A LIPOSOME COMPRISING BRONZAPHYRIN AND METHOD OF PREPARATION THEREOF

Abstract

The present disclosure relates to a liposome comprising a) Bronzaphyrin; and b) a phospholipid selected from the group consisting of soya lecithin, phosphatidylethanolamine (PE), Phosphatidylserine (PS), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC) or a combination thereof. The present disclosure also relates to a method of preparation of the nanocarrier composition.

Specification

Description:FIELD OF THE INVENTION:
[0001] The present disclosure relates to a liposome composition. The present disclosure particularly relates to the liposome of Bronzaphyrin and its method of preparation.

BACKGROUND OF THE INVENTION
[0002] The Bronzaphyrin (BP) is a NIR dye used in treating Cancer. A patent has already been granted for the Bronzaphyrin compound (Patent Application Number 202341004000).
[0003] However, it has not been possible to develop any formulation of Bronzaphyrin due to hydrophobic nature of the compound, it has low bioavailability in cancer tissue and thus low therapeutic efficacy in in cancer treatment. Moreover, BP's high solubility in DCM limits its direct use in cell culture studies because of its toxic nature, which are crucial for evaluating its anti-cancer properties.
[0004] Currently, the formulations and dyes, particularly those in the near-infrared (NIR) region, are available for imaging and therapeutic applications. These include various types of molecular probes and fluorophores. However, there are very few dyes that operate in the shortwave infrared (SWIR) region with peak emissions beyond 1000 nm, and none of these have received FDA approval. Existing cancer therapies also utilize different compounds and delivery systems, but they may not offer the same level of tissue penetration or imaging capabilities as SWIR dyes.
[0005] -The products, especially those based on NIR dyes, have limitations in tissue penetration and spatiotemporal resolution. They also suffer from higher levels of autofluorescence and scattering, which can reduce the clarity and accuracy of imaging. Additionally, existing formulations for anti-cancer compounds may not be compatible with cell culture studies or may not deliver the same level of effectiveness in inducing cancer cell apoptosis or disrupting mitochondrial membrane potential The lack of available and approved SWIR dyes further limits the options for high-resolution imaging and therapeutic applications in this wavelength range.
[0006] Thus, there is an unmet need exits for developing a nanocarrier composition of Bronzaphyrin, that address the compound's inherent limitations, such as low bioavailability and poor solubility, enhances cellular internalization, and increases the encapsulation, specific drug delivery, and the photothermal performance of the NIR dyes to overcome the issues of repeated dosage for treating cancer and fungal infections simultaneously.

OBJECTS OF THE INVENTION
[0007] Some of the objectives of the present disclosure, with at least one embodiment herein satisfied, are listed herein below:
[0008] It is the primary objective of the present disclosure to provide a liposomal composition of Bronzaphyrin (BP) so that BP is suitable for use in cell culture studies.
[0009] It is another objective of the present disclosure to overcome solubility issues as to address the problem of BP's high solubility in dichloromethane (DCM), which is incompatible with cell culture environments.
[0010] It is yet another objective of the present disclosure to enhance the bioavailability of BP in aqueous cell culture media to facilitate its use in biological applications.
[0011] It is another objective of the present disclosure to improve therapeutic effectiveness: of BP in anti-cancer applications by utilizing the liposomal formulation to target and kill cancer cells more efficiently.
[0012] It is yet another objective of the present disclosure to provide a liposomal composition that enables High-Resolution Imaging: for advanced imaging applications, including high-resolution and high-signal-to-noise ratio for biological imaging.
[0013] It is further objective of the present disclosure to provide a liposomal composition for reducing toxicity and ensuring compatibility with standard cell culture practices.
[0014] It is yet another objective of the present disclosure to provide a simple and cost-effective method for the preparation of liposomal composition.

SUMMARY OF INVENTION
[0015] The present disclosure relates to a liposome comprising
a) Bronzaphyrin; and
b) a phospholipid selected from the group consisting of soya lecithin, phosphatidylethanolamine (PE), Phosphatidylserine (PS), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC) or a combination thereof.
[0016] The present disclosure also relates to a method of preparing a liposome of Bronzaphyrin comprising:
a) dissolving Soya Lecithin lipid and Bronzaphyrin in a solvent to obtain a solution;
b) drying the solution obtained at a temperature range of 30°C to 50°C in a rotary evaporator under vacuum to obtain lipid film;
c) hydrating the lipid film with a phosphate buffer saline solution by rotating a round bottom flask at about 150 rpm to 190 rpm at temperature range of 50֠° C to 90 ° C to obtain a liposome suspension;
d) sonicating the liposome suspension was then sonicated using a probe sonicator to obtain small unilamellar liposomes of Bronzaphyrin.

BRIEF DESCRIPTION OF DRAWINGS
[0017] The present disclosure contains the following drawings that simply illustrates certain selected embodiments of the nanocarrier composition and processes that are consistent with the subject matter as claimed herein, wherein:
[0018] Figure 1 depicts Hydrodynamic size of A) blank Liposome , B) Liposome of Bronzaphyrin (LBP) analysis using DLS. C) UV-Vis absorbance spectra of Bronzaphyrin in different solvents.
[0019] Figure 2 depicts Photothermal transduction efficiency of LBP. A) Thermal Images, B) Thermal rise graph.
[0020] Figure 3 depicts Cell viability study in L929 cell lines with different doses of LBP for 24 h by MTT assay
[0021] Figure 4: depicts Cellular uptake of LBP in HeLa cells (Scale bar: 100 μ m). Red fluorescence in cells corresponds to Nile Red, green fluorescence in cells corresponds to Bronzaphyrin, blue fluorescence in cells corresponds to DAPI. Merge image depicts the cytoplasmic localization of nanoparticles.
[0022] Figure 5 depicts representative images of A) timewise uptake of LBP in HeLa Cells (Scale bar 100 µm), B) Reduction of the green fluorescence after laser irradiation in HeLa Cells (Scale bar 50 µm).
[0023] Figure 6 depicts HeLa cells cytotoxicity of LBP without and with NIR laser irradiation using MTT assay
[0024] Figure 7 depicts HeLa cells cytotoxicity of LBP without and with NIR laser irradiation using FDA/PI staining. Green fluorescence of FDA represents the live cell populations and red fluorescence of PI represents the dead cell populations (Scale bar: 100 μ m).
[0025] Figure 8 depicts the alteration of mitochondrial membrane potential analysis in HeLa cells of LBP without and with NIR laser irradiation using JC-1 staining. Green fluorescence represents the monomer or altered membrane potential and red fluorescence indicates the aggregates or functional membrane potential (Scale bar: 100 μ m).
[0026] Figure 9 depicts HeLa cells cytotoxicity of LBP without and with NIR laser irradiation using DCFDA analysis for ROS without and with NIR laser irradiation (Scale bar 100 µm).
[0027] Figure 10 depicts the nucleus fragment assay of HeLa cells after LBP treatment (Scale bar 100 µm). Yellow arrows indicate the fragmented nucleus.
[0028] Figure 11 depicts Apoptosis assay in Hela cells of LBP without and with NIR laser irradiation using Acridine Orange/Ethidium Bromide (AO/EB) dual staining (Scale bar 100 µm).

DESCRIPTION OF THE INVENTION:
[0029] A detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such detail as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
[0030] The terminology used herein is to describe particular embodiments only and is not intended to be limiting to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising", or "includes" and/or "including" or "has" and/or "having" when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
[0031] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0032] As used herein, the term "cancer" refers to a cell that displays uncontrolled growth and division, invasion of adjacent tissues, and often metastasizes to other locations of the body. Herein the cancer is cervical cancer.
[0033] The present disclosure relates to a liposome comprising
a) Bronzaphyrin; and
b) a phospholipid selected from the group consisting of soya lecithin, phosphatidylethanolamine (PE), Phosphatidylserine (PS), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC) or a combination thereof.
[0034] In an embodiment of the present disclosure, weight ratio of the Bronzaphyrin; and the phospholipid is in a ratio range of 1: 79 to 1: 119.
[0035] In another embodiment of the present disclosure, amount of the Bronzaphyrin is in range of 190 µg to 210 µg.
[0036] In another embodiment of the present disclosure, wherein amount of the soya lecithin lipid is in the range of 15 mg to 25 mg.
[0037] In an embodiment of the present disclosure, the liposome is prepared by thin film hydration method.
[0038] In an embodiment of the present disclosure, the Bronzaphyrin is encapsulated into the lipid bilayer of the liposome.
[0039] In yet another embodiment of the present disclosure, the liposome has a diameter in range of 80 to 160nm.
[0040] The present disclosure also relates to a method of preparing a liposome of Bronzaphyrin comprising:
a) dissolving Soya Lecithin lipid and Bronzaphyrin in a solvent to obtain a solution;
b) drying the solution obtained at a temperature range of 30°C to 50°C in a rotary evaporator under vacuum to obtain lipid film;
c) hydrating the lipid film with a phosphate buffer saline solution by rotating a round bottom flask at about 150 rpm to 190 rpm at temperature range of 50֠° C to 90 ° C to obtain a liposome suspension;
d) sonicating the liposome suspension was then sonicated using a probe sonicator to obtain small unilamellar liposomes of Bronzaphyrin.

[0041] In an embodiment of the present disclosure, the solvent is selected from chloroform, methanol or a combination thereof.
[0042] In another embodiment of the present disclosure, the solvent is chloroform methanol mixture in ratio of 2: 1.
[0043] In an embodiment, the present disclosure provides liposome of Bronzaphyrin that addresses the challenge of making BP a compound with strong potential for anticancer applications, compatible with cell culture studies. BP's high solubility in DCM limits its direct use in cell culture studies, which are crucial for evaluating its anti-cancer properties. The liposome formulation overcomes this challenge, making BP suitable for such studies while enhancing its effectiveness in killing cancer cells and its potential as a bioimaging agent.
[0044] The liposomal formulation enables BP to be used in anti-cancer applications by making it compatible for cell culture studies. It also exhibits green fluorescence, suggesting its potential as a bioimaging agent. The liposomes are 102.6 nm in size with a PDI of 0.345 and can achieve a hyperthermic condition of 45°C within 5 minutes at a concentration of 50 µg/mL. In HeLa cells, the formulation shows the highest cellular uptake of BP within 6 hours, causes greater damage to mitochondrial membrane potential, and induces more apoptosis compared to free BP.
[0045] In an embodiment of the present disclosure, the LBP shows Laser-Based Anti-cancer Efficacy:
• MTT: HeLa cells treated with LBP and exposed to NIR laser showed more significant cell death compared to those treated with free BP or untreated controls.
• FDA/PI: The LBP -treated group demonstrated significantly more cell death in HeLa cells compared to untreated controls, indicating the therapeutic potential of LBP NPs.
• JC-1: Treatment with LBP led to a shift in fluorescence from red to green in the JC-1 assay, indicating a disruption in mitochondrial membrane potential and suggesting the induction of apoptosis in HeLa cells.
• DCFDA: LBP induced significant reactive oxygen species (ROS) generation in HeLa cells, highlighting their role in causing oxidative stress and potential DNA damage.
• Nucleus Fragmentation Study: A significant increase in nuclear fragmentation was observed in HeLa cells treated with LBP, confirming their ability to induce apoptosis and genomic instability in cancer cells.
• Acridine Orange / Ethidium Bromide (AO/EB) Dual Staining: The LBP treated group exhibited elevated red fluorescence and fragmented nuclei, consistent with apoptosis-induced cell death, further validating the apoptotic effect of the LBP on cancer cells.
ADVANTAGES OF THE PRESENT INVENTION
[0046] In accordance with the present disclosure. a liposomal formulation of Bronzaphyrin (BP has the following advantages:
- Improved Therapeutic Delivery: The liposomal formulation enhances the solubility and bioavailability of BP, making it suitable for effective cellular uptake and targeted anti-cancer treatment.

- Enhanced Imaging Capabilities: The green fluorescence of BP, combined with the ability to induce a photothermal effect, provides high-resolution imaging and tracking capabilities, offering valuable insights into cellular and tissue dynamics.

- Optimized Treatment Protocols: The time-dependent fluorescence and subsequent quenching of BP post-laser treatment allow for precise optimization of treatment protocols, improving the efficacy of therapeutic interventions.

- Increased Efficacy in Cancer Therapy: The formulation's ability to target cancer cells and induce apoptosis, along with its photothermal effects, enhances its potential as a powerful anti-cancer agent.

- Advanced Research Tools: The findings support the use of BP as a bioimaging agent, expanding the toolkit for researchers studying cellular processes and therapeutic outcomes.

[0047] The present disclosure will be explained using the following examples:

EXAMPLE
[0048] Materials: The components involved in the synthesis of liposome of Bronzaphyrin are:
• (DMSO), (MTT), and Soya lecithin were acquired from SRL Chemicals Limited, India.
• Fluorescein diacetate (FDA), Propidium iodide (PI), 4 ,6-diamidino-2-phenylindole (DAPI), and 2 ′-7'-Dichlorodihydro fluorescein diacetate (DCFH-DA) were obtained from Sigma Aldrich.
• JC-1 dye were supplied by Invitrogen by Thermo Fisher Scientific, USA.
• Cell culture media-DMEM, RPMI-1640, FBS, Trypsin-EDTA, PBS, and antibiotics were obtained from HiMedia laboratories in India.

[0049] Maintenance of cell lines: L929 (mouse embryonic healthy fibroblast), and HeLa (human cervical cancer) were collected from NCCS, Pune, India. L929 and HeLa both were supported to grow in high glucose DMEM with FBS (10 %), gentamicin, and penicillin-streptomycin. The cells were kept in a sterile CO2 incubator at 5 % CO2 and 37 ◦C for further experiments.

Example 1
Preparation of Liposome of Bronzaphyrin:
[0050] The liposomes were prepared by the thin film hydration method. Briefly, lipids Soya Lecithin (20 mg) and Bronzaphyrin (BP) (200µg) was dissolved in a 2: 1 chloroform: methanol mixture. The solution was then dried in a rotary evaporator under vacuum to obtain a thin lipid film at 40◦ C. The lipid film was hydrated with a phosphate buffer saline solution by rotating the round bottom flask at about 180 rpm at 60◦ C until the lipid film was completely hydrated and a homogeneous dispersion was formed. The liposome suspension was then sonicated using a probe sonicator to obtain small bronzaphyrin-encapsulated unilamellar liposomes.

Example 2
Characterization of Liposome of Bronzaphyrin:
A. UV-Visible Analysis
[0051] The absorbance spectra of BP after dissolving into chloroform were recorded by a UV-VIS spectrophotometer (Shimadzu UV-1800, Japan).
Result
[0052] BP exhibits three major absorbance peaks at 497 nm, 807 nm, and 987 nm when dissolved in dichloromethane (DCM). During the synthesis of the liposomal Bronzaphyrin (LBP) formulation, BP was dissolved in chloroform, and it was observed that the absorbance peaks remained unaffected by this solvent change (Figure 1C).

B. Diameter and Polydispersity Index Analysis:
[0053] The diameter, polydispersity index of blank Liposome and Liposome of Bronzaphyrin (LBP) were measured using dynamic light scattering (Particle Sizing Systems, Inc., Santa Barbara, California, U.S. A.)
Result
[0054] ). DLS was employed for assessing the hydrodynamic diameter of the LBP. The hydrodynamic diameter of blank liposome was observed to be 102.6 nm with 0.345 PDI, (Figure 1 A). After loading BP, the size of LBP increased slightly to 108.4 nm with 0.317 PDI (Figure 1B), attributed to the successful loading of BP, with homogenous distributions.

C. Photothermal Transduction Efficiency:
[0055] The PTT efficiency of LBP, along with control groups, were investigated by placing in plastic well plates and exposing them to NIR irradiation using an 1064 nm NIR laser with a 650 mW output for 0, 1, 3, 5, and 7 min. A thermal camera was used to capture the thermal images throughout the NIR irradiation. Plots of each sample's temperature at various intervals were made.
Result
[0056] The LBP s were subsequently exposed to 1064 nm (650 mW) NIR laser radiation. The temperature of the LBP at 50 µg/mL concentration is higher than the temperature at 20 µg/mL (Figure 2). In 50 µg/mL LBP increased beyond 44 ◦C (within 5 min) due to an enhanced photothermal effect resulting from NIR laser irradiation. They maintained a steady temperature increase till 7 min after being exposed to a continuous NIR laser beam. These results revealed that LBP is a better photothermal agent for cancer therapy.

Example 3:
Biocompatibility testing on L929 cells:
[0057] In vitro, cyto-compatibility of LBP was tested on L929 cell lines using MTT assay. Cells (1 × 104 cells/well) were nurtured in 96 well plates and kept for 24 h in a CO2 incubator at 37 ◦C, allowing the adherent of cells to the surface. On the Next day, cells were incubated with different doses of LBP and further incubated for 24 h. After that, MTT assay was conducted by adding MTT (5 mg/mL) solution to each well and incubating for 3 h. Formazan crystals were allowed to be solubilized with the help of DMSO. The absorbance was recorded at 570 nm using a plate reader.
Result
[0058] MTT assay was carried out in normal cell lines L929 to demonstrate the reduction in cell viability with LBP incubation. L929 was treated with LBP at concentrations of 5, 10, 25, 50, 100, and 200 μg mL−1. LBP showed excellent biocompatibility up to 50 μg mL−1 in L929 (Fig. 3).

Example 4
Intracellular uptake study in HeLa cells:
[0059] In 6 well plates, HeLa cells (6 × 10 5 cells/well) were supported to grow on top of UV-sterile, clean coverslips. After seeding, cells were kept in a CO2 incubator for 24 h to reach confluency. Cells were incubated with LBPs for 3 h. After that, cells were fixed with 4 % PFA before being washed in PBS. To see the cell nucleus, cells in each well were treated with 4′,6-diamidino-2-phenylindole (DAPI) (1 μ g/mL) for 30 min. Biorad ZOE™ Fluorescent Cell Imager microscope was used to take the images.
Result
[0060] Intracellular uptake is one of the key experiments to determine the effectiveness of therapeutics. The cellular internalization of hydrophobic therapeutics is incredibly low and fall short of the standard for effectiveness. The therapeutic efficacy and cellular internalization of target-specific nanoparticles and dye/drugs with greater hydrophilicity can be quite high.
[0061] Nile Red, (NR) a hydrophobic dye with red fluorescence (stain the cell cytoplasm), was loaded into LBP to track cellular internalization. It was observed that HeLa cells with LBP have significant red fluorescence due to the presence of NR and have significant green fluorescence due to the presence of BP (Figure 4).
[0062] Cells were also stained with DAPI that stains the core of the nuclear region by emitting blue fluorescence. These observations demonstrated that the cellular uptake process of LBP may prove to be effective in treating cancer cells. Moreover, the green fluorescence of BP indicates its use as a bioimaging agent.
[0063] Time-dependent fluorescence analysis of Bronzaphyrin (BP) in HeLa cells was conducted to assess its intracellular accumulation. The results indicated that BP exhibited the highest level of green fluorescence at 6 hours post-uptake, suggesting maximum intracellular concentration at this time point (Figure 5 A). Based on this observation, laser treatment was administered to HeLa cells 6 hours after BP treatment to optimize the therapeutic effect, leveraging the peak intracellular presence of BP for enhanced efficacy.
[0064] Based on the photothermal effect exhibited by Bronzaphyrin (BP), it was hypothesized that laser irradiation would result in the degradation of BP, leading to a reduction in green fluorescence. This was supported by the observed results, where a significant decrease in BP fluorescence was noted after laser treatment compared to the fluorescence levels before irradiation (Figure 5 B), confirming the anticipated quenching effect. This observation supports the conclusion that the fluorescence observed was attributable solely to Bronzaphyrin (BP), as the quenching effect following laser treatment confirmed the direct correlation between BP presence and fluorescence intensity

Example 5
Laser-Based Cell Cytotoxicity in HeLa Cells:
[0065] The in vitro anti-cancer efficacy of the synthesized LBP was evaluated using HeLa cell lines. The photothermal killing of HeLa cells using LBP was initially assessed using the MTT assay. Briefly, 1 x 104 HeLa cells/well were seeded in a 96-well plate, and up on attachment, the cells were treated with BP and LBP. The cells were then irradiated with a 1064 nm laser for 7 min. the cell viability was quantified at 24 hours post treatment.
Result
[0066] To investigate the cell viability reduction without/with NIR laser irradiation, we incubated HeLa cells with LBP and control groups. Cells were irradiated with 1064 nm (650 mW) NIR laser for 5 min. More cell death was observed in LBP treated groups in comparison to free BP treated groups and untreated control groups (Figure 6).

Example 6
Assessment of cell viability using fluorescence imaging:
[0067] The qualitative assessment of NIR laser-based cytotoxicity was evaluated by FDA-PI staining. HeLa cells were cultured in 96 well plates and incubated till complete attachment to the surface of each well. Cells were treated with LBP and control groups, similar to the cytotoxicity study. Cells were exposed to 1064 nm NIR laser (650 mW) irradiation for 5 min and incubated further for 12 h. FDA and PI (both in 1 μ M concentration) were added to the respective wells. Live and dead cell images without and with NIR laser irradiation were taken by Biorad ZOE™ Fluorescent Cell Imager.
Result
[0068] FDA/PI analysis was performed in HeLa cells to evaluate the live and dead cell populations. After 24 h of treatment of HeLa cells with LBP, cells are subjected to FDA/PI staining and observed under a fluorescence microscope. No significant cell death was observed in untreated group (Figure 7). The LBP group showed more significant cell death than untreated, depicting that LBP is promising therapeutics to kill the HeLa cells.

Example 7
Analysis of mitochondrial membrane potential:
[0069] After liposomal treatment, the mitochondrial membrane potential change without and with NIR laser irradiation was examined using JC-1 dye. HeLa cells (1 × 10 4 cells/well) were seeded in 96 well plate and then incubated to attach to the surface of the well. After 2 h of incubation of cells with nanoparticles, the cells were given 5 min NIR laser treatment followed by a further 5 h of incubation. Cells were co-loaded with JC-1 dye (20 μ M) and incubated for 45 min. PBS was used to rinse the cells before capturing the images by ZOE™ cell imager using fluorescence filters. Red fluorescence indicates an active membrane potential, while green fluorescence indicates an alteration in membrane potential.
Result
[0070] Mitochondria control multiple cellular activities like ATP gene ration, redox balance maintenance, cell cycle regulation, and apoptosis. The mitochondrial membrane potential is a crucial marker for mitochondrial function. Impaired membrane potential leads to a series of consequences corresponding to apoptosis. Alteration of mitochondrial membrane potential was assayed using a membrane potential sensitive dye named JC1, which showed a shift of red fluorescence (aggregates) to green fluorescence (monomer) on the distortion of mitochondrial membrane potential. HeLa cells were treated with LBP showed the highest green fluorescence intensity, suggesting the impaired mitochondrial membrane potential of HeLa cells (Figure 8).

Example 8
Reactive oxygen detection by DCFDA analysis:
[0071] Free radical hyperaccumulations (ROS) in HeLa cells without and with NIR laser irradiation were measured using a fluorescence dye DCFH-DA (dichlorodihydrofluorescin diacetate). The DCFH-DA rapidly oxidized into DCF (2 ′ ,7 ′ 3 -dichlorofluorescein), sensing the presence of ROS. First, HeLa cells (1 × 104 cells/well) were seeded in 96 well plates, and after individual nanoparticles treatment, cells were incubated in an incubator for 2 h. Then, each well-containing cell was exposed to 1064 nm NIR laser radiation (650 mW), followed by an additional 5 h of incubation. Following this, cells were stained with DCFH-DA (20 μ M) and left to stand for 20 min. A fluorescent microscope was used to look at the cells.
Result
[0072] HeLa cells are incubated with respective control and treated groups to determine the DNA damage response. After 24 h, the cellular ROS generation was analysed using DCFDA with green fluorescence. A significant ROS level (green fluorescence) was observed in LBP in comparison with only liposome and untreated groups (Figure 9).

Example 9
D. Nucleus Fragmentation Study:
[0073] In 96 well plates, 4T1 cells were seeded, and after complete adherence, the cells were incubated with LBP for 6 hr in a CO2 incubator. Then, cells were undergone laser therapy by using 1064 laser and kept for 12 hr in a CO2 incubator. Further, DAPI staining (1 μg/ml) was done and kept for 1h incubation. The nucleus fragmentation was observed using a ZOETM fluorescence cell imager.
Result:
[0074] Nuclear fragmentation studies were performed to provide valuable insights into cell death mechanisms, treatment responses, and genomic stability. Nuclear fragmentation is a hallmark of apoptosis, a major programmed cell death mechanism. When cells undergo apoptosis, their DNA undergoes controlled cleavage into smaller fragments. Assessing nuclear fragmentation can confirm the presence of apoptotic cells within a population, validating the cell death mechanisms. We have observed that HeLa cells treated with LBP showed a significant number of fragmented nuclei compared to untreated and liposome control groups (Figure 10). This observation depicts that LBP can cause cell death by enhancing genomic instability or damaging the DNA of cancer cells.

Example 10
Detection of cell death by acridine orange/ethidium bromide dual staining:
[0075] Cell death induced by LBP was observed in ZOETM fluorescence cell imager. Briefly, 1 x 104 cells were seeded in each well of the 96 well plates. The cells were treated with LBP and the control group which received media only. After 6 hours of incubation Laser was put to generate the heat and again cells were kept for 24 hours inside the incubator. Thereafter the media were removed, and the cells were incubated with acridine orange (5 µg/ml) and ethidium bromide (5 µg/ml) for 15 min. The cells were washed with PBS and were then observed in ZOETM fluorescence cell imager using the filters of acridine orange DNA (green) and EtBr (Orange).
Statistical analysis:
[0076] The control and test groups were compared using Dunnett's & Tukey's test (One-Way ANOVA) (***P 0.001, **P 0.01, *P 0.05).
Result
[0077] AO/EB stain are crucial techniques for detecting apoptosis and fragmented nuclei. AO and EB bind to DNA in cells. AO intercalates into the DNA double helix, emitting green fluorescence in live cells, while EB, penetrating damaged cell membranes, emits red fluorescence when bound to fragmented or apoptotic cell DNA, allowing for the distinction between live and apoptotic cells based on nuclear morphology. The nuclear fragmentation assay complements this by specifically visualizing chopped or fragmented nuclei, providing valuable insights into cellular apoptosis processes. In the group treated with LBP, we noted elevated red fluorescence and fragmented nuclei, indicating apoptosis-induced cell death. These observations underscore the ability of LBP to induce DNA damage and apoptosis-mediated cell death (Figure 11).
 

SPECIFIC EMBODIMENTS OF THE PRESENT INVENTION
[0078] The present disclosure relates to a liposome comprising
a) Bronzaphyrin; and
b) a phospholipid selected from the group consisting of soya lecithin, phosphatidylethanolamine (PE), Phosphatidylserine (PS), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC) or a combination thereof.
[0079] Such a liposome, wherein weight ratio of the Bronzaphyrin; and the phospholipid is in a ratio range of 1: 79 to 1: 119.
[0080] Such a liposome, wherein an amount of the Bronzaphyrin is in range of 190 µg to 210 µg.
[0081] Such a liposome, wherein an amount of the soya lecithin lipid is in range of 15 mg to 25 mg.
[0082] Such a liposome, wherein the liposome is prepared by thin film hydration method.
[0083] Such a liposome, wherein the Bronzaphyrin is encapsulated into the lipid bilayer of the liposome.
[0084] Such a liposome, wherein the liposome has a diameter in range of 80 to 160nm.
[0085] The present disclosure also relates to a method of preparing a liposome of Bronzaphyrin comprising:
a) dissolving Soya Lecithin lipid and Bronzaphyrin in a solvent to obtain a solution;
b) drying the solution obtained at a temperature range of 30°C to 50°C in a rotary evaporator under vacuum to obtain lipid film;
c) hydrating the lipid film with a phosphate buffer saline solution by rotating a round bottom flask at about 150 rpm to 190 rpm at temperature range of 50֠° C to 90 ° C to obtain a liposome suspension;
d) sonicating the liposome suspension was then sonicated using a probe sonicator to obtain small unilamellar liposomes of Bronzaphyrin.
[0086] Such a method, wherein the solvent is selected from chloroform, methanol or a combination thereof.
[0087] Such a method, wherein the solvent is chloroform-methanol mixture in a ratio of 2: 1.
, Claims:WE CLAIM
1. A liposome comprising
a) Bronzaphyrin; and
b) a phospholipid selected from the group consisting of soya lecithin, phosphatidylethanolamine (PE), Phosphatidylserine (PS), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC) or a combination thereof.

2. The liposome as claimed in claim 1, wherein weight ratio of the Bronzaphyrin; and the phospholipid is in a ratio range of 1: 79 to 1: 119.

3. The liposome as claimed in claim 1, wherein an amount of the Bronzaphyrin is in range of 190 µg to 210 µg.

4. The liposome as claimed in claim 1, wherein an amount of the soya lecithin lipid is in range of 15 mg to 25 mg.

5. The liposome as claimed in claim 1, wherein the liposome is prepared by thin film hydration method.

6. The liposome as claimed in claim 1, wherein the Bronzaphyrin is encapsulated into the lipid bilayer of the liposome.

7. The liposome as claimed in claim 1, wherein the liposome has a diameter in range of 80 to 160nm.

8. A method of preparing a liposome of Bronzaphyrin comprising:
a) dissolving Soya Lecithin lipid and Bronzaphyrin in a solvent to obtain a solution;
b) drying the solution obtained at a temperature range of 30°C to 50°C in a rotary evaporator under vacuum to obtain lipid film;
c) hydrating the lipid film with a phosphate buffer saline solution by rotating a round bottom flask at about 150 rpm to 190 rpm at temperature range of 50֠° C to 90 ° C to obtain a liposome suspension;
d) sonicating the liposome suspension was then sonicated using a probe sonicator to obtain small unilamellar liposomes of Bronzaphyrin.

9. The method as claimed in claim 8, wherein the solvent is selected from chloroform, methanol or a combination thereof.

10. The method as claimed in claim 9 wherein the solvent is chloroform methanol mixture in ratio of 2: 1.

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