Targeted long-acting heat-generating nanocomposites for enhancing photothermal tumor therapy and applications thereof

Abstract

The application discloses a targeted long-acting heat-generating nanocomposite for enhancing photothermal therapy of tumors and application thereof. The heat-generating nanocomposite is a core-shell structure, the inner core is an oleic acid modified metal nanoparticle loaded with a photothermal material, the outer shell is a tissue compatible degradable material, and the outer shell surface is connected with a tumor targeting molecule. The application of the heat-generating nanocomposite in the preparation of tumor treatment drugs, especially in the preparation of tumor photothermal treatment drugs. The application utilizes a degradable biological material, internally wraps an oleic acid modified ferroferric oxide loaded with a photothermal conversion agent, and connects a targeting molecule with a therapeutic function on the surface, realizes local photothermal and antibody targeted therapy in tumor tissues, synergistically resists tumors, reduces toxic side effects, and the material used has high tissue compatibility, is suitable for in-vivo application, and has great conversion application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials application technology, specifically relating to the preparation of a targeted, long-acting thermogenic nanocomposite with enhanced tumor photothermal therapy function. Background Technology

[0002] Cancer is a major disease that seriously threatens human health. Its incidence and mortality rates continue to rise year by year, and there is currently no effective treatment.

[0003] Photothermal therapy (PTT) is a novel tumor treatment technology. Its basic principle is to use near-infrared (NIR) lasers to irradiate a photothermal converter, converting light energy into heat energy. This raises the local temperature of the tumor, "burning" or even thermally ablating and eliminating tumor cells, thus achieving the goal of treating the tumor. However, traditional photothermal therapy still has many shortcomings, mainly the lack of photothermal converters that can generate heat for a long time and have precise targeting, affecting the therapeutic effect and causing high temperatures in some normal tissues, leading to side effects such as burns, blisters, discomfort, or pain.

[0004] Over the past few decades, significant progress has been made in the application of nanotechnology to prepare nanomaterials with photothermal conversion capabilities for photothermal therapy of tumors. However, effective photothermal therapy requires reaching an effective treatment temperature of 41-43°C and maintaining it for 45-90 minutes. Most nanomaterials lack stability and cannot generate heat for extended periods, leading to suboptimal efficacy in photothermal therapy (PTT) and difficulties in clinical translation. CN202011175117.5 discloses a chemotherapy-photothermal-immunotherapy synergistic anti-tumor targeted nanoparticle. This nanoparticle uses a biocompatible biodegradable material as a carrier, internally encapsulating chemotherapeutic drugs, photothermal materials, and immunotherapeutic drugs, with tumor-targeting molecules linked to the nanoparticle surface. However, the photothermal material in this nanoparticle is any one of polydopamine, polyaniline, or polypyrrole, and its ability to maintain temperature and generate heat for extended periods needs improvement. Furthermore, the poor targeting of the photothermal conversion material further limits its application in PTT. Furthermore, although there has been considerable research on each component of photothermal therapy drugs, and many new materials, targeting molecules, or compounds have indeed emerged, the safety of these new materials and compounds still requires further observation and verification. Therefore, developing nanocomposites that can generate heat for a long time, specifically target tumors, and also have high safety will be beneficial for the rapid commercialization of PTT drugs. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of short duration of continuous irradiation and lack of targeting in the application of traditional photothermal conversion agents, and to provide a tumor-targeting nanocomposite with a long-lasting heat generation function.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A thermogenic nanocomposite, wherein the thermogenic nanocomposite has a core-shell structure, the core being oleic acid-modified metal nanoparticles loaded with photothermal materials, the outer shell being a tissue-compatible biodegradable material, and tumor-targeting molecules are attached to the surface of the outer shell.

[0008] As a preferred embodiment of the present invention, the photothermal material includes, but is not limited to, at least one of indocyanine green (ICG), IR825, IR780, and Cypate.

[0009] As a preferred embodiment of the present invention, the metal nanoparticles are iron(III) oxide nanoparticles.

[0010] As a further preferred embodiment of the present invention, the oleic acid-modified metal nanoparticles have a particle size of 5-50 nm, preferably 5-10 nm.

[0011] As a preferred embodiment of the present invention, the oleic acid-modified metal nanoparticles loaded with photothermal materials are prepared by an emulsification method.

[0012] As a preferred embodiment of the present invention, the biocompatible biodegradable material is selected from any one or more of polylactic acid glycolic acid, polylactic acid, or liposomes.

[0013] As a preferred embodiment of the present invention, the tumor-targeting molecules include, but are not limited to, tumor-specific antibodies or other tumor-targeting molecules.

[0014] As a further preferred embodiment of the present invention, the anti-tumor specific antibody includes, but is not limited to, monoclonal antibodies capable of specifically binding to tumor antigens, preferably at least one of anti-tumor specific protein 70 (SP70) monoclonal antibody, anti-Her2 monoclonal antibody, and anti-EGFR monoclonal antibody; the other tumor-targeting molecules include, but are not limited to, at least one of transferrin, RGD peptide, octreotide, folic acid, and hyaluronic acid.

[0015] The method for preparing the thermogenic nanocomposite of the present invention includes the following steps:

[0016] (1) Photothermal conversion materials are loaded onto the oleic acid layer of oleic acid-modified metal nanoparticles through hydrophobic interactions;

[0017] (2) A nanocomposite was prepared by the double emulsion solvent evaporation method, with oleic acid-modified iron oxide loaded with photothermal conversion material as the core and biocompatible degradable material as the shell.

[0018] (3) The tumor-targeting molecule is attached to the surface of the nanocomposite prepared in step (2) to obtain the thermogenic nanocomposite.

[0019] The application of the thermogenic nanocomposite described in this invention in the preparation of tumor therapeutic drugs.

[0020] As a preferred embodiment of the present invention, the application of the thermogenic nanocomposite described herein in the preparation of tumor photothermal therapy drugs is described herein.

[0021] A drug for treating tumors, comprising the long-acting thermogenic nanocomposite described in this invention.

[0022] As a preferred embodiment of the present invention, the drug further comprises a buffer solution.

[0023] All features disclosed in the specification, or all steps in the disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0024] Beneficial effects:

[0025] This invention relates to a thermogenic nanocomposite using a tissue-compatible biodegradable material coupled with tumor-targeting molecules as a carrier and oleic acid (OA)-modified metal nanoparticles loaded with photothermal materials as a core. The particle size of the thermogenic nanocomposite is between 80 and 120 nm. In a preferred embodiment of this invention, OA-Fe3O4 nanoparticles with a diameter of 5–10 nm were selected. Under equal mass conditions, compared with larger-sized OA-Fe3O4, these nanoparticles loaded with more photothermal material ICG molecules. ICG is linked to oleic acid through hydrophobic bonds, and the encapsulation of the ICG-loaded OA-Fe3O4 by the tissue-compatible biodegradable material PLGA further enhances the stability of this thermogenic nanocomposite. Furthermore, Fe3O4, as a heat storage element, can synergistically generate heat with ICG, thereby enabling this nanocomposite to possess a relatively stable and sustained heat-generating capacity under near-infrared irradiation. This nanocomposite can specifically bind to lung cancer cells and be endocytosed into the cytoplasm. In a mouse model of orthotopic lung tumors, the nanocomposite, when nebulized and inhaled, specifically accumulates within the lung cancer tissue and is phagocytosed by cancer cells. Under near-infrared light irradiation, it generates prolonged heat, precisely killing tumor cells, inhibiting tumor growth, and reducing damage to normal tissues and organs, thus minimizing toxic side effects. Furthermore, the ICG, nano-iron oxide, and PLGA used in the preparation of the nanocomposite are all FDA-approved drugs or excipients, exhibiting good biocompatibility, strong compliance, and high safety for in vivo application, demonstrating significant translational application value. Attached Figure Description

[0026] Figure 1Preparation and characterization of nanocomposites. (A) Schematic diagram of the preparation of targeted nanocomposites. (B) Loading capacity of ICG in nanocomposites with OA-Fe3O4@PLGA cores of equal mass and different particle sizes. (C) Sustained photothermal conversion capability of nanocomposites with OA-Fe3O4 cores of different particle sizes. (D) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of ICG-OA-Fe3O4@PLGA (non-targeted nanocomposites). (F) Particle size distribution and (G) Zeta potential of ICG-OA-Fe3O4@PLGA. (H) Scanning electron microscopy (SEM) and (I) TEM image of ICG-OA-Fe3O4@PLGA-NJ001 (targeted nanocomposites). (J) Particle size distribution and (K) Zeta potential of ICG-OA-Fe3O4@PLGA-NJ001. (L) 808 nm, 1 W / cm 2 Continuous heat generation capacity of different nanoparticles under continuous laser irradiation. (M) 808nm, 1W / cm 2 Intermittent laser irradiation and the heat generation capacity of different nanoparticles.

[0027] Figure 2 To assess the SP70 binding ability of the targeting nanocomposite. (A) The nanocomposite was incubated with the supernatant of SPC-A1 culture for 30 min, and the residual amount of SP70 in the supernatant was measured. The binding ability of the nanocomposite to SP70 was calculated; a higher binding rate of the nanocomposite to SP70 indicates a lower residual amount of SP70 in the supernatant. (B) ICG-OA-Fe3O4@PLGA-NJ001 and (C) ICG-OA-Fe3O4@PLGA were co-incubated with SP70-positive SPC-A1 cells, and (D) ICG-OA-Fe3O4@PLGA-NJ001 was co-incubated with HBE cells (SP70-negative). Fluorescence was observed using a laser confocal microscope. White arrows indicate the binding sites of the targeting nanocomposite to SPC-A1 cells.

[0028] Figure 3 To investigate the in vitro killing effect of targeted nanocomposites on lung cancer cells, different nanoparticles were co-incubated with SPC-A1 cells for different times, followed by irradiation with an 808 nm laser at 1 W / cm² for 10 min. Annexin V / PI double staining and flow cytometry were used to assess cell viability. Incubation times were 0.5 h (AB), 12 h (CD), and 24 h (EF). (G) Non-viable cell statistics are shown in the figure. *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 4The distribution, retention, and heat generation of the targeted nanocomposite in a mouse model are shown. (A) Schematic diagram of the therapeutic mechanism of the targeted nanocomposite ICG-OA-Fe3O4@PLGA-NJ001. After inhalation, the targeted nanocomposite specifically binds to SP70 via NJ001 and accumulates in the lung cancer lesion. 808nm near-infrared irradiation induces heat generation at the tumor site, resulting in excessively high tumor temperature and achieving precise photothermal therapy. (B) Fluorescence images of mice detected by a small animal in vivo imaging system at different time points; green circles indicate the location of the lungs. Lung temperature of mice under NIR irradiation was monitored at 24h (C) and 48h (D) after inhalation of the nanocomposite.

[0030] Figure 5 This study assesses the efficacy of targeted nanocomposites in photothermal therapy on a mouse model of lung cancer. (A) Experimental groups. (B) Bioluminescence imaging of the lung cancer model mice before treatment (day 0) and after inhalation (days 7, 14, and 21). (C) Micro-CT scan on day 20. (D) Lung photograph on day 21. (E) HE staining of lung tissue. (F) Bioluminescence intensity statistics. (G) Relative body weight of mice during treatment. (H) Micro-CT showing the number of lung nodules. **p<0.01, ***p<0.001.

[0031] Figure 6 This study aimed to detect the expression of SP70 in the serum and lung tissue of mice with lung cancer. (A) ELISA was used to detect the level of SP70 in mouse serum. (B) Immunohistochemical staining was used to detect SP70 expression in lung tissue; a brownish-yellow color indicated the presence of SP70 protein.

[0032] Figure 7 Different nanoparticles at 808nm, 1W / cm 2 In vitro thermograms under continuous laser irradiation. Different nanoparticles (808 nm) were used at a concentration of 1 W / cm². 2 The laser irradiated the area continuously for 18 hours, and temperature images were collected at corresponding time points. Figure 1 L is the temperature statistics graph for this figure.

[0033] Figure 8 Different nanoparticles at 808nm, 1W / cm 2 In vitro thermograms under intermittent laser irradiation. Different nanoparticles were intermittently irradiated (3 minutes irradiation, 7 minutes rest), and temperature maps were captured during the first cycle and the peak temperature maps for the remaining 11 cycles. Figure 1 M is the temperature statistics graph for this figure.

[0034] Figure 9 This is a temperature measurement image of the lungs of a mouse model of orthotopic lung cancer. Near-infrared laser light was used to irradiate the mouse lungs, and temperature images were collected at specified time points. Figure 4 C and D are temperature statistics graphs for this diagram.

[0035] Figure 10 HE staining was performed on the major organs of mice treated with the targeted nanocomposite. The heart, liver, spleen, kidney, and lungs of mice treated with the targeted nanocomposite were stained with HE, while mice treated with physiological saline served as controls. Detailed Implementation

[0036] The objectives, features, and advantages of the present invention will be further described below with reference to the embodiments and accompanying drawings.

[0037] Example 1

[0038] The preparation of PLGA-targeted nanoparticles with a targeted, long-lasting thermogenic nanocomposite that enhances tumor photothermal therapy in this invention is carried out in four steps. Figure 1 A).

[0039] S1, Selection of OA-Fe3O4 Particle Size. Dissolve 1, 2, or 3 mg of ICG in 1 mL of deionized water. Add equal masses of OA-Fe3O4 with different particle size ranges (5-10 nm or 30-50 nm) to the ICG aqueous solution. Sonicate at 100 W for 2 minutes using a microprobe ultrasonic emulsifier, stir for 4 hours, and collect the OA-Fe3O4 loaded with ICG molecules using a magnetic rack (ICG-OA-Fe3O4). Dissolve 100 mg of PLGA in 2 mL of dichloromethane and add ICG-OA-Fe3O4. Prepare a primary emulsion by ultrasonic emulsification (sonication at 100 W for 60 seconds). Add 30 mL of 2% PVA solution to the primary emulsion and ultrasonic emulsify again. (Ultrasonication at 100W for 100 seconds) A double emulsion was prepared by adding 10 mL of 0.5% PVA solution to the double emulsion and stirring for 4 h to allow dichloromethane to evaporate and solidify the microspheres. The microspheres were washed with deionized water and centrifuged three times to obtain the nanocomposite ICG-OA-Fe3O4@PLGA, which was stored at 4℃ for later use. 10 mg of ICG-OA-Fe3O4@PLGA was taken, the nanoparticles were lysed, and the supernatant was collected by centrifugation. The ICG concentration was determined by spectrophotometry. Under the same mass conditions, the ICG loading capacity of OA-Fe3O4 (5-10 nm) was approximately four times that of OA-Fe3O4 (30-50 nm). Figure 1 B). Under NIR irradiation, the peak temperature of the nanocomposite material with OA-Fe3O4 (5-10nm) core was 48.1℃, while the peak temperature of the nanocomposite material with OA-Fe3O4 (30-50nm) core was only 36.1℃. Figure 1 C).

[0040] S2. Loading ICG into the oleic acid layer of OA-Fe3O4. First, dissolve 5 mg of ICG in 1 mL of deionized water. After complete dissolution, add 1 mg of OA-Fe3O4 (5-10 nm, dissolved in chloroform, purchased from Nanjing Shennuoqing Biotechnology Co., Ltd.) to the above ICG aqueous solution, and sonicate for 2 minutes using a microprobe ultrasonic emulsifier at 100W. Stir for 4 hours to evaporate the organic solvent chloroform, and then magnetically separate the OA-Fe3O4 loaded with ICG molecules (denoted as ICG-OA-Fe3O4).

[0041] S3. Preparation of PLGA nanocomposite encapsulated with ICG-OA-Fe3O4 by double emulsion solvent evaporation method. 1 mg of OA-Fe3O4 loaded with ICG was dissolved in 200 μl of deionized water. Simultaneously, 10 mg of PLGA polymer was dissolved in 2 ml of dichloromethane. Then, 200 μl of the ICG-OA-Fe3O4 aqueous phase was injected into 2 ml of the PLGA oil phase, and homogenized at high speed (or ultrasonically) to form a primary emulsion. Then, 30 ml of 2% polyvinyl alcohol (PVA) was added, and homogenized at high speed or ultrasonically to form a secondary emulsion. 10 ml of 0.5% PVA was added to the resulting secondary emulsion solution, and the mixture was stirred for 4 h to evaporate the dichloromethane. Finally, the nanocomposite was collected by centrifugation, washed repeatedly with deionized water, named (ICG-OA-Fe3O4@PLGA), and stored at 4℃ for later use.

[0042] S4. Preparation of PLGA nanocomposites with long-lasting heat generation and tumor-targeting functions

[0043] 10 mg of ICG-OA-Fe3O4@PLGA was resuspended in 1 ml of MES buffer (50 mM, pH 6.0) containing EDC (2 mM) and NHS (5 mM), and the mixture was rotated for 30 minutes to activate the carboxyl groups on the surface of the nanocomposite. The targeted nanocomposite was collected by centrifugation at 10,000 rpm for 5 min, and washed three times with deionized water to obtain carboxyl-activated PLGA nanoparticles. The nanocomposite was resuspended in 1 mL of the novel tumor marker SP70 monoclonal antibody NJ001 (monoclonal antibody NJ001-1 was secreted by hybridoma cell line NM001-1 with accession number CCTCC NO: C201172; preparation method detailed in CN201110344683.9) (pH 7.4), reacted at room temperature for 2 hours, and collected by centrifugation at 10,000 rpm for 5 min to obtain the PLGA nanocomposite with long-lasting thermogenic and tumor-targeting functions, named ICG-OA-Fe3O4@PLGA-NJ001. Scanning electron microscopy, transmission electron microscopy, particle size analyzer, and Zeta potential analyzer were used to analyze the ICG-OA-Fe3O4@PLGA nanocomposite. Figure 1 DG) and ICG-OA-Fe3O4@PLGA-NJ001 were characterized ( Figure 1HK); subsequently, the photothermal conversion performance of the nanocomposite solution was evaluated by continuous near-infrared irradiation for 18 h. The results showed that the temperatures of the ICG-OA-Fe3O4@PLGA and ICG-OA-Fe3O4@PLGA-NJ001 solutions rose to 51.8℃ and 48.7℃, respectively, within 3 minutes. Figure 1 L, Figure 7 Under continuous irradiation, both maintained above 41°C for approximately 8 hours. ICG / Fe3O4@PLGA (10 mg PLGA dissolved in 2 ml dichloromethane as the oil phase, 1 mg Fe3O4 and 3 mg ICG simultaneously dissolved in 200 μl deionized water and added to 2 ml PLGA oil phase; homogenized by high-speed shearing or ultrasonication to form a primary emulsion; then added 30 ml 2% polyvinyl alcohol (PVA) and homogenized by high-speed shearing or ultrasonication to form a secondary emulsion. 10 ml 0.5% PVA was added to the resulting secondary emulsion solution, stirred for 4 h, and the dichloromethane was volatilized. Finally, the nanocomposite was collected by centrifugation, washed repeatedly with deionized water, named ICG / Fe3O4@PLGA, and stored at 4°C for later use), ICG@PLGA (10 mg PLGA dissolved in 2 ml dichloromethane as the oil phase, 1 mg Fe3O4 and 3 mg ICG simultaneously dissolved in 200 μl deionized water and added to 2 ml PLGA oil phase; the primary emulsion was prepared by high-speed shearing or ultrasonication, and then 30 ml 2% polyvinyl alcohol (PVA) was added. The secondary emulsion was then prepared by high-speed shearing or ultrasonication. 10 ml 0.5% PVA was added to the resulting secondary emulsion solution, stirred for 4 h, and the dichloromethane was volatilized. Finally, the nanocomposite was collected by centrifugation, washed repeatedly by centrifugation with deionized water, named ICG / Fe3O4@PLGA, and stored at 4°C for later use), ICG@PLGA (10 mg PLGA dissolved in 2 ml dichloromethane as the oil phase, 1 mg Fe3O4 and 3 mg ICG simultaneously dissolved in 200 μl deionized water and added to 2 ml 3 mg PLGA was dissolved in 2 ml of dichloromethane as the oil phase, and 3 mg ICG was dissolved in 200 μl of deionized water and added to the 2 ml PLGA oil phase. The mixture was homogenized using high-speed shearing or sonication to prepare a primary emulsion. Then, 30 ml of 2% polyvinyl alcohol (PVA) was added, and the mixture was homogenized using high-speed shearing or sonication to prepare a secondary emulsion. 10 ml of 0.5% PVA was added to the resulting secondary emulsion solution, and the mixture was stirred for 4 h to allow the dichloromethane to evaporate. Finally, the nanocomposite was collected by centrifugation, washed repeatedly with deionized water, and named ICG / Fe3O4@PLGA (stored at 4℃ for later use). The temperatures of the free ICG reached 49.9℃, 48.1℃, and 47.4℃, respectively, but were maintained for only a few minutes. Throughout the near-infrared irradiation process, the temperature rise of the Blank@PLGA (PLGA nanoparticle) solution was minimal. Furthermore, to investigate the photothermal conversion stability of the nanocomposite solution, intermittent near-infrared irradiation was applied to the solution. Irradiation was performed for 3 minutes to raise the temperature to its peak, followed by 7 minutes of irradiation to allow the temperature to return to near room temperature. This constituted one cycle, and a total of 12 cycles were performed. The results showed that the heat production capacity of ICG-OA-Fe3O4@PLGA and ICG-OA-Fe3O4@PLGA-NJ001 did not show a significant decrease throughout the entire process. Figure 1 M, Figure 8 This indicates that the PLGA nanocomposite with ICG-OA-Fe3O4 as its core can withstand multiple rounds of near-infrared irradiation without a decrease in photothermal conversion efficiency. The highest temperatures of ICG / Fe3O4@PLGA, ICG@PLGA, and free ICG decreased to below 41℃ in the 9th, 3rd, and 3rd cycles, respectively. No photothermal conversion effect was observed in Blank@PLGA.

[0044] Example 2: In vitro targeting and killing of tumor cells by ICG-OA-Fe3O4@PLGA-NJ001

[0045] First, the binding ability of the targeted nanocomposite to free SP70 in the SPC-A1 cell culture supernatant was determined. ICG-OA-Fe3O4@PLGA-NJ001 was co-incubated with the SPC-A1 cell culture supernatant for 30 min. Blank@PLGA and ICG-OA-Fe3O4@PLGA served as controls. After centrifugation, the residual amount of SP70 in the supernatant was detected by ELISA.

[0046] Next, the binding ability of the targeted nanocomposite to SP70-positive cancer cells was investigated. The binding of the targeted nanocomposite to SPC-A1 cells highly expressing SP70 was observed using laser confocal microscopy (Lsm710). Non-targeted nanocomposite ICG-OA-Fe3O4@PLGA and HBE cells (SP70 negative) were used as controls. After incubating the cells with the nanocomposite for 30 min, non-specifically bound particles were washed off, and the cells were cultured for another 1 or 2 hours. After cell fixation, Hoechst staining was used to stain the nuclei, and Dio staining was used to stain the cell membrane. The cell-particle binding state was observed using laser confocal microscopy.

[0047] ELISA results showed that with increasing input of the targeted nanocomposite ICG-OA-Fe3O4@PLGA-NJ001, the concentration of SP70 in SPC-A1 cell culture medium decreased in a dose-dependent manner. However, neither ICG-OA-Fe3O4@PLGA nor Blank@PLGA could reduce the level of SP70. Figure 2 A). This result indicates that the targeted nanocomposite can neutralize free SP70 molecules.

[0048] The targeting nanocomplex was co-cultured with SPC-A1 cells highly expressing SP70 to observe its binding ability to lung cancer cells. Confocal microscopy analysis showed that the targeting nanocomplex could bind to the SPC-A1 cell membrane and be endocytosed into the cytoplasm. Figure 2 B). However, the non-targeted nanocomposite ICG-OA-Fe3O4@PLGA could not bind to and enter SPC-A1 cells (B). Figure 2 C). Furthermore, ICG-OA-Fe3O4@PLGA-NJ001 also failed to bind and enter HBE cells (SP70 negative). Figure 2 D).

[0049] SPC-A1 cells were co-incubated with Blank@PLGA, ICG-OA-Fe3O4@PLGA, Blank@PLGA-NJ001, and ICG-OA-Fe3O4@PLGA-NJ001 in 96-well plates, respectively. After incubation for half an hour, the non-specifically bound nanocomplexes were washed. After washing, the cells were irradiated with near-infrared light at 808 nm, 1.0 W / cm² for 10 min. Cells were collected and stained with Annexin V-FITC / PI apoptosis kit. Fluorescence signals were recorded by flow cytometry, and the proportion of live and dead cells was quantitatively analyzed using FlowJoV10 software. Flow cytometry analysis showed that after incubation with the ICG-OA-Fe3O4@PLGA-NJ001 targeted nanocomplex group for half an hour, the proportion of non-living cells reached 30.3 ± 4.11%. Figure 3 B, G). As incubation time increased to 12 h and 24 h, the percentage of non-viable cells increased to 67.1 ± 2.28% and 83.3 ± 2.45%, respectively. Figure 3 (D, F, G). The non-targeted nanocomposite ICG-OA-Fe3O4@PLGA group showed very low rates of cell-free incubation after 0.5, 12, and 24 hours, at (3.67±0.39%), (6.31±0.65%), and (6.93±0.96%), respectively. Figure 3 B, D, F, G).

[0050] We also investigated the anticancer activity of the nanomaterials under radiation-free conditions. After incubation for 0.5, 12, and 24 hours, the percentages of viable cells in the ICG-OA-Fe3O4@PLGA-NJ001 group reached (3.22±0.25%), (28.8±2.35%), and (35.6±3.67%), respectively. Figure 3 A, C, E, G). However, in any group treated with non-targeted nanoparticles, the percentage of non-viable cells never exceeded 6.0% ( Figure 3 (Graphs A, C, E, and G).

[0051] Example 3: Accumulation and Heat Generation of Targeted Nanocomposites in Lung Cancer Lesions

[0052] The nanocomposite was administered to mice via inhalation, followed by NIR irradiation to stimulate thermogenicity. Figure 4 A). The distribution of the nanocomposite in mice was monitored on days 0, 1, 2, and 9. Figure 4 B). The results showed that in mice with orthotopic lung cancer that had inhaled the targeting complex ICG-OA-Fe3O4@PLGA-NJ001, there was a strong fluorescent signal of ICG (B). Figure 4Ba,b). Strong fluorescence remained in the lungs 48 hours after inhalation of 100 μg of the targeted nanocomposite, and fluorescence was still detectable on day 9, indicating good in vivo retention. In contrast, the nanocomposite was rapidly cleared from lung cancer model mice inhaled with ICG-OA-Fe3O4@PLGA and tumor-free mice inhaled with ICG-OA-Fe3O4@PLGA-NJ001 after 24 hours. Figure 4 (Bc,d). It is evident that the NJ001 monoclonal antibody not only exerted a targeting effect but also promoted the endocytosis of the ICG-OA-Fe3O4@PLGA-NJ001 nanocomposite by cells, thereby prolonging the retention time of the nanocomposite in cancer cells.

[0053] Lung temperature in mice after NIR irradiation revealed that the lung temperature of mice with an in situ lung cancer model who inhaled 50 μg of ICG-OA-Fe3O4@PLGA-NJ001 rose to a maximum of 41.3℃ within 6 minutes and remained above 40.0℃. In mice with an in situ lung cancer model who inhaled 100 μg of ICG-OA-Fe3O4@PLGA-NJ001, the lung temperature rose to a maximum of 48.6℃ within 6 minutes and remained above 48.0℃ throughout the irradiation process. When mice with in situ lung cancer were given 100 g of ICG-OA-Fe3O4@PLGA and mice without lung cancer were given 100 g of ICG-OA-Fe3O4@PLGA-NJ001, the temperature rose to 47.1℃ and 46.8℃, respectively. However, after 6 minutes, the temperature dropped sharply, and the total time the temperature remained above 41℃ did not exceed 10 minutes. Figure 4 C, Figure 9 ).

[0054] Because the photothermal therapy duration in mice reported in the literature was all within 20 minutes, 20-minute intervals were chosen for each irradiation session, and no longer sessions were administered. However, irradiation was repeated on the second day, bringing the total heat production time of the two irradiations to 40 minutes. Compared to the first irradiation, the second irradiation on the second day showed a lower temperature increase, with only the lungs of mice with an in situ lung cancer model exhibiting temperature elevation after inhaling the targeted nanocomposite. Figure 4 D and Figure 9 As shown, the highest lung temperature in mice with orthotopic lung cancer after inhaling 50 μg of ICG-OA-Fe3O4@PLGA-NJ001 reached 40.8℃, and the temperature gradually decreased slowly after 6 minutes. The highest lung temperature in mice with the model after inhaling 100 μg of ICG-OA-Fe3O4@PLGA-NJ001 reached 46.6℃, and remained above 45.0℃ throughout the irradiation process. The temperature in the non-targeted nanocomposite treatment group and the tumor-free group did not increase significantly.

[0055] Example 4: Photothermal Therapy of Tumors in Vivo Using ICG-OA-Fe3O4@PLGA-NJ001

[0056] Four-week-old male BALB / c nude mice (purchased from the Experimental Animal Center of Nanjing Medical University) were housed in a sterile environment, and all procedures were performed according to the guidelines of the Animal Protection and Use Committee of Nanjing Medical University. Logarithmically growing SPC-A1-luc cells were collected, counted, and resuspended in physiological saline to a final concentration of 5 × 10⁻⁶. 7 / mL, 5×10 6 A mouse model of orthotopic lung tumor was established by injecting 0.1 mL of SPC-A1-luc cell suspension into mice. Bioluminescence imaging showed that the fluorescence intensity of the lung cancer lesions was approximately 9 × 10⁻⁶. 5 photons / s / cm 2 During / sr, mice were randomly divided into 5 groups as follows: Group A: 50 μg / mouse, wavelength 808 nm, power 2 W / cm 2 Group A: Near-infrared irradiation for 20 min; Group B: 50 μg / mouse, 808nm, 2W / cm² near-infrared irradiation for 20 min twice; Group C: 100 μg / mouse ICG-OA-Fe3O4@PLGA-NJ001, 808nm, 2W / cm² near-infrared irradiation for 20 min once; Group D: 100 μg / mouse ICG-OA-Fe3O4@PLGA-NJ001, 808nm, 2W / cm² near-infrared irradiation for 20 min twice; Group E: 100 μg / mouse ICG-OA-Fe3O4@PLGA, 808nm, 2W / cm² near-infrared irradiation for 20 min, for a total of 2 irradiations. Additionally, cancer-free mice inhaled 100 μg ICG-OA-Fe3O4@PLGA-NJ001 and were irradiated at 808nm, 2W / cm² for 20 min. 2 The group was irradiated with near-infrared spectroscopy (NIR) for 20 minutes and served as the control group (group f). Figure 5 A shows the mice divided into 6 groups based on the type of nanocomposite, inhalation dose, and irradiation frequency.

[0057] Next, we evaluated the anticancer effect of the targeted nanocomposite in mice with orthotopic lung cancer. Tumor growth was monitored by bioluminescence imaging every 7 days (days 0, 7, 14, and 21). Figure 5 As shown in B and 5F, tumor growth was rapid in the non-targeted nanocomposite ICG-OA-Fe3O4@PLGA group, while tumor growth was inhibited in all targeted nanocomposite ICG-OA-Fe3O4@PLGA-NJ001 groups. The tumor shrinkage was most significant in the 100μg ICG-OA-Fe3O4@PLGA-NJ001 two-irradiation group. Micro-CT scans showed that the 100μg ICG-OA-Fe3O4@PLGA-NJ001 two-irradiation group had the fewest lung cancer nodules and the smallest volume. Figure 5C, H). Nude mice were sacrificed 21 days after treatment, and their lungs were examined. The lung tissue morphology of the group irradiated with 100μgICG-OA-Fe3O4@PLGA-NJ001 twice was basically normal, with a smooth surface except for a few small nodules. Figure 5 D). HE staining showed that the lung tissue of this group of mice was morphologically intact, with single-celled alveoli, similar to the lung tissue of mice without cancer, while the alveolar structure of the ICG-OA-Fe3O4@PLGA group was severely damaged. Figure 5 E). Furthermore, the body weight of mice in the ICG-OA-Fe3O4@PLGA group decreased rapidly and progressively, while the body weight of mice in the 100μg ICG-OA-Fe3O4@PLGA-NJ001 irradiation group showed no significant decrease (E). Figure 5 G).

[0058] To further verify the inhibitory effect of the targeted nanocomposite on lung tumor growth, we examined the expression of SP70 in serum and lung tissue. The group irradiated twice with 100 μg ICG-OA-Fe3O4@PLGA-NJ001 showed the greatest reduction in serum SP70. Figure 6 A). Immunohistochemical analysis also showed that SP70 expression was significantly reduced in the lung tissue of this group. Figure 6 B).

[0059] Example 5: Biocompatibility of ICG-OA-Fe3O4@PLGA-NJ001 in vivo

[0060] Compared with mice that inhaled saline solution via nebulization, no visible damage was observed in the heart, spleen, kidneys, liver, and lungs of mice after inhalation and irradiation of the targeted nanocomposite ICG-OA-Fe3O4@PLGA-NJ001. Figure 10 This indicates that the targeted nanocomposite material did not cause significant toxic side effects in vivo.

[0061] Although this invention only provides an example of preparing a thermogenic nanocomposite using NJ001 as a tumor-targeting molecule, Example 1 demonstrates that ICG-OA-Fe3O4@PLGA possesses sustained heating characteristics. Those skilled in the art can, based on the concept and method of this invention, replace NJ001 with other tumor-targeting molecules to achieve a targeting effect. Similarly, those skilled in the art can also, based on the concept and method of this invention, replace ICG with other photothermal materials such as IR825, IR780, and Cypate. These alternative solutions can produce the same or similar effects as the embodiments of this invention. Therefore, these alternative technical solutions also fall within the protection scope of this invention.

Claims

1. A thermogenic nanocomposite, characterized in that, The aforementioned thermogenic nanocomposite has a core-shell structure. The core is oleic acid-modified metal nanoparticles loaded with photothermal material, and the outer shell is polylactic acid-glycolic acid, a biocompatible and biodegradable material. Tumor-targeting molecules are attached to the surface of the outer shell. The particle size of the thermogenic nanocomposite is 80-120 nm. The photothermal material is selected from indocyanine green (ICG). The metal nanoparticles are iron oxide nanoparticles. The oleic acid-modified metal nanoparticles have a particle size of 5-10 nm. The tumor-targeting molecule is an anti-tumor-specific protein 70 monoclonal antibody. The anti-tumor-specific protein 70 monoclonal antibody is monoclonal antibody NJ001-1, secreted by hybridoma cell line NM001-1 with accession number CCTCC NO: C201172. The aforementioned thermogenic nanocomposite was prepared through the following steps: (1) Photothermal materials are loaded onto the oleic acid layer of oleic acid-modified metal nanoparticles through hydrophobic interactions; (2) A nanocomposite with oleic acid-modified iron oxide loaded with photothermal material as the core and biocompatible degradable material as the shell was prepared by the double emulsion solvent evaporation method; (3) The tumor-targeting molecule is attached to the surface of the nanocomposite prepared in step (2) to obtain the thermogenic nanocomposite.

2. The method for preparing the thermogenic nanocomposite according to claim 1, characterized in that, Includes the following steps: (1) Photothermal materials are loaded onto the oleic acid layer of oleic acid-modified metal nanoparticles through hydrophobic interactions; (2) A nanocomposite with oleic acid-modified iron oxide loaded with photothermal material as the core and biocompatible degradable material as the shell was prepared by the double emulsion solvent evaporation method; (3) The tumor-targeting molecule is attached to the surface of the nanocomposite prepared in step (2) to obtain the thermogenic nanocomposite.

3. The use of the thermogenic nanocomposite according to claim 1 in the preparation of photothermal therapy drugs for lung tumors.

4. A drug for treating lung tumors, characterized in that... It comprises the thermogenic nanocomposite of claim 1.

5. The drug according to claim 4, characterized in that, It also contains a buffer solution.

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