HYBRID NANOPARTICLES COMPRISING MANGANESE OXIDE AND HIGHLY REDUCED GRAPHENE OXIDE FOR THERANOSTIC APPLICATIONS

Abstract

The present disclosure provides HRG-Mn.sub.3O.sub.4 hybrid nanoparticles. The HRG-Mn.sub.3O.sub.4 hybrid nanoparticles do not pose any cytotoxicity at normal physiological conditions and therefore they are nontoxic and biocompatible at physiological conditions. The HRG-Mn.sub.3O.sub.4 hybrid nanoparticles under exposure of laser light cause massive cellular damage indicating their potential use for photodynamic therapy of cancer. The HRG-Mn.sub.3O.sub.4 hybrid nanoparticles enhance the magnetic resonance signals from cancer cells and exhibit excellent MRI contrast property for tumor imaging and are therefore useful contrast agent.

Claims

1. Hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) as photodynamic therapy agent and imaging agent for cancer.

2. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) are round with the average diameter of about 8 nm to about 16 nm, preferably from about 9 nm to about 15 nm.

3. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) exhibits X-ray photoelectron spectrum comprising the peaks at about 0.65 keV, 5.88 keV, and 6.62 keV, and 0.0-0.5 keV.

4. The hybrid nanoparticles of claim 1, wherein the elements present in the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) e.g., as detected by an energy dispersive X-ray detector (EDX) are manganese, carbon, oxygen, and. In more preferred embodiments, the nanocomposite is characterized by an energy-dispersive X-ray spectrum as shown in FIG. 1(B).

5. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) is characterized by UV-visible spectrum comprising absorption bands at ˜220 and ˜270 nm respective to Mn.sub.3O.sub.4 and HRG.

6. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) is characterized by FT-IR spectrum comprising bands at ˜1630 cm.sup.−1, ˜1209 cm.sup.−1, ˜1050 cm.sup.−1 and a broad band at ˜3440 cm.sup.−1.

7. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) is characterized by an FT-IR spectrum comprising absence of band at ˜1740 cm.sup.−1, the decrease in the intensity of the bands at ˜3440 cm.sup.−1 and presence of absorption bands at ˜624 cm.sup.−1 and ˜525 cm.sup.−1.

8. The hybrid nanoparticles of claim 1, wherein hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) is characterized by The XRD pattern showing a broad peak at ˜22.4° (002) confirming the reduction of graphene oxide.

9. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) is characterized by the weight loss of about 20% after heating up to 800° C.

10. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) are hemocompatible.

11. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) do not cause cytotoxicity at normal physiological conditions.

12. The hybrid nanoparticles of claim 1, wherein the hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) cause cellular damage to cancer cells upon exposure of laser light of 670 nm wavelength at a light intensity of 4 mW cm.sup.2.

13. A method of synthesis of hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) comprising steps of: (i). synthesising manganese oxide (Mn.sub.3O.sub.4) nanoparticles; (ii). synthesising highly reduced graphene oxide (HRG) nanoparticles; and (iii). preparing highly reduced graphene-Mn.sub.3O.sub.4 (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles comprising steps of milling (Mn.sub.3O.sub.4) nanoparticles and highly reduced graphene oxide (HRG) nanoparticles.

14. The method of synthesis of hybrid nanoparticle of claim 13, wherein the synthesis of manganese oxide (Mn.sub.3O.sub.4) nanoparticles comprises steps of: (i). dissolving manganese (II) acetylacetonate in oleylamine in a molar ratio of 1:20 to 1:30 to provide a slurry; (ii). heating the slurry at about 150° C. to 170° C. for a period of about 8 hours to about 18 hours under a nitrogen atmosphere to provide a suspension; (iii). separating a brown precipitate by centrifuging the suspension at about 7000 rpm to about 12000 rpm for about 5 mins to about 30 mins; and (iv). washing the precipitate with a C1-C3 alcohol multiple times to obtain manganese oxide (Mn.sub.3O.sub.4) nanoparticles.

15. The method of synthesis of hybrid nanoparticle of claim 13, wherein the synthesis of highly reduced graphene oxide (HRG) nanoparticles comprises steps of: (i). synthesizing a graphene oxide (GRO) from graphite powder; (ii). converting the graphene oxide (GRO) to a highly reduced graphene oxide (HRG) comprising steps: a) dispersing GRO in water and sonicating for about 10 mins to about 60 mins to provide a suspension; b) heating the suspension up to 100° C. and adding about 1 ml to about 5 ml of hydrazine hydrate and continuing the reaction under reduced temperature of about 95° C. to about 98° C. under stirring for a period of about 18 hours to about 28 hours to provide a suspension; c) centrifuging the suspension at about 2000 rpm to about 5000 rpm for about 2 mins to about 10 mins to obtain a filtrate; d) washing the filtrate several times with water and drying under vacuum to provide a black powder of HRG.

16. The method of synthesis of hybrid nanoparticle of claim 13, wherein the preparing (HRG-(Mn.sub.3O.sub.4)) hybrid nanoparticles comprises steps of: (i). milling manganese oxide (Mn.sub.3O.sub.4) nanoparticles and highly reduced graphene oxide (HRG) nanoparticles in a ratio of about 1:0.5 to about 1:2, preferably at a ratio of about 1:1; and (ii). continuing milling for a period of about 12 hours to 20 hours with intermittent pause(s).

17. Use of (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles as photodynamic therapy agent for treatment of cancer and/or as contrast agent for imaging of cancer cells.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0019] The accompanying drawings, which are incorporated herein, and constitute a part of this invention, illustrate exemplary embodiments of the disclosed methods and systems in which like reference numerals refer to the same parts throughout the different drawings. Components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention. Some drawings may indicate the components using block diagrams and may not represent the internal circuitry of each component. It will be appreciated by those skilled in the art that the invention of such drawings includes the invention of electrical components, electronic components, or circuitry commonly used to implement such components.

[0020] FIG. 1 illustrates characterization of HRG-Mn.sub.3O.sub.4 nanoparticles as per an exemplary embodiment of the present disclosure: (A) Transmission electron microscopy image, (B) Energy-dispersive X-ray spectra, (C) UV-visible spectroscopy spectra, (D) FT-IR spectra, (E) X-ray diffraction analysis spectra; and (F) thermogravimetric analysis graph.

[0021] FIG. 2 illustrates quantitative determination of hemolysis by HRG-Mn.sub.3O.sub.4 nanoparticles as per an exemplary embodiment of the present disclosure: (A) A graph depicting hemolysis (%) by measuring the absorbance of the supernatants at 540 nm of H.sub.2O, PBS, and HRG-Mn.sub.3O.sub.4 nanoparticles at 100, 50, 25, and 12 μg/ml; and (B) digital image of tubes (inside) of said samples.

[0022] FIG. 3 illustrates cellular uptake of HRG-Mn.sub.3O.sub.4 nanoparticles with different concentrations exposure determined by ICP-MS.

[0023] FIG. 4 illustrates cytotoxicity analysis showing cell viability of A549 cells treated with different concentrations of Mn.sub.3O.sub.4 and HRG-Mn.sub.3O.sub.4 nanoparticles.

[0024] FIG. 5 illustrates cell viability of A549 cells incubated with different concentrations of PBS (control), Triton X100 (negative control), Mn.sub.3O.sub.4 and HRG-Mn.sub.3O.sub.4 nanoparticles in presence of 670 nm laser irradiation (0.1 W/cm.sup.2) for 5 min. Data are represented as mean±standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001 versus respective control group.

[0025] FIG. 6 illustrates fluorescence microscopy images of A549 cells co-stained with fluorescein diacetate (green emission for live cells) and propidium iodide (red emission for dead cells) with PBS (control), HRG-Mn.sub.3O.sub.4 nanoparticles with/without laser irradiation (670 nm, 0.1 W/cm.sup.2) for 5 min.

[0026] FIG. 7 illustrates T.sub.1-weighted MR imaging of HRG-Mn.sub.3O.sub.4 nanoparticles in aqueous solution and the T1 relaxivity plot of aqueous suspension of HRG-Mn.sub.3O.sub.4 nanoparticles.

[0027] FIG. 8 illustrates cellular T.sub.1-weighted MR imaging of HRG-Mn.sub.3O.sub.4 nanoparticles. T.sub.1-weighted MR images of HRG-Mn.sub.3O.sub.4 nanoparticles in A549 cells after 24 h incubation time.

[0028] The foregoing shall be more apparent from the following more detailed description of the invention.

DETAILED DESCRIPTION OF INVENTION

[0029] In the following description, for the purposes of explanation, various specific details are set forth in order to provide a thorough understanding of embodiments of the present disclosure. It will be apparent, however, that embodiments of the present disclosure may be practiced without these specific details. Several features described hereafter can each be used independently of one another or with any combination of other features. An individual feature may not address all of the problems discussed above or might address only some of the problems discussed above. Some of the problems discussed above might not be fully addressed by any of the features described herein.

[0030] The ensuing description provides exemplary embodiments only, and is not intended to limit the scope, applicability, or configuration of the disclosure. Rather, the ensuing description of the exemplary embodiments will provide those skilled in the art with an enabling description for implementing an exemplary embodiment. It should be understood that various changes may be made in the function and arrangement of elements without departing from the spirit and scope of the invention as set forth.

[0031] Specific details are given in the following description to provide a thorough understanding of the embodiments. However, it will be understood by one of ordinary skill in the art that the embodiments may be practiced without these specific details. For example, circuits, systems, networks, processes, and other components may be shown as components in block diagram form in order not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, algorithms, structures, and techniques may be shown without unnecessary detail in order to avoid obscuring the embodiments.

[0032] Also, it is noted that individual embodiments may be described as a process which is depicted as a flowchart, a flow diagram, a data flow diagram, a structure diagram, or a block diagram. Although a flowchart may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be re-arranged. A process is terminated when its operations are completed but could have additional steps not included in a figure. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a function, its termination can correspond to a return of the function to the calling function or the main function.

[0033] The word “exemplary” and/or “demonstrative” is used herein to mean serving as an example, instance, or illustration. For the avoidance of doubt, the subject matter disclosed herein is not limited by such examples. In addition, any aspect or design described herein as “exemplary” and/or “demonstrative” is not necessarily to be construed as preferred or advantageous over other aspects or designs, nor is it meant to preclude equivalent exemplary structures and techniques known to those of ordinary skill in the art. Furthermore, to the extent that the terms “includes,” “has,” “contains,” and other similar words are used in either the detailed description or the claims, such terms are intended to be inclusive—in a manner similar to the term “comprising” as an open transition word—without precluding any additional or other elements.

[0034] Reference throughout this specification to “one embodiment” or “an embodiment” or “an instance” or “one instance” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0035] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0036] The phrase “Theranostic application” means combined diagnostic and therapeutic applications by the same nanoparticles having dual functionality.

[0037] The present disclosure provides hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) that are nontoxic and biocompatible at physiological conditions for theranostic applications for example diagnosis and treatment of cancer.

[0038] In an embodiment, the present disclosure provides (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles useful in photodynamic therapy.

[0039] In an embodiment, the present disclosure provides (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles useful as contrast agent to enhance the magnetic resonance signals from cancer cells.

[0040] In an embodiment, the present disclosure provides (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles as photodynamic therapy and imaging agent for cancer.

[0041] In an embodiment, the hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) in accordance of the present disclosure are round with the average diameter of about 8 nm to about 16 nm, preferably from about 9 nm to about 15 nm.

[0042] In an embodiment, the hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) have the average diameter of about 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, or 16 nm.

[0043] In an embodiment, the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure exhibits X-ray photoelectron spectrum comprising the peaks at about 0.65 keV, 5.88 keV, and 6.62 keV, and 0.0-0.5 keV.

[0044] In one embodiment, the elements present in the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) e.g., as detected by an energy dispersive X-ray detector (EDX) are manganese, carbon, and oxygen. In more preferred embodiments, the nanocomposite is characterized by an energy-dispersive X-ray spectrum as shown in FIG. 1(B).

[0045] In an embodiment, the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure is characterized by UV-visible spectrum comprising absorption bands at ˜220 and ˜270 nm respective to Mn.sub.3O.sub.4 and HRG for e.g., as shown in FIG. 1(C).

[0046] In an embodiment, the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure is characterized by FT-IR spectrum comprising bands at ˜1630 cm.sup.−1, ˜1209 cm.sup.−1, ˜1050 cm.sup.−1 and a broad band at ˜3440 cm.sup.−1 respectively for C—O—C stretching, C—O stretching, C═C stretching, representing oxygen-containing functional groups selected from carbonyl, carboxylic, epoxy, and hydroxyl groups present in graphene oxide.

[0047] In an embodiment, the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure is characterized by an FT-IR spectrum comprising absence of band at ˜1740 cm.sup.−1, the relative decrease in the intensity of the bands at ˜3440 cm.sup.−1 respectively representing the removal of oxygen-containing groups of graphene oxide in HRG, reduction of graphene oxide to HRG and presence of absorption bands at ˜624 cm.sup.−1 and ˜525 cm.sup.−1 respective to manganese representing the formation of HRG-Mn.sub.3O.sub.4 nanocomposite for e.g. as shown in FIG. 1(D).

[0048] In an embodiment, the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure is characterized by the XRD pattern showing the characteristics peaks of both the entities (HRG as well as Mn.sub.3O.sub.4); the appearance of a broad peak at ˜22.4° (002) represent the reduction of graphene oxide for e.g., as shown in FIG. 1(E).

[0049] In an embodiment, the hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure is characterized by the weight loss of about 20% after heating up to 800° C. representing the presence of substantial oxygen functionalities for e.g., as shown in FIG. 1(F).

[0050] The hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure have excellent hemocompatibility with negligible RBC lysis.

[0051] The hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure do not pose any cytotoxicity at normal physiological conditions. Thus, the hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure are devoid of cytotoxicity at normal physiological conditions and are therefore biocompatible.

[0052] The hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure with exposure of laser light of specific wavelength resulted in massive cellular damage by HRG-Mn.sub.3O.sub.4 nanoparticles suggesting their potential for photodynamic therapy. The hybrid nanoparticles (HRG-Mn.sub.3O.sub.4) in accordance with the present disclosure also exhibit excellent MRI contrast property for tumor imaging.

[0053] In another embodiment, the present disclosure provides a method of synthesis of hybrid nanoparticle (HRG-Mn.sub.3O.sub.4) comprising steps of [0054] (i). synthesising manganese oxide (Mn.sub.3O.sub.4) nanoparticles; [0055] (ii). synthesising highly reduced graphene oxide (HRG) nanoparticles; and [0056] (iii). preparing highly reduced graphene-Mn.sub.3O.sub.4 (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles comprising steps of milling (Mn.sub.3O.sub.4) nanoparticles and highly reduced graphene oxide (HRG) nanoparticles.

[0057] In one embodiment, the synthesis of manganese oxide (Mn.sub.3O.sub.4) nanoparticles comprises steps of: [0058] (i). dissolving manganese (II) acetylacetonate in oleylamine in a molar ratio of at least about 1:20 to about 1:30 to provide a slurry; [0059] (ii). heating the slurry at least about 150° C. to about 170° C. for a period of at least about 8 hours to about 18 hours under a nitrogen atmosphere to provide a suspension; [0060] (iii). separating a brown precipitate by centrifuging the suspension at least at about 7000 rpm to about 12000 rpm for at least about 5 mins to about 30 mins; and [0061] (iv). washing the precipitate with a C1-C3 alcohol multiple times to obtain manganese oxide (Mn.sub.3O.sub.4) nanoparticles.

[0062] In one embodiment, the molar ratio of manganese (II) acetylacetonate to oleylamine can be selected from at least about 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 and 1:30.

[0063] In one embodiment, the temperature for heating the slurry obtained in step (ii) can be at least at about 150° C., 155° C., 160° C., 165° C., 170° C., or any temperature in between said temperatures.

[0064] In one embodiment, the period for heating the slurry obtained in step (ii) can be at least about 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours or 18 hours.

[0065] In one embodiment, the separating of the brown precipitate by centrifuging the suspension can be carried out at speed of at least about 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm or 12000 rpm.

[0066] In a preferred embodiment, the C1-C3 alcohol used for washing the precipitate is ethyl alcohol.

[0067] In one embodiment, the synthesised manganese oxide (Mn.sub.3O.sub.4) nanoparticles can be re-dispersed in organic solvent selected from but not limiting to hexane, toluene, and dichloromethane.

[0068] In one embodiment, the synthesised manganese oxide (Mn.sub.3O.sub.4) nanoparticles can be dried in a vacuum before use.

[0069] In one embodiment, the synthesis of highly reduced graphene oxide (HRG) nanoparticles comprises steps of: [0070] (i). synthesizing a graphene oxide (GRO) from graphite powder; [0071] (ii). converting the graphene oxide (GRO) to a highly reduced graphene oxide (HRG) comprising steps: [0072] a) dispersing GRO in water and sonicating for at least about 10 mins to about 60 mins to provide a suspension; [0073] b) heating the suspension up to 100° C. and adding about 1 ml to about 5 ml of hydrazine hydrate and continuing the reaction under reduced temperature of at least about 95° C. to about 98° C. under stirring for a period of about 18 hours to about 28 hours to provide a suspension; [0074] c) centrifuging the suspension at least at about 2000 rpm to about 5000 rpm for about 2 mins to about 10 mins to obtain a filtrate; [0075] d) washing the filtrate several times with water and drying under vacuum to provide a black powder of HRG.

[0076] In one embodiment, sonication of GRO dispersed in water in step (iii)(a) can be carried out at least for about 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, or about 60 mins to provide a suspension.

[0077] In one embodiment, in step (iii)(b), the amount of hydrazine hydrate is added is about 1 ml, 2 ml, 3 ml, 4 ml, or 5 ml.

[0078] In one embodiment, in step (iii)(b), the reduced temperature for carrying out the reaction upon addition of hydrazine hydrate is selected from about 95° C., 96° C., 97° C., or 98° C.

[0079] In one embodiment, in step (iii)(b), the reaction after adding hydrazine hydrate is continued for a period selected from of about 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, or 28 hours to provide a suspension.

[0080] In one embodiment, in step (iii)(c), the centrifugation of the suspension is carried out for at least at about 2000 rpm, 3000 rpm, 4000 rpm, or 5000 rpm.

[0081] In one embodiment, in step (iii)(c), the centrifugation of the suspension is carried out at least for about 2 mins, 3, mins, 4 mins, 5 mis, 6 mins, 7 mins, 8 mins, 9 mis and 10 mins.

[0082] In one embodiment, the preparing (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles comprises steps of: [0083] (i). milling manganese oxide (Mn.sub.3O.sub.4) nanoparticles and highly reduced graphene oxide (HRG) nanoparticles in a ratio of about 1:0.5 to about 1:2, preferably at a ratio of about 1:1; and [0084] (ii). continuing milling for a period of at least about 12 hours to 20 hours with intermittent pause(s).

[0085] In one embodiment, the milling of manganese oxide (Mn.sub.3O.sub.4) nanoparticles and highly reduced graphene oxide (HRG) nanoparticles is continued for a period selected from about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours or 20 hours.

[0086] In an embodiment, the milling of manganese oxide (Mn.sub.3O.sub.4) nanoparticles and highly reduced graphene oxide (HRG) nanoparticles is carried out in the presence of stainless steel balls of about 5 mm diameter with the ball to powder weight ratio of about 1:1 to provide highly reduced graphene-Mn.sub.3O.sub.4 (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles.

[0087] The highly reduced graphene-Mn.sub.3O.sub.4 (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles in accordance with the present disclosure are useful for theranostic applications for example diagnosis and treatment of cancer. The highly reduced graphene-Mn.sub.3O.sub.4 (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles of the present disclosure are useful for optical and MRI imaging technique for both in-vivo animal and clinical cancer diagnosis. The highly reduced graphene-Mn.sub.3O.sub.4 (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles in accordance with the present disclosure are useful for photodynamic therapy.

[0088] In another embodiment, the present disclosure provides a use of (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles for photodynamic therapy of cancer.

[0089] In another embodiment, the present disclosure provides a use of (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles as MRI imaging agent or contrast agent to enhance the magnetic resonance signals from cancer cells.

[0090] In another embodiment, the present disclosure provides use of (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles as photodynamic therapy agent for treatment of cancer and/or as contrast agent for imaging of cancer cells.

[0091] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

EXAMPLES

[0092] The present invention is illustrated in further details by the following non-limiting examples.

Example 1

[0093] Preparation of (HRG-Mn.sub.3O.sub.4) Hybrid Nanoparticles

Chemicals and Reagents:

[0094] All chemicals including solvents used for the synthesis of nanoparticles were procured from Sigma Aldrich (St. Louis, Mo., USA). Graphite powder (99.999%, 200 mesh) was purchased from Alfa Aesar, Kandel, Germany. Deionized water was prepared from a Millipore Milli-Q system and used in all experiments.

A) Synthesis of Mn.sub.3O.sub.4 Nanoparticles:

[0095] A slurry of Manganese (II) acetylacetonate was dissolved in oleylamine (molar ratio of Manganese (II) acetylacetonate:oleylamine=1:25) in a 100 mL three-neck flask. The mixture was heated at 162° C. for 11 hrs. under a nitrogen atmosphere, and then the resulting mixture was cooled down to room temperature to form a brown suspension. After centrifugation at 9000 rpm for 15 mins, the supernatant was removed and a brown precipitate was obtained. The brown precipitate was washed with ethanol five times to acquire pure Mn.sub.3O.sub.4 nanoparticles. Finally, Mn.sub.3O.sub.4 nanoparticles were dried in a vacuum before use.

B) Synthesis of Highly Reduced Graphene Oxide (HRG):

[0096] Initially graphite oxide (GO) was synthesized from graphite powder and then using a modified Hummers method (Hummers W S, Offeman R E., Preparation of graphitic oxide, J Am Chem Soc. 1958; 80: 1339; and Alam S N, Sharma N, Kumar L., Synthesis of graphene oxide (GRO) by modified Hummers method and its thermal reduction to obtain highly reduced graphene oxide (HRG), Graphenes 2017; 6: 1-18) and then it was converted to highly reduced graphene oxide (HRG) following several steps of centrifugation, washing and finally sonication. GRO was reduced according to a previously reported method (Assal M E, Shaik M R, Kuniyil M, Khan M, Alzahrani A Y, Al-Warthan A, Siddiqui M R H, Adil S F., Mixed zinc/manganese on highly reduced graphene oxide: A highly active nanocomposite catalyst for aerial oxidation of benzylic alcohols, Catalysts 2017; 7: 391). Briefly, GRO was dispersed in water and sonicated for 30 min. The resulting suspension was allowed to heat up to 100° C. and subsequently 3 ml of hydrazine hydrated were added. The temperature was slightly reduced (98° C.), and the suspension was kept under stirring for 24 h. Finally, a black powder was obtained which was filtered and washed several times with water. The resultant suspension was centrifuged at 4,000 rpm for several 3 min, and the final product was collected via filtration and dried under vacuum.

C) Preparation of Highly Reduced Graphene-Mn.sub.3O.sub.4 (HRG-Mn.sub.3O.sub.4) Hybrid Nanoparticles:

[0097] Approximately 200 mg of Mn.sub.3O.sub.4 nanoparticles and 200 mg of highly reduced graphene (HRG) powder were milled using Fritsch Pulverisette P7 (Idar-Oberstein, Germany) planetary ball mill. The nanocomposite 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 hours. The milling process was paused at regular intervals to give (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles.

Example 2

[0098] Characterization of (HRG-Mn.sub.3O.sub.4) Hybrid Nanoparticles

[0099] The synthesized (HRG-Mn.sub.3O.sub.4) hybrid nanoparticles were characterized for size, elemental composition, physicochemical properties and stability using high resolution transmission electron microscopy (JEM-2100F, JEOL, Japan), energy-dispersive X-ray spectroscopy (EDX), UV-Vis spectroscopy (Perkin Elmer lambda 35, Waltham, Mass., USA), FT-IR spectroscopy (Perkin Elmer 1,000 FT-IR spectrometer), X-ray diffraction analysis (D2 Phaser X-ray diffractometer, Bruker, Germany) and thermogravimetric analysis (TGA/DSC1, Mettler Toledo AG, Analytical, Schwerzenbach, Switzerland).

[0100] The shape of hybrid nanoparticles appeared as round with the average diameter of 12±2.21 nm (FIG. 1(A)).

[0101] In EDX analysis, the intense signals at 0.65, 5.88, and 6.62 keV strongly suggests that ‘Mn’ was the major element, which has an optical absorption in this range owing to the surface plasmon resonance (SPR). The other signals found in the range 0.0-0.5 keV signify the absorption of carbon and oxygen, confirming the formation of HRG-Mn.sub.3O.sub.4 nanocomposite (FIG. 1(B)).

[0102] UV-visible spectrum of HRG-Mn.sub.3O.sub.4 nanoparticles showed respective absorption bands at ˜220 (Mn.sub.3O.sub.4) and ˜270 nm (HRG) indicating the formation of HRG-Mn.sub.3O.sub.4 (FIG. 1(C)).

[0103] FT-IR spectrum of HRG-Mn.sub.3O.sub.4 displayed the graphene oxide bands at ˜1630 cm.sup.−1 (for C═C stretching), ˜1209 cm.sup.−1 (for C—O—C stretching), ˜1050 cm.sup.−1 (for C—O stretching), and a broad band at around 3440 cm.sup.−1 for hydroxyl groups indicated the presence of various oxygen-containing functional groups, such as carbonyl, carboxylic, epoxy, and hydroxyl groups in graphene oxide. The removal of oxygen-containing groups of graphene oxide in HRG was indicated by the disappearance of some of the bands such as the band at ˜1740 (which is present in HRG only; spectrum not shown). Also, the relative decrease in the intensity of some of the bands, like the decrease in intensity of broad band at 3440 cm.sup.−1 points towards the reduction of graphene oxide. The existence of other absorption bands of Mn at 624 and 525 cm.sup.−1 clearly indicated the formation of HRG-Mn.sub.3O.sub.4 nanocomposite (FIG. 1(D)).

[0104] The XRD pattern of HRG-Mn.sub.3O.sub.4 nanoparticles showed the characteristics peaks of both the entities (HRG as well as Mn.sub.3O.sub.4); the appearance of a broad peak at ˜22.4° (002) confirmed the reduction of graphene oxide (FIG. 1(E)).

[0105] TGA analysis of HRG-Mn.sub.3O.sub.4 nanoparticles displays the weight loss of about 20% after heating up to 800° C. indicating the presence of substantial oxygen functionalities (FIG. 1(F)).

Example 3

[0106] Biological Assays of (HRG-Mn.sub.3O.sub.4) Hybrid Nanoparticles

Statistics:

[0107] All the cell based analyses were performed in triplicate and the results reported as means±standard deviation. The data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test. P values<0.05 were considered as statistically significant.

I. Hemolysis Assay:

[0108] Hemolysis assay was performed by collecting 2 mL of blood from Sprague Dawley rat (6 weeks, Female) through cardiac puncture. The blood drawn from the rat was immediately centrifuged at 1500 rpm for 10 min. The pellet was washed with PBS three times and finally suspended in 20 mL of PBS. Then, 500 μL of freshly prepared RBCs were added to different concentrations (12, 25, 50 and 100 μg mL-1) of nanoparticles (500 μL), as well as positive (PBS) and negative control (H.sub.2O) tubes. All of samples were prepared in triplicate. The samples were then placed in a 37° C. incubator for 4 hours and then centrifuged at 1500 rpm for 10 min. The absorbance of the supernatant was recorded at 540 nm and collected the digital image. Hemolysis percent was calculated by diving each sample's hemoglobin concentration by the total blood hemoglobin as per the following equation:

Hemolysis %=(Hemoglobin in test sample/Total Blood Hemoglobin)×100.

[0109] No obvious hemolysis was observed when the RBCs were incubated even with a higher concentration (100 μg mL.sup.−1) of nanoparticles. In addition, the extent of lysis was similar to PBS implied that developed HRG-Mn.sub.3O.sub.4 nanoparticles had excellent hemocompatibility with negligible RBC lysis (FIG. 2). This result confirmed that the HRG-Mn.sub.3O.sub.4 nanoparticles are biocompatible.

II. Cytotoxicity Assay:

[0110] In-vitro cytotoxicity assay to investigate the toxicity profile of newly synthesized HRG-Mn.sub.3O.sub.4 hybrid nanoparticles was performed. In vitro cytotoxicity studies of nanoparticles are preferred as they are simple, cost-effective and faster than in-vivo models.

[0111] The cytotoxicity of Mn.sub.3O.sub.4 and HRG-Mn.sub.3O.sub.4 nanoparticles was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. A549 cells (a non-small cell lung cancer cell line comprising lung carcinoma epithelial cells) were seeded in the 96-well plate (4×10.sup.4 cells per well) in RPMI medium and incubated in the atmosphere of 5% CO.sub.2 at 37° C. for 24 hrs. Different concentrations (6.25, 12.5, 25, 50 and 100 μg/ml) of Mn.sub.3O.sub.4 and HRG-Mn.sub.3O.sub.4 were added to the respective wells of micro plate.

[0112] For laser-induced phototoxicity analysis, the nanoparticles and further incubated for 4 h. Phosphate buffer saline (PBS) and triton X-100 were used as control and negative control, respectively. Then cells were treated with a 670 nm laser irradiation at 0.1 W/cm.sup.2 for 5 min and further incubated for 24 h. MTT aqueous solution (50 ml) 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 absorbance at 590 nm, which was converted to cell viability based on absorbance of dissolved formazan. The cellular uptake was performed by ICP-MS. The viable quantity of cells was then calculated using the following equation:

Cell viability (%)=(absorbance of sample cells/absorbance of control cells)×100

[0113] The results showed that developed nanoparticles are capable of cellular interactions to A549 cells at different concentrations (100, 50, 25 μg mL.sup.−1) while the cellular uptake was directly proportional with the concentration of nanoparticles (FIG. 3).

[0114] The cytotoxicity analysis using MTT assay showed that more than 98% of A549 cells survived even after the exposure of a high concentration (100 μg/ml) of nanomaterials indicating the biocompatibility of both Mn.sub.3O.sub.4 and HRG-Mn.sub.3O.sub.4 nanoparticles (FIG. 4).

[0115] These results confirmed that the HRG-Mn.sub.3O.sub.4 nanoparticles can easily be taken up by cells (FIG. 3) and are nontoxic (FIG. 4) under physiological conditions.

[0116] Almost 100% cells were viable when treated with phosphate buffered saline (PBS) or Mn.sub.3O.sub.4 nanoparticles in presence of 670 nm wavelength laser irradiation (0.1 W/cm.sup.2) for 5 min (FIG. 5). However, laser irradiation resulted in significant and concentration-dependent cellular damage by HRG-Mn.sub.3O.sub.4 nanoparticles (FIG. 5). These results confirm a significant and concentration-dependent cytotoxicity of HRG-Mn.sub.3O.sub.4 after laser irradiation as compared to Mn.sub.3O.sub.4 nanoparticles.

III. In-Vitro Photodynamic Therapy Using Fluorescence Microscopy of Live and Dead Cells:

[0117] The live/dead assay kit containing fluorescein diacetate (FDA) and propidium iodide (PI) to visualize live and dead cells, respectively was used and cells were visualized under fluorescence microscope. A549 cells (2×10.sup.4 cells per well) were seeded on a 24 well plate and incubated in the atmosphere of 5% CO.sub.2 at 37° C. for 24 hrs. Mn.sub.3O.sub.4 and HRG-Mn.sub.3O.sub.4 nanoparticles (50 μg/ml) were added to the 24 well plate. PBS was used as a control and the plate was incubated for 4 h. Then cells were exposed to a 670 nm wavelength laser irradiation at 0.1 W/cm.sup.2 for 5 min and further incubated for 24 h. FDA and PI were added to treated cells and incubated for 5 min. Then cells were washed with PBS thrice to remove excess FDA/PI and fluorescence images were acquired by fluorescence microscope with 490 nm excitation and emission at 525 nm.

[0118] To study the interactions between cells and the nanoparticles, we used the visible red optical imaging of A549 cells after incubation in PBS, Mn.sub.3O.sub.4 nanoparticles, HRG-Mn.sub.3O.sub.4 nanoparticles and HRG-Mn.sub.3O.sub.4 nanoparticles with and without laser irradiation for 5 min. (FIG. 6). After 5 min, propidium iodide (PI) (red emission for dead cells) fluorescent dots were observed in HRG-Mn.sub.3O.sub.4 nanoparticles plus laser treated group when compared to HRG-Mn.sub.3O.sub.4 nanoparticles treated cells. However, no red fluorescent dots were observed in PBS and Mn.sub.3O.sub.4 treated A549 groups of cells. On the other hand, A549 cells incubated in PBS, Mn.sub.3O.sub.4 nanoparticles, HRG-Mn.sub.3O.sub.4 nanoparticles showed abundant green emission indicating the presence of live cells (FIG. 6). This result confirm that hybrid nanoparticles produced cytotoxicity only after laser irradiation suggesting their potential for PDT of cancer.

IV. In-Vitro MRI Imaging:

[0119] A series of aqueous solutions of HRG-Mn.sub.3O.sub.4 nanoparticles (with Mn from 0 to 1 mM) were prepared and imaged in a 0.2 Eppendorf tube on a 3 T MRI instrument. A series of cell culture medium of HRG-Mn.sub.3O.sub.4 nanoparticles (0 to 50 mg/mL) were treated with A549 cells (1*104) per well. After 4 h incubation, cells were washed with PBS and collected for MRI imaging using a 3 T MRI scanner (BioSpec 47/40; Bruker, Germany) at Korea Basic Science Institute with 72 mm and 35 mm volume coils for phantom.

[0120] The results of MRI analysis demonstrated a significant enhancement of signal intensity of nanoparticles with increasing Mn concentration using nanoparticles suspension (FIG. 7) as well as cells exposed to nanoparticles (FIG. 8). The specific relaxivity (r1) was calculated from linear curve generated from concentration of Mn versus 1/T.sub.1 (s.sup.−1). The r1 value was found to be 0.06 mM.sup.−1s.sup.−1. The results of MRI showed concentration dependent contrast property of HRG-Mn.sub.3O.sub.4 nanoparticles in both aqueous suspension (FIG. 7) and cancer cells (FIG. 8). Thus, they have higher T1 or T2 relaxivity and meet the requirements for clinical usefulness.

[0121] These results confirmed that the HRG-Mn.sub.3O.sub.4 nanoparticles do not pose any cytotoxicity at normal physiological conditions and therefore they are biocompatible. However, exposure of laser light of specific wavelength resulted in massive cellular damage by HRG-Mn.sub.3O.sub.4 nanoparticles suggesting their potential for photodynamic therapy. The newly synthesized nanoparticles also showed excellent MRI contrast property for tumor imaging. Thus, these results show capability of the HRG-Mn.sub.3O.sub.4 nanoparticles for theranostic applications.

[0122] While considerable emphasis has been placed herein on the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the invention. These and other changes in the preferred embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter to be implemented merely as illustrative of the invention and not as a limitation.