Bionic inorganic nanoparticles with antigen capture ability and preparation method thereof

Abstract

The present invention discloses a biomimetic inorganic nanoparticle with antigen capture capability and a preparation method thereof. The biomimetic inorganic nanoparticle comprises a bacterial outer membrane vesicle, wherein the bacterial outer membrane vesicle is coated with Prussian blue-manganese dioxide inorganic nanoparticles, the vesicle wall of the bacterial outer membrane vesicle is embedded with a photosensitizer Ce6, and the outer surface of the vesicle wall of the bacterial outer membrane vesicle is modified with maleimide. After the nanomaterial is injected into the tumor, MnO2 catalyzes H2O2 to produce O2 to solve the problem of hypoxia at the tumor site. PB and Ce6 respectively play a PTT / PDT synergistic role in killing tumor cells, inducing cell immunogenic death and releasing tumor antigens. Mal captures the released antigens by forming a thioether bond. In addition, OMV contains danger signals derived from the bacterial outer membrane, stimulates dendritic cells to mature, and stimulates a strong tumor immune response. The above mechanism can synergistically and efficiently kill tumors and prevent their metastasis and recurrence, thereby achieving efficient treatment of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of colorectal cancer drugs, and in particular to a biomimetic inorganic nanoparticle with antigen-capturing capability and a preparation method thereof. Background Art

[0002] The treatments for colorectal cancer mainly include chemotherapy, radiotherapy, surgery, immunotherapy, targeted therapy, etc. However, due to limitations such as tumor resistance, limited surgical sites, and large toxic side effects, the treatment effect is difficult to be satisfactory. It is urgent to explore an efficient, targeted, low-side-effect and non-invasive treatment method. Surgery combined with radiotherapy is a commonly used treatment for colorectal cancer. Surgical treatment can remove metastatic lesions of rectal cancer, and radiotherapy is usually used as an adjuvant treatment for colorectal cancer. Radiotherapy can reduce the tumor stage, improve the anal preservation rate, and reduce the local recurrence rate. However, colorectal cancer surgery combined with radiotherapy still faces two major problems: easy recurrence and metastasis of tumors after surgery and low postoperative survival rate. Therefore, a better therapeutic drug is urgently needed clinically. Summary of the Invention

[0003] The purpose of the present invention is to address the deficiencies in the prior art and provide a biomimetic inorganic nanoparticle with antigen-capturing capability and a preparation method thereof.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] The first aspect of the present invention is to provide a bionic inorganic nanoparticle with antigen capture ability, including bacterial outer membrane vesicles, the bacterial outer membrane vesicles are coated with Prussian blue-manganese dioxide inorganic nanoparticles, the vesicle wall of the bacterial outer membrane vesicles is embedded with photosensitizer Ce6, and the outer surface of the vesicle wall of the bacterial outer membrane vesicles is modified with maleimide.

[0006] The second aspect of the present invention is to provide a method for preparing the above-mentioned biomimetic inorganic nanoparticles, comprising the following steps:

[0007] Step 1: Obtain bacterial outer membrane vesicles from Escherichia coli, denoted as OMVs;

[0008] Step 2: The OMVs obtained in step 1 were dispersed in PBS buffer and treated with N-ethylmaleimide to deplete free thiols on the OMVs.

[0009] Step 3: The maleimide-tetraethylene glycol-succinimidyl acrylate solution is mixed with the OMV treated in step 2, incubated, washed and centrifuged to obtain maleimide-modified bacterial outer membrane vesicles, which are recorded as OMV-Mal;

[0010] Step 4: Stirring the dihydrochlorin Ce6 and the OMV-Mal obtained in step 3 in PBS buffer to complete the loading of Ce6 on the OMV, which is recorded as OMV-Mal / Ce6. The obtained OMV-Mal / Ce6 is resuspended in PBS buffer and stored for later use;

[0011] Step 5: Dissolve polyvinyl pyrrolidone in hydrochloric acid solution, add potassium ferrocyanide and potassium permanganate in sequence, and collect the resulting reaction mixture by centrifugation to obtain Prussian blue-manganese dioxide, denoted as PBMn. The purified PBMn nanoparticles are redispersed in water and stored for later use.

[0012] Step six: The PBMn suspension and the OMV-Mal / Ce6 suspension were vortexed, and the ultrasonic probe was used to coat the OMV on the PBMn to obtain PBMn@OMV-Mal / Ce6.

[0013] Furthermore, the step 1 is specifically as follows: Escherichia coli is inoculated into LB culture medium, placed in a shaking incubator at 35-37° C., and bacterial outer membrane vesicles are obtained by ultrasonic disruption, which are recorded as OMVs.

[0014] Furthermore, in the step 2, the treatment time with N-ethylmaleimide at 5-35° C. is 2-6 hours.

[0015] Furthermore, in the step 3, the incubation temperature is 5-35° C., the incubation time is 2-6 hours, and the concentration of the maleimide-tetraethylene glycol-succinimidyl acrylate solution is 0.6-5 mg / mL.

[0016] Furthermore, in step 4, the temperature during stirring is 5-35°C, and the stirring time is 2-8 hours; the storage temperature of the OMV-Mal / Ce6 suspension is 0-4°C.

[0017] Furthermore, in the step 4, the mass ratio of dihydrochlorin Ce6 to OMV-Mal is 1:(1-10).

[0018] Furthermore, in the step 5, the hydrochloric acid solution is obtained by dissolving concentrated hydrochloric acid with a concentration of 36 w / v% in deionized water, and the volume ratio of the concentrated hydrochloric acid to the deionized water is 1:(480-600).

[0019] Furthermore, in the step 5, the reaction temperature is 80-90° C., and the reaction time is 24-36 hours.

[0020] Furthermore, in step six, the mass ratio of PBMn to OMV-Mal / Ce6 is 1:(2-10).

[0021] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0022] The bionic inorganic nanoparticles with antigen-capturing ability prepared by the present invention are bacterial outer membrane vesicles (OMVs) embedded with the photosensitizer Ce6 and coated with inorganic nanoparticles Prussian blue-manganese dioxide (PB-MnO2, PBMn). In addition, maleimide (Mal) is modified on the surface of the OMVs through chemical bonds. After the nanomaterial is injected into the tumor, MnO2 catalyzes H2O2 to produce O2 to solve the problem of hypoxia in the tumor site. PB and Ce6 respectively exert PTT / PDT to synergistically kill tumor cells, induce cell immunogenic death, and release tumor antigens. Mal captures the released antigens by forming thioether bonds. In addition, OMVs contain danger signals derived from the bacterial outer membrane, which stimulate dendritic cells to mature and stimulate a strong tumor immune response. The above mechanisms can synergistically and efficiently kill tumors and prevent their metastasis and recurrence, thereby achieving efficient treatment of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart for the preparation of biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 of the present invention;

[0024] Figure 2 This is a transmission electron microscopy image of the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 of the present invention;

[0025] Figure 3 Figure 2 is a particle size diagram of the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 and PBMn of the present invention;

[0026] Figure 4 Zeta potential diagram of the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 and OMV, PBMn, and PBMn@OMV of the present invention;

[0027] Figure 5 This is the SDS-PAGE image of the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6, OMV and PBMn of the present invention;

[0028] Figure 6 This is a photothermal effect diagram of the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 of the present invention;

[0029] Figure 7 This is a diagram showing the effect of the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 producing ROS;

[0030] Figure 8 This is a statistical diagram of the killing effect of the bionic inorganic nanoparticles PBMn@OMV-Mal / Ce6 of the present invention on colorectal cancer cells. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0032] refer to Figure 1 The present invention provides a bionic inorganic nanoparticle with antigen capture ability, including bacterial outer membrane vesicles, the bacterial outer membrane vesicles are coated with Prussian blue-manganese dioxide inorganic nanoparticles, the vesicle wall of the bacterial outer membrane vesicles is embedded with photosensitizer Ce6, and the outer surface of the vesicle wall of the bacterial outer membrane vesicles is modified with maleimide.

[0033] Continue to refer Figure 1 The present invention also provides a method for preparing the above-mentioned biomimetic inorganic nanoparticles, comprising the following steps:

[0034] Step 1: Obtain bacterial outer membrane vesicles from Escherichia coli, denoted as OMVs;

[0035] Step 2: The OMVs obtained in step 1 were dispersed in PBS buffer and treated with N-ethylmaleimide to deplete free thiols on the OMVs.

[0036] Step 3: The maleimide-tetraethylene glycol-succinimidyl acrylate solution is mixed with the OMV treated in step 2, incubated, washed and centrifuged to obtain maleimide-modified bacterial outer membrane vesicles, which are recorded as OMV-Mal;

[0037] Step 4: Stirring the dihydrochlorin Ce6 and the OMV-Mal obtained in step 3 in PBS buffer to complete the loading of Ce6 on the OMV, which is recorded as OMV-Mal / Ce6. The obtained OMV-Mal / Ce6 is resuspended in PBS buffer and stored for later use;

[0038] Step 5: Dissolve polyvinyl pyrrolidone in hydrochloric acid solution, add potassium ferrocyanide and potassium permanganate in sequence, and collect the resulting reaction mixture by centrifugation to obtain Prussian blue-manganese dioxide, denoted as PBMn. The purified PBMn nanoparticles are redispersed in water and stored for later use.

[0039] Step six: The PBMn suspension and the OMV-Mal / Ce6 suspension were vortexed, and the ultrasonic probe was used to coat the OMV on the PBMn to obtain PBMn@OMV-Mal / Ce6.

[0040] As a preferred example, step 1 specifically includes: inoculating Escherichia coli into LB culture medium, culturing in a shaking incubator at 35-37°C, and obtaining bacterial outer membrane vesicles by ultrasonic disruption, which are recorded as OMVs.

[0041] As a preferred example, in step 2, the treatment time with N-ethylmaleimide at 5-35° C. is 2-6 hours, more preferably 2 hours.

[0042] As a preferred example, in step 3, the incubation temperature is 5-35° C., the incubation time is 2-6 hours, more preferably 2 hours; the concentration of the maleimide-tetraethylene glycol-succinimide acrylate solution is 0.6-5 mg / mL, more preferably 0.6 mg / mL.

[0043] As a preferred example, in step 4, the stirring temperature is 5-35°C, the stirring time is 2-8 hours, more preferably 2 hours; the storage temperature of the OMV-Mal / Ce6 suspension is 0-4°C, more preferably 4°C.

[0044] As a preferred example, in step 4, the mass ratio of dihydrochlorin Ce6 to OMV-Mal is 1:(1-10), more preferably 1:1.

[0045] As a preferred example, in step five, the hydrochloric acid solution is obtained by dissolving concentrated hydrochloric acid with a concentration of 36 w / v% in deionized water, and the volume ratio of the concentrated hydrochloric acid to the deionized water is 1:(480-600).

[0046] As a preferred example, in step five, the reaction temperature is 80-90°C, more preferably 86°C oil bath, and the reaction time is 24-36 hours, more preferably 24 hours.

[0047] As a preferred example, in step six, the mass ratio of PBMn to OMV-Mal / Ce6 is 1:(2-10), more preferably 1:2.

[0048] Example 1

[0049] This embodiment provides a method for preparing biomimetic inorganic nanoparticles with antigen capture capability, comprising the following steps:

[0050] Step 1: Inoculate Escherichia coli into 250mL of LB medium and place it in a shaker at 37℃; when the OD600 of the medium reaches 1.2, collect the bacteria by centrifugation at 5000rpm for 3min; after washing, redisperse the bacteria in 12mL of OMV extract (10mMTris-HCl, 10mMEDTA, 150mMNaCl, pH7.4), and then crush them in an ultrasonic cell disruptor (ultrasound on for 5 seconds, off for 10 seconds, total time is 90 minutes, 0℃), pay attention to ice bath to prevent the temperature from being too high; after ultrasonication, centrifuge at 14000g, 4℃ for 20min, and collect the supernatant as OMV; then freeze-dry the OMV and store at -80℃ for subsequent experiments.

[0051] Step 2: Treat OMVs with N-ethylmaleimide (0.5 mg / mL) at room temperature for 2 hours to deplete free thiols on the OMVs (the purpose is that if the OMVs have too many thiols (i.e., sulfhydryl groups), they may deplete the Mal on Mal-PEG4-NHS, thus causing its inactivation and loss of its ability to adsorb tumor antigens).

[0052] Step 3: Mix 100 μL of Mal-PEG4-NHS solution (concentration of 0.6 mg / mL) with 600 μL of treated OMVs (containing 600 μg of protein) and incubate at room temperature for 2 hours (reaction principle: NHS, i.e., N-hydroxysuccinimide, can react with NH2 primary amino groups to form amide bonds); ultracentrifuge at 150,000 × g, 4°C, for 3 hours to remove excess Mal-PEG4-NHS.

[0053] Step 4: Stir 100 μg / mL Ce6 and 100 μg / mL OMV-Mal in 2 mL PBS buffer at room temperature for 2 h to complete the loading of Ce6 on OMVs; centrifuge and wash with PBS (14,000 g, 20 min) to remove free Ce6; suspend the obtained OMV-Mal / Ce6 again in 2 ml PBS and store at 4°C for use.

[0054] Step 5: Dissolve 3 g of PVP in 50 mL of deionized water containing 104 μL of concentrated HCl (36%, w / v) at room temperature, and add 132 mg of K3[Fe(CN)6] and 52 mg of KMnO4 in sequence; after KMnO4 is completely dissolved, immediately transfer the resulting brown-black solution to an oil bath preheated to 86°C, and continue the reaction for 24 hours. The resulting reaction mixture is centrifuged (16,000 rpm, 30 min) to collect the PBMn nanoparticles; wash the PBMn nanoparticles with water five times to remove PVP; and redisperse the purified PBMn nanoparticles in water and store at room temperature for use.

[0055] Step 6: Vortex 1 mL of PBMn suspension (1 mg / mL) and 1 mL of OMV-Mal / Ce6 suspension (2 mg / mL); use an ultrasonic probe (50 W, 3 min) to coat the OMV on the composite inorganic nanoparticles. Centrifuge PBMn@OMV-Mal / Ce6 at 6000 × g, 15 min, 4°C, and resuspend in PBS to obtain the final material.

[0056] The transmission electron microscope (TEM) Figure 2 The particle size diagram is shown in Figure 3 The results show that the hydrated particle size of PBMn is about 120nm, and the hydrated particle size of PBMn@OMV-Mal / Ce6 is about 200nm. The Zeta potential is shown in Figure 4The results show that the potential of PBMn@OMV-Mal / Ce6 is negative and has the largest absolute value. Figure 5 The results showed that the characteristic proteins of PBMn@OMV-Mal / Ce6 were consistent with those of OMV. The above results can all indicate that PBMn@OMV-Mal / Ce6 has been successfully synthesized.

[0057] Its photothermal effect is Figure 6 , showing that the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 of the present invention can increase the temperature from 28.1°C to 52.7°C within 5 minutes, indicating that it has good photothermal performance.

[0058] The effect of producing ROS is shown in Figure 7 , showing that after 660nm laser irradiation and incubation with DCFH-DA probe, a large amount of green fluorescence was observed under a fluorescence microscope for the biomimetic inorganic nanoparticles PBMn@OMV-Mal / Ce6 of the present invention, which indicates that the material has a strong ROS production ability and can be used for PDT.

[0059] The killing effect of different concentrations on colorectal cancer cells is shown in Figure 8 The material's cytotoxicity against colorectal cancer cells was measured at concentrations of 0, 5ug / mL, 10ug / mL, 25ug / mL, and 50ug / mL. One group received PTT, one received PDT, and one received PDT + PTT. The results showed that PDT combined with PTT had a stronger cytotoxic effect than either PTT or PDT alone, and the cytotoxicity increased with increasing concentration.

[0060] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A biomimetic inorganic nanoparticle with antigen capture capability, characterized in that: The method comprises bacterial outer membrane vesicles, wherein the bacterial outer membrane vesicles are coated with Prussian blue-manganese dioxide inorganic nanoparticles, and the Prussian blue-manganese dioxide is denoted as PBMn; the vesicle wall of the bacterial outer membrane vesicles is embedded with photosensitizer Ce6, and the outer surface of the vesicle wall of the bacterial outer membrane vesicles is modified with maleimide, denoted as OMV-Mal / Ce6; the mass ratio of PBMn to OMV-Mal / Ce6 is 1:(2-10).

2. The method for preparing the biomimetic inorganic nanoparticles according to claim 1, characterized in that: The steps include: Step 1: Obtain bacterial outer membrane vesicles from Escherichia coli, denoted as OMVs; Step 2: The OMVs obtained in step 1 were dispersed in PBS buffer and treated with N-ethylmaleimide to deplete free thiols on the OMVs. Step 3: The maleimide-tetraethylene glycol-succinimidyl acrylate solution is mixed with the OMV treated in step 2, incubated, washed and centrifuged to obtain maleimide-modified bacterial outer membrane vesicles, which are recorded as OMV-Mal; Step 4: Stirring the dihydrochlorin Ce6 and the OMV-Mal obtained in step 3 in PBS buffer to complete the loading of Ce6 on the OMV, which is recorded as OMV-Mal / Ce6. The obtained OMV-Mal / Ce6 is resuspended in PBS buffer and stored for later use; Step 5: Dissolve polyvinyl pyrrolidone in hydrochloric acid solution, add potassium ferrocyanide and potassium permanganate in sequence, and collect the resulting reaction mixture by centrifugation to obtain Prussian blue-manganese dioxide, denoted as PBMn. The purified PBMn nanoparticles are redispersed in water and stored for later use. Step six: The PBMn suspension and the OMV-Mal / Ce6 suspension were vortexed, and the ultrasonic probe was used to coat the OMV on the PBMn to obtain PBMn@OMV-Mal / Ce6.

3. The preparation method according to claim 2, characterized in that The step 1 specifically includes: inoculating Escherichia coli into LB culture medium, placing it in a shaking incubator at 35-37° C., and obtaining bacterial outer membrane vesicles by ultrasonic disruption, which are recorded as OMVs.

4. The preparation method according to claim 2, characterized in that In the step 2, the treatment time with N-ethylmaleimide is 2-6 hours at 5-35°C.

5. The preparation method according to claim 2, characterized in that In the step 3, the incubation temperature is 5-35° C., and the incubation time is 2-6 hours; the concentration of the maleimide-tetraethylene glycol-succinimidyl acrylate solution is 0.6-5 mg / mL.

6. The preparation method according to claim 2, characterized in that In the step 4, the temperature during stirring is 5-35°C, and the stirring time is 2-8 hours; the storage temperature of the OMV-Mal / Ce6 suspension is 0-4°C.

7. The preparation method according to claim 2, characterized in that In the step 4, the mass ratio of dihydrochlorin Ce6 to OMV-Mal is 1:(1-10).

8. The preparation method according to claim 2, characterized in that In the step 5, the hydrochloric acid solution is obtained by dissolving concentrated hydrochloric acid with a concentration of 36 w / v% in deionized water, and the volume ratio of the concentrated hydrochloric acid to the deionized water is 1:(480-600).

9. The preparation method according to claim 2, characterized in that In the step 5, the reaction temperature is 80-90° C. and the reaction time is 24-36 hours.

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