Integrated bionic nanoplatform based on hollow polydopamine nanoparticles and its preparation and application

Abstract

An integrated bionic nano-platform based on hollow polydopamine nanoparticles, with a general formula of [AVPt@HP@M], is prepared as follows: ZIF‑8 is synthesized by zinc nitrate and dimethylimidazole, and Pt@ZIF‑8 is synthesized by adding PVP-modified Pt nanoparticles. Dopamine is added to the Pt@ZIF‑8 system to form hollow polydopamine nanoparticles Pt@HP embedded with platinum. The 100‑109 functional fragment of apoptin and verteporfin are added to form AVPt@HP, and AVPt@HP is mixed with cancer cell membrane fragments and stirred in an ice bath, and extruded with a nano-extruder so that AVPt@HP is camouflaged by cancer cell membrane. The nano-platform of the present invention has excellent targeting and strong drug loading capacity, and its combined radiotherapy can kill tumor cells by improving hypoxia at the tumor site, directly promoting tumor cell apoptosis, and producing photodynamic therapy effects, thereby playing a radiotherapy sensitization role.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to an integrated bionic nano-platform based on hollow polydopamine nano-particles and a preparation method and application thereof. Background Art

[0002] Cancer is the leading cause of death. Cancer treatment requires an individualized approach, such as surgery, radiotherapy, and chemotherapy. Recently, the effects of immunotherapy and photodynamic therapy have been verified in clinical trials. Radiotherapy is widely used in clinical practice, and 70% of cancer patients require radiotherapy. However, the radioresistance of tumor tissues and the dose limitation of normal tissues severely limit the efficacy of radiotherapy. In addition, radiotherapy alone can cause serious side effects, including fibrosis or induction of advanced cancer. Therefore, in cancer treatment, improving radiosensitivity is an ongoing challenge.

[0003] The extent of cancer cell damage induced by radiotherapy depends mainly on the oxygen content of the tumor tissue. Reactive oxygen species can be used to stabilize radiation-induced DNA damage and prevent cells from repairing DNA, thereby improving the efficiency of radiotherapy. However, hypoxia is a hallmark of malignant tumors because of their inadequate oxygen supply system. The abnormal vascular system cannot deliver enough oxygen to rapidly proliferating cancer cells, leading to radioresistance. Therefore, alleviating tumor hypoxia is crucial to enhance the efficacy of RT. Recently, researchers have provided many strategies to overcome tumor tissue hypoxia, including direct oxygen delivery, increasing blood flow in tumor tissue, and in situ oxygen production. Among them, in situ oxygen generation has been applied in many fields of nanomedicine. But the efficiency of in situ oxygen production is currently a challenge.

[0004] Apoptin (AP), a protein encoded by the VP3 gene from chicken anemia virus (CAV), can selectively cause cell apoptosis. AP aggregates in filaments and is then degraded by the proteasome without harming normal cells. However, AP can be activated in malignant cells, preventing DNA repair in dividing cancer cells and then inducing apoptosis in malignant cells. Our early studies found that apoptin100-109 (AP) has the potential to be a radiosensitizer. So, how to make it targeted and play a better radiosensitizing role is a technical challenge.

[0005] Traditional photodynamic therapy (PDT) has shown great efficacy in tumor treatment and has been used clinically. However, the main disadvantage of PDT is the limited light penetration depth. Due to the high tissue penetration of X-rays, researchers have proposed that X-ray induced photodynamic therapy (X-PDT) can overcome the shortcomings of PDT and improve the treatment of deep-seated cancers. In the classic X-PDT model, the energy of X-rays used for clinical radiotherapy ranges from hundreds of keV to MeV, accompanied by in situ generated Cherenkov radiation and a series of high-energy secondary electrons. Traditional photosensitizers can absorb X-ray radiation energy and transfer it to produce cytotoxic singlet oxygen ( 1 O2), which is required for tumor destruction. The researchers found that X-PDT had a higher efficacy than radiotherapy alone, indicating that X-PDT is a unique radiosensitization method. Both PDT and X-PDT generate cytotoxic reactive oxygen species (ROS), especially singlet oxygen ( 1 O2), these ROS can directly or indirectly cause cancer cell death. So how to present photosensitizers to the appropriate site to produce more precise photodynamic effects is the current challenge.

[0006] Advances in nanotechnology provide new ideas for drug delivery. Hollow nanomaterials with excellent performance (such as hollow mesoporous silica nanoparticles, hollow manganese dioxide, and hollow gold nanospheres) show great potential in the field of drug delivery. Among them, hollow polydopamine nanoparticles (HP) polymerized by dopamine (DPA) have excellent biocompatibility and have received increasing attention. However, for targeted drug delivery, nanocarriers without surface modification can be easily recognized by the reticuloendothelial system (RES); therefore, most nanocarriers accumulate in organs such as the liver and spleen and cannot effectively reach the tumor site. How to achieve active targeting, help the delivery system locate the tumor site, and improve the efficacy is the difficulty faced by the current nano drug delivery system. Summary of the invention

[0007] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide an integrated bionic nanoplatform based on hollow polydopamine nanoparticles and its preparation and application, which can be used to improve hypoxia in tumor sites, promote tumor cell apoptosis, produce photodynamic therapy effects, and improve tumor radiotherapy sensitivity.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] An integrated bionic nanoplatform based on hollow polydopamine nanoparticles has homologous targeting and a general formula of [AVPt@HP@M], wherein A is the 100-109 functional fragment of the radiosensitizing fusion polypeptide apoptin, whose amino acid sequence is LKESLITTTP, V is verteporfin, Pt@HP is a hollow polydopamine nanoparticle embedded with platinum, and @M represents a cell membrane wrapped on the surface of the nanoparticle.

[0010] In one embodiment, the verteporfin can be used as a photosensitizer and activated by the Cerenkov light generated by X-rays, thereby producing a photodynamic therapy effect.

[0011] The present invention also provides a method for preparing an integrated bionic nano-platform based on hollow polydopamine nanoparticles, comprising the following steps:

[0012] Step 1, synthesizing zeolite imidazolate framework 8 nanoparticles, namely ZIF-8, with zinc nitrate and dimethylimidazole;

[0013] Step 2, adding polyvinylpyrrolidone (PVP)-modified Pt nanoparticles into ZIF-8 to synthesize Pt@ZIF-8;

[0014] Step 3, dopamine is added to Pt@ZIF-8, and dopamine replaces dimethylimidazole by chelation competition-induced polymerization to synthesize platinum-embedded hollow polydopamine nanoparticles, namely Pt@HP;

[0015] Step 4, adding the 100-109 functional fragment of the radiosensitizing fusion peptide apoptin and verteporfin to Pt@HP to synthesize AVPt@HP;

[0016] Step 5, after mixing AVPt@HP with cancer cell membrane fragments and stirring in an ice bath, a nano-extruder is used to extrude the AVPt@HP so that the AVPt@HP is camouflaged by the cancer cell membrane, forming an integrated bionic nano-platform based on hollow polydopamine nanoparticles, namely AVPt@HP@M.

[0017] Specifically, in step 1, a methanol solution of dimethylimidazole and a methanol solution of zinc nitrate are mixed and stirred until they turn white, the supernatant is discarded by centrifugation, and the precipitate is resuspended with methanol to obtain ZIF-8, wherein the molar ratio of dimethylimidazole to zinc nitrate is 1:4-1:6.

[0018] Specifically, in step 2, the Pt nanoparticles modified with polyvinyl pyrrolidone (PVP) are prepared by the following method:

[0019] A double-distilled aqueous solution of polyvinyl pyrrolidone, a double-distilled aqueous solution of platinum chloride and a double-distilled aqueous solution of sodium borohydride are mixed and stirred until they turn dark brown, and double-distilled water is added after standing, and then dialyzed with a dialysis bag, and the dialyzed liquid is rotary evaporated until a dry precipitate is obtained, and the precipitate is resuspended with anhydrous ethanol to obtain polyvinyl pyrrolidone (PVP) modified Pt nanoparticles, wherein the molar ratio of polyvinyl pyrrolidone, platinum chloride and sodium borohydride is 9:3:1-8:4:1.

[0020] Specifically, in step 2, Pt nanoparticles modified with polyvinyl pyrrolidone (PVP) and ZIF-8 are added to methanol, stirred and centrifuged to discard the supernatant, and the precipitate is resuspended with methanol to obtain Pt@ZIF-8, wherein the molar ratio of Pt nanoparticles modified with polyvinyl pyrrolidone (PVP) and ZIF-8 is 1:1-1:5.

[0021] Specifically, in step 3, dopamine hydrochloride and Pt@ZIF-8 are added to methanol, and the reaction is heated in an oil bath. The supernatant of the product is discarded, and the precipitate is resuspended with PBS buffer and freeze-dried with a freeze dryer to obtain Pt@HP, wherein the molar ratio of dopamine hydrochloride to Pt@ZIF-8 is 1:1-1:3.

[0022] Specifically, in step 4, the 100-109 functional fragment of the radiotherapy sensitizing fusion polypeptide apoptin, verteporfin and Pt@HP are added to methanol, stirred in the dark, centrifuged and the supernatant is discarded, the precipitate is resuspended with PBS buffer, and freeze-dried with a freeze dryer to obtain AVPt@HP; wherein the molar ratio of the 100-109 functional fragment of the radiotherapy sensitizing fusion polypeptide apoptin, verteporfin and Pt@HP is 1:1:3-1:1:5.

[0023] The integrated bionic nano-platform based on hollow polydopamine nanoparticles of the present invention can be used to prepare drugs that target and improve hypoxia in tumor sites or promote tumor cell apoptosis or produce photodynamic therapy effects or improve tumor radiotherapy sensitivity.

[0024] Compared with the prior art, the nano-platform obtained by the present invention combined with radiotherapy can improve hypoxia in the tumor site, directly promote tumor cell apoptosis, produce photodynamic therapy effect and kill tumor cells together, thus playing the role of radiotherapy sensitization. The nano-platform prepared by the preparation method of the present invention has the advantages of excellent targeting, pH responsiveness and strong drug loading capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a synthetic schematic diagram of AVPt@HP@M.

[0026] Figure 2 , Figure 3 is the preparation and characterization of AVPt@HP@M.

[0027] Figure 2 A is the transmission electron microscopy (TEM) image of AVPt@HP@M.

[0028] Figure 2 B is the elemental analysis result of AVPt@HP@M.

[0029] Figure 2 C is the transmission electron microscopy (TEM) image of the cell membrane on the surface of AVPt@HP@M.

[0030] Figure 2 D is the change of Zeta potential during the synthesis of AVPt@HP@M.

[0031] Figure 2 E is WB detection of cell membrane on the surface of AVPt@HP@M

[0032] Figure 3 AB is the X-ray diffraction (XRD) of ZIF-8, Pt@ZIF-8, and Pt@HP.

[0033] Figure 3 C is the UV-Vis absorption spectra of AP, VP, Pt@HP, and AVPt@HP.

[0034] Figure 3 D is the FT-IR absorption spectra of AP, VP, Pt@HP, and AVPt@HP.

[0035] Figure 3 E is the drug loading capacity test of AVPt@HP@M.

[0036] Figure 3 F is the hydrodynamic diameter distribution of AVPt@HP@M.

[0037] Figure 4 For targeted detection of AVPt@HP@M.

[0038] Figure 5 Detection of Pt@HP@M's ability to improve tumor cell hypoxia.

[0039] Figure 6 To detect the ability of AVPt@HP@M to promote tumor cell apoptosis.

[0040] Figure 7 Produce photodynamic therapy effect detection for AVPt@HP@M.

[0041] Figure 8 A is a schematic diagram of the in vivo treatment experiment.

[0042] Figure 8 B is the weight statistics of mice after treatment in different treatment groups.

[0043] Figure 8 C is the statistics of mouse tumor volume after treatment in different treatment groups.

[0044] Figure 8 D is the statistics of mouse tumor weight after treatment in different treatment groups.

[0045] Figure 8 E shows HE staining and Tunel staining of tumor tissues after treatment in different treatment groups.

[0046] Fig. 9 A shows the results of WB detection of marker proteins of DNA damage repair after treatment in different treatment groups.

[0047] Fig. 9 Observation and statistics of γ-H2AX foci formation after BD was treated with different treatment groups

[0048] Fig. 9 E shows the changes in the expression of γ-H2AX and HIF-1a verified by immunofluorescence and immunohistochemistry after treatment in different treatment groups. DETAILED DESCRIPTION

[0049] The embodiments of the present invention are described in detail below with reference to the accompanying drawings and examples.

[0050] Hollow nanomaterials show great potential for cancer treatment due to their excellent performance in drug delivery. For example, hollow mesoporous silica nanoparticles, hollow manganese dioxide and hollow gold nanoparticles. Dopamine (DA) as a bioactive molecule is widely present in the body, has good biocompatibility, is easy to aggregate and drug, and has attracted widespread attention from researchers. In addition, bionic cell membrane smart nanoparticles are also gaining more and more attention. This kind of smart nanoparticles has high stability, biocompatibility and targeting.

[0051] Apoptin is a small apoptosis-inducing protein derived from chicken anemia virus (CAV) containing 121 amino acids. It has specific cytotoxic effects independent of p53 in a variety of human malignant transformed cells, but apoptin has little or no cytotoxic effects in many normal human cell lines. Another laboratory has previously demonstrated that apoptin can enhance the in vitro radiotherapy response of tumor cell lines, indicating that it has the potential to be a radiosensitizer. Studies have shown that the 100-109 functional fragment of apoptin plays a key role in radiosensitization.

[0052] Verteporfin is a clinically approved photosensitizer for the treatment of macular degeneration. It has a strong absorption band in the near ultraviolet (UV)-blue region and a second weak absorption band around 700nm. It can be excited by 6MeV X-rays through the induced Cherenkov light, electrons, or both in biological media, thereby producing photodynamic effects.

[0053] The present invention attempts to synthesize platinum-embedded hollow polydopamine nanoparticles with a stable size, utilize their strong drug-loading capacity, encapsulate apoptin100-109 (AP) functional fragments and verteporfin (VP), and modify the tumor cell membrane with tumor targeting effect, and finally construct platinum-embedded targeted hollow polydopamine nanoparticles, providing experimental materials for the next step of radiotherapy sensitization combined with photodynamic therapy research.

[0054] Specifically, the present invention provides an integrated bionic nanoplatform based on hollow polydopamine nanoparticles, whose general formula is [AVPt@HP@M], the sequence of A is LKESLITTTP, and the corresponding functional fragments and their synthesis have been publicly recorded in patent CN105330749B, which will not be described in detail in the present invention.

[0055] The present invention also discloses a preparation method thereof, which comprises the following steps: synthesizing zeolite imidazolate framework 8 (ZIF-8) nanoparticles by zinc nitrate and dimethylimidazole, and adding polyvinylpyrrolidone (PVP)-modified Pt nanoparticles to synthesize Pt@ZIF-8 during the reaction. Then, the binding ability of polydopamine to Zn2+ is higher than that of dimethylimidazole. When dopamine is added to the Pt@ZIF-8 system, dopamine replaces dimethylimidazole by chelation competition-induced polymerization, thereby forming hollow polydopamine nanoparticles (Pt@HP) embedded with platinum. Apoptin's 100-109 functional fragment (AP) and verteporfin (VP) are added during the synthesis process to form AVPt@HP. Finally, AVPt@HP is mixed with cancer cell membrane fragments and stirred in an ice bath, and then extruded by a nano-extruder so that AVPt@HP is camouflaged by the cancer cell membrane, and finally an integrated bionic nano-platform (AVPt@HP@M) based on hollow polydopamine nanoparticles is formed, such as Figure 1 shown.

[0056] The application of this integrated bionic nano-platform based on hollow polydopamine nanoparticles improves hypoxia in the tumor site, directly promotes tumor cell apoptosis, and produces a photodynamic therapy effect to kill tumor cells, thereby playing a role in radiotherapy sensitization. The following is a verification test of AVPt@HP@M:

[0057] 1. Synthesis of AVPt@HP@M

[0058] 1) Synthesis of platinum nanoparticles (Pt)

[0059] (1) Use a measuring cylinder to measure 51.4 mL of double distilled water into a 100 mL beaker, and weigh 86 mg of PVP to dissolve in the beaker;

[0060] (2) Prepare a 15 mL EP tube, use a measuring cylinder to measure 8.6 mL of double distilled water and pour it into the EP tube, weigh 6 mM (27.13 mg) chloroplatinic acid and dissolve it in the EP tube (chloroplatinic acid is highly corrosive to metals, so use a plastic spoon when weighing and be careful not to spill it on the balance);

[0061] (3) Prepare a 1.5 mL EP tube, inject 1 mL of double distilled water, weigh 10 mg of NaBH4, and dissolve it in the EP tube;

[0062] (4) Place the beaker containing dissolved PVP on a magnetic stirrer and stir at a low speed. Use a plastic dropper to drop the dissolved chloroplatinic acid into the beaker drop by drop to mix well.

[0063] (5) Use a plastic dropper to drop the dissolved NaBH4 into the beaker drop by drop, seal the beaker with plastic wrap, and continue stirring. A large number of bubbles will be generated during the stirring process, and the solution will turn from light yellow to dark brown;

[0064] (6) Observe until bubbles stop being generated, turn off the magnetic stirrer, and leave the beaker at room temperature for 24 h;

[0065] (7) Prepare a 1 L beaker, add 9000 mL of double distilled water, and place it on a magnetic stirrer;

[0066] (8) Prepare a dialysis bag, soak it in double distilled water for 1 min, put the rested liquid into the dialysis bag, and seal it with a sealing clip;

[0067] (9) Move the dialysis bag to a beaker, turn on the magnetic stirrer, and start dialysis. Change the water at intervals of 2 h, 4 h, 6 h, and 8 h, for a total of 20 h.

[0068] (10) After connecting the rotary evaporator to the condenser, turn on the heating switch and raise the temperature to 60°C for standby use;

[0069] (11) Transfer the dialyzed liquid to a 100 mL round-bottom flask, connect it to a rotary evaporator, turn on the condenser, and start rotary evaporation until the precipitate is dry;

[0070] (12) Pour 5 mL of anhydrous ethanol into a round-bottom flask, resuspend the precipitate, and then pour in 5 mL of acetone. Ultrasonic mixing is performed until the precipitate on the wall of the flask is completely dissolved, and then rotary evaporation is performed again. This process is repeated three times;

[0071] (13) Resuspend the precipitate in 3 mL of ethanol and store at room temperature.

[0072] 2) Synthesis of ZIF-8

[0073] (1) Prepare two 500 mL beakers, measure 100 mL of methanol using a measuring cylinder, pour it into the two beakers respectively, seal the beakers with plastic wrap, and place them on a magnetic stirrer for later use;

[0074] (2) Weigh 5 mg of dimethylimidazole and 1 mg of zinc nitrate, add them into a beaker, and stir rapidly until all the particles are dissolved;

[0075] (3) Pour the dissolved dimethylimidazole into the zinc nitrate solution and stir rapidly. During the process, the mixed solution changes from transparent to milky white and finally to white.

[0076] (4) After the mixed solution turned pure white, it was transferred to four 50 mL EP tubes and centrifuged at 10,000 rpm / min for 1 min;

[0077] (5) Discard the supernatant, inject 3 mL of methanol into each tube, shake well while ultrasonicating until the precipitate is completely dissolved, transfer the suspension to a 15 mL EP tube, centrifuge at 10,000 rpm / min for 1 min, and repeat 3 times;

[0078] (6) Resuspend the precipitate in 12 mL of methanol and store at room temperature.

[0079] 3) Synthesis of Pt@ZIF-8

[0080] (1) Prepare a 50 mL beaker, inject 20 mL of methanol, seal the beaker with plastic wrap, and place it on a magnetic stirrer for later use;

[0081] (2) Take 1 mL of synthesized platinum nanoparticles and 4 mL of synthesized ZIF-8, mix them in a beaker, turn on the magnetic stirrer, and stir for 2 h;

[0082] (3) Transfer the mixture in the beaker to two 15 mL EP tubes and centrifuge at 10,000 rpm / min for 1 min;

[0083] (4) Discard the supernatant, inject 5 mL of methanol into each tube, shake well while ultrasonicating until the precipitate is completely dissolved, transfer the suspension to a 15 mL EP tube, and centrifuge at 10,000 rpm / min for 1 min;

[0084] (5) Discard the supernatant, inject 5 mL of methanol, and shake well while ultrasonicating until the precipitate is completely dissolved. Repeat twice;

[0085] (6) Resuspend the precipitate in 5 mL of methanol and store at room temperature.

[0086] 4) Synthesis of Pt@HP

[0087] (1) Open the oil bath and heat to 60°C for later use;

[0088] (2) Prepare a 10 mL EP tube, inject 5 mL of methanol, weigh 0.02 g of dopamine hydrochloride, pour it into the EP tube, and ultrasonically dissolve it for later use;

[0089] (3) Prepare a 250 mL three-necked flask, connect the condenser, open one cap, inject 30 mL methanol, 5 mL Pt@ZIF-8 and 5 mL dopamine hydrochloride solution, and then close the cap;

[0090] (4) Fix the three-necked flask in an oil bath and stir at a constant speed for 4-6 hours;

[0091] (5) After stirring, collect the liquid in a 50 mL EP tube and centrifuge at 10,000 rpm / min for 1 min;

[0092] (6) Discard the supernatant, inject 10 mL of methanol, shake well while ultrasonicating until the precipitate is completely dissolved, transfer the suspension to a 15 mL EP tube, centrifuge at 10,000 rpm / min for 1 min, and repeat 3 times;

[0093] (7) Resuspend the precipitate in 1 mL of PBS, freeze-dry it, and store it at -20°C.

[0094] 5) Synthesis of AVPt@HP

[0095] (1) Prepare two 1.5 mL EP tubes and inject 1 mL of methanol;

[0096] (2) Weigh 1 mg AP, 1 mg VP and 1 mg Pt@HP, add them into EP tubes respectively and dissolve them by ultrasonication;

[0097] (3) Prepare a 20 mL light-proof sample bottle, inject 10 mL of methanol, and place it on a magnetic stirrer for later use;

[0098] (4) Add AP, VP and Pt@HP solutions into the sample bottle and stir for 24 h;

[0099] (5) Collect the liquid in a 15 mL EP tube and centrifuge at 10,000 rpm / min for 1 min;

[0100] (6) Discard the supernatant, inject 10 mL of PBS, shake well while sonicating until the precipitate is completely dissolved, centrifuge at 10,000 rpm / min for 1 min, and repeat twice;

[0101] (7) Resuspend the precipitate in 1 mL of PBS, freeze-dry it, and store it at -20°C.

[0102] 6) Cell membrane and membrane protein extraction

[0103] (1) Prepare reagents

[0104] a) Thaw and mix membrane protein extraction reagents A and B at room temperature, and place them on an ice bath immediately after melting;

[0105] b) Take appropriate amounts of membrane protein extraction reagents A and B for use, and add PMSF a few minutes before use to make the final concentration of PMSF 1 mM.

[0106] (2) Collect cells

[0107] a) Wash the adherent cells with PBS, scrape the cells with a cell scraper or treat the cells with a cell digestion solution containing EDTA but not trypsin, and blow the cells with a pipette to obtain a cell suspension;

[0108] b) Collect the cell suspension in a sterile enzyme-free 15 mL EP tube, centrifuge at 1000 rpm / min for 3 min, remove the supernatant, retain the cell pellet, and resuspend in 2 mL pre-cooled PBS for later use.

[0109] (3) Identification of cell disruption and disruption effects

[0110] a) Add PMSF membrane protein extraction reagent A to the cells, (2-5)×10 7 cells / 1mL PMSF, fully suspend the cells, gently place on ice for 10-15 minutes;

[0111] b) Transfer the cell suspension to a 10 mL pre-cooled glass homogenizer and homogenize for about 30-50 times in an ice bath;

[0112] c) After 30 homogenizations, take about 2-3 μl of the cell homogenate and drop it on a coverslip and observe under a microscope. If a halo around the nucleus or intact cell morphology is seen, it means that the cells are still intact and need to be homogenized again;

[0113] d) Continue to observe while homogenizing until 70-80% of the cells have no perinuclear halo and intact cell morphology, indicating that the cells have been fully disrupted;

[0114] e) Record the number of homogenizations for reference in subsequent experiments.

[0115] (4) Obtaining cell membrane fragments

[0116] a) Centrifuge the broken cells at 4°C, 1000 rpm / min for 10 min to remove the nuclei and unbroken cells;

[0117] b) Carefully collect the supernatant into a new centrifuge tube. To ensure the purity of the supernatant, do not touch the precipitate when collecting the supernatant. It is acceptable to leave about 30-50 μl of supernatant uncollected.

[0118] c) The collected supernatant was centrifuged at 4°C, 14,000 g for 30 minutes to obtain the precipitate, i.e., the cell membrane fragments.

[0119] (5) Extraction of cell membrane proteins

[0120] a) Add 200 μl of membrane protein extraction reagent B to the cell membrane fragment pellet, resuspend the pellet, and place at 4°C for 12 h;

[0121] b) Centrifuge the suspension at 14,000 g for 5 min at 4°C, discard the precipitate, collect the supernatant, and store at -80°C.

[0122] 7) Synthesis of AVPt@HP@M

[0123] (1) Prepare two 5 mL EP tubes and inject 2 mL of pre-cooled PBS into each tube;

[0124] (2) Weigh 5 mg of Pt@HP and add it to an EP tube. After sonication, dissolve it and place it on ice for later use.

[0125] (3) Add the frozen cell membrane fragments to another EP tube and gently blow with a 1 mL pipette until the fragments are evenly distributed;

[0126] (4) Mix the liquids in the two EP tubes evenly. The whole process is carried out on an ice bath.

[0127] (5) Place a polycarbonate membrane with a pore size of 200 nm in the nanoextruder, inject the homogenous liquid into the needle of the nanoextruder, 1 mL each time, and after about 20 times of aspiration, collect the liquid with a new 5 mL EP tube;

[0128] (6) After all the liquid is collected, centrifuge at 1000 rpm / min for 1 min, discard the supernatant, collect the precipitate, and store it at -80°C.

[0129] The parameters involved in the above preparation process can be adaptively adjusted according to actual needs, and the above parameters are not limitations of the present invention.

[0130] 2. Characterization of AVPt@HP@M

[0131] 1) Electron Microscopy Sample Preparation

[0132] (1) Take a 1.5 mL EP tube, take 1 mL of the prepared nanoparticle solution and place it in a centrifuge tube, and use a vortexer or ultrasound to evenly disperse the nanoparticles;

[0133] (2) Take another 1.5 mL EP tube, add 100 μL of ethanol solution, then inject 1 μL of the original solution, and ultrasonically disperse for 3 minutes to evenly disperse the nanoparticles;

[0134] (3) Take out the carbon film copper mesh with sample tweezers and place it on the laboratory table with the front side facing up;

[0135] (4) Use a 10 μL pipette to drop the dispersed nanoparticle solution onto the carbon film copper grid, dry it in air, repeat 4-5 times, and observe it using a transmission electron microscope with an accelerating voltage of 200 KV and a test temperature of 23+2°C;

[0136] (5) After observation, the sample can be subjected to EDS analysis to detect the chemical elements contained in the nanoparticles.

[0137] 2) Determination of particle size, dispersibility and zeta potential

[0138] (1) Particle size and dispersibility determination:

[0139] a) Take a 1.5 mL EP tube, add 1 mL of ethanol solution, inject 1 μL of the prepared nanoparticle solution into a centrifuge tube, and perform ultrasound to evenly disperse the nanoparticles;

[0140] b) Prepare the cuvette, turn on the instrument to preheat, use a 1mL pipette to transfer the prepared sample into the cuvette, and test it on the instrument. Make sure the cuvette is placed with the triangular part facing forward;

[0141] (2) Potential measurement:

[0142] a) Take a 1.5 mL EP tube, add 1 mL of ethanol solution, inject 5 μL of the prepared nanoparticle solution into a centrifuge tube, and perform ultrasound to evenly disperse the nanoparticles;

[0143] b) Prepare a disposable folded capillary sample cell and add the sample into the cell using a 2mL syringe. Be careful not to generate bubbles during the process and make sure that the liquid level exceeds the conductive part of the sample cell.

[0144] c) Turn on the instrument to preheat, place the sample pool with the Malvern logo first, and start the test.

[0145] 3) Ultraviolet absorption spectrum detection

[0146] (1) Turn on the UV-visible spectrometer and preheat for half an hour;

[0147] (2) Prepare PBS for baseline scanning and timely cleaning of cuvettes;

[0148] (3) Take a 5 mL EP tube, add 3 mL PBS solution, and then inject 10 μL of the prepared nanoparticle solution into the centrifuge tube;

[0149] (4) Use a 1 mL pipette to transfer the prepared sample into a cuvette, place it in a UV-visible spectrophotometer, use the buffer solution as the blank solution, and scan the absorption spectrum in the wavelength range of 250-800 nm.

[0150] 4) Infrared spectroscopy analysis

[0151] (1) Turn on the UV-visible spectrometer and preheat for half an hour;

[0152] (2) freeze-drying the prepared nanoparticle solution in vacuum, and after the sample becomes dry powder, wrap it with weighing paper and press it lightly with a glass slide to make the sample more uniform;

[0153] (3) Scan after loading.

[0154] 5) X-ray diffraction test

[0155] (1) The prepared nanoparticle solution is freeze-dried in a vacuum, and after the sample becomes a dry powder, it is wrapped with weighing paper and gently pressed with a glass slide to make the sample more uniform;

[0156] (2) Use a small spoon to put the sample into the slot, and then use a large glass plate to flatten the sample;

[0157] (3) Press down the spring sheet of the sample chamber, place the card slot into the sample chamber and push it to the innermost position;

[0158] (4) When the circulating water system is turned on, the water temperature cannot exceed 20°C. If it exceeds this temperature, the equipment will fail and must be stopped. The water pressure cannot exceed 0.4.

[0159] (5) Turn on the X-ray diffractometer, wait for the power indicator to light up, and then turn on the computer after a half-minute self-check;

[0160] (6) Open the test software and check the door of the sample chamber. If the small box in the dialog box is green, it indicates that the door is closed; if it is red, it indicates that the door is not closed;

[0161] (7) After the test, set the parameters and scan the sample.

[0162] 6) High performance liquid chromatography analysis

[0163] (1) Instrument preparation:

[0164] a) The chromatographic column and mobile phase were selected according to the analysis object. A C18 column (Nova-Pak 3.9×250 mm, Waters, Milford, MA) was used. The mobile phase included water and acetonitrile in a volume ratio of 1:9.

[0165] b) Filter the mobile phase, select different filter membranes according to needs, and perform ultrasonic degassing on the filtered mobile phase for 30 minutes;

[0166] c) Connect the mobile phase pipeline, turn on the HPLC workstation (including computer software and chromatograph), and connect the detection system;

[0167] d) Flush the pump and injection valve. To flush the pump, use a needle to draw water directly from the pump outlet. To flush the injection valve, click purge in the manual menu, then click start. The flushing speed should not exceed 10 ml / min.

[0168] e) Design the sampling method, click file, select select users and methods, click new method, and select the required accessories (injection valve, pump, detector, etc.). A complete sampling method needs to include:

[0169] (a) Steady flow before injection, usually 2-5 minutes;

[0170] (b) baseline zeroing;

[0171] (c) loading-inject conversion of injection valve;

[0172] (d) The skewing time varies with different samples.

[0173] (2) Sample preparation:

[0174] a) Prepare a 2mL syringe, a 0.45um filter, and a sample bottle;

[0175] b) Remove the syringe needle, draw in the sample, inject it into the filter and filter it into the sample bottle. The sample volume is about 200 μL;

[0176] c) Observe the sample bottle. There must be no bubbles. If there are bubbles, shake the sample bottle until the bubbles are discharged.

[0177] (3) Sample injection

[0178] a) Select the sampling method and click start to start sampling;

[0179] b) First introduce a pure solvent as the baseline, then start to introduce the test samples. After all samples are tested, use the same method to create a baseline and clean the system.

[0180] (4) Shutdown

[0181] Shut down the computer first, then the HPLC.

[0182] 3. In vivo application of AVPt@HP@M

[0183] Healthy nude mice aged 5 weeks and weighing about 18 g were selected and cultured in a laminar flow chamber for 1 week. HCT-116 cells (2×10 6 / mouse) to establish a nude mouse xenograft model of human colorectal cancer cells. The nude mice were raised until the tumor volume reached about 100 mm3 The nude mice were used as models for subsequent experiments.

[0184] 1) Grouping: Control, IR, AVPt@HP@M, APt@HP@M+IR, VPt@HP@M+IR, AVPt@HP@M+IR;

[0185] 2) Tail vein injection of each group of nanodrugs

[0186] (1) Fix the mouse with a mouse fixer, exposing the mouse tail; prepare a 1 ml syringe;

[0187] (2) Dissolve each group of nanomedicines in PBS and filter them with a 0.22 filter membrane;

[0188] (3) Find the three veins on the mouse's tail. There is one on each side and they are relatively shallow and easy to puncture. The middle one is deep and difficult to puncture. After finding the veins on the left and right sides, wipe them repeatedly with 75% alcohol to make them full and soften the stratum corneum of the skin, which is convenient for puncture.

[0189] (4) Use the index and middle fingers of your left hand to pinch the side of the selected blood vessel close to the body. Use the ring finger and little finger to hold a piece of gauze or tissue. Use the thumb to press the tip of the selected blood vessel. The distance between the blood vessels should be such that it does not affect the movement of the needle held by the right hand.

[0190] (5) Hold the syringe in your right hand, draw an appropriate amount of nano-drug solution, and insert the needle at the lower 2 / 3 to 1 / 2 of the tail with the bevel of the needle facing upward. The needle should form a slight angle with the skin (about 10°). Push the needle tip slightly upward and insert it. If there is no resistance, the needle is successfully inserted and the solution is pushed into the blood vessel.

[0191] (6) Pull out the syringe and press the puncture site with gauze to stop bleeding;

[0192] 3) Radiotherapy of mice

[0193] (1) Radiotherapy planning was done in the physics room. The radiation source was a linear accelerator 4MV-X-ray, the source skin distance (SSD) was 100 cm, the radiation field size was 2 cm × 2 cm, and the dose was set to 2 Gy;

[0194] (2) The tumor-bearing mice injected with nanodrugs (APt@HP@M, VPt@HP@M, and AVPt@HP@M) for 2 consecutive days were subjected to gas anesthesia;

[0195] (3) Take the anesthetized mouse to the radiotherapy room, place it in the center of the irradiation field, and start irradiation.

[0196] Results and Discussion:

[0197] like Figure 2As shown in Figure A, the synthesized ZIF-8 is a three-dimensional hexagon with uniform size and good dispersibility. When Pt nanoparticles are modified on the surface of ZIF-8, the formed Pt@ZIF-8 is still a three-dimensional hexagon with uniform size and good dispersibility. Pt@ZIF-8 is further modified with dopamine to form Pt@HP with a hollow ring shape. Such a structure not only has better biosafety but also makes the drug loading space of nanoparticles larger. EDS analysis of nanoparticles with energy dispersive spectrometer shows that Pt@HP@M mainly contains N, O, Zn, and Pt elements, and is distributed in a ring shape, which coincides with the shape of Pt@HP@M, indicating that Pt particles are successfully loaded on the hollow polydopamine ring ( Figure 2 B). After the Pt@HP surface was modified with cell membrane to form Pt@HP@M by a nano-squeezer, it was observed that Pt@HP@M was still hollow and ring-shaped, but the surface was covered with a thin film of about 8 nm ( Figure 2 C). The changes in surface potential during the preparation of the nanocomposite were detected, and the results showed that: after the cell membrane was modified on the surface of AVPt@HP, the AVPt@HP@ obtained was negatively charged at about -24.5mV. This further confirmed that the film on the surface of AVPt@HP was a cell membrane. Further WB results showed that the membrane protein was successfully expressed on the surface of AVPt@HP@M and was consistent with the purified cell membrane. This showed that the cell membrane was indeed successfully coated on the surface of AVPt@HP.

[0198] Figure 3 The XRD analysis results shown in AB show the crystal structure of ZIF-8. The Pt@ZIF-8 formed after modification with Pt is highly consistent with the single ZIF-8, and four additional specific peaks are observed at large angles of 30°–90°, corresponding to Pt (111), (200), (220) and (310). As for Pt@HP, the four peaks of Pt are retained, while the peaks of ZIF-8 disappear and are replaced by the scattered diffraction of the amorphous polymer shell. This shows that the modification of Pt does not affect the crystal structure of ZIF-8, and when Pt@ZIF-8 is further modified with dopamine to form Pt@HP, the crystal structure of ZIF-8 disappears. In summary, hollow polydopamine (HP) nanoparticles were successfully synthesized.

[0199] The UV-visible absorption spectrum shows that the UV-visible absorption peak λmax of AP is located near 220nm; the UV-visible absorption peak λmax of VP is located near 420nm; while Pt@HP does not have a UV-visible absorption peak; when Pt@HP is loaded with AP and VP, the UV-visible absorption peak λmax appears red-shifted, and the maximum absorption peaks are located near 240nm and 440nm, respectively ( Figure 3C). This shows that Pt@HP has good drug loading performance and can successfully load AP and VP. The infrared spectrum analysis results show that compared with AP, VP and pure nanocarrier Pt@HP, the absorption peak of AVPt@HP after loading AP and VP has changed, indicating the generation of new functional groups. This further proves that Pt@HP can successfully load AP and VP ( Figure 3 D).

[0200] Figure 3 E By comparing the concentrations before and after drug loading, it was determined that the encapsulation efficiency of AP alone was 65.3% ± 1.32%, and the encapsulation efficiency of VP alone was 84.6% ± 1.22%. When the concentration ratio of AP and VP was 1:1, the co-encapsulation efficiency of AP and VP was the best, which was about 44.6 and 83.2, respectively. Therefore, the concentration ratio of AP and VP was 1:1 as the synthesis condition of the nanocomplex in subsequent experiments. Figure 3 F shows that the final synthesized AVPt@HP particle size is concentrated at 250 nm.

[0201] To verify the targeting of Pt@HP coated with tumor cell membrane, we coated AVPt@HP with HCT-116 cell membrane (AVPt@HP@M) and then co-incubated with different types of cancer cells (MCF-7, Hela and HCT-116 cells). The results showed that AVPt@HP@M had a stronger specific self-recognition ability for HCT-116 cells than MCF-7 and Hela cells ( Figure 4 ).

[0202] [Ru(dpp)3]Cl2 is an intracellular O2 level indicator used to detect O2 levels under normoxic and hypoxic conditions before and after treatment. The red fluorescence of [Ru(dpp)3]Cl2 is negatively correlated with the intracellular O2 concentration. Under normoxic conditions, the intracellular red fluorescence was significantly weaker than that in the hypoxic group. When treated with HP@M without platinum, there was no significant change in the red fluorescence of the normoxic and hypoxic groups compared with the control group within 4 hours. In contrast, when treated with Pt@HP@M for 4 hours, the intracellular red fluorescence of both the normoxic and hypoxic groups was significantly reduced, indicating that Pt significantly catalyzed the production of oxygen and alleviated hypoxia in both the normoxic and hypoxic groups ( Figure 5 ).

[0203] Flow cytometry apoptosis results showed that the apoptosis rate of the control group was 5.7%, while the apoptosis rates of the IR or AVPt@HP@M treatment groups were 20.0% and 6.9%, respectively. For the combined treatment groups: APt@HP@M+IR, VPt@HP@M+IR, and AVPt@HP@M+IR, the cell death rates increased to 34.1%, 32.6%, and 62.1%, respectively. Figure 6). These results confirm that AVPt@HP@M combined with IR can effectively promote cell apoptosis and improve the sensitivity of radiotherapy.

[0204] DCFH-DA is a reactive oxygen species probe that is commonly used to detect intracellular H2O2 and oxidative stress. The probe is cell permeable and is hydrolyzed intracellularly to the DCFH carboxylate anion, which is retained within the cell. The two-electron oxidation of DCFH results in the formation of a fluorescent product called dichlorofluorescein (DCF) and can be easily monitored by fluorescence-based techniques. When treated with IR or AVPt@HP@M, a weak green fluorescence appeared in the cells, similar to the control group, which may be due to the presence of endogenous ROS in living cells. When the cells were treated with nanoparticles (APt@HP@M, VPt@HP@M, and AVPt@HP@M) in combination with IR, the green fluorescence in the cells increased significantly, and the strongest green fluorescence was shown in the AVPt@HP@M+IR treatment group, indicating that a large amount of ROS was generated ( Figure 7 ). Previous studies have shown that deep penetration X-ray irradiation is accompanied by the in situ generation of Cherenkov radiation and a cascade of high-energy secondary electrons. AP is a radiosensitizer that promotes the generation of ROS after IR. At the same time, VP is a photosensitizer that can be activated by Cherenkov radiation to produce X-ray-induced photodynamic therapy (X-PDT) to generate cytotoxic ROS. Therefore, the AVPt@HP@M combined with IR group showed greater reactive oxygen species generation capacity than other groups (P<0.05). These data show that AVPt@HP@M nanoparticles effectively increase the level of intracellular ROS and have the potential to improve radiosensitivity in vivo.

[0205] To determine the in vivo effects of AVPt@HP@M, we randomly divided HCT-116 tumor-bearing mice into six groups: control group, IR group, AVPt@HP@M, APt@HP@M+IR, VPt@HP@M+IR, and AVPt@HP@M+IR. Figure 8 As shown in A, mice were treated with IR. The body weight and tumor growth of each group were recorded every 2 days to evaluate the therapeutic effect. Body weight measurements showed that mice in each group had similar growth curves ( Figure 8 B); Tumor growth measurements showed that the tumor growth in the AVPt@HP@M+IR treatment group was significantly slower than that in the other treatment groups ( Figure 8 C); The tumor weight results showed that the AVPt@HP@M+IR treatment group had the lowest tumor weight ( Figure 8 D). As shown in the figure Figure 8E, HE tissue staining results of tumor sections showed that the AVPt@HP@M+IR treatment group had more necrosis in the tumor tissue compared with other groups. TUNEL fluorescence staining results showed that a small number of TUNEL-positive apoptotic cells (green dots) were detected in the tumor sections of the control group, IR group and AVPt@HP@M. In contrast, there were more green dots in the tumor sections of the AVPt@HP@M+IR treatment group, indicating that its apoptosis rate was significantly higher than that of the control group, APt@HP@M+IR and VPt@HP@M+IR groups, indicating that APt@HP@M can kill solid tumors, and its tumor killing effect is more obvious after combined with radiotherapy.

[0206] Fig. 9 The results of WB experiments in A showed that the expression levels of p-BRCA1, p-ATM, and γ-H2AX increased in the APt@HP@M+IR, APt@HP@M+IR, and AVPt@HP@M+IR treatment groups compared with the control and IR groups, while BRCA1 and ATM did not change. These results indicate that APt@HP@M and AVPt@HP@M inhibit HR repair after DSBs. These results suggest that AP plays a radiosensitizing role in IR by inhibiting the HR signaling pathway after DSBs. Immunofluorescence (IF) and immunohistochemistry (IHC) staining were used to evaluate the expression levels of γ-H2AX and HIF-1α to further investigate the role of AVPt@HP@M in alleviating hypoxia and improving radiosensitivity in vivo. Immunofluorescence showed that the tumor had the brightest green fluorescence in the AVPt@HP@M+IR group, indicating the highest expression level of γ-H2AX. In contrast, the expression of HIF-1α (red fluorescence) was significantly reduced in all treatment groups, especially in the AVPt@HP@M+IR group. Consistent with the immunofluorescence results, the immunohistochemical staining results showed that the expression level of γ-H2AX was the highest and the expression level of HIF-1α was the lowest in the AVPt@HP@M+IR group.

[0207] In summary, the one-piece bionic nano-platform [AVPt@HP@M] based on hollow polydopamine nanoparticles proposed in the present invention can improve hypoxia in the tumor site, directly promote tumor cell apoptosis, produce photodynamic therapy effects and kill tumor cells together, thus playing a role in radiotherapy sensitization. The nano-platform of the present invention can also carry other drugs to achieve a variety of therapeutic effects. The nano-platform prepared by the preparation method of the present invention has the advantages of excellent targeting and strong drug loading capacity.

[0208] Sequence Listing <110> The First Affiliated Hospital of Xi'an Jiaotong University School of Medicine <120> Integrated bionic nanoplatform based on hollow polydopamine nanoparticles and its preparation and application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <400> 1 Leu Lys Glu Ser Leu Ile Thr Thr Thr Pro 1 5 10

Claims

1. A method for preparing an integrated bionic nanoplatform based on hollow polydopamine nanoparticles, characterized in that: The steps include: Step 1, synthesizing zeolite imidazolate framework 8 nanoparticles, namely ZIF-8, with zinc nitrate and dimethylimidazole; Step 2, adding polyvinylpyrrolidone (PVP)-modified Pt nanoparticles into ZIF-8 to synthesize Pt@ZIF-8; Step 3, dopamine is added to Pt@ZIF-8, and dopamine replaces dimethylimidazole by chelation competition-induced polymerization to synthesize platinum-embedded hollow polydopamine nanoparticles, namely Pt@HP; Step 4, adding the 100-109 functional fragment of the radiosensitizing fusion peptide apoptin and verteporfin to Pt@HP to synthesize AVPt@HP; Step 5, after mixing AVPt@HP with cancer cell membrane fragments and stirring in an ice bath, the mixture is extruded using a nano-extruder so that AVPt@HP is camouflaged by the cancer cell membrane, thereby forming an integrated bionic nano-platform based on hollow polydopamine nanoparticles, namely AVPt@HP@M; The bionic nano-platform has a general formula of [AVPt@HP@M], wherein A is the 100-109 functional fragment of the radiosensitizing fusion polypeptide apoptin, the amino acid sequence is LKESLITTTP, V is verteporfin, Pt@HP is a hollow polydopamine nanoparticle embedded with platinum, and @M represents a cell membrane wrapped on the surface of the nanoparticle; the verteporfin can be used as a photosensitizer and activated by the Cherenkov light generated by X-rays, thereby producing a photodynamic therapy effect; the nano-platform has homologous targeting; In the step 1, a methanol solution of dimethylimidazole and a methanol solution of zinc nitrate are mixed and stirred until they turn white, the supernatant is discarded by centrifugation, and the precipitate is resuspended with methanol to obtain ZIF-8, wherein the molar ratio of dimethylimidazole to zinc nitrate is 1:4-1:6; In step 2, the Pt nanoparticles modified with polyvinyl pyrrolidone (PVP) are prepared by the following method: A double-distilled aqueous solution of polyvinyl pyrrolidone, a double-distilled aqueous solution of platinum chloride and a double-distilled aqueous solution of sodium borohydride are mixed and stirred until they turn dark brown, and double-distilled water is added after standing, and then dialyzed with a dialysis bag, and the dialyzed liquid is rotary evaporated until a dry precipitate is obtained, and the precipitate is resuspended with anhydrous ethanol to obtain polyvinyl pyrrolidone (PVP) modified Pt nanoparticles, wherein the molar ratio of polyvinyl pyrrolidone, platinum chloride and sodium borohydride is 9:3:1-8:4:1; In the step 4, the 100-109 functional fragment of the radiotherapy sensitizing fusion polypeptide apoptin, verteporfin and Pt@HP are added to methanol, stirred in the dark, centrifuged and the supernatant is discarded, the precipitate is resuspended with PBS buffer, and freeze-dried with a freeze dryer to obtain AVPt@HP; wherein the molar ratio of the 100-109 functional fragment of the radiotherapy sensitizing fusion polypeptide apoptin, verteporfin and Pt@HP is 1:1:3-1:1:

5.

2. The preparation method according to claim 1, characterized in that: In the step 2, Pt nanoparticles modified with polyvinyl pyrrolidone (PVP) and ZIF-8 are added to methanol, stirred and centrifuged to discard the supernatant, and the precipitate is resuspended with methanol to obtain Pt@ZIF-8, wherein the molar ratio of Pt nanoparticles modified with polyvinyl pyrrolidone (PVP) to ZIF-8 is 1:1-1:

5.

3. The preparation method according to claim 1 or 2, characterized in that: In the step 3, dopamine hydrochloride and Pt@ZIF-8 are added to methanol, and the reaction is heated in an oil bath. The supernatant of the product is discarded, and the precipitate is resuspended with PBS buffer and freeze-dried with a freeze dryer to obtain Pt@HP, wherein the molar ratio of dopamine hydrochloride to Pt@ZIF-8 is 1:1-1:

3.

4. Use of the integrated bionic nanoplatform based on hollow polydopamine nanoparticles as described in claim 1 for preparing drugs that target improving hypoxia in tumor sites or promoting tumor cell apoptosis or producing photodynamic therapy effects or improving tumor radiotherapy sensitivity.

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