Mesoporous polydopamine loaded trichinella anti-tumor protein nanoparticle, and preparation method and application thereof

Abstract

The application discloses mesoporous polydopamine loaded trichinella anti-tumor protein nanoparticles and a preparation method and application thereof, wherein mesoporous polydopamine (MPDA) is prepared first, and then trichinella anti-tumor protein (NTspATP) is loaded to obtain MPDA@NTspATP. Under the irradiation of near-infrared light at 808 nm, 500 μg / mL of the nanoparticles can be heated to 48.4 DEG C within 5 minutes, can not only inhibit the proliferation and migration of tumor cells, but also can induce tumor cell apoptosis and stimulate immunogenic cell death (ICD). When ICD occurs, related signal pathways are activated, thereby causing the release of damage-associated molecular patterns DAMPs, and the DAMPs can be combined with pattern recognition receptors on antigen presenting cells, thereby causing a protective anti-cancer immune response of the body, but will not inhibit the immune cells at the tumor site, and will not damage the healthy tissues near the tumor.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanosynthesis technology, and specifically relates to a method for preparing mesoporous polydopamine-loaded Trichinella spiralis antitumor protein with photothermal effect. Background Technology

[0002] Globally, liver cancer is the most common and deadliest malignant tumor, and its incidence has been increasing year by year in recent years. Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers and has become a major global health problem. Currently available cancer treatments include surgery, radiotherapy, chemotherapy, and immunotherapy. Although these treatments have achieved certain efficacy, they have not yet effectively and completely eliminated liver cancer-inducing factors. Therefore, research on tumor biopharmaceuticals will help overcome the bottlenecks in cancer treatment.

[0003] Photothermal therapy (PTT) is an emerging cancer treatment strategy that utilizes the photothermal effect of photothermal converters (PTAs) to harvest energy from light and convert it into heat, raising the temperature of the surrounding environment, inhibiting tumor metastasis, and inducing cancer cell death. Polydopamine (PDA) is a biomimetic polymer obtained by polymerizing dopamine monomers under alkaline conditions. It possesses unique physical and chemical properties, including low invasiveness, high biocompatibility and biodegradability, and effective drug loading capacity.

[0004] Trichinella spiralis (T. spiralis) is an important zoonotic parasitic nematode. Studies have confirmed that Trichinella spiralis proteins contain active proteins that regulate tumor cell apoptosis, and these proteins participate in regulating apoptosis in the human hepatocellular carcinoma line H7402 through mitochondrial and death receptor apoptosis pathways. A type of Trichinella spiralis protein nanoparticle with photothermal conversion capabilities can be rapidly heated under the action of a near-infrared laser, enhancing the antitumor effect of the Trichinella spiralis antitumor protein. Currently, PDA has not been applied to nano-drug delivery systems for Trichinella spiralis proteins; therefore, developing a feasible solution for loading Trichinella spiralis antitumor protein nanoparticles with mesoporous polydopamine is a crucial problem that needs to be solved. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles, their preparation method, and applications. These nanoparticles can achieve rapid heating under the action of a near-infrared laser, thereby enhancing the antitumor effect of the Trichinella spiralis antitumor protein.

[0006] The present invention discloses a method for preparing mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles, the specific steps of which are as follows:

[0007] I. Preparation of mesoporous polydopamine (MPDA):

[0008] 1) Mix 25 mL of ethanol and 25 mL of water, and add 0.2–0.6 g of dopamine hydrochloride and 0.5–1 g of poloxamer (Pluronic F-127) to the solution;

[0009] 2) Slowly add 0.4–1 mL of 1,3,5-trimethylbenzene (TMB) and stir at room temperature for 30 minutes;

[0010] 3) Slowly add 1.5-2.5 mL of ammonia solution with a concentration of 25-28%, and stir the mixture at room temperature for 4-6 hours;

[0011] 4) Collect the product by centrifugation and wash it three times with ethanol and water;

[0012] 5) Wash three times with a 2:1 volume ratio ethanol / acetone mixture, collect the MPDA nanoparticles by centrifugation and dry them.

[0013] II. Mesoporous polydopamine (MPDA) loaded with Trichinella spiralis antitumor protein (NTspATP):

[0014] 1) Disperse 0.5–1 mg MPDA in 1 mL of deionized water and sonicate to mix.

[0015] 2) Add 5 mg of Trichinella spiralis antitumor protein to the above solution and stir overnight at 4°C.

[0016] 3) After the reaction was completed, the supernatant was removed by centrifugation, and the precipitate was washed three times with PBS buffer to obtain mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles (MPDA@NTspATP).

[0017] The present invention also provides mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles prepared by the above method, and the use of the mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles in the preparation of antitumor therapeutic drugs.

[0018] The beneficial effects of this invention are:

[0019] The MPDA obtained by the method of this invention, when heated to 48.4°C within 5 minutes by irradiation with near-infrared light at 808 nm using 500 μg / mL nanoparticles, and loaded with Trichinella spiralis antitumor protein (NTspATP), yields MPDA@NTspATP that not only inhibits tumor cell proliferation and migration but also induces tumor cell apoptosis and triggers immunogenic cell death (ICD). During ICD, related signaling pathways are activated, leading to the release of damage-associated molecular patterns (DAMPs), such as calreticulin (CRT) exposed to the outer cell membrane and high-mobility group box 1 (HMGB1) from the cell nucleus. After DAMPs are produced, they can bind to pattern recognition receptors (PRRs) on antigen-presenting cells (APCs), thereby eliciting a protective anti-cancer immune response. However, it does not inhibit immune cells at the tumor site, nor does it damage healthy tissue near the tumor. Attached Figure Description

[0020] Figure 1 This is a PCR identification diagram of the pET28a-NTspATP recombinant plasmid constructed in this invention; M: marker. 1: target band.

[0021] Figure 2 This is an SDS-PAGE image of the Trichinella spiralis antitumor protein (NTspATP) expressed in this invention; M: marker, 1: uninduced bacterial supernatant, 2: uninduced bacterial precipitate, 3: induced bacterial supernatant, 4: induced bacterial precipitate.

[0022] Figure 3 This is a Western blot image of the Trichinella spiralis antitumor protein (NTspATP) expressed in this invention; M: marker, 1: target protein band.

[0023] Figure 4 This is a transmission electron microscope (TEM) image of the MPDA prepared in this invention.

[0024] Figure 5 This is the Fourier transform infrared (FTIR) spectrum of the MPDA@NTspATP prepared in this invention.

[0025] Figure 6 The effects of MPDA, NTspATP, MPDA@NTspATP, and MPDA@NTspATP+NIR on the migration of HepG2 cells in vitro. Detailed Implementation

[0026] The technical solution of the present invention will be further explained and described below with reference to specific embodiments.

[0027] Example 1

[0028] Based on the anti-tumor gene of Trichinella spiralis, primers were designed using pET-28a as the prokaryotic expression vector and BamHI and XhoHI as multiple cloning restriction sites. The target gene was amplified by PCR using cDNA from adult or newly formed larvae of Trichinella spiralis as a template. The nucleotide sequences of the primers are shown below:

[0029] Upstream primer F (sequence 1): 5'-CGC GGA TCC CTG ATG ACA TCT AAT GAA TTT-3', (the "GGATCC" part at the 5' end of the sequence is the BamHI restriction site)

[0030] Downstream primer R (sequence 2): 5'-TTA CTC GAG ATC AGA CAA TGT TGG TGA CAC CTT-3', (the "CTCGAG" part at the 5' end of the sequence is the XhoI restriction site)

[0031] 1) Extract the target gene and pET-28a plasmid separately, and identify them by enzyme digestion with BamHI and XhoI;

[0032] 2) Adjust the concentration of vector pET-28a and target gene fragment (sequence 3) to a molar ratio of 1:3 to 1:9, and ligate overnight in a 16°C water bath;

[0033] 3) The recombinant plasmid pET28a-NTspATP was transformed into Escherichia coli expression strain BL21(DE3) and sequenced for identification;

[0034] 4) The correctly identified bacteria were induced to express their expression by adding isopropyl-β-D-thiogalactoside (IPTG) at 37°C for 5 h.

[0035] 5) Collect the bacterial cells, sonicate them, and remove impurities and proteins using a low concentration of urea. Finally, dissolve the bacteria in 8 mol / L urea.

[0036] 6) The purified and refolded recombinant protein was collected using Ni column affinity chromatography. First, the protein dissolved in 8 mol / L urea was centrifuged, and the supernatant was filtered.

[0037] 7) Inject the protein sample and slowly replace the solvent 8 mol / L urea with refolding buffer over 120 min to complete the on-column refolding.

[0038] 8) Elute the refolded protein with elution buffer, collect in 1.5 mL centrifuge tubes, and store at -80°C.

[0039] Figure 1 This is a PCR identification image of the recombinant plasmid pET28a-NTspATP constructed in this invention. As can be seen from the image, after the target gene is ligated to the vector, a bright band appears around 457 bp, consistent with the size described in the literature, proving that the recombinant plasmid pET28a-NTspATP was successfully constructed.

[0040] Figure 2 This is an SDS-PAGE image of the Trichinella spiralis antitumor protein (NTspATP) expressed in this invention. The image shows that the protein appears in the precipitate after bacterial induction, with a size of approximately 21 kDa, demonstrating that the recombinant protein is expressed in inclusion bodies.

[0041] Figure 3 This is a Western blot image of the Trichinella spiralis antitumor protein (NTspATP) expressed in this invention. The image shows that after incubation with a His-tagged antibody, a band appears around 21 kDa, proving that the protein has been successfully tagged with the His tag.

[0042] Example 2

[0043] 1) Mix 25 mL of ethanol and 25 mL of water, and add 0.2 g of dopamine hydrochloride and 0.5 g of poloxamer (Pluronic F-127) to the solution;

[0044] 2) Slowly add 1 mL of 1,3,5-trimethylbenzene (TMB, purity 97%) and stir at room temperature for 30 minutes;

[0045] 3) Slowly add 2.5 mL of ammonia solution with a concentration of 25-28%, and stir the mixture at room temperature for 4 hours;

[0046] 4) Collect the product by centrifugation and wash it three times with ethanol and water respectively;

[0047] 5) Wash three times with a mixture of ethanol / acetone (v / v = 2:1), collect the MPDA nanoparticles by centrifugation and dry them.

[0048] 6) Disperse 1 mg MPDA in 1 ml of deionized water and sonicate to mix.

[0049] 7) Add 5 mg of the Trichinella spiralis antitumor protein obtained in Example 1 to the above solution and stir overnight at 4°C.

[0050] 8) After the reaction is complete, centrifuge to remove the supernatant, wash the precipitate three times with PBS buffer to obtain MPDA@NTspATP.

[0051] Figure 4 This is a transmission electron microscope (TEM) image of the MPDA prepared in this invention. The image shows that the MPDA particles are relatively uniform in size and have a mesoporous surface structure.

[0052] Figure 5 This is the Fourier transform infrared (FTIR) spectrum of the MPDA@NTspATP prepared in this invention. As can be seen from the figure, at 1063 cm⁻¹... -1 At this point, the characteristic absorption peak of the recombinant protein appeared in MPDA@NTspATP, proving that MPDA@NTspATP was successfully prepared.

[0053] Verification Experiment

[0054] Cell scratch assays demonstrated the inhibitory effect of the MPDA@NTspATP prepared in this invention on the migration of HepG2 cells.

[0055] HepG2 cells were spaced at 1×10⁶ cells per well. 5 Cells were seeded at a density of [insert cell density here] into 6-well plates and incubated for 24 hours. The culture medium was changed after incubation. When the cell density reached 90%, the cells were scratched with a 10 μl pipette tip. Cells were washed with PBS to remove dead cells. 2 mL of serum-free culture medium was added to each well to minimize the impact of cell proliferation on the experiment. Separate groups were set up: PBS group, MPDA group, NTspATP group, MPDA@NTspATP group, and MPDA@NTspATP+NIR group. Cells in the photothermal therapy group were treated with 808 nm near-infrared light for 5 minutes. Cells were incubated for 24 hours. In vitro wound healing was observed under an inverted fluorescence microscope at 0 h, 6 h, 12 h, and 24 h.

[0056] Figure 6 The study demonstrated the inhibitory effects of different drug groups on HepG2 cell migration in vitro. As shown in the figure, compared with the PBS control group and the MPDA group, the NTspATP group, MPDA@NTspATP, and MPDA@NTspATP+NIR group showed varying degrees of inhibition on HepG2 cell migration, with the degree of inhibition gradually increasing. In the MPDA@NTspATP and MPDA@NTspATP+NIR groups, a small number of cells died.

Claims

1. A method for preparing mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles, characterized in that, The specific steps of this method are as follows: 1) Disperse 0.5–1 mg of mesoporous polydopamine nanoparticles in 1 mL of deionized water and mix by ultrasonication to obtain a solution; 2) Add 5 mg of Trichinella spiralis antitumor protein to the above solution and stir at 4°C for 12-16 hours; 3) After the reaction was completed, the supernatant was removed by centrifugation, and the precipitate was washed three times with PBS buffer to obtain mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles. The preparation method of the Trichinella spiralis antitumor protein in step 2) is as follows: Using the Trichinella spiralis antitumor protein gene as the target gene, pET-28a as the prokaryotic expression vector, and BamHI and XhoHI as polyclonal restriction enzyme sites, primers were designed. The target gene was amplified by PCR using cDNA from adult or newly formed larvae of Trichinella spiralis as a template. The nucleotide sequences of the primers are shown below: Upstream primer F has the sequence 1: 5'-CGC GGA TCC CTG ATG ACA TCT AAT GAA TTT-3', where the "GGATCC" portion at the 5' end is the BamHI restriction site. Downstream primer R is sequence 2: 5'-TTA CTC GAG ATC AGA CAA TGT TGG TGA CAC CTT-3', where the "CTCGAG" portion at the 5' end is the XhoI restriction site. (1) Extract the target gene and pET-28a plasmid respectively, and identify them by enzyme digestion with BamHI and XhoI; (2) Adjust the concentration of vector pET-28a and target gene fragment, i.e. sequence 3, to make their molar concentration ratio 1:3~1:9, and ligate them in a 16℃ water bath for 12~16 hours; (3) The recombinant plasmid pET28a-NTspATP was transformed into Escherichia coli expression strain BL21 and sequenced for identification; (4) The correctly identified bacteria were induced to express by adding isopropyl-β-D-thiogalactoside at a final concentration of 0.6 mM for 5 h at 37 °C. (5) Collect the bacterial cells, sonicate them, and remove impurities and proteins using a low concentration of urea. Finally, dissolve them in 8 mol / L urea. (6) Centrifuge the protein dissolved in 8 mol / L urea, filter the supernatant to obtain the protein sample; (7) The purified and refolded recombinant protein was collected by Ni column affinity chromatography. The protein sample was injected and loaded. The solvent 8 mol / L urea was slowly replaced with refolding buffer over 120 min to complete the on-column refolding. (8) Use elution buffer to elute the refolded protein, collect it in a 1.5 mL centrifuge tube, and store it at -80°C.

2. The method for preparing mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles according to claim 1, characterized in that, Step 1) The preparation method of mesoporous polydopamine is as follows: a. Mix 25 mL of ethanol and 25 mL of water, and add 0.2–0.6 g of dopamine hydrochloride and 0.5–1 g of poloxamer F-127 to the solution; b. Slowly add 0.4–1 mL of 1,3,5-trimethylbenzene and stir at room temperature for 30 minutes; c. Slowly add 1.5–2.5 mL of ammonia solution with a concentration of 25–28%, and stir the mixture at room temperature for 4–6 hours; d. Collect the product by centrifugation and wash it three times with ethanol and water; e. Wash three times with a 2:1 volume ratio ethanol / acetone mixture, collect the mesoporous polydopamine nanoparticles by centrifugation and dry them.

3. Mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles prepared by the method described in claim 1 or 2.

4. The use of the mesoporous polydopamine-loaded Trichinella spiralis antitumor protein nanoparticles as described in claim 3 for the preparation of antitumor therapeutic drugs.

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