf extract that adhere to the surface of the nanoparticles.

[0046] The drawings and the foregoing description provide examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of the processes described herein may be changed and are not limited to the manner described herein. Furthermore, the actions of any flowchart need not be implemented in the order shown; nor do all actions necessarily need to be performed. Also, those actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and material use, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0047] Advantages, other benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, benefits, solutions to problems, and any components that may cause an advantage or solution to occur or become more apparent are not to be construed as a critical, required, or essential feature or component of any or all of the claims. REFERENCES 100 A system for the phytosynthetic synthesis of anisotropic metal nanoparticles. 102 Leaf vacuum cleaner 104 Metal Precursor Processing Unit 106 Nanoparticle synthesis unit 108 Characterization Unit 110 Unit for the Assessment of Anticancer Activity

Claims

[1] A system for the phytosynthetic synthesis of anisotropic metal nanoparticles, comprising: a) a leaf extraction unit for the production of Bombax Ceiba leaf extract; b) a metal precursor production unit configured to produce metal salt solutions; c) a nanoparticle synthesis unit configured to combine the leaf extract with metal salt solutions at room temperature and neutral pH; d) a characterization unit comprising several analytical instruments for analyzing the synthesized nanoparticles; and (e) an anticancer activity evaluation unit configured to test the synthesized nanoparticles on cancer cell lines. [2] The system of claim 1, wherein the leaf extraction unit comprises: a) a leaf washing subsystem for cleaning Bombax Ceiba leaves; b) a leaf drying subsystem for drying the leaves in the shade; c) a grinding mechanism for converting dried leaves into powder; d) an extraction vessel for boiling leaf powder in distilled water; e) a filter mechanism for separating the leaf extract; and (f) a centrifuge device operated at 2000 rpm for a specified period of time, the device centrifuging the filtered leaf extract to separate heavy biomass. [3] The system of claim 1, wherein the metal precursor preparation unit is configured to prepare 0.001 M solutions of AgNO3 and HAuCl 4 herzustellen . [4] The system of claim 1, wherein the nanoparticle synthesis unit comprises: a) a stirring vessel with a permanent stirring mechanism, the stirring mechanism comprising a magnetic stirrer; b) a dropwise addition system for introducing leaf extract into the mixing vessel containing prepared solutions of AgNO3 and HAuCl4; c) a temperature control mechanism to maintain the room temperature; and d) a pH monitoring system that ensures a neutral medium. [5] The system of claim 1, wherein the nanoparticle synthesis unit is configured to produce anisotropic silver and gold nanoparticles within 5 minutes of addition of the leaf extract. [6] The system of claim 1, wherein the characterization unit comprises: a) a UV-Vis-NIR spectrophotometer for optical characterization; b) an X-ray diffractometer for crystallinity analysis; c) a transmission electron microscope for determining shape and size; (d) a dynamic light scattering instrument for measuring particle size distribution; (e) a zeta potential analyzer to evaluate the stability of nanoparticles; f) a Fourier transform infrared spectrometer for composition analysis. [7] The system of claim 1, wherein the anticancer activity evaluation unit is configured to test nanoparticles on the cell lines HIO180 and HeyA8 and determine IC50 values.

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