Patent Application
20230256116
The present disclosure generally relates to nanocomposites. Specifically, the present disclosure relates to a nitrogen doped graphene (NDG)-Mn3O4 nanocomposite comprising a nitrogen doped graphene (NDG) and Mn3O4 nanoparticles. The NDG-Mn3O4 nanocomposite is useful in bimodal performance including photodynamic therapy (PDT) and magnetic resonance imaging (MRI). The NDG-Mn3O4 nanocomposites of the present invention cause significant cell death under laser irradiation.
Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Cancer is one of the deadliest and costliest diseases and is the second leading cause of death worldwide. Photodynamic therapy (PDT) is a promising treatment modality for cancer with minimal side effects and is expected to replace traditional chemotherapy, which is associated with numerous adverse effects. PDT involves combination of light and a photosensitizer (PS), which is activated by absorption of light of a specific wavelength, causing the generation of potentially toxic reactive oxygen species (ROS) that induce a cascade of intracellular molecular events resulting in targeted tissue damage [Avci, P.; Erdem, S.S.; Hamblin, M.R. J. Biomed. Nanotechnol. 2014, 10, 1937-1952]. Sun et al. have reviewed the application of metal-based nanoparticles (NPs) for PDT of cancer [Sun J et al. Molecules, 2018, 23, 1704]. Metal oxide-based nanomaterials have also significantly impacted the landscape of healthcare, including in the areas of diagnosis and therapeutic applications. Metal oxides have demonstrated great potential in PDT and magnetic resonance imaging (MRI) in diagnostic radiology. A majority of the transition metals-based oxides offer several advantages in the field of biomedicines due to their biocompatibility and non-toxicity. Even some of them, such as iron oxide, have been approved as an MRI contrast agent by concerned authorities [Wang, D. et al, ACS Nano 2014, 8, 6620-6632]. Therefore, metal oxide NPs have the potential to serve as both therapeutic and imaging agents, particularly, manganese oxide (Mn3O4) nanoparticles are considered as effective in tumor diagnosis and treatment due to their decent biocompatibility, in-vivo imaging performance and tumor microenvironment (TME) responsiveness. Notably, Mn3O4 is consists of Mn2+ and Mn3+, due to which it is extremely sensitive to the redox environment in the cell and rapidly decomposes upon exposure to glutathione (GSH). Tumor specific antibodies functionalized Mn3O4 NPs were applied as T1 MRI contrast agent for selective imaging of cancer cells [Na, H.B. et al, Angew. Chem. 2007, 46, 5397-5401]. To avoid some limitations, such as aggregation, poor water dispersibility, high dermal toxicity and low clearance of these NPs, several stabilizing ligands have applied to the surfaces of NPs which make them stable and suitable for therapeutic applications. For instance, folic acid (FA) has been used as ligand for targeting folate receptors (FR), a tumor-associated protein over-expressed in cancer cells having high binding affinity toward folic acid. Recently, transitional metal oxide NPs including Mn3O4, have been effectively combined with a variety of 2D materials, specially graphene, which has received promising attention for phototherapy due to its excellent photosensitizer properties.
Graphene is made up of a single layer of carbon atoms arranged in a honeycomb structure, demonstrating specific combination of physiochemical properties, such as, high surface area (2630 m2g-1), optimal thermal conductivity (~5000 Wm K-1), and remarkable optical transparency, which make it excellent candidate for drug delivery and therapeutic applications. However, its hydrophobicity causes irreversible agglomeration, which is a great obstacle for utilizing its drug career properties. On the other hand, the oxidation of graphene into graphene oxide (GO) significantly reduces its aggregation tendency. GO exhibits amphiphilic nature due to the presence of hydrophobic graphene moiety and hydrophilic edges; the former property is important for carrying water-insoluble drugs through non-covalent bonding, π-π stacking or hydrophobic interaction or hydrogen bonding whereas the latter property not only provides anchor sites for functionalization but also maintains colloidal stability due to negative surface charge. When dispersed in water, GO attains a negative surface charge due to ionization of hydroxyl and carboxylic groups. The magnitude of this negative charge is sufficient to cause electrostatic repulsion resulting in stable dispersion of GO in water [Li, D et al, Nat. Nanotechnol. 2008, 3, 101-105].
The water dispersibility of GO is considered better than the water dispersibility of carbon nanotubes (CNTs). However, GO contains a variety of oxygen containing random functional groups, which inhibit the homogeneous binding of the NPs on its surface. Biological investigations of GO, both in-vitro and in-vivo have no consensus results and sometimes the results are in contradiction [Kiew, S.F. et al, J. Control. Release, 2016, 226, 217-228].
There is therefore a need in the art to develop nanocomposite, which is useful in photodynamic therapy as well for magnetic resonance imaging (MRI). The present invention satisfies the existing needs, as well as others, and generally overcomes the side effects of the chemotherapy.
Primary objective of the present disclosure is to provide a novel nanocomposite for photodynamic therapy and magnetic resonance imaging.
Another objective of the present disclosure is to provide a nanocomposite comprising nitrogen doped graphene oxide (NGO) and Mn3O4 nanoparticles.
Another objective of the present disclosure is to provide a method of preparation of nanocomposite comprising nitrogen doped graphene oxide (NGO) and Mn3O4 nanoparticles.
Another objective of the present disclosure is to provide a method of treating tumors or imaging targeted tissue by photodynamic therapy using nanocomposite comprising nitrogen doped graphene oxide (NGO) and Mn3O4 nanoparticles.
The present disclosure relates to a NDG-Mn3O4 nanocomposite comprising a nitrogen doped graphene (NDG) and Mn3O4 nanoparticles. The NDG-Mn3O4 nanocomposite is useful in bimodal performance including photodynamic therapy (PDT) and magnetic resonance imaging (MRI). The NDG-Mn3O4 nanocomposites of the present invention caused significant cell death under laser irradiation, while control and Mn3O4 nanoparticles showed negligible cell death.
In an aspect, the present invention relates to a nanocomposite comprising a nitrogen doped graphene oxide conjugated with Mn3O4 nanoparticle.
In another aspect of the present invention, particle size of the nanocomposite is in a range of 5 nm to 15 nm.
In another aspect of the present invention, the nitrogen doped graphene oxide and the Mn3O4 nanoparticle is present in the nanocomposite in a ratio of 1:1.
In another aspect of the present invention, the nanocomposite is obtained using milling process.
In yet another aspect, the present invention relates to a process of preparation of a nanocomposite as claimed in claim 1, wherein the process comprises the steps of:
In another aspect of the present invention, the manganese (II) acetylacetonate and oleylamine used in the process of preparing nanocomposite is present in a molar ratio of 1:25.
In another aspect of the present invention, the base used in the process of preparing nanocomposite is ammonium hydroxide or potassium hydroxide.
In another aspect of the present invention, the nitrogen doped graphene oxide and the Mn3O4 nanoparticles used in the process of preparation of nanocomposite is present in 1:1 ratio.
In another aspect of the present invention, the temperature in step (a) is 160° C. and in step (b) is 90° C.
In yet another aspect, the present invention relates to a method of treating a cancer or imaging targeted tissue in a subject, comprising:
In another aspect of the present invention, the cancer is lung cancer, breast cancer, prostate cancer, brain cancer, colorectal cancer, pancreatic cancer, ovarian cancer, cervical cancer, liver cancer, head/neck/throat cancer, skin cancer, bladder cancer and a hematologic cancer.
In another aspect of the present invention, the imaging is Magnetic Resonance Imaging (MRI).
The following extensive discussion of preferred embodiments will reveal several objects, features, characteristics, and advantages of the inventive subject matter.
The embodiments herein and the various features and advantageous details thereof are explained more comprehensively with reference to the non-limiting embodiments that are detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of the ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention.
As used in the description herein, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
As used herein, the terms “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are meant to be non- limiting, i.e., other steps and other ingredients which do not affect the end of result can be added. The above terms encompass the terms “consisting of” and “consisting essentially of”.
The present disclosure relates to a NDG-Mn3O4 nanocomposite comprising a nitrogen doped graphene (NDG) and Mn3O4 nanoparticles. The NDG-Mn3O4 nanocomposite is useful in bimodal performance including photodynamic therapy (PDT) and magnetic resonance imaging (MRI). The NDG-Mn3O4 nanocomposites of the present invention caused significant cell death under laser irradiation.
In an embodiment, the present invention relates to a nanocomposite comprising a nitrogen doped graphene oxide conjugated with Mn3O4 nanoparticle.
In another embodiment of the present invention, particle size of the nanocomposite is in a range of 5 nm to 15 nm. Preferably, the particle size of the nanocomposite is 10±1.7 nm.
In another embodiment of the present invention, the nitrogen doped graphene oxide and the Mn3O4 nanoparticle is present in the nanocomposite in a ratio of 1:1.
In another embodiment of the present invention, the nanocomposite is obtained using milling process.
In yet another embodiment, the present invention relates to a process of preparation of a nanocomposite as claimed in claim 1, wherein the process comprises the steps of:
In another embodiment of the present invention, the manganese (II) acetylacetonate and oleylamine used in the process of preparing nanocomposite is present in a molar ratio of 1:25.
In another embodiment of the present invention, the base used in the process of preparing nanocomposite is ammonium hydroxide or potassium hydroxide.
In another embodiment of the present invention, the nitrogen doped graphene oxide and the Mn3O4 nanoparticles used in the process of preparation of nanocomposite is present in 1:1 ratio.
In another aspect of the present invention, the temperature in step (a) is 160° C. and in step (b) is 90° C.
In yet another embodiment, the present invention relates to a method of treating a cancer or imaging targeted tissue in a subject, comprising:
In another embodiment of the present invention, the cancer is lung cancer, breast cancer, prostate cancer, brain cancer, colorectal cancer, pancreatic cancer, ovarian cancer, cervical cancer, liver cancer, head/neck/throat cancer, skin cancer, bladder cancer and a hematologic cancer.
In another aspect of the present invention, the imaging is Magnetic Resonance Imaging (MRI).
According to the present invention, NDG—Mn3O4 nanocomposites did not cause any cytotoxicity unless activated by laser irradiation that resulted in concentration dependent cytotoxicity in lung cancer cells (
In an embodiment of the present invention, the 670 nm laser was used in order to keep the wavelength within the red optical window (620-750 nm).
In another embodiment of the present invention, the NDG-Mn3O4 nanocomposites killed 68% of cancer cells which is more effective than GQD-PDA-Mn3O4 nanoparticles (51% cell death). The mechanism of laser-induced toxicity of NDG—Mn3O4 nanocomposites can be multifactorial. The laser irradiation during PDT triggered the disruption of cellular membranes resulting in a higher cellular uptake of the GQD-PDA-Mn3O4 nanoparticles compared to graphene quantum dots. This selective transport across the cell membrane might have been influenced by the size, shape and surface chemistry of nanoparticles.
In another embodiment of the present invention, the NDG-Mn3O4 nanocomposites caused 1O2 generation under laser irradiation in a time-dependent manner and longer exposure to laser irradiation produced excessive ROS generation (
In another embodiment of the present invention, the results of MRI demonstrated a concentration dependent enhancement of signal intensity with increasing concentration of NDG-Mn3O4 nanocomposites (
The present disclosure may be more fully understood by reference to the following examples:
Manganese (II) acetylacetonate was dissolved in oleylamine (molar ratio of manganese (II) acetylacetonate: oleylamine = 1:25) and the mixture was heated at 160 C for 10 h under a nitrogen cover. The resulting product was cooled to room temperature to form a brownish suspension, which was centrifuged at 9000 rpm for 15 min and the supernatant was removed to obtain a brown residue. The precipitate was washed multiple times with ethanol to acquire pure Mn3O4 nanoparticles, which were dried under vacuum before use.
Initially, graphite oxide (GO) was synthesized from graphite powder using a modified Hummers method. Briefly, graphite powder (0.5 g) and NaNO3 (0.5 g) were added to 23 mL of H2SO4 and the mixture was stirred for 10 min in an ice bath. Subsequently, KMnO4 (3 g) was slowly added and after proper mixing, the ice bath was replaced with water bath (35) for 1 h, resulting in the formation of a thick paste. Thereafter, 40 mL of deionized water was added, and the mixture was stirred for 30 min at 90 C. Finally, 100 mL of deionized water was added, followed by the slow addition of 3 mL of H2O2. The mixture was allowed to cool, filtered and washed with deionized water. The resulting thick brown paste was dispersed in water and centrifuged at 1000 rpm for 2 min. This step was repeated 4-5 times, until all unsettled particles were removed. The resultant paste was dispersed in water with mild sonication to obtain a suspension of graphene oxide (GO). For nitrogen doping, the resulting suspension was taken in a round bottom flask, to which 4 mL of NH4OH and 4 mL hydrazine hydrate were added simultaneously. The mixture was stirred for a few minutes, and the flask (equipped with cooling condenser) was put in a water bath controlled at 90° C. for 3 h. The product was collected after been filtered through micropore filters (Whatman filter paper, pore size-20 µm, W&R Balston Limited, Maidstone, Kent, UK), washed by deionized water and freeze-dried.
Equal amounts of Mn3O4 nanoparticles and NDG were milled using a Fritsch Pulverisette P7 planetary ball mill (Idar-Oberstein, Germany). The nanomaterials powder and stainless steel balls (5 mm diameter) with the ball to powder weight ratio of 1:1 were introduced into the stainless steel container. The milling of the powder was performed for 16 h, with intermittent pausing of milling process at regular intervals.
The synthesized nanoparticles were characterized for size and physicochemical properties using high resolution transmission electronmicroscopy (JSM-7610F, JEOL, Tokyo, Japan), X-ray diffraction analysis (D2 Phaser X-ray diffractometer, Bruker, Ettlingen, Germany) and FT-IR spectroscopy (Perkin Elmer 1000 FT-IR spectrometer,Waltham, MA, USA). A microplate reader determined the absorbance at 570 nm (Molecular Devices, USA). Percentage cell viability was calculated, and cell-survival curves were constructed.
The results of high resolution transmission electron microscopy (HRTEM) displayed the existence of spherical shaped Mn3O4 nanoparticles on the surface of NDG within the range of 5-15 nm (
The XRD pattern of Mn3O4NPs shown in
FT-IR spectra of Mn3O4 NPs displayed the characteristic peak of Mn-O, stretching mode in the range of 624 cm-1 while the vibrational frequency associated to the Mn-O distortion vibration poisoned at 525 cm-1 (
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used for testing the cytotoxicity of Mn3O4 and NDG—Mn3O4 nanoparticles. A549 lung cancer cells were seeded into 96-well plate (4 × 104 cells per well) in RPMI and incubated at 37° C. for 4 h in a 5% CO2 incubator. Different concentrations (6.25-100 µg/mL) of Mn3O4 and NDG—Mn3O4 nanoparticles were added to the 96-well plate. Phosphate buffer saline (PBS) was used as a control whereas triton-X100 was used as negative control. The cells were treated with a 670 nm laser irradiation at 0.1 W/cm2 for 5 min and further incubated for 24 h. Aqueous solution of MTT (50 µL) was added to each well in the 96-well plate 4 h before the termination of 24 h incubation. The upper layer of the solution was discarded. The MTT solubilization solution, DMSO (100 µL) was added to each well to dissolve the formazan crystals by pipette stirring and then observed the absorbance at 590 nm, which was converted to cell viability using the following equation. Cell viability (%) = (absorbance of sample cells/absorbance of control cells) × 100
The results of cell viability analysis using MTT assay showed that exposure of Mn3O4 and NDG-Mn3O4 in the concentration range of 6.25-100 µg/mL did not cause any cytotoxicity (
Fluorescence microscopy was used for morphological analysis of cancer cells following treatment with nanoparticles and laser irradiation. Fluorescein diacetate (FDA) and propidium iodide (PI) were used to visualize the live and dead cells, respectively. A549 cells (2 × 104 cells per well) were seeded in a 24 well plate and incubated at 37° C. for 24 h in an atmosphere of 5% CO2. Mn3O4 and NDG-Mn3O4 nanoparticles (50 µg/mL) were added to the wells and the plate was incubated for 4 h. After incubation, the cells were irradiated for 5 min with a 670 nm laser, followed by another incubation for 24 h. Both the dyes were added to wells and the plate was incubated for 5 min. Then, the cells were washed three times with PBS to remove excess dyes, and the fluorescence images were acquired by fluorescence microscope with 490 nm excitation and 525 nm emission wavelengths.
The results of in-vitro photodynamic therapy are shown in
1,3-Diphenylisobenzofuran (DBPF) was used to detect singlet oxygen (1O2) generation by NDG-Mn3O4 nanocomposites under 670 nm laser irradiation (0.1 W/cm2). Fifty microliters of ethanolic solution of DPBF (1 mg/mL) were added to the nanocomposites solution under stirring and irradiated with laser for different time points. The absorbance of solution was measured by UV-Visible spectrophotometer. The decrease in absorbance at 426 nm indicated the degradation of DPBF in presence of 1O2 which was generated by laser-induced activation of NDG-Mn3O4 nanocomposites.
To evaluate the 1O2 generation from NDG-Mn3O4 nanocomposites under laser irradiation, we measured the absorbance of 1,3-diphenylisobenzofuran (DPBF) after laser irradiation (670 nm, 0.1 W/cm2) at different time points (
A series of aqueous suspensions of NDG-Mn3O4 nanoparticles (with Mn concentration from 0 to 1 mM) were prepared and imaged in 0.2 mL Eppendorf tubes using a 3T clinical MRI instrument (GE Signa Excite Twin-Speed, GE Healthcare, Milwaukee, WI, USA). The specific relaxivity (r1) was calculated from linear curve generated from concentration of NDG-Mn3O4 nanocomposites versus 1/T1 (s-1).
For testing the effectiveness of NDG-Mn3O4 nanocomposites toward diagnostic standpoint, we investigated whether these nanoparticles have MRI contrast properties or not. Various concentrations of nanoparticles were subjected to imaging by 3T MRI scanner. The result demonstrated a concentration dependent enhancement of signal intensity with increasing concentration of NDG-Mn3O4 nanocomposites. The r1 value was found to be 0.09 mM-1s-1 (
The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
The present invention provides a nanocomposite comprising nitrogen doped graphene oxide conjugated with Mn3O4 nanoparticle for the effective treatment of cancer using photodynamic therapy.
The present disclosure provides a nanocomposite comprising nitrogen doped graphene oxide conjugated with Mn3O4 nanoparticle for the magnetic resonance imaging (MRI).
