Graphene oxide nanocomposite for photothermal and photodynamic treatment of multidrug-resistant cancer and method for its preparation

Abstract

The present invention relates to a reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells capable of simultaneously performing enhanced photothermal therapy and photodynamic therapy, and a method for manufacturing the same. The nanocomposite of the present invention comprises reduced graphene, poly(N-isopropylacrylamide)-acrylic acid, and folic acid, thereby exhibiting excellent targeting function for multiple drug-resistant cancer cells. In particular, when an anticancer agent including indocyanine green is encapsulated internally, it exhibits a synergistic effect of photothermal and photodynamic therapy against multiple drug-resistant cancer.

Description

Technology Field

[0001] This specification relates to a reduced graphene oxide nanocomposite for targeting multidrug-resistant cancer cells capable of simultaneously performing photothermal therapy and photodynamic therapy, and a method for manufacturing the same. Background Technology

[0003] In the treatment of cancer, nanomedicines are known to effectively accumulate at the tumor site through enhanced permeability and retention (EPR) effects, thereby reducing the distribution of chemotherapy drugs in non-specific tissues and mitigating side effects caused by excessive local drug concentrations.

[0004] Recently, new cancer treatments such as photothermal therapy and photodynamic therapy have emerged. Photothermal therapy is a method that eliminates cancer cells by applying near-infrared light from outside the body, exploiting the weakness of cancer cells, which are more susceptible to heat than normal cells. In this method, a photothermal agent converts light into heat under near-infrared light to kill the cancer cells. Photodynamic therapy is a method in which a photosensitizer accumulates selectively only in cancer tissue; by irradiating it with light of a specific wavelength, the photosensitizer is activated, generating reactive oxygen species that act on target cancer cells to specifically eliminate only the cancerous tissue.

[0005] Since there are limitations to completely curing cancer with either single photothermal therapy or single photodynamic therapy alone, recent trends in cancer treatment are moving away from performing these methods individually. Research is now being conducted on fusion therapies that enhance the advantages of both photothermal therapy and photodynamic therapy while compensating for their respective disadvantages.

[0006] Multidrug resistance (MDR), in which cancer cells develop high resistance to the cytotoxicity of chemotherapy agents, gradually increases the survival and recurrence rates of cancer cells, leading to ineffective chemotherapy treatment. Among the potential mechanisms of MDR, the overexpression of P-glycoprotein (P-gp) is closely associated with reduced chemotherapy efficacy in MDR cancer cells. This is because this protein inhibits drug accumulation and induces the release of therapeutic agents from cancer cells. These drawbacks can be addressed through co-delivery systems containing chemotherapy drugs and P-gp inhibitors (e.g., small molecules, small interfering RNA (siRNA)) on various nanoscale carriers (e.g., polymers, liposomes, organic-inorganic hybrid nanoparticles). However, existing nanoscale drug delivery systems (DDS) face difficulties in development due to low drug loading doses, low targeting ability for specific cancer cells, and uncontrolled release of encapsulated factors from nanoparticles. Additionally, the encapsulation ability of P-gp-related small molecules or siRNA is greatly influenced by the internal structure and surface charge of the nanoscale carrier.

[0007] Therefore, combination phototherapy using existing graphene oxide-based nanoparticles for the treatment of multidrug-resistant cancer cells still requires further research, and there are limitations that hinder clinical use due to issues such as unwanted drug release and the absence of targeting functions. Prior art literature

[0009] Republic of Korea Registered Patent No. 2096469 The problem to be solved

[0010] In exemplary embodiments of the present invention, in one aspect, a nanocomposite capable of drug release and photothermal therapy by temperature change and near-infrared irradiation and a method for manufacturing the same are provided.

[0011] In exemplary embodiments of the present invention, in another aspect, a nanocomposite and a method for manufacturing the same are provided, which exhibit synergistic effects in multidrug-resistant cancer targeting, photothermal and photodynamic therapy mediated by near-infrared irradiation. means of solving the problem

[0013] In exemplary embodiments of the present invention, a reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy is provided, wherein the nanocomposite comprises reduced graphene oxide; poly(N-isopropylacrylamide)-acrylic acid; and folic acid.

[0014] In an exemplary embodiment, the poly(N-isopropylacrylamide)-acrylic acid is preferably bonded to the surface of reduced graphene oxide.

[0015] In an exemplary embodiment, the folic acid is preferably bonded to the surface of the poly(N-isopropylacrylamide)-acrylic acid.

[0016] In an exemplary embodiment, the shape of the nanocomposite is preferably spherical.

[0017] In an exemplary embodiment, the diameter of the nanocomposite is preferably 400 to 600 nm at 30 to 38.5 °C.

[0018] In an exemplary embodiment, the zeta potential of the nanocomposite is preferably -21.5 to -18.5 mV.

[0019] In an exemplary embodiment, it is preferable that an anticancer agent is loaded onto the nanocomposite.

[0020] In an exemplary embodiment, the anticancer agent is preferably indocyanine green (ICG).

[0021] In an exemplary embodiment, the nanocomposite preferably contains 8 to 20 weight percent of the indocyanine green based on the total weight of the nanocomposite loaded with the indocyanine green.

[0022] In an exemplary embodiment, the zeta potential of the nanocomposite is preferably -33.5 to -29.5 mV.

[0023] In an exemplary embodiment, when near-infrared rays are irradiated onto the nanocomposite, it is preferable that the temperature rises by 5 to 25°C.

[0024] In an exemplary embodiment, the nanocomposite preferably targets multidrug-resistant breast cancer cells.

[0025] In exemplary embodiments of the present invention, a method for preparing a reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy comprises: a first step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid nanocomposite (GO-PNIPAM-AAc) by binding poly(N-isopropylacrylamide)-acrylic acid (PNIPAM-AAc) to graphene oxide (GO); and a second step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (GO-PNIPAM-AAc-FA) by binding folic acid to GO-PNIPAM-AAc. A method for preparing a reduced graphene oxide nanocomposite is provided, comprising: a third step of reducing the above GO-PNIPAM―AAc-FA to provide a reduced graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (rGO-PNIPAM―AAc-FA).

[0026] In an exemplary embodiment, the method preferably further comprises a fourth step of loading an anticancer agent onto the rGO-PNIPAM-AAc-FA.

[0027] In an exemplary embodiment, the first step is preferably to introduce a carboxyl group (-COOH) onto the surface of GO and then bind PNIPAM-AAc.

[0028] In an exemplary embodiment, the second step preferably involves combining the folic acid by conjugating it with folic acid-polyethylene glycol-amine (FA-PEG-NH2). Effects of the invention

[0030] The reduced graphene oxide nanocomposites of exemplary embodiments of the present invention contain reduced graphene oxide, poly(N-isopropylacrylamide)-acrylic acid, and folic acid, thereby exhibiting excellent targeting ability against multidrug-resistant cancer cells. Furthermore, when an anticancer drug is loaded into the nanocomposites of the present invention, synergistic effects as photothermal and photodynamic therapeutic agents can be achieved. In particular, the photothermal therapeutic effect is excellent even when loaded with a low concentration of anticancer drug, and the indiscriminate release of the loaded drug and side effects caused by the loaded drug can be minimized, and the loaded drug can be selectively released only at temperatures above a certain temperature. Brief explanation of the drawing

[0032] Figure 1 is a schematic diagram of the synthesis process of an ICG-encapsulated rGO-PNIPAM-AAc-FA nanocomposite and an overview of photothermal-photodynamic therapy in multidrug-resistant breast cancer cells using the same. Figure 2A is a TEM image of GO-COOH, rGO-PNIPAM-AAc-FA, and ICG@rGO-PNIPAM-AAc-FA. Figure 2B shows the results of DLS analysis of the rGO-PNIPAM-AAc-FA nanocomposite at different temperatures. Figure 2C is a graph showing the zeta potential of GO, GO-COOH, GO-PNIPAM, GO-PNIPAM-AAc-FA, rGO-PNIPAM-AAc-FA, and ICG@rGO-PNIPAM-AAc-FA nanocomposites. Figure 3A is the FT-IR spectrum of the rGO-PNIPAM-AAc-FA nanocomposite. Figure 3B shows the UV-VIS spectra of FA, rGO-PNIPAM-AAc-FA, ICG, and ICG@rGO-PNIPAM-AAc-FA nanocomposites. Figure 4A is a graph showing the results of emitting 808 nm near-infrared light to rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA to compare the photothermal effect by indocyanine green. Figures 4B and 4C show the concentration of the nanocomposite (50, 100, 200 μg / ml) and the intensity of 808 nm near-infrared light (1.0, 1.5, 2 W / cm²). 2 This is a graph showing the results of observing the temperature rise according to ). Fig. 4D is 808nm NIR (1W / cm²) 2 This is the result of the photostability evaluation of the ICG@rGO-PNIPAM-FA (100μg / ml) nanocomposite after irradiation with 3 on / off cycles for 10 minutes. Figure 5A is the UV-VIS spectrum of a DPBF solution containing an ICG@rGO-PNIPAM-FA nanocomposite after 808 nm NIR irradiation over time. Figure 5B shows the ROS generation curves of GO-PNIPAM-FA and ICG@rGO-PNIPAM-FA nanocomposites. Figure 6A is the thermal reactive ICG release profile of the ICG@rGO-PNIPAM-AAc-FA nanocomposite at 25, 37, and 50°C. Figure 6B is a graph showing the thermal + NIR laser responsive ICG emission behavior of the ICG@rGO-PNIPAM-AAc-FA nanocomposite. Figure 7 is an image of cell uptake of the ICG@rGO-PNIPAM-AAc-FA nanocomposite and cultured cells. Figure 8 is an image of intracellular ROS detection in MCF-7 / ADR cells treated with ICG@rGO-PNIPAM-AAc-FA nanocomposite with or without NIR laser irradiation. Figure 9A shows the results of irradiating with an 808 nm NIR laser (3 W / cm²) after treatment with rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA nanocomposites at different concentrations. 2 This is a graph showing the quantitative viability of MCF-7 / ADR cells (5 min). Fig. 9B shows the results of irradiating with an NIR laser (3W / cm²) after treatment with 80 μg / mL of ICG@rGO-PNIPAM-AAc-FA nanocomposite to confirm the synergistic photothermal therapy (PTT) and photodynamic therapy (PDT) effects. 2 Fluorescence image of a Live / Dead assay of unexamined MCF-7 / ADR cells (green: survival, red: death). Specific details for implementing the invention

[0033] Definition of Terms

[0034] In this specification, nano means 100 nm or less.

[0035] In this specification, GO means graphene oxide.

[0036] In this specification, rGO means reduced graphene oxide.

[0037] In this specification, PNIPAM means poly(N-isopropylacrylamide).

[0038] In this specification, AAc means acrylic acid.

[0039] In this specification, PNIPAM-AAc means that PNIPAM and acrylic acid are copolymerized and bonded.

[0040] FA in this specification means folic acid.

[0041] In this specification, GO-PNIPAM-AAc-FA means graphene oxide, PNIPAM-AAc, and folic acid combined.

[0042] In this specification, rGO-PNIPAM-AAc-FA means that GO-PNIPAM-AAc-FA has been reduced.

[0043] In this specification, ICG means indocyanine green.

[0044] In this specification, ICG@rGO-PNIPAM-AAc-FA means that indocyanine green is supported within rGO-PNIPAM-AAc-FA.

[0046] Description of exemplary implementations

[0047] The present invention will be described in detail below.

[0048] In one aspect, the present invention provides a reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy, wherein the nanocomposite comprises reduced graphene oxide; poly(N-isopropylacrylamide)-acrylic acid; and folic acid.

[0049] The above term for multiple drug-resistant cancer cells refers to cancer cells that are resistant to two or more drugs.

[0050] In an exemplary embodiment, the poly(N-isopropylacrylamide)-acrylic acid can be bonded to the surface of reduced graphene oxide.

[0051] In an exemplary embodiment, the folic acid may be bound to the surface of the poly(N-isopropylacrylamide)-acrylic acid. For example, the folic acid may be introduced to the surface of the poly(N-isopropylacrylamide)-acrylic acid in the form of folic acid-polyethylene glycol-amine (FA-PEG-NH2). The nanocomposite of the present invention enables folic acid receptor-mediated endocellular translocation by including the folic acid.

[0052] In an exemplary embodiment, the shape of the nanocomposite may be spherical.

[0053] In an exemplary embodiment, the diameter of the nanocomposite may be 400 to 600 nm at 30 to 38.5 °C, preferably 410 to 650 nm, 420 to 600 nm, 430 to 550 nm, and more preferably 420 to 525 nm.

[0054] In an exemplary embodiment, the zeta potential of the nanocomposite may be -21.5 to -18.5 mV, preferably -21.4 to -18.75 mV, -21.3 to -19 mV, and more preferably -21.2 to -19.25 mV.

[0055] In an exemplary embodiment, an anticancer agent may be loaded onto the nanocomposite. For example, the anticancer agent may include an anticancer agent known in the art.

[0056] In an exemplary embodiment, the anticancer agent may be indocyanine green (ICG).

[0057] In an exemplary embodiment, the nanocomposite may contain 8 to 20 weight% of the indocyanine green based on the total weight of the nanocomposite loaded with the indocyanine green, preferably 9 to 18 weight%, 10 to 16 weight%, and more preferably 11 to 14 weight%.

[0058] In an exemplary embodiment, the zeta potential of the nanocomposite supporting the indocyanine green may be -33.5 to -29.5 mV, preferably -33.25 to -29.75 mV, -33 to -30 mV, and more preferably -33 to -30.25 mV.

[0059] In an exemplary embodiment, when near-infrared rays are irradiated onto the nanocomposite, the temperature may rise by 5 to 25°C. For example, the near-infrared rays may preferably be an 808 nm NIR laser. Additionally, when near-infrared rays are irradiated onto the nanocomposite, the temperature may preferably rise by 7.5 to 22.5°C or 10 to 20°C, and more preferably by 12.5 to 17.5°C.

[0060] In an exemplary embodiment, the nanocomposite may target multidrug-resistant breast cancer cells. For example, the multidrug-resistant breast cancer cells may be human MCF-7 / ADR.

[0061] In addition, the release of ICG from the nanocomposite of the present invention can be effectively promoted above the Lower Critical Solution Temperature (LCST) by NIR laser irradiation. The ICG@rGO-PNIPAM-FA nanocomposite is internalized into MDR breast cancer cells (MCF-7 / ADR) through folate receptor-mediated endocytosis, showing excellent synergistic effects in photothermal and photodynamic therapy, thereby maximizing the anticancer effect.

[0062] In an exemplary embodiment, a method for preparing a reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy comprises: a first step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid nanocomposite (GO-PNIPAM) by binding poly(N-isopropylacrylamide)-acrylic acid (PNIPAM-AAc) to graphene oxide (GO); and a second step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (GO-PNIPAM-AAc-FA) by binding folic acid to GO-PNIPAM-AAc. A method for preparing a reduced graphene oxide nanocomposite is provided, comprising a third step of reducing the above GO-PNIPAM-AAc-FA to provide a reduced graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (rGO-PNIPAM-AAc-FA).

[0063] In an exemplary embodiment, a fourth step of loading an anticancer agent onto the rGO-PNIPAM-AAc-FA may be further included.

[0064] In an exemplary embodiment, the first step may involve introducing a carboxyl group (-COOH) onto the surface of GO and then bonding PNIPAM-AAc.

[0065] In an exemplary embodiment, the second step may involve conjugating the folic acid by conjugating it with folic acid-polyethylene glycol-amine (FA-PEG-NH2).

[0067] The present invention will be described in more detail below through examples and the like. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples. Anything having a configuration substantially identical to the technical concept described in the claims of the present invention and achieving the same functional effect is included within the technical scope of the present invention.

[0069] Definition of Terms

[0070] In this specification, nano means 100 nm or less.

[0071] In this specification, GO means graphene oxide.

[0072] In this specification, rGO means reduced graphene oxide.

[0073] In this specification, PNIPAM means poly(N-isopropylacrylamide).

[0074] In this specification, AAc means acrylic acid.

[0075] In this specification, PNIPAM-AAc means that PNIPAM and acrylic acid are copolymerized and bonded.

[0076] FA in this specification means folic acid.

[0077] In this specification, GO-PNIPAM-AAc-FA means graphene oxide, PNIPAM-AAc, and folic acid combined.

[0078] In this specification, rGO-PNIPAM-AAc-FA means that GO-PNIPAM-AAc-FA has been reduced.

[0079] In this specification, ICG means indocyanine green.

[0080] In this specification, ICG@rGO-PNIPAM-AAc-FA means that indocyanine green is supported within rGO-PNIPAM-AAc-FA.

[0082] Description of exemplary implementations

[0083] The present invention will be described in detail below.

[0084] In one aspect, the present invention provides a reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy, wherein the nanocomposite comprises reduced graphene oxide; poly(N-isopropylacrylamide)-acrylic acid; and folic acid.

[0085] The above term for multiple drug-resistant cancer cells refers to cancer cells that are resistant to two or more drugs.

[0086] In an exemplary embodiment, the poly(N-isopropylacrylamide)-acrylic acid can be bonded to the surface of reduced graphene oxide.

[0087] In an exemplary embodiment, the folic acid may be bound to the surface of the poly(N-isopropylacrylamide)-acrylic acid. For example, the folic acid may be introduced to the surface of the poly(N-isopropylacrylamide)-acrylic acid in the form of folic acid-polyethylene glycol-amine (FA-PEG-NH2). The nanocomposite of the present invention enables folic acid receptor-mediated endocellular translocation by including the folic acid.

[0088] In an exemplary embodiment, the shape of the nanocomposite may be spherical.

[0089] In an exemplary embodiment, the diameter of the nanocomposite may be 400 to 600 nm at 30 to 38.5 °C, preferably 410 to 650 nm, 420 to 600 nm, 430 to 550 nm, and more preferably 420 to 525 nm.

[0090] In an exemplary embodiment, the zeta potential of the nanocomposite may be -21.5 to -18.5 mV, preferably -21.4 to -18.75 mV, -21.3 to -19 mV, and more preferably -21.2 to -19.25 mV.

[0091] In an exemplary embodiment, an anticancer agent may be loaded onto the nanocomposite. For example, the anticancer agent may include an anticancer agent known in the art.

[0092] In an exemplary embodiment, the anticancer agent may be indocyanine green (ICG).

[0093] In an exemplary embodiment, the nanocomposite may contain 8 to 20 weight% of the indocyanine green based on the total weight of the nanocomposite loaded with the indocyanine green, preferably 9 to 18 weight%, 10 to 16 weight%, and more preferably 11 to 14 weight%.

[0094] In an exemplary embodiment, the zeta potential of the nanocomposite supporting the indocyanine green may be -33.5 to -29.5 mV, preferably -33.25 to -29.75 mV, -33 to -30 mV, and more preferably -33 to -30.25 mV.

[0095] In an exemplary embodiment, when near-infrared rays are irradiated onto the nanocomposite, the temperature may rise by 5 to 25°C. For example, the near-infrared rays may preferably be an 808 nm NIR laser. Additionally, when near-infrared rays are irradiated onto the nanocomposite, the temperature may preferably rise by 7.5 to 22.5°C or 10 to 20°C, and more preferably by 12.5 to 17.5°C.

[0096] In an exemplary embodiment, the nanocomposite may target multidrug-resistant breast cancer cells. For example, the multidrug-resistant breast cancer cells may be human MCF-7 / ADR.

[0097] In addition, the release of ICG from the nanocomposite of the present invention can be effectively promoted above the Lower Critical Solution Temperature (LCST) by NIR laser irradiation. The ICG@rGO-PNIPAM-FA nanocomposite is internalized into MDR breast cancer cells (MCF-7 / ADR) through folate receptor-mediated endocytosis, showing excellent synergistic effects in photothermal and photodynamic therapy, thereby maximizing the anticancer effect.

[0098] In an exemplary embodiment, a method for preparing a reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy comprises: a first step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid nanocomposite (GO-PNIPAM) by binding poly(N-isopropylacrylamide)-acrylic acid (PNIPAM-AAc) to graphene oxide (GO); and a second step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (GO-PNIPAM-AAc-FA) by binding folic acid to GO-PNIPAM-AAc. A method for preparing a reduced graphene oxide nanocomposite is provided, comprising a third step of reducing the above GO-PNIPAM-AAc-FA to provide a reduced graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (rGO-PNIPAM-AAc-FA).

[0099] In an exemplary embodiment, a fourth step of loading an anticancer agent onto the rGO-PNIPAM-AAc-FA may be further included.

[0100] In an exemplary embodiment, the first step may involve introducing a carboxyl group (-COOH) onto the surface of GO and then bonding PNIPAM-AAc.

[0101] In an exemplary embodiment, the second step may involve conjugating the folic acid by conjugating it with folic acid-polyethylene glycol-amine (FA-PEG-NH2).

[0103] The present invention will be described in more detail below through examples and the like. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples. Anything having a configuration substantially identical to the technical concept described in the claims of the present invention and achieving the same functional effect is included within the technical scope of the present invention.

[0105] [Preparation Example]

[0107] 1. Preparation of materials

[0108] GO solution was purchased from Graphene Supermarket, Inc (Ronkonkoma, NY, USA). NIPAM, N,N'-Methylenebisacrylamide (MBA), AAc, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AIBA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Folic acid-polyethyleneglycol-amine (FA-PEG-NH2, 5 kDa) was purchased from Nanocs, Inc. (New York, NY, USA). Methoxy PEG amine (CH3-PEG-NH2, 5 kDa) was purchased from SunBio Co. (Gunpo, Korea). ICG, 1,3-diphenylisobenzofuran (DPBF), and 3-(4,5-dimethylthiozole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Mouse fibroblasts (NIH-3T3), human lung carcinoma (A549), human MCF-7, and MCF-7 / ADR cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco Phosphate Buffered Saline (DPBS), Fetal Bovine Serum (FBS), 40,6-diamidino-2-phenylindole (DAPI), antibiotics, and a live / dead double staining kit were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

[0110] 2. Synthesis of GO-PNIPAM-AAc Nanocomposite

[0111] To synthesize the GO-PNIPAM-AAc nanocomposite, 50 mL of GO solution (1 mg / mL) was sonicated for 1 hour. 2.4 g of NaOH and 2.0 g of chloroacetic acid were added, and the solution was sonicated for 3 hours to introduce carboxyl groups to the surface of GO. The resulting solution was dialyzed in deionized water (DW) for 3 days using a MWCO (Molecular Weight Cut Off) 3.5 kDa membrane to remove residual chemicals. To introduce PNIPAM, 20 mg of GO-COOH was dissolved in 20 mL of DW, and 20 mg of AIBA was added. The pH was adjusted to 10 using ammonium hydroxide, and the solution was stirred at 70°C. After 2 hours, 20 mg of PNIPAM, 2 mg of MBA, and 2 μL of AAc were added to the solution, and the mixture was stirred at 80°C for 3 hours under a nitrogen atmosphere. The GO-PNIPAM-AAc nanocomposite was obtained by purifying DW for 2 days using an MWCO 6-8 kDa dialysis membrane and freeze-drying.

[0113] 3. Synthesis of rGO-PNIPAM-AAc-FA nanocomposite

[0114] Targeting ability against MDR breast cancer cells and enhanced colloidal stability can be provided by conjugating NH2-PEG-FA to the surface of GO-PNIPAM using an EDC / NHS coupling reaction. Initially, 10 mg of GO-PNIPAM-AAc was dissolved in PBS (pH 5.8) and sonicated for 30 minutes. 40 mg of EDC and 44 mg of NHS were added, and the solution was sonicated for 1 hour. Next, 5 mg of FA-PEG-NH2 was added and stirred for 18 hours. To remove unreacted chemicals, the GO-PNIPAM-AAc-FA nanocomposite was purified through a dialysis membrane (MWCO: 6-8 kDa) for 48 hours. The reduction process of GO-PNIPAM-AAc-FA was performed by treating the nanocomposite with 0.05% hydrazine monohydrate followed by heating at 85°C for 15 minutes. rGO-PNIPAM-AAc-FA was obtained by lyophilizing after purification for 1 day using a dialysis membrane (3.5 kDa). As a control, rGO-PNIPAM-AAc-PEG nanocomposites were synthesized using the same protocol.

[0116] 4. Synthesis of ICG-encapsulated rGO-PNIPAM-AAc-FA nanocomposites

[0117] To encapsulate an NIR laser-mediated photodetector in the rGO-PNIPAM-AAc-FA nanocomposite, 10 mg of rGO-PNIPAM-AAc-FA (10 mL, 1 mg / mL) was sonicated for 10 minutes. 5 mg / mL of ICG was added dropwise to the rGO-PNIPAM-AAc-FA solution and vigorously stirred in a dark room for 24 hours. Unencapsulated ICG was removed by centrifugation (14,000 rpm, 10 min) and washed with DW. The ICG-loaded rGO-PNIPAM-AAc-FA nanocomposite was obtained by freeze-drying. The presence of ICG in the rGO-PNIPAM-AAc-FA nanocomposite was estimated by UV-vis spectroscopy at 780 nm (UV 1800, Tokyo, Japan).

[0119] [Experimental Example]

[0121] 1. Characterization of rGO-PNIPAM-AAc-FA Nanocomposites

[0122] (1) Experimental method

[0123] The size, morphology, and surface roughness of the rGO-PNIPAM-AAc-FA nanocomposite were observed using a transmission electron microscope (TEM, JEM-ARM200F, Tokyo, Japan). The surface charge and LCST of the rGO-PNIPAM-AAc-FA nanocomposite were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). Fourier transform infrared spectroscopy (FT-IR, Vertex 70, Bruker Inc., Billerica, MA, USA) was used to confirm the chemical structure of the rGO-PNIPAM-AAc-FA nanocomposite. ICG encapsulated in the rGO-PNIPAM-AAc-FA nanocomposite was identified by UV-vis spectroscopy (UV 1800, Tokyo, Japan).

[0125] (2) Experimental results

[0126] To provide a high loading capacity and temperature response of ICG under 808 nm NIR laser irradiation, NIPAM monomers were conjugated to GO nanosheets by radical polymerization, and then AAc was added to the NIPAM-GO mixed solution to increase the LCST. Through the conjugation of ICG encapsulated in a PNIPAM structure and PEG-FA, the nanocomposite demonstrated that intracellular uptake by MDR breast cancer cells was possible, and the therapeutic effect on MDR breast cancer could be enhanced under NIR laser irradiation (Fig. 1).

[0127] The size and morphology of the thermally reactive GO nanocomposites were evaluated by TEM (Fig. 2A). The GO-COOH nanocomposites measured 209 ± 5.43 nm and exhibited the characteristic spherical shape of GO nanosheets. However, the morphology of the rGO-PNIPAM and ICG@rGO-PNIPAM-AAc-FA nanocomposites changed to densely aggregated dark spots because the PNIPAM-AAc chains extended from the rGO surface. Additionally, the diameters of the nanocomposites increased dramatically to 230 ± 5.32 and 233.4 ± 15.92 nm, respectively, indicating that NH2-PEG-FA was conjugated to the ends of the rGO-PNIPAM nanocomposites and that ICG molecules were encapsulated within the rGO-PNIPAM-AAc-FA nanocomposites. The thermal reactivity of the PNIPAM-AAc formed on the rGO nanocomposites was investigated by DLS analysis. Changes in the diameter of the rGO-PNIPAM-AAc-FA nanocomposites were observed as the temperature increased.

[0128] As shown in Fig. 2B, the diameter of the nanocomposite remained unchanged at approximately 470 nm at 30–38°C. In contrast, the diameter of the rGO-PNIPAM nanocomposite decreased significantly to 150–170 nm at 39°C or higher. This result indicates that the increase in hydrophilic groups caused by the copolymerized AAc in the PNIPAM chain leads to a phase change of PNIPAM-AAc. Therefore, the LCST of the rGO-PNIPAM-AAc-FA nanocomposite was fixed at 39°C. Furthermore, while the LCST of the conventional temperature-sensitive polymer PNIPAM without AAc is known to be around 32°C, the nanocomposite of the present invention exhibited a higher LCST, confirming that it has excellent control over drug release behavior according to temperature. Therefore, the ICG@rGO-PNIPAM-AAc-FA developed in this study enables selective release at temperatures above 39°C, which reduces side effects associated with drug release and can maximize photothermal and photodynamic therapy effects on multidrug-resistant cancers using indocyanine green.

[0129] Figure 2C shows the zeta potentials of the rGO-PNIPAM-AAc-FA nanocomposites obtained through each deformation process. Compared to the original GO (-27.3 ± 1.48 mV), GO-COOH exhibited a strong negative charge (-32.7 ± 1.16 mV) due to additional carboxyl groups on the GO surface caused by chloroacetic acid. The GO-PNIPAM and GO-PNIPAM-AAc-FA nanocomposites were recorded as -36.2 ± 2.35 mV and -24.1 ± 2.03 mV, respectively. These changes in values ​​were derived from the conjugation of negatively charged PNIPAM-AAc with positively charged NH2-PEG-FA. After the reduction process, the rGO-PNIPAM-AAc-FA nanocomposite showed a slight decrease to -20.2 ± 0.85 mV, supporting the removal of residual hydroxyl groups from the GO-PNIPAM-AAc-FA nanocomposite. The ICG@rGO-PNIPAM-AAc-FA nanocomposite was measured at -31.6 ± 1.27 mV, which is attributed to the negatively charged ICG molecules encapsulated in the rGO-PNIPAM-AAc-FA nanocomposite.

[0130] The chemical structure of the rGO-PNIPAM-AAc-FA nanocomposite was analyzed by FT-IR spectroscopy in Fig. 3A. GO-COOH corresponds to the C=O and OH vibrations, respectively, at 1735 cm⁻¹. -1 and 3400cm -1 Two major peaks were observed. In the spectrum of the GO-PNIPAM nanocomposite, peaks at 1100, 1381, and 1587 cm⁻¹ were attributed to the PNIPAM-AAc domain. -1 Stretching vibrations of CN, NH, and C=O were observed. After NH2-PEG-FA binding, the GO-PNIPAM-AAc-FA nanocomposite exhibited 1350, 1460, 940, and 1080 cm⁻¹. -1A new vibrational peak was exhibited, indicating the presence of -CH2, -CH3 backbone stretching and -COC- asymmetric and symmetric vibrations in the PEG chain. Upon chemical reduction, the spectrum of the rGO-PNIPAM-AAc-FA nanocomposite was at approximately 3400 cm⁻¹. -1 It decreased in the OH stretching vibration. However, FA molecules could not be detected as specific peaks in the rGO-PNIPAM-AAc-FA spectrum, because FA and PEG are composed of similar chemical structures. UV-vis spectroscopy was used to confirm the presence of FA in the rGO-PNIPAM-AAc-FA nanocomposite, and an absorption peak at 280 nm was observed in FA and rGO-PNIPAM-AAc-FA in Fig. 3B. In addition, ICG encapsulated in the nanocomposite was evaluated using the same analytical method. The rGO-PNIPAM-AAc-FA nanocomposite did not exhibit an absorption peak in the NIR region (750-1100 nm), but the rGO-PNIPAM-AAc-FA nanocomposite encapsulated with ICG exhibited a strong absorption peak in the same wavelength range. These results verified the conjugation of FA as well as encapsulated ICG in the rGO-PNIPAM-AAc-FA nanocomposite through the intrinsic absorption peaks of FA and ICG at 280 and 780 nm, respectively. Additionally, the absorption peak at 780 nm in the UV-vis spectrum provided the amount of ICG molecules encapsulated in the rGO-PNIPAM-AAc-FA nanocomposite, and the loading capacity of ICG was determined to be 12.2%. This value was relatively higher than the loading capacity of ICG directly loaded onto pure rGO nanosheets (6%), indicating that the PNIPAM-AAc in the rGO nanocomposite improved complexation with ICG through hydrophobic interactions and demonstrated enhanced drug carrying capacity.

[0132] 2. Analysis of Photothermal and Photodynamic Effects of rGO-PNIPAM-AAc-FA Nanocomposites Under NIR Laser Irradiation

[0133] (1) Experimental method

[0134] The NIR laser-mediated photothermal properties of ICG@rGO-PNIPAM-AAc-FA nanocomposites were evaluated through real-time temperature monitoring. rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA nanocomposites were dissolved in DW at various concentrations (50, 100, and 200 μg / mL), and an 808 nm NIR laser (BWF2, BWF Inc., Sweden) was applied at 1 W / cm² 2 Irradiation was performed for 10 minutes at power densities. The effect of laser power density was [indicated by] power densities (1.0, 1.5, and 2.0 W / cm²). 2 The temperature of different nanocomposite solutions was increased and monitored. In addition, the photothermal stability of the ICG@rGO-PNIPAM-AAc-FA solution (100 μg / mL) was investigated against an 808 nm NIR laser. The solution was exposed to NIR laser irradiation for 10 minutes and then cooled for 3 cycles. The temperature change of the nanocomposite was recorded using a thermocouple digital thermometer (DTM-318, Tecpel Co., Taiwan). Furthermore, the photodynamic properties of the ICG@rGO-PNIPAM-AAc-FA nanocomposite were demonstrated through ROS generation induced by the NIR laser. DPBF was used to determine ROS generation from the ICG@rGO-PNIPAM-AAc-FA nanocomposite. An ICG@rGO-PNIPAM-AAc-FA solution (200 μL, 0.1 mg / mL in PBS) was homogeneously mixed with 10 μL of a DPBF solution in DMSO (1 mg / mL). The mixed solution at different time intervals at 1W / cm 2 It was exposed to an 808 nm NIR laser at a power density. For comparison, an rGO-PNIPAM-AAc-FA solution was treated in the same way. The corresponding absorption spectrum of DPBF was measured using UV-vis spectroscopy (410 nm wavelength).

[0136] (2) Experimental results

[0137] 1) Photothermal and photostability efficiency of rGO-PNIPAM-AAc-FA nanocomposites

[0138] The photothermal and photostability properties of the ICG@rGO-PNIPAM-AAc-FA nanocomposite were confirmed through real-time monitoring of temperature changes under 808 nm NIR laser irradiation (Fig. 4).

[0139] To confirm the PTT enhanced by ICG encapsulated in rGO-PNIPAM-AAc-FA nanocomposites, rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA nanocomposites were treated with an 808 nm NIR laser (1 W / cm²). 2 ) was exposed to the investigation for 10 minutes.

[0140] To compare the photothermal effect of indocyanine green, rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA were prepared and irradiated with 808 nm near-infrared light. As a result, rGO-PNIPAM-AAc-FA showed a temperature increase of 9°C due to the photothermal effect of graphene itself, whereas the ICG@rGO-PNIPAM-AAc-FA nanocomposite loaded with indocyanine green showed a temperature increase of up to approximately 15.1°C under the same experimental conditions, confirming that the indocyanine green loaded on the nanocomposite exhibited a synergistic effect (Fig. 4A).

[0141] Also, 808nm NIR lasers of various intensities (1.0, 1.5, 2 W / cm²) 2 While investigating the temperature change of ICG@rGO-PNIPAM-AAc-FA nanocomposites at various concentrations (50, 100, 200 μg / mL), (Figs. 4B and 4C).

[0142] To observe heat loss due to near-infrared irradiation, the nanocomposite was repeatedly irradiated with near-infrared light. As a result, no heat loss occurred, confirming the possibility of using it for photothermal therapy (Fig. 4D).

[0144] 2) Photodynamic properties of rGO-PNIPAM-AAc-FA nanocomposites under NIR laser irradiation

[0145] To investigate the NIR laser-mediated photodynamic properties of the nanocomposite, a DPBF probe was applied to the ROS detection of the ICG@rGO-PNIPAM-AAc-FA nanocomposite. In Fig. 5A, after mixing the ICG@rGO-PNIPAM-AAc-FA nanocomposite and the DPBF probe, an 808 nm NIR laser was irradiated every 2 minutes (1 W / cm²). 2 UV-vis spectroscopy was performed after irradiation. When the DPBF probe reacts with ROS, the probe degrades and o-dibenzoylbenzene is formed, inducing a reduced absorption intensity at 410 nm. It was confirmed that the absorption peak at 410 nm gradually decreased with NIR laser irradiation time. As shown in Figure 5B, under 808 nm NIR laser irradiation, the rGO-PNIPAM-AAc-FA nanocomposite without ICG did not change its relative absorption, whereas the ICG@rGO-PNIPAM-AAc-FA nanocomposite with the DPBF probe showed a continuous decrease in relative absorption. Furthermore, when the mixture of ICG@rGO-PNIPAM-AAc-FA and the DPBF probe were not exposed to NIR laser irradiation, the absorption at 410 nm remained unchanged. This is interpreted as meaning that while no reactive oxygen species were generated in rGO-PNIPAM-AAc-FA, continuous reactive oxygen species were generated in ICG@rGO-PNIPAM-AAc-FA upon near-infrared irradiation. These results indicate that the ICG@rGO-PNIPAM-AAc-FA nanocomposite can effectively generate a large amount of ROS by irradiation with an 808nm NIR laser.

[0147] 3. Analysis of NIR and Thermoreactive ICG Emission of rGO-PNIPAM-AAc-FA Nanocomposites

[0148] (1) Experimental method

[0149] To investigate the thermally responsive ICG release of the rGO-PNIPAM-AAc-FA nanocomposite, 1 mL of ICG@rGO-PNIPAM-AAc-FA solution (1 mg / mL) was prepared and stirred at 25, 37, and 50°C. After predetermined time intervals (0–72 hours), the supernatant of each sample was collected by centrifugation (14,000 rpm, 5 min) and replaced with an equal volume of fresh PBS. The ICG released from the rGO-PNIPAM-AAc-FA nanocomposite was estimated by fluorescence spectroscopy. Additionally, NIR laser-mediated ICG release from the rGO-PNIPAM-AAc-FA nanocomposite was confirmed. The ICG@rGO-PNIPAM-AAc-FA solution (1 mg / mL) was placed at 37°C and irradiated for 10 minutes at a predetermined time point. As a control, ICG@rGO-PNIPAM-AAc-FA solution placed at 25 and 37°C without NIR laser irradiation was used. The released ICG was determined by the method described above.

[0151] (2) Experimental results

[0152] To elucidate ICG release via thermal reaction, the ICG release behavior of the rGO-PNIPAM-AAc-FA nanocomposite was observed for 72 hours at various temperatures (25, 37, and 50°C). In Figure 6A, when the ICG@rGO-PNIPAM-AAc-FA nanocomposite was placed at 25°C and 37°C, encapsulated ICG was released at 19% and 24%, respectively. The LCST of the rGO-PNIPAM-AAc-FA nanocomposite was already obtained in Figure 2B (between 38 and 40°C). Since the entire polymer network of PNIPAM-AAc in the rGO-PNIPAM-AAc-FA nanocomposite remained in a swollen state through hydrogen bonding, cumulative ICG release from the nanocomposite was suppressed under the LCST. Compared to the release profile below the LCST, the ICG released from the nanocomposite increased to 49% at 50°C. This result is more than twofold higher, This indicates that PNIPAM-AAc is modified into a hydrophobic state with reduced hydrogen bonding, which can accelerate ICG emission from the ICG@rGO-PNIPAM-AAc-FA nanocomposite through the disruption of the polymer network on the LCST, as previously described. Additionally, the thermal and NIR laser-responsive ICG emission capabilities of the rGO-PNIPAM-AAc-FA nanocomposite were investigated in Fig. 6B. The ICG@rGO-PNIPAM-AAc-FA nanocomposite was irradiated with an NIR laser (1 W / cm²) for 5 minutes at 1-hour intervals. 2It was exposed to NIR and placed at 37°C. The total amount of ICG emitted by NIR laser irradiation over 5 hours reached 36%, whereas the ICG emitted from the nanocomposite at 25°C and 37°C without NIR laser irradiation was negligible (9.6% and 11.7%). The thermal and NIR laser-responsive rGO-PNIPAM-AAc-FA nanocomposite showed a large amount of ICG emission compared to the control group, indicating that NIR laser irradiation could accelerate the ICG release of the rGO-PNIPAM-AAc-FA nanocomposite by inducing a temperature increase in the LCST of the nanocomposite through rGO. Therefore, the ICG@rGO-PNIPAM-AAc-FA nanocomposite of the present invention can be used in a drug release system controlled by dual stimulation through thermal and NIR laser irradiation, and it was confirmed that not only temperature-dependent drug release control but also near-infrared control is possible through the introduction of the temperature-sensitive polymer (PNIPAM-AAc).

[0154] 4. Analysis of the cytotoxicity of the rGO-PNIPAM-AAc-FA nanocomposite

[0155] (1) Experimental method

[0156] Cell viability testing of the rGO-PNIPAM-AAc-FA nanocomposite was performed using the MTT assay. Normal (NIH-3T3 fibroblasts), human lung carcinoma (A549), and human breast cancer (MCF-7, MCF-7 / ADR) cells were placed in a 96-well plate at a density of 1 x 10⁶ cells. 4The cells were inoculated at a density of 100 μL and cultured in a CO2 incubator for 24 hours. 100 μL of rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA nanocomposites at various concentrations (10-100 μg / mL) were added to each well, and 96-well plates were cultured for 24 hours. The cells were washed with DPBS, fresh medium containing MTT preparation (0.5 mg / mL) was added, and the cells were cultured for an additional 4 hours. The culture medium was carefully removed, and 200 μL of DMSO was added. Cell viability of NIH-3T3, A549, MCF-7, and MCF-7 / ADR cells was quantified using an iMark™ microplate reader (Bio-rad, Hercules, CA, USA).

[0158] (2) Experimental results

[0159] The cytotoxicity analysis of the rGO-PNIPAM-AAc-FA nanocomposite was performed using normal (NIH-3T3 fibroblasts), breast cancer (MCF-7), and MDR breast cancer (MCF-7 / ADR) cells. The cell viability of NIH-3T3, MCF-7, and MCF-7 / ADR cells treated with ICG@rGO-PNIPAM-AAc-FA nanocomposites at various concentrations (20–100 μg / mL) was demonstrated. The survival rate of NIH-3T3, MCF-7, and MCF / ADR cells did not decrease below 85% even at high concentrations of the nanocomposite (100 μg / mL). Furthermore, cell viability treated with the rGO-PNIPAM-AAc-FA nanocomposite without ICG was observed in the same manner. Similar to previous results, cells treated with the rGO-PNIPAM-AAc-FA nanocomposite without ICG also exhibited high cell viability, exceeding 85% at all concentrations. These results demonstrated that the rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA nanocomposites exhibited excellent biocompatibility.

[0161] 5. Analysis of uptake of rGO-PNIPAM-AAc-FA nanocomposite into MDR breast cancer target cells

[0162] (1) Experimental method

[0163] The targeting ability of the ICG@rGO-PNIPAM-AAc-FA nanocomposite on breast cancer (MCF-7, MCF-7 / ADR) cells was evaluated by confocal microscopy (CLSM 900, Carl Zeiss, Germany). NIH-3T3, A549, MCF-7, and MCF-7 / ADR cells were cultured at 2 × 10⁴ per well in 8-well culture plates. 4 Cells were seeded at cell density and cultured overnight. Cells were treated with 100 μg / mL of ICG@rGO-PNIPAM-AAc-FA nanocomposite in serum-free medium for 4 hours. To remove residual nanocomposite, cells were washed several times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. Subsequently, cells were treated with 0.1% Triton X-100 and 4% BSA at room temperature for 15 minutes. Cells were stained with Alexa Fluor 594 phalloidin (1:200, Invitrogen, USA) at 4°C for 1 day and with 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, USA) for 15 minutes. Compared to FA-mediated cellular uptake of the ICG@rGO-PNIPAM-AAc-FA nanocomposite, methoxy-PEG-amine (CH3-PEG-NH2) was conjugated to rGO-PNIPAM and ICG was subsequently encapsulated (ICG@rGO-PNIPAM-CH3). Cells were stained using the same protocol, and cellular uptake images were obtained using a confocal microscope.

[0165] (2) Experimental results

[0166] The selective cellular uptake of the ICG@rGO-PNIPAM-AAc-FA nanocomplex was further investigated in folate receptor-negative cells (NIH-3T3, A549) and positive cells (MCF-7, MCF-7 / ADR) (Fig. 7). Fluorescence images were obtained after incubation with the ICG@rGO-PNIPAM-AAc-FA nanocomplex. Cell nuclei were stained blue (DAPI), cytoplasm was labeled green (phalloidin 594), and intracellularly localized ICG was labeled red. Relatively strong red fluorescence was distributed in folate receptor-positive (MCF-7, MCF-7 / ADR) cells containing the ICG@PNIPAM-FA nanocomplex. However, normal (NIH-3T3) and lung cancer (A549) cells treated with the ICG@rGO-PNIPAM-AAc-FA nanocomplex did not exhibit fluorescence of ICG. In addition, an ICG@rGO-PNIPAM-CH3 (FA-free) nanocomplex was prepared as a control and treated to folate receptor-positive cells. As expected, MCF-7 and MCF-7 / ADR cells did not exhibit any fluorescence associated with ICG because the nanocomplex was free of FA. Therefore, these results suggest that the rGO-PNIPAM-AAc-FA nanocomplex was selectively internalized into breast cancer cells through folate receptor-mediated endocytosis.

[0168] 6. Analysis of Intracellular NIR Laser-Mediated ROS Generation by rGO-PNIPAM-AAc-FA Nanocomposite

[0169] (1) Experimental method

[0170] ROS production in breast cancer cells (MCF-7, MCF-7 / ADR) treated with the ICG@rGO-PNIPAM-AAc-FA nanocomposite was quantified by the DCFDA assay. MCF-7 and MCF-7 / ADR cells were seeded at a density of 1 x 10⁴ cells and cultured with GO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA (60 μg / mL) for 6 hours. To evaluate intracellular ROS production, cells were irradiated with an 808 nm NIR laser (3 W / cm²).2 The cells were exposed to irradiation for 5 minutes. Subsequently, the cells were washed several times with DPBS and cultured for 45 minutes with fresh medium containing DCFDA (20 μM). Fluorescence signals of MCF-7 and MCF-7 / ADR cells were detected using an inverse fluorescence microscope (Olympus IX73, Tokyo, Japan).

[0172] (2) Experimental results

[0173] NIR laser-mediated ROS generation by the ICG@rGO-PNIPAM-AAc-FA nanocomposite provided oxidative stress to MDR breast cancer cells and induced effective immunogenic apoptosis. To evaluate intracellular ROS in breast cancer cells via NIR laser irradiation, breast cancer cells were treated with the commercial intracellular ROS indicator DCFDA probe mixed with the ICG@rGO-PNIPAM-AAc-FA nanocomposite and exposed to 808 nm NIR laser irradiation. Figure 8 confirmed that the fluorescence intensity of MCF-7 / ADR cells irradiated with the NIR laser was different. No green signal was displayed when MCF-7 / ADR cells treated with the ICG@rGO-PNIPAM-AAc-FA nanocomposite were not exposed to NIR laser irradiation. In contrast, the highest fluorescence intensity was observed in cells treated with the ICG@rGO-PNIPAM-AAc-FA nanocomposite and near-infrared laser irradiation. In addition, intracellular ROS generation was observed as breast cancer cells (MCF-7) were treated with the ICG@PINPAM-FA nanocomposite and irradiated with an NIR laser (Figure S4). As expected, no green fluorescence intensity was observed in MCF-7 cells that were not irradiated with a near-infrared laser, but a green signal was significantly expressed in MCF-7 cells irradiated with an 808 nm near-infrared laser.

[0174] Therefore, it was confirmed that the ICG@PNIPAM-FA nanocomposite can be internalized into breast cancer cells and generate a large amount of cytotoxic ROS through NIR laser irradiation.

[0176] 7. Analysis of Synergistic Therapeutic Efficacy of rGO-PNIPAM-AAc-FA Nanocomposites

[0177] (1) Experimental method

[0178] The combined PTT-PDT effect of the rGO-PNIPAM-AAc-FA nanocomposite upon NIR laser irradiation on breast cancer cells was evaluated by MTT assay. MCF-7 / ADR cells were plated in a 96-well plate (1 x 10⁶ 4 Canine cells / well) were seeded and cultured for 24 hours. Subsequently, cells were treated with rGO-PNIPAM-AAc-FA and ICG@rGO-PNIPAM-AAc-FA nanocomposites at various concentrations (20-80 μg / mL). After culturing for 4 hours, the cells were treated with an 808 nm NIR laser (3 W / cm²). 2 The cells were irradiated for 5 minutes. Additionally, live / dead analysis was performed to visualize synergistic therapeutic effects under NIR laser irradiation. MCF-7 and MCF-7 / ADR cells were cultured with the rGO-PNIPAM-AAc-FA nanocomposite for 4 hours and exposed to NIR laser irradiation for 5 minutes. Cells were stained with Calcein AM and Ethidium Homodimer-1 for 20 minutes and washed several times. Finally, live / dead images were obtained using an Olympus IX73 microscope.

[0180] (2) Experimental results

[0181] To confirm the efficacy of combination therapy with NIR laser-mediated PTT and PDT on MDR breast cancer cells, MCF-7 / ADR cells were cultured with various concentrations of ICG@rGO-PNIPAM-AAc-FA nanocomposites (20–80 μg / mL), and cytotoxicity tests were performed (Fig. 9A). In the control group, MCF-7 / ADR cells treated with rGO-PNIPAM-AAc-FA nanocomposites without ICG were irradiated with an NIR laser (5 min, 3 W / cm²). 2The cells were exposed to ). The viability of MCF-7 / ADR cells showed a dose-dependent effect on the nanocomposite and gradually decreased to 51%, which is because the rGO-PNIPAM-AAc-FA nanocomposite was internalized in MCF-7 / ADR cells through folate receptor endoclinization, and MCF-7 / ADR cells died from NIR laser-mediated rGO-based PTT alone. However, when MCF-7 / ADR cells cultured with the ICG@rGO-PNIPAM-AAc-FA nanocomposite were irradiated with an 808 nm NIR laser, cell viability decreased significantly, reaching approximately 22%. This potent therapeutic efficacy of MCF-7 / ADR cells is attributed to enhanced PTT via ICG and rGO, and abundant ROS generation by the NIR laser-mediated ICG@rGO-PNIPAM-AAc-FA nanocomposite. Additionally, a live / dead analysis was performed to visualize the synergistic therapeutic efficacy of MDR breast cancer cells. As shown in Fig. 9B, although MCF-7 cells were exposed only to 808 nm NIR laser irradiation, most cells exhibited green fluorescence, indicating that NIR laser irradiation alone did not induce cytotoxicity in the cells. In MCF-7 / ADR cells treated with both the rGO-PNIPAM-AAc-FA nanocomposite and NIR laser irradiation, a majority of MCF-7 / ADR cells changed to red fluorescence due to the NIR laser-mediated PTT effect. Finally, when the ICG@rGO-PNIPAM-AAc-FA nanocomposite treated in MCF-7 / ADR cells was exposed to near-infrared laser irradiation, most MCF-7 cells were killed due to synergistic therapeutic efficacy through enhanced PTT and PDT effects.

[0183] In conclusion, through these experiments, heat-responsive rGO nanocomposites were fabricated, and the efficacy of the combined treatment of PTT and PDT against MDR breast cancer cells was evaluated. To fabricate a versatile nanoscale carrier, it was confirmed that the direct introduction of heat-responsive PNIPAM-AAc into GO nanosheets via radical polymerization can provide excellent LCST. By conjugating NH2-PEG-FA to the ends of rGO-PNIPAM-AAc, the rGO-PNIPAM nanocomposites enhanced nanoscale dispersion in aqueous solutions and demonstrated targeting ability against MDR breast cancer cells. To enhance phototherapeutic efficacy, it was confirmed that ICG molecules, encapsulated within the rGO-PNIPAM-AAc-FA nanocomposites through hydrophobic interactions, can induce abundant ROS generation and localized temperature elevation upon irradiation with an 808 nm NIR laser. The ICG@rGO-PNIPAM-AAc-FA nanocomposite exhibited cellular uptake through folate receptor-mediated endocytosis in breast cancer cells and also enhanced the therapeutic efficacy of MCF-7 / ADR cells through synergistic PTT and PDT effects.

[0184] Therefore, the rGO-PNIPAM-AAc-FA nanocomposite of the present invention can be used as a multifunctional carrier for MDR cancer treatment through the combined therapeutic effects of photothermal and photodynamic therapy.

Claims

Claim 1 A reduced graphene oxide nanocomposite for targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy, wherein the nanocomposite comprises reduced graphene oxide; poly(N-isopropylacrylamide)-acrylic acid; and folic acid, and is characterized by indocyanine green (ICG) being supported on the nanocomposite. Claim 2 A reduced graphene oxide nanocomposite according to claim 1, characterized in that the poly(N-isopropylacrylamide)-acrylic acid is bonded to the surface of the reduced graphene oxide. Claim 3 A reduced graphene oxide nanocomposite according to claim 1, characterized in that the folic acid is bonded to the surface of the poly(N-isopropylacrylamide)-acrylic acid. Claim 4 A reduced graphene oxide nanocomposite according to claim 1, wherein the shape of the nanocomposite is spherical. Claim 5 A reduced graphene oxide nanocomposite according to claim 1, characterized in that the diameter of the nanocomposite is 400 to 600 nm at 30 to 38.5 ℃. Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 The reduced graphene oxide nanocomposite according to claim 1, wherein the nanocomposite comprises 8 to 20 weight percent of the indocyanine green based on the total weight of the nanocomposite loaded with the indocyanine green. Claim 10 A reduced graphene oxide nanocomposite according to claim 1, characterized in that the zeta potential of the nanocomposite is -33.5 to -29.5 mV. Claim 11 A reduced graphene oxide nanocomposite according to claim 1, characterized in that the temperature rises by 5 to 25°C when near-infrared rays are irradiated onto the nanocomposite. Claim 12 A reduced graphene oxide nanocomposite according to any one of claims 1 to 5 and claims 9 to 11, wherein the nanocomposite targets multidrug-resistant breast cancer cells. Claim 13 A method for preparing a reduced graphene oxide nanocomposite targeting multiple drug-resistant cancer cells for photothermal therapy and photodynamic therapy, the method comprising: a first step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid nanocomposite (GO-PNIPAM-AAc) by binding poly(N-isopropylacrylamide)-acrylic acid (PNIPAM-AAc) to graphene oxide (GO); a second step of providing a graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (GO-PNIPAM-AAc-FA) by binding folic acid to GO-PNIPAM-AAc; and a third step of providing a reduced graphene oxide-poly(N-isopropylacrylamide)-acrylic acid-folic acid nanocomposite (rGO-PNIPAM-AAc-FA) by reducing GO-PNIPAM-AAc-FA. A method for preparing a reduced graphene oxide nanocomposite, comprising: a fourth step of loading indocyanine green (ICG) onto the rGO-PNIPAM-AAc-FA. Claim 14 delete Claim 15 A method for preparing a reduced graphene oxide nanocomposite according to claim 13, wherein the first step involves introducing a carboxyl group (-COOH) onto the surface of GO and then bonding PNIPAM-AAc. Claim 16 A method for preparing a reduced graphene oxide nanocomposite according to claim 13, wherein the second step involves binding the folic acid by conjugating it with folic acid-polyethylene glycol-amine (FA-PEG-NH2).

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