An acid-responsive shell structure nano-preparation, and a preparation method and application thereof

Abstract

The application discloses an acid-responsive shell structure nano preparation and a preparation method and application thereof, and belongs to the technical field of biological medicine. The acid-responsive shell structure nano preparation and the preparation method thereof are provided, and the acid-responsive shell structure nano preparation comprises mesoporous polydopamine, a platinum drug, a bacterial membrane vesicle expressing lactic acid oxidase and a metal-polyphenol network shell layer; the mesoporous polydopamine is used for loading the platinum drug, and the outer layer of the mesoporous polydopamine is sequentially coated with the bacterial membrane vesicle expressing lactic acid oxidase and the metal-polyphenol network shell layer. The acid-responsive shell structure nano preparation has the ability of active tumor targeting, can respond to a tumor micro-acid environment, integrates platinum drug chemotherapy, lactic acid metabolism regulation, cGAS-STING pathway activation and tumor immune microenvironment remodeling into a single nano preparation, can effectively activate an anti-tumor immune response, and significantly inhibits tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an acid-responsive shell-structured nano-formulation, its preparation method, and its application. Background Technology

[0002] Chemotherapy, with its potent and broad-spectrum anti-tumor activity, remains a key method in clinical cancer treatment. Platinum-based drugs, represented by cisplatin (CDDP), are among the most widely used traditional chemotherapy agents and have been incorporated into standard treatment regimens for various solid tumors. With the advent of precision medicine and immunotherapy in cancer treatment, the combined strategy of chemotherapy and immunotherapy shows synergistic potential, promising to overcome the limitations of single-therapy approaches and improve overall efficacy.

[0003] However, tumor cells evade platinum-based drugs through various resistance mechanisms. Lactate (LA), a key metabolite of tumor aerobic glycolysis, enhances homologous recombination-mediated DNA damage repair, thereby reducing tumor sensitivity to chemotherapeutic drugs such as cisplatin. Furthermore, lactate can also shape an immunosuppressive tumor microenvironment (TME) by inhibiting dendritic cell (DC) antigen presentation, weakening effector T cell activation and cytokine secretion, promoting regulatory T cell (Treg) proliferation, and inducing macrophage polarization towards the M2 phenotype. Therefore, targeting and reducing TME lactate levels has become a potential strategy for reversing cisplatin resistance and synergistically enhancing anti-tumor immune responses.

[0004] Currently, strategies for reducing lactate levels in the total metabolic enzyme (TME) mainly focus on three levels: inhibiting glycolysis to block lactate production, interfering with lactate transporter function to inhibit its efflux, and directly degrading lactate using lactate oxidase (LOX). With the rapid development of synthetic biology, expressing LOX using engineered bacterial systems has become a feasible approach. Bacterial membrane vesicles (BMVs) retain similar functions and possess inherent immunostimulatory properties because they carry bioactive components derived from the parent bacteria. Compared to live bacteria, BMVs lack replication capacity, thus exhibiting higher biocompatibility, making them attractive therapeutic delivery vectors. However, how to effectively integrate lactate metabolism regulation, platinum-based chemotherapy, and immunotherapy into a single delivery system to achieve multi-mechanism synergistic therapy remains an ideal solution.

[0005] Therefore, developing a multifunctional nanoformulation that can simultaneously reverse platinum drug resistance, activate immunity, and reshape the tumor immune microenvironment has become an urgent need in this field. Summary of the Invention

[0006] The purpose of this invention is to effectively integrate lactate metabolism regulation, platinum-based chemotherapy, and immunotherapy into a single delivery system, providing an acid-responsive shell-structured nanoformulation and its preparation method.

[0007] In a first aspect, the present invention provides an acid-responsive shell-structured nanoformulation comprising mesoporous polydopamine, platinum-based drugs, bacterial membrane vesicles, and a metal-polyphenol network shell. The mesoporous polydopamine is a carrier for loading the platinum-based drug, and the outer layer of the mesoporous polydopamine sequentially coats the bacterial membrane vesicles and the metal-polyphenol network shell. The platinum-based drug is cisplatin; the metal-polyphenol network shell comprises metal ions and polyphenolic compounds, wherein the metal ions are Mn. 2+ Ions; the polyphenolic compounds include at least one of tannic acid, gallic acid, and catechol; The bacterial membrane vesicles are derived from Escherichia coli and express a cytolysin A-lactate oxidase fusion protein; the amino acid sequence of cytolysin A is shown in SEQ ID NO:3, and the amino acid sequence of lactate oxidase is shown in SEQ ID NO:1.

[0008] Preferably, the *Escherichia coli* contains a nucleotide sequence encoding a cytolysin A-lactate oxidase fusion protein; the nucleotide sequence of cytolysin A is shown in SEQ ID NO:4, and the nucleotide sequence of the gene encoding lactate oxidase is shown in SEQ ID NO:2.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned acid-responsive shell structure nanoparticles, comprising the following steps: (1) The engineered bacteria that have been introduced with the gene encoding lactate oxidase were cultured, and after induction of expression, the bacterial cells were collected and the bacterial membrane vesicles expressing the cytolysin A-lactate oxidase fusion protein were extracted. (2) The template agent and dopamine hydrochloride were dissolved in a mixed solvent of ethanol and water, an organic pore-expanding agent was added, and then an alkaline reagent was added to adjust to alkalinity. The polymerization reaction yielded mesoporous polydopamine nanoparticles. The mesoporous polydopamine nanoparticles were mixed with cisplatin to obtain drug-loaded mesoporous polydopamine nanoparticles. The template agent includes either Pluronic F127 or Pluronic P123; the mass ratio of the template agent to dopamine hydrochloride is 1:0.2~1; The organic pore-expanding agent is 1,3,5-trimethylbenzene, and the mass ratio of the organic pore-expanding agent to the template agent is 0.5~2:1; The amount of cisplatin added is based on the amount of platinum element, with 0.6~8 μmol of cisplatin added per milligram of mesoporous polydopamine nanoparticles; (3) Mix and incubate the bacterial membrane vesicles in step (1) and the drug-loaded mesoporous polydopamine nanoparticles in step (2), and extrude them through a microporous filter membrane to obtain drug-loaded mesoporous polydopamine nanoparticles coated with membrane vesicles. The mass ratio of the drug-loaded mesoporous polydopamine nanoparticles to the membrane proteins in the bacterial membrane vesicles is 1:0.5~3; (4) Dissolve the drug-loaded mesoporous polydopamine nanoparticles and polyphenolic compounds coated with membrane vesicles from step (3) in water, and add Mn 2+ An ionic salt solution was used to adjust the pH of the system to a weakly acidic level, resulting in an acid-responsive shell-structured nano-formulation. The polyphenolic compounds include at least one of tannic acid, gallic acid, and catechol; The mass ratio of the drug-loaded mesoporous polydopamine nanoparticles coated with the membrane vesicles to the polyphenolic compound is 1:0.2~2; the Mn 2+ The ion feed amount is 10~20 μmol per milligram of drug-loaded mesoporous polydopamine nanoparticles coated with the membrane vesicles.

[0010] Preferably, in step (2), the volume ratio of ethanol to water in the mixed solvent of ethanol and water is 1:0.5~2; the reaction time after adding the organic pore expander is 0.2~2 h; the pH of the system is adjusted to 7.5~12.0 by the alkaline reagent, and the reaction time after alkalinization is 1~6 h.

[0011] Preferably, in step (3), the microporous filter membrane includes a polycarbonate membrane with a pore size of 400~800 nm.

[0012] Preferably, in step (4), the Mn² + The concentration of the ion salt solution is 50-70 mM, and the pH of the system is 4-7.

[0013] Thirdly, the present invention provides the application of the above-mentioned acid-responsive shell structure nanoformulation in the preparation of drugs for treating and / or alleviating tumors.

[0014] Compared with the prior art, the present invention has the following advantages: The acid-responsive mesoporous polydopamine shell-structured nanoformulation provided by this invention possesses the ability to actively target tumors and responds to the tumor microacidic environment. It integrates platinum-based chemotherapy, lactate metabolism regulation, cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene-stimulating protein) pathway activation, and tumor immune microenvironment remodeling into a single nanoformulation, overcoming the efficacy limitations of traditional monotherapy or simple combination therapy. This nanoformulation can effectively activate anti-tumor immune responses and significantly inhibit tumor growth.

[0015] The acid-responsive mesoporous polydopamine shell-structured nanoformulation provided by this invention has the ability to restore the sensitivity of tumors to chemotherapeutic drugs. By utilizing engineered bacterial membrane vesicles to deliver lactate oxidase (LOX), lactate is continuously consumed in the tumor microenvironment, inhibiting homologous recombination-mediated DNA damage repair; this relieves the dual inhibition of the cGAS-STING pathway by lactate, thereby effectively reversing platinum-based drug resistance and restoring the chemotherapeutic sensitivity of tumor cells.

[0016] The acid-responsive mesoporous polydopamine shell-structured nanoformulation provided by this invention achieves acid-responsive release through a metal-polyphenol network shell, improving drug delivery precision and reducing toxic side effects. The metal-polyphenol network shell used in this invention is structurally stable under physiological conditions, effectively reducing premature cisplatin leakage. Upon reaching the acidic tumor microenvironment, the shell undergoes responsive degradation, achieving precise release of the active ingredient at the target site. Simultaneously, the metal-polyphenol network shell can shield pathogen-associated molecular patterns (PAMPs) on the surface of bacterial membrane vesicles during delivery, avoiding non-specific inflammatory responses in vivo and significantly improving the safety and targeting of the formulation. Simultaneously, cisplatin induces DNA damage to produce cytoplasmic double-stranded DNA (dsDNA), while lactate oxidase-mediated lactate consumption blocks DNA repair and promotes dsDNA accumulation, providing ample activating ligands for cGAS. Metal ions, as co-activators of cGAS, can significantly enhance cGAS's affinity for double-stranded DNA and amplify downstream STING signals. The synergistic effect of these three factors achieves potent activation of the cGAS-STING pathway, efficiently triggering type I interferon production and T cell immune responses.

[0017] The acid-responsive mesoporous polydopamine shell-structured nanoformulation provided by this invention utilizes synthetic biology techniques to obtain engineered bacteria expressing lactate oxidase. Bacterial membrane vesicles are extracted, and their enzymatic activity and immunostimulatory properties enable both lactate degradation and immune activation. Simultaneously, it avoids the infection risks associated with live bacterial preparations, exhibiting improved biosafety.

[0018] The acid-responsive mesoporous polydopamine shell-structured nano-formulation and its preparation method provided by this invention are simple and convenient to prepare, and the prepared nano-formulations are stable, powerful, and have excellent tumor treatment effects. Attached Figure Description

[0019] Figure 1 A flowchart of a method for preparing acid-responsive mesoporous polydopamine shell-structured nanoparticles; Figure 2 This is a schematic diagram illustrating the construction of PPLM and its anti-tumor mechanism; among which, Figure 2 A is a schematic diagram of the PPLM build process. Figure 2 B is a schematic diagram of the anti-tumor mechanism of PPLM; Figure 3 The image shown is an electron microscope image from Experiment Example 1; in which, Figure 3 A is a scanning electron micrograph of PP. Figure 3 B is a transmission electron microscope image of PP. Figure 3 C is a transmission electron microscope image of PPL; Figure 4 This is a scanning electron microscope image of PP in Experiment Example 1; Figure 5 The hydrated particle size of PPL in Experiment Example 1; Figure 6 This is a transmission electron microscope image of the PPLM in Experiment Example 2; Figure 7 The structure and characterization of the acid-responsive mesoporous polydopamine shell nanoformulation in Experimental Example 2; among which, Figure 7 A is a transmission electron microscope image obtained using a PPLM microscope. Figure 7 B represents the particle size test result. Figure 7 C is the Zeta potential. Figure 7 D is the mapping diagram of PPLM. Figure 7 E represents the X-ray photoelectron spectroscopy (XPS) analysis of the PPLM. Figure 7 F represents the particle size change of PPLM over 7 days in pure water and cell culture medium containing 10% serum. Figure 8 This section characterizes the pH-responsive drug release behavior of PPLM in Experiment Example 3; among which, Figure 8 A represents PPLM in PBS buffers at pH 5.5 and pH 7.4, with Mn. 2+ The cumulative release curve, Figure 8 B represents PPLM in PBS buffers at pH 5.5 and pH 7.4. 2+ The cumulative release curve; Figure 9 The cytotoxicity and lactate-consuming capacity of PPLM in Experiment Example 4; among which, Figure 9 A represents the cytotoxicity of PPLM on 4T1 cells at different concentrations. Figure 9 B represents the cytotoxicity of different nanoparticles to 4T1 cells at the same cisplatin concentration. Figure 9 C represents the LA content in the supernatant of 4T1 cell culture after different nanoparticle treatments; Figure 10 This refers to the in vivo antitumor effect of PPLM in Experimental Example 5; among which, Figure 10 A represents the results of changes in tumor size in mice. Figure 10 B represents the mouse's weight change. Figure 10 C represents the survival curve of tumor-bearing mice. Detailed Implementation

[0020] To better understand the present invention, the following embodiments are further illustrations of the invention, but the scope of the invention is not limited to these embodiments. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods. The reagents and materials used in the embodiments are all commercially available, and the quantitative experiments involved in the embodiments are all performed in at least three replicates.

[0021] The present invention adopts the following technical solution: This invention provides an acid-responsive mesoporous polydopamine shell-structured nanoformulation, one of which is prepared as follows: Figure 1 As shown, it includes: S1: Engineered bacteria modification and bacterial membrane vesicle extraction An exogenous gene encoding lactate oxidase (LOX) was introduced into host bacteria, and bacterial cells were collected after induction of expression. The bacterial cells were resuspended in buffer, lysed, and undisturbed cells and large fragments were removed by differential centrifugation. The supernatant containing bacterial membrane vesicles was collected and further enriched by ultracentrifugation to obtain engineered bacterial membrane vesicles expressing the target protein.

[0022] The host bacterium is a Gram-negative bacterium, preferably Escherichia coli Nissle 1917 (EcN).

[0023] The amino acid sequence of the lactate oxidase (LOX) is SEQ ID NO:1, and the nucleotide sequence is SEQ ID NO:2.

[0024] To enable the host bacteria to correctly express lactate oxidase (LOX) on their surface, the nucleotide sequence encoding the cytolysin A (ClyA)-lactic acid oxidase (LOX) fusion protein was introduced into the host bacteria. ClyA is a membrane surface protein; through ClyA localization, the fusion protein ensures the correct expression of LOX on the bacterial membrane surface. The amino acid sequence of ClyA is shown in SEQ ID NO:3; the nucleotide sequence of ClyA is shown in SEQ ID NO:4.

[0025] The method for lysing the bacterial cells is not particularly limited, and includes chemical lysis, ultrasonic disruption, high-pressure homogenization, etc.

[0026] S2: Preparation of mesoporous polydopamine nanoparticles The template agent and dopamine hydrochloride were dissolved in a mixed solvent of ethanol and water, and an organic pore-expanding agent was added. The mixture was then adjusted to alkalinity to obtain the reaction product, mesoporous polydopamine nanoparticles.

[0027] The volume ratio of ethanol to water in the mixed solvent of ethanol and water is 1:0.5~2.

[0028] The template agent is any one or a combination of at least two of the block copolymer and surfactant; preferably, it is a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (Pluronic series), including Pluronic F127 or Pluronic P123.

[0029] The organic pore-expanding agent includes 1,3,5-trimethylbenzene, o-xylene, etc. The mass ratio of the organic pore-expanding agent to the template agent is 0.5~2:1, and the reaction time is 0.2~2 hours.

[0030] The type of reagent used to adjust alkalinity is not particularly limited, as long as it can adjust the acidity or alkalinity of the solution without affecting the formation of nanoparticles. This includes ammonia, sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, tris(hydroxymethyl)aminomethane buffer (Tris buffer), etc. The system is adjusted to pH 7.5-12, preferably 8.0-10.0, and the reaction time is 1-6 hours.

[0031] S3: Drug-carrying mesoporous polydopamine nanoparticles Platinum-based drugs were added dropwise to mesoporous polydopamine solution nanoparticles to obtain drug-loaded mesoporous polydopamine nanoparticles.

[0032] The platinum-based drugs include cisplatin (CDDP), carboplatin, oxaliplatin, nedaplatin, lobaplatin, etc. The amount of platinum-based drugs added is based on the amount of platinum element, with 0.6~8 μmol of platinum element added for every 1 mg of mesoporous polydopamine nanoparticles.

[0033] Preferably, the mass ratio of cisplatin to mesoporous polydopamine is 0.2 to 2:1; and the stirring time is 1 to 6 hours.

[0034] S4: Bacterial membrane vesicle encapsulation of drug-loaded mesoporous polydopamine nanoparticles The drug-loaded mesoporous polydopamine nanoparticles obtained in S3 were dispersed in a buffer solution containing engineered bacterial membrane vesicles obtained in S1 and incubated at 20-35℃ for 10-30 min. This allowed the bacterial membrane vesicles to be coated onto the surface through physical adsorption or intermolecular interactions. The coated nanoparticles were then extruded through a polycarbonate membrane to obtain the drug-loaded mesoporous polydopamine nanoparticles coated with membrane vesicles, i.e., membrane vesicle coated product nanoparticles.

[0035] The mass ratio of the drug-loaded mesoporous polydopamine nanoparticles to the membrane proteins in the membrane vesicles is 1:0.5~3.

[0036] The polycarbonate membrane has a pore size of 400~800nm.

[0037] S5: Coated metal-polyphenol network shell The membrane vesicle-coated nanoparticles obtained in S4 were dissolved in water along with polyphenolic compounds. A metal ion salt solution was added, and then an alkaline reagent was added to adjust the solution to a weakly acidic state. Utilizing the coordination and complexation effect between the polyphenolic compounds and metal ions, a metal-polyphenol network shell was formed in situ cross-linked on the particle surface. After centrifugation and washing, an acid-responsive mesoporous polydopamine shell-structured nanoformulation was obtained.

[0038] The polyphenolic compounds include tannic acid (TA), gallic acid, catechol, etc.

[0039] The metal ion is manganese ion (Mn²⁺). + The concentration of the manganese ion salt solution is 50-70 mM, and the amount of manganese ions fed is 10-20 μmol per milligram of drug-loaded mesoporous polydopamine nanoparticles coated with the membrane vesicles.

[0040] The mass ratio of the membrane vesicle-coated product nanoparticles to polyphenolic compounds is 1:0.2~2.

[0041] The alkaline reagent is not particularly limited and includes ammonia, sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, tris(hydroxymethyl)aminomethane buffer (Tris buffer), etc., to adjust the pH value to 4-7, preferably 5.5-6.5.

[0042] The present invention provides the construction of an acid-responsive mesoporous polydopamine shell-structured nanoformulation and its anti-tumor mechanism, as follows: Figure 2 As shown, it includes: After entering tumor cells, acid-responsive mesoporous polydopamine shell-structured nanoparticles undergo responsive degradation of the metal-polyphenol network shell in an acidic microenvironment, releasing metal ions, bacterial membrane vesicles (BMVs-EcL), and platinum-based drugs. The released platinum-based drugs induce DNA damage in tumor cells, producing cytosolic double-stranded DNA (dsDNA). Simultaneously, LOX carried by BMVs-EcL continuously catalyzes the decomposition of lactate in the tumor microenvironment, reducing lactate levels and significantly amplifying the downstream STING (interferon gene-stimulating protein) signaling pathway, triggering type I interferon production and anti-tumor immune responses. Furthermore, LOX-mediated lactate consumption helps remodel the tumor immune microenvironment, relieving lactate's inhibition of immune effector cells and further enhancing the anti-tumor immune response, achieving synergistic treatment through multiple mechanisms. The effects of the present invention will be described in detail below with reference to the embodiments.

[0043] Example 1: Preparation of shell-structured nanoformulations of acid-responsive mesoporous polydopamine 1. Construction and culture of engineered bacteria A recombinant plasmid (pJ23119-ClyA-6×His-LOX) encoding the ClyA-LOX fusion protein was designed using the nucleotide sequences encoding cytolysin A (ClyA) and lactate oxidase (LOX). The recombinant plasmid (pJ23119-ClyA-6×His-LOX) was transformed into *E. coli* (Nissle 1917), successfully obtaining an engineered LOX-expressing bacterium (EcL).

[0044] The amino acid sequence of the lactate oxidase (LOX) is SEQ ID NO:1, and the nucleotide sequence is SEQ ID NO:2; the amino acid sequence of the cytolysin A is shown in SEQ ID NO:3; the nucleotide sequence of the cytolysin A is shown in SEQ ID NO:4; and the nucleotide sequence of the recombinant plasmid (pJ23119-ClyA-6×His-LOX) is shown in SEQ ID NO:5.

[0045] Escherichia coli (Nissle 1917) was purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC). The engineered LOX expression strain (EcL) was prepared and provided by Wuhan Miaoling Biotechnology Co., Ltd. The wild-type strain Nissle 1917 (EcN) was cultured in LB medium, while the engineered strain EcL was cultured in LB medium containing 100 μg / mL ampicillin sodium. Both culture conditions were 200 rpm and 37 °C.

[0046] 2. Preparation of mesoporous polydopamine nanoparticles 100 mg of Pluronic F127 (poloxam 407) and 60 mg of DA·HCl (dopamine hydrochloride) were dissolved in 60 mL of a mixed solvent of anhydrous ethanol and water in a 1:1 volume ratio and mixed well. 100 mg of TMB (1,3,5-trimethylbenzene) was added dropwise with stirring. After reacting for 30 minutes, 100 μL of NH3·H2O was added, and the reaction continued for 3 hours. The mixture was washed three times each with water and anhydrous ethanol to obtain mesoporous dopamine nanoparticles (PDA).

[0047] 3. Drug loading of mesoporous polydopamine nanoparticles Cisplatin (CDDP) was then dissolved in DMSO (dimethyl sulfoxide) to obtain a stock solution of 10 mg / mL, which was diluted with water to 1 mg / mL. 1 mL of the cisplatin solution was slowly added dropwise to 2 mL of an aqueous solution of PDA (1 mg / mL) with stirring for 3 hours. After stirring, the solution was centrifuged at 12000 rpm and washed twice with pure water to obtain drug-loaded mesoporous polydopamine nanoparticles (PP).

[0048] 4. Preparation of bacterial membrane vesicles The engineered bacteria obtained in step 1 were dispersed in an extraction buffer (containing 20 mM Tris-HCl, 50 mM NaCl, and 5 mM EDTA) and sonicated for 120 minutes. The resulting liquid was then centrifuged at 12,000 rpm for 30 minutes at 4°C, and the supernatant was collected. The supernatant was further centrifuged at high speed, and the precipitate was collected to obtain bacterial membrane vesicles (BMVs-Ecl), which were then dissolved in water to obtain a BMVs-Ecl solution.

[0049] 5. Bacterial membrane vesicle encapsulation of drug-loaded mesoporous polydopamine nanoparticles Drug-loaded mesoporous polydopamine nanoparticles (PP) were dispersed in water to obtain a PP solution with a concentration of 1 mg / mL. This PP solution was then slowly added dropwise to a BMVs-Ecl solution with a membrane protein concentration of 2 mg / mL, resulting in a final PP to BMVs-Ecl mass ratio of membrane protein of 1:1. After incubation at 25°C for 10 min, the solution was extruded through a 400 nm polycarbonate membrane to obtain membrane vesicle-coated nanoparticles (PPL). Using the BCA protein concentration assay kit (P0012) from Beyotime Biotechnology Co., Ltd., and following the product instructions, the supernatant obtained after centrifugation of lysed bacteria was used as the test bacterial membrane solution. Bovine serum albumin was used as a standard, and the absorbance of each well was measured at a wavelength of 562 nm. The membrane protein concentration of the bacterial membrane solution was calculated based on the standard curve.

[0050] 6. Coated Mn 2+ - Tannic acid network shell Add 30 μL of tannic acid (TA, 20 mg / mL) to 1 mL of PPL (1 mg / mL), then add 250 μL of MnSO4·H2O (10 mg / mL), vortex, add NaOH solution, adjust the pH to 5.5, and stir to obtain an acid-responsive mesoporous polydopamine shell structure nanoformulation (PPLM).

[0051] Example 2 The only difference between this embodiment and Example 1 is that the dopamine hydrochloride (DA·HCl) used in step 1 is 40 mg.

[0052] Example 3 The only difference between this embodiment and Example 1 is that the dopamine hydrochloride (DA·HCl) used in step 1 is 80 mg.

[0053] Example 4 The only difference between this embodiment and Example 1 is that the mass ratio of membrane proteins in PP and BMVs-EcL in step 3 is 1:0.5.

[0054] Example 5 The only difference between this embodiment and Example 1 is that the mass ratio of membrane proteins in PP and BMVs-EcL in step 3 is 1:3.

[0055] Example 6 The only difference between this embodiment and Embodiment 1 is that in step 2, 100 mg of Pluronic P123 is used instead of Pluronic F127.

[0056] Example 7 The only difference between this embodiment and Embodiment 1 is that 50 μL of NH3·H2O is used in step 2.

[0057] Example 8 The only difference between this embodiment and Embodiment 1 is that the amount of NH3·H2O used in step 2 is 200 μL.

[0058] Example 9 The only difference between this embodiment and Example 1 is that 1 ml of cisplatin solution with a concentration of 0.5 mg / mL is added in step 3.

[0059] Example 10 The only difference between this embodiment and Example 1 is that 1 ml of cisplatin solution with a concentration of 2 mg / mL is added in step 3.

[0060] Example 11 The only difference between this embodiment and Example 1 is that the amount of tannic acid (TA, 20 mg / mL) added in step 6 is 60 μL.

[0061] Example 12 The only difference between this embodiment and Example 1 is that the amount of tannic acid (TA, 20 mg / mL) added in step 6 is 100 μL.

[0062] Example 13 The only difference between this embodiment and Example 1 is that the amount of MnSO4·H2O (10 mg / mL) added in step 6 is 100 μL.

[0063] Example 14 The only difference between this embodiment and Example 1 is that the amount of MnSO4·H2O (10 mg / mL) added in step 6 is 500 μL.

[0064] Example 15 The only difference between this embodiment and Embodiment 1 is that gallic acid is added in step 6 instead of tannic acid.

[0065] Example 16 The only difference between this embodiment and Embodiment 1 is that catechol is added in step 6 to replace tannic acid.

[0066] Example 1: Structural characterization of drug-loaded polydopamine nanoparticles (PP) and membrane vesicle-coated product nanoparticles (PPL) The drug-loaded polydopamine nanoparticles (PP) and membrane vesicle-coated product nanoparticles (PPL) from Example 1 were used to observe their structural characterization using scanning electron microscopy and transmission electron microscopy.

[0067] Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of drug-loaded polydopamine nanoparticles (PP) are shown below. Figure 3 A, Figure 3 As shown in Figure B, the synthesized PP is roughly spherical with a regular morphology and a size of approximately 90 nm.

[0068] Transmission electron microscopy images of membrane vesicle-coated product nanoparticles (PPL) are shown below. Figure 3 As shown in Figure C, the PPL nanoparticles are spherical with a regular morphology and a size of approximately 120 nm. A layer of low-contrast structure is visible at the edge, indicating that bacterial membrane vesicles (BMVs-EcL) have been successfully encapsulated.

[0069] The drug-loaded polydopamine nanoparticles (PP) obtained in Examples 1, 2, 3, 6, 7, and 8 were subjected to scanning electron microscopy to observe their structure. The PP nanoparticles in Examples 1, 2, 3, 6, 7, and 8 are PP-1, PP-2, PP-3, PP-6, PP-7, and PP-8, respectively, and their scanning electron microscopy images are shown below. Figure 4 As shown, the prepared PP is spherical in shape, with only slight differences in size.

[0070] The hydrated particle size of the membrane vesicle-coated nanoparticles (PPL) obtained in Examples 1, 4, 5, 9, and 10 was measured. The PPLs from Examples 1, 4, 5, 9, and 10 are PPL-1, PPL-4, PPL-5, PPL-9, and PPL-10, respectively, and their hydrated particle sizes are as follows: Figure 5 As shown, the particle size of PPL is between 100-200nm.

[0071] Experimental Example 2: Structural and Performance Characterization of Acid-Responsive Mesoporous Polydopamine Shell-Structured Nanoformations (PPLM) Unless otherwise specified, the drug-loaded polydopamine nanoparticles (PP), membrane vesicle-coated product nanoparticles (PPL), and acid-responsive mesoporous polydopamine shell-structured nanoformulations (PPLM) used in this experimental example were all prepared in Example 1.

[0072] 1. Observation using transmission electron microscopy The shell-structured nanoformulations of acid-responsive mesoporous polydopamine (PPLM) were taken and their structure was observed using transmission electron microscopy. The structures of the shell-structured nanoformulations of acid-responsive mesoporous polydopamine (PPLM) obtained in Examples 11, 12, 13, and 14 were also observed using transmission electron microscopy.

[0073] The PPLMs used in Examples 11, 12, 13, and 14 are PPLM-1, PPLM-11, PPLM-12, PPLM-13, and PPLM-14, respectively, and their transmission electron microscope images are as follows: Figure 6 As shown; a transmission electron microscope image of the acid-responsive mesoporous polydopamine shell-structured nanoformulation (PPLM) in Example 1 is shown below. Figure 7 As shown in Figure A, it can be seen that the size of PPLM is slightly increased, and it is spherical similar to PP and PPL. The edge regularity is slightly reduced, indicating that the addition of membrane vesicles and shell structures has led to slightly irregular edges.

[0074] 2. Dynamic scattering (DLS) instrument for measuring dimensions and potentials. Mesoporous dopamine nanoparticles (PDA), drug-loaded polydopamine nanoparticles (PP), membrane vesicle-coated product nanoparticles (PPL), and acid-responsive mesoporous polydopamine shell-structured nanoformations (PPLM) were taken and their size and potential were measured using dynamic scattering (DLS).

[0075] Dynamic scattering instrument measured PP, PPL, PPLM as follows Figure 7 As shown in B and 7C, the hydrated particle size of PPLM is further increased to 227.3 nm compared to PP and PPL; the Zeta potential of PPLM is increased to -17.3 mV compared to PPL.

[0076] 3. Material elemental composition analysis Acid-responsive mesoporous polydopamine shell-structured nanoformulation (PPLM) was obtained, and its elemental surface distribution (mapping) and physicochemical properties were analyzed using X-ray photoelectron spectroscopy and field emission scanning electron microscopy (SEM) with its attached energy dispersive spectroscopy (EDS).

[0077] Element surface distribution (Mapping) analysis, such as Figure 7 As shown in Figure D, N, Pt, S, P and Mn elements are uniformly distributed on PPLM nanoparticles, indicating that cisplatin drug loading, BMVs-EcL coating and Mn-TA coating modification were successful.

[0078] X-ray photoelectron spectroscopy (XPS) analysis, such as Figure 7As shown in Figure E, characteristic signals of N, Pt, S, and Mn were detected, indicating that the BMVs-EcL coating and Mn-TA coating modification were successful.

[0079] 4. Dispersion stability test Acid-responsive mesoporous polydopamine shell-structured nanoparticles (PPLM) were dispersed in water and RPMI-1640 cell culture medium containing 10% serum, respectively, and cultured at 25°C for 7 days. The particle size changes were monitored using a dynamic scattering instrument.

[0080] PPLM particle size variation as follows Figure 7 As shown in Figure F, the particle size of PPLM did not change significantly, indicating that it has good dispersion stability.

[0081] Experimental Example 3: Characterization of the acid responsiveness of acid-responsive mesoporous polydopamine shell-structured nanoformulations (PPLM) The acid-responsive mesoporous polydopamine shell-structured nanoparticles (PPLM) from Example 1 were dispersed in phosphate-buffered saline (PBS) at pH 5.5 and pH 7.4, respectively, to prepare solutions with a final concentration of 2 mg / mL. These solutions were placed in dialysis bags with a molecular weight cutoff of 1000 and released at 37°C and 200 rpm. At predetermined time points, extradialysate was collected, and the Mn content in the release medium at different time points was determined using inductively coupled plasma mass spectrometry (ICP-MS). 2+ and Pt 2+ The cumulative release amount is plotted as a release curve.

[0082] Mn 2+ The release curve is as follows Figure 8 As shown in Figure A, it can be seen that Mn 2+ The release rate at pH 5.5 was significantly higher than that at pH 7.4, indicating that the Mn-TA metal-phenol coordination network shell possesses excellent acid-responsive degradation characteristics. Pt 2+ The release curve is as follows Figure 8 As shown in B, Pt can be seen 2+ The release behavior of Mn2+ and Pt showed a similar trend, with the release rate being significantly faster in an acidic environment at pH 5.5 than at pH 7.4. Furthermore, under both pH conditions, the release rates of Mn2+ and Pt... 2+ The release gradually leveled off after approximately 8 hours. This indicates that the coordination bonds in the Mn-TA shell break under acidic conditions, leading to rapid disintegration of the coating and the release of Mn from the nano-formulation. 2+ and Pt 2+ PPLM nanoparticles possess excellent acid-responsive release properties, which facilitates the specific release of drugs in the acidic microenvironment of tumors, thereby reducing off-target toxicity of drugs to normal tissues.

[0083] Experimental Example 4: Detection of the antitumor properties of PPLM in 4T1 tumor cells 1. Detection of antitumor properties at different PPLM concentrations The acid-responsive mesoporous polydopamine shell-structured nanoformulation (PPLM) from Example 1 was used to prepare RPMI 1640 culture medium with PPLM nanoparticle concentrations of 25, 50, 75, 100, 150, and 200 μg / mL.

[0084] Mouse breast cancer cells (4T1) were purchased from the China Center for Type Culture Collection (CCTCC, GDC0294) and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). The medium was supplemented with 1% penicillin-streptomycin (10,000 U / mL), and the cells were cultured at 37°C and 5% CO2. All cell lines were confirmed to be free of mycoplasma contamination. 4T1 cells were cultured at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates, adhered overnight, and then the original culture medium was discarded.

[0085] 4T1 cells after adherent culture were added to RPMI 1640 medium containing 20 mM lactate (LA) and different concentration gradients (0, 25, 50, 75, 100, 150, 200 μg / mL) of PPLM nanoparticles, and incubated at 37°C for 48 hours. The medium was then replaced with fresh medium containing 10% CCK-8 reagent, and incubation continued for 1.5 hours. The absorbance at 450 nm was measured using a microplate reader.

[0086] Cell viability as detected by the CCK-8 assay, such as Figure 9 As shown in Figure A, PPLM exhibits concentration-dependent cytotoxicity against 4T1 cells, with cell viability gradually decreasing as the concentration of nanoparticles increases; when the concentration reaches 150 μg / mL, cell viability drops below 50%.

[0087] 2. Detection of antitumor properties of different nanoparticles Take the acid-responsive mesoporous polydopamine shell-structured nanoparticle formulation (PPLM) from Example 1 and prepare RPMI 1640 medium with a PPLM nanoparticle concentration of 150 μg / mL; take the membrane vesicle-coated product nanoparticles (PPL) from Example 1 and prepare RPMI 1640 medium containing different nanoparticles, maintaining the CDDP addition dose in the medium at the same level as the RPMI 1640 medium containing 150 μg / mL PPLM.

[0088] The mouse breast cancer cell line 4T1 was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and routinely passaged in a constant temperature incubator at 37°C and 5% CO2. 4T1 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well, allowed to adhere overnight, and then the original medium was discarded.

[0089] 4T1 cells after adherent culture were taken and RPMI 1640 medium containing 20 mM lactate (LA) and the different nanoparticles mentioned above were added and incubated for 48 hours. The original medium was discarded and replaced with 100 μL of fresh medium containing 10% CCK-8 reagent and incubated for another 1.5 hours. The absorbance (OD) value at 450 nm was measured using a microplate reader.

[0090] Separately, adherent 4T1 cells were cultured and incubated with RPMI 1640 medium containing 20 mM lactate (LA) and the aforementioned different nanoparticles for 48 hours. The cell culture supernatant from each group was collected, centrifuged at 3000 rpm for 10 min at 4°C to remove cell debris, and the lactate concentration was measured according to the instructions of the lactate assay kit (Nanjing Jiancheng Co., Ltd.).

[0091] Cell viability as detected by the CCK-8 assay, such as Figure 9 As shown in Figure B, the PPLM-treated group had the lowest cell survival rate, indicating that it had the strongest killing effect on 4T1 cells.

[0092] The lactic acid content in the cell supernatant is as follows Figure 9 As shown in Figure C, the LA content in the supernatant of 4T1 cells in the PPLM group was the lowest, indicating that it had the strongest LA clearance ability.

[0093] Based on the above experiments, it can be shown that PPLM can be taken up by tumor cells, has significant toxicity to tumor cells, and has a strong lactic acid clearance ability.

[0094] Experimental Example 5: Detection of the antitumor properties of PPLM in mice All mice used in this experiment were Balb / c mice, 6-8 weeks old, female, and weighing between 18 and 22 g. The mice were housed in an SPF-grade laboratory animal center with air cleanliness level 7, room temperature of 22-25°C, humidity of 50%-60%, and light intensity of 15-20 lx, with a 12-hour / 12-hour light-dark cycle. The mice were housed in individually ventilated cages (IVCs), six mice per cage, with free access to food and water. Feed was sterilized by cobalt-60 irradiation, and drinking water was autoclaved.

[0095] All animal experiments in this case were conducted in accordance with the charter of the Experimental Animal Ethics Committee of Wuhan University.

[0096] 1.4 Construction of T1 tumor-bearing mouse model Female Balb / c mice were subcutaneously injected with 1×10⁻⁶ spores into the right leg side. 6 4T1 cells were used to construct a subcutaneous breast tumor model. The tumor was allowed to grow to approximately 100 mm in size. 3 The mice were randomly divided into 3 groups and then subjected to further procedures.

[0097] 2. Antitumor performance testing The acid-responsive mesoporous polydopamine shell-structured nanoparticles (PPLM) and membrane vesicle-coated product nanoparticles (PPL) from Example 1 were dissolved in PBS solution to prepare a solution with a nanoparticle concentration of 100 μg / mL.

[0098] Using day 0 as the time when mice were inoculated with tumor cells, 100 μL of the above-prepared nanoparticle solutions and the same volume of PBS solution were injected via the tail vein on days 7 and 13, respectively. The tumor volume and body weight of the mice were measured every two days, and the survival status of the mice was recorded over 60 days.

[0099] Changes in tumor volume in mice, such as Figure 10 As shown in Figure A, it can be seen that after treatment with acid-responsive mesoporous polydopamine shell-structured nanoparticles (PPLM), tumor growth in tumor-bearing mice was significantly inhibited, and the tumor volume was significantly smaller than that of other materials. This indicates that PPLM has a good inhibitory effect on tumor growth, and the scavenging effect of PPLM on tumor cells is enhanced due to the clearance of lactic acid.

[0100] Mouse weight changes as follows Figure 10 As shown in Figure B, there was no significant fluctuation in the body weight of mice in each group, indicating that PPLM has good biocompatibility.

[0101] Mouse survival analysis results are as follows Figure 10 As shown in Figure C, about one-third of the mice in the PPLM group survived on day 60, while all mice in the other groups died. This indicates that PPLM treatment can significantly prolong the survival time of tumor-bearing mice and has a good anti-tumor effect.

[0102] The acid-responsive shell structure nanoformulations obtained in Examples 2-16 were subjected to the verification experiments as in Examples 1-5, and the results were similar to those of the acid-responsive shell structure nanoformulations obtained in Example 1.

[0103] SEQ ID NO:1 MVDAVKADPFGKVNAIDVLDLASLEARAEKVLGRGEFGYISEGSDDGYTMHRNTTAFQDVHMLPRVLQGVENPDQSTTFMGAKLASPLLTAPIASNTLAHPSGELGLAKGAKEAGIMMSQSTFASKTIAETAAVSDGAPYMFQLYMPKDWEYCQYLLDEAKQAGALAIILTADSTLGGYREKDVMNHYHLKGRLANLEGYNTGQSGVGAGGLFKESMQKLDLGLISKLASYSGLPIIIKGIQHPADAVAAITAGAAGIYVSNHGGRQLDGAPGAIEQLPAIAAAVDHRVPIIFDGGVQRGTHVLKALALGADLVGIGRPFSYGLALGGWQGVKDVADHLKMEINIAMQLTGCQTMADVKQMKVKTTFA SEQ ID NO:2 SEQ ID NO:3 MTEIVADKTVEVVKNAIETADGALDLYNKYLDQVIPWQTFDETIKELSRFKQEYSQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAAYILLFDEYNEKKASAQKDILIKVLDDGITKLNEAQKSLLVSSQSFNNASGKLLAL DSQLTNDFSEKSSYFQSQVDKIRKEAYAGAAAGVVAGPFGLIISYSIAAGVVEGKLIPELKNKLKSVQNFFTTLSNTVKQANKDIDAAKLKLTTEIAAIGEIKTETRFYVDYDDLMLSLLKEAAKKMINTCNEYQKRHGKKTLFEVPEV SEQ ID NO.4 ATGACTGAAATCGTTGCAGATAAAACGGTAGAAGTAGTTAAAAACGCAATCGAAACCGCAGATGGAGCATTAGATCTTTATAATAAATATCTCGATCAGGTCATCCCCTGGCAGACCTTTGATGAAACCATAAAAGAGTTAAGTCGCTTTAAACAAGAGTATTCACAGGCAGCCAGCGTTTTAGTTGGCGATATTAAAACCTTACTTATGGATAGCCAAGATAAGTATTTTGAAGCAACCCAAACAGTGTATGAATGGTGTGGTGTTGCGACGCAATTGCTCGCAGCGTATATTTTGCTATTTGATGAGTACAATGAGAAGAAAGCATCCGCCCAAGAAGACATTCTCATTAAAGTACTGGATGACGGCATCACGAAGCTGAATGAAGCGCAAAAATCCCTGCTGGTAAGCTCACAAGTTTCAACAACGCATTCCGGGAAACTGCTGGCGTTAGATAGCCAGTTAACCAATGATTTTTCAGAAAAAAGCAGCTATTTCCAGTCACAGGTAGATAAAATCAGGAAGGAAGCATATGCCGGTGCCGCAGCCGGTGTCGTCGCCGGTCCATTTGGATTAATCATTTCTTATTCTATTGCTGCGGGCGTAGTTGAAGGAAAACTGATCCCAGAATTGAAGAACAAGTTAAAATCTGTGCAGAATTTCTTTACCACCCTGTCTAACACGGTTAAACAAGCGAATAAAGATATCGATGCCGCCAAATTGAAATTAACCACCGAAATAGCCGCCATCGGTGAGATAAAAACGGAAACTGAAACCACAAGATTCTAC SEQ ID NO.5 The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. An acid-responsive shell-structured nano-formulation, characterized in that, Including mesoporous polydopamine, platinum-based drugs, bacterial membrane vesicles, and metal-polyphenol network shells; The mesoporous polydopamine is a carrier for loading the platinum-based drug, and the outer layer of the mesoporous polydopamine sequentially coats the bacterial membrane vesicles and the metal-polyphenol network shell. The platinum-based drug is cisplatin; the metal-polyphenol network shell comprises metal ions and polyphenolic compounds, wherein the metal ions are Mn. 2+ Ions; the polyphenolic compounds include at least one of tannic acid, gallic acid, and catechol; The bacterial membrane vesicles are derived from Escherichia coli and express a cytolysin A-lactate oxidase fusion protein; the amino acid sequence of cytolysin A is shown in SEQ ID NO:3, and the amino acid sequence of lactate oxidase is shown in SEQ ID NO:

1.

2. The acid-responsive shell-structured nanoformulation according to claim 1, characterized in that, The *E. coli* contains a nucleotide sequence encoding a cytolysin A-lactate oxidase fusion protein; the nucleotide sequence of cytolysin A is shown in SEQ ID NO:4, and the nucleotide sequence of the gene encoding lactate oxidase is shown in SEQ ID NO:

2.

3. A method for preparing an acid-responsive shell-structured nanoparticle formulation as described in any one of claims 1 to 2, characterized in that, Includes the following steps: (1) The engineered bacteria that have been introduced with the gene encoding lactate oxidase were cultured, and after induction of expression, the bacterial cells were collected and the bacterial membrane vesicles expressing the cytolysin A-lactate oxidase fusion protein were extracted. (2) The template agent and dopamine hydrochloride were dissolved in a mixed solvent of ethanol and water, an organic pore-expanding agent was added, and then an alkaline reagent was added to adjust to alkalinity. The polymerization reaction yielded mesoporous polydopamine nanoparticles. The mesoporous polydopamine nanoparticles were mixed with cisplatin to obtain drug-loaded mesoporous polydopamine nanoparticles. The template agent includes either Pluronic F127 or Pluronic P123; the mass ratio of the template agent to dopamine hydrochloride is 1:0.2~1; The organic pore-expanding agent is 1,3,5-trimethylbenzene, and the mass ratio of the organic pore-expanding agent to the template agent is 0.5~2:1; The amount of cisplatin added is based on the amount of platinum element, with 0.6~8 μmol of cisplatin added per milligram of mesoporous polydopamine nanoparticles; (3) Mix and incubate the bacterial membrane vesicles in step (1) and the drug-loaded mesoporous polydopamine nanoparticles in step (2), and extrude them through a microporous filter membrane to obtain drug-loaded mesoporous polydopamine nanoparticles coated with membrane vesicles. The mass ratio of the drug-loaded mesoporous polydopamine nanoparticles to the membrane proteins in the bacterial membrane vesicles is 1:0.5~3; (4) Dissolve the drug-loaded mesoporous polydopamine nanoparticles and polyphenolic compounds coated with membrane vesicles from step (3) in water, and add Mn 2+ An ionic salt solution was used to adjust the pH of the system to a weakly acidic level, resulting in an acid-responsive shell-structured nano-formulation. The polyphenolic compounds include at least one of tannic acid, gallic acid, and catechol; The mass ratio of the drug-loaded mesoporous polydopamine nanoparticles coated with the membrane vesicles to the polyphenolic compound is 1:0.2~2; the Mn 2+ The ion feed amount is 10~20 μmol per milligram of drug-loaded mesoporous polydopamine nanoparticles coated with the membrane vesicles.

4. The preparation method according to claim 3, characterized in that, In step (2), the volume ratio of ethanol to water in the mixed solvent of ethanol and water is 1:0.5~2; the reaction time after adding the organic pore expander is 0.2~2 h; the pH of the system is adjusted to 7.5~12.0 by the alkaline reagent, and the reaction time after alkalinization is 1~6 h.

5. The preparation method according to claim 3, characterized in that, In step (3), the microporous filter membrane includes a polycarbonate membrane with a pore size of 400~800 nm.

6. The preparation method according to claim 3, characterized in that, In step (4), the Mn² + The concentration of the ion salt solution is 50-70 mM, and the pH of the system is 4-7.

7. The use of an acid-responsive shell structured nanoformulation as described in any one of claims 1 to 2 in the preparation of drugs for treating and / or alleviating tumors.

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