LECTIN NANOCAPSULES FOR TARGETED NANO DRUG DELIVERY IN CANCEROUS TUMORS AND METHOD OF PREPARATION THEREOF

Abstract

The present invention discloses the concanavalin-A nanocapsules comprising concanavalin-A and procyanidolic oligomers, wherein shell of nanocapsule comprises concanavalin-A and procyanidolic oligomers loaded with in the nanocapsules and its method of preparation thereof. Concanavalin-A nanocapsules are spherical in shape with an average size of 52.20±24.99 nm. Nanocapsules shows anticancer, antioxidative and antimicrobial activity, which fulfil all the essential requirement to combat the cancer and bacterial infections associated with the deadly disease. The technology is robust, easy-to-scale-up and products made are physically and chemically stable.

Specification

Description:FIELD OF INVENTION
The present invention is in the field of Nano Science & Technology related to pharmaceutical field. More particularly, the invention relates to the concanavalin A nanocapsules and its method of preparation for targeted nano drug delivery in cancerous tumors.
BACKGROUND OF INVENTION
Cancer accounts for approximately 0.3 million deaths annually in India, making it second most common disease in India with the highest death toll, which posed a major global health threat to human beings.
By 2025, India's cancer burden will rise to 2.98 million from 2.67 million.
Cancer remains a major global health threat to human beings, despite the significant advancements in detection, diagnosis and treatment of Cancer.
All types of cancers have been reported in India, including cancer of skin, lung, breast, stomach, prostate, rectum cancer, liver, cervix, esophagus, bladder, mouth, blood etc. In women, breast, cervix and ovarian cancers are the most prevalent, while in men, lung, mouth and prostate cancers are common.
Breast cancer is the deadliest type of cancer reported in women globally. The year 2022 saw 670 000 breast cancer deaths worldwide and 21.3 million new cases of the disease among women. All over the world, breast cancer affects women at any age after adolescence, though its prevalence rises with increasing age [WHO].
In comparison to 2020, there will likely be a projected 12.8% rise in cancer incidence by 2025. According to projections, India's cancer burden will increase from 26.7 million DALYs (adjusted mortality to incidence) in 2021 to 29.8 million in 2025, with the north and northeast experiencing the greatest burden [www.ncdirindia.org]. In India, breast cancer is the most common type of cancer among women, making up 14 % of all cancers among female population. According to reports, an Indian woman receives a breast cancer diagnosis every four minutes. In both rural and urban India, breast cancer is becoming more common. In India, the prevalence of breast cancer is rising at an alarming rate, especially in the younger age groups of the 30s and 40s. The majority of women with breast cancer are between the ages of 20 and 59. The healthcare sector has benefited greatly from the introduction and subsequent advancement of nanotechnology in recent years, particularly in the field of cancer treatment.
Despite tremendous advancements in cancer detection, diagnosis and treatment, cancer remains a major threat to people worldwide. Cancer accounts for approximately 0.3 million deaths annually in India, making it the second most common disease that causes the highest death toll.
By 2025, India's cancer burden will rise to 2.98 million from 2.67 million. The "Cancer capital of the world" tag has captured the global attention, causing nationwide alarm. India's reputation as the "Cancer Capital of the World" has drawn global attention and caused national alarm. Rising cancer rates in India have made it the "Cancer capital of the world", surpassing the global rate.
An estimated 2,001,140 new cases and 611,720 cancer related deaths are predicted in the United States in 2024. Among all types of cancers, lung cancer poses the greatest risk to an individual's health because it is frequently detected at an advanced stage when there are few therapeutic options.
In the US, it is expected that in 2024, there will be 125070 lung cancer deaths and 234580 new cases of lung and bronchus cancer among men and women. The incidence and mortality of lung cancer are highest in Asia compared with Europe and USA [Lam DC et al. 2023].
The most common types of lung cancer are non-small cell carcinoma (NSCLC) and small cell carcinoma (SCLC). For different types of cancer, chemotherapy remains an important therapeutic option despite advances in surgical and radiation treatment. However, the inherent and acquired resistance of cancer cells to anticancer medications significantly restricts the effectiveness of chemotherapy. High dose, nonspecific distribution, extreme toxicity to healthy cells, insufficient drug concentration at cancerous site is the main cause of emergence of multidrug resistance.
In cancer patients, bacterial infections pose a significant challenge, which is difficult to manage. The primary factors that impede the progression of lung cancer are bacterial infections, ear, nose, throat, and gastrointestinal tract infections. Throughout the course of cancer development and treatment, bacteria and cancer cells have a unique symbiotic relationship. The bacteria like E. coli, P. aeruginosa, S. aureus etc. frequently invade cancer patients and result in infections.
However, when receiving the right combination of antimicrobial therapy and cytostatic therapy, patients with malignant diseases can live longer [Yan T, et al 2023]. Cancer treatment is closely associated with antibiotic use. Thus, it is necessary to consider new and safe alternatives, which can be accomplished by combining nanotechnology with naturally occurring bioactive molecules.
In various fields, including physics, chemistry, biology, engineering and medicine, nanotechnology has a wide range of applications. The field of Clinical medicine have found the application of nanotechnology in developing nanocarrier systems delivering therapeutic compounds to treat various chronic diseases including cancer in safe and controlled manner. Nanocarrier also helps in improving the stability and bioavailability of the drug.
The advancements in nanotechnology have profoundly impacted the development of targeted drug delivery systems. As a promising new paradigm in cancer treatment, targeted drug delivery systems deliver drugs directly to cancer cells while causing minimal harm to healthy cells.
In order to enhance the bioavailability, biodistribution and accumulation of drugs, nanocarriers are regarded as popular targeted drug delivery systems.
Using targeted drug delivery strategies, most drugs can be delivered precisely to tumor cells rather than to normal cells or tissues. Nanotechnology can be used to achieve such delivery strategies. Nanoparticle based drug delivery has gained attention due to the fact that nanoparticles can accumulate at tumor sites based on the EPR effect.
Herbal remedies are preferred by 70-80 % of people worldwide to treat any illness, making them the most prevalent type of traditional medicine. A novel fusion of nature and science could result in a medicinal revolution. Polyphenols are bioactive compounds present in plants, which have a number of potential therapeutic applications making it a revolutionary idea.
Protection of bioactive polyphenols for therapeutic purposes using nanotechnology is the basis of this potentially ground breaking concept. This study explores plant based anticancer candidate, which is effective against cancer and microbes, so we can use it as supportive therapy for cancer.
Plant based polyphenols accounts for antioxidant as well as anticancer activity.
Grape seed extract is one of the most widely consumed fruits globally, which consists of approximately 90 % proanthocyanidins and 7 % other polyphenols (flavonoids). Procyanidolic oligomers (PCOs) also known as Oligomeric proanthocyanidins (OPCs) present in plants is an effective & powerful antioxidant garnered attention recently [Peng et al., 2005]
The present invention involves the Concanavalin-A (Con- A) lectin as the encapsulating agent in synthesizing nanoformulations of Procyanidolic oligomers for targeted drug delivery in cancerous tumors.
Lectins are the proteins which have ability to bind with carbohydrates present on tumors and these are of non-immunological origin present mostly in plants (and present in some animals and microbes also). Lectins possess two or more sugar binding sites i.e. multivalent molecules which can agglutinate plant and animal cells. The carbohydrates moieties present on tumor can be used for targeted drug delivery in cancer therapy.
More than hundred lectins are known to be isolated from the plants. Concanavalin-A (Con- A) has been the first among these and was discovered in 1919, it is most abundant and low-cost plant lectin Con-A was extracted from Jack bean and it has high binding affinity with glucose and mannose, it is used as a model lectin to investigate the multivalent binding of glycopolymers [ Yilmaz G. et al 2015].
It has long been known that the most researched plant lectin, concanavalin- A (Con-A) is a strong antineoplastic agent. Determining the precise molecular mechanism of lectin has garnered a lot of attention since the first reports about its ability to destroy cancer cells. Concanavalin A has been shown to bind to a number of receptors on cancerous cells and alter the associated signalling cascades [Huldani H et al 2022].
At present, there are no studies on synthesis of lectin nanocapsules i.e. no one has directly encapsulated the bioactive compound e.g. procyanidolic oligomers in concanavalin-A (lectin). However, many researchers have loaded the active compounds in other polymers e.g. PLGA, Chitosan etc. and then conjugate these nanoparticles with concanavalin-A by carbodiimide method. OBJECTIVES OF THE INVENTION
The principal objective of present invention is to provide procyanidolic oligomer loaded concanavalin A nanocapsules.
Another objective of present invention is to provide a facile process for preparation of concanavalin A nanocapsules.
Further objective of present invention is to use concanavalin-A nanocapsules for anticancer activity.
These and other features and advantages of the present invention may be incorporated into certain embodiments of the invention and will become more fully apparent from the following description or may be learned by the practice of the invention as set forth hereinafter. The present invention does not require that all the advantageous features and all the advantages described herein be incorporated into every embodiment of the invention.
SUMMARY OF THE INVENTION
The present invention discloses the concanavalin A nanocapsules comprising concanavalin A and procyanidolic oligomer, wherein shell of nanocapsule comprises concanavalin A and procyanidolic oligomer loaded with in the nanocapsules.
The method of preparation of concanavalin A nanocapsules comprising of
dissolve the concanavalin A in 5 ml double distilled water and sonicate for 20 minutes;
adjust the pH of solution to 2.8;
add dropwise 5ml ethanol;
add dropwise 5 mg procyanidolic oligomers (PCOs) solution dissolved in 500 µl double distilled water.
add dropwise 500µl of calcium chloride (0.2% w/v);
after 20 minutes add 500 µl of freshly prepared solution of pluronic F-68 & polyethylene imine (PF+PEI); (0.5% w/v aq. solution of pluronic F-68was prepared previously and then in this solution 4% v/v polyethyleneimine was added).
after 20 minutes add dropwise 500µl of aqueous solution of tween-80 (2% v/v);
stir the solution 6-10 hours at room temperature;
store the formulation after freeze drying.
concanavalin A nanocapsules are spherical in shape with an average size of 52.20±24.99 nm. The antioxidative activity of the of nanocapsules are even higher than the Vitamin C in equivalent concentration. Concanavalin A nanocapsules shows anticancer, antioxidative and antimicrobial activity, which fulfil all the essential requirement to combat the cancer and bacterial infections associated with the deadly disease.
BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES
The present invention will become more understandable from the description given herein and the accompanying drawings below. These are given by way of illustration only and therefore not limited to present invention and wherein:
Figure 1 illustrates the (a) Micrograph obtained from particles size analysis of Lec-PCO-NC, (b) Zeta potential curve of Lec-PCO-NC
Figure 2 illustrates the FESEM image of Lec-PCO-NC nanoformulation, (a & b) before lyophilization i.e thin film of Lec-PCO-NC without lyophilization, (c & d) lyophilized Lec-PCO-NC nanoformulation.
Figure 3 illustrates the TEM image of Lec-PCO-NC nanoformulation
Figure 4 illustrates the FTIR spectra: (a) of Concanavalin-A, (b) Procyanidolic oligomers (PCOs), and (c) concanavalin A nanocapsules nanocapsules (Lec-PCO-NC).
Figure 5 illustrates the Graph demonstrating dose dependent DPPH scavenging activity of Vitamic C, Pure PCOs and concanavalin A nanocapsules (Lec-PCO-NCs).
Figure 6 illustrates the In-vitro anticancer activity of different concentrations of PCOs and Lec-PCO-nanocapsules on SK-MES-1 cancer cell lines.
Figure 7 illustrates the In-vitro anticancer activity of different concentrations of PCOs and LEC-PCO-NC on MCF-7 cancer cell lines.
Figure 8 illustrates the microscopic images of the morphological alterations that PCOs (8, b-e) and Lec-PCO-NC (8,g-j) at different concentrations caused in SK-MES-1 cancer cell lines following a 24 h treatment period, compared to untreated controls (8,a) as well as standard drug (8,f).
Figure 9 illustrates the microscopic images of the morphological alterations that PCOs (9,b-e) and Lec-PCO-NC (9,g-j) at different concentrations caused in MCF-7 cancer cell lines following a 24 h treatment period, compared to untreated controls 9,a) as well as standard drug (9,f).
Figure 10 illustrates the Graph showing comparative size of zone of inhibition of PCOs and Lec-PCO-NC against the bacterial strains used
Table 1. The colloidal stability of the Lec-PCO-NC over different time intervals and environmental conditions is shown according to particle size, Polydispersity index and zeta potential
Table 2. In vitro anticancer activity of PCOs, Lec-PCO-NC against SK-MES-1 cancer cell line
Table 3. In vitro anticancer activity of PCOs, Lec-PCO-NC against MCF-7 cancer cell line
Table 4. Comparative size of zone of inhibition of PCOs and Lec-PCO-NC against the respective bacterial strains used

DETAILED DESCRIPTION OF THE INVENTION
The following presents a simplified description of the invention in order to provide a basic understanding of some aspects of the invention. This description is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form.
Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.
Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the invention as defined by the appended claims and their equivalents.
It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
By the term "substantially" it is meant that the recited characteristic, parameter, or value need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement error, measurement accuracy limitations and other factors known to those of skill in the art, may occur in amounts that do not preclude the effect the characteristic was intended to provide.
It should be emphasized that the term "comprises/comprising" when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
It should be emphasized that the term "Lec-PCO-NC" when used in this specification is taken to specify the concanavalin A nanocapsules.
It should be emphasized that the term "PCOs" when used in this specification is taken to specify the procyanidolic oligomers.
The present invention discloses the concanavalin-A nanocapsules comprising concanavalin-A and procyanidolic oligomers, wherein shell of nanocapsule comprises concanavalin -A and procyanidolic oligomers loaded with in the nanocapsules.
Best Method of preparation
The method of preparation of concanavalin- A nanocapsules comprising of
dissolve the concanavalin-A in 5 ml double distilled water and sonicate for 20 minutes;
adjust the pH of solution to 2.8;
add dropwise 5ml ethanol;
Then 5 mg PCOs previously dissolved in 500 µl double distilled water was added dropwise
add dropwise 500µl of calcium chloride (0.2% w/v);
after 20 minutes add 500 µl of freshly prepared solution of pluronic F-68 & polyethylene imine (PF+PEI); (0.5% w/v aq. solution of Pluronic F-68was prepared previously and then in this solution 4% v/v polyethyleneimine was added).
after 20 minutes add dropwise 500µl of aqueous solution of tween-80 (2% v/v);
stir the solution 6-10 hours at room temperature;
store the formulation after freeze drying.
Embodiments
Characterization: Concanavalin -A nanocapsules were characterized using a diverse range of advanced techniques. The size distribution, dispersity and zeta potential were measured by performing the dynamic light scattering (DLS). The surface morphology and accurate size were determined through Field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM). To study the composition and interaction within the nanoformulation the infrared spectrum was obtained on the FTIR spectrophotometer.
Encapsulation efficiency: The encapsulation efficiency of Lec-PCO-NC was determined by calculating the free drug concentration in the supernatant relative to total drug.
To separate the free drug from the nanoformulation (Lec-PCO-NC), centrifugation was done at 10,000 rpm (4 °C) for 30 minutes by using centrifuge. To analyze the amount of drug (PCOs) in the supernatant at 280 nm wavelength.
To calculate the percent encapsulation efficiency the following formula was used:-
% Encapsulation Efficiency=(Total drug-Free drug)/(Total drug)×100
Evaluation of Antioxidative Potential of Lec-PCO-NC: In vitro DPPH (1,1-diphenyl-B-picryl-hydrazyl) free radical scavenging assay was used as mentioned in our previous research to study the antioxidant activity of Lec-PCO-NC in comparision with blank PCOs [Lal S. et al 2024]. The different concentrations of PCOs and Lec-PCO-NC (25-200 µg/ml) and DPPH solution (1ml: 3.9 mg/100 ml) containing reaction mixture were incubated in dark for 30 minutes at room temperature. By using UV-Vis spectrophotometer the absorbance of reaction mixture was taken at 517 nm. DPPH without sample was used as control and percent inhibition was measured by using the below mentioned formula:
% inhibition=((Absorbance of Control-Absorbance of Sample))/(Absorbance of Control) × 100
IC50 was calculated by plotting the graph between percentage of DPPH scavenging against nanoformulation concentration and the value obtained was used to express the antioxidant potential of Lec-PCO-NC.
2.6 Evaluation of Anticancer Activity of Lec-PCO-NC by using SRB assay: SRB i.e. Sulforhodamine B is an aminoxanthene dye which is bright pink in color having two sulfonic groups that bind to basic amino acid in mildly acidic environments and dissociates in basic condition. The mass of the cell directly relates to the amount of dye extracted from stained cells. SRB binds to basic amino acid residues in proteins in Tri chloroacetic acid (TCA) fixed cells under mild conditions to provide a sensitive index of the protein content of the cells. Obtention of rapid, stable and visible color was measured using either spectrophotometer or 96 plate reader. The cells fixed with TCA were stained for 30 min with SRB 0.4% (w/v) and dissolved in 1% acetic acid. TCA fixed SRB stained samples were air dried . After removing the SRB, the cultures were washed four times with 1% acetic acid to get rid of any remaining unbound dye.
In vitro cytotoxicity assay on SK-MES-1 cell line and MCF-7 cell line by Sulforhodamine B (SRB) assay:The human lung cancer cell line (SK-MES-1) and human breast cancer cell line (MCF-7) were maintained in a 5% CO2 atmosphere at 37° C in RPMI 1640 medium, supplemented with 20% fetal bovine serum (FBS), 2µM glutamine and antibiotics. After 24 h of time a partial monolayer was formed, the supernatant was decanted off and the monolayer was once rinsed with 100µl of the medium. To examine the effects of Lec-PCO-NC solution in DMSO, PURE PCOs and the control drug, the cells were sub cultured twice a week. 1000 µg/ml solution of PCOs was prepared and the corresponding similar concentration of Lec-PCO-NC was prepared. The following concentration of pure PCOs and Lec-PCO-NC were used viz. 10 µg/ml, 20 µg/ml, 40 µg/ml and 80 µg/ml. Adriamycin was used as positive control and all the samples were added to the culture medium containing 0.1 ml of diluted cell suspension (10,000 cells approximately) per well of the 96-well microplate
The plates were incubated in a CO2 incubator at 37° C for 3 days. TCA (25µl of 50% conc.) was added to the wells slowly in such a way that a thin layer was formed over the drug dilution to make overall concentration 10%. The culture plates incubated at 4°C for 1 h and after that the plates were rinsed five times to remove any traces of medium and the nanoparticles thus obtained were air dried.
2.7 Antimicrobial activity: Lec-PCO-NC was evaluated for its antimicrobial activity by using agar well diffusion method. The bacterial strains viz. Escherichia coli (MTCC 048), Staphylococcus aureus (MTCC 6908), Pseudomonas aeruginosa (NCDC 105) and Bacillus subtilis (MTCC 441) were used to assess the antimicrobial efficacy of the Lec-PCO-NC, using ceftriaxone sodium as positive control as described in earlier studies [Jangra SL 2012]. The luria agar medium in molten state was poured aseptically into the sterile petri plates and fresh bacterial culture (107 CFU) was inoculated into the plates and allowed to solidify. Five mm diameter wells were excavated on each plate at evenly spaced intervals with the help of a sterile cork-borer. The respective test and control samples (80µl) were added (following proper sonication) in the specified wells. The petri plates were then incubated at 37° C for 24h. The clearly visible inhibition zones were obtained post incubation and were measured using ruler and expressed in mm.
Statistical Analysis: All the experiments were performed in triplicates and data were analyzed using One-way ANOVA followed by Tukey's post hoc analyses (Microsoft Excel, 365). P<0.05 indicated the acceptance of a significant difference. All the findings are presented as average ± standard deviation (SD).
In an embodiment the Z-average of the concanavalin-A nanocapsules nanoformulation is 147.9 as per the DLS measurement, while the Pdi and zeta potential were 0.397 and -25.0 mV respectively (Figure 1 a & b).
The uniform distribution of the nanoparticle's attributes to low PDI value obtained in DLS of concanavalin-A nanocapsules while strongly negative zeta potential value of the nanoparticles is the indicator of high colloidal stability.
DLS provides the information about hydrodynamic diameter hence it gives only rough estimation of the nanoparticle size. Therefore, the electron microscopy techniques viz. SEM and TEM were used to observe the actual size and morphology of concanavalin A nanocapsules
In an embodiment the concanavalin - A nanocapsules are spherical nanoparticles having highly monodisperse nature having smooth surface with size range 34.78 nm to 266.64 nm, with an average size 92.84±52.62 nm as shown in FESEM micrograph (Figure 2).
The micrographs in figure 2 a and b represents FESEM images of concanavalin A nanocapsules without lyophilization, while the FESEM images of lyophilized concanavalin-A nanocapsules are shown in figure 2c, d.
In an embodiment the concanavalin- A nanocapsules have an average particle size 52.2±24.99 nm and are in non-aggregated form in TEM imcrograph (Figure 3).
In the FESEM and TEM micrograph the difference in the sizes might be due to the difference in sample preparation technique and hydration due to the presence of surfactant. Nonetheless, the distribution and morphologies of nanoparticles found in TEM and SEM are complementary.
In an embodiment the encapsulation efficiency of concanavalin A nanocapsules is 90.71%.
FTIR spectral analysis was used to perform the FTIR analysis in the range 4000 to 400 cm-1. Fourier transform infrared spectroscopy (FTIR) spectrum of the compound revealed the information related to functional group as well as chemical bond. The spectra of the various samples are shown in figure 4.
In an embodiment the Concanavalin-A exhibit various bands at 3439.93 cm-1, 3286.61 cm-1, 2961.42 cm-1, 2923.49 cm-1, 1830 cm-1, 1640.48 cm-1 and 692.34 cm-1. The peak at 1382.26 cm-1 corresponds to C-O or C-H vibration and amide group. At 1640.48 cm-1 the peak shows the presence of carbonyl structure. The peaks at 3286.61 cm-1 and 1382.26 cm-1 pertaining to hydroxyl groups [Moghaddam M.M. et al 2023].
In an embodiment the Pure procyanidolic oligomers exhibited a broad absorption band from 3400 to 3150 cm-1 centered at 3429.24 cm-1 due to the H bond effect between the phenolic hydroxyl groups in procyanidolic oligomers.
In an embodiment the Procyanidolic oligomers (PCOs) displays functional groups at 1621.62 cm-1 and 1107.06 cm-1 that are typically associated to polyflavonoid moiety.
In an embodiment the Polysaccharide structures is linked to the C-O stretch at 1054.31 cm-1 and the out of plane bending of CH3 at 1390.47 cm-1.
In an embodiment the in the procyanidolic oligomers' structure, the aromatic rings's skeletal stretching modes and the CH out of plane deformation containing two adjoining free hydrogen atoms exhibited peaks at 1526.86 cm-1 and 770.51 cm-1 respectively [Deng S, et al 2021].
The spectra show distinctive bands at 1159 cm-1 and 823.65 cm-1 which are shifted in the concanavalin A nanocapsules, confirming that PCOs interacts with concanavalin-A.
As a result of hydrogen bond formation between phenolic groups of PCOs and the amide groups of concanavalin-A, the peaks of -OH were moved to 3449.61 cm-1 in the spectrum of PCOs nanoformulation.
Apart from the significant variation in the -OH stretching band, the finger print region showed a noticeable variation at wavenumbers 1500 to 400 cm-1, particularly at following peaks 1463.98 cm-1, 1379.84 cm-1, 1096.43 cm-1, 1022.92 cm-1, 938.79 cm-1, 886.53 cm-1 and 602.24 cm-1. These measurements confirmed the presence of PCOs in the nanoformulation and also the interaction between PCO and Concanavalin-A.
Colloidal stability: The concanavalin- A nanocapsules were kept 4 °C and 25 °C for 12 weeks in order to investigate the colloidal stability, the results are shown in table 1.
In an embodiment no significant differences are observed in PS, PDI and ZP of the concanavalin- A nanocapsules.
Antioxidant activity: The DPPH antioxidant assay was used to assess the free radical scavenging ability of PCOs and concanavalin A nanocapsules.
In an embodiment the DPPH assay results that concanavalin A nanocapsules exhibited excellent potential when compared with pure PCOs and Vitamin C (Figure 5). The IC50 of the concanavalin-A nanocapsules, pure PCOs and Vitamin C obtained from the dose dependent curve are 9.04, 14.51 and 10.24 µg/ml respectively.
The IC50 is a measure of antioxidant potential and inversely proportional to the scavenging potential.
In an embodiment the concanavalin A nanocapsules exhibits an IC50 even lower than the Vitamin C, which confirmed the extraordinary therapeutic potential of the as synthesized Lec-PCO-NC.
3.4 In vitro anticancer activity: In an embodiment the concanavalin-A nanocapsules were screened for anticancer potential against SK-MES-1 (Figure 6) and MCF-7 cell lines (Figure 7) using SRB assay and the results are summarized in table 2 and 3. Adriamycin, a positive control, PCOs and concanavalin-A nanocapsules (Lec-PCO-NC) exhibited linear cytotoxic response against the cancer cell lines with respect to drug concentration. Lec-PCO-NC displayed even slightly higher anticancer activity as compared to Adriamycin. The decrease in percent cell viability was observed with increase in concentration of the tested compound. PCOs and Lec-PCO-NC exhibited dose dependent behavior on cell viability with a concentration range 10-80 µg/ml against both cancer cell lines. Higher cellular uptake of nano drug as compared to pure drug makes the nanoformulation more effective than the free drug. The present study suggests that sustained drug release behavior is responsible for the enhanced effect of drugs encapsulated into nanoparticles, despite the fact that free drugs have the ability to diffuse directly into the cell nucleus [Guo XY, et al 2015].
3.5 Antimicrobial Evaluation: In an embodiment the antibacterial activity of concanavalin-A nanocapsules is 70µg/ml against B. subtilis and S. aureus (gram-positive)
In an embodiment the antibacterial activity of concanavalin A nanocapsules is 100µg/ml against P. aeruginosa and E. coli (gram-negative) bacterial pathogenic strains.
The Lec-PCO-NC displayed significant antibacterial activity against all the test strains (Figure 10). The comparative zone of inhibition has been shown in graph (Figure 10). The maximum zone of inhibition at 100 µg/ml (22 mm) was obtained against E. coli and minimum zone of inhibition (15 mm) was obtained against P. aeruginosa (Table 4). This implies that the Lec-PCO-NC depicted broad spectrum of bactericidal activity. The exact mechanism by which PCOs work as antibacterial agents is not well understood.
Nonetheless, recent research suggests that primary mechanism behind antibacterial activity of plant flavonoids may be the inhibition of DNA gyrase in gram-negative bacteria.
while in case of gram positive bacteria the antibacterial activity is mainly based on their membrane action [Yan Y, et al 2024].
Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the invention with modifications. However, all such modifications are deemed to be within the scope of the invention.
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, Claims:1. Concanavalin A nanocapsules comprising concanavalin A and procyanidolic oligomer, wherein shell of nanocapsule comprises concanavalin A and procyanidolic oligomer loaded with in the nanocapsules.
2. The concanavalin A nanocapsules as claimed in claim 1, prepare by a method comprising;
i. dissolve the concanavalin A in 5 ml double distilled water and sonicate for 20 minutes;
ii. adjust the pH of solution to 2.8;
iii. add dropwise 5ml ethanol;
iv. add dropwise 500µl of calcium chloride (0.2% w/v);
v. after 20 minutes add 500 µl of freshly prepared solution of pluronic F-68 & polyethylene imine (PF+PEI);
vi. after 20 minutes add dropwise 500µl of aqueous solution of tween-80 (2% v/v);
vii. stir the solution 6-10 hours at room temperature;
viii. store the formulation after freeze drying.

3. The concanavalin A nanocapsules as claimed in claim 1, are spherical in shape.
4. The concanavalin A nanocapsules as claimed in claim 1, has size ranges from 34.78 nm to 266.64 nm.

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