Hyaluronic acid targeted manganese-phenol nano-modulator and preparation and application thereof

Abstract

The application discloses a hyaluronic acid-targeted manganese-phenol nano-regulator and a preparation and application thereof. 2+ The hyaluronic acid-targeted manganese-phenol nano-regulator is prepared by combining phenol and Mn 2+ The application integrates phenol and Mn in the same nano platform to realize bidirectional regulation of the PI3K / AKT pathway and the STING pathway, releases the inhibition of the AKT pathway on the STING, and combines the activation of the STING, so that the immune activation effect is significantly better than that of a single STING agonist. The nano structure is endowed with the POD-like activity to generate ROS in situ in a tumor microenvironment, efficiently induces immunogenic cell death, releases a large amount of tumor antigens, increases the 'antigen supply' dimension on the basis of the pathway regulation, and realizes the deep amplification of the immune response. Through the CD44 active targeting mediated by hyaluronic acid, tumor-specific enrichment is realized, the proportion of central memory T cells and intratumoral CD8 + T cell infiltration is significantly increased, tumor growth is significantly inhibited, and diagnosis and treatment integration is realized, thereby providing a new scheme for TNBC immunotherapy.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomedicine, specifically to a hyaluronic acid-targeted manganese-phenol nanomodulator and its preparation and application. Background Technology

[0002] Triple-negative breast cancer (TNBC) is the most aggressive and metastatic subtype of breast cancer due to the lack of estrogen receptor, progesterone receptor and HER2 expression. Treatment options are extremely limited, and traditional chemotherapy is ineffective and has significant side effects.

[0003] The cGAS-STING pathway is a key bridge connecting innate and adaptive immunity. Activation of this pathway can induce the production of type I interferon (IFN), promote the maturation of dendritic cells (DCs), enhance antigen cross-presentation, and drive CD8. + T-cell infiltration. Several STING agonists have entered clinical trials, such as cyclic dinucleotides (MK-1454, ADU-S100) and non-CDN small molecules (MSA-2, diABZI).

[0004] However, the efficacy of STING agonists is severely limited by the intrinsic inhibitory mechanisms of tumor cells. In breast cancer, the overactivated PI3K / AKT signaling pathway is a key negative regulator that can directly phosphorylate TBK1, disrupt the formation of the STING-TBK1 complex, and thus inhibit STING signal transduction. More than 70% of TNBC patients have gene mutations in the PI3K / AKT pathway, leading to abnormal activation of this pathway. Existing STING agonist monotherapy or simple combination therapy with AKT inhibitors has the following main drawbacks: (1) Asynchronous delivery and insufficient synergy. AKT inhibitors and STING agonists are two independent drug molecules with very different physicochemical properties. Their pharmacokinetic behavior, tissue distribution, and cellular uptake efficiency are different. There is a lack of a single carrier platform that can simultaneously encapsulate and deliver two drugs with different properties, making it difficult to ensure that they reach the same tumor cells synchronously and work synergistically. (2) Lack of active tumor targeting ability. Free STING agonists or AKT inhibitors lack the ability to actively recognize tumor tissues and mainly rely on passive diffusion. The accumulation efficiency at the tumor site is low, the non-specific exposure to normal organs is high, and the therapeutic window is narrow. (3) Difficulty in inducing ICD and insufficient antigen release. Existing STING agonists or AKT inhibitors have limited direct killing effects on tumor cells. Current strategies focus only on single pathway regulation and do not integrate functional modules that can efficiently kill tumor cells and induce ICD, thus severing the immune cycle chain of "antigen release" and "immune activation." This makes it difficult to induce a strong ICD effect, resulting in insufficient release of tumor antigens and DAMPs, and hindering the generation of the "first signal" (antigen peptide-MHC-I complex) required for cross-presentation. This makes the "second signal" provided by downstream STING activation unable to effectively drive CD8+ T cell responses due to the lack of specific antigen anchoring, limiting the strength and persistence of the immune response. Currently, few strategies that can simultaneously target and inhibit PI3K / AKT and activate the STING pathway have been reported. Summary of the Invention

[0005] The purpose of this invention is to provide a manganese-phenol nano-regulator (MnPae@HA) and its preparation, so as to solve the problems existing in the prior art.

[0006] A manganese-phenol nanomodulator (MnPae@HA) with synergistic regulation of PI3K / AKT and STING pathways, which is composed of paeonol (Pae) ​​and Mn 2+ A nanocore was constructed through metal-phenol coordination self-assembly and then modified with hyaluronic acid (HA). The inhibition of STING signal transduction by paeonol on the PI3K / AKT pathway was relieved, while Mn... 2+Activating the STING pathway, synergistically enhancing the anti-tumor immune response, and inducing immunogenic cell death (ICD) through POD-like activity, the three mechanisms work synergistically to provide a new strategy for TNBC immunotherapy.

[0007] The manganese-phenol nano-regulator (MnPae@HA) of the present invention achieves the following synergistic effects: (1) Active targeting and specific accumulation of tumors: HA can specifically recognize CD44 receptors that are highly expressed on the surface of tumor cells, mediate active targeting delivery, enhance tumor accumulation and endocytosis efficiency, and reduce non-specific distribution to normal tissues.

[0008] (2) Spatiotemporal synchronization and coordinated regulation: ensuring Pae and Mn through a nano-co-delivery system 2+ Simultaneously reaching tumor cells and being released, Pae inhibits the PI3K / AKT pathway to relieve its inhibition of STING signaling, Mn 2+ Activate the STING pathway to maximize the enhancement of downstream immune responses.

[0009] (3) Immunogenic cell death induction and immune amplification: The POD-like activity of MnPae@HA catalyzes the production of ROS from H2O2 in the tumor microenvironment, directly killing tumor cells and inducing ICD (CRT exposure, HMGB1 release, ATP secretion), releasing a large amount of tumor antigens and DAMPs, providing the necessary conditions for dendritic cell maturation and T cell activation, and further amplifying the effect of immunotherapy.

[0010] The preparation method of the above-mentioned hyaluronic acid-targeted manganese-phenol nano-regulator includes the following steps: (1) Dissolve MnCl2 in an ethanol solution containing polyvinylpyrrolidone (PVP), wherein PVP acts as a stabilizer for metal ions; (2) Add the ethanol solution containing paeonol dropwise to the solution obtained in step (1), stir, and then add it dropwise to the CAPs solution under ultrasound. Adjust the pH of the system to alkaline and stir at room temperature to form MnPae nanocores. (2) Then add sodium hyaluronate aqueous solution to make the final concentration of the solution 1 mg / mL, stir at room temperature, and coat the MnPae surface with hyaluronic acid.

[0011] Preferably, the mass ratio of MnCl2 to paeonol is 2:1.

[0012] Preferably, the pH value is adjusted to 10 in step (2).

[0013] Preferably, the Mw of the sodium hyaluronate aqueous solution in step (3) is 3000.

[0014] The above-mentioned hyaluronic acid-targeted manganese-phenol nanomodulator is used in the preparation of antitumor drugs. The tumor includes triple-negative breast cancer.

[0015] A treatment for triple-negative breast cancer comprising the aforementioned hyaluronic acid-targeted manganese-phenol nanomodulator.

[0016] Compared with existing technologies, this invention combines natural small molecule paeonol (Pae, a PI3K / AKT inhibitor) with manganese ions (Mn) 2+ A novel nanomodulator, MnPae@HA, was constructed by coordinating and self-assembling a STING agonist and then surface-modifying it with hyaluronic acid (HA, targeting the CD44 receptor). It offers the following main advantages: (1) This invention utilizes a metal-phenol coordination self-assembly strategy to combine paeonol (a PI3K / AKT inhibitor) with Mn 2+ The integration of STING agonists into the same nanoplatform enables bidirectional regulation of the STING and PI3K / AKT pathways, resulting in significantly better immune activation than either a single STING agonist or paeonol alone.

[0017] (2) This invention utilizes the peroxidase-like (POD) activity imparted by the nanostructure to generate ROS in situ in the tumor microenvironment, efficiently inducing immunogenic cell death and releasing a large amount of tumor antigens, including HMGB1, CRT, and ATP. Based on pathway regulation, it adds the dimension of "antigen supply," providing a sufficient first signal (antigen library), while simultaneously combining dual-pathway regulation to activate STING and provide a second signal (adjuvant). This achieves a deep amplification of the immune response.

[0018] (3) This invention achieves tumor-specific enrichment through active CD44 targeting mediated by hyaluronic acid (HA). In a TNBC-bearing mouse model, MnPae@HA significantly inhibited tumor growth, reducing tumor volume and weight by 3.5-fold and 4.5-fold, respectively, compared to the control group, and significantly increased the proportion of central memory T cells and intratumoral CD8+. + T-cell infiltration was detected, and the MRI imaging properties of manganese metal enabled integrated diagnosis and treatment. This approach achieved an organic combination of tumor-targeted enrichment, pathway synergistic regulation, and ICD induction, providing a new strategy for TNBC immunotherapy. Attached Figure Description

[0019] Figure 1(a) Schematic diagram of MnPae@HA synthesis; (b) Particle size and polydispersity index under different manganese ion to paeonol feed ratios; (c) Transmission electron microscopy image of MnPae@HA; (d) Dynamic light scattering particle size distribution of MnPae@HA in different media; (e) Ultraviolet-visible absorption spectrum; (f) High-resolution X-ray photoelectron spectroscopy of manganese 2p; (g) High-resolution X-ray photoelectron spectroscopy of oxygen 1s; (h) Peroxidase-like activity assay; (i) Mn 2+ Comparison of POD-like activities of Pae and MnPae@HA; (j) Mn at different concentrations 2+ The ability to catalyze the generation of hydroxyl radicals; (k) The ability of different concentrations of MnPae@HA to catalyze the generation of hydroxyl radicals.

[0020] Figure 2 (a) qPCR detection of genes related to the STING pathway; (b) qPCR detection of genes related to the PI3K / AKT pathway; (c) Western blot detection of pathway-related proteins; (d) Western blot quantitative analysis of phosphorylated protein levels; (e) Killing effect of free paeonol on 4T1 cells; (f) Killing effect of free paeonol on 231 cells; (g) Killing effect of equal amounts of Pae, MnCl2, and MnPae@HA on 4T1 cells; (h) Killing effect of equal amounts of Pae, MnCl2, and MnPae@HA on 231 cells; (i) Determination of the half-maximal inhibitory concentration (IC50) of 4T1 cells; (j) Determination of the half-maximal inhibitory concentration (IC50) of 231 cells; (k) Flow cytometry detection of apoptosis rate under different treatments; (l) Immunofluorescence staining of calreticulin (CRT) exposure and release of high-mobility group box 1 (HMGB1); (m) Flow cytometry detection of the fluorescence intensity of CRT expression on 4T1 cells after treatment with different concentrations of MnPae@HA, scale bar 25 μm; (n) ELISA detection of ATP content released by 4T1 cells after different treatments; (o) Flow cytometry detection of dendritic cell (DC) maturation markers (CD80+MHCII+); (p) Statistical graph of DC maturity after different treatments.

[0021] Figure 3 (a) Measurement of longitudinal relaxation rate (r1); (b) Measurement of transverse relaxation rate (r2); (c) T1-weighted magnetic resonance imaging at different time points after tail vein injection in 4T1 tumor-bearing mice; (d) In vivo fluorescence imaging at different time points; (e) Fluorescence imaging of major organs ex vivo 24 h after injection.

[0022] Figure 4(a) Tumor volume change curves in each group of mice; (b) Statistical analysis of ex vivo tumor weight in each group; (c) Photographs of ex vivo tumors in each group; (d) Body weight change curves in each group of mice during treatment; (e) H&E, TUNEL, and Ki67 staining of tumor tissues; (f) Immunofluorescence staining of p-AKT and p-IRF3 in tumor tissues; (g) ELISA quantitative detection of IFN-β and CXCL10 in tumor tissues; (h) Activated CD8 in the spleen. + T cells (CD) + CD69 + (i) Flow cytometry detection of the proportion of CD8+ in tumors; + Flow cytometry detection of T cell infiltration ratio; (j) CD8 in tumor tissue + Immunofluorescence staining for T cell and NK cell infiltration; (k) ELISA quantitative detection of IFN-γ levels in tumor tissue. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0024] Example 1 Synthesis of a hyaluronic acid-targeted manganese-phenol nanomodulator, such as Figure 1 As shown in (a), the steps include: First, paeonol (Pae) ​​and manganese ions (Mn) are combined. 2+ In the presence of PVP, a metal-phenol coordination self-assembly process was used to form a nanocore. The specific preparation process was as follows: 40 mg Pae was dissolved in 1 mL of ethanol, and 20 mg MnCl2 was dissolved in 5 mL of ethanol solution containing 66 mg PVP. After stirring for 5 min, the Pae solution was added dropwise, and the mixture was stirred for 4 h. Then, 1 mL of the above mixture was added dropwise to 10 mL of CAPs buffer (pH=10) under ultrasonication to adjust the pH to 10, promoting the deprotonation of the phenolic hydroxyl groups in Pae to enhance its coordination ability. The reaction was stirred at room temperature for 8 h to form the MnPae nanocore. Subsequently, an aqueous solution of sodium hyaluronate (Mw 3k) was added to bring the final concentration to 1 mg / mL, and the mixture was stirred at room temperature for 8 h. Hyaluronic acid (HA) was then coated onto the MnPae surface through electrostatic / coordination interactions, yielding MnPae@HA.

[0025] By changing the feed ratio to 1:2, 1:1, and 2:1, the manganese ion (Mn) concentration was determined. 2+ The optimal feed ratio of phenolic drugs to phenolic drugs is 2:1. Figure 1 (b)

[0026] 1. Characterization of basic physicochemical properties TEM images show that MnPae@HA are uniformly spherical with a particle size of approximately 40 nm. Figure 1 (c) Fluid dynamics revealed that this nanomaterial exhibits good dispersibility in water, physiological saline, and culture media, with a hydrated particle size of approximately 100 nm. Figure 1 (d) The UV absorption spectrum shows that the characteristic UV peaks of Pae alone are at 274 nm and 316 nm, while MnPae@HA also shows broad absorption peaks at these two locations, with a significant increase in absorbance at approximately 316 nm, indicating the formation of a metal-phenol complex and confirming the successful encapsulation of paeonol. Figure 1 (e) XPS confirms the presence of Mn and O elements. Figure 1 In (f), Mn exists in the +2 valence state. Figure 1 In the middle (g) , the O 1s spectrum confirmed that Mn is coordinated with the phenolic hydroxyl / carbonyl group of Pae. Figure 1 In the middle (h). MnPae@HA can catalyze the production of hydroxyl radicals from H2O2, leading to the oxidative discoloration of TMB, while an equal amount of Mn alone... 2+ Neither paeonol nor paeonol can change its color, such as Figure 1 In the middle (i), the catalytic performance of MnPae@HA is significantly better than that of Mn alone. 2+ The specific data is as follows: 800 µM Mn 2+ The absorbance of the catalytic product was 0.13 ( Figure 1 (j)), while the absorbance of the 200 µM MnPae@HA catalyzed product was 0.93 (j). Figure 1 The presence of POD (k) indicates that the unique nanostructure endows MnPae@HA with excellent peroxidase (POD)-like activity. POD can catalyze the highly expressed H2O2 at tumor sites to generate highly toxic hydroxyl radicals. The highly active ·OH radicals attack important biomolecules such as lipids, proteins, and DNA in tumor cells, causing irreversible oxidative damage, inducing cell death, thereby promoting the release of tumor antigens and activating immunity.

[0027] 2. Dual pathway regulation of MnPae@HA, immunogenic cell death, and in vitro antitumor effects. Quantitative polymerase chain reaction (PCR) results showed that MnPae@HA significantly upregulated the expression of downstream genes in the STING pathway and downregulated genes related to the PI3K / AKT pathway, such as... Figure 2 (a) and (b). Western blotting confirmed that MnPae@HA effectively inhibited AKT phosphorylation and significantly promoted the phosphorylation of STING, TBK1, and IRF3, such as... Figure 2 In (c) and (d), the above results confirm the dual-pathway regulatory role of MnPae@HA. Regarding its in vitro antitumor activity, such as... Figure 2As shown in (e) and (f), Pae alone only exhibited significant killing effects on both types of tumor cells at high concentrations >1 mM, and showed no significant killing effect on tumor cells at the same concentration as MnPae@HA. Under the same concentration conditions, MnPae@HA showed a significant synergistic effect on both types of tumor cells, such as... Figure 2 In (g) and (h). Specifically, MnPae@HA significantly reduced the half-maximal inhibitory concentration (IC50) of 4T1 and 231 cells compared to MnCl2, and significantly reduced the IC50 of MnCl2 compared to the same concentration of MnCl2. 2+ In comparison, MnPae@HA significantly reduced the IC50 value of tumor cells, specifically from 60.55 μM to 30.66 for murine 4T1 tumor cells (nearly half) and from 117.6 μM to 50.2 for human 231 tumor cells (more than half). Figure 2 In (i) and (j), flow cytometry further confirmed that the MnPae@HA treatment group induced the highest number of apoptosis, such as... Figure 2 In terms of inducing immunogenic cell death and promoting tumor antigen release, immunofluorescence showed that MnPae@HA significantly increased calreticulin membrane exposure and promoted the release of high-mobility group box 1, such as... Figure 2 (l); Flow cytometry further demonstrated that MnPae@HA-induced calreticulin exposure was concentration-dependent, such as Figure 2 (m); Meanwhile, the release of ATP from tumor cells after different treatments was quantified by enzyme-linked immunosorbent assay (ELISA), showing that MnPae@HA induced the highest ARP release, such as... Figure 2 In the co-culture experiment with dendritic cells, MnPae@HA significantly promoted the growth of dendritic cells (CD80). + MHCII + The maturation and activation of [the substance] were higher than in other treatment groups, such as [other substances]. Figure 2 The presence of (o) and (p) indicates that MnPae@HA has the ability to activate immunity.

[0028] 3. Magnetic resonance imaging capabilities of MnPae@HA and tumor-targeted accumulation Mn 2+ MnPae@HA possesses magnetic resonance imaging (MRI) properties, which were therefore utilized to evaluate its potential as an in vivo MRI contrast agent. In vitro relaxation rate testing showed that MnPae@HA exhibits T1 and T2 imaging potential, with high transverse and longitudinal relaxation rates. Its imaging performance is comparable to clinically used T1 contrast agents such as gadolinium and commercially available T2 contrast agents such as iron, indicating its suitability as a T1 / T2 contrast agent. Figure 3(a) and (b). Following tail vein injection into 4T1 tumor-bearing mice, dynamic T1-weighted magnetic resonance imaging was performed at different time points. The results showed that the signal at the tumor site gradually increased over time, reaching a peak at 6 hours post-injection. Figure 3 (c) shows that HA-mediated active targeting enables MnPae@HA to effectively accumulate in tumor tissue. Fluorescence in vivo imaging and ex vivo organ imaging further confirm its good tumor targeting and biodistribution, such as... Figure 3 (d) and (e). It can be seen that MnPae@HA can significantly inhibit the growth of tumors in mice, reducing tumor volume and weight by 3.5 times and 4.5 times, respectively, compared with the control group.

[0029] 4. In vivo anti-tumor efficacy and immune activation assessment Subsequently, a 4T1 tumor-bearing mouse model was constructed to evaluate the in vivo antitumor effect of MnPae@HA. Compared to paeonol and Mn alone... 2+ In the treatment group, MnPae@HA significantly inhibited tumor growth, specifically manifested as a reduction in tumor volume and weight. Figure 4 (a), (b), and (c). Throughout the treatment process, there was no significant difference in body weight among the treatment groups. Figure 4 The result (d) indicates good biocompatibility. Subsequently, hematoxylin-eosin, TUNEL, and KI67 staining were performed on tumors in mice from each treatment group. Extensive necrosis, significantly increased apoptosis, and decreased proliferation were observed in the MnPae@HA group, as shown in [example data]. Figure 4 (e) Immunofluorescence and enzyme-linked immunosorbent assays confirmed that MnPae@HA effectively reduced p-AKT levels and significantly upregulated p-IRF3, IFN-β, and CXCL10. Figure 4 The results (f) and (g) indicate that MnPae@HA can still bidirectionally regulate the PI3K-AKT and STING pathways in vivo. Regarding the immune response, flow cytometry showed that the spleen in the MnPae@HA treatment group neutralized and activated CD8+. + T cells (CD8) + CD69 + The proportion has increased significantly. Figure 4 (h) and intratumoral CD8 + T cells, Figure 4 Increased infiltration of CD8 in the tumor (i). Immunofluorescence and enzyme-linked immunosorbent assay confirmed the presence of CD8 in the tumor. + Increased T cell and NK cell infiltration and significantly elevated IFN-γ levels, such as Figure 4 In (j) and (k), these results indicate that MnPae@HA activates a systemic antitumor immune response in mice.

Claims

1. A method for preparing a hyaluronic acid-targeted manganese-phenol nano-regulator, characterized in that, Includes the following steps: (1) Dissolve MnCl2 in an ethanol solution containing polyvinylpyrrolidone; (2) Add the ethanol solution containing paeonol dropwise to the solution obtained in step (1), stir, adjust the pH of the system to alkaline, and stir at room temperature to form MnPae nanocores; (3) Then add sodium hyaluronate aqueous solution to make the final concentration of the solution 1 mg / mL, stir at room temperature, and coat the MnPae surface with hyaluronic acid.

2. The preparation method according to claim 1, characterized in that, The mass ratio of MnCl2 to paeonol added is 2:

1.

3. The preparation method according to claim 1, characterized in that, In step (2), the pH value is adjusted to 10.

4. The preparation method according to claim 1, characterized in that, In step (3), the Mw of the sodium hyaluronate aqueous solution is 3000.

5. The hyaluronic acid-targeted manganese-phenol nano-regulator obtained by the preparation method according to claim 1.

6. The application of the hyaluronic acid-targeted manganese-phenol nanomodulator according to claim 5 in the preparation of antitumor drugs.

7. A treatment drug for triple-negative breast cancer, characterized in that, It contains the manganese-phenol nanomodifier targeting hyaluronic acid as described in claim 5.

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