Near-infrared II nanoparticles, their preparation method and applications

Abstract

This invention discloses near-infrared II nanoparticles, their preparation method, and applications, belonging to the field of biomedical technology. The nanoparticles include a polymer shell and a near-infrared II fluorescent molecular core; the polymer shell comprises distearate phosphatidylethanolamine-polyethylene glycol. This invention achieves the batch preparation and performance optimization of near-infrared II nanoparticles through microfluidic technology. By adjusting the ratio and flow rate, the particle size and distribution can be effectively controlled. Furthermore, this invention enables precise temporal and spatial synergy between "drug" and "light," ultimately achieving complete eradication of lung infection lesions / tumors while maximizing the protection of surrounding normal tissues, thus improving treatment safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a near-infrared II nanoparticle, its preparation method and application. Background Technology

[0002] Lung infections, including viral, bacterial, fungal, and mycoplasmal infections, remain a leading cause of death from infectious diseases worldwide. Hospital-acquired pneumonia has a profound impact on individual health and public healthcare systems, often leading to persistent chronic infections and high mortality rates even with appropriate treatment. In recent years, the overuse of broad-spectrum antibiotics has resulted in drug resistance, which has evolved into a major global public health crisis. The efficacy of many first-line clinical drugs has significantly declined or even become completely ineffective, and the pace of traditional drug development is far behind the rate of pathogen mutation. Therefore, developing novel lung infection strategies that do not rely on traditional mechanisms of action and can effectively overcome drug resistance has become an urgent problem to be solved in the field of lung infection treatment.

[0003] Cancer, including malignant solid tumors, hematologic malignancies, neuroendocrine tumors, and stromal tumors, remains one of the leading causes of death worldwide. Cancer diagnosed in hospitals or communities has a profound impact on individual health and public healthcare systems. Even with appropriate surgery, radiotherapy, or chemotherapy, it often leads to recurrence, metastasis, chronic progression, and high mortality rates. In recent years, with the widespread use of chemotherapy and targeted drugs, cancer drug resistance has evolved into a major global public health crisis. The efficacy of many first-line clinical drugs has significantly declined or even completely failed, and the pace of traditional drug development is far behind the heterogeneous evolution and mutational escape of tumor cells. Therefore, developing novel anti-tumor strategies that do not rely on traditional mechanisms of action and can effectively overcome drug resistance has become an urgent problem to be solved in the field of cancer treatment.

[0004] Among numerous emerging therapies, phototherapy (including photodynamic therapy and photothermal therapy) demonstrates enormous application potential and clinical translation prospects due to its unique therapeutic mechanism. This therapy uses a specific wavelength of laser light to excite a photosensitizer, generating a large amount of reactive oxygen species or heat, thereby damaging biomolecules (such as DNA, RNA, proteins, and lipids) in pathogens or cancer cells, thus efficiently killing pathogens / cancer cells. Compared to the mechanisms of action of traditional drugs, phototherapy's multi-target killing mode makes it difficult for bacteria / cancer cells to develop drug resistance. In clinical treatment, phototherapy has achieved significant results in superficial tissue infections (such as skin wound infections and oral infections) or superficial tumors (such as skin cancer). However, due to the insufficient tissue penetration of traditional phototherapy, its application in deep lesions such as lung infections and liver cancer is limited.

[0005] Therefore, how to provide a novel treatment system for lung infections / tumors that is efficient, precise, minimally invasive, and has good clinical translation potential is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a near-infrared II nanoparticle, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A near-infrared II nanoparticle comprising a polymer shell and a near-infrared II fluorescent molecular core; The polymer shell comprises distearylphosphatidylethanolamine-polyethylene glycol.

[0008] Beneficial effects: Polyethylene glycol on the surface of nanoparticles can effectively enhance the stability of nanoparticles in aqueous solution, thereby improving their blood circulation performance and prolonging their metabolic half-life in vivo.

[0009] Preferably, the structure of the near-infrared II fluorescent molecular core is shown in formula (I): (I); Wherein, R1 is independently selected from one of H, CH3 and OCH3; R2 and R3 are independently selected from H, , , , and One of them; R4 is independently selected from either S or Se.

[0010] Beneficial effects: The near-infrared II molecules loaded in the nanoparticle core have high reactive oxygen species yield and high photothermal conversion efficiency. Under laser irradiation, they can convert light energy into reactive oxygen species or heat energy, achieving precise elimination of lesions. At the same time, the near-infrared II molecules have aggregation-induced emission properties, exhibiting high fluorescence quantum yield in the aggregated state, enabling precise diagnosis of lesions and guiding treatment.

[0011] Preferably, the polymer shell includes modifying groups; The modifying group includes one of small molecule ligands, targeting peptides, and monoclonal antibodies.

[0012] More preferably, the small molecule ligand includes one of phenylboronic acid, folic acid, and mannose.

[0013] Beneficial effects: Phenylboronic acid can form dynamic covalent bonds with polysaccharides on the surface of bacteria or in biofilms, achieving effective enrichment in lung infection lesions; folic acid can bind to folic acid receptors overexpressed on the surface of cancer cells or M1 macrophages, achieving effective enrichment in tumors or inflammatory sites; mannose can bind to mannose receptors overexpressed on the surface of M2 macrophages, achieving effective enrichment in tumors.

[0014] More preferably, the sequence of the targeting peptide is one of RLYLRIGRR, GLRRLLRKIR GRWK, ELTPEERMLRLIFGRK, ADGVGDAESRTR, and RDG.

[0015] Beneficial effects: Targeting peptides RLYLRIGRR, GLRRLLRKIR GRWK, ELTPEERMLRLIFGRK, and ADGVGDAESRTR can improve the targeting of nanoparticles to infectious lesions, achieving effective enrichment of nanoparticles in lung infectious lesions; RDG can achieve efficient enrichment in tumor tissues by targeting integrin receptors overexpressed on the surface of cancer cells.

[0016] More preferably, the monoclonal antibody is one of Panobacumab, Gremubamab, Tosatoxumab, Pse-MAB1, and anti-KL64 capsular antibody.

[0017] Beneficial effects: The above-mentioned monoclonal antibodies can improve the targeting of nanoparticles to infectious lesions, and achieve effective enrichment of nanoparticles in lung infectious lesions.

[0018] A method for preparing near-infrared II nanoparticles includes the following steps: Near-infrared II fluorescent molecules and polymers dissolved in the solution were used as the organic phase, and pure water was used as the aqueous phase. The organic phase and the aqueous phase were mixed and reacted using a microfluidic system to prepare the near-infrared II nanoparticles.

[0019] Preferably, in the organic phase, the concentration of near-infrared II fluorescent molecules is 0.1-0.5 mg / mL, the concentration of polymer is 5 mg / mL, and the solvent is tetrahydrofuran (THF). In the microfluidic system, the total flow rate of the organic phase and the aqueous phase is 4-20 mL / min, and the flow rate ratio is 1:3.

[0020] Beneficial effects: This invention enables the batch preparation and performance optimization of near-infrared II nanoparticles through microfluidic technology. By adjusting the ratio and flow rate, the particle size and distribution of nanoparticles can be effectively controlled.

[0021] Preferably, the polymer is one of DSPE-mPEG, DSPE-PEG-PBA, DSPE-PEG-FA, DSPE-PEG-Man, DSPE-PEG-Mal, and DSPE-PEG-COOH.

[0022] More preferably, the DSPE-mPEG is DSPE-PEG2000.

[0023] Beneficial effects: The nanoparticles assembled in this invention have a surface of polyethylene glycol (PEG), which can effectively enhance the stability of the nanoparticles in aqueous solution, thereby improving their blood circulation performance and prolonging their metabolic half-life in vivo.

[0024] Preferably, when the polymer is DSPE-PEG-Mal, the reaction after the organic phase and aqueous phase are mixed further includes the following steps: After the reaction was completed, the product was mixed with the target peptide and stirred before dialyzing to obtain near-infrared II nanoparticles modified with the target peptide.

[0025] More preferably, the amount of the product obtained after the reaction and the amount of the target peptide added are based on the molar ratio of maleimide in the polymer to cysteine ​​(Cys) in the target peptide of (5-1):(1-5), and more preferably 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0026] More preferably, the mixing and stirring is carried out at room temperature for 12 hours.

[0027] More preferably, the dialysis is performed for 12 hours using a dialysis bag with a molecular weight cutoff of 5000 Da.

[0028] Beneficial effects: In this invention, the maleimide (Mal) modified at the PEG end can undergo a click reaction with the thiol group on the cysteine ​​residue at the end of the peptide, so as to efficiently modify the target peptide onto the surface of the nanoparticle.

[0029] Preferably, when the polymer is DSPE-PEG-COOH, the reaction after the organic phase and aqueous phase are mixed further includes the following steps: After the reaction was completed, the product was activated with carbodiimide and N-hydroxythiosuccinimide to activate the surface carboxyl groups. Then, it was mixed with a monoclonal antibody solution for covalent coupling to obtain near-infrared II nanoparticles modified with monoclonal antibody.

[0030] More preferably, the surface carboxyl group activation is carried out under ice-water bath conditions and pH 4.5-7.2. More preferably, the molar ratio of the surface carboxyl group to carbodiimide and N-hydroxythiosuccinimide of the product obtained after the reaction is 1:2:4.

[0031] More preferably, the concentration of the monoclonal antibody solution is 5 µg / mL.

[0032] More preferably, the mass ratio of the product obtained at the end of the reaction to the monoclonal antibody is 10:1.

[0033] Beneficial effects: The nanoparticles in this invention modify the antibody surface through an amidation reaction. The reaction conditions are mild, allowing for quantitative and controllable modification, minimizing the damage to the antibody structure and function during the reaction process, and maintaining the antibody's high targeting ability.

[0034] Application of near-infrared II nanoparticles in the preparation of drugs for treating lung infections.

[0035] Preferably, during the application process, fiber-optic-assisted near-infrared II nanoparticle therapy is used.

[0036] More preferably, the treatment method for the lung infection is puncture interventional therapy, specifically, the use of intravenous injection of near-infrared second-zone nanoparticles, which are distributed to the whole body and lung infection foci through blood circulation. Under the real-time guidance of high-resolution CT, a fine needle is used to directly puncture the lung infection foci or abscess cavity through the chest wall and pleural cavity, and the photosensitizer solution is directly injected to achieve the highest concentration of drug enrichment in the lesion. After inserting an optical fiber into the lesion using a puncture needle, a laser is connected and the lesion is irradiated according to preset parameters, which assists in the treatment of lung infection by near-infrared nanoparticles in the second zone.

[0037] Beneficial effects: This invention uses a puncture needle and optical fiber to precisely deliver excitation to the lesion site, effectively overcoming the problem of insufficient tissue penetration of light and achieving effective treatment of lung infection lesions.

[0038] Application of near-infrared II nanoparticles in the preparation of drugs for treating tumors.

[0039] Preferably, during the application process, fiber-optic-assisted near-infrared II nanoparticle therapy is used.

[0040] Preferably, the treatment method for the tumor is puncture interventional therapy, which involves intravenous injection of near-infrared second-zone nanoparticles, which are distributed throughout the body and to the pulmonary infection foci via blood circulation, thereby achieving the highest concentration of drug enrichment within the lesion. After inserting an optical fiber into the lesion using a puncture needle, a laser is connected and the lesion is irradiated according to preset parameters, which assists in the treatment of lung infection by near-infrared nanoparticles in the second zone.

[0041] Beneficial effects: This invention uses a puncture needle and optical fiber to precisely deliver excitation to the lesion site, effectively overcoming the problem of insufficient tissue penetration of light and achieving effective treatment of tumor tissue.

[0042] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides near-infrared II nanoparticles, their preparation method, and applications. These nanoparticles can be combined with minimally invasive puncture interventions to achieve precise treatment of various lung infections and liver cancer. This invention utilizes microfluidic technology to achieve the batch preparation and performance optimization of near-infrared II nanoparticles. By adjusting the ratio and flow rate, the particle size and distribution of the nanoparticles can be effectively controlled. Furthermore, this invention safely, stably, and precisely delivers near-infrared lasers (808 nm, 980 nm, 1064 nm) with stronger tissue penetration capabilities to the lesion area, achieving precise temporal and spatial synergy between "drug" and "light," ultimately achieving complete removal of deep-seated infected lesions / tumors while maximizing the protection of surrounding normal lung tissue. Attached Figure Description

[0043] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The diagram shows the structures of the near-infrared II nanoparticles (a) obtained in Examples 1-7, the near-infrared II nanoparticles modified with small molecule ligands (b) obtained in Examples 8-10, the near-infrared II nanoparticles modified with peptides (c) obtained in Examples 11-15, and the near-infrared II nanoparticles modified with monoclonal antibodies (d) obtained in Examples 16-20 of the present invention. Figure 2 The particle size distribution (a) and stability characterization (b) of the near-infrared II nanoparticles obtained in Example 1 of this invention are shown. Figure 3 This is a photograph of the near-infrared II nanoparticle formulation obtained in Example 1 of the present invention. Figure 4 The image shows the bactericidal effect of near-infrared II nanoparticles obtained in Example 1 of the present invention at different concentrations. Figure 5 The image shows the bactericidal effect of near-infrared II nanoparticles obtained in Example 1 under different light exposure times according to the present invention. Figure 6 The results of the safety evaluation of near-infrared II nanoparticles obtained in Example 1 of this invention in a pig model; Figure 7 This is a flowchart illustrating the fiber optic interventional treatment of acute bacterial pulmonary infection in pigs according to the present invention; Figure 8 This refers to the bacterial load in the local tissue of the fiber optic interventional treatment model for lung infection of this invention; Figure 9These are H&E staining images of local lung tissues from different treatment groups in the fiber optic interventional treatment model for lung infection of this invention. Figure 10 The images show the staining of TNF-α, IL-10, and IL-1b in local lung tissues from different treatment groups in the fiber optic interventional treatment model for lung infection of this invention. Figure 11 This invention describes the treatment process of fiber optic interventional therapy for real liver cancer. Figure 12 The therapeutic effect of fiber optic interventional therapy for real liver cancer is shown in this invention. Among them, a is a diagram showing the actual liver cancer treatment process, b is an image of immunohistochemistry (HE, Caspase 3 and CD31) and immunofluorescence staining (Tunel and Ki67), and c is a statistical representation of immunohistochemistry and immunofluorescence. Figure 13 HE (a) and TUNEL (b) staining for fiber optic interventional treatment of real liver cancer according to the present invention; Figure 14 HE (a), TUNEL staining (b), and apoptosis rate statistics (c) of real liver cancer “tumor-adjacent tissue-normal tissue” for fiber optic interventional treatment according to the present invention. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels; The near-infrared II fluorescent molecule was prepared according to the patent application document with reference application number CN202511606453.3.

[0047] The multidrug-resistant Klebsiella pneumoniae strain was purchased from the China Industrial Microbial Culture Collection Center, ATCC number BAA-2146; DSPE-mPEG is DSPE-PEG2000; The near-infrared II fluorescent molecular structure used is: Unless otherwise specified, room temperature or normal temperature in the embodiments of the present invention refers to 25±3℃.

[0048] Example 1 A method for mass production of near-infrared II nanoparticles includes the following steps: (1) Accurately weigh 10 mg of DSPE-mPEG and mix it with 1.0 mg of near-infrared II fluorescent molecules. Dilute to 2.00 mL with THF to prepare a series of organic phase solutions with a near-infrared II fluorescent molecule concentration of 0.5 mg / mL. Place them in syringe A; use ultrapure water as the aqueous phase and place it in syringe B.

[0049] (2) The syringe type of the rapid nanomedicine preparation system is CN, the chip type is OBM, and the total volume is 8.00 mL; the total flow rate is set to 4 mL / min, and the flow rate ratio A:B is fixed at 1:3; the initial waste liquid volume is set to 1 mL, and the subsequent waste liquid volume is set to 0.5 mL; syringe A is 5 mL, and syringe B is 10 mL; the temperature is set to room temperature (25 ℃), and preparation is started after the system stabilizes. After the system stabilizes and the sample is dispensed, the subsequent nanoparticle dispersion is collected to obtain near-infrared II nanoparticles. The structural schematic diagram of the near-infrared II nanoparticles is shown below. Figure 1 As shown in part (a).

[0050] Example 2 A method for the batch preparation of near-infrared II nanoparticles differs from Example 1 only in that the concentration of near-infrared II fluorescent molecules in the organic phase solution in step (1) is 0.5 mg / mL, and the total flow rate in step (2) is 6 mL / min. The remaining process steps and parameters are the same as in Example 1.

[0051] Example 3 A method for the batch preparation of near-infrared II nanoparticles differs from Example 1 only in that the concentration of near-infrared II fluorescent molecules in the organic phase solution in step (1) is 0.5 mg / mL, and the total flow rate in step (2) is 8 mL / min. The remaining process steps and parameters are the same as in Example 1.

[0052] Example 4 A method for the batch preparation of near-infrared II nanoparticles differs from Example 1 only in that the concentration of near-infrared II fluorescent molecules in the organic phase solution in step (1) is 0.5 mg / mL, and the total flow rate in step (2) is 10 mL / min. The remaining process steps and parameters are the same as in Example 1.

[0053] Example 5 A method for the batch preparation of near-infrared II nanoparticles differs from Example 1 only in that the concentration of near-infrared II fluorescent molecules in the organic phase solution in step (1) is 0.5 mg / mL, and the total flow rate in step (2) is 12 mL / min. The remaining process steps and parameters are the same as in Example 1.

[0054] Example 6 A method for the batch preparation of near-infrared II nanoparticles differs from Example 1 only in that the concentration of near-infrared II fluorescent molecules in the organic phase solution in step (1) is 0.5 mg / mL, and the total flow rate in step (2) is 16 mL / min. The remaining process steps and parameters are the same as in Example 1.

[0055] Example 7 A method for the batch preparation of near-infrared II nanoparticles differs from Example 1 only in that the concentration of near-infrared II fluorescent molecules in the organic phase solution in step (1) is 0.5 mg / mL, and the total flow rate in step (2) is 20 mL / min. The remaining process steps and parameters are the same as in Example 1.

[0056] Example 8 A method for the mass production of near-infrared II nanoparticles is disclosed, differing from Example 1 only in that DSPE-mPEG is replaced with an equal mass of DSPE-PEG-PBA; all other process steps and parameters are the same as in Example 1. PBA-modified near-infrared II nanoparticles are obtained, and their structural schematic diagram is shown below. Figure 1 As shown in section (b).

[0057] Example 9 A method for mass production of near-infrared II nanoparticles is disclosed, differing from Example 1 only in that DSPE-mPEG is replaced with an equal mass of DSPE-PEG-FA; all other process steps and parameters are identical to those in Example 1. FA-modified near-infrared II nanoparticles are obtained, and their structural schematic diagram is shown below. Figure 1 As shown in section (b).

[0058] Example 10 A method for mass production of near-infrared II nanoparticles is disclosed, differing from Example 1 only in that DSPE-mPEG is replaced with an equal mass of DSPE-PEG-Man; all other process steps and parameters are identical to those in Example 1. Man-modified near-infrared II nanoparticles are obtained, and their structural schematic diagram is shown below. Figure 1 As shown in section (b).

[0059] Example 11 A method for mass production of peptide-modified near-infrared II nanoparticles includes the following steps: (1) Accurately weigh 10 mg of distearate phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-Mal), mix it with 1 mg of near-infrared II fluorescent molecules, and dilute to 2.00 mL with THF to prepare an organic phase solution with a near-infrared II fluorescent molecule concentration of 0.5 mg / mL. Place it in syringe A; use ultrapure water as the aqueous phase and place it in syringe B.

[0060] (2) The syringe type of the rapid nanomedicine preparation system is CN, the chip type is OBM, and the total volume is 8.00 mL; the total flow rate is set to 10 mL / min, and the flow rate ratio A:B is fixed at 1:3; the initial waste liquid volume is set to 1 mL, and the subsequent waste liquid volume is set to 0.5 mL; the A syringe specification is 5 mL, and the B syringe specification is 10 mL; the temperature is set to room temperature (25 ℃), and preparation is started after the system stabilizes. After the system stabilizes and dispenses the sample, the subsequent near-infrared II nanoparticle dispersion is collected.

[0061] (3) The near-infrared II nanoparticle dispersion obtained in step (2) was mixed with Cys-RLYLRIGRR at a molar ratio of maleimide to cysteine ​​(Cys) in DSPE-PEG-Mal of (1-5):(5-1). The mixture was stirred at room temperature for 12 hours, followed by dialyzing for 12 hours using a dialysis bag with a molecular weight cutoff of 5000 Da. After dialysis, peptide-modified near-infrared II nanoparticles were obtained. The structural schematic diagram is shown below. Figure 1 As shown in section (c).

[0062] Example 12 A method for mass production of peptide-modified near-infrared II nanoparticles differs from Example 11 only in that Cys-RLYLRIGRR in step (3) is replaced with Cys-GLRRLLRKIRGRWK, and the molar ratio of maleimide to cysteine ​​(Cys) in DSPE-PEG-Mal is (1-5):(5-1). The remaining process steps and parameters are the same as in Example 11.

[0063] Example 13 A method for mass production of peptide-modified near-infrared II nanoparticles differs from Example 11 only in that Cys-RLYLRIGRR in step (3) is replaced with Cys-ELTPEERMLRLIFGRK, and the molar ratio of maleimide to cysteine ​​(Cys) in DSPE-PEG-Mal is (1-5):(5-1). The remaining process steps and parameters are the same as in Example 11.

[0064] Example 14 A method for mass production of peptide-modified near-infrared II nanoparticles differs from Example 11 only in that Cys-RLYLRIGRR in step (3) is replaced with Cys-ADGVGDAESRTR, and the molar ratio of maleimide to cysteine ​​(Cys) in DSPE-PEG-Mal is (1-5):(5-1). The remaining process steps and parameters are the same as in Example 11.

[0065] Example 15 A method for mass production of peptide-modified near-infrared II nanoparticles differs from Example 11 only in that Cys-RLYLRIGRR in step (3) is replaced with RGD, and the molar ratio of maleimide to cysteine ​​(Cys) in DSPE-PEG-Mal is (1-5):(5-1). The remaining process steps and parameters are the same as in Example 11.

[0066] Example 16 A method for the mass production of near-infrared II nanoparticles modified with monoclonal antibodies includes the following steps: (1) Accurately weigh 10 mg of distearate phosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH), mix it with 1 mg of near-infrared II fluorescent molecules, and dilute to 2.00 mL with THF to prepare an organic phase solution with a near-infrared II fluorescent molecule concentration of 10 mg / mL. Place it in syringe A; use ultrapure water as the aqueous phase and place it in syringe B.

[0067] (2) The syringe type of the rapid nanomedicine preparation system is CN, the chip type is OBM, and the total volume is 8.00 mL; the total flow rate is set to 10 mL / min, and the flow rate ratio A:B is fixed at 1:3; the initial waste liquid volume is set to 1 mL, and the subsequent waste liquid volume is set to 0.5 mL; the A syringe specification is 5 mL, and the B syringe specification is 10 mL; the temperature is set to room temperature (25 ℃), and preparation is started after the system stabilizes. After the system stabilizes and dispenses the sample, the subsequent near-infrared II nanoparticle dispersion is collected.

[0068] (3) Under ice-water bath and pH 4.5-7.2 conditions, 0.021 g of carbodiimide and 0.045 g of N-hydroxythiosuccinimide were added to 0.1 g of near-infrared II nanoparticle dispersion. The carboxyl groups on the surface of the carboxyl nanoparticles were activated by carbodiimide and N-hydroxythiosuccinimide for 0.5 h. According to the molar ratio of DSPE-PEG-COOH to monoclonal antibody (1-5):1, 0.1 g of activated nanoparticles were mixed with 0.01 g of monoclonal antibody (Panobacumab) solution with a concentration of 5 µg / mL at 0 ℃. The mixture was slowly heated to room temperature and the reaction was continued for 12 h. Covalent coupling was completed through carboxyl-amino amidation reaction to obtain near-infrared II nanoparticles modified with monoclonal antibody. The structural schematic diagram is shown below. Figure 1 As shown in section (d).

[0069] Example 17 The only difference from Example 16 is that the monoclonal antibody (Panobacumab) solution in step (3) is replaced with a monoclonal antibody (Gremubamab) solution, and the molar ratio of DSPE-PEG-COOH to monoclonal antibody is (1-5):1. The remaining process steps and parameters are the same as in Example 16.

[0070] Example 18 The only difference from Example 16 is that the monoclonal antibody (Panobacumab) solution in step (3) is replaced with the monoclonal antibody (Tosatoxumab) solution, and the molar ratio of DSPE-PEG-COOH to monoclonal antibody is (1-5):1. The remaining process steps and parameters are the same as in Example 16.

[0071] Example 19 The only difference from Example 16 is that the monoclonal antibody (Panobacumab) solution in step (3) is replaced with a monoclonal antibody (Pse-MAB1) solution, and the molar ratio of DSPE-PEG-COOH to monoclonal antibody is (1-5):1. The remaining process steps and parameters are the same as in Example 16.

[0072] Example 20 The only difference from Example 16 is that the monoclonal antibody (Panobacumab) solution in step (3) is replaced with a monoclonal antibody (anti-KL64 capsular antibody) solution, and the molar ratio of DSPE-PEG-COOH to monoclonal antibody is (1-5):1. The remaining process steps and parameters are the same as in Example 16.

[0073] Comparative Example 1 The only difference from Example 1 is that in step (1), the THF solvent is replaced with methanol, and the rest of the process steps and parameters are the same as in Example 1.

[0074] Comparative Example 2 The only difference from Example 1 is that the concentration of near-infrared II fluorescent molecules in the organic phase solution in step (1) is 1 mg / mL, while the other process steps and parameters are the same as in Example 1.

[0075] Comparative Example 3 The only difference from Example 1 is that in step (2), the total flow rate is set to 10 mL / min and the flow rate ratio A:B is fixed at 1:5; the other process steps and parameters are the same as in Example 1.

[0076] Technical effects: 1. The hydrated particle size, polydispersity index (PDI), and zeta potential were determined using dynamic light scattering (DLS). Measurements were repeated three times for each condition, and the average value was taken as the characterization result under that condition. The results are as follows: Figure 2 As shown in Table 1: Table 1 Depend on Figure 2 As can be seen from Table 1, the prepared nanoparticles have a particle size between 120 and 150 nm, a uniform particle size distribution, and a weak negative charge on their surface.

[0077] 2. Verification of the in vitro antibacterial properties of near-infrared II nanoparticles (1) Take multidrug-resistant Klebsiella pneumoniae culture stored at -80 ℃, streak it on LB agar plates, and activate it overnight at 37 ℃. Pick a single colony and inoculate it into 10 mL of LB liquid medium, and culture at 37 ℃ and 200 rpm for 10 h until the logarithmic growth phase. Centrifuge the bacterial culture at 4500 rpm for 2 min, discard the supernatant, resuspend and wash with sterile PBS, and repeat 3 times. Adjust OD with PBS. 600 Up to 1.0 (approximately 1×10) 8 (CFU / mL), then diluted with MH broth to 1×10⁻⁶. 6 CFU / mL was used to obtain a bacterial suspension for later use.

[0078] (2) The near-infrared II nanoparticles obtained in Examples 1-20 and Comparative Examples 1-4 were serially diluted with MH broth to 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 µM to obtain near-infrared II nanoparticle broth solutions. 100 µL of the near-infrared II nanoparticle broth solution was added to each well of a 96-well plate, followed by 100 µL of the bacterial suspension obtained in step (1) (final concentration 5 × 10⁻⁶). 5CFU / mL, total volume 200 µL). A positive control without nanoparticles and a blank control with only MH broth were included, with 3 replicates per group.

[0079] (3) The initial absorbance at 600 nm was read using an ELISA reader. After incubating the 96-well plate at 37 °C and 170 rpm for 1 h, the light group was then exposed to an 808 nm near-infrared laser (0.5 W / cm²). 2 Vertical irradiation for 5 min, with the non-light-illuminated group serving as a control. After 16 h of continued shaking incubation, OD values ​​were recorded. 600 The minimum concentration of nanoparticles required to inhibit 90% of bacterial growth is defined as the MIC. 90 .

[0080] like Figure 3 The results showed that under 808 nm laser irradiation, the near-infrared II nanoparticles obtained in Example 8 exhibited a high MIC against Klebsiella pneumoniae. 90 It is 25 µM.

[0081] 3. Concentration-dependent bactericidal experiment (1) Same as the above-mentioned near-infrared II nanoparticle in vitro antibacterial performance verification step (1) to prepare multidrug-resistant Klebsiella pneumoniae suspension (1×10 6 (CFU / mL) To each well of a 96-well plate, add 100 µL of near-infrared II nanoparticle broth solution with concentrations of 0, 12.5, 25, 50, 100, and 200 µM, respectively, followed by 100 µL of bacterial suspension. Incubate the plate at 37°C on a shaker for 1 hour to obtain the incubated bacteria-nanoparticle mixture.

[0082] (2) Using an 808 nm laser (0.5 W / cm²) 2 All wells containing the incubated bacterial-nanoparticle mixture (except for the 0 µM control well) were uniformly irradiated for 5 minutes. Immediately after irradiation, 100 µL of the mixture was aspirated from each well and diluted 10-fold with sterile PBS. 100 µL of the diluted mixture was then evenly spread onto LB agar plates. The plates were incubated upside down at 37°C for 24 hours. Colony forming units (CFU) were counted on each plate, and the viable bacterial concentration in the original mixture was calculated.

[0083] like Figure 4 The results showed that when the concentration of near-infrared II nanoparticles was 25µM, it already had excellent bactericidal effect, reducing the bacterial content in liquid by nearly 10,000 times.

[0084] 4. Time-dependent sterilization experiment The concentration of the near-infrared II nanoparticle broth solution was fixed at 25 µM. The operation steps were the same as those in Part 3, the concentration-dependent sterilization experiment. The bacteria-nanoparticle mixture obtained in step (1) of Part 3 was subjected to an 808 nm laser (0.5 W / cm²). 2 Irradiate for 0, 5, 10, 15, or 20 minutes. Immediately after irradiation, take samples, dilute, plate, incubate, and count.

[0085] The results are as follows Figure 5 As shown, the sterilization effect increases with the extension of light exposure time. After 5 minutes of light exposure, the sterilization rate is 99.99%; after 10 minutes of light exposure, the sterilization rate can reach nearly 100%.

[0086] 5. Organ toxicity verification of near-infrared II nanoparticles Animal preparation and sampling: Healthy three-way crossbred pigs, weighing approximately 15 kg, were selected and injected with the near-infrared II nanoparticles obtained in Example 8 via the marginal ear vein at a dose of 5 mg / kg. After 24 hours, organs from different treatment groups were selected for H&E staining to assess morphological changes in the tissues.

[0087] The results are as follows Figure 6 As shown, intravenous injection of near-infrared II nanoparticles does not affect the morphological characteristics of the heart, liver, spleen, lungs, and kidneys in pigs, and there is no obvious material toxicity.

[0088] 6. Construction of an animal model of acute bacterial pneumonia in pigs like Figure 7 As shown, it includes the following steps: (1) Preparation and anesthesia of experimental animals: Healthy three-way crossbred pigs weighing about 15kg were selected. They were fasted for 12 hours before the operation and allowed free access to water. After intramuscular injection of dexmedetomidine and 50 for mixed anesthesia, the animals were turned into right lateral decubitus position, the hair on the left thoracic surgical area was shaved, and the area was disinfected with iodine and 75% alcohol in sequence. A sterile drape was then laid on top.

[0089] (2) Preparation of pathogen suspension: The multidrug-resistant Klebsiella pneumoniae strain stored at -80℃ was streaked onto Luria-Bertani (LB) solid agar plates and activated overnight at 37℃. Single colonies were picked and inoculated into 10 mL of LB liquid medium and cultured at 37℃ and 200 rpm for 10 hours until the logarithmic growth phase. The bacterial suspension was centrifuged at 4500 rpm for 2 minutes, the supernatant was discarded, and the bacterial pellet was gently resuspended and washed with sterile phosphate-buffered saline (PBS). This process was repeated three times. Finally, the bacterial cells were resuspended in PBS, and the absorbance (OD600) of the bacterial suspension at 600 nm was adjusted to 1.0 using a UV-Vis spectrophotometer. At this point, the bacterial concentration was approximately 1 × 10⁻⁶. 8CFU / mL. Collect bacterial cells by centrifugation, wash with an equal volume of sterile PBS and resuspend, finally preparing a concentration of 1×10⁻⁶ CFU / mL. 8 CFU / mL bacterial suspension.

[0090] (3) Precise intralobar injection of infection: Locate the 4th and 5th intercostal spaces on the left side of the pig by palpation. At the selected intercostal space, use a sterile 5 mL syringe to draw 1 mL of the bacterial suspension obtained in step (2). Hold the syringe perpendicular to the skin surface and puncture the skin, intercostal muscles, and pleura in sequence. After feeling a sense of loss, advance the needle tip forward steadily by about 1.8 cm to ensure that the needle tip enters the lung parenchyma, and slowly inject the bacterial suspension into the lung tissue. A slight bulge in the local lung tissue can be observed. After the injection is completed, withdraw the needle quickly and steadily. Immediately apply sterile gauze to the puncture site for 1-2 minutes to stop the bleeding.

[0091] (4) Two days after infection, the animals began to show typical acute infection symptoms such as high fever (>41℃), rapid and difficult breathing, lethargy, and refusal to eat. The sick pigs were randomly divided into four groups: control group, light group, material group, and material + light group.

[0092] Evaluation of the therapeutic efficacy of fiber optic minimally invasive interventional therapy for lung infections (1) Grouping and Treatment: Animals that successfully developed the model were randomly divided into 4 groups (n=3): (A) Control group (infection only, PBS treatment); (B) Light group (infection + PBS + fiber optic light); (C) Material group (infection + near-infrared II nanoparticles, no light); (D) Material + Light group (infection + near-infrared II nanoparticles + fiber optic light). In the treatment group, 48 hours after infection, the near-infrared II nanoparticle solution obtained in Example 8 was injected via the marginal ear vein at a concentration of 1 mg / mL and a dose of 5 mg / kg. Twelve hours after injection, in the light group and the material + light group, a sterile optical fiber was percutaneously inserted into different regions of the lung near the center of the lung infection lesion using a syringe needle. The optical fiber was connected to an 808 nm laser at 0.5 W / cm². 2 Irradiate the lesion area with a power density of 10 minutes.

[0093] (2) Evaluation of efficacy: All animals were euthanized 24 hours after treatment. Immediately, they were dissected, and approximately 0.5 g of lung tissue was accurately collected from the central region of the fiber optic irradiation site / infected lesion. After weighing, sterile PBS was added, and the tissue was thoroughly homogenized using a tissue homogenizer under ice bath conditions. The tissue homogenate was serially diluted 10-fold, and 100 µL of an appropriate dilution was spread onto LB agar plates. After incubation at 37°C overnight, colonies were counted. The bacterial load per gram of lung tissue was calculated.

[0094] Treatment results as follows Figure 8As shown, compared with the control group, the light group or the material group had a limited effect on the bacterial load in the local tissue of pig lungs, while the bacterial load in the lung tissue of the material + light group decreased by more than 2 log values, which was significantly lower than that of the other groups (p < 0.001), proving that the scheme has a strong in vivo bactericidal ability.

[0095] Lung tissue samples from each group were subjected to H&E staining, and the results are as follows: Figure 9 As shown, the control group exhibited typical acute necrotizing inflammation. In the material + light irradiation group, the infiltration of inflammatory cells within the lesions was significantly reduced, the area of ​​hemorrhage and necrosis was significantly reduced, the alveolar structure began to repair, and new epithelial cells were visible, showing a clear tendency for tissue repair. However, the improvement was not significant in the light irradiation group or the material group.

[0096] Lung tissues from each group were stained for TNF-α, IL-10, and IL-1b. The results are as follows: Figure 10 As shown, compared with the control group, the light irradiation group and the material group, the fluorescence signals of TNF-α, IL-10 and IL-1b in the material + light irradiation group were significantly reduced, indicating that fiber optic interventional therapy has a good anti-inflammatory effect.

[0097] 7. Evaluation of the therapeutic effect of fiber optic minimally invasive intervention in a real ex vivo perfusion model of liver cancer like Figure 11 As shown, an in vitro perfusion model was constructed to simulate intravenous drug administration, and clinical trials were conducted. The specific steps are as follows: The near-infrared II nanoparticle solution obtained in Example 8 was injected into the circulating perfused blood at a concentration of 1 mg / mL and an injection dose of 5 mg / kg. After 12 hours of circulation, the solution was enriched in the tumor. An optical fiber was positioned in the tumor area using a puncture needle. After confirming the needle tip position was correct, the puncture needle sheath was withdrawn, leaving the optical fiber in the lesion. A laser was connected, and irradiation was performed on the lesion according to preset parameters. After treatment, perfusion continued for 24 hours. Immunohistochemistry and immunofluorescence staining were used to evaluate the therapeutic and anti-inflammatory effects and safety.

[0098] The results are as follows Figure 12-14 As shown, compared to the un-illuminated areas, the laser-irradiated tumor tissue exhibited significant necrosis. Section results revealed that after laser irradiation, cancer cells in the tumor tissue underwent significant apoptosis, cell proliferation factors were significantly reduced, and blood vessels within the tumor were extensively destroyed. Notably, no significant damage was observed to normal liver tissue before and after treatment. The fiber optic intervention strategy can precisely kill cancer cells in tumor tissue without damaging cells in normal tissue, demonstrating good controllability and safety.

[0099] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A near-infrared two-region nanoparticle, characterized in that, Includes a polymer shell and a near-infrared II fluorescent molecular core; The polymer shell comprises distearylphosphatidylethanolamine-polyethylene glycol.

2. The near-infrared two-region nanoparticle of claim 1, wherein, The structure of the near-infrared II fluorescent molecular core is shown in formula (I): （I）； Wherein, R1 is selected from one of H, CH3 and OCH3; said R2and R3are independently selected from one of H, , , , and . R4 is selected from S and Se.

3. The near-infrared II nanoparticle according to claim 1, characterized in that, The polymer shell includes modifying groups; The modifying group includes one of small molecule ligands, targeting peptides, and monoclonal antibodies.

4. A method for preparing near-infrared II nanoparticles as described in any one of claims 1-3, characterized in that, Includes the following steps: Near-infrared II fluorescent molecules and polymers dissolved in the solution were used as the organic phase, and pure water was used as the aqueous phase. The organic phase and the aqueous phase were mixed and reacted using a microfluidic system to prepare the near-infrared II nanoparticles.

5. The method for preparing near-infrared II nanoparticles according to claim 4, characterized in that, In the organic phase, the concentration of near-infrared II fluorescent molecules is 0.1-0.5 mg / mL, the concentration of polymer is 5 mg / mL, and the solvent is tetrahydrofuran; In the microfluidic system, the total flow rate of the organic phase and the aqueous phase is 4-20 mL / min, and the flow rate ratio is 1:

3.

6. The method for preparing near-infrared II nanoparticles according to claim 4, characterized in that, The polymer is one of DSPE-mPEG, DSPE-PEG-PBA, DSPE-PEG-FA, DSPE-PEG-Man, DSPE-PEG-Mal, and DSPE-PEG-COOH.

7. A method for preparing near-infrared II nanoparticles according to claim 4 or 6, characterized in that, When the polymer is DSPE-PEG-Mal, the following steps are included after the organic phase and aqueous phase are mixed and reacted: After the reaction was completed, the product was mixed with the target peptide and stirred before dialyzing to obtain near-infrared II nanoparticles modified with the target peptide.

8. A method for preparing near-infrared II nanoparticles according to claim 4 or 6, characterized in that, When the polymer is DSPE-PEG-COOH, the following steps are included after the organic phase and aqueous phase are mixed and reacted: After the reaction was completed, the product was activated with carbodiimide and N-hydroxythiosuccinimide to activate the surface carboxyl groups. Then, it was mixed with a monoclonal antibody solution for covalent coupling to obtain near-infrared II nanoparticles modified with monoclonal antibody.

9. The use of near-infrared II nanoparticles as described in any one of claims 1-3 in the preparation of a medicament for treating lung infections.

10. The use of near-infrared II nanoparticles as described in any one of claims 1-3 in the preparation of medicaments for treating tumors.

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