CN120242074A - A supramolecular nanogene carrier and its preparation method and application - Google Patents

Abstract

The invention relates to a supermolecule nano gene vector, a preparation method and application thereof. The supermolecule nano-carrier comprises beta-cyclodextrin grafted polyethyleneimine, 8-adamantane grafted polyamide-amine dendrimer, adamantane grafted polyethylene glycol and adamantane grafted polyethylene glycol modified by using cell penetrating peptide (TAT). The prescription of the nano material monomer is screened by self-assembled load CRISPR/Cas9 system, and then the research on cells and retina organoids proves that the nano material monomer has good transfection efficiency and RS1 protein expression quantity. The supermolecule nanoparticle loaded gene editing system has good potential for treating X-linked retinal cleavage. In addition, the supramolecular nanoparticle gene vector has high biological safety, high transfection efficiency, simple and easily obtained preparation mode and good potential for clinical application in the future.

Description

Supermolecule nano gene vector and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a supermolecule nano gene vector, a preparation method and application thereof.

Background

The gene editing treatment of diseases mainly aims at treating diseases by targeted knocking out or knocking in exogenous genes so as to interfere the expression of specific genes in target cells. Gene therapy is widely used, involving cancer, hemophilia, retinal disease, and the like. However, the key point of gene medicine is that naked nucleic acid is easy to be degraded by nuclease, exogenous gene is easy to be identified and cleared by immune system, and strong electrostatic repulsion exists between DNA or RNA and phospholipid bilayer with negative charge, so that cell uptake of gene is quite challenging. Therefore, the development of safe and effective gene delivery vectors to improve targeting delivery efficiency and transfection efficiency is not only the key to successful gene therapy, but also the current key technical problem.

Currently, two broad classes of viral vectors and non-viral vectors have been developed. The problems of high efficiency of the virus vector, potential biosafety risk, high production cost and the like greatly reduce the clinical application. The non-viral vector mainly has lipid nano-particles (LNP), and has the unique advantages of simple preparation, low immunogenicity, difficult integration with host genes and the like, and becomes an important candidate for nucleic acid drug delivery. However, LNP systems also suffer from drawbacks such as toxicity, poor biodistribution, and inefficient transfection. And LNP delivery is primarily limited to the liver, with insufficient targeting capability for organs other than the liver. The rapid development of nanotechnology provides a new idea for the solution of gene delivery. The advantages of various nano particles are reasonably utilized to design a nano carrier structure, so that a plurality of excellent gene medicine delivery platforms are combined together, the gene medicine delivery efficiency is improved, the treatment effect of the gene medicine is optimized, and the nano carrier structure has great potential for future gene-based treatment. Therefore, a delivery system with high biological safety, low toxicity, high targeting and high transfection efficiency is designed, and has important application potential in the fields of gene therapy, gene editing, gene vaccine and the like.

Eyes are very important organs of a human body, vision is the most important sense of the human body, and vision diseases can deeply affect the quality of life of the human body. X-linked retinal detachment (X-Linkedretinoschisis, XLRS) is a clinically common genetic disorder. XLRS is cleaved from retina protein 1

The RS1 gene is responsible for encoding a 224 amino acid protein, called RS1 protein, caused by (Retinoschisin 1, RS 1) genetic variation. The interaction between the RS1 protein and cells is related to cell adhesion, so that the retina cell interlayer structure can be maintained, normal connection of synapses can be ensured, and the transduction of visual signals can be promoted. There is no clear and effective treatment for this disease to date. Currently, gene therapy approaches are widely focused on by researchers, but the use of viral vector-loaded genes has been shown to produce inflammatory responses in clinical trials. Therefore, the development of supramolecular nanodelivery systems, around the effectiveness and safety of the treatment, has positive significance for the treatment of XLRS.

Disclosure of Invention

Aiming at the clinical requirement of treating X-linked retinal cleavage, the invention aims to provide a supermolecule nano gene vector, and a preparation method and application thereof. The supermolecule nano gene carrier provided by the invention consists of four parts, including beta-cyclodextrin grafted polyethyleneimine (beta-CD-PEI), 8-adamantane grafted polyamide-amine dendrimer (8-Ad-PAMAM), adamantane grafted polyethylene glycol (Ad-PEG) and adamantane grafted polyethylene glycol (Ad-PEG-TAT) modified by using cell penetrating peptide (TAT). The supermolecule nano gene vector can load target genes into nano particles to form a stable and efficient gene delivery vector. By evaluating its loading rate, the highest loading rate was 92%. The prepared supermolecule nano gene vector has high transfection efficiency through the transfection of HEK293T cells. RS1 gene delivery in retinal organoids of patients with retinal cleavage was found to have good fluorescent protein expression and expression levels of RS1 protein and photoreceptor-related proteins, with the potential to cure the disease at the genetic level. The transfection reagent provided by the invention has the advantages of low toxicity, high transfection property, high targeting property and the like, can effectively overcome the problems of the existing gene delivery system, and has important application prospects in the field of realizing accurate and efficient gene knocking-in.

The invention is realized by the following technical scheme:

The first object of the present invention is to provide a supramolecular nanogene vector comprising beta-cyclodextrin grafted polyethyleneimine, 8-adamantane grafted polyamide-amine dendrimer, adamantane grafted polyethylene glycol and adamantane grafted polyethylene glycol modified with cell penetrating peptide.

In one embodiment of the invention, the 8-adamantane grafted polyamide-amine dendrimer is prepared by the following method:

The preparation method comprises the steps of dissolving PAMAM in an organic solvent to form PAMAM solution, adding 1-adamantane isocyanate, stirring at room temperature for reaction, removing the solvent, adding diethyl ether into the reaction residue to generate white precipitate, and collecting, washing and drying the white precipitate through filtration to obtain 8-adamantane grafted polyamide-amine dendrimer (white solid, 8-Ad-PAMAM).

In one embodiment of the invention, the adamantane grafted polyethylene glycol is prepared by the following method:

Dissolving 1-amantadine hydrochloride in organic solvent, sequentially adding triethylamine and mPEG-NHS, stirring at room temperature, removing solvent in vacuum, adding water to the reaction residue, centrifuging to remove unreacted 1-amantadine, filtering, and using Dialysis cartridges were dialyzed overnight and lyophilized to give adamantane grafted polyethylene glycol (white powder, ad-PEG).

In one embodiment of the invention, the adamantane grafted polyethylene glycol modified with a cell penetrating peptide is prepared by the following method:

Adding triethylamine and MAL-PEG-NHS to the organic solution of 1-amantadine hydrochloride, stirring at room temperature, removing the solvent in vacuo after the reaction, and adding PBS buffer containing CGRKKRRQRRR (TAT peptide) to the reaction residue, stirring the resultant mixture at room temperature, centrifuging, dialyzing and lyophilizing to obtain adamantane-grafted polyethylene glycol modified with cell penetrating peptide (white powder, ad-PEG-TAT).

In one embodiment of the invention, the beta-cyclodextrin grafted polyethyleneimine is prepared by the following method:

The branched polyethylenimine is dissolved in an organic solvent, 6-p-toluenesulfonyl-beta-cyclodextrin (6-OTs-beta-CD) is added for reaction, after dialysis is finished, the obtained mixture is filtered to remove unreacted 6-OTs-beta-CD (white precipitate), and the filtrate is freeze-dried overnight to obtain beta-cyclodextrin grafted polyethylenimine (white soft solid, beta-CD-PEI).

In one embodiment of the invention, the mass ratio of the 8-adamantane grafted polyamide-amine dendrimer to the beta-cyclodextrin grafted polyethyleneimine is 3.75:1 to 15:1, preferably 6:1.

In one embodiment of the invention, the mass ratio of the beta-cyclodextrin grafted polyethyleneimine to the adamantane grafted polyethyleneglycol is 1:2.5-1:10, preferably 1:4.

In one embodiment of the invention, the mass ratio of the adamantane grafted polyethylene glycol to the adamantane grafted polyethylene glycol modified with a cell penetrating peptide is from 8:1 to 12:1, preferably 10:1.

In one embodiment of the invention, the particle size of the supramolecular nanogenetic vector is 30nm-140nm.

The second object of the present invention is to provide a preparation method of the supramolecular nano-gene vector, comprising the following steps:

(1) Respectively preparing beta-cyclodextrin grafted polyethyleneimine, 8-adamantane grafted polyamide-amine dendrimer, adamantane grafted polyethylene glycol and adamantane grafted polyethylene glycol modified by using a cell penetrating peptide;

(2) Dissolving 8-adamantane grafted polyamide-amine dendrimer in an organic solvent to obtain 8-adamantane grafted polyamide-amine dendrimer solution, adding the solvent, vortex oscillating and mixing uniformly, ice-bath, adding beta-cyclodextrin grafted polyethyleneimine and adamantane grafted polyethylene glycol, vortex oscillating and mixing uniformly, ice-bath, continuously adding adamantane grafted polyethylene glycol modified by cell penetrating peptide, oscillating and mixing uniformly, and ice-bath to obtain the supermolecule nano gene vector.

In one embodiment of the invention, in step (2), the solvent is enzyme-free water and/or PBS. Preferably enzyme-free water.

A third object of the present invention is to provide a nano-gene delivery formulation comprising the supramolecular nano-gene vector and a plasmid loaded in nanoparticles.

In one embodiment of the invention, the plasmid comprises one or more of a Donor RS1/GFP plasmid, a Cas9/sgRNA plasmid, and a GFP plasmid.

In one embodiment of the invention, the nano-gene delivery preparation has the particle size of about 120nm, uniform particle size distribution and good dispersibility, and is suitable for gene drug delivery.

The fourth object of the invention is to provide the application of the nano-gene delivery preparation in preparing the medicine for treating monogenic genetic diseases.

In one embodiment of the invention, the genetic disorder includes treatment of one or more of X-linked retinal splitting, sickle-cell anemia, beta thalassemia, and duchenne muscular failure.

The invention can obviously improve the cell transfection efficiency through the Ad-PEG-TAT modified supermolecule nano particles.

Compared with the prior art, the technical scheme of the invention has the following advantages:

(1) The raw materials for synthesizing the nano material monomer are convenient and easy to obtain;

(2) The self-assembly process of the supermolecule nanometer is simple, rapid and efficient;

(3) The nano gene delivery preparation provided by the invention comprises a supermolecule nano gene vector and plasmids loaded in nanoparticles, wherein the supermolecule nano gene vector is preferably coated with the supermolecule nano gene vector with the particle size of 100nm in consideration of the fact that the supermolecule nano particles can effectively coat the plasmids and can be taken up by cells through endocytosis, so that the nano gene delivery preparation with the particle size of about 120nm is obtained, and the dispersibility is good;

(4) The encapsulation rate of the genes is high;

(5) Low cytotoxicity, high biological safety and easy metabolism and discharge;

(6) The transfection efficiency is high, and the expression level of the RS1 protein on the retina organoids of the XLRS patients is good, so that the vector is a good gene delivery vector.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,

FIG. 1 is a transmission electron microscope image of a supramolecular nanocarrier of the present invention;

FIG. 2 is an in vitro stability evaluation of a supramolecular nanocarrier of the present invention, wherein A is stability of temperature change, B is stability of time change, C is stability before and after ultrasonic treatment;

FIG. 3 is a graph showing fluorescence of transfected cells of example 4 and comparative example 1 of two SMNPs of the present invention, wherein (a) is a graph showing fluorescence of three components SMNPs after transfection and (b) is a graph showing fluorescence of four components SMNPs after transfection;

FIG. 4 shows fluorescence of supramolecular nanocarrier cell transfection in accordance with the present invention;

FIG. 5 shows the results of inhibition of the activity of HEK293T cells at various concentrations SMNPs in the present invention;

FIG. 6 shows the fluorescent protein expression of Day30 and Day39 in XLRS patients after the third gene knock-in according to the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used, unless otherwise specified, are commercially available.

The materials and reagent sources used in the present invention are as follows:

PAMAM is available from shanghai michel biochemical technology limited under the accession number 536709;

1-adamantane isocyanate is purchased from Shanghai Michael Biochemical technology Co., ltd, and the product number is M54534;

1-amantadine hydrochloride was purchased from Shanghai Michael Biochemical technologies Co., ltd, under the product number A1260;

Triethylamine is purchased from national pharmaceutical group chemical reagent company, the product number is 80134318;

mPEG-NHS is purchased from Shanghai Michlin Biochemical technologies Co., ltd, and the product number is MKL-P968648;

The dialysis cassette is purchased from the company Siemens Feier technology, USA, under the product number 2KD 66230;3.5KD 66110;20KD 66030;

MAL-PEG-NHS was purchased from ponsure organisms under the trade designation PS2-MH-5K;

TAT peptide was purchased from Kirschner Biotechnology Inc. under the designation RP20256;

branched polyethylenimine was purchased from Shanghai Biyun biotechnology company under the designation C0539 and 100mL;

6-p-toluenesulfonyl-beta-cyclodextrin was purchased from Shanghai Michlin Biochemical technologies Co.Ltd M830137-5g;

GFP plasmid was purchased from Shanghai Biyun biotechnology company under the accession number D2626;

The Donor-RS1/GFP plasmid was purchased from Shanghai Bioengineering company for customization;

The Cas9/sgRNA plasmid was synthesized by Shanghai Biotechnology Co;

HEK293T cells were purchased from shanghai bi yun biotechnology company under the accession number C6008;

RMM-2 medium was prepared from DMEM high-sugar medium (commercially available from Thermo Fisher company 11965092, U.S.), 200mL, DMEM/F12 medium (Thermo Fisher company 11320033, U.S.), 200mL, NEAA (China Biyun biotechnology C0332-100 mL), 4mL, double antibody (China Biyun biotechnology C0222), 4mL, B27 additive (Thermo Fisher company 17504044, U.S.), 8mL, glutamax solution (Thermo Fisher company A1286001, U.S.) and 4mL.

The retinal organoids ROs are obtained by differentiation culture of human induced pluripotent stem cells of healthy and X-linked retinal detachment patients;

Lipo3000 is available from Thermo Fisher, inc., U.S., under the designation L3000001;

DMEM high sugar complete culture medium is prepared according to the proportion of DMEM to FBS to double antibodies of 100:10:1, and FBS is purchased from Thermo Fisher company of America, and the product number is 10099141C.

The synthesis modes of the beta-CD-PEI, the Ad-PEG, the 8-Ad-PAMAM and the Ad-PEG-TAT used in the embodiment of the invention are as follows:

(1) Synthesis of 8-Ad-PAMAM

A methanol solution containing PAMAM (20 wt%,100mg,0.07 mmol) was added to a round bottom flask, methanol was evaporated in vacuo and dissolved in 10mL dry DMF to form a PAMAM solution. 1-adamantane isocyanate (244.6 mg,1.4 mmol) in 10mL dry DMF was added to the PAMAM solution. After the mixture was stirred at room temperature for 2 hours, the solvent was removed in vacuo. Diethyl ether (100 mL) was added to the reaction residue to give a white precipitate, which was collected by filtration, washed with diethyl ether (100 mL. Times.3) and dried to give 8-Ad-PAMAM as a white solid.

(2) Synthesis of Ad-PEG

To a solution of 1-adamantanamine hydrochloride (187.7 mg,1.0mmol,5.0 eq.) in 10mL CH ₂Cl₂, triethylamine (105.0 mg,1.0mmol,5.1 eq.) and mPEG-NHS (1.0 g,0.2mmol,1.0 eq.) were added sequentially. Stirring was carried out at room temperature for 2h, then the solvent was removed in vacuo, and water was added to the reaction residue. The solution was transferred to a centrifuge tube and centrifuged at 10000rpm for 10min to remove unreacted 1-adamantanamine. Filtered through a 0.45 μm filter and then usedThe dialysis cassette (MWCO, 2 kD) was dialyzed overnight and lyophilized to give a white powder.

(3) Synthesis of Ad-PEG-TAT

To a solution of 1-adamantanamine hydrochloride (0.94 mg, 5.0. Mu. Mol,5.0 eq.) and CH ₂Cl₂ (1.0 mL) were added triethylamine (0.6 mg, 5.0. Mu. Mol,5.0 wt.) and MAL-PEG-NHS (5.0 mg,1.0mmol,1.0 eq.). The reaction mixture was stirred at room temperature for 2h. After the reaction was completed, the solvent was removed in vacuo, and a PBS buffer (1 mL) containing CGRKKRRQRRR (TAT peptide, 7.5mg, 5.0. Mu. Mol,5.0 eq) was added to the reaction residue. The mixture was stirred at room temperature for a further 2h. The solution was transferred to a centrifuge tube and centrifuged at 10000rpm for 10min to remove unreacted 1-adamantanamine. The solution was passed through a 0.45 μm filter and then dialyzed against Slide-A-LyzerThe dialysis cassette (MWCO, 3.5 kD) was overnight and lyophilized to give a white powder Ad-PEG-TAT.

(4) Synthesis of beta-CD-PEI

Branched polyethylenimine (100 mg, 10.0. Mu. Mol) was dissolved in 100mL dimethyl sulfoxide and 6-p-toluenesulfonyl- β -cyclodextrin (6-OTs- β -CD) (1.29 g,1.0 mmol) was added. After 3 days of reaction at 70 ℃, the mixture was transferred toThe cartridge (MWCO, 10 kDa) was dialyzed against deionized water for 6 days. After the dialysis was completed, the reaction mixture was filtered to remove unreacted 6-OTs- β -CD (white precipitate), and the filtrate was freeze-dried overnight to give β -CD-PEI as a white soft solid product.

EXAMPLE 1 preparation of supramolecular nanogene vector

The embodiment provides a preparation method of a supermolecule nano-gene vector, wherein the mixing mass ratio of 8-Ad-PAMAM, beta-CD-PEI and Ad-PEG is 6:1:4, and the preparation method comprises the following steps:

Accurately weighing 7.5 mug of 8-Ad-PAMAM in a centrifuge tube, adding 10 mug LDMSO to dissolve the 8-Ad-PAMAM to form 8-Ad-PAMAM solution, then adding enzyme-free water (constructing a 50 mug system) in the centrifuge tube, and mixing uniformly by vortex oscillation. Adding 1.25 μg of beta-CD-PEI and 5 μg of Ad-PEG, mixing, stirring, ice-bathing for 30min, adding 0.5 μg of Ad-PEG-TAT, stirring, and ice-bathing for 30min. The supermolecule nano gene carrier (SMNPs) with the grain diameter of 100nm is obtained.

Example 2

The preparation method of the supermolecule nano-gene vector is similar to that of the embodiment 1, and the difference is that the mixing mass ratio of 8-Ad-PAMAM, beta-CD-PEI and Ad-PEG is 15:4:10, and the rest operations are consistent, so that the supermolecule nano-gene vector (SMNPs) with the particle size of 30nm is obtained.

Example 3

The preparation method of the supermolecule nano-gene vector is similar to that of the embodiment 1, and the difference is that the mixing mass ratio of 8-Ad-PAMAM, beta-CD-PEI and Ad-PEG is 15:1:10, and the rest operations are consistent, so that the supermolecule nano-gene vector (SMNPs) with the particle size of 140nm is obtained.

Test case

(1) Stability with temperature to understand the thermal stability of SMNPs, the resulting SMNPs real-time DLS measurements of 30nm, 100nm and 140nm particle sizes were monitored for size changes of SMNPs at different temperatures from 10 ℃ to 50 ℃. The samples were equilibrated at a given temperature for 20min.

(2) The in vitro stability test conditions were stability over time. After mixing the four molecular building blocks in the respective proportions, SMNPs were prepared with particle sizes of 30nm, 100nm and 140nm, and real-time DLS measurements were used to monitor the size change of SMNPs at different times. The SMNPs sizes were recorded every 4 minutes for 36 minutes. The results show SMNPs that the nanoparticles exhibit good stability over time.

(3) Dynamic stability-sonication was used to provide the energy barrier required to overcome SMNPs recombinations. SMNPs of 30nm, 100nm and 140nm particle sizes were prepared, the size of SMNPs was measured in solution, and monitored by DLS before and after sonication (1 MHz,50W,10 min) at room temperature for SMNPs min each. No significant size change was observed under experimental conditions, indicating that only sonication alone was unable to break down SMNP. (the results are shown in FIG. 2)

Example 4

8-Ad-PAMAM (7.5. Mu.g) was dissolved in 10. Mu.L DMSO, 1.5. Mu.g of GFP plasmid was added, enzyme-free water (50. Mu.L system was built up), mixed by shaking and ice-bath for 10min, and Ad-PEG (5.0. Mu.g) and beta-CD-PEI (1.25. Mu.g) were added, mixed by shaking and ice-bath for 30min. Continuously adding Ad-PEG-TAT (0.5 μg), shaking, mixing, and ice-bathing for 30min to obtain GFP PLASMIDSMNPs。

Comparative example 1

Dissolving 8-Ad-PAMAM (7.5 mug) in 10 mug of DMSO, adding 1.5 mug of GFP plasmid, adding enzyme-free water (constructing a 50 mug system), shaking and mixing uniformly, and ice-bathing for 10min, adding Ad-PEG (5.0 mug) and beta-CD-PEI (1.25 mug), shaking and mixing uniformly, and ice-bathing for 30min, thus obtaining the three-component supermolecule nanoparticle loaded with GFP plasmid.

Cells were seeded in 24-well plates at a cell density of 5×10 ⁴ cells/well, old medium was discarded after 18h, both SMNPs obtained in example 4 and comparative example 1 were added to the well plates (3 groups per SMNPs, 6 wells total) and 1 mL/well of serum-containing medium was replenished. After 48 hours, the result is shown in figure 3 by using a confocal fluorescence microscope, and the result shows that the fluorescence efficiency of cells is higher after the Ad-PEG-TAT component is added, and the transfection efficiency is improved from 55% to 75%. Thus, the Ad-PEG-TAT component is essential in SMNPs for gene delivery.

EXAMPLE 5Donor RS1/GFPSMNPs and Cas 9/sgRNASMNPs co-delivery transfection HEK293T cells:

The synthesized four nano material monomers are self-assembled into the supermolecule nano particles by adjusting the following 3 parameters of 8-Ad-PAMAM, beta-CD-PEI and Ad-PEG with the mass ratio of 6:1:4. The specific synthesis scheme is as follows:

8-Ad-PAMAM (7.5. Mu.g) was weighed and dissolved in 10. Mu.L DMSO, 1.5. Mu. gDonor-RS1/GFP plasmid was added, enzyme-free water (50. Mu.L system was constructed), mixed well with shaking and ice-bath for 10min, ad-PEG (5.0. Mu.g), beta-CD-PEI (1.25. Mu.g) were added, mixed well with shaking and ice-bath for 30min, ad-PEG-TAT (0.5. Mu.g) was added, mixed well with shaking and ice-bath for 30min. The Donor RS1/GFP was then obtained SMNPs. After the mixture was allowed to stand at 4℃for 30min, the Donor RS1/GFP was subjected to Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM)SMNPs particle size.

Preparation of Cas9/sgRNA Using similar methodsSMNPs the only difference was that the Donor-RS1/GFP plasmid was replaced with a Cas9/sgRNA plasmid, and the remaining procedures were identical. After storing the mixture for 1h at 4 ℃, the nanoparticle size was characterized using Dynamic Light Scattering (DLS), transmission Electron Microscopy (TEM).

Characterized by Donor RS1/GFPSMNPs and Cas9/sgRNASMNPs has particle diameter of about 120nm, uniform particle diameter distribution and good form dispersibility. HEK293T cells are co-transfected, and the fluorescent microscope shows that the HEK293T cells have better transfection efficiency which reaches 65 percent. MTT experiments showed little cytotoxicity.

EXAMPLE 6 Donor RS1/GFP & Cas 9/sgRNASMNPs transfection of HEK 293T cells:

and synthesizing the synthesized nano material monomer into the supermolecule nano particles through self-assembly. Preparation of Donor RS1/GFP & Cas 9/sgRNA Using a similar self-assembled approach SMNPs. The synthesis formula is as follows:

8-Ad-PAMAM was weighed and dissolved in 10. Mu.L DMSO, cas9/sgRNA plasmid (0.75 ug), donor RS1/GFP plasmid (0.75 ug), enzyme-free water (50 uL system was constructed), vortexed and mixed well and ice-bathed for 10min, ad-PEG (5.0 ug), beta-CD-PEI (1.25 ug) were added and dissolved in shaking and ice-bathed for 30min, ad-PEG-TAT (0.5 ug) was added and mixed well and ice-bathed for 30min. Subsequent Donor RS1/GFP & Cas9/sgRNA was obtained SMNPs. After storing the mixture for 1h at 4 ℃, the nanoparticle size was characterized using Dynamic Light Scattering (DLS), transmission Electron Microscopy (TEM). The particle size is about 120nm, the particle size is uniformly distributed, and the form dispersibility is good. HEK293T cells were transfected, and the fluorescence microscope showed a better transfection efficiency, up to 75%, as shown in FIG. 4.

MTT experiments showed that the delivery system was less cytotoxic:

The MTT method is adopted to examine the activity inhibition effect on the HEK293T of the cells under the optimal prescription and at different concentrations SMNPs (25 mu L)/mL-200 mu L/mL). As can be seen in fig. 5, SMNPs delivery systems at different concentrations exhibited a concentration-dependent inhibition. There was still 80% cell viability at 150. Mu.L/mL, and at 200. Mu.L/mL, cell viability was reduced to 60%. However, in the study, when 50. Mu.L/mL SMNPs was used, there was a higher transfection efficiency. Therefore, SMNPs used in the present invention is substantially non-toxic to cells, and SMNPs delivery system is better biosafety.

Comparative example 2

Preparation DonorRS/GFP respectivelyLNP,Cas9/sgRNALNP,Donor RS

1/GFP&Cas 9/sgRNALNP was lipofected by transfecting cells in 24-well plates seeded with cells during log phase growth. Wherein DonorRS/GFPLNP、Cas9/sgRNALNP and Donor RS1/GFP & Cas 9/sgRNALNP was prepared similarly to example 5 and example 6 by taking a 1.5mL centrifuge tube, adding 2. Mu.L of liposome reagent Lipo3000 and diluting with 100. Mu.L of serum-free DMEM medium, vortexing, mixing well, taking another 1.5mL centrifuge tube, adding 1.5. Mu.g of plasmid (1.5. Mu. gCas9/sgRNA plasmid, 1.5. Mu. gdonor RS1/GFP plasmid or 0.75. Mu. gCas9/sgRNA plasmid and 0.75. Mu. gdonorRS1/GFP plasmid) and diluting with 100. Mu.L of serum-free medium, vortexing. Mixing the two tubes, and incubating for 20min in dark to obtain Donor RS1/GFP respectivelyLNP、Cas9/sgRNALNP、DonorRS1/GFP&Cas 9/sgRNALNP。

Taking out the inoculated cells, discarding the old culture medium, washing with PBS, respectively adding the incubated reagents, mixing uniformly with light shaking, placing in an incubator, discarding the culture solution after 6 hours, replacing the culture solution with the complete culture medium, culturing for 48 hours, and carrying out fluorescence quantification by using a confocal fluorescence microscope. Transfection efficiencies ranged from 45% -55% below the efficiency of delivery using SMNPs.

EXAMPLE 7Donor RS1/GFP & Cas9/sgRNASMNPs transfection of retinal organoids:

The ROs were incubated in Matrigel treated 24 well plates at a density of 1000 ROs per well, cultured using RMM-2 medium, and XLRS was used as a blank (no delivery was performed, fresh RMM-2 medium was changed every 2 days). Treatment group was prepared by taking ROS of XLRS and performing Donor RS1/GFP & Cas9/sgRNA three times every 14 days (Day 0, day14, day 28) SMNPs (prepared in example 6) and significant green fluorescent protein expression was observed at Day30, day39 after the third knock-in. The results show that the RS1/GFP gene delivery by the supramolecular nanoparticles has significant fluorescent protein expression under a fluorescent microscope, as shown in fig. 6. The supermolecule nanometer delivery system has good potential for carrying out accurate gene knock-in treatment on X-linked retinal cleavage.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. A supramolecular nanogenetic vector comprising a beta-cyclodextrin grafted polyethyleneimine, an 8-adamantane grafted polyamide-amine dendrimer, an adamantane grafted polyethylene glycol, and an adamantane grafted polyethylene glycol modified with a cell penetrating peptide.

2. The supramolecular nanogenetic vector according to claim 1, wherein the mass ratio of 8-adamantane grafted polyamide-amine dendrimer to beta-cyclodextrin grafted polyethyleneimine is 3.75:1-15:1.

3. The supramolecular nanogenetic vector according to claim 1, wherein the mass ratio of beta-cyclodextrin grafted polyethyleneimine to adamantane grafted polyethyleneglycol is 1:2.5-1:10.

4. The supramolecular nanogenetic vector according to claim 1, wherein the mass ratio of adamantane grafted polyethylene glycol to adamantane grafted polyethylene glycol modified with cell penetrating peptide is from 8:1 to 12:1.

5. The supramolecular nanogene vector of claim 1, wherein the particle size of the supramolecular nanogene vector is 30nm-140nm.

6. The method for preparing the supermolecule nano-gene vector as claimed in any one of claims 1 to 5, comprising the steps of:

(1) Respectively preparing beta-cyclodextrin grafted polyethyleneimine, 8-adamantane grafted polyamide-amine dendrimer, adamantane grafted polyethylene glycol and adamantane grafted polyethylene glycol modified by using a cell penetrating peptide;

(2) Dissolving 8-adamantane grafted polyamide-amine dendrimer in an organic solvent to obtain 8-adamantane grafted polyamide-amine dendrimer solution, adding the solvent, vortex oscillating and mixing uniformly, ice-bath, adding beta-cyclodextrin grafted polyethyleneimine and adamantane grafted polyethylene glycol, vortex oscillating and mixing uniformly, ice-bath, continuously adding adamantane grafted polyethylene glycol modified by cell penetrating peptide, oscillating and mixing uniformly, and ice-bath to obtain the supermolecule nano gene vector.

7. A nano-gene delivery formulation comprising the supramolecular nano-gene vector of any one of claims 1-5 and a plasmid loaded in nanoparticles.

8. The nano-gene delivery formulation of claim 7, wherein the plasmid comprises one or more of a Donor RS1/GFP plasmid, a Cas9/sgRNA plasmid, and a GFP plasmid.

9. Use of a nano-gene delivery formulation according to any one of claims 7-8 for the manufacture of a medicament for the treatment of monogenic genetic diseases.

10. The use of claim 9, wherein the genetic disorder comprises treatment of one or more of X-linked retinal splitting, sickle cell anemia, beta thalassemia, and duchenne muscular failure.