TLR agonist / organic photosensitizer protein nanocomposite and preparation method and application thereof

Abstract

The invention discloses a Toll-like receptor (TLR) agonist / organic photosensitizer protein nano-composite as well as a preparation method and application of the Toll-like receptor (TLR) agonist / organic photosensitizer protein nano-composite. In order to overcome the defects of short blood half-life period, low bioavailability and the like of the existing TLR agonist, the protein nano-composite is obtained by forming a compound in a protein cavity by using the TLR agonist and an organic photosensitizer, and has tumor and lymph node dual-targeting characteristics. The protein nano-composite is simple in preparation method, mild in condition, free of an organic solvent, uniform in particle size and has a pH-responsive drug release behavior, and the protein nano-composite is prepared by a one-step method with water as a solvent. Based on an active targeting mechanism mediated by an albumin binding receptor and an active uptake mechanism of antigen presenting cells to albumin, the nano-composite has tumor and lymph node dual-targeting characteristics, has immunotherapy and light therapy effects under irradiation of near-infrared light, and has a good application prospect from two aspects of short-term quick action and long-term body immunity improvement. Tumor growth is effectively restrained, and tumors are expected to be radically treated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to protein nanocomposites of TLR agonists and organic photosensitizers, their preparation methods, and applications. Background Technology

[0002] Effective antitumor immune responses typically involve a cascade of events: neoantigens generated by tumor cell disruption (which can be induced by chemotherapy, phototherapy, radiotherapy, etc.) are phagocytosed by dendritic cells (DCs); DCs process tumor-derived antigens and mature in lymph nodes; DCs present the captured antigens to T cells via major histocompatibility complex molecules; and activated effector T cells migrate and infiltrate the tumor core to kill tumor cells. Therefore, tumors and lymph nodes are two major battlegrounds in the antitumor immune response. Studies have shown that Toll-like receptor (TLR) agonists (biologics that trigger innate immune responses by activating Toll-like receptors, demonstrating significant application value in tumor immunotherapy, vaccine adjuvant development, and antiviral therapy), and phototherapy (a treatment method where photosensitizers reach the site of action and selectively destroy diseased tissue under specific wavelength light sources, avoiding excessive damage to healthy tissue) both have the ability to reshape the immune microenvironment at the tumor site and activate immune cells in the lymph nodes. Therefore, targeting TLR agonists to lymphocytes is necessary to better leverage the effects of immunotherapy. It enhances the immune system's recognition and killing of tumor cells by altering lymphocyte activity, thus restoring the body's immunity (curing tumors). Simultaneously, by targeting the photosensitizer to tumor cells, once excited by near-infrared light, it exerts photothermal effects and generates reactive oxygen species (ROS), resulting in photothermal therapy (PTT) and photodynamic therapy (PDT) (rapidly treating tumors). Therefore, the combination of TLR agonists and photosensitizers in immunotherapy / phototherapy can significantly improve tumor efficacy by addressing both rapid treatment effectiveness and radical tumor cure.

[0003] TLR agonists are typically nonionic organic complexes, including mod TLR agonists (such as the clinically used drugs retinomod R848 and imiquimod R837) and mot class TLR agonists.

[0004] Phototherapy combined with photosensitizers is a widely used clinical treatment method. Common organic photosensitizers are mostly macrocyclic heterocyclic or conjugated dyes, typically possessing a planar multi-conjugated system in their structure. The main categories include: 1) Porphyrins: such as protoporphyrin IX (PpIX) and hematoporphyrin derivatives (Photofrin) commonly used in photodynamic therapy (PDT); 2) Chlorin derivatives / chloropyridines: such as chlorochlorophyll e6 (Ce6); 3) Phthalocyanines: such as aluminum tetrasulfonate phthalocyanine (AlPcS4), zinc chloride phthalocyanine (ZnPc), etc.; 4) Rhodamine and similar conjugated fluorescent dyes: such as Rhodamine B, 6G, etc.; 5) Carbazole dyes; 6) Indocyanine / cyanine dyes, etc. Indocyanines include indocyanine green (ICG) and neo-indocyanine green (IR820). ICG, a near-infrared dye photosensitizer, is composed of conjugated polycycline chains. Its absorption spectrum shifts significantly towards the near-infrared, with a peak absorption at ~800 nm. It can be excited by light at wavelengths of 750–810 nm and emits fluorescence at approximately 840 nm. Its penetration depth in tissues can reach 0.5–1.0 nm. It possesses photothermal effects and the ability to generate reactive oxygen species (ROS), making it suitable for photothermal therapy (PTT) and photodynamic therapy (PDT), and also for tissue imaging. ICG's advantages include good water solubility and near-infrared light excitation; it is an FDA-approved clinical drug. However, it suffers from poor photostability (prone to photobleaching) and a short half-life in blood.

[0005] In recent years, there have been numerous reports on the development of nanomedicines combining targeted immunotherapy with phototherapy. Many nanocarriers, including inorganic and organic materials, have been used for the co-delivery of TLR agonists / photosensitizers. Chemical coupling or physical encapsulation are common strategies for drug co-delivery. One researcher introduced carboxyl groups into sodium alginate to promote esterification between alginate and the hydroxyl groups of the TLR agonist R848, followed by self-assembly to form nanoparticles. In the tumor microenvironment, the ester bonds of these nanoparticles degrade, releasing R848, further enhancing antigen presentation by dendritic cells (DCs) and the killing ability of T cells. Another researcher utilized the active amino groups of R848 to covalently link them to human serum albumin (HSA) via a glutathione-activated linker, thus establishing a series of R848-HSA conjugates. RS-HSA with an R848:HAS molar ratio of 1.6:1 was selected and assembled with active peptides from tumor cell membranes to form immune nano-agonists. These studies have shown good effects in regulating the tumor immunosuppressive microenvironment, disrupting the extracellular matrix, and enhancing anti-tumor immunity, effectively reducing the systemic toxicity of TLR agonists. However, these studies only involve single-component TLR agonist encapsulation. Other researchers have used chemical modification methods to modify R848 with hydrophilic polyethylene glycol and responsive thioacetate linkers to obtain amphiphilic prodrugs. These prodrugs, along with photosensitizers, were then encapsulated in nanoparticles via nanoprecipitation, achieving precise photoimmunotherapy guided by NIR-II fluorescence imaging. However, the complex preparation methods have limited further clinical translation. Some researchers have covalently coupled photosensitizers to human serum albumin and simultaneously loaded R848 non-covalently to construct tumor-targeting albumin nanocomposites. Others have prepared PLGA nanoparticles, simultaneously encapsulating the hydrophobic near-infrared photosensitizing dye ICG and the TLR7 / 8 agonist R848 using a double emulsification technique, thus combining photothermal therapy with immunotherapy. Still others have employed a method of covalently coupling TLR agonists, photosensitizers, and albumin, but this approach has not avoided problems such as complex preparation processes and difficulties in quality control. Furthermore, when preparing albumin nanoparticles by physically encapsulating TLR agonists and photosensitizers, the two are typically co-encapsulated within a nanocarrier through hydrophobic interactions to form nanomedicines. This cannot avoid the problem of poor biocompatibility caused by organic solvents, and cross-linking is required to enhance nanoparticle stability. Moreover, low encapsulation efficiency often leads to poor targeting, unsatisfactory efficacy, and significant side effects.

[0006] In summary, existing drug delivery systems for chemically coupled or physically encapsulated TLR agonists / photosensitizers have complex formulations and preparation processes, making it difficult to achieve highly efficient tumor treatment that targets both tumors and lymph nodes.

[0007] Therefore, combining TLR agonist immunotherapy with photosensitizer phototherapy, and developing a simple drug delivery system that delivers TLR agonists and photosensitizers to lymph nodes (cells) and tumor cells, can achieve: 1) rapid inhibition and elimination of growing tumors; and 2) radical cure of tumors by restoring the function of the immune system. This strategy is of great significance for improving the current level of cancer treatment research. Summary of the Invention

[0008] To address the aforementioned challenges, this invention expands the conventional single-drug mineralization technology of albumin to dual-drug co-encapsulation by encapsulating TLR agonists and photosensitizers in albumin to form nanoparticles, thus laying the foundation for the development of a dual-drug albumin delivery system with promising clinical translation prospects.

[0009] This invention fully utilizes the tunable structure of albumin and the intermolecular interactions of drugs to obtain a dual-drug nanocomposite with a simple preparation process, high efficiency in drug encapsulation, and controllable size. It possesses characteristics such as good stability, controllable release, low immunogenicity, high targeting to lymph nodes and tumors, and good therapeutic effects, demonstrating potential for future clinical translation.

[0010] The present invention addresses the limitations of existing technologies by providing a TLR agonist and organic photosensitizer protein nanocomposite, its preparation method, and its applications. This invention utilizes clinically approved albumin and other proteins as single-molecule protein carriers, leveraging the interaction between a TLR agonist and an organic photosensitizer to prepare a protein nanocomposite. The constructed lymphocyte-targeting and tumor-targeting TLR agonist and organic photosensitizer protein nanocomposite is prepared using water as a solvent. The method is simple and mild, exhibiting pH-responsive release characteristics and effectively improving drug pharmacokinetics and tissue distribution behavior. This achieves lymphocyte (nodule) targeting and tumor targeting, exerting immunotherapy and phototherapy effects. It effectively inhibits tumor growth and holds promise for curing tumors by providing rapid short-term efficacy and long-term enhancement of the body's immune function.

[0011] To achieve this objective, the present invention adopts the following technical solution: A TLR agonist / organic photosensitizer protein nanocomplex includes a TLR agonist and an organic photosensitizer, wherein the TLR agonist and the organic photosensitizer form a complex within a protein cavity.

[0012] In this invention, the TLR agonist and the organic photosensitizer form a complex in the protein cavity, which further induces the TLR agonist and the organic photosensitizer to nucleate and grow in the protein cavity, thereby constructing a lymphocyte and tumor-targeting TLR agonist and organic photosensitizer protein nanocomplex.

[0013] The TLR agonist and organic photosensitizer protein nanocomposite disclosed in this invention comprises a TLR agonist, an organic photosensitizer, and a protein. Preferably, the TLR agonist / organic photosensitizer protein nanocomposite is composed of a TLR agonist, an organic photosensitizer, and a protein, and its drug loading is 1% to 20%. The drug loading refers to the mass of TLR agonist in the purified sample divided by (mass of TLR agonist + mass of protein).

[0014] This invention discloses a method for preparing the aforementioned TLR agonist / organic photosensitizer protein nanocomplex. The method involves mixing a protein aqueous solution with a pH of 6-8 and a TLR agonist solution, stirring, and then adding an organic photosensitizer solution to react and obtain the TLR agonist / organic photosensitizer protein nanocomplex. Further, under mild conditions in an aqueous solution, the method involves mixing a protein aqueous solution with a pH of 6-8 and a TLR agonist solution, stirring, and then adding an organic photosensitizer solution to react and obtain the TLR agonist / organic photosensitizer protein nanocomplex solution. Specifically, the method involves mixing a TLR agonist solution and a protein aqueous solution (pH 6-8), adjusting the pH of the mixture to 6-8, adding an organic photosensitizer solution, reacting under stirring for a certain time, and then purifying by ultrafiltration to obtain the TLR agonist / organic photosensitizer protein nanocomplex solution.

[0015] In this invention, the TLR agonist includes mod-type TLR agonists and mot-type TLR agonists, such as one or more of Resiquimod (R848), Imiquimod (R837), Motolimod, Vesatolimod, and Telratolimod; the organic photosensitizer includes indocyanine green photosensitizers, such as one or more of Indocyanine Green (ICG) and New Indocyanine Green (IR820); and the protein includes proteins with lymph node targeting activity, such as one or more of albumin, ferritin, and low-density lipoprotein.

[0016] In this invention, the molar ratio of TLR agonist to protein is (10~160):1; the molar ratio of TLR agonist to organic photosensitizer is (1~16):1; and the concentration of the protein solution is 5~25 mg / mL.

[0017] Preferably, the reaction temperature is 25-55℃, the reaction is carried out in an aqueous solution with a pH of 6-8, and the reaction time is 0.5-4 hours. After the reaction, the product is purified by centrifugation in an ultrafiltration tube or by separation by gel chromatography.

[0018] Preferably, the reactants are purified to obtain the TLR agonist / organic photosensitizer protein nanocomplex; the post-reaction purification is carried out by centrifugation in an ultrafiltration tube or separation by gel chromatography column.

[0019] In the TLR agonist / organic photosensitizer protein nanocomposite disclosed in this invention, the drug loading of the TLR agonist is 1% to 20%.

[0020] The TLR agonist / organic photosensitizer protein nanocomposite disclosed in this invention has a transmission electron microscope size of 5-50 nm and a hydrated particle size of 20-150 nm.

[0021] The TLR agonist / organic photosensitizer protein nanocomposite disclosed in this invention is a drug that has the combined effect of immunotherapy and phototherapy to enhance immune activation and effectively inhibit tumor growth.

[0022] Unlike traditional drug-encapsulated nanocarriers, this invention unexpectedly introduces a TLR agonist and an organic photosensitizer to create a protein nanocomplex, significantly simplifying the preparation method. In contrast, existing methods require complex procedures involving the chemical modification of small molecule fragments to further introduce other drugs when coupling the protein with R848. Furthermore, the simultaneous introduction of the photosensitizer into the nanocomplex allows for the combined effect of immunotherapy and phototherapy, ensuring therapeutic efficacy.

[0023] The TLR agonist and organic photosensitizer protein nanocomposite provided by this invention has the characteristics of long in vivo circulation and tumor- and lymphocyte-targeting, which promotes the entry of TLR agonist and organic photosensitizer into tumor cells and lymphocytes. Combined with far-infrared light irradiation, it exerts the combined effect of immunotherapy and phototherapy, reverses the tumor immune microenvironment and effectively treats tumors.

[0024] The TLR agonist and organic photosensitizer protein nanocomposite of the present invention can be formulated into an injection, and further formulated into various semi-solid preparations, solid preparations, etc.

[0025] In this invention, the reaction temperature is 25~55 °C, the reaction time is 0.5~24 hours, preferably 0.5~4 hours, and the purification after the reaction is performed by centrifugation in an ultrafiltration tube. Centrifugation is performed at a speed of 1000~10000 rpm for 8~15 minutes. Preferably, the centrifugation speed is 1500~3000 rpm.

[0026] In this invention, the molar ratio of TLR agonist to protein is (10~160):1; the molar ratio of TLR agonist to organic photosensitizer is (1~16):1; and the concentration of the protein aqueous solution is 5~25 mg / mL. Preferably, the molar ratio of TLR agonist to protein is (20~80):1; the molar ratio of TLR agonist to organic photosensitizer is (2~8):1; and the concentration of the protein aqueous solution is 10~20 mg / mL.

[0027] This invention discloses a Toll-like receptor (TLR) agonist / organic photosensitizer protein nanocomposite, its preparation method, and its applications. To address the shortcomings of current TLR agonists, such as short blood half-life and low bioavailability, this invention designs an albumin nanocomposite with dual tumor and lymph node targeting properties. The protein nanocomposite is obtained by forming a complex between a TLR agonist and an organic photosensitizer within a protein cavity. The preparation method for this protein nanocomposite is simple and mild, requiring no organic solvents; it is prepared in a one-step process using water as the solvent. The nanocomposite exhibits uniform particle size and pH-responsive drug release behavior. Based on the albumin-binding receptor-mediated active targeting mechanism and the active uptake mechanism of albumin by antigen-presenting cells, this nanocomposite possesses dual tumor and lymph node targeting properties. Under near-infrared light irradiation, it can exert immunotherapeutic and phototherapy effects, effectively inhibiting tumor growth and potentially curing tumors by providing rapid short-term efficacy and long-term enhancement of the body's immune function.

[0028] This invention discloses the use of the protein nanocomposite of the above-mentioned TLR agonist and organic photosensitizer in the preparation of tumor therapeutic drugs and their application in tumor treatment.

[0029] In this invention, deionized water is preferred as the solvent.

[0030] In this invention, the TLR agonist and the organic photosensitizer bind by forming a complex within the protein cavity.

[0031] In this invention, the pH value of the protein aqueous solution is adjusted using NaOH aqueous solution, preferably with a NaOH aqueous solution concentration of 0.2 mM.

[0032] The preparation method of the TLR agonist and organic photosensitizer protein nanocomposite of the present invention can be illustrated as follows: An aqueous albumin solution is prepared, and the pH is adjusted to 6-8 with 0.2 mM NaOH solution. Then, a TLR agonist solution is added, and the mixture is stirred for 5 min. Subsequently, a photosensitizer solution is added. The mixture is heated in a water bath at a set temperature, and after a certain reaction time, the reaction solution is transferred to an ultrafiltration tube and centrifuged to remove free Toll-like receptor agonists and photosensitizers, thus obtaining the TLR agonist and organic photosensitizer protein nanocomposite. The transmission electron microscopy (TEM) size of this protein nanocomposite is 5-50 nm, and the hydrated particle size is 50-150 nm.

[0033] This invention provides the application of the above-mentioned TLR agonist and organic photosensitizer protein nanocomposite in the preparation of tumor therapeutic drugs.

[0034] This invention, for the first time, employs a preparation method involving the formation of a complex of two organic molecules, successfully preparing a protein nanocomplex of a TLR agonist and an organic photosensitizer. The resulting protein nanocomplex is uniformly dispersed and has a consistent particle size, exhibiting excellent tumor / lymph node targeting activity. This invention uses a TLR agonist stably incorporated into the aqueous cavity of albumin as a precursor complex, followed by the introduction of an organic photosensitizer to induce a ligand exchange reaction. This induces the formation of a TLR agonist-organic photosensitizer complex within the protein cavity, thereby solving the problem of poor solubility and difficulty in effectively encapsulating TLR agonists. Furthermore, the introduction of a photosensitizer provides a synergistic effect. By controlling the particle size, drug release behavior, and other physicochemical properties through publicly available synthetic conditions such as feed ratio and reaction time, a protein nanocomplex loaded with a TLR agonist and an organic photosensitizer, exhibiting controllable drug release and good targeting, was obtained. Compared with other TLR agonist-loaded nanoparticles, the TLR agonist and organic photosensitizer protein nanocomposite provided by this invention has the advantages of simple one-step preparation process under isothermal conditions with water as solvent, controllable size uniformity, good biocompatibility, good water solubility, pH-responsive drug release behavior, long circulation time in blood, and high targeting of tumor lymph nodes, laying the foundation for efficient tumor treatment. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the technical solution of the present invention.

[0036] Figure 2 The solution appearance of physical complexes of TLR agonists and organic photosensitizers with different feed ratios is shown in the diagram.

[0037] Figure 3 Particle size distributions of protein nanocomposites formed with two TLR agonists (R848, R837) and two organic photosensitizers (ICG, IR820) are shown. Figure 3 A is the particle size distribution of R848 / ICG@HSA in Example 1; Figure 3 B is the particle size distribution of R848 / IR820@HSA in Example 2; Figure 3 C is the particle size distribution of R837 / ICG@HSA in Example 3.

[0038] Figure 4 These are transmission electron microscope (TEM) images. Among them, Figure 4 Transmission electron microscopy image of the TLR agonist and organic photosensitizer protein nanocomposite in Example 1; Figure 4 B is a transmission electron microscope image of R848 / ICG@HSA in Example 5; Figure 4C is a transmission electron microscope image of R848 / ICG@HSA in Example 6.

[0039] Figure 5 This is a bar chart showing drug loading. Among them, Figure 5 A represents the particle size and transmission electron microscopy dimensions of R848 / ICG@HSA prepared in Examples 1 and 4, as well as the drug loading of the TLR agonist. Figure 5 B represents the particle size and transmission electron microscopy dimensions of R848 / ICG@HSA prepared in Examples 1, 5, and 6, as well as the drug loading of the TLR agonist.

[0040] Figure 6 The release curve of the TLR agonist contained in the protein nanocomposite prepared in Example 1.

[0041] Figure 7 The particle size stability of the protein nanocomposite prepared in Example 1.

[0042] Figure 8 The results show the investigation of the chemical stability of the protein nanocomposite prepared in Example 1. Among them, Figure 8 A determination of TLR agonists, Figure 8 B. Determination of organic photosensitizers.

[0043] Figure 9 The cell viability of CT26 cells is shown in the figure for the protein nanocomposite prepared in Example 1.

[0044] Figure 10 The figure shows the results of the uptake of the protein nanocomposite prepared in Example 1 by CT26 cells, DC2.4 cells, and J774A.1 cells.

[0045] Figure 11 The image shows the effect of the protein nanocomposite prepared in Example 1 on the maturation of DC2.4 cells.

[0046] Figure 12 In the diagram: A is the polarization diagram (M1) of the protein nanocomposite and free drug prepared in Example 1 on the J774A.1 cell line; B is the polarization diagram (M2) of the protein nanocomposite and free drug prepared in Example 1 on the J774A.1 cells.

[0047] Figure 13 In the figures: A shows the distribution of the protein nanocomposite prepared in Example 1 compared to the free drug (ICG) in the tumors of BALB / c mice; B shows the distribution of the protein nanocomposite prepared in Example 1 compared to the free drug (ICG) in the lymph nodes of BALB / c mice.

[0048] Figure 14In the figures: A shows the inhibition curve of the protein nanocomposite prepared in Example 1 on BALB / c mice with CT26 tumors over 30 days; B shows the survival rate of the protein nanocomposite prepared in Example 1 on BALB / c mice with CT26 tumors over 100 days; C shows the weight change of the protein nanocomposite prepared in Example 1 on BALB / c mice with CT26 tumors over 30 days. Detailed Implementation

[0049] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0050] Existing technologies have very limited types of proteins that can bind to TLR agonists and organic photosensitizers, making it difficult to significantly improve the tumor and lymph node targeting effects and therapeutic efficacy of TLR agonists and organic photosensitizers. In this invention, albumin, ferritin, low-density lipoprotein, and other proteins are biocompatible carrier materials with lymph node targeting capabilities. The TLR agonist in solution is introduced into the albumin aqueous cavity as a precursor complex, and then an organic photosensitizer is introduced to induce a ligand exchange reaction, thereby inducing the formation of a complex between the TLR agonist and the organic photosensitizer within the protein cavity. This solves the problem of effectively encapsulating TLR agonists. The preparation method of the protein nanocomplex of the TLR agonist and organic photosensitizer in this invention is as follows: After adjusting the pH of the protein aqueous solution to 6-8, the TLR agonist aqueous solution is mixed and stirred with the protein aqueous solution for 2-6 min. Then, the organic photosensitizer solution is added, reacted at 20-60℃, and purified to obtain the TLR agonist and organic photosensitizer protein nanocomplex. (See [link to relevant documentation]). Figure 1 .

[0051] The following experiments illustrate the technological advancements of this invention. The raw materials used are existing products, and the specific preparation operations and performance tests all follow conventional techniques. Statistical analysis employs conventional methods, and animal experiments meet the relevant requirements of Soochow University. Stirring during the reaction of the TLR agonist and organic photosensitizer is a prior art technique, and can be performed at 100-500 rpm, ensuring stable stirring with vortex formation without splashing. The following experiments used 250 rpm.

[0052] Comparison Example TLR agonist aqueous solutions with organic photosensitizer solutions in different molar ratios (1:1, 2:1, 4:1, 8:1, 16:1) were mixed, vortexed thoroughly, and centrifuged at 3000 rpm. The formation of a precipitate at the bottom was observed, yielding a TLR agonist-organic photosensitizer complex. Figure 2 ).

[0053] Example 1 The specific steps for preparing the TLR agonist (R848) / organic photosensitizer (ICG) protein nanocomposite are as follows: Take 5 mL of 10 mg / mL albumin aqueous solution and adjust the pH of the mixture to 6.5 with 0.2 mM NaOH solution. Mix 1 mL of 3 mM TLR agonist (R848) aqueous solution with albumin aqueous solution at a volume ratio of 1:5, with a molar ratio of Toll-like receptor agonist to albumin of 20:1. Then add 1 mL of 2.2 mg / mL organic photosensitizer indocyanine green (ICG) aqueous solution to obtain a mixture with a molar ratio of TLR agonist to photosensitizer of 4:1. React the mixture at 37 °C for 4 hours, and then add the resulting mixture to an ultrafiltration tube. Centrifuge at 2000 rpm to remove impurities to obtain an aqueous solution of TLR agonist / organic photosensitizer protein nanocomposite (R848 / ICG@HSA).

[0054] The hydrated particle size of the prepared protein nanocomposite aqueous solution was measured, and the results are as follows: Figure 3 As shown in Figure A, the particle size of the prepared protein nanocomposite is approximately (73.8 ± 1.6) nm (n = 3). Transmission electron microscopy (TEM) images of the prepared protein nanocomposite are shown in the figure. Figure 4 As shown in Figure A, the prepared protein nanocomposite was uniformly dispersed and had a uniform particle size, with an average particle size of 12.6 ± 1.8 nm (n = 3) and a drug loading of 12.9% (n = 3). Figure 5 A). The drug content determination results showed that the mass ratio of R848 to ICG in the prepared nanocomposite was 3:2 (molar ratio approximately 4:1).

[0055] Referring to Example 1, the difference lies in that: the photosensitizer used in the reaction is not ICG, but neo-indocyanine green (IR820); the TLR agonist used in the reaction is not R848, but imiquimod (R837); the mixture is reacted at 37 °C for 3.5 hours; and the remaining processes remain unchanged. As a result, the prepared protein nanocomposite is uniformly dispersed and has a uniform particle size.

[0056] Example 2 An aqueous solution of a TLR agonist / organic photosensitizer protein nanocomplex was prepared by replacing the photosensitizer ICG in Example 1 with the photosensitizer neo-indocyanine green (IR820) and keeping the other conditions the same as in Example 1.

[0057] The hydrated particle size of the prepared protein nanocomposite was measured, and the results are as follows: Figure 3 As shown in B, the particle size of the prepared protein nanocomposite is approximately 68 ± 1.9 nm.

[0058] Example 3 The TLR agonist (R848) in Example 1 was replaced with a clinically used TLR agonist (R837, imiquimod), and the other conditions were the same as in Example 1, to prepare an aqueous solution of TLR agonist / organic photosensitizer protein nanocomplex.

[0059] The hydrated particle size of the prepared protein nanocomposite was measured, and the results are as follows: Figure 3 As shown in Figure C, the particle size of the prepared protein nanocomposite is approximately 78 ± 2.1 nm.

[0060] Example 4 The molar ratio of R848 to ICG in Example 1 was adjusted from 4:1 to 2:1, while other conditions remained unchanged. The prepared protein nanocomposite was then analyzed. The results showed an average hydrated particle size of 122.4 nm, and a transmission electron microscopy (TEM) yield of 38.6 ± 8.0 nm, with a drug loading of 7.7% (e.g., ...). Figure 5 A).

[0061] Example 5 In this embodiment, the solution pH was changed from pH 6.5 in Example 1 to pH 6.0, while other parameters remained unchanged. The prepared protein nanocomposite was analyzed. The results showed that its hydrated particle size was approximately 88.1 nm, and transmission electron microscopy revealed an average particle size of 13.2 ± 2.4 nm, with a drug loading of 9.5% (see...). Figure 4 B, 5B).

[0062] Example 6 In this embodiment, the solution pH was changed from pH 6.5 in Example 1 to pH 7.0, while other parameters remained unchanged. The prepared protein nanocomposite was analyzed. The results showed that its hydrated particle size was approximately 122.4 nm. Transmission electron microscopy revealed an average particle size of 19.7 ± 6.9 nm and a drug loading of 10.5% (see...). Figure 4 C, 5B).

[0063] Example 7 Referring to Example 1, the reaction time was changed from 4 hours to 2 hours, while the rest remained the same; the prepared protein nanocomposite was tested, and the result showed that its average hydrated particle size was 74.3 ± 2.6 nm.

[0064] Example 8 Referring to Example 1, the heating reaction at 37 °C for 4 hours was modified to heating reaction at 55 °C for 2 hours, while the rest remained the same; the prepared protein nanocomposite was tested, and the result showed that its average hydrated particle size was 73.8 nm.

[0065] Example 9 Referring to Example 1, the temperature was changed from 37 °C to 55 °C, while all other conditions remained the same, and the prepared protein nanocomposite was tested. The result showed that its average hydrated particle size was 74.3 nm.

[0066] Comparative Example Referring to Example 1, the difference is that the molar ratio of Toll-like receptor agonist to ICG is changed from 4:1 to 1:0.5, while the rest of the process remains unchanged. As a result, the solution is clear, but the nanoscale size cannot be measured, making it difficult to prepare protein nanocomposites.

[0067] Referring to Example 1, the difference is that the reaction pH is not 6.5, but 5, while the rest of the process remains unchanged. As a result, the solution is clear, but the nanoscale size cannot be measured, making it difficult to prepare protein nanocomposites.

[0068] Referring to Example 1, the difference is that the reaction pH is not 6.5, but 9, while the rest of the process remains unchanged. As a result, the solution is clear, but the nanoscale size cannot be measured, making it difficult to prepare protein nanocomposites.

[0069] Referring to Example 1, the difference is that the photosensitizer used in the reaction is not ICG, but zinc chloride phthalocyanine (ZnPc), while the rest of the process remains unchanged. As a result, the solution is clear, but the nanoscale size cannot be measured, making it difficult to prepare protein nanocomposites.

[0070] Referring to Example 1, the difference is that the photosensitizer used in the reaction is not ICG, but Rhodamine 6G, while the rest of the process remains unchanged. As a result, the solution is clear, but the nanoscale size cannot be measured, making it difficult to prepare protein nanocomposites.

[0071] Referring to Example 1, the difference is that the TLR agonist used in the reaction is not R848, but lipid monophosphate A (MPLA), while the rest of the process remains unchanged. As a result, the solution is clear, but the nanoscale size cannot be measured, making it difficult to prepare protein nanocomposites.

[0072] Referring to Example 1, the difference is that the TLR agonist used in the reaction is not R848, but CU-CPT17e, while the rest of the process remains unchanged. As a result, the solution is clear, but the nanoscale size cannot be measured, making it difficult to prepare protein nanocomposites.

[0073] Example 10 The performance of the protein nanocomposite containing the Toll-like receptor agonist (R848) and organic photosensitizer (ICG) prepared in Example 1 was tested using the following methods: 1. In vitro release study of the prepared protein nanocomplexes: R848 and R848 / ICG@HSA (1 mL) solutions were placed in dialysis bags (MWCO 8000-14000 Da), and the dialysis bags were placed in 15 mL centrifuge tubes pre-filled with buffer solutions. The buffer solutions were phosphate buffer (pH 7.4) and acetate buffer (pH 5.5), 10 mL of each solution was prepared in triplicate. The centrifuge tubes were placed in a constant temperature shaker (37℃, 120 rpm), and the solutions in the centrifuge tubes were removed at 0.5, 1, 2, 4, 8, and 24 h, and fresh buffer solutions (10 mL) were added. The content of R848 was determined by UV-Vis, the cumulative drug release was calculated, and in vitro release curves were plotted. Results: In vitro release chromatograms ( Figure 6 The results indicate that the prepared protein nanocomposite has a sustained-release effect, with release rates of 32% and 60% at pH 5.0 and pH 7.4, respectively, both lower than the 62% (pH 5.0) and 90% (pH 7.4) of free R848, demonstrating that the nanoparticles of this invention have excellent sustained-release properties.

[0074] 2. Stability study of the prepared protein nanocomposite. Particle size stability study: 200 μL of the prepared protein nanocomposite was diluted to 1 mL with deionized water, and the change in hydrated particle size was monitored over one day (1, 2, 4, 10, 24 h) using a nanoparticle size and potential analyzer. The results showed that ( Figure 7 The protein nanocomposite exhibited good particle size stability within 24 hours.

[0075] Chemical stability study: 0.5 mL of each of the same concentrations of R848, ICG, and R848 / ICG@HSA solutions were mixed thoroughly with 3.5 mL of environmental solvents: deionized water, acetate buffer (pH 5.5), phosphate buffer (pH 6.5, pH 7.4), and complete cell culture medium, respectively. Three replicates were prepared for each group of solutions. At 0, 6, 12, 24, 36, and 48 h, 0.5 mL of each mixed solution was taken and mixed with 4.5 mL of the corresponding environmental solvent. The UV absorption spectra of each solution were detected by UV-Vis, and stability curves were plotted. The results showed that the prepared protein nanocomplexes exhibited good in vitro stability, and the change in R848 concentration was minimal in both groups within 48 h. Figure 8 A), while the UV absorption of the free ICG group was significantly lower than that of the R848 / ICG@HSA group, with the decrease being approximately 10-30% greater. Figure 8 B).

[0076] The R848 / ICG physical mixture was prepared according to the control example. The specific steps were to mix the TLR agonist aqueous solution with the organic photosensitizer solution (mass ratio of 3:2) without centrifugation.

[0077] Protein nanoparticles containing a single layer of R848 were prepared, specifically following Example 1, except that the TLR agonist was mixed with a sodium sucrose octasulfate solution, while the rest of the process remained unchanged. Protein nanoparticles containing a single layer of ICG were also prepared, specifically following Example 1, except that the ICG was mixed with an ammonium sulfate solution, while the rest of the process remained unchanged. As a result, both solutions were clear, but the nanoscale size could not be measured, making it difficult to prepare protein nanocomposites containing a single drug layer. Therefore, subsequent examples only involve protein nanocomposites co-encapsulating a TLR agonist and an organic photosensitizer.

[0078] 3. Cytotoxicity test of the protein nanocomposite prepared in Example 1: CT26 and DC2.4 cells in logarithmic growth phase were seeded in 96-well plates at a density of 6 × 10⁻⁶. 3 100 μL / mL was added to each well and incubated in a cell culture incubator for 12 hours. After confirming cell adhesion, the culture medium was discarded, and the cells were washed 1-2 times with phosphate-buffered saline (pH 7.4). The following procedures were then followed: For the experimental group, 100 μL of protein nanocomplex prepared with culture medium was added to each well, with 5 replicates per concentration. For the control group, a physical mixture of the same concentration of Toll-like receptor agonist R848 and organic photosensitizer ICG was added at a mass ratio of 3:2 (w / w). After incubation for 24 hours, the culture medium was replaced with 10 µL of PBS solution containing 5 mg / mL MTT. After 4 hours, the culture medium was discarded, 200 µL of LDMSO was added, and the mixture was shaken for 10 minutes. The absorbance was measured at 490 nm using a microplate reader.

[0079] The light-treated group (experimental group) was incubated with the drug for 24 h. After removing the culture medium, the cells were washed three times with PBS. Fresh culture medium was added, and the liquid was irradiated with a 785 nm laser for 5 min (light intensity 0.5 W / cm²). 2 After culturing for another 24 hours, MTT assay was added to detect cell phototoxicity.

[0080] result( Figure 9 The results showed that, under non-light conditions, the protein nanocomposite had almost no killing effect on CT26 cells, while under light conditions, the protein nanocomposite could effectively kill CT26 cells, with an IC50 value of [missing value]. 50 The concentration was 12.58 µg / mL, while the IC50 of the physically mixed group was 12.58 µg / mL. 50 It is 19.66 µg / mL.

[0081] result( Figure 9The results showed that under non-light conditions, R848 / ICG@HSA was safe and harmless to tumor cells and had no killing effect; after light exposure, R848 / ICG@HSA caused significant damage to CT26 cells, and the cell death rate was positively correlated with ICG concentration. 50 The concentration was 12.58 µg / mL, while the IC50 of the physical mixture was... 50 The concentration was 19.66 µg / mL. Furthermore, at an ICG concentration of 50 mg / mL, the cell viability in the R848 / ICG light-treated group was 27%, while the cell viability in the R848 / ICG@HSA light-treated group decreased to 12.05%. This indicates that nanomedicines effectively enhance the cell-killing effect of drugs, suggesting that R848 / ICG@HSA can be considered a good phototherapy drug.

[0082] 4. Investigation on the uptake of the protein nanocomposite prepared in Example 1 by CT26, DC2.4, and J774A.1 cells: CT26, DC2.4, and J774A.1 cells in logarithmic growth phase were seeded in 6-well plates at a seeding density of 1×10⁻⁶. 6 Cells were cultured at 2 mL / well for 2, 6, 12, and 24 h. At the corresponding time points, digestion was terminated with 700 μL trypsin, followed by dilution with PBS to 1 mL. Cell counts were performed using a cell counting chamber. Cells were then fragmented using an ultrasonic cell disruptor, and 1 mL of DMSO was added to the lysis buffer for extraction. The extract was quantified using high-performance liquid chromatography (HPLC) to determine the R848 content, and the drug uptake in each group at different time points was calculated. Results ( Figure 10 The results showed that all three cell lines could effectively take up the protein nanocomposite, and further, the uptake at 24 h was higher than that of the free drug, increasing by about 1.8, 1.3, and 1.7 times, respectively.

[0083] 5. Examination of cell maturation induced by the protein nanocomposite prepared in Example 1: DC2.4 cells were uniformly seeded in 6-well plates overnight at a density of 1 × 10⁻⁶ cells per well. 6 The next day, R848, ICG, a physical mixture of R848 / ICG, or culture medium containing R848 / ICG@HSA (10 μg / mL ICG) were added and incubated for 24 h. After removing the culture medium, the samples were washed three times with PBS. For the light-illuminated group, the liquid was irradiated with a 785 nm laser for 5 min, with the light intensity adjusted to 0.3 W / cm². 2 After culturing in an incubator for 24 hours, the culture medium was changed, cells were collected, and anti-CD86 (a marker of dendritic cell maturation) was added. The mean fluorescence intensity was measured using flow cytometry. Figure 11 The results showed that, compared with the untreated group, the protein nanocomposite combined with light irradiation group could induce dendritic cell maturation more effectively, increasing it by about 2.8 times.

[0084] 6. Cell polarization study of the protein nanocomposite prepared in Example 1: J774A.1 cells were uniformly seeded in 6-well plates overnight at a density of 1 × 10⁻⁶ cells per well. 6 The next day, each sample was incubated with a physical mixture of R848 (15 μg / mL), ICG (10 μg / mL), or R848 (15 μg / mL) / ICG (10 μg / mL), or a culture medium containing R848 / ICG@HSA (10 μg / mL ICG) for 24 h. After removing the culture medium, the sample was washed three times with PBS. For the light-treated group, the liquid was irradiated with a 785 nm laser for 5 min, with the light intensity adjusted to 0.5 W / cm². 2 After the light exposure ended, the cells were incubated for another 12 h and stained with anti-CD86 and anti-CD206 antibodies (1:500) for 30 min. After washing three times with PBS, the cells were collected and resuspended in 0.5 mL PBS. Flow cytometry was used to detect the expression of CD86 (a macrophage M1 phenotypic marker) and CD206 (a macrophage M2 phenotypic marker) on the cell surface. The groups were the same as in the DC2.4 maturation assay. Figure 12 The results showed that the protein nanocomposite light irradiation group induced macrophage polarization to M1 type (increased by about 3.1 times) while reducing the expression of M2 type macrophages (decreased by about 2 times).

[0085] Protein nanocomposites were added to physiological saline to prepare a tumor therapeutic drug for the following analysis.

[0086] 7. The in vivo tissue distribution experiment of the protein nanocomposite prepared in Example 1 was investigated in mice. The specific steps are as follows: (1) Establishment of tumor model: CT26 tumor cells were cultured and digested to prepare 1×10⁻⁶ tumor cells. 7 A cell suspension of 80 μL / mL was used to ensure uniform cell dispersion. Tumors were implanted in the axillae of nude mice, with each mouse receiving a subcutaneous injection. Tumor volume was observed and recorded daily. Tumor volume formula: Tumor volume = (Length × Width) 2 ) / 2; (2) The protein nanocomposite obtained in Example 1 was injected via tail vein into two groups (light-exposed group and non-light-exposed group) of BALB / c mice (R848 dosage was 5 mg / kg). At 1, 2, 6, 12, 18, 24, and 48 h after injection, the heart, liver, spleen, lung, kidney, tumor, and lymph nodes of the mice were removed. Each tissue was accurately weighed and placed in a 10 mL centrifuge tube. 1.5 mL of physiological saline was added, and the mixture was homogenized until no tissue fragments were found. 4 mL of chloroform and 1 mL of methanol were added, and the mixture was vortexed for 5 min, centrifuged at 3000 rpm for 10 min, and 3 mL of the lower chloroform layer was collected in a 5 mL centrifuge tube. The mixture was then evaporated in a fume hood, and 100 μL DDMSO was added to reconstitute the mixture. The R848 content in each tissue was quantified by HPLC. Figure 13 It is known that the protein nanocomposite described in this invention has good dual targeting of tumor and lymph nodes in the CT26 tumor model. The accumulation of R848 in the tumor site is the highest at 12 h after administration (approximately 7% ID / g), and the drug accumulation in the lymph nodes is the highest at 6 h (2.5% ID / g) and gradually decreases over time.

[0087] 8. To investigate the tumor-suppressing effect of the protein nanocomposite prepared in Example 1 on colon cancer in mice: A mouse tumor-bearing model was constructed using the method established under "7. (1)". The tumor on the right side was allowed to grow to 80-100 mm. 3 Mice were randomly divided into 7 groups (since single-coated nanoparticles could not be produced, the protein nanocomposites used in subsequent experiments were all double-coated drugs): (G1) PBS, (G2) R848 / ICG, (G3) R848 / ICG+Tumor Laser, (G4) R848 / ICG+Tumor / LNLaser, (G5) R848 / ICG@HSA, (G6) R848 / ICG@HSA+Tumor Laser, (G7) R848 / ICG@HSA+Tumor / LN Laser, with 7 mice in each group. On days 1, 3, and 5, mice were injected intravenously with R848 / ICG or R848 / ICG@HSA (2 mg / kg ICG, 3 mg / kg R848). The PBS group was injected with PBS. 12 h later, a 785 nm laser (power 0.5 W / cm²) was used. 2 The tumor was irradiated with light for 5 minutes, followed by a 785 nm laser (0.3 W / cm²) at a 6-hour interval. 2 The lymph nodes were exposed to light for 5 minutes, and the experiment lasted for 100 days. During this period, the tumor size, body weight, and survival curve of the mice were recorded. Tumor volume exceeded 1500 mm. 3 The mouse dies at this point. Figure 14The study on tumor changes in mice showed that the PBS injection group had no effect on tumor growth in the CT26 tumor model (median survival: 22 days); however, compared with the non-light-exposed group (median survival: 40 days), the protein nanocomplex injection combined with dual-light-exposed group significantly inhibited tumor growth, with an inhibition rate of 92%. Furthermore, the mice remained in good condition during the observation period, with no significant change in body weight, and some mice survived for up to 100 days (*). P <0.001 indicates that the protein nanocomposite of the present invention can effectively inhibit tumor growth; at the same time, the tumor inhibition effect of the free R848 / ICG group and the combined light irradiation group was poor, and all of them died within 30 days (median survival: 26 / 28 days).

[0088] This invention relates to protein nanocomposites of TLR agonists and organic photosensitizers, their preparation methods, and applications. To address the shortcomings of current TLR agonist / photosensitizer methods, such as short blood half-life and low bioavailability, this invention designs albumin nanocomposites with dual tumor and lymph node targeting properties. The TLR agonist and organic photosensitizer form a complex within a protein cavity, thus simultaneously delivering two drugs. The preparation method for this protein nanocomposite is simple and mild, requiring no organic solvents; it is prepared in a one-step process using water as the solvent, with a particle size of approximately 12.7 nm and exhibiting pH-responsive drug release behavior. The protein nanocomposites provided by this invention have significant advantages in the co-delivery of TLR agonists and organic photosensitizers. Based on the active targeting mechanism mediated by albumin-binding receptors and the active uptake mechanism of albumin by antigen-presenting cells, this nanocomposite has dual targeting characteristics of tumors and lymph nodes. The synergistic tumor and lymph node immune activation mechanism can exert phototherapy effects under near-infrared light irradiation, rapidly inhibiting and eliminating tumors in a short period of time; and significantly enhancing the body's immune activation effect, fundamentally inhibiting tumor growth and truly and efficiently treating tumors.

Claims

1. A TLR agonist / organic photosensitizer protein nanoparticle complex comprising a TLR agonist, an organic photosensitizer, characterized in that, The TLR agonist and the organic photosensitizer form a complex in the protein cavity.

2. The TLR agonist / organic photosensitizer protein nanoparticle complex of claim 1, wherein, The TLR agonist comprises one or more of the following: a resiquimod, an imiquimod, a motolimod, a vesatimod, a tiramod; the organic photosensitizer comprises one or more of the following: an indocyanine green photosensitizer; the protein comprises one or more of the following: an albumin, a ferritin, a low-density lipoprotein.

3. The TLR agonist / organic photosensitizer protein nanoparticle complex of claim 2, wherein, The TLR agonist comprises one or more of the following: a resiquimod, an imiquimod, a motolimod, a vesatimod, a tiramod; the organic photosensitizer comprises one or more of the following: an indocyanine green photosensitizer; the protein comprises one or more of the following: an albumin, a ferritin, a low-density lipoprotein.

4. The method of producing a TLR agonist / organic photosensitizer protein nanoparticle complex of claim 1, wherein, The TLR agonist / organic photosensitizer protein nanocomplex is prepared by mixing a protein aqueous solution with a pH value of 6-8 and a TLR agonist solution, stirring, and then adding an organic photosensitizer solution.

5. The method of claim 4, wherein the TLR agonist / organic photosensitizer protein nanoparticle complex is prepared by, The molar ratio of the TLR agonist to the protein is (10-160):1; the molar ratio of the TLR agonist to the organic photosensitizer is (1-16):1; the concentration of the protein solution is 5-25 mg / mL.

6. The method for preparing the TLR agonist / organic photosensitizer protein nanocomposite according to claim 4, characterized in that, The reaction temperature is 25-55℃, the reaction is carried out in an aqueous solution with a pH value of 6-8, and the reaction time is 0.5-4 hours.

7. The TLR agonist / organic photosensitizer protein nanocomplex of claim 1 is used for preparing a medicament.

8. The TLR agonist / organic photosensitizer protein nanocomplex of claim 1 is used for preparing a medicament for enhancing immune activation effect.

9. The TLR agonist / organic photosensitizer protein nanocomplex of claim 1 is used for preparing an anti-tumor medicament.

10. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The active ingredient of the anti-tumor medicament comprises the TLR agonist / organic photosensitizer protein nanocomplex of claim 1.

Images