A GOLD-COATED BIMETALLIC NANOCARRIER COMPOSITION AND ITS METHOD OF PREPARATION

Abstract

A nanocarrier composition comprising a core comprising bimetallic nanoparticles comprising copper and iron ions, linked with an organic linker; and a coating material comprising gold metal, wherein the core is coated with the coating material. The present disclosure also relates to the method of preparation thereof.

Specification

Description:FIELD OF THE INVENTION:
[0001] The present disclosure relates to a nanocarrier composition. More particularly, the disclosure relates to the gold-coated bimetallic nanocarrier composition. The present disclosure also relates to a method of preparing gold-coated bimetallic nanocarrier composition.

BACKGROUND OF THE INVENTION
[0002] Cancer is one of the major causes of global mortality, the treatment options are restricted by the side effects caused. Conventional treatment modalities such as surgery, chemo, and radiotherapies are widely used to treat cancers; however, are falling short owing to the undesirable side effects caused such as off-targeted toxicity, development of resistance, the requirement of multiple dosages, poor quality, to name some. Several metal based nanocarriers, such as gold, silver, copper, iron, cobalt, platinum, ruthenium are being studied for their anti-cancer efficacy.
[0003] Photothermal therapy is one of the recently developed, affordable, non-invasive cancer therapies that exploits near infra-red lasers (NIR) lasers to cause death due to heat generation and the subsequent reactive oxygen species (ROS) generation. The interaction of NIR lasers with photoresponsive/photosensitizers generates heat that causes the cell death.
[0004] Metal-based nanosystems are being widely explored for their anti-cancer efficacy as given below:
[0005] Chen K et al., "Cellular Trojan Horse initiates bimetallic Fe-Cu MOF-mediated synergistic cuproptosis and ferroptosis against malignancies"4 describes disruptions in metal balance can trigger a synergistic interplay of cuproptosis and ferroptosis, offering promising solutions to enduring challenges in oncology. Cellular Trojan Horse, named MetaCell, uses live neutrophils to stably internalize thermosensitive liposomal bimetallic Fe-Cu MOFs (Lip@Fe-Cu-MOFs).
[0006] Qiao C et al "Virus‐Inspired Metal-Organic Frameworks (MOFs) for Targeted Imaging and Chemotherapy of Glioma" discloses the development and evaluation of the dimetal-organic framework-based nanocarrier (MILB@LR), which closely mimicked both the bullet-shape structure and surface functions of natural rabies virus (RABV). MILB@LR benefited from a more comprehensive RABV-mimic strategy than mimicking individual features of RABV and exhibited significantly enhanced blood brain barrier (BBB) penetration and brain tumor targeting. MILB@LR also displayed superior inhibition of tumor growth when loaded with oxaliplatin.
[0007] However, they are limited by their high dosage and multiple dosage requirements.
[0008] Hence, there is need for developing a nanocarrier composition that is composed of materials that are already a part of the human body, with potential anti-cancer effects, better efficacy and help in overcoming the above-mentioned disadvantage.

OBJECTS OF THE INVENTION
[0009] Some of the objectives of the present disclosure, with at least one embodiment herein satisfied, are listed herein below:
[0010] It is the primary objective of the present disclosure to provide a biocompatible bimetallic nanocarrier composition comprising iron and copper.
[0011] It is another objective of the present disclosure to provide gold coating on the bimetallic nanocarrier to enhance the photothermal efficacy.
[0012] It is yet another objective of the present disclosure to provide the synergism between the anticancer efficacy of bimetallic nanoparticles and the gold coating.
[0013] It is yet another objective of the present disclosure to provide a simple and cost-effective method for preparing the gold-coatedgold-coated bimetallic nanocarrier composition.

SUMMARY OF INVENTION
[0014] The present disclosure relates to a nanocarrier composition comprising
a. a core comprising bimetallic nanoparticles comprising copper and iron ions, linked with an organic linker; and
b. a coating material comprising gold metal,
wherein the core is coated with the coating material.

[0015] The present disclosure also relates to a method of preparing the nanocarrier composition, comprising
a) mixing copper chloride dihydrate, iron (III) chloride hexahydrate, 2-amino teraphthalic acid in a solvent;
b) stirring the mixture obtained in step a) at a temperature range of 40 °C to 70 °C for a time period of 30 minutes to 60 minutes to obtain a solution;
c) centrifuging the solution at 10,000 to 15,000 rpm for 10 to 20 minutes to obtain the core comprising bimetallic nanoparticles;
d) adding and mixing gold chloride solution to the bimetallic nanoparticles to form the coating; and
e) adding ascorbic acid to obtain the nanocarrier composition

BRIEF DESCRIPTION OF DRAWINGS
[0016] 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:
[0017] Figure 1 depicts the DLS of the gold-coated bimetallic nanoparticles comprising copper and iron ions.
[0018] Figure 2 depicts the SEM images of gold-coated bimetallic nanoparticles comprising copper and iron ions.
[0019] Figure 3 depicts the UV-Vis spectroscopy of gold-coated bimetallic nanoparticles comprising copper and iron ions.
[0020] Figure 4 depicts the Thermal transduction and pictures of temperature raise.
[0021] Figure 5 depicts the biocompatibility of gold-coated bimetallic nanoparticles comprising copper and iron ions in L929 cells and NIH 3T3 cells.
[0022] Figure 6 depicts the cytotoxicity studies in MCF-7 and B16 cells
[0023] Figure 7 depicts laser-based cytotoxicity studies in B16 cells
[0024] Figure 8 depicts laser-based cytotoxicity studies in MCF-7 cells
[0025] Figure 9 depicts Live/Dead assay performed in MCF-7 cells
[0026] Figure 10 depicts Live/Dead assay performed in B16 cells.

DESCRIPTION OF THE INVENTION:
[0027] 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.
[0028] 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.
[0029] 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.
[0030] As used herein the term "nanocarrier" refers to the nanosized transport agents that carries drug or IR dye to the target tissue. Herein the nanocarrier is a gold-coated bimetallic nanoparticles.
[0031] As used herein the term "cancer" refers to a cell that displays uncontrolled growth and division, invasion of adjacent tissue and often metastasizes to other location of body. Herein the cancer is breast cancer or melanoma.
[0032] As used herein the term "Photothermal therapy (PTT)" is a therapy for local treatment of cancer that uses heat generated from absorbed light energy (laser) to destroy tumor cells.
[0033] In an embodiment, the present disclosure relates to a nanocarrier composition comprising
a) a core comprising bimetallic nanoparticles comprising copper and iron ions, linked with an organic linker; and
b) a coating material comprising gold metal,
wherein the core is coated with the coating material.
[0034] In an embodiment of the present disclosure, the concentration of the copper ion is in range of 2mM - 5mM.
[0035] In another embodiment of the present disclosure, the concentration of the iron is in range of 2mM - 4mM.
[0036] In yet another embodiment of the present disclosure, the concentration of the gold metal in the coating material is in range of 1mM - 2mM.
[0037] In a further embodiment of the present disclosure, the source of the copper ion is copper chloride dihydrate and the source of the iron ion is iron (III) chloride hexahydrate.
[0038] In an embodiment of the present disclosure, the ratio of copper ion and iron ion is in the range of 0.5:1 - 1:1
[0039] In another embodiment of the present disclosure, the organic linker is 2-amino terephthalic acid.
[0040] In an embodiment of the present disclosure, the gold-coatedgold-coated bimetallic nanocarrier composition comprising iron and copper shows anticancer activity against solid tumors (e.g., Breast Cancer, Melanoma). In another embodiment, the nanocarrier composition could release the metal ions at the tumor site owing to the acidic environment. Also, the gold coating on the bimetallic nanoparticles shows synergistic cell death.
[0041] In another embodiment of the present disclosure, the gold coating on the bimetallic nanoparticles surprisingly shows the Photothermal Transduction (PTT) effect.
[0042] In an embodiment, the present disclosure relates to a method of preparing the nanocarrier composition comprising
a) mixing copper chloride dihydrate, iron (III) chloride hexahydrate, 2-amino teraphthalic acid in a solvent;
b) stirring the mixture obtained in step a) at a predefined temperature for a predefined time to obtain a solution;
c) centrifuging the solution to obtain a core comprising bimetallic nanoparticles comprising copper and iron;
d) adding and mixing gold chloride solution to the bimetallic nanoparticles; and
e) adding ascorbic acid to form the gold coating on the bimetallic nanoparticles.
[0043] In an embodiment of the present disclosure, the solution is centrifuged at 10,000 to 15,000 rpm for 10 to 20 minutes
[0044] In another embodiment of the present disclosure, the predefined temperature is in range of 40 °C to 70 °C.
[0045] In yet another embodiment of present disclosure, the predefined time is in range 30 minutes to 60 minutes
[0046] In another embodiment of the present disclosure, the solvent is selected from ethanol.
[0047] In yet another embodiment of the present disclosure, the molar ratio of copper chloride dihydrate, iron (III) chloride hexahydrate, and 2-amino terephthalic acid is from 1:1:1, 1:2:1, 2:1:1, 3:1:1, and 1:3:1.
[0048] In another embodiment of the present disclosure, the gold coating on the nanoparticles is achieved by reducing chloroauric acid using ascorbic acid.
[0049] In an embodiment of the present disclosure, gold nanoparticle alone has very less PPT effect (Zhang et al., 2019). As shown herein, the nanoparticles when coated with gold alone surprisingly showed increased PPT effect and increased cell death.

ADVANTAGES OF THE PRESENT INVENTION
[0050] In accordance with the present disclosure nanocarrier composition and its method of preparation has the following advantages:
• Increased potothermal efficiency
• Targeted delivery
• Cost effective
[0051] The present disclosure will be explained using the following examples:

EXAMPLE
[0052] Materials: The components involved in the synthesis of gold-coated bimetallic nanoparticles include:
• Copper chloride dihydrate procured from Sigma
• Iron (II) chloride hexahydrate procured from Sigma
• 2-aminotheraphthalic acid from TCI Chemicals Pvt Ltd
• Ethanol procured from RANKEM
• Gold chloride solution is procured from Sigma
• Ascorbic acid is procured from Sigma
• The cell culture and the cell lines involved are:
• MCF-7 cells procured from NCCS Pune
• BC-16 cells procured from NCCS Pune
• L929 cells procured from NCCS Pune
• NIH3T3 cells procured from NCCS Pune

Example 1
A. Preparation of bimetallic Nanocarrier Composition:
[0053] Equimolar of copper chloride dihydrate, iron (III) chloride hexahydrate, 2- aminotheraphthalic acid were mixed in 10mL of ethanol and let to stir at 50 °C for 1 hour. The solution was then centrifuged at 12,000rpm for 10 minutes, washes were repeated for 3 more times, with ethanol and water to obtain iron copper bimetallic nanoparticles (FC NPs).
B. Gold Coating:
[0054] 2mM gold chloride solution was added to 2mg/mL of FC NPs and mixed well. Further, 20mM ascorbic acid was added to the above mixture and the purple liquid formation indicated successful gold coating
Example 2
Characterization of bimetallic Nanocarrier Composition
A. Size and Morphology
[0055] The size and morphology of the nanocarrier composition is analysed by SEM (JOEL-JSM-7600F (Joel, Japan)), and DLS (PSS Z3000 NICOMP NANOz)
[0056] The DLS revealed the hydrodynamic size of around 185nm (figure 1) and SEM also showed the nanoparticles of size around 130nm (figure 2). The morphology of the nanocarrier system had hexagonal shape as observed from the SEM images.

B. UV-Visible Spectroscopy
[0057] The UV-Vis spectrophotometer (SHIMADZU - UV1800) showed a broad peak around 600-800nm, indicating the successful coating of the nanosystem with gold (Figure 3).
C. Thermal transduction Analysis:
[0058] The thermal transduction experiments were performed using 100µl 200 µg/mL of the gold coated nanoparticles taken in a 96 well plate and was irradiated with 690nm laser for 10mins and the temperature was recorded using a thermal camera at regular time intervals.
[0059] The gold-coated bimetallic nanocarrier composition showed temperature raise up to 51.5° C in 10 minutes (Figure 4).

Example 3
Biocompatibility Analysis
[0060] The biocompatibility of the nanocarrier composition was evaluated using MTT assay. Briefly, L929 cells and NIH 3T3 cells were seeded in a 96 well plate and were treated with 25, 50, 100, 150, 200, 250 µg/mL of gold-coated bimetallic nanocarrier composition for 24 hrs and an MTT assay was performed to understand the biocompatibility.
[0061] The gold-coated bimetallic nanocarrier composition showed excellent biocompatibility till 250 µg/mL (80%) on L929 cells and NTH3T3 cells (Figure 5).

Example 4:
Cytotoxicity analysis
[0062] The cytotoxicity of the gold-coated bimetallic nanoparticles was assessed using MTT assay in breast cancer (MCF-7) and melanoma (B16) cells. The cytotoxicity with Plain Fe-Cu NPs was also evaluated
[0063] There was an increased reduction in compatibility of the cells after treatment with 200 µg/mL of gold-coated bimetallic nanoparticles upto 60% and 76% cell survival in MCF-7 and B16 cells, respectively as compared to plain Fe-Cu NPs (Figure 6).
[0064] Further, laser-mediated cytotoxicity was assessed in MCF-7 and B16 cells, using 690nm laser. The cells after treatment with 200 µg/mL of gold-coated nanoparticles were irradiated with laser for 10 minutes. The cells were incubated for 24 hrs post irradiation and the results revealed a significant reduction in cell survival for gold-coated nanoparticles as compared to plain Fe-Cu NPs (Figure 7 and 8).

Example 5
Live Dead Assay:
[0065] Live/Dead assay using FDA/PI was further performed to qualitatively assess the cytotoxicity. The cells were treated with 200 µg/mL of gold-coated bimetallic nanoparticles and were irradiated with 690nm laser for 10 minutes. The cells were incubated for 24 hrs post irradiation and then stained with 1 µg/mL of FDA and PI. The cells were then imaged using live cells imager. The results showed a significant cell death in cells treated with laser when compared to the control groups (Figure 9 and 10)

 


SPECIFIC EMBODIMENTS OF THE PRESENT INVENTION
[0066] In an embodiment, the present disclosure relates to a nanocarrier composition comprising
a. a core comprising bimetallic nanoparticles comprising copper and iron ions, linked with an organic linker; and
b. a coating material comprising gold metal,
wherein the core is coated with the coating material.
[0067] Such a nanocarrier composition wherein concentration of the copper ion is in range of 2mM - 5mM.
[0068] Such a nanocarrier composition wherein concentration of the iron is in range of 2mM - 4mM.
[0069] Such a nanocarrier composition wherein concentration of the gold metal in the coating material is in range of 1mM - 2mM.
[0070] Such a nanocarrier composition wherein source of the copper ion is copper chloride dihydrate and the source of iron ion is iron (III) chloride hexahydrate.
[0071] Such a nanocarrier composition wherein the ratio of copper ion and iron ion is in range of 0.5:1 - 1:1
[0072] Such a nanocarrier composition wherein the organic linker is f 2-amino terephthalic acid.
[0073] The present disclosure relates to a method of preparing the nanocarrier composition, comprising
a. mixing copper chloride dihydrate, iron (III) chloride hexahydrate, 2-amino teraphthalic acid in a solvent;
b. stirring the mixture obtained in step a) at a temperature range of 40 °C to 70 °C for a time period of 30 minutes to 60 minutes to obtain a solution;
c. centrifuging the solution at 10,000 to 15,000 rpm for 10 to 20 minutes to obtain the core comprising bimetallic nanoparticles comprising copper and iron;
d. adding and mixing gold chloride solution to the bimetallic nanoparticles; and
e. adding ascorbic acid to form gold coating on the bimetallic nanoparticles.
[0074] Such a method wherein the solvent is selected from ethanol.
[0075] Such a method, wherein molar ratio of copper chloride dihydrate, iron (III) chloride hexahydrate2-amino terephthalic acid is from 1:1:1, 1:2:1, 2:1:1, 3:1:1, and 1:3:1.
, Claims:We Claim:
1. A nanocarrier composition comprising
a. a core comprising bimetallic nanoparticles comprising copper and iron ions, linked with an organic linker; and
b. a coating material comprising gold metal,
wherein the core is coated with the coating material.

2. The nanocarrier composition as claimed in claim 1, wherein concentration of the copper ion is in range of 2mM - 5mM.

3. The nanocarrier composition as claimed in claim 1, wherein concentration of the iron is in range of 2mM - 4mM.

4. The nanocarrier composition as claimed in claim 1, wherein concentration of the gold metal in the coating material is in range of 1mM - 2mM.

5. The nanocarrier composition as claimed in claim 1, wherein source of the copper ion is copper chloride dihydrate and the source of iron ion is iron (III) chloride hexahydrate.

6. The nanocarrier composition as claimed in claim 1, wherein the ratio of copper ion and iron ion is in range of 0.5:1 - 1:1

7. The nanocarrier composition as claimed in claim 1, wherein the organic linker is 2-amino terephthalic acid.

8. A method of preparing the nanocarrier composition as claimed in claim 1, comprising
a) mixing copper chloride dihydrate, iron (III) chloride hexahydrate, 2-amino teraphthalic acid in a solvent;
b) stirring the mixture obtained in step a) at a temperature range of 40 °C to 70 °C for a time period of 30 minutes to 60 minutes to obtain a solution;
c) centrifuging the solution at 10,000 to 15,000 rpm for 10 to 20 minutes to obtain the core comprising bimetallic nanoparticles comprising iron and copper;
d) adding and mixing gold chloride solution to the bimetallic nanoparticles; and
e) adding ascorbic acid to form gold coating on the bimetallic nanoparticles.

9. The method as claimed in claim 8, wherein the solvent is selected from ethanol.

10. The method as claimed in claim 8, wherein molar ratio of copper chloride dihydrate, iron (III) chloride hexahydrate, 2-amino terephthalic acid is from 1:1:1, 1:2:1, 2:1:1, 3:1:1, and 1:3:1.

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