CN113876736B

CN113876736B - Near-infrared response bionic nano preparation and preparation method and application thereof

Info

Publication number: CN113876736B
Application number: CN202111160742.7A
Authority: CN
Inventors: 王丹; 陈柳先; 袁睿; 李承碧; 罗艳秋; 汤青霞; 文飞; 李滔
Original assignee: Chongqing Rongchang District People's Hospital
Current assignee: Chongqing Rongchang District People's Hospital
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2022-11-29
Anticipated expiration: 2041-09-30
Also published as: CN113876736A

Abstract

The invention provides a near-infrared response bionic nano preparation. The bionic nano preparation comprises a cell membrane of an HBV hepatocyte model and a nano carrier wrapped in the cell membrane, wherein the nano carrier comprises near-infrared response up-conversion nanoparticles and CRISPR/Cas9, avidin proteins are modified on the surfaces of the up-conversion nanoparticles, and biotin-modified photolysis molecules are connected between the avidin proteins and the CRISPR/Cas9. The method solves the problems that a CRISPR/Cas9 delivery system in the prior art is high in immunogenicity, low in targeting property, small in carrying load and incapable of realizing remote space-time operation. A high-efficiency CRISPR/Cas9 delivery system with low immunogenicity, high targeting, large carrying load and simple and convenient production and synthesis is established, and accurate remote space-time operation is carried out on the CRISPR/Cas9.

Description

Near-infrared response bionic nano preparation and preparation method and application thereof

Technical Field

The invention relates to the technical field of DNA recombination, in particular to a near-infrared response bionic nano preparation and a preparation method and application thereof.

Background

Hepatitis B Virus (HBV) infection is one of the major public health problems facing the world. There are currently about 3 hundred million HBV infected people worldwide, with nearly 1 hundred million in china, reported by WHO; nearly 100 million deaths occur annually from HBV infection, half of which are in china. The current clinical drugs for hepatitis B patients mainly include two major classes of interferon and nucleoside (acid) analogs, aiming at reducing HBV DNA in serum and liver tissues of patients. However, in the long treatment process, patients have the risks of drug-resistant mutation and transformation to liver fibrosis, cirrhosis and liver cancer, and although the drugs can significantly inhibit HBV replication, none of the drugs can directly act on HBV replication template, namely covalently closed circular DNA (cccDNA), in the hepatocyte nucleus, and thus cannot completely remove the covalently closed circular DNA in vivo, resulting in difficulty in completely curing hepatitis b. HBV cccDNA is the source of chronic infection and relapse of HBV, and the expression level of the HBV cccDNA is an important index for clinically monitoring the curative effect of antiviral treatment and guiding a treatment scheme.

Currently, research on HBV cccDNA target therapy is mainly performed from two aspects, namely, inhibition of HBV cccDNA formation, such as negative regulation of cccDNA using target viral proteins HBx and HBc to inhibit its formation. However, this approach may target host proteins, resulting in more toxic side effects. Another aspect is to eliminate HBV cccDNA, epigenetically modify HBV cccDNA within the hepatocyte nucleus or target elimination by gene editing, such as histone modification and DNA methylation modification of targeted HBV cccDNA, etc. and gene editing techniques. However, no effective method for completely eliminating cccDNA in the liver cell nucleus of hepatitis b patients has been found so far, and complete elimination of HBV cccDNA is the key point for complete cure of hepatitis b.

The CRISPR/Cas (clustered regularly interrupted short palindromic repeats/CRISPR-associated proteins) system is an immune system which is found in most bacteria and all archaea at present, and is now a research hotspot in the field of life science at present. Researches show that the CRISPR/Cas9 system can effectively destroy HBV expression templates and has no obvious cytotoxicity. It could be a potential method to eradicate persistent HBV cccDNA in chronic HBV infected patients.

However, the CRISPR/Cas9 system has a troublesome problem to enter cells to play a role, namely how to efficiently deliver two macromolecules simultaneously: cas9 (about 160 kDa) and single guide RNA (sgRNA, over 100 base pairs). The current vectors for delivering CRISPR/Cas9 are mainly divided into two major categories, viral vectors and non-viral vectors. Viral vectors (e.g., lentiviruses, adenoviruses, etc.) are commonly used by researchers as a means of delivery to introduce foreign substances into cells depending on the nature of the delivery. The method is convenient, quick and easy to implement, but the method is very easy to cause canceration, insertion mutation or host immune response in organisms, and brings difficulties for subsequent research and treatment. In recent years, researchers have searched for non-viral vector delivery methods, and a non-viral vector delivery system which has no endogenous virus recombination, low immunogenicity, large carrying load, small adverse reaction and simple production and synthesis is expected to be developed. To date, safe delivery of CRISPR/Cas9 has been preliminarily achieved in vitro and in vivo experiments for non-viral vectors including gold nanoparticles, graphene oxide, polymer nanoparticles, and other nanomaterials. However, an efficient CRISPR/Cas9 delivery system with low immunogenicity, high targeting, large carrying load and simple and convenient production and synthesis is not established at present, and how to implement precise remote spatio-temporal operation on the CRISPR/Cas9 is a major problem faced by researchers.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a near-infrared response bionic nano-carrier and a preparation method and application thereof, and solves the problems that a CRISPR/Cas9 delivery system in the prior art is high in immunogenicity, low in targeting property, small in carrying load and incapable of realizing remote space-time operation.

The invention provides a near-infrared response bionic nano preparation, which comprises a cell membrane of a hepatocyte model and a nano carrier wrapped in the cell membrane, wherein the nano carrier comprises near-infrared response up-conversion nanoparticles and CRISPR/Cas9, avidin proteins are modified on the surfaces of the up-conversion nanoparticles, and biotin-modified photolysis molecules are connected between the avidin proteins and the CRISPR/Cas9.

A Biotin-modified photolytic molecule (PCB) is a uv-irradiated photocleavable linker comprising Biotin, PC (Photo-cleavable) and a Photo-responsive group; the surface of the Upconversion nanoparticles (UCNPs) is modified with Avidin to form Avidin modified Upconversion nanoparticles (UCNPs-Avidin); due to the strong affinity between Avidin and biotin, the PCB can be easily combined with UCNPs-Avidin to form nanoparticle UNCPs-Avidin/PCB, and then combined with CRISPR/Cas9 to generate UCNPs-Cas9; finally, UCNPs-Cas9 is wrapped by the cell membrane of the liver cell model to form the nano preparation. Upon irradiation by Near Infrared (NIR) light, UCNPs convert high-energy NIR light to low-energy UV light, which cleaves PC bonds, thereby releasing CRISPR/Cas9 for controllable gene editing.

Preferably, the hepatocyte model is one of HBV replication hepatocyte model, HBV infected hepatocyte model, primary hepatocyte model.

Preferably, the near-infrared response upconversion nanoparticles are of a core-shell structure, ca ²⁺ Doped NaYF ₄ :Yb ³⁺ /Tm ³⁺ As a nucleus, naYF ₄ Yb/Nd is the shell.

Preferably, the biotin-modified photolytic molecule is a PC biotin-nhs ester.

The PC Biotin-NHS Ester (PC Biotin-NHS Ester) comprises an o-nitrophenyl and an N-hydroxysuccinimide (NHS) group which are in photoresponse, the NHS group is connected with an amino group of a Cas9 protein in CRISPR/Cas9, and under the regulation of certain light, the o-nitrophenyl is broken, so that the CRISPR/Cas9 is released.

Preferably, the CRISPR/Cas9 comprises a Cas9 protein and sgrnas, and the corresponding DNA sequences of the sgrnas are shown in SEQ ID nos. 1 to 3.

In another aspect of the present invention, a method for preparing a near-infrared response biomimetic nano-preparation is provided, comprising the steps of:

1) Construction of CRISPR/Cas9: designing and synthesizing sgRNA, and co-incubating the sgRNA and Cas9 protein to prepare CRISPR/Cas9;

2) Preparing a nano-carrier UCNPs-Cas9: synthesizing up-conversion nanoparticles, carrying out hydrophilic modification and Avidin surface modification on the up-conversion nanoparticles to obtain UNCPs-Avidin, then carrying out a connection reaction with a biotin-modified photolysis molecule to form nanoparticle UNCPs-Avidin/PCB, and co-incubating the nanoparticle UNCPs-Avidin/PCB with the CRISPR/Cas9 prepared in the step 1) to obtain a nano-carrier UCNPs-Cas9;

3) Preparing a bionic nano preparation UCNPs-Cas9@ CM: extracting HBV cell membranes of the copied liver cancer cell model, mixing the cell membranes with the UCNPs-Cas9 prepared in the step 2), and repeatedly extruding through a polycarbonate filter membrane to obtain the UCNPs-Cas9@ CM.

Preferably, in step 1), the molar mass ratio of sgRNA to Cas9 protein is 1.

Preferably, in step 2), the surface of the up-conversion nanoparticle comprises an oleic acid ligand, the modifier used in the hydrophilic modification is polyacrylic acid, and the method for surface modification of avidin comprises: connecting avidin on the surface of the upconversion nanoparticles by using EDC/NHS; the temperature of the connection reaction is 37 ℃, and the time is 2h; the mass ratio of Avidin to biotin-modified photolytic molecules in UNCPs-Avidin is 1; the temperature of the co-incubation is 4 ℃ and the time is 8-12h.

Preferably, in step 3), the pore size of the polycarbonate filter membrane is 200nm or 400nm.

In another aspect, the invention provides an application of a near-infrared response bionic nano preparation in preparing a medicament for treating hepatitis.

The technical principle of the invention is as follows:

UCNPs do not have tissue targeting ability, thereby reducing their therapeutic effect at the site of action. In the prior art, the surface of UCNPs is modified by chitosan, PEG and other groups, and targeting ligands (including antibodies, peptides or aptamers and the like) are added to enhance the targeting property of the UCNPs and prolong the circulation time of the UCNPs in blood. However, this chemical modification is readily recognized by the human immune system and eliminated by the mononuclear phagocyte system. And because the targeting mechanisms of different tissues or cells are greatly different, the application of the artificial micro-nano transport carrier in the biomedical field is severely limited. Through repeated experiments, the inventor unexpectedly discovers that the bionic nano preparation obtained by wrapping a cell membrane bionic shell of a hepatocyte model outside UCNPs has better stability, longer blood circulation time and better homotypic fine targeting capability. Meanwhile, avidin (Avidin) is modified on the surface of UCNPs, the Avidin is combined with biotin in the PCB, and the PCB is connected with CRISPR/Cas9 to form UCNPs-Cas9; and wrapping with cell membrane of hepatocyte model to obtain UCNPs-Cas9@ CM. The homologous targeting of the cell membrane of the hepatocyte model accurately delivers UCNPs-Cas9@ CM to the hepatocyte, and the UCNPs-Cas9 enters the hepatocyte after the UCNPs-Cas9 is internalized by the hepatocyte. Under NIR irradiation, UCNPs convert the light into UV emitted light, and because the PCB comprises a light response group, the absorption spectrum of the light response group is superposed with the emission spectrum of the UCNPs, the light response group is broken, CRISPR/Cas9 is released, the CRISPR/Cas9 enters cell nucleus under the guide of a nuclear localization signal, the gene editing function aiming at HBV is exerted, the aim of the cooperative treatment of cell membrane and CRISPR/Cas9 on the anti-HBV is realized, and the remote space-time operation of the bionic nano preparation is realized.

Compared with the prior art, the invention has the following beneficial effects:

(1) The bionic nano preparation not only has the physical and chemical properties of a nano carrier, but also has a biological activity function similar to that of a target cell without additionally modifying a natural cell membrane of the nano preparation, and has longer blood circulation time and better isomorphous fine targeting capability under physiological conditions; experiments show that the hydrodynamic diameter change of the bionic nano preparation is almost constant within 7 days, and the bionic nano preparation has high stability; the bionic nano preparation (UCNPs-Cas9 @ CM2.2.15) coated by the HepG2.2.15 cell membrane is preferentially accumulated in HepG2.2.15 cells, is obviously superior to the accumulation in 293T (human embryonic kidney cells), heLa (human cervical cancer cells) and other heterotypic cells, and indicates that the bionic nano preparation has high homotypic cell targeting capability; the bionic nano preparation can escape from the uptake of macrophages, which shows that the nano preparation has the anti-phagocytosis capacity, long blood circulation time and low immunogenicity; and the drug is released under the irradiation of laser to achieve the purpose of synergistic treatment. In the prior art, the up-conversion nanoparticles coated by Polyethyleneimine (PEI) and silicon and PEG do not have targeting property, and have lower anti-phagocytosis performance and stability, so that the treatment effect is poorer;

(2) UCNPs adopt a core-shell structure, and Ca is doped in the core ²⁺ Compared with UCNPs without other ion doping, the fluorescence intensity of the UCNPs can be improved; the emission spectrum of the UCNPs is overlapped with the absorption spectrum of the photosensitive molecules at 360nm, so that the UCNPs transfer energy to the photosensitive molecules well through fluorescence resonance energy transfer, the release efficiency of CRISPR/Cas9 is improved, remote control is realized, and the treatment effect is improved;

(3) The surface of UCNPs is modified with avidin, and avidin and biotin have high affinity, so that more biotin-modified photosensitive molecules can be connected, more Cas9/sgRNA is loaded, a cascade amplification effect is achieved, and the effect of enhancing the treatment effect is further achieved; in the prior art, upconversion nanoparticles (such as NaYF) are adopted ₄ Yb/Tm) and photosensitive molecules (such as 4-hydroxymethyl-3-nitrobenzoic acid), the amount of Cas9/sgRNA loaded can be significantly reduced, resulting in limited therapeutic efficacy;

(4) The off-target effect of the bionic nano preparation can be almost ignored, so that the fixed-point editing efficiency of the bionic nano preparation is improved, the targeting efficiency and the knockout efficiency are improved, and the off-target rate is reduced; the influence on the functional area where the target point is removed is reduced, so that the influence on the expression of other genes is avoided, and the safety of the bionic nano preparation is improved;

(5) The UCNPs are subjected to hydrophilic-hydrophobic assembly, and hydrophilic modification is carried out to remove surface hydrophobic ligands and endow the UCNPs with water solubility, so that the UCNPs can be stably dispersed in water, and have good biocompatibility;

(6) The UCNPs have small particle size, the average diameter is 64nm, and the smaller the particle size, the less the UCNPs are difficult to be phagocytosed as foreign matters by macrophages, so that the treatment effect is improved;

in conclusion, the invention firstly triggers CRISPR/Cas9 to remove HBV cccDNA in a targeted way based on biomembrane mediated near-infrared light control, has good antiviral effect, can fundamentally remove hepatitis B virus, enables a hepatitis B patient to be cured, and provides a new treatment strategy for curing hepatitis B, and the bionic nano preparation has low immunogenicity, high targeting property, large carrying load and simple and convenient production and synthesis, can realize the remote operation of accurate time and space of CRISPR/Cas9, improves the treatment effect and efficiency, and is expected to be developed into a new method for treating hepatitis B with simplicity, reliability and strong practicability.

Drawings

Fig. 1 is an agarose gel electrophoresis of Cas9/sgRNA in vitro sgRNA targeting gene sequences in example 2 of the present invention;

FIG. 2 is a diagram of the characteristic analysis of UCNPs in example 2 of the present invention, wherein A is NaYF ₄ : SEM image of Yb/Tm/Ca; b is the size histogram thereof; c is NaYF ₄ :Yb/Tm/Ca@NaYF ₄ SEM image of Yb/Nd (UCNPs); d is a size histogram of the D; e is an HRTEM image of UCNPs; f is SAED of UCNPs;

FIG. 3 is a diagram of the characteristic analysis of UCNPs in example 2 of the present invention, wherein A is NaYF ₄ : TEM image of Yb/Tm/Ca; b is NaYF ₄ :Yb/Tm/Ca@NaYF ₄ TEM images of Yb/Nd (UCNPs); c is XRD pattern of UCNPs: d is UCNPs and an EDX element analysis chart thereof; e is UCNPs, UCNPs-PAA of any unit,Ultraviolet-visible absorption spectra of Avidin and UCNPs-Avidin; f is the Fourier infrared spectrum of UCNPs, UCNPs-PAA and UCNPs-Avidin; g is fluorescence spectrum of UCNPs and UCNPs-PAA activated by 808nm laser and ultraviolet absorption spectrum of PC Biotin-NHSester (PCB), and the inset is bright field (left) of UCNPs and fluorescence picture (right) under NIR excitation;

FIG. 4 is a representation of cell membranes in example 3 of the present invention: a is a transmission electron microscope image of CMs, B is the hydrodynamic diameter of CMs, and C is the zeta potential of CMs;

FIG. 5 is a representation of NIR-controlled UCNPs-Cas9@ CM: a is a TEM image of UCNPs-Cas9@ CM; b is an SEM image of UCNPs-Cas9@ CM; c is hydrodynamic size distribution of CMs, UCNPs and UCNPs-Cas9@ CM; d is zeta potential of Cas9/sgRNA, UCNPs-PAA, UCNPs-Avidin/PCB, UCNPs-Cas9 and UCNPs-Cas9@ CM; e is the SDS-PAGE protein analysis of total cellular protein, UCNPs-Cas9@ CM, avidin and Cas9 protein (36 pmol) (from left to right, samples stained with Coomassie Brilliant blue; F is WB analysis of (I) cell lysates and (II) CMs; G is the change in hydrodynamic diameter of UCNPs-Cas9@ CM with time; H is the determination of the UV absorption of UCNPs-Cas9 supernatant with 808nm laser at different irradiation times (280 nm); I is the CLSM image of cells incubated with UCNPs-Cas9@ CM after NIR- + irradiation (white: GFP-labeled Cas9; light grey: DAPI);

FIG. 6 is a graph showing the effect of NIR on the controlled release of CRISPR/Cas9 after UCNPs-Cas9@ CM is internalized by cells by CLSM: a is the effect profile of cells incubated with UCNPs-Cas9@ CM in the absence of NIR irradiation; b is a graph of the effect of cells incubated with UCNPs-Cas9@ CM under NIR irradiation;

FIG. 7 is a graph of the results of immune escape and isotype targeting experiments for UCNPs-Cas9@ CM: a is a CLSM image and a flow cytometer map of RAW264.7 cells cultured together with UCNPs-Cas9 or UCNPs-Cas9@ CM for 6 hours; b is HepG2.2.15, 293T, hela, L929 cell and UCNPs-Cas9@ CM _2.2.15 CLSM images and flow cytometer profiles after 6 hours of co-culture;

FIG. 8 is a CLSM image of Raw264.7 cells after 3 hours or 6 hours of co-culture of Raw264.7 cells with UCNPs-Cas9 and UCNPs-Cas9@ CM;

FIG. 9 is a CLSM image after 1h, 3h or 6h of co-culture of four cell lines with UCNPs-Cas9@ CM;

FIG. 10 is a graph of the results of cell viability assays: a is cell viability after irradiation with different NIR power (fixed 20min, no UCNPs-Cas9@ CM); b is fixed NIR power of 2.0W/cm ² Cell viability at different irradiation times (no UCNPs-Cas9@ CM); c is cell viability after different concentrations of UCNPs-Cas9@ CM (no NIR) treatment; d is UCNPs-Cas9@ CM (NIR power: 2.0W/cm) at different concentrations ² Fixing for 20 min) cell viability after irradiation; wherein, the concentration of UCNPs-Cas9@ CM is marked in the form of Cas9 concentration (uM);

FIG. 11 is a graph of the temperature change of UCNPs-Cas9@ CM solution with and without NIR irradiation;

FIG. 12 shows the gene editing effect of UCNPs-Cas9@ CM on HBV in vitro: a is the experimental result of T7EI in HepG2.2.15 and HepAD38 cells after/without NIR irradiation treatment of UCNPs-Cas9@ CM; B. c is a sequencing result graph of a fragment of the sgRNA17 effect targeting gene amplified by PCR in HepG2.2.15 cells; D-F is qRT-PCR analysis UCNPs-Cas9@ CM after NIR irradiation treatment, HBV3.5kb mRNA, intracellular HBV DNA and HBV cccDNA levels in HepG2.2.15 and HepAD38 cells, and PBS treatment group as negative control; G-H is the ELISA result of the HBsAg and HBeAg levels in the supernatant of HepG2.2.15 and HepAD38 cells after treatment with NIR irradiation by UCNPs-Cas9@ CM;

FIG. 13 is a graph showing the results of in vivo biocompatibility and toxicity analysis of UCNPs-Cas9@ CM: a is the hemolytic effect of different concentrations of UCNPs-Cas9@ CM on mouse erythrocytes, and the concentration of UCNPs-Cas9@ CM is marked in the form of Cas9 concentration (uM); b is the blood normal and blood biochemical levels of the mice 14 days after treatment with UCNPs-Cas9@ CM and PBS, respectively; c is the change in body weight of mice treated with UCNPs-Cas9@ CM and PBS for 14 days, respectively; d is lanthanide ion (Y) in feces and urine within 7 days after intravenous UCNPs-Cas9@ CM injection ³⁺ ) Content (c); e is H in the major organs of mice treated with UCNPs-Cas9@ CM and PBS for 14 days&E staining, scale bar: 60um;

FIG. 14 shows the gene editing effect of UCNPs-Cas9@ CM on HBV in vivo: a is the HBV DNA load of serum in the serum of an HBV-Tg mouse detected by adopting qRT-PCR; B-C is the level of HBsAg and HBeAg in the serum of the mouse measured by a chemiluminescence immunoassay method; d is a representative image of HBsAg and HBeAg expression levels in the liver of HBV-Tg mice after 14 days of treatment with UCNPs-Cas9@ CM (immunohistochemical staining, measuring bar: 30 um);

FIG. 15 is the off-target effect of UCNPs-Cas9@ CM in HepG2.2.15 cells in vitro (A-B) and mouse liver in vivo (C-D): 15A, 15B are shown in light gray with a list of off-target sequences with mismatched sites and mismatched bases.

Detailed Description

The technical solution of the present invention is further illustrated by the following examples.

Example 1 design to synthesize sgRNA against targeted HBV genome

From the published literature, 3 HBV-specific sgrnas (RT, 6 and 17) were selected, and the sgrnas were transcribed ex vivo from the DNA template using T7RNA polymerase. sgRNA was synthesized using hisscribe t7 rapid high-yield RNA synthesis kit (NEB, # E2040S), and then the template DNA was removed using dnase i (Takara, # 2270A) at 37 ℃ for 15 minutes. Finally, the resulting RNA was purified using the HiPureRNA kit (magenta, # R2144). The sgrnas obtained were measured using a Nano-500 spectrophotometer (auston) and stored at-80 ℃ until further use. The DNA sequences corresponding to sgRNA RT, sgRNA 6 and sgRNA17 are shown in SEQ ID NO. 1-3. The in vitro transcribed DNA template sequences are shown in Table 1, SEQ ID NO. 4-6.

TABLE 1 DNA template sequences

Note: the underlined part is the target gene sequence, the 5 'end is the T7 promoter, and the 3' end is sgRNA Scaffold.

Example 2 construction and characterization of Cas9/sgRNA System

1. The synthesized sgRNA was incubated with Cas9 protein with Nuclear Localization Signal (NLS) and GFP expression (EGFP-Cas 9-NLS, purchased from tokyo jinsry technologies) at 37 ℃ for 10min in a molar mass ratio of 1 to form a complex Cas9/sgRNA.

Activity detection of in vitro cutting HBV cccDNA by Cas9/sgRNA

2.1 PCR amplifying target segment containing sgRNA target gene sequence by using PCH9/3091 plasmid as template. The primer sequences used are shown in Table 2, SEQ ID NO. 7-12.

TABLE 2 primer sequences

2.2 sgRNA, cas9 and 10 Xreaction buffer (200mM HEPES,1M NaCl,50mM MgCl) ₂ 1mM EDTA, pH 6.5) the reaction system was prepared as shown in Table 3. Then 5ul of the purified full-length PCR Fragment (FL) with sgRNA target sequence was added and reacted at 37 ℃ for 2h. And finally detecting the enzyme digestion efficiency by using 1% agarose gel electrophoresis.

TABLE 3 reaction System for in vitro cleavage of HBV cccDNA by Cas9/sgRNA

The results of the experiment are shown in FIG. 1. The results show that: cas9/sgRNA17 has higher cleavage efficiency.

Example 3 preparation and characterization of nanocarrier UCNPs-Cas9

1. Preparation of calcium oleate precursor

6.09g of sodium oleate (NaOA) was dissolved in 20mL of absolute ethanol and mixed with 10mL of a calcium nitrate solution (10 mM) in a round-bottom flask. Then, 35ml of cyclohexane was added, and the mixed solution was heated under reflux at 80 ℃ for 4 hours. After completion, the mixture was cooled to room temperature and transferred to a separatory funnel. The upper layer was collected and washed with water. And finally, removing residual cyclohexane through rotary evaporation to obtain a white calcium oleate precursor.

2.Ca ²⁺ Doped NaYF ₄ :Yb ³⁺ /Tm ³⁺ (NaYF ₄ Yb/Tm/Ca) synthesis

0.5mM of naked nucleus precursor LnCl ₃ The aqueous solution was added to a three-necked flask and heated to 100 ℃ until solid chloride, lnCl, was formed ₃ The aqueous solution comprises 0.1205g of yttrium chloride (YCl) ₃ ·6H ₂ O), 0.03874g ytterbium chloride (YbCl) ₃ ·6H ₂ O) and 0.000958g of thulium chloride (TmCl) ₃ ·6H ₂ O). Then, 30mg of calcium oleate precursor, 3.75mL of Oleic Acid (OA), and 7.5mL of Octadecene (ODE) were added to give mixture 1. Mixture 1 was heated to 150 ℃ and stirred for 1.5h to form a homogeneous solution, which was then cooled to room temperature. Then, 0.05g of NaOH and ammonium fluoride (NH) were added ₄ F) 5mL of 0.074g methanol solution was added dropwise to the reaction, stirred at 50 ℃ for 30min, the solution was heated to 100 ℃ for 30min, and methanol was evaporated. The solution was then heated at 300 ℃ for 1.5h and then cooled to room temperature. All experiments were performed under argon. Finally, 2 volumes of absolute ethanol were added for precipitation, centrifuged at 12000rpm for 5min, washed several times with ethanol/cyclohexane (1 v/v), and 4mL of cyclohexane was dispersed to give Ca ²⁺ Doped NaYF ₄ :Yb ³⁺ /Tm ³⁺ And (3) solution.

3.NaYF ₄ :Yb/Tm/Ca@NaYF ₄ Synthesis of Yb/Nd core-shell type up-conversion nano material (UCNPs)

0.4mM of core-shell precursor LnCl ₃ Aqueous solution (containing 0.04852g YCl) ₃ ·6H ₂ O、0.07172g NdCl ₃ ·6H ₂ O、0.01549g YbCl ₃ ·6H ₂ O) was added to a three-necked flask and heated to 100 ℃ until solid chloride was formed. Then, OA 7.5mL and ODE 15mL were added to obtain mixture 2. Mixture 2 was heated to 150 ℃ for 1.5h, stirred to form a homogeneous solution, and then cooled to room temperature. Then, 4mL of pre-prepared Ca was added ²⁺ Doped NaYF ₄ :Yb ³⁺ /Tm ³⁺ Solution and containing NaOH 0.04g and NH ₄ F0.0592 g of 5mL of methanol solution are added dropwise to the reaction and stirred vigorously at 50 ℃ for 30min. The solution was heated to 100 ℃ for 30min and cyclohexane and methanol were evaporated. The solution was then heated at 300 ℃ for 1.5h and then cooled to room temperature. All experiments were performed under argon. Finally, 2 times volume of absolute ethyl alcohol is added for precipitation, centrifugation is carried out at 12000rpm for 5min, and ethanol/cyclohexane is usedAlkane (1. Finally, drying at 60 ℃ for 12h to obtain UCNPs.

Preparation of UCNPs-Cas9

A mixture of polyacrylic acid (PAA) 0.5g and diethylene glycol 10mL was heated to 110 ℃ for 1 hour. 2mL of cyclohexane containing 30mg of UCNPs was added dropwise to the reaction, and stirred at 110 ℃ for 1h. The solution was then heated to 240 ℃ for 1 hour and then cooled to room temperature. Dilute hydrochloric acid (pH 4-5) was added, followed by vortexing and centrifugation at 12000rpm for 5min. Carboxyl-functionalized UCNPs (UCNPs-PAA) are obtained.

Avidin was bound to the surface of UCNPs-PAA using 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC)/N-hydroxysuccinimide (NHS) covalent chemistry. The specific method comprises the following steps: 1mg of UNCPs-PAA, 50mg of EDC and 25mg of NHS were dispersed in 1mL of 2- (n-morpholine) glycolic acid buffer (MES, 0.1M, pH 5.5) and incubated at 37 ℃ for 1h with gentle rotation. Then, 1mg of avidin was immediately added and the pH was adjusted to 8. The reaction solution was kept incubated overnight at 4 ℃. The obtained UCNPs-Avidin was washed 3 times. 1mg of the obtained UCNPs-Avidin (calculated as Avidin) was resuspended in 600ul PBS, then mixed with 4mg of PC Biotin-NHS Ester (resuspended in 400ul PBS, purchased from sigma) solution, rotated for 2h at 37 ℃, and nanoparticles UNCPs-Avidin/PCB were collected and washed 3 times with PBS; simultaneously mixing NLS-Cas9-EGFP protein and sgRNA in PBS for 10min (37 ℃) to form CRISPR/Cas9 (or called Cas9 RNP and Cas 9/sgRNA); then, the CRISPR/Cas9 and the obtained nanoparticle UNCPs-Avidin/PCB are rotated at 4 ℃ overnight for 12h to obtain UCNPs-Cas9. The mixture was then dialyzed using an ultrafiltration spin column (vivaspin 500, 300kd) and unbound CRISPR/Cas9 was discarded. The recovered UCNPs-Cas9 was resuspended in500 ul PBS and stored briefly at 4 ℃.

The above products were subjected to spectral analysis and characterization of morphology, size, charge, etc. by uv-vis spectrophotometer, negative staining Electron Microscope, transmission Electron Microscope (TEM), scanning Electron Microscope (SEM), dynamic Light Scattering (DLS), etc.

The experimental results are shown in fig. 2, 3, and 5D. The results show that: calcium doped NaYF ₄ :Yb ³⁺ /Tm ³⁺ Particles with an average diameter of 41nm (FIGS. 2A-B, FIG. 3A). NaYF ₄ :Yb/Tm/Ca@NaYF ₄ Yb/Nd is a core-shell structure with an average diameter of 64nm (FIGS. 2C-D, FIG. 3B). HRTEM and SAED showed that the lattice fringe spacing of UCNPs was 0.52nm, corresponding to a typical (100) plane of hexagonal structure (fig. 2E-F). XRD showed that UCNPs have hexagonal phase because diffraction peaks can be well indexed to hexagonal beta-NaYF ₄ (JCPDS card No. 28-1192) (FIG. 3C). Transmission electron microscopy elemental analysis plots show the detailed UCNPs composite structure (fig. 3D). These results indicate that NaYF ₄ :Yb/Tm/Ca@NaYF ₄ Yb/Nd core-shell UCNPs have been successfully synthesized.

Uv-vis spectroscopy, FITR spectroscopy (fig. 3E-F) and zeta potential (fig. 5D) measurements demonstrate the surface modification of water-soluble carboxyl functionalized UCNPs-PAA. As shown in fig. 3E-F, the major uv absorbance maxima of avidin blue-shifted after UCNPs-PAA modification. FTIR at 1737cm ^-1 And 1558cm ^-1 The visible absorption peaks at (A) indicate tensile vibration of the C = O and COO-groups, indicating effective modification of the carboxyl groups, and at 1643cm ^-1 N-H bending vibration was also observed at the (amide) site. FIG. 5D shows that the zeta potential of UCNPs is about-46.46 mV after PAA modification; after coupling of avidin, the zeta potential is approximately 22.02mV; these results indicate that the synthesis of UCNPs-Avidin was successful. The fluorescence intensity of UCNPs-PAA was not reduced compared to UCNPs. In addition, the ultraviolet-visible absorption light (UV-vis) absorption of the PCB partially overlaps with the emission spectrum of UCNPs (fig. 3G).

Example 3 preparation and characterization of biomimetic NanoPrepration UCNPs-Cas9@ CM

1. Extraction of cell membrane fragments

HepG2.2.15 and HepAD38 cells were selected as hepatocyte models and cell membrane debris (CMs) were collected. The following procedures were carried out according to the cell membrane protein and cytoplasmic protein extraction kit (Biyuntian, product code P0033): first, cells were scraped from the culture dish using a cell scraper, collected in an EP tube, the cell pellet gently resuspended with an appropriate amount of ice-cooled PBS, the cells counted, and then centrifuged at 600g for 5 minutes at 4 ℃ to pellet the cells. 1ml of membrane protein extraction reagent A added with PMSF before use is added into 2000-5000 ten thousand cells, the cells are gently and fully suspended, and the mixture is placed in ice bath for 10-15 minutes. The cell suspension was transferred to a glass homogenizer pre-cooled in an ice bath and homogenized at about 30-50 deg.f. The supernatant was then carefully collected into a new EP tube by centrifugation at 700g for 10 minutes at 4 ℃. Finally centrifugation was carried out at 14000g for 30 minutes at 4 ℃ to pellet cell membrane debris. The supernatant was discarded, and the precipitate was freeze-dried and stored at-80 ℃. And the extracted cell membrane fragments were characterized, and the results are shown in fig. 4.

2. Preparation of UCNPs-Cas9@ CM

And (3) sequentially extruding the mixed solution of the UCNPs-Cas9 and the cell membrane fragments by using polycarbonate filter membranes with the pore diameters of 400nm and 200 nm. After repeated 21 cycles of squeezing, the cell membrane remaining in the supernatant was removed by centrifugation, and the obtained UCNPs-Cas9@ CM was placed in PBS buffer again and stored at 4 ℃.

The characterization of the cell membrane was performed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). First, protein electrophoresis was performed according to standard protocols. Then, staining with Coomassie brilliant blue, and destaining with glacial acetic acid overnight, protein bands were visible. For Western blot analysis, cell membrane proteins were electrophoretically blotted onto nitrocellulose membranes. Detection was performed using the indicated antibodies, including GAPDH (Proteintetech, # 60004-1-Ig), na +/K + -ATPase (Proteintetech, # 14418-1-AP), histone H3 (bimake, # A5737), cytochrome c (bimake, # A5184), pan-cadherin (bimake, # A5614), and secondary antibody goat anti-rabbit IgG (Proteintetech, # SA 000001-2). The purity of the cell membrane extraction was analyzed by observing the signal with ECL reagent (Millipore).

The release of Cas9 RNP was studied using an NIR laser (Vinca laser technology, inc., china) at a wavelength of 808 nm. The UV absorption spectrum was measured with a Nano-500 spectrophotometer.

The results of the experiment are shown in FIGS. 4-6. From the results, it was found that the extracted cell membrane had no nucleus, mitochondria, and other organelles, indicating that the cell membrane had a high purity (FIG. 4A), and the particle size and zeta potential of the cell membrane were measured by Dynamic Light Scattering (DLS) (FIGS. 4B-C); transmission electron microscopy images showed that UCNPs-Cas9@ CM had a core-shell structure with an outer shell thickness of about 10nm (FIG. 5A), with scanning electronThe mirror detects its surface morphology (fig. 5B); particle size and zeta potential were measured by Dynamic Light Scattering (DLS) (fig. 5C-D); figure 5E shows that the membrane proteins in the source cell membrane are widely retained and there are bands corresponding to monomeric avidin (17 kDa) and Cas9 (160 kDa), indicating that avidin and Cas9 are successfully embedded in biomimetic nanopreparations; in 10ul UCNPs-Cas9@ CM solution, the Cas9 protein band is about 1/3 of that of the pure Cas9 protein band (36 pmol); from this, each 10ul of UCNPs-Cas9@ CM solution contained about 12pmol of Cas9; FIG. 5F shows the results for pan cadherin and Na ⁺ /K ⁺ ATPase (membrane marker) retention was good, but not GAPDH (cytoplasmic marker), cytochrome C (mitochondrial marker) and histone H3 (nuclear marker), indicating that the membrane protein has selective retention; therefore, cell membrane fragments are successfully coated on the UCNPs-Cas9 nano-particles; from FIG. 5G, UCNPs-Cas9@ CM also showed good stability, with the hydrodynamic diameter change almost constant over 7 days; FIG. 5H shows that absorbance at 280nm increases with increasing NIR exposure time, whereas the NIR-unexposed group does not have this trend; CLSM in FIG. 6 shows that the location of GFP-tagged Cas9 is located in the cytoplasm after 3h of co-culture of cells with UCNPs-Cas9@ CM; at 6 hours under NIR irradiation, GFP fluorescence was visible in the nucleus by UCNPs-Cas9@ CM, whereas GFP fluorescence of the control group (without NIR) appeared only in the cytoplasm (FIG. 5I); therefore, the nano-preparation of the invention has the potential of remotely controlling the time and space of CRISPR/Cas9 release.

Example 4 study of in vitro immune escape and isotype targeting of UCNPs-Cas9@ CM

UCNPs-Cas9 is used as a control to research the anti-phagocytosis capacity of UCNPs-Cas9@ CM on mouse macrophage RAW 264.7. RAW264.7 (mouse macrophage) cells were seeded in 24-well culture plates, on which cell-climbing sheets were spread. Cells were co-incubated with GFP-labeled UCNPs-Cas9 and UCNPs-Cas9@ CM in DMEM medium. Cells were then fixed with 4% paraformaldehyde for 20min, mounted with DAPI-containing antifluorescent quenchers, and visualized by confocal laser scanning microscopy (CLSM, leica tcssp8, germany). In addition, the level of Cas9-GFP accumulation in cells was analyzed using flow cytometry (beckmann coulter, usa). The results are shown in FIGS. 7A and 8.

From the results, it can be seen that: fluorescence shown in the cells showed that RAW264.7 cells internalized a large amount of UCNPs-Cas9 after co-culture for 6 h. In contrast, RAW264.7 cells co-cultured with UCNPs-Cas9@ CM exhibited very weak fluorescent signals. Furthermore, flow cytometry was used to assess its internalization efficiency, consistent with the results observed with CLSM. These results indicate that the cell membrane-coated particles are effective in inhibiting the uptake behavior of macrophages.

In order to verify the homotypic targeting capability of UCNPs-Cas9@ CM, the invention evaluates the UCNPs-Cas9 nano-carrier (named as UCNPs-Cas9@ CM) coated by HepG2.2.15 cell membranes in HepG2.2.15, 293T (human embryonic kidney cells) and HeLa (human cervical cancer cells) _2.2.15 ) Was evaluated for cellular internalization. The results show that: UCNPs-Cas9@ CM _2.2.15 Preferentially accumulated in HepG2.2.15 cells and showed higher fluorescence intensity. In contrast, the fluorescence intensity of the heterotypic cell group was much weaker (FIG. 7B). The Mean Fluorescence Intensity (MFI) of each cell was then determined by flow cytometry. The data show that the MFI content in hepg2.2.15 cells was about 3.5-4.1 fold compared to other cells (fig. 7B). In addition, the internalization rate of the cells was faster over time (fig. 9). These results indicate that UCNPs-Cas9@ CM has highly targeted specificity for the source cell.

Example 5 in vitro therapeutic Effect of UCNPs-Cas9@ CM

1. Cell viability assay

HepG2.2.15 and HepAD38 cells were cultured in 96-well plates for 12 hours and then treated with different concentrations of UCNPs-Cas9@ CM, NIR power and irradiation time. PBS was the negative control. Then, 10% CCK-8 solution was added to each well and incubated for 4h. By measuring OD ₄₅₀ Values to assess cell viability.

1.1 the effect of different NIR power and irradiation time on cell viability was examined before UCNPs-Cas9@ CM treatment of cells. The results are shown in FIG. 10. From the results, it can be seen that: different NIR power (fixed irradiation time of 20 minutes) and different illumination time (fixed irradiation power of 2W/cm) ² ) Viability was not affected on hepg2.2.15 and HepAD38 cells (fig. 10A, 10B).

1.2 addition of UAfter CNPs-Cas9@ CM treatment of cells, the effect of UCNPs-Cas9@ CM (without NIR) at different concentrations on cell viability was studied; UCNPs-Cas9@ CM of different concentrations at fixed laser power and illumination time (2W/cm) ² 20 min) on cell viability. The results are shown in FIG. 10. From the results, it can be seen that: under the above conditions, there was no significant effect on cell viability (fig. 10C, 10D).

1.3 temperature changes were observed by irradiation of UCNPs-Cas9@ CM solution. The temperature distribution results are shown in fig. 11. From the results, it can be seen that: stopping 2min intervals after 1min irradiation in the experimental process, and the UCNPs-Cas9@ CM can not cause damage to cells due to overhigh heat production.

2. in vitro Effect detection of UCNPs-Cas9@ CM

HepG2.2.15 and HepAD38 cells were cultured in a 96-well plate for 12 hours, then cultured with UCNPs-Cas9@ CM (Cas 9 concentration 0.06 uM) for 4 hours, and irradiated with NIR at a power of 2W/cm ² Irradiation time is 20 minutes, irradiation is 1 minute, interval is 2 minutes, and the cycle is repeated. PBS was the negative control.

2.1 detection of the efficiency of the UCNPs-Cas9@ CM cleavage of HBV cccDNA

After further 3 days of culture, genomic DNA was isolated from HepAD38 cells and HepG2.2.15 cells, respectively, using the AllPureDNA/RNA kit (magenta, china). The sgRNA17 fragment acting on the target gene was amplified by PCR, and the DNA amplified by PCR was digested with T7EI, which was analyzed by agarose gel electrophoresis, and the results are shown in FIG. 12A. Meanwhile, PCR amplification products of HepG2.2.15 cells were cloned into pTOPO-Blunt vector (Chinese Mei5, china) and sequenced, with the results shown in FIGS. 12B and C.

From the results, UCNPs-Cas9@ CM successfully cleaved the gene HBV cccDNA (FIG. 12A), with high cleavage efficiency; the PCR amplicons of the UCNPs-Cas9@ CM + NIR treatment group showed a DNA sequencing doublet confirming that the HBV cccDNA site was mutated (FIGS. 12B-C).

2.2.detection of HBV3.5kbRNA, HBVDNA and HBVcccDNA

HBV3.5kbRNA, intracellular HBV DNA and HBV cccDNA levels are detected by an RT-qPCR method.

HBV DNA was isolated from HepG2.2.15 and HepAD38 cells. Utensil for cleaning buttockThe method comprises the following steps: first, cells were lysed in 0.5ml core lysis buffer (10 mM Tris-HCl pH8.0,1mM EDTA, 1% NP-40, 2% sucrose) at 37 ℃ for 15 minutes. After centrifugation, 4ul DNaseI (5 IU/mL) and 5ul MgCl were used ₂ (1M) treatment at 37 ℃ for 4 hours. Further centrifugation, 200ul 35% PEG8000 was incubated with the supernatant in an ice bath for 1 hour to precipitate HBV core capsids. Then treated with proteinase K at 45 ℃ for 12h. HBV DNA was purified by phenol/chloroform (1. The extracted HBV DNA uses a pair of specific primers (HBV-F) ₂₁₅₀ And HBV-R ₂₃₀₀ ) Quantification was performed by qPCR method.

HBVcccDNA is isolated by using a modified Hirt method. The specific method comprises the following steps: the cells were lysed in 0.5ml of SDS lysis buffer (50 mM Tris-HCl, pH8.0,10mM EDTA,150mM sodium chloride, 1% SDS) at 37 ℃ for 15 minutes, and then the lysates were incubated with 125ul KCl (2.5M) overnight at 4 ℃. After centrifugation, the supernatant was extracted with phenol/chloroform (1), precipitated with isopropanol, washed with ethanol, and resuspended in TE buffer. HBV cccDNA is detected by adopting taqman probe qRT-PCR.

The expression of HBV pgRNA was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The specific method comprises the following steps: cells were seeded overnight in 24-well plates and then treated with PBS and UCNPs-Cas9@ CM (Cas 9 concentration 0.06 uM), respectively. After 3h incubation, an NIR laser (2W/cm) at 808nm was used ² ) Irradiation was carried out for 20min (1 min after 2 min). After 72h, RNA was extracted from the cells using the AllPureDNA/RNA kit, followed by reverse transcription using the cDNA Synthesis kit (Takara). The qRT-PCR was performed using SYBR Green qPCR Master Mix (Bimake, china) using ABI7500 real-time PCR System (applied biosystems, USA). Normalizing transcription of the target gene to GAPDH and using 2 ^-ΔΔCT The results were analyzed by the method. The primer sequences used are shown in Table 4, SEQ ID NO. 13-21.

TABLE 4 primer sequences

2.3 detection of HBsAg and HBeAg

The levels of HBsAg and HBeAg in the cell culture supernatants of HepG2.2.15 and HepAD38 were determined using an ELISA kit (Kowa Biotechnology, shanghai, china).

The results are shown in FIGS. 12D-H. From the results, it was found that HBV3.5kbRNA, intracellular HBVDNA, HBVcccDNA and HBV viral antigen were significantly decreased after the cells were incubated with UCNPs-Cas9@ CM, as compared with the negative control group. The UCNPs-Cas9@ CM can clear HBV cccDNA in cells and has obvious antiviral effect. The detection shows that the effect of the polypeptide is obviously superior to that of UCNPs-Cas9, and CMs has no treatment effect on HBV.

Example 6 antiviral Effect of UCNPs-Cas9@ CM in vivo

HBV-Tg mice with higher HBVDNA, HBsAg level and HBeAg level were selected and randomly divided into two groups of 3 mice each. Mice were then injected continuously for 3 days with PBS or UCNPs-Cas9@ CM at a concentration of 2mg/kg (Cas 9 concentration) per UCNPs-Cas9@ CM injection, PBS groups were injected with equal volume of UCNPs-Cas9@ CM solution, and then the liver was irradiated with NIR laser for 30min (1 min as indicated, 2min stopped, cycle back and forth). On day 14 after injection, blood was collected from the eyeball of the mouse, then the mouse was sacrificed by dislocation of cervical vertebrae, and the liver and other major organs of the mouse were dissected.

Safety assessment of UCNPs-Cas9@ CM

During treatment, body weight was monitored every 2 days after injection. To further analyze the excretion of UCNPs-Cas9@ CM in vivo, ICP-MS was used to determine the lanthanide ion (Y) excreted by feces and kidneys ³⁺ ) And (4) concentration. Blood routine and blood biochemical tests were performed on mouse blood and histological analysis of mouse major organs were performed to evaluate toxicity based on UCNPs-cas9@ cm, respectively. The results of the experiment are shown in FIG. 13.

From the results, it can be seen that: after co-incubation with mouse blood cells with different concentrations of UCNPs-Cas9@ CM, no hemolysis was detected (FIG. 13A). In addition, there was no significant change in blood analysis, both routine and biochemical (fig. 13B) and body weight (fig. 13C) in the mice in both groups. Y in urine and feces of mice ³⁺ The levels gradually decreased over time, suggesting that UCNPs could be efficiently excreted in vivo (fig. 13D). Two groups of mice masterHE staining of the organs showed that no significant differences were found after treatment (fig. 13E). These results again confirmed that UCNPs-Cas9@ CM has good biocompatibility.

In vivo antiviral Effect of UCNPs-Cas9@ CM

HBV DNA, HBsAg and HBeAg in the blood of the mouse and HBsAg and HBe in the liver cells are respectively detected to evaluate the in vivo antiviral effect of UCNPs-Cas9@ CM. The results of the experiment are shown in FIG. 14.

After UCNPs-Cas9@ CM and NIR light treatment, the secretion levels of HBV DNA, HBsAg and HBeAg in mouse serum can be obviously reduced to different degrees (FIG. 14A-C), and the HBsAg and HBe levels of mouse liver cells are also reduced through immunohistochemical detection (FIG. 14D). This is consistent with in vitro experimental results.

Example 7 off-target Effect detection of UCNPs-Cas9@ CM

After treating HepG2.2.15 cells and mice with UCNPs-Cas9@ CM, respectively, according to the methods of examples 5-2 and 6 above, genomic DNA was isolated from HepG2.2.15 cells and mouse liver tissues, respectively, using the AllPureDNA/RNA kit (magenta, china). Potential off-target sites of the sgRNA17 were predicted by bioinformatics software, and off-target genomic sites were amplified using specific primers whose sequences are shown in Table 5 (h: human, m: mouse), SEQ ID nos. 22 to 47. The product was subjected to T7EI detection, and the results are shown in FIG. 15. 15A and B are in vitro off-target effects of UCNPs-Cas9@ CM: the light gray in A is the list of off-target sequences of mismatched sites and mismatched bases; b, detecting by adopting T7EI, and carrying out agarose gel electrophoresis on the obtained product to determine potential off-target effects in different target sequences (Chr #) of the chromosome; 15C, D are the in vivo off-target effect of UCNPs-Cas9@ CM: light gray in C is the list of off-target sequences of mismatched sites and mismatched bases; d is detection by T7EI, and agarose gel electrophoresis is carried out on the obtained product to determine potential off-target effects in different target sequences (Chr #) of chromosomes.

TABLE 5 specific primers

The above results indicate that the off-target effect of UCNPs-Cas9@ CM is negligible. sgRNA17 can specifically recognize and skillfully edit HBV.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention/utility model and not for limiting, although the present invention/utility model is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention/utility model can be modified or substituted equally without departing from the spirit and scope of the technical solutions of the present invention/utility model, and they should be covered within the scope of the claims of the present invention/utility model.

SEQUENCE LISTING

<110> Min Hospital in Rongchang district of Chongqing City

<120> near-infrared response bionic nano preparation and preparation method and application thereof

<130> CQ202110235

<160> 47

<170> PatentIn version 3.5

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Claims

1. A near-infrared response bionic nano preparation is characterized in that: the liver cell model based on the CRISPR/Cas9 gene is characterized by comprising a cell membrane of a liver cell model and a nano-carrier wrapped in the cell membrane, wherein the nano-carrier comprises near-infrared response up-conversion nanoparticles and CRISPR/Cas9, avidin proteins are modified on the surfaces of the up-conversion nanoparticles, and biotin-modified photolysis molecules are connected between the avidin proteins and the CRISPR/Cas9;

the near-infrared response up-conversion nano particles are of a core-shell structure, ca ²⁺ Doped NaYF ₄ :Yb ³⁺ /Tm ³⁺ Is a nucleus, naYF ₄ Yb/Nd is the shell; the biotin-modified photolytic molecule is PC biotin-nhs ester.

2. The near-infrared responsive biomimetic nano-formulation of claim 1, wherein: the hepatocyte model is one of an HBV replication hepatocyte model, an HBV infection hepatocyte model and a primary hepatocyte model.

3. The near-infrared responsive biomimetic nano-formulation of claim 1, wherein: the CRISPR/Cas9 comprises a Cas9 protein and sgRNAs, and DNA sequences corresponding to the sgRNAs are shown in SEQ ID NO. 1-3.

4. A preparation method of a near-infrared response bionic nano preparation is characterized by comprising the following steps: the method comprises the following steps:

2) Preparing a nano-carrier UCNPs-Cas9: synthesizing up-conversion nanoparticles, carrying out hydrophilic modification and Avidin surface modification on the up-conversion nanoparticles to obtain UNCPs-Avidin, then carrying out a connection reaction with biotin-modified photolysis molecules to form nano-particle UNCPs-Avidin/PCB, and co-incubating the nano-particle UNCPs-Avidin/PCB with the CRISPR/Cas9 prepared in the step 1) to obtain a nano-carrier UCNPs-Cas9;

3) Preparing a bionic nano preparation UCNPs-Cas9@ CM: extracting HBV to copy the cell membrane of the liver cancer cell model, mixing the cell membrane with UCNPs-Cas9 prepared in the step 2), and repeatedly extruding through a polycarbonate filter membrane to obtain UCNPs-Cas9@ CM.

5. The method for preparing a near-infrared response biomimetic nano-formulation according to claim 4, characterized in that: in step 1), the molar mass ratio of sgRNA to Cas9 protein is 1 to 1, the co-incubation time is 5 to 15min, and the temperature is 37 ℃.

6. The method for preparing a biomimetic nano-formulation with near-infrared response according to claim 4, wherein the method comprises the following steps: in step 2), the surface of the up-conversion nanoparticle comprises an oleic acid ligand, the modifier used in the hydrophilic modification is polyacrylic acid, and the method for surface modification of avidin comprises the following steps: linking avidin on the surface of the upconversion nanoparticle by using EDC/NHS; the temperature of the connection reaction is 37 ℃ and the time is 2h; the mass ratio of Avidin to biotin-modified photolytic molecules in UNCPs-Avidin is 1; the temperature of the co-incubation is 4 ℃ and the time is 8-12h.

7. The method for preparing a near-infrared response biomimetic nano-formulation according to claim 4, characterized in that: in the step 3), the aperture of the polycarbonate filter membrane is 200nm or 400nm.

8. Use of a near-infrared responsive biomimetic nano-formulation according to any of claims 1-3 in the preparation of a medicament for the treatment of hepatitis.