Drug-loaded nanoparticles, preparation method, and application thereof

Abstract

The present invention discloses a drug-loaded nanoparticle and its preparation method and application. The drug-loaded nanoparticle comprises: a hydrophobic anti-tumor drug, an amphiphilic polymer and a tannic acid-manganese complex; the hydrophobic anti-tumor drug is coated in the amphiphilic polymer; the tannic acid-manganese complex is loaded on the surface of the amphiphilic polymer; wherein the amphiphilic polymer comprises a hydrophilic segment and a hydrophobic segment, the hydrophilic segment is selected from polyethylene glycol; the hydrophobic segment is selected from poly(lactic acid-co-glycolic acid) copolymer. The present invention uses the amphiphilic polymer to encapsulate the hydrophobic anti-tumor drug, and uses tannic acid polyphenol to chelate metal manganese on its surface to form a metal-phenol network covering it, ultimately obtaining drug-loaded nanoparticles; achieving in situ killing of tumor cells and release of TAAs. Thus, a long-term and efficient immune response is achieved. At the same time, the material also has an imaging effect, which facilitates real-time monitoring of tumor tissue during treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a drug-loaded nanoparticle, a preparation method and an application thereof. Background Art

[0002] Immunotherapy based on immune checkpoint inhibitors has opened up new ideas for cancer treatment, but only a few patients can truly benefit from it. One of the important reasons is that the immunosuppressive microenvironment of the tumor itself limits the effect of tumor immunotherapy. Simply put, the tumor immune microenvironment can be divided into "cold" tumors (little immune cell infiltration) and "hot" tumors (much immune cell infiltration). In recent years, the important functions of the cGAS-STING pathway in innate immunity and adaptive immune responses have gradually been revealed, which has aroused people's exploration of the use of cGAS-STING pathway agonists as vaccine adjuvants. After activation, the cGAS-STING pathway has many functions on the immune system, including promoting cross-presentation of antigen-presenting cells, promoting the transformation of M2 macrophages to M1 macrophages, and promoting CD4 + Differentiation and activation of CD8 T cells + T cell immune response, etc. The stimulation of the cGAS-STING pathway by cytoplasmic nucleic acids can activate the transcription factors IRF3 and NF-kB, promote the expression of type I interferon and other proinflammatory factors, thereby enhancing antigen presentation and adaptive immune response.

[0003] Although promising, the direct application of STING agonists in vivo faces considerable challenges. Natural STING agonists include 2'3'-cGAMP, 3'3'-cGAMP, c-di-GMP, and c-di-AMP. cGAMP can bind to the open STING dimer, causing it to undergo conformational changes, subsequently aggregating and activating downstream pathways. However, the use of cGAMP or other natural conventional cyclic dinucleotides (CDNs) in vivo has been greatly hindered due to their poor pharmacokinetic profiles. Their negative charge and highly polar properties result in limited cell penetration. In addition, CDNs have a short half-life because the phosphodiester bond is easily hydrolyzed. Clinically, CDNs are only used for intratumoral injection, which limits their use in the treatment of metastatic tumors. The non-nucleotide small molecule agonist DMXAA, once considered a promising alternative to cGAMP, failed in Phase III clinical trials.

[0004] Therefore, it is necessary to design a new drug-loaded nanoparticle that can activate the STING pathway to reverse the immunosuppressive microenvironment, thereby inducing a long-term and efficient immune response; and has the function of MRI T1 imaging, which can realize real-time monitoring of tumor treatment effects using MRI while treating tumors. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, in a first aspect, the present invention proposes a drug-loaded nanoparticle that can activate the STING pathway to reverse the immunosuppressive microenvironment, thereby inducing a long-term and highly effective immune response. The nanoparticle also has MRI T1 imaging capabilities, enabling real-time monitoring of tumor treatment efficacy using MRI while treating tumors.

[0006] The second aspect of the present invention also provides a method for preparing drug-loaded nanoparticles.

[0007] The third aspect of the present invention also provides an application of drug-loaded nanoparticles.

[0008] According to the first aspect of the present invention, a drug-loaded nanoparticle is provided, comprising:

[0009] hydrophobic antitumor drugs;

[0010] An amphiphilic polymer; the amphiphilic polymer is coated with a hydrophobic anti-tumor drug;

[0011] Tannic acid-manganese complex; the tannic acid-manganese complex is loaded on the surface of the amphiphilic polymer;

[0012] The amphiphilic polymer comprises a hydrophilic segment and a hydrophobic segment, the hydrophilic segment is selected from polyethylene glycol; the hydrophobic segment is selected from at least one of polylactic acid-co-glycolic acid, polylactic acid, polycaprolactone or polyphosphate.

[0013] The drug-loaded nanoparticles according to the embodiments of the present invention have at least the following beneficial effects:

[0014] The present invention uses an amphiphilic polymer to encapsulate a hydrophobic anti-tumor drug, and uses tannic acid polyphenols to chelate metal manganese on its surface to form a metal-phenol network covering it, ultimately obtaining drug-loaded nanoparticles, and utilizing the hydrophobic interaction between the hydrophobic block of the amphiphilic polymer and the hydrophobic anti-tumor drug to effectively encapsulate the drug and form an amphiphilic drug-loaded nanosystem; using the amphiphilic drug-loaded nanosystem as a template, compared with hyaluronic acid, natural tannic acid polyphenols can chelate with metal manganese ions in large quantities on the surface of the hydrophilic layer to form a metal-phenol network, ultimately obtaining drug-loaded nanoparticles that efficiently co-load hydrophobic anti-tumor drugs and hydrophilic manganese ions; the drug-loaded nanoparticles can effectively accumulate at the tumor site and co-deliver two different drugs, achieving in situ killing of tumor cells and release of TAAs. At the tumor site, the hydrophobic anti-tumor drug induces DNA damage, promotes the release of dsDNA into the cytoplasm, initiates the activation of the STING pathway, and Mn 2+As a STING pathway agonist, it can synergistically enhance the DNA-activated STING pathway, thereby promoting the release of large amounts of type I interferon. Type I interferon can promote the cross-presentation of antigens by antigen-presenting cells and activate CD8 + T cells, induced CD4 + The activation of the STING pathway is conducive to the infiltration of T lymphocytes, thereby achieving a long-term and efficient immune response. 2+ It can be used as a contrast agent to perform specific magnetic resonance imaging in tumor tissue. The imaging function of the drug-loaded nanoparticles facilitates real-time monitoring of tumor tissue during treatment. In addition, the drug-loaded nanoparticles also have good biocompatibility and degradability, and have great clinical application prospects.

[0015] According to some embodiments of the present invention, the hydrophobic anti-tumor drug accounts for 3.7-4.9% of the mass of the drug-loaded nanoparticles.

[0016] According to some embodiments of the present invention, the average particle size of the drug-loaded nanoparticles is 60-80 nm.

[0017] According to some embodiments of the present invention, the hydrophobic anti-tumor drug is selected from at least one of camptothecin, paclitaxel, hydrophobic doxorubicin or daunorubicin.

[0018] According to some embodiments of the present invention, the hydrophobic anti-tumor drug is selected from hydrophobic doxorubicin. Hydrophobic doxorubicin, as a topoisomerase inhibitor, interferes with DNA replication, thereby inducing apoptosis in tumor cells. In addition, doxorubicin damages double-stranded DNA, causing it to be released into the cytoplasm, thereby activating STING pathway signaling.

[0019] According to some embodiments of the present invention, the hydrophobic doxorubicin is prepared by the following method:

[0020] Doxorubicin hydrochloride was dissolved in water, triethylamine was added dropwise under stirring, and the mixture was stirred overnight. The mixture was then centrifuged several times until no red color was left in the supernatant. The resulting hydrophobic doxorubicin was lyophilized.

[0021] According to some embodiments of the present invention, the molecular weight of the hydrophilic segment is 2000-8000.

[0022] According to some embodiments of the present invention, the molecular weight of the hydrophobic segment is 9,000 to 15,000.

[0023] According to a second aspect of the present invention, an embodiment provides a method for preparing the above-mentioned drug-loaded nanoparticles, comprising the following steps:

[0024] S1, mixing an amphiphilic polymer dissolved in a first organic solvent, a hydrophobic antitumor drug dissolved in a second organic solvent, and water, and removing the first organic solvent and the second organic solvent by dialysis to obtain an amphiphilic polymer solution coated with the hydrophobic antitumor drug;

[0025] S2. Adjusting the pH of the amphiphilic polymer solution coated with the hydrophobic anti-tumor drug to 7.9-8.2, mixing it with the tannic acid solution and the manganese salt solution, and ultrafiltration to obtain drug-loaded nanoparticles.

[0026] According to some embodiments of the present invention, in step S1, the mass ratio of the amphiphilic polymer to the hydrophobic anti-tumor drug is 8 to 12:1.

[0027] According to some embodiments of the present invention, the concentration of tannic acid in the tannic acid solution is 20-60 mg / mL.

[0028] According to some embodiments of the present invention, the concentration of the manganese salt in the manganese salt solution is 10-30 mg / mL.

[0029] According to some embodiments of the present invention, the manganese salt includes at least one of manganese chloride tetrahydrate, manganese chloride dihydrate, manganese chloride, manganese sulfate heptahydrate, and manganese nitrate tetrahydrate.

[0030] According to some embodiments of the present invention, the first organic solvent and the second organic solvent are independently selected from at least one of dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).

[0031] According to some embodiments of the present invention, the volume ratio of the first organic solvent and the second organic solvent to water is 1:4.5-10.

[0032] According to some embodiments of the present invention, the ultrafiltration speed is 3000-5000 rpm.

[0033] According to some embodiments of the present invention, the ultrafiltration is used to remove tannic acid and manganese ions that do not form a tannic acid-manganese complex.

[0034] The third aspect of the present invention provides the use of the above-mentioned drug-loaded nanoparticles in the preparation of anti-tumor drugs.

[0035] According to some embodiments of the invention, the tumor comprises breast cancer.

[0036] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0038] Figure 1 Schematic diagram of the synthesis route of TMPD NPs in Example 1;

[0039] Figure 2 The particle size diagram and transmission electron microscopy image of TMPD NPs in Example 1;

[0040] Figure 3 is the scanning electron microscopy elemental mapping of TMPD NPs of Example 1;

[0041] Figure 4 Potential diagrams and UV absorption diagrams of PD NPs, TMP NPs, and TMPD NPs;

[0042] Figure 5 To detect the apoptosis and cytotoxicity induced by PD NPs, TMP NPs, and TMPD NPs on 4T1 cells;

[0043] Figure 6 Western blot detection of STING pathway activation induced by PD NPs, TMP NPs, and TMPD NPs and relative quantitative graph;

[0044] Figure 7 ELISA was used to detect the production of type I interferon and flow cytometry was used to detect the maturation of DC cells induced by PD NPs, TMP NPs, and TMPD NPs;

[0045] Figure 8 Schematic diagram of T1-weighted MRI of mice before and after injection of TMPD NPs;

[0046] Figure 9 Figure 2 shows the in vivo therapeutic efficacy of PD NPs, TMP NPs, and TMPD NPs;

[0047] Figure 10 The figure shows the flow cytometry detection of immune cell infiltration in mouse tumors.

[0048] The full names of the abbreviations appearing throughout the text are shown in Table 1:

[0049] Table 1

[0050]

[0051] DETAILED DESCRIPTION

[0052] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0053] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0054] Some of the raw materials used in the following examples are as follows:

[0055] Poly(lactic-co-glycolic acid) polyethylene glycol copolymer PEG 5k -PLGA 11.7k The samples were purchased from Hefei Ruyou Biotechnology Co., Ltd., DMSO from Shanghai Chemical Reagent Co., Ltd., doxorubicin (DOX) from Shanghai MacLean Biochemical Co., Ltd., and tannic acid (TA) from Aladdin Industries. Manganese chloride tetrahydrate (MnCl2·4H2O) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Other biochemical reagents were of conventional domestic analytical grade.

[0056] Reagent preparation:

[0057] 10 mg / mL DOX solution: Weigh 10 mg of hydrophobic DOX and dissolve it in DMSO to 10 mg / mL.

[0058] 10 mg / mL PEG-PLGA solution: Weigh 10 mg of PEG-PLGA and dissolve it in DMSO to 10 mg / mL.

[0059] 19.8 mg / mL MnCl2·4H2O solution: Weigh 19.8 mg of MnCl2·4H2O and dissolve it in ultrapure water to 19.8 mg / mL.

[0060] 40 mg / mL TA solution: Weigh 40 mg of TA and dissolve it in ultrapure water to 40 mg / mL.

[0061] Example 1

[0062] Example 1 provides a drug-loaded nanoparticle (TMPD NPs), the synthesis route of which is shown in FIG. Figure 1 As shown, it is prepared by the following steps:

[0063] S1. Vortex 1 mL of PEG-PLGA (10 mg / mL) solution and 100 μL of DOX (10 mg / mL) solution. Add dropwise to a 25 mL flask containing 5 mL of ultrapure water while stirring. After stirring for 1 hour, remove the solution and dialyze overnight to obtain approximately 8 mL of PD NPs.

[0064] S2. Adjust the pH of the 8 mL PD NPs solution obtained above to 8, add 20 μL of TA (40 mg / mL) solution dropwise while vortexing, and then add 20 μL of MnCl2·4H2O (19.8 mg / mL) solution dropwise while vortexing. Then, ultrafiltration was performed at 4000 rpm for 10 min to obtain TMPD NPs.

[0065] Example 2

[0066] Example 2 provides a drug-loaded nanoparticle (TMPD NPs), the synthesis route of which is shown in FIG. Figure 1 As shown, it is prepared by the following steps:

[0067] S1. Vortex 1 mL of PEG-PLGA (10 mg / mL) solution and 75 μL of DOX (10 mg / mL) solution. Add dropwise to a 25 mL flask containing 5 mL of ultrapure water while stirring. After stirring for 1 hour, remove the solution and dialyze overnight to obtain approximately 8 mL of PD NPs.

[0068] S2. Adjust the pH of the 8 mL PD NPs solution obtained above to 8, add 20 μL of TA (40 mg / mL) solution dropwise while vortexing, and then add 20 μL of MnCl2·4H2O (19.8 mg / mL) solution dropwise while vortexing. Then, ultrafiltration was performed at 4000 rpm for 10 min to obtain TMPD NPs.

[0069] Example 3

[0070] Example 3 provides a drug-loaded nanoparticle (TMPD NPs), the synthesis route of which is shown in FIG. Figure 1 As shown, it is prepared by the following steps:

[0071] S1. Vortex 1 mL of PEG-PLGA (10 mg / mL) solution and 125 μL of DOX (10 mg / mL) solution. Add dropwise to a 25 mL flask containing 5 mL of ultrapure water while stirring. After stirring for 1 hour, remove the solution and dialyze overnight to obtain approximately 8 mL of PD NPs.

[0072] S2. Adjust the pH of the 8 mL PD NPs solution obtained above to 8, add 20 μL of TA (40 mg / mL) solution dropwise while vortexing, and then add 20 μL of MnCl2·4H2O (19.8 mg / mL) solution dropwise while vortexing. Then, ultrafiltration was performed at 4000 rpm for 10 min to obtain TMPD NPs.

[0073] Example 4

[0074] Example 4 provides a drug-loaded nanoparticle (TMPD NPs), the synthesis route of which is shown in FIG. Figure 1 As shown, it is prepared by the following steps:

[0075] S1. Vortex 1 mL of PEG-PLGA (10 mg / mL) solution and 125 μL of DOX (10 mg / mL) solution. Add dropwise to a 25 mL flask containing 5 mL of ultrapure water while stirring. After stirring for 1 hour, remove the solution and dialyze overnight to obtain approximately 8 mL of PD NPs.

[0076] S2. Adjust the pH of the 8 mL PD NPs solution obtained above to 8, add 25 μL of TA (40 mg / mL) solution dropwise while vortexing, and then add 25 μL of MnCl2·4H2O (19.8 mg / mL) solution dropwise while vortexing. Ultrafiltration at 4000 rpm for 10 min yields TMPD NPs.

[0077] Comparative Example 1

[0078] Comparative Example 1 provides a PD NPs, the preparation steps of which are as follows:

[0079] 1 mL of PEG-PLGA (10 mg / mL) solution was vortexed with 100 μL of DOX (10 mg / mL) solution and then added dropwise to a 25 mL flask containing 5 mL of ultrapure water under stirring. After stirring for 1 hour, the solution was removed and dialyzed overnight to obtain approximately 8 mL of PD NPs.

[0080] Comparative Example 2

[0081] Comparative Example 2 provides a TMP NPs, and its preparation method is basically the same as that of Example 1, except that DOX is not added in Comparative Example 2.

[0082] Comparative Example 3

[0083] Comparative Example 3 provides HMPD NPs, which are prepared by a method basically the same as that in Example 1, except that in Comparative Example 3, 20 μL of TA (40 mg / mL) solution in S2 is replaced with 100 μL of sodium hyaluronate (HA-Na, 10 mg / mL) solution, which is added dropwise under vortexing conditions; subsequently, 20 μL of MnCl2·4H2O (19.8 mg / mL) solution is added dropwise under vortexing conditions.

[0084] Performance Testing

[0085] The physicochemical properties of TMPD NPs, PD NPs and TMP NPs prepared in Example 1, Comparative Example 1 and Comparative Example 2, respectively, were identified.

[0086] TMPD NPs (about 1 mg) were suspended in ultrapure water. The average particle size and particle size distribution of TMPD NPs were measured using a dynamic light scattering instrument Zetasizer Nano ZSE (Malvern, UK). Figure 2 As shown in Figure a, it can be seen that the particle size of most TMPD is concentrated in the range of 60 to 100 nm, with the majority of TMPD particles being around 70 nm. The morphology of the prepared nanoparticles was observed using a transmission electron microscope. The suspension of TMPD NPs was placed on a copper grid with a membrane, dried for 10 minutes, and observed using a field emission transmission electron microscope (Talos F200X). The results are shown in Figure 3. Figure 2 As shown in Figure b, the nanoparticles are relatively uniform spherical. Further, the elemental mapping image of TMPD was analyzed by field emission transmission electron microscopy (Talos F200X). Figure 3 As shown in the figure, it can be seen that C, O, Mn, and N elements are evenly distributed in the particles, indicating that the particles are successfully loaded with DOX and Mn. These results show that TA and Mn 2+ PD NPs can be used as templates to form TMPD NPs with consistent size, morphology and other characteristics on their surface.

[0087] In addition, the particle size of HMPD NPs prepared in Comparative Example 3 showed a multimodal distribution with large fluctuations, indicating that HA-Na and Mn 2+ The HMPD NPs formed by complexation on the surface of PD NPs are of uneven size, making it difficult to effectively load drugs, and thus limiting their research potential.

[0088] Determination of drug loading and encapsulation efficiency of nanoparticles: The encapsulation efficiency (EE) of DOX-loaded nanoparticles was determined by ultraviolet-visible (UV-Vis) spectrophotometry at 480 nm. After lyophilization of TMPD particles, 1 mg of nanoparticles was dissolved in 1 mL of dimethyl sulfoxide (DMSO), and the DOX content in the DOX-loaded nanoparticles was determined by UV-visible spectrophotometry at a wavelength of 480 nm.

[0089] The Mn encapsulation efficiency (EE) was determined by ICP-MS. The EE of TMPD was calculated as follows:

[0090] EE (%) = (mass of DOX in nanoparticles / total mass of DOX) × 100%;

[0091] The encapsulation efficiency of DOX was measured to be 39.5%; 2+ The encapsulation efficiency was 75.6%.

[0092] The Zeta potential of PD, TMP, and TMPD was measured using a dynamic light scattering instrument. Figure 4As shown in a. It can be seen that the potentials of TMPD and TMP are close to -30mV, while the PD potential is close to -25mV, indicating that the phenol-metal network formed by TA-Mn has been successfully loaded on the surface of the nanoparticles. The ultraviolet absorption peak of TMPD of the drug-loaded nanoparticles was examined by ultraviolet spectrophotometer, as shown in Figure 4 As shown in (b), there are absorption peaks at 290 nm and 480 nm, which are consistent with the characteristic peak of TMP chelation and the ultraviolet absorption peak of PD, proving that DOX is successfully loaded and the metal-phenol network of TA-Mn is coated on the surface.

[0093] Experimental Example 1: Verification of the in vitro cell-enhanced anti-tumor effect of the drug-loaded nanoparticles TMPD of the present invention:

[0094] 1. Effect of drug-loaded nanoparticles TMPD on inducing apoptosis in 4T1 cells

[0095] Mouse breast cancer cell line 4T1 cells were used as experimental subjects. These cells were incubated in culture medium containing different concentrations of TMPD as the experimental group. Culture medium supplemented with PD and TMP served as the control group. The degree of apoptosis in the incubated cells was detected using the Annexin V-APC / DAPI kit to analyze the killing effect of drug-loaded nanoparticles TMPD on 4T1 cells. The specific experimental process is as follows:

[0096] 4T1 cells were seeded into 24-well plates (1*10 5 After incubation for 24 hours, the supernatant was discarded and PD, TMP, and TMPD diluted in 1640 medium (corresponding concentrations: DOX = 10 μg / mL, Mn = 2.5 μg / mL) were added and incubated for 24 hours. Serum-free RPMI 1640 medium without drug addition was used as the PBS group. Three replicates were set up for each group. The cells were then treated with the apoptosis kit Annexin V-APC / DAPI, and the degree of cell apoptosis was detected by flow cytometry. The results are shown in Figure 2. Figure 5 As shown in a, the TMPD group induced the highest proportion of early and late apoptosis of tumor cells, indicating that TMPD has a better effect of inducing cell apoptosis compared with other groups.

[0097] 2. Cytotoxic effect of drug-loaded nanoparticles TMPD on 4T1 cells

[0098] The MTT assay was then used to detect the activity of the cells after incubation to analyze the killing effect of the drug-loaded nanoparticles TMPD on 4T1 cells by enhancing tumor immunity. The specific experimental process is as follows:

[0099] 4T1 cells were seeded into 96-well plates (8,000 cells / well) and incubated for 24 hours. The supernatant was then aspirated and the cells were added with TMPD, a drug-loaded nanoparticle, diluted to varying concentrations in serum-free RPMI 1640 medium ([DOX] = 30, 15, 7.5, 3.75, 1.825 μg / mL; [Mn] = 7.5, 3.75, 1.88, 0.94, 0.47 μg / mL). A control group was treated with TMP and PD at the same DOX or Mn concentrations, and serum-free RPMI 1640 medium without drug was used as a blank control. Six replicates were set up for each group. Incubation was carried out at 37°C, 5% CO₂ for 24 hours. After the incubation period, dilute the CCK-8 reagent with 1640 medium at a concentration of 10 μL / 1 mL per well. Replace the drug-containing medium in the 96-well plate with the 1640 medium solution containing CCK-8. Incubate at 37°C, 5% CO2 for 1 hour. Measure the absorbance at 450 nm directly using a microplate reader. Calculate the cell viability of the experimental group based on the absorbance ratio of the experimental group to the blank control group. The viability of untreated cells is defined as 100%. Cell viability is expressed by the following equation:

[0100] Cell viability = absorbance of the test group / absorbance of the negative control group × 100%;

[0101] The test results are as follows Figure 5 As shown in Figure b, TMPD is more effective than PD and TMP in killing 4T1 tumor cells, which indicates that the drug-loaded nanoparticles TMPD of the present invention have a good tumor cell killing effect.

[0102] Experimental Example 2: Verification of the activation of the STING pathway induced by the drug-loaded nanoparticles TMPD of the present invention:

[0103] TBK-1 protein and IRF-3 protein are downstream proteins of the STING pathway. Therefore, the present invention uses Western Blot to verify the phosphorylation degree of these two proteins, thereby verifying the activation of the STING pathway. First, bone marrow-derived dendritic cells (BMDCs) were extracted from the tibia and fibula of 6-week-old BALB / c mice and cultured in 24-well plates. The medium was half-changed every two days with 1640 medium containing serum and GM-CSF (20 ng / ml) and IL-4 (10 ng / ml). At the same time, 4T1 cells were plated in 12-well plates (5×10 5) and cultured overnight. Cells were treated with PD, TMP, and TMPD nanoparticles. On day 6 of DC culture, supernatant from tumor cells treated with the nanoparticles was co-cultured with BMDCs for 4 hours. DCs were then centrifuged at 450g for 10 minutes and lysed using RIPA buffer. The cells were then centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was gently aspirated to obtain the protein sample. Protein was quantified using a BCA assay kit. An appropriate volume of sample was mixed with SDS buffer and heated at 99°C for 15 minutes. The sample volume per well was ensured to be 10 μL and 30 μg of protein. Electrophoresis was then performed, the membrane was transferred, and the antibody was blocked using rapid blocking buffer. Primary antibody dilutions for TBK-1, pTBK-1, IRF-3, and pIRF-3 (Cell Signaling Technology, US) were added and incubated overnight at 4°C. The cells were then washed three times with PBST for 10 minutes each on a shaker at room temperature for decolorization. Add the secondary antibody dilution solution to the culture dish, place it on a shaker, and incubate at room temperature for 45 minutes. Wash it three times with PBST for decolorization on a shaker at room temperature, each time for 10 minutes. Then use the ImageQuant LAS 4000mini chemiluminescence imaging system for exposure and development. Figure 6 As shown in the figure, the phosphorylated TBK-1 and IRF-3 proteins in the TMPD group were significantly increased compared with the PD and TMP groups, and the corresponding quantitative results also proved this, indicating that the TMPD group significantly activated the STING pathway.

[0104] Experimental Example 3: Verification of the drug-loaded nanoparticles TMPD of the present invention inducing DC cell maturation:

[0105] 1. Drug-loaded nanoparticles TMPD produce type I interferon effects in vitro:

[0106] The concentration of IFN-β in the supernatant was detected using an ELISA kit (Dakoway, CN). Figure 7 As shown in a, the TMPD group significantly increased the proportion of mature DC cells and effectively promoted the production of type I interferon, indicating that after TMPD treated tumor cells, it activated the STING pathway, promoted the production of type I interferon, and promoted the maturation of DC cells.

[0107] 2. Effect of drug-loaded nanoparticles TMPD on DC cell activation in vitro

[0108] As in Experimental Example 1, bone marrow-derived dendritic cells (BMDCs) were extracted from the tibia and fibula of 6-week-old BALB / c mice and cultured in 24-well plates. The medium was half-changed every two days with serum-containing 1640 medium containing GM-CSF (20 ng / ml) and IL-4 (10 ng / ml). At the same time, 4T1 cells were cultured in 12-well plates (5 × 10 5) and cultured overnight. The cells were treated with PD, TMP and TMPD nanoparticles. On the 6th day of DC cell culture, the tumor cell supernatant treated with the nanoformulation was co-cultured with BMDCs for 24 hours. Subsequently, the DC cells were transferred to a centrifuge tube. After centrifugation (450g, 5min), the supernatant was separated, and CD16 / 32 antibodies were added to each tube of cells for blocking, and then 30μL of antibody mixture (anti-CD3-Per cp-Cy 5.5, anti-CD80-FITC, anti-CD86-APC / Cy7) was added and incubated in the dark at 4°C for 45min. After the incubation, PBS was filled up, centrifuged (3000g, 2min) to remove excess antibodies, 200μL of PBS was added for re-suspending, and the cell suspension was transferred to a flow tube after filtering the membrane, and the DC cell maturity was detected by flow cytometry. As Figure 7 As shown in b, the proportion of DC cells mature in vitro in the TMPD group was significantly higher than that in the other groups, indicating that TMPD can effectively induce the maturation of DCs.

[0109] Experimental Example 4: Magnetic Resonance Imaging Effect of Drug-Loaded Nanoparticles of the Present Invention

[0110] To establish the 4T1 tumor model in BALB / c mice, 1х10 6 4T1 breast cancer cells were injected into the right mammary pad of BALB / c mice, and the tumors were grown to 100 mm. 3 Five 4T1 tumor-bearing mice were injected with TMPD (Mn 2+ Concentration 1.5 mg / kg), scans were performed at room temperature using a 3.0T MRI scanner (Verio, Siemens, Erlangen, Germany) equipped with a soft coil (repetition time: 600 ms, echo time: 12 ms) to obtain T1-weighted images before injection and 1 h, 2 h, 4 h, 8 h, and 12 h after injection. T1-weighted images were analyzed using a built-in workstation, and statistical analysis was performed using the upper limb muscle signal and tumor site signal in the same coronal plane. Figure 8 As shown in the figure, the signal in the mouse tumor changes over time, reaching its highest point at 4 hours and then slowly decreasing, indicating that TMPD can reach the tumor site and achieve tumor-targeted imaging.

[0111] Experimental Example 5: Verification of the in vivo immune-enhancing and anti-tumor effects of the drug-loaded nanoparticles TMPD of the present invention

[0112] 1. Therapeutic effect of drug-loaded nanoparticles TMPD at the animal level

[0113] To establish the 4T1 tumor model in BALB / c mice, 1х10 64T1 breast cancer cells were injected into the right mammary pad of BALB / c mice, and the tumors were grown to 50-100 mm. 3 All mice were randomly divided into 4 groups, with 6 mice in each group. 200 μL of PBS (G1), PD (G2), TMP (G3), and TMPD (G4) (DOX: 4 mg / kg; Mn: 1 mg / kg) were injected into the tail vein. The drugs were given once every two days for a total of three times, and the mice were treated for 22 days. During the entire treatment process, the volume of the tumor was measured with a caliper every two days, and the weight changes of the mice in each experimental group were detected. The formula for calculating the tumor volume is as follows:

[0114] Volume (mm 3 )=0.5×length×width 2

[0115] The test results are as follows Figure 9 As shown, the PBS group had the most rapid tumor growth. The PD and TMP groups had some inhibitory effects on tumor growth. The TMPD group had a more pronounced inhibitory effect on tumor growth. This is because the tumor immunity-enhancing drug-loaded nanoparticles of the present invention achieve a therapeutic effect on tumors by improving immunity at the tumor site.

[0116] 2. Immunoenhancing effect of drug-loaded nanoparticles TMPD at the animal level

[0117] To verify immune system activation at the tumor site, mice were sacrificed and draining lymph nodes were isolated. After grinding the lymph nodes, the cell suspension was transferred to a centrifuge tube through a filter. After centrifugation (450g, 5 minutes), 1 mL of red blood cell lysis buffer was added to each tube of cells and lysed at room temperature for 3 minutes. Lysis was terminated by filling the tube with PBS. After centrifugation (450g, 5 minutes), the supernatant was discarded. CD16 / 32 antibody solution was added to each tube of cells, and the cells were blocked at 4°C for 15 minutes. Then, 30 μL of flow cytometry antibody cocktail (anti-CD45-BV510, anti-CD3-PE, and anti-CD86-APC / Cy7) was added and incubated at 4°C in the dark for 45 minutes. After incubation, each tube was filled with PBS and centrifuged (3000g, 2 minutes) to remove excess antibody. The cells were then resuspended in 200 μL of PBS and filtered into flow cytometry tubes. DC maturation was assessed by flow cytometry.

[0118] Tumor tissue was isolated and minced. Digestion was performed in a digestion solution (containing collagenase IV 1 mg / ml, DNase 100 μg / ml, and hyaluronidase 100 μg / ml) for 45 minutes. The cells were filtered and centrifuged (450 g for 5 minutes), and the supernatant was discarded. Each tube was then resuspended in 6 mL of 40% Percoll solution and centrifuged (800 g for 20 minutes, 6 steps up and 2 steps down), and the supernatant was discarded. Each tube was then lysed with 2 mL of erythrocyte lysis buffer for 3 minutes, topped up with PBS, and centrifuged (450 g for 5 minutes), and the supernatant was discarded. After counting by flow cytometry, all cells were divided into two tubes, A and B. 30 μL of CD16 / 32 antibody solution was added to each tube of cells in tube A, and the cells were blocked for 15 minutes. Then, 30 μL of a flow cytometry antibody cocktail (anti-CD45-BV510, anti-CD3-FITC, and anti-CD8-PE) was added, and the cells were incubated in the dark for 45 minutes. Each tube was filled with PBS solution and centrifuged (3000g, 2min), the cells were resuspended, filtered and detected by flow cytometry. 30μL CD16 / 32 antibody solution was added to each tube of cells in tube B, blocked for 15min, and then 30μL flow cytometry antibody mixture (anti CD45-BV510, anti CD3-FITC, anti APC / Cy7-CD4) was added, and then incubated in the dark for 45min. Each tube was filled with PBS solution and centrifuged (3000g, 2min). 300μL Fluxation working solution was added to each tube to resuspend the cells, incubated in the dark for 45min at room temperature, and then 800μL 1× Perm Buffer was added. After vortexing, the cells were centrifuged at room temperature (first 300-400g, 5min, then 8500g, 2min). 30μL of antibody staining solution (anti Foxp3-BV421) prepared in 1× Perm Buffer was added to each well and incubated in the dark for 45min. 1mL was added to each tube. PBS was added and centrifuged at room temperature (300-400g for 5 minutes, then 8500g for 2 minutes), and the supernatant was discarded. Each tube of cells was resuspended in 200 μL of PBS, filtered, and loaded into flow cytometry tubes. The proportion of immunosuppressive regulatory T (Treg) cells was determined by flow cytometry. The proportion of myeloid-derived suppressor cells (MDSCs) was determined using the same method, except that the antibodies used were anti-CD45-BV510, anti-CD11b-BV421, and anti-Gr-1-FITC. All antibodies were from Biolegend.

[0119] The results are as follows Figure 10 As shown in Figure 3, the proportion of mature DC cells in the lymph nodes of the TMPD group was the highest, 1.9 times that of the PBS group, indicating that TMPD can effectively induce DCs maturation. +The frequency of T lymphocytes was the highest (38.6% ± 5.6%), which was about 1.6 times that of the PBS group (22.8% ± 4.8%). Compared with the PBS group, TMP (32.0% ± 4.3%) and PD (27.8% ± 3.6%) treatments only resulted in the increase of CD8 + T cells increased slightly. Results for Treg cells and MDSCs showed that the proportion of Treg cells in the TMPD group was 6.7% ± 1.4%, significantly lower than that in the PBS group (24.6% ± 6.9%). On the other hand, the incidence of MDSCs was 21.4% ± 6.1%, almost twice as low as that in the PBS group (42.2% ± 7.6%). Therefore, the TMPD nanoparticles of the present invention can induce a strong immune response, thereby significantly inhibiting tumors.

[0120] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. A drug-loaded nanoparticle, characterized in that: include: hydrophobic antitumor drugs; amphiphilic polymers; Tannic acid-manganese complex; The hydrophobic anti-tumor drug is coated in the amphiphilic polymer; the tannic acid-manganese complex is loaded on the surface of the amphiphilic polymer; the average particle size of the drug-loaded nanoparticles is 60-80 nm; The hydrophobic anti-tumor drug is selected from at least one of camptothecin, paclitaxel, doxorubicin or daunorubicin; The amphiphilic polymer comprises a hydrophilic segment and a hydrophobic segment, the hydrophilic segment is selected from polyethylene glycol; and the hydrophobic segment is selected from polylactic acid-glycolic acid copolymer.

2. The drug-loaded nanoparticles according to claim 1, characterized in that The hydrophobic anti-tumor drug accounts for 3.7-4.9% of the mass of the drug-loaded nanoparticles.

3. The method for preparing drug-loaded nanoparticles according to claim 1 or 2, characterized in that: The steps include: S1, mixing an amphiphilic polymer dissolved in a first organic solvent, a hydrophobic antitumor drug dissolved in a second organic solvent, and water, and removing the first organic solvent and the second organic solvent by dialysis to obtain an amphiphilic polymer solution coated with the hydrophobic antitumor drug; S2. The pH of the amphiphilic polymer solution coated with the hydrophobic anti-tumor drug is adjusted to 7.9-8.2, mixed with the tannic acid solution and the manganese salt solution, and ultrafiltered to obtain drug-loaded nanoparticles.

4. The method for preparing drug-loaded nanoparticles according to claim 3, characterized in that: In step S1, the mass ratio of the amphiphilic polymer to the hydrophobic anti-tumor drug is 8-12:

1.

5. The method for preparing drug-loaded nanoparticles according to claim 3, characterized in that: The concentration of tannic acid in the tannic acid solution is 20-60 mg / mL.

6. The method for preparing drug-loaded nanoparticles according to claim 3, characterized in that: The manganese salt includes at least one of manganese chloride tetrahydrate, manganese chloride dihydrate, manganese chloride, manganese sulfate heptahydrate and manganese nitrate tetrahydrate.

7. The method for preparing drug-loaded nanoparticles according to claim 3, characterized in that: The concentration of the manganese salt in the manganese salt solution is 10-30 mg / mL.

8. Use of the drug-loaded nanoparticles according to claim 1 or 2 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that Such tumors include breast cancer, colorectal cancer, and melanoma.

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