Micro-nano structure photo-responsive hydrogel microsphere as well as preparation method and application of micro-nano structure photo-responsive hydrogel microsphere

Abstract

The invention provides a micro-nano structure photo-responsive hydrogel microsphere as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The hydrogel microsphere is composed of a micron shell wrapped by hyaluronic acid and a drug-loaded mesoporous silica core co-modified by a bacterial targeting LPS antibody and a thermal response plate Poly A. The hydrogel microsphere can promote targeting retention and light-operated responsive release of a drug at an osteosarcoma part; and finally, an antibacterial and anti-tumor photoimmunochemotherapy combined effect is realized. The unique structure of the hydrogel microsphere can inhibit osteosarcoma growth, remove bacteria in tumors and improve tumor microenvironment immunogenicity at the same time. According to the hydrogel microspheres provided by the invention, by removing bacteria in tumors and regulating and controlling the microenvironment, a method for achieving the antibacterial and anti-tumor photoimmunochemotherapy combined effect is achieved, and a new method is provided for clinical treatment of osteosarcoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a micro-nano structured light-responsive hydrogel microsphere, a preparation method thereof, and an application thereof in intratumoral bacteria clearance and osteosarcoma treatment. Background Art

[0002] Osteosarcoma is the most common type of malignant bone tumor, mostly seen in children and adolescents, often occurring at the metaphysis of long bones, and is characterized by high malignancy, strong invasiveness, and easy metastasis. Treating the tumor microenvironment is the new direction of current tumor treatment. However, on the one hand, the tumor microenvironment is mainly composed of tumor cells and their surrounding fibroblasts, immune and inflammatory cells, glial cells, as well as the extracellular matrix, microvessels, and biomolecules infiltrated therein, forming a very complex mechanism network, which is crucial for the growth and prognosis of tumors; on the other hand, as a "cold" tumor, the immunosuppressive tumor microenvironment of osteosarcoma further exacerbates the deterioration of the tumor.

[0003] Research shows that insufficient immunogenicity of the tumor microenvironment and resident intratumoral bacteria are important reasons for the poor prognosis of osteosarcoma patients. Therefore, how to clear intratumoral bacteria and improve the immunogenicity of the tumor microenvironment is the key to improving the treatment effect of osteosarcoma.

[0004] In recent years, research on the tumor microenvironment has emerged in an endless stream, especially the research on microorganisms in the tumor microenvironment is one of the hot directions in the current field of tumor treatment. For example, the Deborah Nejman team conducted a comprehensive analysis of the tumor microbiome and studied tumors and their adjacent tissues of 7 cancer types including bone cancer. The study found that most solid tumors contain bacteria, and there are also differences in the proportion of bacteria DNA-positive tumors among different tumor types, among which breast, pancreatic, and bone tumors account for 60%, and each type of tumor has a unique microbiome composition. Another example is that the research group of Professor Susan Bullman found that non-cancer cells (including some bacteria) around the tumor can help the tumor avoid immune system attacks and then metastasize. Another example is that the research team led by Dr. Cai Shang recently isolated live bacteria from tumor tissues, proving the existence of intracellular bacteria in tumors; at the same time, the research team found that the tumor began to metastasize after injecting a single dose of bacteria into the tumor. Therefore, the bacterial population in tumors is likely to be a potential target for preventing early metastasis of osteosarcoma.

[0005] However, a large number of studies have shown that the physiological barrier formed by the tumor microenvironment greatly hinders the delivery of drugs. Therefore, how to design a new drug delivery system to break through the barriers of the osteosarcoma microenvironment and achieve targeted treatment of intracellular bacteria in tumors is one of the key scientific issues for improving the clinical efficacy of osteosarcoma at present.

[0006] In recent years, mesoporous silica nanoparticles (MSN) have developed very rapidly as drug delivery materials. Compared with traditional delivery materials, MSN has the advantages of easy surface functionalization, good biocompatibility, high specific surface area, stable porous structure, and adjustable pore size, allowing a large number of cargo molecules to be carried. For example, the Lu research group developed a new type of mesoporous silica-coated bismuth sulfide nanoparticles loaded with doxorubicin, which showed high specificity for osteosarcoma and good combined killing effect through conjugation with arginine-glycine-aspartic acid. Another example is that the Zhou team designed a simple and practical photothermal controlled release system on the surface of MSN by base modification and using base complementary pairing, realizing the rapid release of drugs at the tumor site. However, unfortunately, when MSN is used as a drug delivery material for the treatment of osteosarcoma, there is a problem that the administration method cannot target and eliminate intratumoral bacteria. For example, when using the systemic administration method, since the flora in the body is not only distributed near the tumor, but there are a large number of microorganisms in other tissues such as the gastrointestinal tract, this will cause serious side effects; while directly injecting in situ, due to the rich blood vessels and large pore size of the blood vessel wall at the tumor site, it is also easy to cause drug loss. Therefore, using a new type of drug carrier to carry mesoporous silica nanoparticles and improving the retention and penetration of drugs at the tumor site is of great significance for enhancing the antibacterial and anti-tumor effects of drugs.

[0007] Hydrogels have received extensive attention in the field of biomedical materials due to their good biocompatibility and have been widely used in the site-specific release of drugs. Wang et al. used hydrogels to load black phosphorus nanosheets and implanted them surgically at the rat skull defect site, effectively promoting skull repair. The Cui team developed a supramolecular hydrogel system that forms in situ in tumors for the local delivery of cyclic diadenosine monophosphate, significantly enhancing the anti-tumor immune response. The Goldberg research group implanted an immune gel during tumor resection surgery, successfully regulating the tumor immunosuppressive microenvironment and inhibiting tumor recurrence. However, conventional hydrogels have the disadvantages of large external size and the need for surgical implantation, while the gelation conditions required for hydrogels that form in situ in tumors are relatively harsh, which greatly limits their application.

[0008] Therefore, there are still deficiencies in the treatment of osteosarcoma, such as the selection of drug delivery carriers and the inability to eliminate intratumoral bacteria, which affect the gelation performance. How to provide a micro-nano structured hydrogel microsphere that can specifically eliminate intratumoral bacteria in tumors and photoreprogrammably reconstruct the tumor microenvironment, in order to change the current situation that the effect of current biomedical materials in the treatment of intratumoral bacteria and osteosarcoma is not ideal and new drug delivery materials are urgently needed to be developed, has become a technical problem to be solved urgently. Summary of the Invention

[0009] The present invention is to solve the above technical problems, and thus provides a micro-nano structured photo-responsive hydrogel microsphere, a preparation method thereof, and an application in intratumoral bacteria clearance and osteosarcoma treatment. The technical object of the present invention is, on the one hand, to solve the problems that the existing hydrogel materials as drug delivery carriers for osteosarcoma require surgical implantation and have harsh gelation conditions, resulting in a large impact on the effect by intratumoral bacteria; on the other hand, to solve the problem that the therapeutic effect of hydrogel drugs on osteosarcoma is not good. The present invention provides a micro-nano structured hydrogel microsphere capable of directionally eliminating intratumoral bacteria in tumors and photo-controlled programmed reconstruction of the tumor microenvironment, which can be well used for intratumoral bacteria clearance and osteosarcoma treatment, and the effect is extremely remarkable.

[0010] In order to achieve the above technical object, the technical solution adopted by the present invention is as follows:

[0011] The present invention first provides a preparation method of a micro-nano structured photo-responsive hydrogel microsphere, comprising the following steps:

[0012] (1) Using EDC and NHS as activators, mixing and stirring with thymine-1-acetic acid, and then adding amino-modified mesoporous silica for reaction to obtain silica-thymine;

[0013] (2) Mixing silica-thymine with a lipopolysaccharide antibody solution, then adding gemcitabine and indocyanine green, after stirring the reaction is completed, washing and removing impurities, adding polyadenylic acid to the obtained product, and continuing to stir the reaction to obtain drug-loaded nanoparticles;

[0014] (3) Mixing methacrylated hyaluronic acid, a photoinitiator and the drug-loaded nanoparticles described in step (2) as the aqueous phase, using mineral oil as the oil phase, preparing hydrogel microspheres through a microfluidic device, and then curing with ultraviolet light to obtain the micro-nano structured photo-responsive hydrogel microsphere.

[0015] The present invention successfully constructs a hydrogel microsphere with a micro-nano structure for the directional elimination of intratumoral bacteria and the photo-controlled programmed reconstruction of the tumor microenvironment. First, a functionalized nanoparticle I / G-LPMSN is developed based on the three-step surface modification of mesoporous silica. Then, the nanoparticle I / G-LPMSN is embedded into a hyaluronic acid hydrogel matrix through microfluidic technology to form a hydrogel microsphere with antibacterial and anti-tumor effects. Due to the binding of hyaluronic acid in the hydrogel to CD44 on the tumor surface, after in-situ injection into the tumor, the hydrogel with a micro-nano structure can achieve targeting and retention of tumor cells. Then, the nanoparticle I / G-LPMSN released from the hydrogel with a micro-nano structure can not only capture intratumoral bacteria by means of anti-LPS antibodies, but also achieve the photosensitive release of gemcitabine (GEM) and indocyanine green (ICG), thereby further producing an antibacterial effect and realizing tumor photothermal chemotherapy. Finally, the immunogenic substances released by apoptotic tumor cells and dead bacteria can recruit T cells to achieve the synergistic photoimmunotherapy of osteosarcoma and tumor bacteria. Therefore, the hydrogel microsphere provided by the present invention realizes the combined effect of antibacterial and anti-tumor photoimmunochemotherapy by clearing intratumoral bacteria and regulating the microenvironment, providing a new idea for the application of injectable hydrogel microspheres in the clinical treatment of osteosarcoma.

[0016] Furthermore, the weight ratio of EDC, NHS, and thymine-1-acetic acid in step (1) is 24:9:0.13.

[0017] Furthermore, the weight ratio of the amino-modified mesoporous silica to NHS in step (1) is 1:1.8.

[0018] Furthermore, the reaction time in step (1) is 12 hours.

[0019] Furthermore, the weight ratio of the lipopolysaccharide antibody to silica-thymine in step (2) is 1:20.

[0020] Furthermore, the weight ratio of silica-thymine, gemcitabine, and indocyanine green in step (2) is 2:2:1.

[0021] Furthermore, the weight ratio of polyadenylic acid to silica-thymine in step (2) is 1:1.

[0022] Furthermore, the concentration of methacrylated hyaluronic acid in step (3) is 5 wt%.

[0023] The second object of the present invention is to provide a micro-nano structured photo-responsive hydrogel microsphere prepared by the method as described above.

[0024] A third object of the present invention is to provide the application of the micro-nano structured photo-responsive hydrogel microspheres as described above in the preparation of drugs for in-tumor bacteria clearance and treatment.

[0025] Specifically, the tumor includes osteosarcoma.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The present invention successfully constructs a hydrogel microsphere with a micro-nano structure for the directional extermination of bacteria in tumors and the photo-controlled programmed reconstruction of the tumor microenvironment. It consists of a micron shell wrapped by hyaluronic acid and a drug-loaded mesoporous silica core co-modified with a bacterial-targeting LPS antibody and a thermo-responsive block Poly A, aiming to promote the targeted retention and photo-controlled responsive release of drugs at the osteosarcoma site, and ultimately achieve the combined effect of antibacterial and anti-tumor photoimmunochemotherapy. The unique structure of this hydrogel microsphere enables it to clear bacteria in the tumor while inhibiting the growth of osteosarcoma and enhancing the immunogenicity of the tumor microenvironment. In vitro and in vivo experiments show that on the one hand, this hydrogel microsphere eliminates bacteria in the tumor through the combined effect of photoimmunochemotherapy, improving the therapeutic effect on osteosarcoma; on the other hand, it activates the expression of CD4 and CD8 in osteosarcoma, regulates the tumor microenvironment, and enhances the anti-tumor effect. The method proposed in the present invention for achieving the combined effect of antibacterial and anti-tumor photoimmunochemotherapy by clearing bacteria in the tumor and regulating the microenvironment provides a new idea for the clinical treatment of osteosarcoma with injectable hydrogel microspheres. Description of the Drawings

[0028] Figure 1 Characterization of I / G-LPMSN nanoparticles; (A) Absorbance of ICG, GEM, MSN-T, and I / G-LPMSN; (B) Particle size distribution of I / G-LPMSN; (C) TEM image of I / G-LPMSN; (D) Zeta potential changes at different synthesis stages (n = 3); (E) Surface element distribution analysis of I / G-LPMSN; (F) Photostability of ICG under 785 nm irradiation; (G) Photostability of I / G-LPMSN under 785 nm light irradiation; (H) Photothermal imaging of I / G-LPMSN under 785 nm light irradiation; (I) Photothermal effect of I / G-LPMSN under 785 nm light irradiation; (J) Photothermal conversion efficiency of I / G-LPMSN under 785 nm irradiation; (K) Drug release behavior of GEM, I / G-LPMSN, and I / G-LPMSN after light irradiation.

[0029] Figure 2For cytotoxicity and intracellular evaluation; cell viability of (A) GEM, (B) GEM-LPMSN, (C) ICG, (D) ICG, (E) ICG-LPMSN, (F) ICG-LPMSN, (G) I / G-LPMSN, (H) I / G-LPMSN under 785 nm irradiation; (I) Live / dead staining of K7M2 cells treated with PBS, GEM-LPMSN, ICG-LPMSN, I / G-LPMSN with or without 785 nm irradiation; Calcein-AM (green) and PI (red); (J) K7M2 cells stained with LysoTracker green DND 26 (green) and Hoechst 33342 (blue) after incubation with I / G-LPMSN under 785 nm irradiation.

[0030] Figure 3 For antibacterial ability study; growth curves of EcN with 50 μg ICG mass in I / G-PMSN and I / G-LPMSN under different drug treatments; (B) Bacterial colony images on agar plates; (C) Live / dead staining of EcN bacteria treated with PBS, I / G-PMSN and I / G-LPMSN under 785 nm light; (D) Colony images on agar plates; (E) CFU images after different drug treatments.

[0031] Figure 4 For characterization and in vivo study of I / G-LPMSN@HAMA; (A) Bright-field and fluorescence images of I / G-LPMSN@HAMA; (B) SEM image of I / G-LPMSN@HAMA; (C) and (D) Element distribution analysis of I / G-LPMSN@HAMA; (E, G) NIRF imaging and fluorescence intensity in K7M2 tumor-bearing mice after injection of free ICG, I / G-LPMSN and I / G-LPMSN@HAMA; (F, H) Infrared thermal imaging and temperature elevation curves of K7M2 tumor-bearing mice treated with PBS, I / G-LPMSN, I / G-LPMSN@HAMA under 785 nm light.

[0032] Figure 5 For antibacterial and antitumor therapy; (A) Schematic diagram of in vivo experiment in mice; (B) Relative volume change of tumors in mice 14 days after injection; (C) Plate images of EcN colonies in tumors under different drug actions; (D) cfu analysis; (E) Immunofluorescence images of CD4+ T cells and CD8+ T cells in tumors; (F) Expression analysis of CD4+ T cells in ImageJ; (G) Expression analysis of CD8+ T cells in ImageJ.

[0033] Figure 6For pathological analysis; (A) H&E staining, Ki67 immunofluorescence, and TUNEL staining images of tumors; (B) Analysis of Ki67 expression in tumors; (C) Analysis of TUNEL staining images.

[0034] Figure 7 It is: (A) Particle size distribution of MSN-T; (B) SEM image of MSN-T.

[0035] Figure 8 It is the elemental analysis diagram of I / G-LPMSN.

[0036] Figure 9 It is the photothermal conversion efficiency of ICG.

[0037] Figure 10 It is: (A) Growth curves of EcN with 100 μg of ICG in I / G-PMSN and I / G-LPMSN under different drug treatments; (B) Bacterial colony images on agar plates.

[0038] Figure 11 It is CRT immunofluorescence staining.

[0039] Figure 12 It is HMGB1 immunofluorescence staining.

[0040] Figure 13 It is the drug release diagram.

[0041] Figure 14 It is the live / dead staining diagram of K7M2 tumor cells.

[0042] Figure 15 It is the live / dead staining diagram of EcN bacteria.

[0043] Figure 16 It is HE staining of normal tissues.

[0044] Figure 17 It is the schematic diagram of the synthesis and mechanism of action of the hydrogel with micro-nano structure developed by the present invention; (A) Synthesis of I / G-LPMSN@HAMA; (B) Antibacterial and antitumor mechanism. Detailed implementation manners

[0045] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content still belong to the protection scope of the present invention.

[0046] Example 1

[0047] I. Experimental materials and methods

[0048] 1. Synthesis of I / G-LPMSN nanoparticles

[0049] Pre-mix 48 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), 18 mg of NHS (N-hydroxysuccinimide) and 0.26 mg of thymine-1-acetic acid in 1 ml of pure water (pH adjusted to neutral) and stir for 1 hour. Subsequently, add 10 mg of MSN-NH2 (aminated silica) to the solution and stir for 12 hours to obtain MSN-T (silica-thymine). Add 100 μg of Anti-LPS (lipopolysaccharide antibody) and 2 mg of MSN-T to 1 mL of pure water respectively and stir at room temperature for 4 h. Subsequently, add 2 mg of GEM (gemcitabine) and 1 mg of ICG (indocyanine green), and stir in pure water for another 12 h. After three thorough washing steps, add 2 mg of PolyA (polyadenylic acid) and continue to stir for 2 h to obtain the final product, denoted as I / G-LPMSN.

[0050] Referring to the above method, the final products obtained by adding ICG alone or GEM alone in the MSN formulation are named ICG-LPMSN or GEM-LPMSN.

[0051] 2. Characteristics and photostability of I / G-LPMSN nanoparticles

[0052] Absorbance was measured using a microplate reader (Multiskan GO). The particle size and Zeta potential were detected using a Zetasizer Nano ZSE. The morphology and elemental analysis map were observed using a transmission electron microscope (TEM).

[0053] Regarding photostability, add I / G-LPMSN and free ICG (3 ml) into quartz test tubes respectively. Then, irradiate the samples with magnetic stirring at 785 nm and 1.0 W / cm 2 for 0.5, 1, 2, 3, 5 and 7 min. The ultraviolet-visible absorbance of the samples was measured at each time point to determine their photostability under light irradiation.

[0054] 3. Photothermal conversion efficiency and photothermal effect of nanoparticles

[0055] First, measure the absorbance of the free ICG solution and the I / G-LPMSN solution at 785 nm using a microplate reader. Then, irradiate the samples at 785 nm and 1.0 W / cm 2 until the temperature no longer rises. Subsequently, the light irradiation is removed. Monitor the sample temperature with a thermometer every 30 s until the sample temperature returns to room temperature. Finally, calculate the photothermal conversion efficiency according to the formula in the existing literature.

[0056] To further study the photothermal effect, the temperature of the samples was monitored using thermal imaging technology. I / G-LPMSN and free ICG (500 μL) were irradiated at concentrations of 5, 10, 20, 50, and 100 μg / mL for 5 min under 785 nm (1.0 W / cm 2 ) irradiation. The temperature and thermal images of the samples were recorded every 1 min.

[0057] 4. Drug release performance

[0058] Dialysis was used to detect the drug release behavior. 1 mL of GEM and 1 mL of I / G-LPMSN (without irradiation) and 1 mL of I / G-LPMSN (with light irradiation) were added into dialysis bags respectively. Then the dialysis bags were immersed in 5 mL of PBS. Next, PBS was collected and replaced at different time points. Finally, the content of GEM in PBS was determined using an enzyme-linked immunosorbent assay (ELISA) reader.

[0059] 5. Cell viability

[0060] The cell viability of the samples was determined using the CCK8 method. K7M2 cells (5000 cells / well) were seeded in 96-well plates overnight. Free ICG, free GEM, GEM-LPMSN, ICG-LPMSN, and I / G-LPMSN at concentrations of 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / mL were added into the wells and incubated for 24 h. Then, the cells were exposed to (785 nm, 1.0 W / cm 2 , 3 min), incubated for another 24 h or not incubated for 24 h, and then incubated with CCK8 solution for 0.5 h. Finally, the cell viability was measured using a microplate reader.

[0061] 6. Live / dead staining

[0062] The cytotoxicity of the samples was further determined using a live / dead staining kit. Calcein-AM shows green fluorescence, representing live cells. PI emits red fluorescence, indicating that the cells are dead. K7M2 cells (5×10 4 / well) were seeded in 24-well plates overnight. GEM-LPMSN, ICG-LPMSN, and I / G-LPMSN were added into the cells and incubated for 24 h. Then the cells were irradiated at 785 nm, 1.0 W / cm 2 for 3 min or not irradiated. After 24 h of irradiation, the cells were treated with the live / dead staining kit and incubated at 37 °C for 30 min. Finally, the cells were washed with PBS.

[0063] 7. Intracellular distribution

[0064] Using Lysotracker Green DND 26 as a probe for lysosomes and Hoechst 33342 as a biomarker for cell nuclei. Seed K7M2 cells in a 6-well plate overnight. Add I / G-LPMSN to the wells and incubate for 2 h. Then irradiate the cells (785 nm, 1.0 W / cm 2 , 3 min) or not. Subsequently, add Hoechst 33342 to the plate and incubate at 37 °C for 10 min. Place Lysotracker Green DND 26 in the culture dish and incubate for another 3 min. Finally, wash the cells 3 times with PBS and observe using a fluorescence imager.

[0065] 8. Bacteriostatic Activity

[0066] To verify the bacteriostatic activity of I / G-LPMSN, inoculate suspensions of PBS, I / G-PMSN, and I / G-LPMSN with EcN in LB medium. After co-incubation for 2 h, irradiate the EcN (785 nm, 1.0 W / cm 2 , 3 min) or not, and measure the OD at time points of 0.5, 1, 2, 4, 6, 12, and 24 h after irradiation. 600 Quantitatively evaluate the bacteriostatic effect using the colony counting method. The treatment method is the same as before. After the irradiation treatment, take out the mixture, dilute it 1000-fold with sterile PBS, pipette 100 μL of the bacterial solution and add it dropwise to the culture dish, spread it evenly, and count the number of growing colonies after overnight culture.

[0067] 9. Live / Dead Staining of Bacteria

[0068] Use a bacterial live / dead staining kit to measure the antibacterial effect. STYO 9 shows green fluorescence, representing live bacteria. PI emits red fluorescence, indicating that the bacteria are dead. Inoculate suspensions of PBS, I / G-PMSN, and I / G-LPMSN with EcN in LB medium. After co-incubation for 2 h, irradiate the EcN (785 nm, 1.0 W / cm 2 , 3 min) or not, and measure the live / dead of bacteria using a bacterial live / dead staining kit.

[0069] 10. In Vitro Intracellular Antibacterial Experiment

[0070] Seed K7M2 cells (1×10 5Inoculate (into the wells) in a 24-well plate and incubate overnight. Gently aspirate the culture medium, perform bacterial infection at an MOI of 10:1, and place it in a carbon dioxide incubator for 2 h. Subsequently, gently wash three times with PBS until no bacteria are observed under an optical microscope, and incubate with complete medium containing 50 μg / mL kanamycin for 1 h under the same conditions. Add PBS, I / G-PMSN, and I / G-LPMSN (at an encapsulated ICG concentration of 100 μg / mL). Then incubate the cells for another 3 h, and then irradiate (785 nm, 1.0 W / cm 2 , for 3 min) or not. Then lyse with 0.1% Triton X-100 and count the CFUs. Use a pipette to aspirate 100 μL of the lysate and add it dropwise to a culture dish, spread it evenly, and count the number of colonies that grow after overnight culture.

[0071] 11. Synthesis and Characterization of I / G-LPMSN@HAMA Hydrogel Microspheres

[0072] Use mineral oil as the oil phase and 5 wt% HAMA and 0.5 wt% photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylacrylic acid as the aqueous phase. Then, use an infusion pump and a microfluidic chip to control the flow rates between the oil phase and the aqueous phase. Ensure the uniform and controllable size of the hydrogel microspheres under a microscope. Finally, expose the product to ultraviolet light for gel curing. Mix HAMA with I / G-LPMSN in pure water to obtain I / G-LPMSN@HAMA. Then observe the morphology of I / G-LPMSN@HAMA using a scanning electron microscope and a fluorescence microscope.

[0073] 12. Near-Infrared Fluorescence Images

[0074] The experiment was carried out under the guidance of the JXMC Laboratory Animal Ethics Committee, ethics number: JUMC2021-128. To study the residual tumors in the samples, an infrared camera was used. Balb / c mice carrying K7M2 tumor cells were injected intratumorally with free ICG, I / G-LPMSN, and I / G-LPMSN@HAMA. Then, the mice were observed using a small animal in vivo imager at 0, 1, 4, and 7 d after injection.

[0075] 13. In Vivo Thermal Imaging

[0076] Balb / c mice carrying K7M2 tumor cells were injected intratumorally with PBS, I / G-LPMSN, and I / G-LPMSN@HAMA. Then, irradiate the tumor (785 nm, 1.0 W / cm 2 ) for 5 min at 24 h after injection. Use an infrared thermal imager to monitor the temperature of the tumor area.

[0077] 14. In Vivo Antitumor Efficacy and Immunostaining Analysis

[0078] PBS, free ICG, I / G-PMSN@HAMA, and I / G-LPMSN@HAMA were injected separately into the tumors. Then, 24 h after injection, the tumors were irradiated at 785 nm (1.0 W / cm 2 , 3 min) or not irradiated. The tumor volume was measured at regular intervals within the next 14 days. Finally, the tumors were excised from the mice for photography 14 d after injection.

[0079] The tumors of mice with different treatments were fixed by soaking in 4 wt% paraformaldehyde. Then the tumors were cut into slides and blocked for 1 h. The tumor slides were incubated with primary antibodies (CD4, CD8, Ki67), and then with secondary antibodies for immunofluorescence staining. Hematoxylin and eosin (H&E) staining was performed, and the sections were incubated with hematoxylin and eosin in turn. Finally, the slides were observed under a fluorescence microscope, and the images were analyzed using ImageJ software.

[0080] 15. CFU counting test in tumors

[0081] One day before drug injection, 100 μL of 5×10 6 CFU / mL EcN was injected into the tumors of mice in situ. The mice were sacrificed on the 14th day after bacterial injection, and the tumors were excised. The tumor tissues were cut into 1-mm pieces and transferred to sterile tubes. 1 mL of tissue preservation solution was added and homogenized by a homogenizer. Finally, 0.1% Triton X-100 was added to lyse the cells, and 100 μL of the lysate was taken on LB agar. Incubated at 37 °C for 24 h, and the CFUs were counted.

[0082] II. Experimental results and discussion

[0083] 1. Preparation and characterization of I / G-LPMSN

[0084] I / G-LPMSN was prepared by three-step surface functionalization on the surface of mesoporous silica. First, MSN-NH2 was chemically coupled with thymine-1-acetic acid to generate MSN-thymine (MSN-T) through an amidation reaction, and then amidated with anti-LPS antibody for chemical coupling to encapsulate ICG and GEM in the pores. Finally, the pores of MSN were sealed with PolyA according to the complementary A-T base pairing principle to obtain I / G-LPMSN. To confirm the successful synthesis, the ultraviolet absorption of GEM, ICG, MSN-T, and I / G-LPMSN was measured ( Figure 1 in A). The results showed that I / G-LPMSN had characteristic absorption peaks of ICG and GEM, indicating the successful preparation of the drug. Dynamic light scattering (DLS) analysis showed that the average size of I / G-LPMSN was 295 nm ( Figure 1In B), this may be due to partial aggregation in pure water. Transmission electron microscopy (TEM) images show that its morphology is uniform, with an average particle size of 70 nm( Figure 1 In C). Compared with MSN-T, the size of I / G-LPMSN is significantly larger( Figure 7 In A and B). To further confirm the successful formation of the nanoparticles, the Zeta potential of various samples was also measured. The change in Zeta potential indicates the change in the composition of the nanoparticles( Figure 1 In D). Elemental analysis of I / G-LPMSN further confirmed the successful synthesis (Si from MSN, F from GEM, P from Poly-A, S from ICG)( Figure 1 In E, Figure 8 ). These all prove the successful construction of I / G-LPMSN.

[0085] Then, the photostability of the nanoparticles was determined by the change in ultraviolet absorption. The results show that I / G-LPMSN exhibits stronger photostability compared with ICG( Figure 1 In F and G). Under irradiation, the absorption peak of GEM at 264 nm remains stable( Figure 1 In G), indicating that the potency of GEM is not affected by light irradiation.

[0086] In addition, to detect the photothermal effect of I / G-LPMSN, we used an infrared thermal imager to monitor the temperature of solutions with different concentrations under 785 nm irradiation( Figure 1 In H), and the results showed a dose-dependent photothermal effect( Figure 1 In I). We also further determined the photothermal conversion efficiency, and the results showed that the photothermal conversion efficiency of I / G-LPMSN was 32.6%, exceeding 20.1% of ICG( Figure 1 In J, Figure 9 ), and it has good anti-tumor and antibacterial effects in vivo.

[0087] The A-T pairing of adenine (A) and thymine (T) is base pairing, which can be observed when DNA strands, RNA strands, or a combination of both form complementary interactions. PolyA (adenine base sequence) pairs with thymine and is designed for photothermal-responsive release behavior, and the release kinetics are regulated using this base pairing mechanism. As Figure 1 shown in K, the cumulative release amount of GEM from I / G-LPMSN under irradiation is greater than that without irradiation, indicating that I / G-LPMSN can control drug release after irradiation.

[0088] 2. Cytotoxicity and intracellular distribution

[0089] To evaluate the cytotoxicity of the nanoparticles, CCK8 assay and live / dead staining were used. AsFigure 2 As shown in A, GEM has high cytotoxicity against K7M2 cells. However, when encapsulated in MSN carriers, its biocompatibility is improved ( Figure 2 as shown in B). In the absence of light, ICG shows excellent biocompatibility both as a monomer and as nanoparticles ( Figure 2 as shown in C and E). When irradiated, ICG and ICG-LPMSN have the same ability to produce photothermal effects in K7M2 cells ( Figure 2 as shown in D and F). Under 785 nm irradiation, the cytotoxicity of I / G-LPMSN is significantly enhanced. ( Figure 2 as shown in G and H), attributed to the cytotoxic effect of irradiation-induced increased release of GEM. In addition, the cell damage of K7M2 cells was further evaluated by live / dead staining ( Figure 2 as shown in I), and it was found that under 785 nm irradiation, the cell mortality rate of cells treated with I / G-LPMSN was higher, further confirming its cytotoxic effect.

[0090] To explore the intracellular distribution of I / G-LPMSN, we used the lysosome marker LysoTracker Green DND-26 and the nuclear dye Hoechst 33342 to visualize lysosomes and the nucleus ( Figure 2 as shown in J). In the absence of light, I / G-LPMSN showed obvious co-localization with lysosomes. However, lysosome rupture was observed in K7M2 cells after light irradiation, resulting in a significant reduction in co-localization. This indicates that ICG-induced photodamage can effectively promote the cytoplasmic release and transport of GEM.

[0091] 3. Antibacterial study in vitro

[0092] To improve the antibacterial activity of nanoparticles, anti-LPS antibodies were modified to achieve tumor-targeting. It has been previously reported that lipopolysaccharide is a typical Gram-negative bacterial biomarker contained in the osteosarcoma microenvironment 13. To verify the antibacterial activity of I / G-LPMSN, we inoculated suspensions of EcN with different formulations (PBS, I / G-PMSN, I / G-LPMSN) into LB medium and measured OD600 at specific times ( Figure 3 as shown in A). After light irradiation, both I / G-PMSN and I / G-LPMSN showed obvious antibacterial effects, and the antibacterial effect of the I / G-LPMSN group after light irradiation was stronger than that of the I / G-PMSN group, which may be related to the targeted anti-LPS antibody. The antibacterial effect was further observed by colony counting method ( Figure 3 as shown in B). Under 785 nm light irradiation, the number of colonies treated with I / G-LPMSN was the least. In addition, we also carried out the same antibacterial experiment at a higher concentration of ICG ( Figure 10In A and B), the results showed that both I / G-PMSN and I / G-LPMSN after light exposure exhibited better antibacterial ability. In addition, we further studied the antibacterial ability of bacteria using a live / dead staining kit ( Figure 3 In C). The highest bacterial mortality rate was observed in the I / G-LPMSN light-exposed group, further confirming its antibacterial ability.

[0093] To study the bactericidal ability of EcN-infected K7M2 cells against intracellular bacteria, different formulations were used under light or non-light conditions, and then CFU counting was performed after lysing with 0.1% Triton X-100 ( Figure 3 In D and E). There was no statistically significant difference in bacteria treated with I / G-LPMSN and those without light exposure to I / G-LPMSN. Under 785 nm irradiation, both I / G-PMSN and I / G-LPMSN-treated bacteria had good killing effects. In addition, I / G-LPMSN had a good antibacterial effect on intracellular bacteria, which may be related to the targeting effect of anti-LPS antibodies.

[0094] 4. In vitro immunogenic cell death study

[0095] To determine in vitro immunogenic cell death, we performed immunofluorescence detection using immunogenic cell death probes. The typical characteristics of immunogenic cell death are increased expression of calreticulin (CRT) on the cell surface and release of high-mobility group box-1 (HMGB-1). As Figure 11 shown, K7M2 tumor cells treated with I / G-LPMSN under 785 nm irradiation expressed higher levels of CRT, and the cell morphology also became fragmented. For the expression of HMGB-1 ( Figure 12 ), under 785 nm irradiation, cells treated with I / G-LPMSN showed the opposite results, which may be due to the release of HMGB-1 into the extracellular matrix.

[0096] 5. Preparation and characterization of hydrogel microspheres

[0097] Since nanomaterials are easily metabolized by organs such as the liver and spleen through systemic administration, they may cause serious side effects due to their cytotoxicity. In addition, due to the large pores in the tumor blood vessel wall, their retention in tumors is poor. Therefore, constructing hydrogels with nanostructures is of great significance for promoting tumor retention and enhancing immune activation. First, we used microfluidic technology to adjust the flow rate ratio of the oil phase and the water phase to synthesize HAMA hydrogel microspheres with uniform and controllable sizes. Using the above mapping conditions, we further synthesized I / G-LPMSN@HAMA with nanostructures and observed I / G-LPMSN@HAMA using a fluorescence microscope ( Figure 4In A). The results showed that the nanoparticles with red fluorescence were uniformly distributed in the microspheres, further proving the uniform loading of the nanoparticles in the hydrogel microspheres with nano-microstructures.

[0098] To further prove the successful loading of the nanoparticles, the morphology of I / G-LPMSN@HAMA was characterized by scanning electron microscopy (SEM). The results showed that a dense layer of nanoparticles covered the surface of I / G-LPMSN@HAMA ( Figure 4 In B). The elemental analysis of I / G-LPMSN@HAMA also proved the successful loading of the nanoparticles ( Figure 4 In C and 4). As Figure 13 shown, the cumulative release of GEM in I / G-LPMSN@HAMA showed a slow release behavior within one month under irradiation, which may contribute to the effective inhibition of tumors. In addition, live / dead staining was used to further evaluate the cell damage of K7M2 cells and EcN ( Figure 14 and Figure 15 ), showing that under 785 nm irradiation, the cell mortality and bacterial mortality of the cells treated with I / G-LPMSN@HAMA were higher.

[0099] 6. Tumor retention and in vivo thermal imaging studies

[0100] To evaluate the retention of tumors in vivo, we detected the fluorescence of free ICG, I / G-LPMSN, and I / G-LPMSN@HAMA in mice bearing K7M2 tumor cells using a near-infrared (NIR) fluorescence imaging system. As Figure 4 shown in E and G, the fluorescence intensity of the mice treated with free ICG decreased continuously, while the fluorescence intensity of the mice injected with I / G-LPMSN and I / G-LPMSN@HAMA showed an explosive increase at 24 h after injection and a decreasing trend within 7 d. However, at 7 d after injection, the fluorescence intensity of the mice treated with I / G-LPMSN@HAMA was higher than that of the mice treated with free ICG and I / G-LPMSN. This indicates that compared with the nanoparticles, the hydrogel with nano-microstructures has better tumor retention ability, which may contribute to prolonging the eradication time of osteosarcoma cells.

[0101] In addition, we used an infrared thermal imager to monitor the in vivo photothermal effect ( Figure 4 in F and H). The mice treated with I / G-LPMSN@HAMA showed a similar photothermal effect to I / G-LPMSN, thus ensuring subsequent effective in vivo tumor inhibition.

[0102] 7. Photoimmunotherapy for osteosarcoma

[0103] To demonstrate their synergistic effect in osteosarcoma rich in intratumoral bacteria, we first constructed a mouse model carrying K7M2 cells and injected bacteria. Then, we measured the tumor volume 14 days after injection ( Figure 5 A and B in). Under 785 nm irradiation, the tumor growth rate of mice in the GEM+ICG group and the I / G-PMSN@HAMA group decreased compared with that of the PBS group within 14 days, indicating the photothermal chemotherapy effect on tumors. The tumor growth curve of mice treated with I / G-LPMSN@HAMA was slower than that of mice treated with I / G-PMSN@HAMA, which was due to the presence of anti-LPS antibodies, indicating enhanced tumor suppression.

[0104] To further confirm the bacteriostatic effect on tumor bacteria, the tumor bacteria were lysed and the bacterial CFU was counted ( Figure 5 C and D in). Under 785 nm irradiation, the bacterial proliferation in mice treated with ICG+GEM decreased slightly. In addition, under 785 nm irradiation, both I / G-PMSN@HAMA and I / G-LPMSN@HAMA had good bacteriostatic effects, indicating that the photothermal effect had good damage to bacteria. However, under 785 nm irradiation, almost no bacteria survived in the osteosarcoma of mice treated with I / G-LPMSN@HAMA, which might be due to the modification of LPS antibodies ( Figure 5 D in).

[0105] Then we performed immunofluorescence staining to further examine the tumor immune microenvironment ( Figure 5 E in). In the tumors treated with I / G-LPMSN@HAMA irradiation, the expressions of CD4+ T cells ( Figure 5 F in) and CD8+ T cells ( Figure 5 G in) were the highest, which was beneficial to the anti-tumor immune response. This might indicate that dead bacteria could also enhance immunogenicity and induce immune activation.

[0106] Finally, to further determine the tumor damage ability, we performed immunohistochemical treatment on the tumors and analyzed the images using imageJ. Hematoxylin and eosin (H&E) staining showed that the tumor cells of mice irradiated with I / G-LPMSN@HAMA were severely damaged ( Figure 6 A in). No obvious damage was seen in normal tissues such as the heart, liver, spleen, lungs, and kidneys ( Figure 16 ). In addition, we measured the immunofluorescence staining and tunnel staining of ki67 ( Figure 6 A in). Ki67 is an indicator of cell proliferation characteristics. As Figure 6As shown in B, after light irradiation, Ki67 was most strongly expressed in the PBS group and least strongly expressed at I / G-LPMSN@HAMA, indicating that the tumor cells at I / G-LPMSN@HAMA hardly proliferated after irradiation. In addition, Tunel staining is an indicator of apoptosis. The results also showed that under 785 nm irradiation, the apoptosis rate of tumor cells in mice treated with I / G-LPMSN@HAMA was the highest ( Figure 6 in C). The above results indicate that the targeted bacterial hydrogel with nano-microstructure causes the most serious damage to tumors under irradiation and is an effective method for treating bacteria-rich tumors. In summary, this indicates that I / G-LPMSN@HAMA can significantly enhance the cytotoxic effect on osteosarcoma cells by inducing immune activation.

[0107] III. Conclusion

[0108] In summary, the present invention established photo-immunogel microspheres with micro-nano structures through microfluidic technology. I / G-LPMSN@HAMA exhibited stronger tumor retention and effective photo-immune activation. In particular, I / G-LPMSN@HAMA in tumors could effectively generate a thermal effect, induce photo-damage, and enhance the immunogenicity of tumor cells. At the same time, the LPS antibody carried on I / G-LPMSN@HAMA and the induced photo-thermal damage could effectively kill the bacteria in the tumor, improve the tumor microenvironment, and thus enhance the anti-tumor effect of the microspheres ( Figure 17 ). It can be seen that the proof-of-concept design of the photo-immunogel microspheres developed in the present invention provides new possibilities for immunosuppressive tumor treatment.

[0109] Comparative Example 1

[0110] According to the experimental steps in Example 1, considering the influence of different raw material ratios on the drug loading rate and encapsulation rate of the nanoparticles, the results are as shown in Table 1 below. It can be seen that when the weight ratio of drug GEM to IGG is 2:1, the prepared nanoparticles have higher drug loading rate and encapsulation rate.

[0111] Table 1

[0112]

[0113]

Claims

1. A preparation method of a micro-nano structured light-responsive hydrogel microsphere, characterized in that, It includes the following steps: (1) Using EDC and NHS as activators, mix and stir with thymine-1-acetic acid, and then add amino-modified mesoporous silica for reaction to obtain silica-thymine; (2) Mix silica-thymine with lipopolysaccharide antibody solution, then add gemcitabine and indocyanine green, after stirring and reacting, wash to remove impurities, add polyadenylic acid to the obtained product, and continue stirring and reacting to obtain drug-loaded nanoparticles; (3) Mix methacrylated hyaluronic acid, photoinitiator and the drug-loaded nanoparticles described in step (2) as the aqueous phase, use mineral oil as the oil phase, prepare hydrogel microspheres through a microfluidic device, and then cure with ultraviolet light to obtain the micro-nano structured light-responsive hydrogel microspheres.

2. The method according to claim 1, characterized in that In step (1), the weight ratio of the EDC, NHS and thymine-1-acetic acid is 24:9:0.

13.

3. The method according to claim 1, characterized in that, In step (1), the weight ratio of the amino-modified mesoporous silica to NHS is 1:1.

8.

4. The method according to claim 1, characterized in that In step (1), the reaction time is 12 hours.

5. The method according to claim 1, characterized in that In step (2), the weight ratio of the lipopolysaccharide antibody to silica-thymine is 1:20; the weight ratio of the polyadenylic acid to silica-thymine is 1:

1.

6. The method according to claim 1, characterized in that In step (2), the weight ratio of the silica-thymine, gemcitabine and indocyanine green is 2:2:

1.

7. The method according to claim 1, characterized in that, In step (3), the concentration of the methacrylated hyaluronic acid is 5 wt%.

8. The micro-nano structured light-responsive hydrogel microspheres prepared by the method according to any one of claims 1-7.

9. Use of the micro-nano structured light-responsive hydrogel microspheres according to claim 8 in the preparation of drugs for in-tumor bacteria clearance and treatment.

10. The application according to claim 9, wherein The tumor includes osteosarcoma.

Images