COMPOSITION FOR DELIVERY OF THERAPEUTIC COMPLEX WITHIN GOLGI APPARATUS AND METHOD OF PRODUCING THE SAME

Abstract

ABSTRACT COMPOSITION FOR DELIVERY OF THERAPEUTIC COMPLEX WITHIN GOLGI APPARATUS AND METHOD OF PRODUCING THE SAME The present invention describes a composition (100) and a method (200) for producing the same, for targeted delivery of a therapeutic complex for the treatment of cells within Golgi apparatus in a human subject. The composition includes a carrier agent (102), a cell binding agent (104), and a staining agent (106). The carrier agent (102) includes a plurality of naturally sourced nanoparticles. The cell binding agent (104) is covalently functionalized to a surface of the plurality of naturally sourced nanoparticles. The staining agent (106) is conjugated with the cell binding agent. The cell binding agent (104) is designed to selectively target the Golgi apparatus whereas the staining agent is designed to (i) trace the nanoparticles via its fluorescent properties, and (ii) bombard the Golgi apparatus in melanoma cells. (Fig. 1)

Specification

Description:[001] FIELD OF THE INVENTION
[002] The present invention relates to therapeutic complex delivery, and particularly, to a composition for targeted delivery of a therapeutic complex for the treatment of cells within Golgi apparatus in a human subject, and a method of producing the same.

[003] BACKGROUND OF THE INVENTION
[004] Background description includes information that may be useful in understanding the present invention.
[005] The current cancer treatment modalities are non-specific, and fill makes chemotherapy most damaging to healthy organs and tissues. Targeting cancer cells specifically while leaving normal cells undisturbed remains a significant challenge in cancer treatment. Recent advances reveal the Golgi apparatus as a potential cancer therapy focus as it is involved in the trafficking and processing of proteins necessary for the growth and replication of cancer cells. However, the B16 melanoma cell line is aggressive and resistant to most treatments, making conventional therapies more difficult.
[006] Several strategies and products are currently available for the treatment of melanoma, including:
[007] Immunotherapy
[008] Anti-PD-1/PD-L1 (e.g., Nivolumab, Pembrolizumab): It enhances the ability of the immune system to attack melanoma by blocking the PD-1/PD-L1 pathway used by the tumor cells to evade immune recognition.
[009] Anti-CTLA-4 (e.g., Ipilimumab): It unleashes activity in T cells within the body to better find and kill melanoma by blocking the CTLA-4 receptor, which inhibits the body's immune response.
[0010] Targeted Therapy
[0011] BRAF Inhibitors (e.g., Vemurafenib, Dabrafenib): They target the BRAF gene, which MEK Inhibitors (e.g., Trametinib): Generally combined with BRAF inhibitors for downstream signaling inhibition, causing a reduction in proliferation that would otherwise occur in the tumor.
[0012] Oncolytic Viruses
[0013] Talimogene Laherparepvec (T-VEC): A herpes virus genetically engineered to selectively replicate inside melanoma cells and kill them, all while inducing a generalized anti-tumor immune attack.
[0014] These epitomize the most selective and effective treatments for managing B16 melanoma by leveraging the immune system, targeting genetic differences, and using viral therapies.
[0015] A patent application US20070190056A1 titled "Chondroitin Sulfate Carriers for Drug Delivery" describes novel conjugates of chondroitin sulfate with utility for targeted drug delivery, especially in cancer therapy.
[0016] A patent US9457014B2 titled "Nanoparticles Formulations for Targeted Drug Delivery" concerns the nanoparticle formulation process for drug delivery using natural extracts and possibly some Spirulina nanoparticles. These nanoparticles are developed to make a therapeutic agent more stable, bioavailable, and targeted.
[0017] Another patent application US20130058925A1 introduces glycan-coated nanoparticles as a new agent for Golgi apparatus targeting. The glycan-coated nanoparticles, including those functionalized with GalNAc target the Golgi apparatus in cells.
[0018] The current treatments for B16 melanoma have some problems, such as:
[0019] Lack of Target Specificity: Chemotherapy (e.g., Dacarbazine): The non-selective action of chemotherapy leads to generalized injury of the normal cells, and this is why it results in severe side effects like vomiting and damage to body organs thus limiting the dose that can be administered safely.
[0020] Rapid Development of Resistance: BRAF Inhibitors (e.g., Vemurafenib): B16 melanoma cells quickly develop resistance through secondary mutations or activation of alternative signaling pathways, thereby causing treatment failure and tumor return.
[0021] Severe Autoimmune Reactions: Checkpoint Inhibitors (e.g., Nivolumab, Ipilimumab): The oversensitivity of the immune system causes immune-based side effects like colitis and hepatitis by some drugs, which sometimes attack healthy tissues alongside the tumor.
[0022] Resistance to Radiation therapy: B16 melanoma cells are usually intrinsically resistant to radiation; hence, incomplete control over the tumor is required in higher doses that may hurt neighboring normal tissues.
[0023] Short Duration of Effectiveness: Oncolytic Viruses (e.g., T-VEC): Unfortunately, due to speedy neutralization by the immune system and unresponsiveness from most patients, such interventions' therapeutic effects do not last long, meaning they are ineffective in treating B16 melanoma cases.
[0024] Therefore, there is long desired need of a drug-delivery composition for improved specific targeting with high specificity and reduced systemic toxicity, which not only overcomes the drug resistance toward melanoma cells but simultaneously tracks the drug delivery in real-time facilitating precision treatment vis-à-vis significant accumulation at tumor tissues.

[0025] OBJECTS OF THE INVENTION:
[0026] Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are listed herein below.
[0027] The primary objective of the present invention is to provide a drug-delivery composition for improved specific targeting with high specificity and reduced systemic toxicity, which not only overcomes the drug resistance toward melanoma cells but simultaneously tracks the drug delivery in real-time facilitating precision treatment vis-à-vis significant accumulation at tumor tissues.
[0028] These and other objects and advantages will become more apparent when reference is made to the following description and accompanying drawings.

[0029] SUMMARY OF THE INVENTION
[0030] This summary is provided to introduce concepts related to a composition for targeted delivery of a therapeutic complex for the treatment of cells within Golgi apparatus in a human subject, and a method of producing the same. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
[0031] In an aspect of the present invention, a composition for targeted delivery of a therapeutic complex for the treatment of cells within Golgi apparatus in a human subject is described. The composition includes a carrier agent, a cell binding agent, and a staining agent. The carrier agent includes a plurality of naturally sourced nanoparticles. The cell binding agent is covalently functionalized to a surface of the plurality of naturally sourced nanoparticles. The staining agent is conjugated with the cell binding agent. The cell binding agent is designed to selectively target the Golgi apparatus whereas the staining agent is designed to (i) trace the nanoparticles via its fluorescent properties, and (ii) bombard the Golgi apparatus in melanoma cells.
[0032] In an embodiment of the present invention, the plurality of naturally sourced nanoparticles is obtained from an microalgae.
[0033] In another embodiment of the present invention, the microalgae is a lipid extract of Spirulina sp.
[0034] In another embodiment of the present invention, an average particle size of the plurality of naturally sourced nanoparticles is 193nm.
[0035] In another embodiment of the present invention, the cell binding agent is chondroitin sulfate.
[0036] In another aspect of the present invention, the cell binding agent consists of chondroitin sulfate and Nacetylgalactosamine, GalNAc.
[0037] In another aspect of the present invention, a concentration of the cell binding agent is 1mg/ml.
[0038] In another aspect of the present invention, the cell binding agent attaches selectively with CD44 receptors overexpressed on the Golgi apparatus on a surface of B16 melanoma cells.
[0039] In another aspect of the present invention, the staining agent is Coumarin 6.
[0040] In another aspect of the present invention, a concentration of the staining agent is 50µg/ml.
[0041] In another aspect of the present invention, the staining agent is capable of subsequent spatial detection of the plurality of naturally sourced nanoparticles in the subject during the therapeutic complex's delivery, using a fluorescence microscope.
[0042] In another aspect of the present invention, the staining agent is capable of subsequent temporal detection of the plurality of naturally sourced nanoparticles in the subject during the therapeutic complex's delivery, using a fluorescence microscope.
[0043] In another aspect of the present invention, the staining agent is capable of disrupting processes in the Golgi apparatus once inside the melanoma cells.
[0044] In another aspect of the present invention, a method of producing a composition for targeted delivery of a therapeutic complex for the treatment of cells within the Golgi apparatus in a human subject is described. The method includes the step of obtaining a carrier agent, wherein the carrier agent consists of a plurality of naturally sourced nanoparticles. The method further includes the step of mixing the plurality of naturally sourced nanoparticles with a staining agent in an organic solvent. The method further includes the step of coating a surface of the plurality of naturally sourced nanoparticles with a cell binding agent. The coating enables the cell binding agent to covalently functionalized with the plurality of naturally sourced nanoparticles. The method further includes the step of loading the staining agent by conjugating with the cell binding agent. The cell binding agent is designed to selectively target the Golgi apparatus, whereas the staining agent is designed to (i) trace the distributed nanoparticles via its fluorescent properties, and (ii) bombard the Golgi apparatus in melanoma cells.
[0045] In an embodiment of the present invention, the plurality of naturally sourced nanoparticles are extracted from a microalgae.
[0046] In another embodiment of the present invention, the microalgae is a lipid extract of Spirulina sp.
[0047] In another embodiment of the present invention, the organic solvent is a combination of Chloroform and Methanol in a ratio of 2:1.
[0048] In another embodiment of the present invention, the organic solvent is evaporated at 50-55 ºC before coating the surface of the plurality of naturally sourced nanoparticles with the cell binding agent.
[0049] In another embodiment of the present invention, the cell binding agent is Chondroitin Sulfate.
[0050] In another embodiment of the present invention, the Chondroitin Sulfate taken for coating is 2 ml of 1mg/ml concentration.
[0051] In another embodiment of the present invention, the plurality of naturally sourced nanoparticles coated with the cell binding agent is sonicated for 3 cycles, with 10 minutes per each cycle.
[0052] In another embodiment of the present invention, the staining agent is coumarin 6.
[0053] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

[0054] BRIEF DESCRIPTION OF DRAWINGS:
[0055] The illustrated embodiments of the subject matter will be understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description is intended only by way of example and simply illustrates certain selected embodiments of devices, apparatus, systems, and methods that are consistent with the subject matter as claimed herein, wherein:
[0056] FIG. 1 illustrates a structural diagram of a composition for targeted delivery of a therapeutic complex for the treatment of cells within Golgi apparatus in a human subject, in accordance with an exemplary embodiment of the present disclosure;
[0057] FIG. 2 illustrates a block diagram depicting a method of producing a composition for targeted delivery of a therapeutic complex for the treatment of cells within the Golgi apparatus in a human subject, in accordance with an exemplary embodiment of the present disclosure;
[0058] FIGs. 3a & 3b illustrate a comparison of hydrodynamic diameter of the nanoalgsomes with sample 1 of a plain algosome, sample 2 of Algosome-Chondroitin sulphate complex, and sample 3 of Algosome-Chondroitin SulphateCoumarin 6 complex, in accordance with an exemplary embodiment of the present disclosure;
[0059] FIG. 4 illustrates UV-Vis spectra of the final algosome nanoparticles loaded with coumarin 6, in accordance with an exemplary embodiment of the present disclosure;
[0060] FIG. 5 illustrates a comparison of cell viability of plain algosome (Alg)-based nanoparticle, algosome with chondroitin sulfate (CS), and addition of coumarin 6 (C6) under B16 cell lines, in accordance with an exemplary embodiment of the present disclosure; and
[0061] FIGs. 6a & 6b illustrate a live dead assay for B16 cell line for the particle synthesized with algosome coated with chondroitin sulfate and coumarin 6 where Control indicates the untreated one, in accordance with an exemplary embodiment of the present disclosure.
[0062] The figures depict embodiments of the present subject matter for the purposes of illustration only. A person skilled in the art will easily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.

[0063] DESCRIPTION OF THE INVENTION:
[0064] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered 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 as defined by the appended claims.
[0065] While the embodiments of the disclosure are subject to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the figures and will be described below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure.
[0066] The terms "comprises", "comprising", or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, or assembly that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such system, or assembly, or device. In other words, one or more elements in a system or device proceeded by "comprises… a" does not, without more constraints, preclude the existence of other elements or additional elements in the system or device.
[0067] The present invention is a drug delivery composition and a method of producing the same, aimed at improving the treatment of B16 melanoma (skin cancer) through a novel approach using a nanoparticle carrier agent derived from Spirulina algae. The composition includes three different components: a cell binding agent (e.g., chondroitin sulfate), a carrier agnt (e.g., Nacetylgalactosamine (GalNAc)), and a staining agent (e.g., coumarin 6). Each component specifically targets melanoma cells and interferes with cellular processes, allowing cancer cell survival.
[0068] The present invention concentrates the nanoparticle carrier agent to melanoma cells because systemic administration of cell binding agent functionalized biocompatible carbon dots may bind to CD44 receptors that overexpress on the cancer cell surface and deliver the cargo through the GalNAc-ASGPR pathway, leading endosome escape followed by disruption of Golgi-apparatus based cellular metabolic processes indispensable for tumor cell survival.
[0069] The present invention provides the above-mentioned components as drugs to enhance the best possible treatment of cancer affecting the Golgi apparatus within the cell, which is a crucial organelle for processes involved in protein synthesis, processing, and trafficking. The composition is based on the additive effects of the cell binding agent and the staining agent incorporated in a biocompatible encapsulated nanoparticle carrier agent. Using the composition improves drug and gene transfer efficiency and safety by reducing side effects and increasing the selectivity of drug action towards the Golgi apparatus, a pivotal organelle responsible for the post-translational modification and sorting of proteins destined to be active within the cancer cell.
[0070] For better understanding, one or more embodiments of the present invention shall be described with respect to the earlier-mentioned drawings.
[0071] FIG. 1 illustrates a structural diagram of a composition (100) for targeted delivery of a therapeutic complex for the treatment of cells within Golgi apparatus in a human subject, in accordance with an exemplary embodiment of the present disclosure.
[0072] The composition (100) is engineered to overcome the unique delivery challenges of melanoma cancer cell lines. As illustrated, the composition (100) includes a carrier agent (102), a cell binding agent (104), and a staining agent (106). The carrier agent (102) includes a plurality of naturally sourced nanoparticles. The cell binding agent (104) is covalently functionalized to a surface of the plurality of naturally sourced nanoparticles. The staining agent (106) is conjugated with the cell binding agent. The cell binding agent (104) is designed to selectively target the Golgi apparatus. The staining agent (106) is designed to (i) trace the nanoparticles via its fluorescent properties, and (ii) bombard the Golgi apparatus in melanoma cells.
[0073] In an embodiment of the present invention, the plurality of naturally sourced nanoparticles is obtained from a microalgae.
[0074] In another embodiment of the present invention, the microalgae is a lipid extract of Spirulina sp.
[0075] In another embodiment of the present invention, an average particle size of the plurality of naturally sourced nanoparticles is 193nm.
[0076] In another embodiment of the present invention, the cell binding agent (104) is chondroitin sulfate.
[0077] In another aspect of the present invention, the cell binding agent (104) consists of chondroitin sulfate and Nacetylgalactosamine, GalNAc.
[0078] In another aspect of the present invention, a concentration of the cell binding agent (104) is 1mg/ml.
[0079] In another aspect of the present invention, the cell binding agent (104) attaches selectively with CD44 receptors overexpressed on the Golgi apparatus on a surface of B16 melanoma cells.
[0080] In another aspect of the present invention, the staining agent (106) is Coumarin 6.
[0081] In another aspect of the present invention, a concentration of the staining agent (106) is 50µg/ml.
[0082] In another aspect of the present invention, the staining agent (106) is capable of subsequent spatial detection of the plurality of naturally sourced nanoparticles in the subject during the therapeutic complex's delivery, using a fluorescence microscope.
[0083] In another aspect of the present invention, the staining agent (106) is capable of subsequent temporal detection of the plurality of naturally sourced nanoparticles in the subject during the therapeutic complex's delivery, using a fluorescence microscope.
[0084] In another aspect of the present invention, the staining agent (106) is capable of disrupting processes in the Golgi apparatus once inside the melanoma cells.
[0085] The present formulation employs the cell binding agent and the staining agent encapsulated in the nanoparticle carrier agent obtained from algosome-a lipid extract of the algae Spirulina. Therefore, the present formulation appears promising for selective targeting of the Golgi apparatus in tumor cells, as the lipid nature of the algosome particles increases the delivery and efficacy of the encapsulated agents.
[0086] The cell binding agent i.e. Chondroitin Sulfate, an endogenously available glucosamine goodness, a family of polysaccharides, is known to bind selectively with specific cell membrane receptors, especially over-expressed in cancer cells. In the present invention, the cell binding agent acts as a targeting component that only attaches to specific receptors found on the surface of B16 melanoma cells and delivers the therapeutical complex into the cells. Once in the cell, the cell binding agent naturally, being within the cell, directs drugs targeting the Golgi apparatus to its location. The staining agent i.e. Coumarin 6, a fluorescent dye, forms part of the invention as a tracer for the expression of other components and fixation of the therapeutic complex into target tissues, as well as the cytotoxicity effects it exerts.
[0087] The present formulation is targeted to the Golgi apparatus of the B16 melanoma cells through a dual-ligand strategy by overexpression of CD44 receptors and Asialoglycoprotein receptor (ASGPR). The conjugation of cell binding agent to algosome nanoparticle carrier agent is performed because it has a high affinity for CD44, a receptor commonly overexpressed on the surface of melanoma cells. Upon interaction with CD44, the nanoparticles (NPs) undergo endocytosis via the receptor-mediated pathway, moving further through the endosomal route toward the Golgi. This pathway is significant for conferring specific localization of the therapeutic agents to the Golgi, where perturbation of protein processing and trafficking functions essential for the survival of cancer cells is hindered in action.
[0088] Meanwhile, the nanoparticle carrier agents are further functionalized with N-acetylgalactosamine (GalNAc), which is noted to specifically bind with ASGPR and mediates direct trafficking to the Golgi. This interaction between GalNAc and ASGPR serves as a secondary targeting mechanism to ensure that the nanoparticles are efficiently delivered into the Golgi. Once released into the Golgi, the therapeutic moieties can interfere sufficiently and disrupt the normal functioning of the Golgi's post-translational modifications and vesicular transport processes, which halt the protein secretory pathway and, therefore, melanoma cell proliferation. This would be with enhanced specificity and maximal therapeutic impact at this crucial intracellular target involved in cancer progression.
[0089] The present Nano formulation for melanoma treatment includes several crucial features that contribute to its effectiveness:
[0090] 1. Algosome Nanoparticles carrier agent from Spirulina: These nanoparticles are derived from "Spirulina" algae and thus represent a natural, lipid-based drug carrier. Using a natural source such as Spirulina significantly ensures the particles are safe, biocompatible, and have fewer side effects. The algosome structure also has inherent advantages for encapsulating and facilitating the controlled release of the drug, enhancing the treatment's overall efficacy while reducing potential harm to the patient.
[0091] 2. Chondroitin sulfate cell binding agent for targeting cancer cells: The Chondroitin Sulfate is incorporated into the algosome nanoparticles to specifically target the CD44 receptor, which is known to be elevated in B16 melanoma cells. The Chondroitin Sulfate is a glycosaminoglycan that occurs naturally in the extracellular matrix and binds to CD44 receptors -at higher levels on many cancer cells, such as melanoma. It is innovative in the design-based nanoparticle formulation for cancer treatment, as this prevailing natural ligand-receptor interaction can be exploited and could provide high specificity to target only cancer cells. Such specificity is important for minimizing off-target effects, decreasing systemic toxicity, and enhancing the therapeutic index.
[0092] 3. Coumarin 6 staining agent (dual function) for visualizing treatment in real-time & therapeutic activity: Coumarin 6 is used as a fluorescent marker to visualize drug delivery in real-time for improved monitoring. Traditionally, coumarin 6 has shown strong fluorescent function and has been successfully employed as a tracking agent via its fluorescent properties to trace distributed nanoparticles in biology. In the present invention, the use of coumarin 6 is not limited to visualization purposes. Instead, coumarin 6 is designed to bombard the Golgi apparatus in melanoma cells as a therapeutic effect, causing inhibition and, ultimately, cancer cell destruction. The ability of coumarin 6 to function as a dye and a therapeutic agent is innovative, as the simultaneous ability to track and treat patients is unprecedented. The dual-functioning capability of coumarin 6 provides a response and tool for precise, localized cancer therapy by targeting the Golgi apparatus, which is a less frequently targeted organelle.
[0093] The therapeutic response to treatment with coumarin 6 is a new means of altering the course of chemoresistance by visualizing the treatment response and possibly changing the treatment approach to overcome drug resistance. Incorporating chondroitin sulfate and coumarin 6 in the present invention will undoubtedly improve the efficacy of treating cancers more goal oriented. Using the receptor CD44, the Chondroitin Sulfate further serializes targeting to melanoma cells, ensuring that n leaf takes only in cancerous and minimized side effects and when their safety profile was exquisite. Given the real-time imaging trace provided by coumarin 6, the treatment can be observed and regulated to improve according to individual responses. Combined, these form a highly advantageous and novel strategy for melanoma therapy aimed at overcoming the general drawbacks of current treatment strategies by improving both tumor specificity and efficacy in addition to biological individualization.
[0094] FIG. 2 illustrates a block diagram depicting a method (200) of producing a composition (100) for targeted delivery of a therapeutic complex for the treatment of cells within the Golgi apparatus in a human subject, in accordance with an exemplary embodiment of the present disclosure.
[0095] As illustrated, the method (200) includes the step of obtaining (202) a carrier agent. The carrier agent consists of a plurality of naturally sourced nanoparticles. The method (200) further includes the step of mixing (204) the plurality of naturally sourced nanoparticles with a staining agent in an organic solvent. The method (200) further includes the step of coating (206) a surface of the plurality of naturally sourced nanoparticles with a cell binding agent. The coating enables the cell binding agent to covalently functionalized with the plurality of naturally sourced nanoparticles. The method (200) further includes the step of loading (208) the staining agent by conjugating with the cell binding agent. The cell binding agent is designed to selectively target the Golgi apparatus. The staining agent is designed to (i) trace the distributed nanoparticles via its fluorescent properties, and (ii) bombard the Golgi apparatus in melanoma cells.
[0096] In an embodiment of the present invention, the plurality of naturally sourced nanoparticles are extracted from a microalgae.
[0097] In another embodiment of the present invention, the microalgae is a lipid extract of Spirulina sp.
[0098] In another embodiment of the present invention, the organic solvent is a combination of Chloroform and Methanol in a ratio of 2:1.
[0099] In another embodiment of the present invention, the organic solvent is evaporated at 50-55 ºC before coating the surface of the plurality of naturally sourced nanoparticles with the cell binding agent.
[00100] In another embodiment of the present invention, the cell binding agent is Chondroitin Sulfate.
[00101] In another embodiment of the present invention, the Chondroitin Sulfate taken for coating is 2 ml of 1mg/ml concentration.
[00102] In another embodiment of the present invention, the plurality of naturally sourced nanoparticles coated with the cell binding agent is sonicated for 3 cycles, with 10 minutes per each cycle.
[00103] Now, the production of different components of the composition shall be described.
[00104] Formation of Algosome Nanoparticles as a carrier agent
[00105] Algosome is extracted from Spirulina Sp. where it is mixed with Chloroform and Methanol (Chloroform and Methanol are taken in ratio 2:1), and solvent evaporation is done completely at 50-55 ºC, which takes around 40-45 minutes. It is then mixed with 2 ml of distilled water. Dialysis is done for two hours under 250rpm at room temperature. The final particle was obtained at the transfer to the tube and stored for further use.
[00106] Algosome carrier agent loaded with chondroitin sulfate cell binding agent
[00107] Similar to the above, the plain algosome is extracted from the microalgae (Spirulina sp.). The extracted algosome is mixed with a solvent of Chloroform: Methanol (Chloroform: Methanol taken in ration 2:1). It evaporates with solvent evaporation at 50-55 ºC. After the complete evaporation, 2 ml of 1mg/ml (Chondroitin Sulfate) is used to extract and coat the outer layer of the algosome. It is collected and transferred in the Eppendorf tube and sonicated for 3 cycles, with 10 minutes per each cycle. Finally, the component was aggregated, and the hydrophobic algosome was surrounded and coated with chondroitin sulfate. The coumarin 6 was loaded, which acts as a therapeutic and fluorescent dye. Initially, coumarin 6 is green in most organic solvents, but the final color changes to yellow after the particle synthesis. It also indicates that there is bonding and a combination of these components. Since there was very little pellet. Dialysis was done for two hours under 250rpm at room temperature. The final particle was obtained at the transfer to the Eppendorff tube and stored for further use.
[00108] Inclusion of Coumarin 6 as a staining agent
[00109] Initially, the plain algosome is extracted from the microalgae (Spirulina sp.), which was used using chloroform. The extracted algosome is mixed with coumarin 6 as both are soluble in organic solvent; here, the solvent used is Chloroform and Methanol taken in ratio 2:1. It evaporates with solvent evaporation at 50-55ºC. After the complete evaporation, 2 ml of 1mg/ml (i.e., Chondroitin Sulfate) is used to extract and coat the outer layer of the algosome. It is collected and transferred in the Eppendorf tube and sonicated for 3 cycles, with 10 minutes per each cycle. Finally, the component was aggregated, and the hydrophobic algosome was surrounded and coated with chondroitin sulfate. The coumarin 6 was loaded, which acts as a therapeutic and fluorescent dye. Initially, coumarin 6 is green in most organic solvents, but the final color changes to yellow after the particle synthesis. It also indicates that there is bonding and a combination of these components. Since there was very little pellet. Dialysis was done for two hours under 250rpm at room temperature. The final particle was obtained at the transfer to the Eppendorff tube and stored for further use.
[00110] Coumarin 6, the stating agent, has a dual purpose:
[00111] 1. Fluorescent Marker - Coumarin 6 allows real-time analysis of the drug system within a biological system. It fluoresces for subsequent detection with a fluorescence microscope, allowing where the algosome nanoparticles to be spread (spatial) and specifically located (temporal) within the sample during the drug delivery process.
[00112] 2. Therapeutic Agent: The general introduction of coumarin 6 into melanoma cells disrupts the Golgi apparatus. This disruption leads to the loss of normal cellular functions such as protein processing and trafficking and is followed by cancer cell death.
[00113] Targeting and Therapeutic Action:
[00114] After administration, the functionalized algosome nanoparticles will circulate in the bloodstream. The chondroitin sulfate will help guide the nanoparticle to melanoma by binding with the CD44 receptor. Once inside melanoma cells, coumarin 6 disrupts the Golgi apparatus, which leads to cellular damage and cell death.
[00115] The therapeutic agent can be directed to the Golgi apparatus of malignant cells while being tracked in real-time. Enhanced delivery of targeted therapies is achieved while systemic side effects are minimized. This inventive technology advances the field of directed therapies in melanoma and addresses some of the current gaps in treatment, such as resistance and non-specific toxicity in treatment.
[00116] Interaction of Components
[00117] Algosome Nanoparticles as the carrier agent and Chondroitin Sulfate as the cell binding agent:
[00118] The algosome nanoparticles are specifically functionalized with chondroitin sulfate to bond with CD44 receptors found on B16 melanoma cells. Through interaction between chondroitin sulfate and CD44 receptors on B16 melanoma cells, the algosome nanoparticles can specifically accumulate at the cancer cell origin.
[00119] Algosome Nanoparticles as the carrier agent and Coumarin 6 as the staining agent:
[00120] Coumarin 6 is included in the algosome nanoparticles to ensure the dye is delivered along the nanoparticles to the target cells. The fluorescent signature associated with coumarin 6 allows for tracking nanoparticles in real-time. Coumarin 6 also has a therapeutic interaction to disrupt processes in the Golgi apparatus in melanoma cells due to the significance of the Golgi apparatus as a classic non-mitochondrial mediating of cell death.
[00121] Chondroitin Sulfate as the cell binding agent and Coumarin 6 as the staining agent:
[00122] The targeting capability of chondroitin sulfate ensures nanoparticles carrying coumarin 6 will specifically concentrate at B16 melanoma cells. Following cellular uptake of nanoparticles, coumarin 6 will also further disrupt cellular processes in the Golgi apparatus, while the fluorescent signature confirms the effectiveness and monitoring of nanoparticle targeting and distribution.
[00123] Overall, the cumulative actions of chondroitin sulfate targeting and the dual roles of coumarin 6 (tracked for monitoring - and therapeutic) improve treatment specificity in this approach. Algosome nanoparticles can deliver, target, and confirm the delivery of coumarin 6 to cancer cells and potentially utilize coumarin 6 for efficient and specific disruption of cellular processes in the Golgi apparatus.
[00124] As discussed below, the present invention implements targeted, tracking, and therapeutic functions at an enhanced single nanoparticle platform representing a technological innovation. The specificity of targeted delivery of therapeutic complex and real-time assessment of efficacy because of the increased tracking capabilities represent true advancement in cancer treatment state-of-the-art technologies.
[00125] a. Algosome Nanoparticles as the carrier agent with Chondroitin Sulfate as the cell binding agent
[00126] The targeted delivery of the algosome nanoparticles with chondroitin sulfate represents novel targeted capabilities. Chondroitin sulfate is a glycosaminoglycan that directly binds to CD44 receptors, which are known to be highly expressed in B16 melanoma cells. The novel interaction between the nanoparticles and chondroitin sulfate allows for increased functionality because the nanoparticles can be preferentially drawn to the cells. Chondroitin sulfate is a natural biomolecule effectively used as a target functionalization capacity for lipid-based nanoparticles. Using a natural biomolecule allows highly selective delivery while minimizing damage to healthy tissues.
[00127] b. Coumarin 6 as the staining agent embedded in Algosome Nanoparticles as the carrier agent
[00128] Incorporating coumarin 6 within the algosome nanoparticles effectively combined two levels of functionality-tracking and therapeutic function, into one system. The fluorescence of coumarin 6 allowed for real-time monitoring of the nanoparticle distribution and uptake at the cellular level. By embedding coumarin 6 within the lipid-based nanoparticle, researchers can visualize the aimed location of nanoparticles in a biological system. Meanwhile, coumarin 6 contributes to the therapeutic action by disrupting the Golgi apparatus of the melanoma cells.
[00129] c. Combination of Chondroitin Sulfate as the cell binding agent and Coumarin 6 as the staining agent
[00130] The synergy between chondroitin sulfate and coumarin 6 improves the overall effectiveness by providing specificity and spatial understanding while utilizing theragnostic. Coumarin's capacity was limited to tracking function, but the presence of chondroitin sulfate ensured the targeted delivery. The combination demonstrated novelty in its ability to:
[00131] Target Specificity: Direct targeted therapy of melanoma cells through CD44 binding versus typical nanoparticles that may interact with multiple receptors or without specificity.
[00132] Determine the location of therapeutics: Capture real-time brightness fluorescence tracking for distribution and potentially assess therapeutic success in real-time.
[00133] Interaction with Therapeutic: Disrupt drugs (e.g., Golgi apparatus) by effective cellular damage resulting in cell death of cancer cells.
[00134] FIGs. 3a & 3b illustrate a comparison of hydrodynamic diameter of the nano-algsomes with sample 1 of a plain algosome, sample 2 of Algosome-Chondroitin sulphate complex, and sample 3 of Algosome-Chondroitin Sulphate - Coumarin 6 complex, in accordance with an exemplary embodiment of the present disclosure.
[00135] As illustrated, the algosome nanoparticles were characterized for size using dynamic light scattering (DLS). The average particle size was 193nm for plain algosome, whereas the final particle size was around 242244nm, with a uniform spherical morphology. The size is ideal for cellular uptake and biodistribution. Here, the hydrodynamic diameter is identified for all these samples with plain algosome, algosome formed with coated chondroitin sulfate and finally the nanoparticle, i.e., algosome with chondroitin sulfate and coumarin 6.
[00136] FIG. 4 illustrates UV-Vis spectra of the final algosome nanoparticles loaded with coumarin 6, in accordance with an exemplary embodiment of the present disclosure.
[00137] As illustrated, the UV-Vis spectra of the final algosome nanoparticles loaded with coumarin 6 showed a distinct absorption peak around 420-450 nm, characteristic of coumarin 6. This peak confirms the successful incorporation of the dye into the nanoparticles. The other peak shows for the chondroitin sulfate around 210 nm. The presence of the coumarin 6 absorption peak indicates effective loading and integration within the algosome nanoparticles, while baseline absorbance reflects the inherent properties of the lipid-based nanoparticles. The UV-Vis spectrophotometer readings thus validate the presence and successful encapsulation of coumarin 6 in the final nanoparticle formulation.
[00138] FIG. 5 illustrates a comparison of cell viability of plain algosome (Alg)-based nanoparticle, algosome with chondroitin sulfate (CS), and addition of coumarin 6 (C6) under B16 cell lines, in accordance with an exemplary embodiment of the present disclosure.
[00139] As illustrated, the nanoparticles' therapeutic efficacy was assessed in cytotoxicity assay, including the MTT assay. The nanoparticles demonstrated a dose-dependent decrease in cell viability, while higher concentrations of coumarin 6 produced significant cytotoxic effects. Disruption of the Golgi apparatus was shown using confocal microscopy, confirming impaired Golgi structure and function in treated cells. Here, it can be seen that the cell viability is decreased to 48.2% with the synergistic approach of nanoparticle. It is higher to that of other two formulations. The cytotoxicity of three distinct nanoparticle formulations was assessed against B16 melanoma (derived from mouse melanoma, specifically the C57BL/6 strain) cells, a cell line known for its aggressive nature and resistance to apoptosis, making it a pertinent choice for evaluating the potential therapeutic efficacy of anticancer agents. The nanoparticles were tested at varying concentrations (100, 200, 300, 400, and 500 µg/ml). For lipid algosome-based CS loaded with C6, the cell viability percentages remained lower at most concentrations, with values of 48.25, 47.28, 45.31, 44.01, and 42.61%, respectively, indicating that the nanoparticle loaded with carrier and tracker in the form of chondroitin sulfate and coumarin 6 exert cytotoxic effects on the B16 cells. This result is crucial as it establishes algosome as a negative control, confirming that any observed cytotoxic effects in the other nanoparticle formulations are due to their specific components and not the algo-based lipid nanoparticle structure itself. In contrast, plain algosome-based lipid particles exhibited a very low decrease in cell viability with increasing concentrations, showing percentages of 98.62, 87.56, 83.13, 79.59, and 75.57%, respectively. Also, just the carrier and targeting agent of chondroitin sulfate with algosome lipid particle showcased a concentration dependent increase in cytotoxicity, with cell viability percentages of 95.12, 92.54, 88.24, 84.28, and 80.63%, respectively, demonstrating the enhanced therapeutic potential of combining algosome with synergistic approach of chondroitin sulfate along with coumarin 6.
[00140] FIGs. 6a & 6b illustrate a live dead assay for B16 cell line for the particle synthesized with algosome coated with chondroitin sulfate and coumarin 6, where Control indicates the untreated one, in accordance with an exemplary embodiment of the present disclosure.
[00141] As illustrated, in the live-dead assay, the data suggest that the algosome nanoparticles have a marked effect on the mortality of B16 melanoma cells compared with the control group. Live/dead assay (viable green/nonviable red) on B16 cell lines treated with algosome chondroitin sulphate and coumarin 6-based nanoparticles reveals the potential of nanoparticles targeting melanoma cells. The results of the assay calculated cell viability; treatment with FDA/PI (Fluorescein Diacetate/ Propidium Iodide) led to many dead cells (red fluorescence) and a decrease in cell ability compared to the comparison group. This implies that the nanoparticles can elicit cytotoxicity in B16 cells and are a potentially attractive option for therapeutic advancement. For dead cells, PI binds to DNA and emits red fluorescence when excited by a specific wavelength of light around 535nm. In contrast, live cells with intact membranes will exclude PI and only fluoresce green due to the conversion of FDA to fluorescein inside the cells. This differential staining distinguishes between live (green) and dead (red) cells. FDA/PI staining was done after treatment with coumarin 6 and Chondroitin Sulfate (CS) loaded algosome. Almost all the cells are dead after 24 hrs of treatment. Relative Fluorescence unit (RFU), which is a ratio of the mean of (FDA and PI) for both the control and the particle (Alg_CS@Co6), was calculated and obtained about 6.208 and 0.575, respectively.
[00142] A head-to-head comparison between the present nanoparticles and otherwise conventional therapy procedures was made. The characterization and efficacy of the targeted nanoparticles appeared to be considerably higher than those of the traditional therapies: the nanoparticles also demonstrated a higher reduction in melanoma cell viability and better targeting. The characterization showed that algosome nanoparticles could be effectively prepared and functionalized by Chondroitin sulfate and coumarin 6. The in vitro efficacy evaluation further supported the pivotal improved targeting capacity and marked an immense therapeutic effect, thus setting the foundation for applying this innovative system for effective cancer therapy.
[00143] The present invention is focused on developing a new type of nanoparticle for cancer treatment. The nanoparticles were made from algae-based materials and loaded with a combination of anti-cancer and tracking agents. The size and shape of these nanoparticles were found to be ideal for entering and moving within cells. The anti-cancer effect of the nanoparticles was tested on melanoma cells, showing a significant reduction in their survival compared to untreated cells. The nanoparticles were also found to be safe for healthy cells. Additionally, the tracking agent allowed researchers to visualize the nanoparticles interacting with the cancer cells, confirming their targeting ability. These new nanoparticles hold promise as a potential cancer therapy due to their effectiveness, safety, and ability to be tracked within the body.
[00144] A few of the major advantage of the present invention can be summarized as below:
[00145] Targeted Delivery: Conjugation of chondroitin sulfate proves useful in the targeting of CD44 receptors normally associated with melanoma cells, and this results in increased affected site delivery and minimum effects on other parts of the body.
[00146] Enhanced Efficacy: Coumarin 6 is useful not only for tracking the nanoparticles but also for destroying the Golgi apparatus in cancer cells, thereby enhancing the chances of cell death and, therefore, better therapeutic outcomes.
[00147] Biocompatibility: Algosome nanoparticles derived from Spirulina have certain advantages, such as non-toxic and biocompatibility, reducing the chances of an adverse reaction to the patient.
[00148] Controlled Release: It also guarantees protracted delivery of the active therapeutic moiety and minimizes the chances of giving the patient frequent doses.
[00149] Reduced Side Effects: The selectivity averted towards necrotic cells assists in attaining higher uptake by cancer cells while preserving the healthy tissues, and this keeps the side effects of the traditional treatments at a low level.
[00150] Innovative Combination: The multifunctional property of coumarin 6, which can act as a dye and therapeutic moiety, and the targeting potential of chondroitin sulfate provide a novel and efficient strategy for cancer treatment.
[00151] The present invention offers significant utility in multiple areas of cancer therapy and research:
[00152] Targeted Cancer Therapy: Melanoma Treatment: The primary use of this invention lies in treating melanoma, especially regarding the B16 melanoma cell line. CD44 receptor targeting property of chondroitin sulfate makes the designed nanoparticles deliver the therapeutic agents efficiently towards the melanoma cells and inhibits the growth of tumors and metastatic properties but have minimal impact on the normal cells.
[00153] Drug Delivery System: Precision Medicine: This indicates that the nanoparticle system is quite flexible and can be designed to deliver therapeutic agents other than coumarin 6, thus making it suitable for precision medicine. This ability also ensures that the drug gets to the precise areas required, improving the efficiency of any treatment required to treat different types of cancers.
[00154] Real-Time Imaging and Tracking: Theranostics: Coumarin 6 has both therapeutic properties and fluorescence properties; hence, the distribution of the nanoparticles within the body can be monitored in real-time. These two benefits make this system extremely useful in theranostics, where there is a synergy between diagnosing and treating diseases.
[00155] Research and Development: Preclinical Studies: They can be employed in preclinical research to estimate the efficacy of developed drugs concerning the selective destruction of certain cancer cells. Thus, it is considered a perfect model for examining the drug delivery processes, internalization, and therapeutic effects in cell culture and animal models.
[00156] Advanced Cancer Therapies: Combination Therapies: In EM therapy, this nanoparticle system can be further applied with other treatment modalities- immunotherapy or chemotherapy, for instance, to boost the therapy's effectiveness. Moreover, the system may enhance the overall response of well-recognized cancers, further overcoming resistance when the multiple agents are co-delivered or combined with immune checkpoint inhibitors.
[00157] It is understood that any specific order or hierarchy of blocks in the processes disclosed is an illustration of example approaches. Based upon design preferences, it is understood that the specific order or hierarchy of blocks in the processes may be rearranged, or that all illustrated blocks be performed. Any of the blocks may be performed simultaneously. In one or more implementations, multitasking and parallel processing may be advantageous.
[00158] It should be noted that the description and figures merely illustrate the principles of the present subject matter. It should be appreciated by those skilled in the art that conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present subject matter. It should also be appreciated by those skilled in the art by devising various systems that, although not explicitly described or shown herein, embody the principles of the present subject matter and are included within its spirit and scope.
[00159] Furthermore, all examples recited herein are principally intended expressly to be for pedagogical purposes to aid the reader in understanding the principles of the present subject matter and the concepts contributed by the inventor(s) to further the art and are to be construed as being without limitation to such specifically recited examples and conditions. The novel features which are believed to be characteristic of the present subject matter, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures.
[00160] Although embodiments for the present subject matter have been described in language specific to package features, it is to be understood that the present subject matter is not necessarily limited to the specific features described. Rather, the specific features and methods are disclosed as embodiments for the present subject matter. Numerous modifications and adaptations of the system/device of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the scope of the present subject matter.
 
, Claims:We claim:
1. A composition (100) for targeted delivery of a therapeutic complex for the treatment of cells within Golgi apparatus in a human subject, consisting of:
• a carrier agent (102) consisting of a plurality of naturally sourced nanoparticles;
• a cell binding agent (104) covalently functionalized to a surface of the plurality of naturally sourced nanoparticles; and
• a staining agent (106) conjugated with the cell binding agent (104),
wherein the cell binding agent is designed to selectively target the Golgi apparatus, and wherein the staining agent is designed to (i) trace the nanoparticles via its fluorescent properties, and (ii) bombard the Golgi apparatus in melanoma cells.
2. The composition (100) as claimed in claim 1, wherein the plurality of naturally sourced nanoparticles is obtained from an microalgae.
3. The composition (100) as claimed in claim 2, wherein the microalgae is a lipid extract of Spirulina sp.
4. The composition (100) as claimed in claim 1, wherein an average particle size of the plurality of naturally sourced nanoparticles is 193nm.
5. The composition (100) as claimed in claim 1, wherein the cell binding agent is chondroitin sulfate.
6. The composition (100) as claimed in claim 1, wherein the cell binding agent (104) consists of chondroitin sulfate and Nacetylgalactosamine, GalNAc.
7. The composition (100) as claimed in claim 1, wherein a concentration of the cell binding agent (104) is 1mg/ml.
8. The composition (100) as claimed in claim 1, wherein the cell binding agent (104) attaches selectively with CD44 receptors overexpressed on the Golgi apparatus on a surface of B16 melanoma cells.
9. The composition (100) as claimed in claim 1, wherein the staining agent (106) is Coumarin 6.
10. The composition (100) as claimed in claim 1, wherein a concentration of the staining agent (106) is 50µg/ml.
11. The composition (100) as claimed in claim 1, wherein the staining agent (106) is capable of subsequent spatial detection of the plurality of naturally sourced nanoparticles in the subject during the therapeutic complex's delivery, using a fluorescence microscope.
12. The composition (100) as claimed in claim 1, wherein the staining agent (106) is capable of subsequent temporal detection of the plurality of naturally sourced nanoparticles in the subject during the therapeutic complex's delivery, using a fluorescence microscope.
13. The composition (100) as claimed in claim 1, wherein the staining agent (106) is capable of disrupting processes in the Golgi apparatus once inside the melanoma cells.
14. A method (200) of producing a composition for targeted delivery of a therapeutic complex for the treatment of cells within the Golgi apparatus in a human subject, the method comprising:
• obtaining (202) a carrier agent, wherein the carrier agent consists of a plurality of naturally sourced nanoparticles;
• mixing (204) the plurality of naturally sourced nanoparticles with a staining agent in an organic solvent;
• coating (206) a surface of the plurality of naturally sourced nanoparticles with a cell binding agent, wherein the coating enables the cell binding agent to covalently functionalized with the plurality of naturally sourced nanoparticles; and
• loading (208) the staining agent by conjugating with the cell binding agent,
wherein the cell binding agent is designed to selectively target the Golgi apparatus, and wherein the staining agent is designed to (i) trace the distributed nanoparticles via its fluorescent properties, and (ii) bombard the Golgi apparatus in melanoma cells.
15. The method (200) as claimed in claim 14, wherein the plurality of naturally sourced nanoparticles are extracted from a microalgae.
16. The method (200) as claimed in claim 15, wherein the microalgae is a lipid extract of Spirulina sp.
17. The method (200) as claimed in claim 14, wherein the organic solvent is a combination of Chloroform and Methanol in a ratio of 2:1.
18. The method (200) as claimed in claim 14, wherein the organic solvent is evaporated at 50-55 ºC before coating the surface of the plurality of naturally sourced nanoparticles with the cell binding agent.
19. The method (200) as claimed in claim 14, wherein the cell binding agent is Chondroitin Sulfate.
20. The method (200) as claimed in claim 19, wherein the Chondroitin Sulfate taken for coating is 2 ml of 1mg/ml concentration.
21. The method (200) as claimed in claim 19, wherein the plurality of naturally sourced nanoparticles coated with the cell binding agent is sonicated for 3 cycles, with 10 minutes per each cycle.
22. The method (200) as claimed in claim 19, wherein the staining agent is coumarin 6.

Dated this 5th day of November, 2024

[SONAL MISHRA]
-DIGITALLY SIGNED-
IN/PA-3929
OF L.S. DAVAR & CO.
ATTORNEY FOR THE APPLICANT(S)

Documents