A vector-free nanoparticle based on chemotherapy-gene therapy synergy and its preparation and anti-liver cancer application

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a carrier-free nanoparticle based on chemotherapy-gene therapy synergy and preparation and anti-liver cancer application thereof. The carrier-free nanoparticle comprises an inner core and a shell coated outside the inner core. The inner core is co-assembled by ursolic acid, sorafenib and a CRISPR system Cas9 / RNP complex through multiple coordination, pi-pi stacking, electrostatic and hydrophobic force, with protamine sulfate as an auxiliary agent. The shell is a HepG2 cell membrane. The CRISPR system Cas9 / RNP complex comprises a Cas9 protein and sgRNA which specifically targets a PD-L1 gene. The carrier-free nanoparticle can be specifically accumulated at a liver cancer site through cell membrane homologous recognition, and reaches the cell nucleus under the action of a gene drug nuclear localization sequence to perform gene editing, realizes chemotherapy-gene therapy combined anti-liver cancer effect, has a significant inhibitory effect on the growth of liver cancer, and has a wide application prospect in the preparation of anti-liver cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a carrier-free nanoparticle based on chemotherapy-gene therapy synergy, its preparation, and its application in treating liver cancer. Background Technology

[0002] Liver cancer is insidious in its onset, difficult to diagnose in its early stages, and challenging to treat. It has a high recurrence rate and malignancy, with consistently high numbers of new cases and deaths each year, posing a long-standing threat to human health. Currently, clinical treatment for liver cancer primarily involves a comprehensive approach combining surgical resection, chemotherapy, and radiotherapy. With the discovery and development of various small-molecule drugs, several chemotherapy options are now available for liver cancer. However, due to the low targeting efficiency and significant side effects of most of these drugs, the therapeutic efficacy of traditional chemotherapy for liver cancer remains unsatisfactory.

[0003] Sorafenib (SF) is a small-molecule targeted multi-kinase inhibitor with multiple mechanisms to inhibit the proliferation of liver cancer cells, demonstrating significant anti-liver cancer efficacy and becoming the first-line chemotherapy drug for intermediate and advanced liver cancer. Ursolic acid (UA) is a triterpenoid compound derived from natural plants, which has played a significant role in the field of anti-tumor treatment in recent years. It is low in toxicity, highly effective, and has a mild effect, making it a safe potential drug for cancer treatment. However, both drugs still have drawbacks such as poor water solubility, low bioavailability, and a tendency to induce drug resistance. Researchers have explored methods for combining two or more chemotherapy drugs to reduce the dosage of a single drug and thus lower toxicity and drug resistance. Chinese patent CN201610518377.5 discloses a pharmaceutical composition containing ursolic acid and sorafenib and its application in the preparation of anti-tumor drugs. This composition demonstrates the advantages of low toxicity and high efficacy in the treatment of liver cancer. Furthermore, Chinese patent CN202111598274.1 discloses the preparation and use of carrier-free dual-drug self-assembled nanoparticles, which self-assemble ursolic acid and sorafenib into nanoparticles, effectively avoiding the potential toxicity of traditional polymer or inorganic carriers. Therefore, multi-drug combined self-assembled nanoparticles may become an effective treatment strategy for liver cancer.

[0004] Gene therapy involves introducing therapeutic nucleic acids into target cells or organisms to correct or compensate for diseases caused by defective or abnormal genes, thereby achieving therapeutic goals. This therapy has gradually become a research focus in recent years, providing a new treatment strategy for cancer. Among these, the CRISPR / Cas9 system is a system that can rapidly and conveniently perform precise genome editing, directly targeting tumor cells to achieve targeted knockout of key oncogenes. In recent years, extensive research has been conducted on combining this technology with traditional cancer treatment methods, leveraging the synergistic effects of chemotherapy and gene therapy to significantly improve cancer treatment outcomes, making it a highly promising anti-cancer strategy.

[0005] The development of nanomedicine technology has brought new opportunities and challenges to cancer treatment. By constructing nanodelivery systems, the bioavailability of various anti-tumor drugs can be greatly improved, significantly enhancing the therapeutic effect. However, most exogenous nanocarriers still suffer from problems such as low drug loading capacity and poor biocompatibility. The concept of carrier-free self-assembly provides a new approach for anticancer drug development. Delivery systems prepared through drug self-assembly have complexes that act as both carriers and drugs, offering advantages such as high drug loading capacity and low toxicity. Currently, the construction of nanomedicine systems based on gene-chemotherapy combinations often uses exogenous materials as drug carriers. Chinese Patent 201810781142.4 discloses the preparation and application of a multifunctional nanomedicine based on aptamer-modified hollow mesoporous silica co-loaded plasmids. Hollow mesoporous silica is used as a drug carrier, simultaneously encapsulating sorafenib and EGRF-sgRNA / Cas9 plasmids to achieve synergistic anti-tumor effects. Chinese patent CN202210130435.2 discloses a gene editing delivery system, its preparation method, and its application, which uses a membrane-fused lipid exfoliant to encapsulate the surface of a calcium-precipitated hybrid gene editing plasmid core to achieve CRISPR / Cas9 plasmid delivery. However, there are no reports on the use of carrier-free self-assembly technology-based gene-chemotherapy nanomedicine systems for the synergistic treatment of liver cancer.

[0006] Based on the aforementioned research background, this invention aims to construct a carrier-free nanodelivery system based on synergistic chemotherapy-gene therapy. This system utilizes the intermolecular forces of two hydrophobic drugs to spontaneously assemble into a nanocore, and incorporates CRISPR / Cas9 system components to achieve effective synergy between gene therapy and chemotherapy. Furthermore, the nanoparticles are coated with a biomimetic cell membrane derived from liver cancer cells, enabling them to achieve tumor homology targeting and immune evasion. The carrier-free nanoparticles based on synergistic chemotherapy-gene therapy provided by this invention combine chemotherapy and gene therapy, overcoming the limitations of traditional monotherapy and avoiding the potential problems of traditional nanocarriers, thus showing broad application prospects in the treatment of liver cancer. Summary of the Invention

[0007] The purpose of this invention is to provide a carrier-free nanoparticle based on synergistic chemotherapy-gene therapy, its preparation method, and its application in treating liver cancer. This invention constructs a nanocore 1 with synergistic dual-drug effects through the self-assembly of hydrophobic drug molecules. Using a positively charged protein as an adjuvant, the nanocore 1 is co-assembled with gene-editing components via π-π stacking, electrostatic interactions, hydrophobic interactions, and other means to obtain a nanocore 2 that combines synergistic dual-drug effects and gene therapy capabilities. Then, a tumor cell-derived cell membrane is coated onto the outer layer of the nanocore 2, giving it good tumor recognition and immune escape capabilities, thus obtaining the carrier-free nanoparticle based on synergistic chemotherapy-gene therapy. This carrier-free nanoparticle integrates the combined application strategy of chemotherapy and gene therapy, achieving better tumor suppression effects.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A carrier-free nanoparticle based on chemotherapy-gene therapy synergy, comprising a core and a shell encapsulating the core, wherein the core is co-assembled with a positively charged protein as an adjuvant, a hydrophobic natural product, a hydrophobic antitumor drug, and a CRISPR system Cas9 / RNP complex through multiple coordination, π-π stacking, electrostatic and hydrophobic interactions, and the shell is a tumor cell membrane; wherein the hydrophobic natural product is ursolic acid, the hydrophobic antitumor drug is sorafenib, the positively charged protein is protamine sulfate, the CRISPR system Cas9 / RNP complex comprises Cas9 protein and sgRNA specifically targeting the PD-L1 gene, and the tumor cell membrane is the cell membrane of HepG2 human liver cancer cells; wherein the sequence of the sgRNA is 5'-CACCGCAAGGCCGAAGTCATCTGGA-3', and the amino acid sequence of the Cas9 protein is shown in SEQ ID NO.2.

[0010] The particle size of the carrier-free nanoparticles is 170–200 nm.

[0011] The above-mentioned method for preparing carrier-free nanoparticles based on chemotherapy-gene therapy synergy specifically includes the following steps:

[0012] (1) Dissolve a certain amount of ursolic acid and sorafenib in methanol and ethanol respectively to obtain ursolic acid-methanol solution and sorafenib-ethanol solution;

[0013] (2) Mix a certain amount of ursolic acid-methanol solution and sorafenib-ethanol solution evenly, and add them dropwise to deionized water to obtain US NPs solution;

[0014] (3) Dissolve a certain amount of Cas9 / RNP complex and protamine sulfate in water to obtain aqueous solutions of Cas9 / RNP complex and protamine sulfate.

[0015] (4) Mix a certain amount of Cas9 / RNP complex aqueous solution with sulfuric acid protamine aqueous solution evenly, and add the mixture dropwise to the US NPs solution prepared in step 2) to obtain USPR NPs solution; freeze-dry the obtained USPR NPs solution to obtain USPRNPs lyophilized powder.

[0016] (5) A certain amount of tumor cell membrane lyophilized powder and a certain amount of USPR NPs lyophilized powder are mixed and dissolved together in deionized water, filtered through an aqueous microporous filter membrane, and centrifuged to obtain the carrier-free nanoparticles based on chemotherapy-gene therapy synergy.

[0017] In step (1), the concentration of ursolic acid in the ursolic acid-methanol solution is 4 mg / mL, and the concentration of sorafenib in the sorafenib-ethanol solution is 4 mg / mL.

[0018] In step (2), the volume ratio of the ursolic acid-methanol solution and the sorafenib-ethanol solution is 1:1.

[0019] In step (3), the concentrations of the Cas9 / RNP complex aqueous solution and the protamine sulfate aqueous solution are both 1 mg / mL;

[0020] In step (4), the volume ratio of the Cas9 / RNP complex aqueous solution to the protamine sulfate aqueous solution is 3:1; in step (5), the method for preparing the lyophilized tumor cell membrane powder is as follows: the cell membrane of HepG2 human liver cancer cells is extracted by differential centrifugation, and the resulting precipitate is freeze-dried to obtain the lyophilized tumor cell membrane powder.

[0021] This invention also provides the application of the aforementioned carrier-free nanoparticles based on chemotherapy-gene therapy synergy in the treatment of liver cancer.

[0022] The advantages of this invention are:

[0023] (1) The core of the carrier-free nanoparticles prepared in this invention is composed of natural product ursolic acid and clinical first-line anti-tumor drug sorafenib, and is constructed by co-assembly with PD-L1-Cas9 RNP using FDA-approved protamine sulfate with good biocompatibility as an excipient. It does not contain exogenous synthetic carriers and has good biological safety.

[0024] (2) The carrier-free nanoparticles prepared in this invention utilize the self-recognition ability of liver cancer cells and are encapsulated by cell membranes derived from HepG2 liver cancer cells, giving the nanomedicine good tumor targeting and lysosomal escape capabilities.

[0025] (3) The preparation process of this invention is simple and efficient, and the prepared carrier-free nanoparticles have good stability under various physiological conditions.

[0026] (4) The carrier-free nanoparticles prepared in this invention integrate chemotherapy and gene therapy strategies into the same nanodrug delivery system, which effectively improves the limitations of single chemotherapy in tumor suppression and shows positive application potential in the field of liver cancer treatment. Attached Figure Description

[0027] Figure 1 This is a structural diagram of ursolic acid and sorafenib used in this invention. A, ursolic acid; B, sorafenib.

[0028] Figure 2 TEM images of US NPs, USPRNPs, and USPR@M NPs.

[0029] Figure 3 Stability graphs of US NPs, USPRNPs and USPR@M NPs aqueous solutions stored at room temperature for 7 days.

[0030] Figure 4 This diagram shows the inhibitory effect of free drugs on cell proliferation of each group of nanoparticles.

[0031] Figure 5 This is a graph showing the uptake of nanoparticles by HepG2 liver cancer cells.

[0032] Figure 6 To detect the PD-L1 gene knockout effect of USPR@M NPs on HepG2 liver cancer cells. Detailed Implementation

[0033] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0034] The Cas9 RNP complexes involved in the following examples can be prepared in-house or purchased from commercial suppliers. Specifically, the preparation method of the Cas9 RNP complex described in Example 1 is as follows:

[0035] A specific sgRNA (5'-CACCGCAAGGCCGAAGTCATCTGGA-3') targeting the PD-L1 gene was designed. The sgRNA was mixed with Cas9 protein (SEQ ID NO.2) at a mass ratio of 1:1 and incubated at 220 rpm in a shaker at room temperature for 6 h to form a Cas9 RNP complex.

[0036] Example 1

[0037] This embodiment provides a method for preparing carrier-free nanoparticles based on chemotherapy-gene therapy synergy, the preparation method specifically including the following steps:

[0038] (1) Dissolve ursolic acid (UA) in methanol to prepare solution A with a UA concentration of 4 mg / mL; dissolve sorafenib (SF) in ethanol to prepare solution B with an SF concentration of 4 mg / mL; mix 50 μL of solution A and 50 μL of solution B evenly to obtain a mixed solution AB; add 100 μL of the mixed solution AB to 900 μL of deionized water under vortex shaking to obtain a solution of nanoparticles US NPs;

[0039] (2) Dissolve protamine sulfate (PS) in deionized water to prepare a solution C with a PS concentration of 1 mg / mL; using Cas9 concentration as a standard, dilute the Cas9 RNP complex with deionized water to a solution D with a concentration of 1 mg / mL; mix 20 μL of solution C and 60 μL of solution D evenly, add 20 μL of deionized water to obtain a CD mixed solution; slowly add 100 μL of CD mixed solution to 900 μL of US NPs solution under vortex shaking to obtain a nanoparticle USPR NPs solution; freeze-dry the nanoparticle USPRNPs solution to obtain nanoparticle USPRNPs lyophilized powder.

[0040] Example 2

[0041] This embodiment provides a method for preparing HepG2 cell lysate and extracting HepG2 cell membrane, as follows:

[0042] (1) Culture HepG2 cells to a growth density of 80-90%, digest with 0.25wt% trypsin containing EDTA for 3-5 min to obtain cell suspension, centrifuge at 1500 rpm for 5 min to obtain cell pellet, wash the cell pellet 3 times with physiological saline and disperse it in 0.25x PBS containing phenylmethylsulfonyl fluoride (PMSF) (PBS:PMSF = 100:1), and incubate at 4℃ for 0.5 h to obtain HepG2 cell lysate.

[0043] (2) Use a cell homogenizer to slowly grind the cell lysate for 5-10 minutes to fully break the cells into fragments. Then, centrifuge the cell lysate at 1500g at 4℃ for 5 minutes to remove impurities and larger organelles. Take the supernatant and centrifuge at 12000g at 4℃ for 30 minutes, then centrifuge at 100000g at 4℃ for 1 hour to remove the supernatant. Freeze-dry the resulting precipitate at -40℃ and 13.33Pa. The resulting powder is HepG2 cell membrane (CM) freeze-dried powder.

[0044] Example 3

[0045] This embodiment provides a method for preparing carrier-free nanoparticles (USPR@M NPs) based on chemotherapy-gene therapy synergy. The preparation method steps are as follows:

[0046] The HepG2 cell membrane lyophilized powder collected in Example 2 was mixed evenly with the USPR NPs nanoparticle lyophilized powder prepared in Example 1 and dissolved together in 1 mL of deionized water until the final concentration of the HepG2 cell membrane lyophilized powder was 2 mg / mL and the final concentration of the USPR NPs nanoparticle lyophilized powder was 5 mg / mL. The mixture was placed on a shaker at 220 rpm for 30 min, and then sonicated in a water bath at 35 kHz for 10 min at room temperature. After filtration through a 0.22 μm aqueous microporous membrane 6 to 8 times, the mixture was centrifuged at 12000 g for 30 min to remove free tumor cell membranes. The supernatant was discarded, and the resulting precipitate was the USPR@M NPs nanoparticles.

[0047] Example 4

[0048] Images of US nanoparticles NPs and USPRNPs prepared in Example 1, and USPR@M NPs prepared in Example 3, were captured using a transmission electron microscope.

[0049] like Figure 2 As shown, the US NP nanoparticles are uniform spheres; the USPRNP nanoparticles are coated with a layer of material, which is a PS and Cas9 RNP complex bound to the outer layer of the US NP nanoparticles; and a transparent membrane-like material, namely the tumor cell membrane, is visible on the surface of the USPR@M NP nanoparticles, and the thickness of this layer is about 10-20 nm, which is consistent with the known thickness of tumor cell membranes of about 10-30 nm, indicating that the tumor cell membrane is successfully coated on the surface of the nanoparticles.

[0050] Example 5

[0051] The US NPs and USPR NPs prepared in Example 1, as well as the USPR@M NPs prepared in Example 3, were stored in an aqueous solution at room temperature, and their particle size changes were measured for 14 consecutive days to observe their stability.

[0052] like Figure 3 As shown, the particle size of the carrier-free nanoparticles USPR@M NPs did not change significantly within 14 days, demonstrating good stability.

[0053] Example 6

[0054] The inhibitory effects of different groups of nanoparticles on the proliferation of HepG2 liver cancer cells and L02 normal liver cells were investigated using a CCK-8 cytotoxicity assay. The specific steps were as follows:

[0055] (1) HepG2 and L02 cells in logarithmic growth phase were digested with 0.25% trypsin-EDTA (1 mM), and the cell pellet was collected. The pellets were then resuspended in fresh DMEM and RPMI 1640 medium, respectively, and then subjected to 1.0 × 10⁻⁶ mol / L incubators. 4 The cells were seeded at a density of 1 cell per well in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours until the cells were fully adhered to the plate.

[0056] (2) UA monotherapy group (UA), SF monotherapy group (SF), UA and SF co-administration group (UA+SF, with the final concentrations of both drugs being the same as those in the monotherapy groups), UA and SF self-assembled nanoparticle group (US NPs), PS and Cas9RNP complex group (P-RNP), and USPRNPs and USPR@M NP groups were set up. The drugs in each group were diluted with culture medium to a series of concentrations (10, 20, 30, 40, 50 μM). The drugs from different groups were added to 96-well plates containing HepG2 and L02 cells for incubation.

[0057] (3) After 24 hours of drug treatment, wash the cells three times with physiological saline, add fresh cell culture medium, add 10 μL of CCK-8 solution to each well, incubate in a cell culture incubator for 4 hours, and measure the absorbance value at 540 nm using an ELISA reader, and calculate the cell proliferation inhibition rate accordingly.

[0058] The results are as follows Figure 4 As shown, the self-assembled nanoparticles US NPs of UA and SF exhibit a higher killing effect compared to free drugs, demonstrating a synergistic therapeutic effect. The nanoparticles USPR@M NPs showed the most significant proliferation inhibition, indicating that this composite system can enhance the single-drug killing ability of SF. Furthermore, due to its outer layer encapsulating the HepG2 biomimetic membrane, HepG2 cells have increased homologous uptake of it, achieving a toxic effect on liver cancer cells. In addition, compared to liver cancer cells, the killing effect of each treatment group on normal cells was extremely limited, indicating that the nanoparticles have good safety and low toxicity.

[0059] Example 7

[0060] The uptake of USPR@M NPs nanoparticles by liver cancer cells was investigated using laser confocal microscopy. The specific steps were as follows:

[0061] (1) HepG2 liver cancer cells were injected at a dose of 2.0 x 10⁻⁶. 5 The cells were seeded at a density of 1 cells / well in 12-well plates with pre-placed cell spreaders and incubated at 37°C in a 5% CO2 incubator for 24 hours until the cells were fully adhered.

[0062] (2) Using indocyanine green (ICG) as a fluorescent dye, ICG was combined with UA and SF via solvent exchange to prepare US nanoparticles (mass ratio UA:SF:ICG = 4:4:1), which were then used to prepare USPR@M nanoparticles according to Example 3. The USPR@M nanoparticles were diluted to an ICG equivalent concentration of 2 μg / mL and incubated with HepG2 cells at different time points: 0.5 h, 1 h, 2 h, 4 h, and 6 h.

[0063] (3) Remove the old culture medium, wash the cells three times with physiological saline, add Hochest 33342 staining solution and incubate for 10 min, wash the cells twice with physiological saline, mount with anti-fluorescence quenching agent, and use a laser confocal microscope to simultaneously capture images of the cell uptake fluorescence of USPR@M NPs at different time points.

[0064] The results are as follows Figure 5 As shown, the uptake of USPR@M NPs in cells increased over time, reaching its peak at 4 hours. Notably, before 2 hours, the fluorescence images showed that USPR@M NPs were mainly aggregated in the cytoplasm. The Cas9 RNPs within the nanoparticles contain the NLS nuclear localization system, which can guide the CRISPR / Cas9 system into the nucleus. After 2 hours, the fluorescence of the USPR@M NPs overlapped with the blue fluorescence of the cell nucleus, indicating that the USPR@M NPs entered the nucleus. This is strong evidence that Cas RNPs can successfully enter the nucleus and achieve effective PD-L1 gene editing within it.

[0065] Example 8

[0066] The knockout efficiency of USPR@MNPs on the PD-L1 gene was investigated using genomic PCR, T7 endonuclease 1 (T7E1) digestion, and agarose gel electrophoresis. The specific steps were as follows:

[0067] (1) HepG2 cells in the logarithmic growth phase were seeded into 12-well plates and incubated at 37°C in a 5% CO2 incubator until the cells were completely adhered and the cell density reached 50%–70% before the experiment.

[0068] (2) UA monotherapy group (UA), SF monotherapy group (SF), UA and SF self-assembled nanoparticle group (USNPs), PS and Cas9 RNP complex group (P-RNP), and nanoparticle USPR NPs and nanoparticle USPR@M NP groups were set up respectively. HepG2 cells were incubated for 48h and genomic DNA was extracted.

[0069] (3) After digestion with 0.25% trypsin-EDTA (1mM), the cell pellet was collected, the genome was extracted, and the extracted genome was amplified by PCR using the verification primers PD-L1-F and PD-L1-R (PD-L1-F: 5'-TGGAGTATGGCAGCAACG-3'; PD-L1-R: 5'-AATGAGGAACAACAGGATGGAT-3').

[0070] (4) Add 1.1 μL of 10×T7E1 buffer and 4.4 μL of deionized water to 5 μL of PCR product, mix well, heat at 95℃ for 5 min to anneal, and cool naturally to room temperature for annealing and annealing.

[0071] (5) Add 0.5 μL T7E1 enzyme to each group of reaction systems and react at 37°C for 30 min.

[0072] (6) Weigh 0.45g of agarose powder and dissolve it in 30mL of deionized water. Heat and boil it three times to completely dissolve it. Slowly pour it into an plexiglass tank with a comb and let it stand and cool to obtain 1.5% agarose gel. Add each group of DNA fragment samples and electrophoresis at 100V for 15min. The obtained agarose gel is imaged using a chemiluminescence analyzer. The PD-L1 gene knockout efficiency of each drug group is calculated based on the band brightness.

[0073] The results are as follows Figure 6 As shown, in the nanoparticles that did not bind Cas9 RNP, no obvious new DNA fragments appeared in the cell genome after treatment with T7E1 enzyme, indicating that the gene sequence did not mutate. However, after Cas9 RNP, USPR NPs, and USPR@M NPs were applied to the cells, new DNA fragments appeared on the agarose gel, indicating the successful knockout of the PD-L1 gene. Among them, USPR@M NPs can effectively achieve PD-L1 gene knockout and has a high knockout efficiency.

[0074] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A carrier-free nanoparticle based on chemotherapy-gene therapy synergy, characterized in that: The carrier-free nanoparticles comprise a core and a shell encapsulating the core. The core is assembled from a hydrophobic natural product, a hydrophobic antitumor drug, and a CRISPR system Cas9 / RNP complex via multiple coordination, π-π stacking, electrostatic interaction, and hydrophobic forces, using a positively charged protein as an adjuvant. The shell is a tumor cell membrane. The hydrophobic natural product is ursolic acid, the hydrophobic antitumor drug is sorafenib, and the positively charged protein is protamine sulfate. The CRISPR system Cas9 / RNP complex is formed by mixing Cas9 protein and sgRNA specifically targeting the PD-L1 gene in a 1:1 mass ratio. The tumor cell membrane is the cell membrane of HepG2 human liver cancer cells. The sequence of the sgRNA is 5'-CACCGCAAGGCCGAAGTCATCTGGA-3', and the amino acid sequence of the Cas9 protein is shown in SEQ ID NO.

2.

2. The carrier-free nanoparticle based on chemotherapy-gene therapy synergy according to claim 1, characterized in that: The particle size of the carrier-free nanoparticles is 170~200nm.

3. A method for preparing carrier-free nanoparticles based on chemotherapy-gene therapy synergy as described in claim 1, characterized in that: Includes the following steps: 1) Dissolve ursolic acid and sorafenib in methanol and ethanol respectively to obtain ursolic acid-methanol solution and sorafenib-ethanol solution; 2) Mix 1 volume of ursolic acid-methanol solution and 1 volume of sorafenib-ethanol solution evenly, and add to 18 volume of deionized water to obtain US NPs solution; 3) Dissolve the CRISPR system Cas9 / RNP complex and protamine sulfate in deionized water to obtain Cas9 / RNP aqueous solution and protamine sulfate aqueous solution, respectively; 4) Mix 3 volumes of Cas9 / RNP aqueous solution with 1 volume of protamine sulfate aqueous solution, add 1 volume of deionized water, and then add it to the US NPs solution in step 2) to obtain USPR NPs solution. Freeze-dry the USPR NPs solution to obtain USPR NPs lyophilized powder. 5) Mix 2 parts by weight of tumor cell membrane lyophilized powder and 5 parts by weight of USPR NPs lyophilized powder evenly, dissolve them together in deionized water, filter through an aqueous microporous membrane, centrifuge, and the resulting precipitate is the carrier-free nanoparticle based on chemotherapy-gene therapy synergy.

4. The method for preparing carrier-free nanoparticles based on chemotherapy-gene therapy synergy according to claim 3, characterized in that: In step 1), the concentration of ursolic acid in the ursolic acid-methanol solution is 4 mg / mL, and the concentration of sorafenib in the sorafenib-ethanol solution is 4 mg / mL.

5. The method for preparing carrier-free nanoparticles based on chemotherapy-gene therapy synergy according to claim 3, characterized in that: In step 5), the method for preparing the lyophilized tumor cell membrane powder is as follows: HepG2 cells are lysed and broken into fragments by grinding, and then the HepG2 cell membrane is separated by differential centrifugation and freeze-dried to obtain the lyophilized tumor cell membrane powder.

6. The use of the carrier-free nanoparticles as described in claim 1 in the preparation of anti-liver cancer drugs.

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