Preparation and application of metal-polyphenol modified albumin nano-drug

Abstract

The invention discloses preparation and application of a metal-polyphenol modified albumin nano-drug, and belongs to the field of medicines.The preparation method comprises the steps that 1, IR780 dissolved in absolute ethyl alcohol is added into an albumin aqueous solution under magnetic stirring, absolute ethyl alcohol continues to be dropwise added, and albumin wraps IR780 to be separated out to form nanoparticles; and (2) adding a proper amount of glutaraldehyde cross-linked albumin to obtain the stable IR780-loaded albumin nanoparticles. And (3) in an alkaline environment, tannic acid and Mn < 2 + > with proper concentration are introduced into the IR780-loaded albumin nanoparticles, so that a complex is formed to cover the surfaces of the albumin nanoparticles, and the final preparation of the nanoparticles is completed. According to the invention, the IR780-loaded albumin nanoparticles are modified by a metal-polyphenol network, so that the nano-drug has the capability of co-delivering a photosensitizer and metal manganese ions to a tumor site at the same time. The medicine has good effects in the aspects of treating superficial tumors, inducing tumor immunogenic cell death, promoting antigen presentation, enhancing anti-tumor immune response and the like.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the preparation and application of a metal-polyphenol modified albumin nanomedicine. Background Art

[0002] Human papillomavirus is a circular, double-stranded DNA virus. Persistent infection with its high-risk subtypes (such as HPV16 / 18) is closely related to the occurrence and development of a variety of malignant tumors. Among them, cervical cancer is the most representative HPV-related malignant tumor, and the global disease burden continues to remain high. According to statistics, there will be approximately 660,000 new cases of cervical cancer and approximately 350,000 deaths worldwide in 2022. Its mortality rate ranks fourth among female cancers, posing a major challenge to global public health. Traditional therapies such as surgical resection, radiotherapy and chemotherapy can control local lesions, but they can lead to irreversible loss of reproductive function. In addition, complications such as premature ovarian failure, bladder dysfunction and bone marrow suppression may occur, seriously affecting the quality of life of patients. Therefore, the development of innovative treatment strategies that combine efficient tumor cell killing and systemic immune activation is the key to improving patient prognosis.

[0003] IR780 is a near-infrared photosensitizer with excellent penetration into biological tissues. Due to its unique photothermal and photochemical properties, it is being applied in bioimaging, photodynamic therapy, and photothermal therapy. Furthermore, IR780's lipophilic cationic structure allows it to specifically target the mitochondria of tumor cells. Through mitochondria-targeted photodynamic and photothermal therapy, it can induce strong immunogenic cell death in tumor cells, significantly increasing the release of damage-associated molecular patterns (DAMs), which facilitates the initiation of anti-tumor immune responses.

[0004] Although damage-associated molecular patterns, as endogenous danger signals, can promote antigen presentation and immune response, their antigen presentation efficiency is often limited due to factors such as insufficient immunogenicity of tumor-associated antigens and the influence of immunosuppressive microenvironment. Therefore, combined immune adjuvants are needed to enhance the immunogenicity of antigens and the antigen presentation ability of dendritic cells. 2+ ) plays a vital role in initiating and regulating immune responses by activating the cGAS-STING key signaling pathway of innate immunity. In addition, in the tumor microenvironment, Mn 2+ It can catalyze hydrogen peroxide to generate hydroxyl radicals (·OH), exerting a chemodynamic therapeutic effect, synergizing with IR780 to cause oxidative damage to tumor cells and initiate an immune response. These mechanisms of action provide a theoretical basis for the design of nanomedicines that can enhance ICD effects and immune activation.

[0005] In recent years, natural polyphenols have attracted extensive attention in the field of drug delivery due to their unique chemical properties. Tannic acid (TA) is a typical polyphenol compound. The catechol structure in its molecule can coordinate with various metal ions (such as Fe 3+ 、Al 3+ 、Mn 2+ The metal-polyphenol network can form a stable metal-polyphenol network, which can self-assemble on the surface of nanoparticles to form a functional film, providing an ideal modification platform for drug delivery systems. It is worth noting that the metal-polyphenol network can undergo controllable dissociation in an acidic environment, achieving pH-responsive release of metal ions. This feature provides a new idea for the design of intelligent drug delivery systems that respond to the tumor microenvironment. This study constructed a bioengineered nanodelivery platform (IMT@H) based on HSA to deliver IR780 photosensitizer and Mn 2+ Currently, there are no reports on the research of albumin nanoformulations loaded with photosensitizers modified by metal-polyphenol networks. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a method for preparing a metal-polyphenol modified albumin nanomedicine, comprising the steps of:

[0007] S1: 20 mg human serum albumin was dissolved in 2 mL deionized water. Under magnetic stirring, 400 μL of 5 mg / mL IR780 was added to the albumin solution and dissolved in anhydrous ethanol.

[0008] S2: Continuing to add 8 mL of anhydrous ethanol to the solution at a rate of 1 mL / min under magnetic stirring, the albumin is gradually precipitated from the solvent to form nanoparticles.

[0009] S3: Add 100 μL of 2.5% glutaraldehyde solution to the resulting solution and stir at 800 rpm in the dark for 12 hours to crosslink the albumin nanoparticles and improve their stability;

[0010] S4: The cross-linked solution was centrifuged at 13,000 rpm for 10 min, washed twice with deionized water, the supernatant was discarded, and 6 mL of ddH2O was added to resuspend the precipitate to obtain IR780-loaded albumin nanoparticles;

[0011] S5: Add 200 μL of 40 mg / mL tannic acid aqueous solution and 100 μL of 19.8 mg / mL manganese chloride tetrahydrate solution to the nanoparticle solution obtained in S4;

[0012] S6: Add appropriate amount of Tris-HCl to adjust the pH to about 8.0, vortex for 30 seconds to allow tannic acid to react with Mn 2+Mix thoroughly to form a complex that is deposited on the surface of the albumin nanoparticles;

[0013] S7: The obtained solution was centrifuged at 13000 rpm for 10 min, and then washed twice with deionized water. The supernatant was discarded, and deionized water was added to resuspend the precipitate to obtain IMT@H nanoparticles.

[0014] On the one hand, the present invention provides an application of a metal-polyphenol modified albumin nanomedicine. Specifically, the drug prepared by the above method is used to treat superficial HPV infection-related tumors to induce tumor immunogenic cell death, promote antigen presentation and enhance anti-tumor immune response.

[0015] In summary, the present invention has the following beneficial effects compared to the prior art:

[0016] By modifying photosensitizer-loaded albumin nanoparticles with a metal-polyphenol complex, the present invention improves the co-delivery efficiency of the photosensitizer and metal ions at the tumor site. This makes the drug provided by the present invention highly effective in treating superficial tumors, such as HPV-related tumors, by inducing tumor immunogenic cell death, promoting antigen presentation, and enhancing anti-tumor immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 Schematic diagram of the preparation of IMT@H nanoparticles;

[0019] Figure 2 Transmission electron microscopy images of I@H nanoparticles (A) and IMT@H nanoparticles (B) and the magnified fields of view of the boxed areas;

[0020] Figure 3 is the particle size diagram of I@H and IMT@H nanoparticles;

[0021] Figure 4 Potential diagrams of I@H and IMT@H nanoparticles;

[0022] Figure 5 is the particle size stability of IMT@H nanoparticles in different solvents;

[0023] Figure 6 The potential stability of IMT@H nanoparticles in different solvents;

[0024] Figure 7 is the X-ray photoelectron spectrum of IMT@H nanoparticles;

[0025] Figure 8 UV-visible-near-infrared absorption spectra of IR780, HSA, I@H, Mn-TA, and IMT@H;

[0026] Figure 9 SOSG detection of I@H and IMT@H nanoparticles before and after 808nm laser irradiation 1 O2 generation;

[0027] Figure 10 is the hydroxyl radical (·OH) generation ability of IMT@H nanoparticles;

[0028] Figure 11 For I@H and IMT@H nanoparticle solutions at 1.5W / cm 2 Photothermal curve under 808nm near-infrared light irradiation;

[0029] Figure 12 Mn content of IMT@H nanoparticles in PBS+10% FBS solution with different pH values 2+ Drug release profile;

[0030] Figure 13 Figures of orthotopic tumors and distal tumors and tumor growth curves of mice in each group;

[0031] Figure 14 Flow cytometry was used to analyze the expression of costimulatory molecules CD80 and CD86 on the surface of DCs in the spleen cells of mice after treatment in different groups;

[0032] Figure 15 CD11c + CD80 in DCs + CD86 + Cell ratio statistics;

[0033] Figure 16 Flow cytometry analysis of CD3 in spleen cells of mice after treatment in different groups + CD8 + T cell abundance;

[0034] Figure 17 Mouse splenocyte CD3 + CD8 + T cell proportion statistics;

[0035] Figure 18 Flow cytometry analysis of CD3 in spleen cells of mice after treatment in different groups + CD8 + CD44 + Memory T cell abundance;

[0036] Figure 19 Mouse splenocyte CD3+ CD8 + CD44 + Memory T cell statistics;

[0037] Figure 20 Flow cytometry analysis of CD45 in situ tumor tissues of mice after treatment in different groups + CD3 + CD8 + T cells.

[0038] Figure 21 CD45 for tumor in situ + CD3 + CD8 + T cell proportion statistics;

[0039] Figure 22 Flow cytometry analysis of CD45 in distant tumor tissues of mice after treatment in different groups + CD3 + CD8 + T cells.

[0040] Figure 23 CD45 in distant tumors + CD3 + CD8 + T cell proportion statistics;

[0041] Figure 24 ELISPOT was used to detect the number of IFN-γ producing cells in the spleen lymphocytes of different groups of mice under the stimulation of HPV16 E7 (44-62) peptide and the number of cells that could secrete IFN-γ per million spleen lymphocytes of different groups of mice. DETAILED DESCRIPTION

[0042] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0043] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form can also include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0044] Unless otherwise specifically stated, the relative arrangement of the parts and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention. At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to actual proportional relationships. The techniques, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the techniques, methods and equipment should be considered as part of the authorized specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as being merely exemplary and not as limitations. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, and therefore, once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0045] See also Figure 1 As shown, the present invention provides a method for preparing a metal-polyphenol modified albumin nanomedicine, comprising the steps of:

[0046] S1: 20 mg human serum albumin was dissolved in 2 mL deionized water. Under magnetic stirring, 400 μL of 5 mg / mL IR780 was added to the albumin solution and dissolved in anhydrous ethanol.

[0047] S2: Continuing to add 8 mL of anhydrous ethanol to the solution at a rate of 1 mL / min under magnetic stirring, the albumin is gradually precipitated from the solvent to form nanoparticles.

[0048] S3: Add 100 μL of 2.5% glutaraldehyde solution to the resulting solution and stir at 800 rpm in the dark for 12 hours to crosslink the albumin nanoparticles and improve their stability;

[0049] S4: The cross-linked solution was centrifuged at 13,000 rpm for 10 min, washed twice with deionized water, the supernatant was discarded, and 6 mL of ddH2O was added to resuspend the precipitate to obtain IR780-loaded albumin nanoparticles;

[0050] S5: Add 200 μL of 40 mg / mL tannic acid aqueous solution and 100 μL of 19.8 mg / mL manganese chloride tetrahydrate solution to the nanoparticle solution obtained in S4;

[0051] S6: Add appropriate amount of Tris-HCl to adjust the pH to about 8.0, vortex for 30 seconds to allow tannic acid to react with Mn 2+ Mix thoroughly to form a complex that is deposited on the surface of the albumin nanoparticles;

[0052] S7: The obtained solution was centrifuged at 13000 rpm for 10 min, and then washed twice with deionized water. The supernatant was discarded, and deionized water was added to resuspend the precipitate to obtain IMT@H nanoparticles.

[0053] Tannic acid (TA) and Mn 2+ Under weak alkaline conditions (pH about 8.0), a network structure can be formed and wrapped around the surface of albumin nanoparticles, thereby quickly completing the preparation of IMT@H nanoparticles.

[0054] To demonstrate the reliability of the metal-polyphenol modified albumin nanoparticles provided by the present invention, the following experiments were conducted:

[0055] like Figure 2 As shown in the figure, the structure of the nanodrug was observed under a transmission electron microscope. Compared with the IR780 albumin-loaded nanoparticles (I@H), an obvious membrane-like coating structure was observed on the surface of the IMT@H nanoparticles. This phenomenon confirmed that the Mn-TA complex had been successfully modified on the surface of I@H to form a core-shell structure.

[0056] like Figure 3 As shown in the figure, the average particle size of I@H nanoparticles is 233.07±4.50nm, and the PDI is 0.04±0.01. After modification with Mn-TA, the average particle size of IMT@H nanoparticles increases to 246.00±4.29nm, and the PDI is 0.03±0.02.

[0057] like Figure 4 As shown, the average potential of I@H nanoparticles is -28.63±0.70 mV, and the average potential of IMT@H nanoparticles is -31.73±2.75 mV.

[0058] like Figure 5 As shown in the figure, the particle size of IMT@H remained basically stable after 15 days of observation in deionized water, PBS+10% FBS and DMEM+10% FBS solutions.

[0059] like Figure 6 As shown in Figure 3, the potentials of IMT@H in PBS + 10% FBS and DMEM + 10% FBS solutions were -10.20±0.92 mV and -8.16±0.37 mV, respectively. During the 15-day observation period, the potentials of IMT@H nanoparticles in different solvents remained basically stable.

[0060] like Figure 7 As shown in the XPS spectrum of IMT@H nanoparticles, the characteristic peak of Mn 2p orbital is observed, and its binding energy is similar to that of Mn 2+ The valence state matches.

[0061] The UV absorption spectrum results are shown as follows Figure 8As shown, HSA and IR780 exhibit absorption peaks at 280 nm and 780 nm, respectively. The absorption spectrum of I@H combines the characteristic absorption peaks of HSA and IR780, with a slight red shift in the absorption peak of IR780, indicating that IR780 has been successfully incorporated into the HSA nanoparticles. The absorption spectrum of IMT@H combines the characteristic absorption peaks of Mn-TA and I@H, demonstrating the successful preparation of the nanomedicine.

[0062] Singlet oxygen fluorescence probe was used to detect 1 O2 production Figure 9 As shown in the figure, the I@H and IMT@H solutions without laser irradiation have no obvious fluorescence peak at 525nm. After irradiation with 808nm laser, the fluorescence intensity of I@H and IMT@H at 525nm increases significantly, indicating that I@H or IMT@H nanoparticles can produce 1 O2.

[0063] Methylene blue (MB) was used to detect the hydroxyl radical (·OH) generation ability of IMT@H nanoparticles. Figure 10 As shown, when MB and H2O2 solutions were incubated with MnCl2, Mn-TA, or IMT@H, respectively, a decrease in the absorbance of MB was observed, indicating the generation of ·OH through a Mn-mediated Fenton-like reaction.

[0064] like Figure 11 As shown, using 1.5W / cm 2 When laser irradiated IMT@H or I@H solutions with different concentrations (IR780 concentration gradient was 12.07, 33.33, 100 μg / mL), the average temperature of both solutions at 300 seconds increased significantly with the increase of nanoparticle concentration.

[0065] like Figure 12 As shown in Figure 3, the release of manganese ions from IMT@H nanoparticles was significantly increased under acidic conditions.

[0066] like Figure 13 As shown, bilateral subcutaneous tumor models were established in mice, and the primary tumors were grown to (50-100) cm 3 Treatment was started. 24 hours after the mice were injected with nanomedicine in the tail vein, the tumor site was irradiated with 808 nm laser (1.5 W / cm 2 , 5min), with treatment every 3 days for a total of 3 times. IMT@H nanoparticles irradiated with near-infrared light significantly inhibited the growth of in situ tumors and inhibited the growth of distant tumors by stimulating systemic anti-tumor immunity.

[0067] The changes of DCs and T cell subsets in the spleen were analyzed by flow cytometry. Figures 14-19As shown in Figure 2, the proportion of mature dendritic cells in the spleen of the IMT@H+Laser group increased significantly; in addition, CD8 + T cell ratio and CD8 + CD44 + The proportion of memory T cells was also significantly higher than that of the other groups. The significant difference between IMT@H+Laser and I@H+Laser indicates that the presence of manganese adjuvant promotes antigen presentation, thus achieving a good anti-tumor effect.

[0068] like Figures 20-23 As shown in the figure, the infiltration rates of CD8+T cells in the in situ tumor and distant tumor tissues were 57.67±4.51% and 59.67±7.10%, respectively, which were significantly higher than those in the control group and the I@H+laser group. This indicates that near-infrared light-excited IMT@H nanoparticles activated systemic anti-tumor immunity through the treatment of in situ tumors and increased the CD8 + T cell infiltration, thereby inhibiting tumor growth.

[0069] like Figure 24 As shown, mouse splenic lymphocytes were extracted and stimulated with an HPV16 E7 (44-62) peptide-specific antigen. Each spot represents an antigen-specific T cell capable of secreting IFN-γ, and the number of spots reflects the intensity of the T cell's immune response to the specific antigen. The number of cells producing IFN-γ in the IMT@H+laser group was significantly higher than that in the control and I@H+laser groups, confirming that IMT@H nanoparticles can effectively enhance the specific immune response to HPV-related tumors under near-infrared light excitation.

[0070] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing metal-polyphenol modified albumin nanomedicine, characterized in that: Including steps: S1: 20 mg human serum albumin was dissolved in 2 mL deionized water. Under magnetic stirring, 400 μL of 5 mg / mL IR780 was added to the albumin solution and dissolved in anhydrous ethanol. S2: Continuing to add 8 mL of anhydrous ethanol to the solution at a rate of 1 mL / min under magnetic stirring, the albumin is gradually precipitated from the solvent to form nanoparticles. S3: Add 100 μL of 2.5% glutaraldehyde solution to the resulting solution and stir at 800 rpm in the dark for 12 hours to crosslink the albumin nanoparticles and improve their stability; S4: The cross-linked solution was centrifuged at 13,000 rpm for 10 min, washed twice with deionized water, the supernatant was discarded, and 6 mL of ddH2O was added to resuspend the precipitate to obtain IR780-loaded albumin nanoparticles; S5: Add 200 μL of 40 mg / mL tannic acid aqueous solution and 100 μL of 19.8 mg / mL manganese chloride tetrahydrate solution to the nanoparticle solution obtained in S4; S6: Add appropriate amount of Tris-HCl to adjust the pH to 8.0, vortex for 30 seconds to allow tannic acid to react with Mn 2+ Mix thoroughly to form a complex that is deposited on the surface of the albumin nanoparticles; S7: The obtained solution was centrifuged at 13000 rpm for 10 min, and then washed twice with deionized water. The supernatant was discarded, and deionized water was added to resuspend the precipitate to obtain IMT@H nanoparticles.

2. An application of a metal-polyphenol modified albumin nanomedicine, characterized in that: The metal-polyphenol modified albumin nanomedicine is used to treat superficial tumors to induce tumor immunogenic cell death, promote antigen presentation and enhance anti-tumor immune response.

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