CN104311830A - Dendritic gene and drug carrier, and preparation and application thereof - Google Patents

Abstract

The invention provides a class of biodegradable high-efficiency and low-toxicity dendritic polycation materials. The invention utilizes disulfide bonds to link the low-generation dendritic cationic polymers with very low toxicity to the water-soluble inner core with good biocompatibility , forming a high-molecular-weight polymer, which not only has good gene and drug delivery efficiency, but also can be degraded in cells, which effectively alleviates the contradiction between the delivery efficiency and toxicity of dendritic cationic polymers. The present invention selects simple and easy-to-obtain multi-branched water-soluble polymers and low-generation dendritic cationic polymers as raw materials, and the cost is relatively low; and the synthesis method is simple, and most of them are normal temperature reactions, which do not require harsh reactions such as anhydrous and oxygen-free Conditions, the post-treatment generally adopts the separation method of extraction or dialysis, and the operation is simple. Structural formula of the present invention:

Description

A dendritic gene drug carrier and its preparation and application

technical field

The invention belongs to the field of biotechnology, and relates to a class of high-efficiency and low-toxicity biodegradable dendritic gene drug carriers, in particular to a class of dendritic polycation materials and their preparation methods and applications.

Background technique

Gene drug therapy is a method for treating cancer that has emerged in the past two decades. Its basic strategy is to introduce foreign genes or anticancer drugs into target cells through carrier transport and exert their effects, so as to achieve the purpose of treating cancer. In gene drug therapy, the transport of genes and drugs is a very important part. For the transport carrier, it is important to efficiently transport a certain amount of target genes and drugs to target cells so that they can play a role in a safe, effective and stable manner. the bare minimum. The vectors currently used in gene therapy are mainly divided into two categories: viral vectors and non-viral vectors.

Due to their natural tropism, viral vectors can naturally infect cells, so they have high transport efficiency. At present, there are many commonly used viral vectors in gene therapy, and they have entered the stage of clinical trials. The currently used viruses include retroviruses, adenoviruses, poxviruses, herpes simplex viruses, and adeno-associated viruses. In recent years, significant progress has been made in the research of viral vectors, but due to the shortcomings of viral vectors, such as insufficient biological safety, insufficient targeting specificity, incomplete transduction of certain cells, and easy induction of host immune responses, limitations further development of viral vectors (Thomas CE, Ehrhardt A., Kay MA Nat Rev Genet. 2003, 4:346-358). Non-viral gene vectors are not as efficient as viral vectors in gene and drug delivery, but they have unique advantages, such as low toxicity, low immunogenicity, no infection, easy synthesis and preparation, flexible and controllable structure, etc. (AL-Dosari MS,GAO X. Aaps J , 2009 , 11(4):671-681), and thus has been widely used by researchers, and the polycation material is one of the most important and most widely studied non-viral vectors.

Due to the positive charge in the structure of polycation materials, it is easy to form complexes with negatively charged nucleic acids (DNA, RNA, PNA) through electrostatic interaction, so that the nucleic acids are compressed, thereby avoiding degradation by enzymes. Since the complex normally has a positive net charge, it facilitates attachment to the cell surface and subsequent endocytosis and lysosomal escape. Our research group has published a monograph on polycation materials (TANG GP, LU X. Journal of Zhejiang University: Medical Sciences . 2009 , 38(1):1-6). In recent years, research on polycation materials has Many breakthroughs have been made. Currently commonly used polycation materials include chitosan (chitosan), polyethyleneimine (PEI), polylysine (PLL), dendrimer (Dendrimer) etc. (Morille M., Passirani C., Vonarbourg A. , etal. Biomaterials , 2008 , 29(24-25):3477-3496).

As a special polymer, dendrimer has a precisely arranged branch structure, and there are a large number of cationic groups on the surface, which can be used for gene transportation. A strict three-dimensional structure, which forms many supramolecular cavities for drug delivery (Menjoge AR, Kannan RM, Tomalia DA Drug Discov. Today . 2010 , 15:171–185). However, there is a certain contradiction between the transport efficiency and self-toxicity of dendrimers. High-algebra dendrimers have good transport efficiency, but exhibit obvious cytotoxicity due to poor degradability. At the same time, the synthesis of high algebra dendrimers is also very difficult (Cheng YY, Zhao LB, Li YW, Xu TW Chem. Soc. Rev . 2011, 40:2673−2703; Kukowska-latallo JF, Bielinska AU, Johnson J., Spindler R., Tomalia DA, Baker JR, Jr. Proc. Natl. Acad. Sci. USA. 1996, 93:4897−4902).

In this study, a class of water-soluble polymers with controllable size and some low-generation dendritic cationic polymers were connected through degradable chemical bonds, thus obtaining a series of novel biodegradable dendritic polycations with high efficiency and low toxicity. Material. Through research, we found that this kind of material has a good ability to carry and deliver genes and drugs, and has high efficiency, low toxicity, and good biodegradability. It is a kind of gene drug carrier with great potential.

Contents of the invention

The first object of the present invention is to provide a dendritic gene drug carrier, a class of biodegradable high-efficiency and low-toxicity dendritic polycation compounds (Dendritic cationic polymer), has the following structural formula:

in:

R is the second generation AB ₃ type dendritic cationic polymer (AB ₃ -Dendeimer G2), the structural formula is:

n is the number of repeating units in each branch of eight-branch polyethylene glycol;

m is the number of repeating units in linear polyethylene glycol.

The second object of the present invention is to provide the preparation method of this class novel dendritic polycation compound, realize by following steps (with 8armPEG-SS-PEG-Dendrimer G2 as an example):

1. Preparation of the second-generation dendrimer (compound 4): Weigh a certain amount of compound 1 and dissolve it in dichloromethane with a mass-volume ratio of 20-40 times, and add 3-5 times the amount of compound 1 to it. times the amount of activator 1-hydroxy-7-azobenzotriazole (HOAt) and add 2-4 times the amount of catalyst triethylamine of the amount of HOAt substance, after stirring at room temperature for 5-20 minutes, add the amount of compound 1 N-tert-butoxycarbonylethylenediamine 5-10 times the amount of the substance, and then slowly add 1-ethyl-3 in an amount 2-4 times the amount of the HOAt substance -(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDCI), after the addition, keep it at 0-5 degrees Celsius for 0.5-2 hours, and then react at room temperature for 3-5 hours. After the reaction is completed, add a certain volume of ethyl acetate to the reaction solution, wash the organic phase 2-4 times with 0.1-0.5 mol/liter hydrochloric acid, wash 2-4 times with a certain volume of saturated sodium bicarbonate solution, and wash with a certain volume The volume of saturated sodium chloride solution was washed 1-2 times, then dried with a certain amount of anhydrous sodium sulfate, and rotary evaporated.

Then the intermediate product obtained is dissolved in methanol with a mass volume ratio of 10-20 times, nickel chloride hexahydrate 1-2 times the amount of compound 2 is added thereto, and then the compound is slowly added under the condition of 0-5 degrees Celsius 2 The amount of the substance is 3-10 times the sodium borohydride. After the addition, react at 0-5 degrees Celsius for 0.3-0.5 hours, and then react at room temperature for 3-5 hours. After the reaction is completed, adjust the pH value to acidic with 0.5-2 mol/L hydrochloric acid solution, and then adjust the pH value to alkaline with saturated sodium bicarbonate solution. The methanol in the solution was rotary evaporated, then extracted 2-4 times with ethyl acetate, the organic phase was washed 1-2 times with saturated sodium chloride solution, dried with anhydrous sodium sulfate, then concentrated, and separated by column chromatography Methods Compound 2 was obtained.

Weigh a certain amount of compound 2, 0.05-0.08 times the amount of compound 2, compound 3 and 1.5-3 times the amount of compound 2 HOAt dissolved in dichloromethane mass volume ratio 20-40 times, add The amount of HOAt substance is 2-4 times the amount of catalyst triethylamine, and then EDCI is added at 0-5 degrees Celsius, and the amount of HOAt substance is 2-4 times the amount. After the addition, control the reaction at 0-5 degrees Celsius for 1-3 hours , and then react at room temperature for 30-50 hours. After the reaction is completed, a certain volume of ethyl acetate is added to the reaction solution, the organic phase is washed 2-4 times with a certain volume of 0.1-0.5 mol/liter hydrochloric acid, and washed 2-4 times with a certain volume of saturated sodium bicarbonate solution. Wash with a certain volume of saturated sodium chloride solution for 1-2 times, then dry with a certain amount of anhydrous sodium sulfate, filter and rotary evaporate, and separate the remaining residue by column chromatography to obtain an intermediate product.

Then the obtained intermediate product is dissolved in a mixed solvent of trifluoroacetic acid and dichloromethane at a mass volume ratio of 30-50 times the mass volume ratio at 0-5 degrees Celsius, stirred at room temperature for 10-20 hours, and then the dialysis bag with a certain molecular weight cut-off is used in water After dialysis for 20-40 hours, the second-generation dendrimer (compound 4) was obtained after freeze-drying.

2. Preparation of water-soluble core molecule (compound 6) containing disulfide bonds: Dissolve a certain amount of multi-branched water-soluble core compound 5 in dimethyl sulfoxide with a mass-volume ratio of 3-5 times, and then weigh the activated hydroxyl The linking agent, the dosage is 10-30 times of the amount of the multi-branch compound substance, the linking agent is dissolved in dimethyl sulfoxide with a mass volume ratio of 1-5 times, and the amount ratio of the linking agent substance added is 1-2 times of triethylamine, the solution of the multi-branched compound was added dropwise to the solution of the linking agent under the protection of nitrogen and protected from light, and then the stirring reaction was continued for 3-5 hours. Then add the reaction solution to a mixed solution with a volume ratio of 20-50 times diethyl ether: tetrahydrofuran = 3:1-5:1 volume ratio, let it stand at 0-5 degrees Celsius for 1-3 hours, and filter to obtain the precipitate, which is the intermediate product .

Then the intermediate product obtained by filtration is dissolved in dimethyl sulfoxide with a mass volume ratio of 5-10 times, then cystamine dihydrochloride is weighed, and the amount is 15-30 times the amount of the intermediate product substance, and its Dissolve in dimethyl sulfoxide with a mass volume ratio of 3-10 times, and add triethylamine weighing 3-5 times the amount of cystamine dihydrochloride, and stir for a while until completely dissolved. Then the solution of the intermediate product is slowly added dropwise to the solution of cystamine for 3-5 hours, and then the reaction is continued for 3-5 hours. After the reaction is completed, the solution is dialyzed in the dialysis bag for 24-48 hours, and then freeze-dried An intermediate product is obtained.

Then the intermediate product obtained is dissolved in dimethyl sulfoxide with a mass volume ratio of 5-10 times, and then the linear polyethylene glycol activated on one side is weighed, and the consumption is 5-10 times of the amount of the intermediate product substance, Dissolve it in dimethyl sulfoxide with a mass volume ratio of 5-10 times, add the solution of the intermediate product dropwise into the solution of linear polyethylene glycol, stir and react for 2-5 hours, and then add the reaction solution to In a mixed solution with a volume ratio of 20-50 times ether: tetrahydrofuran = 3:1-5:1 volume ratio, let stand at 0-5 degrees Celsius for 1-3 hours, and then filter to obtain a white powder that contains disulfide bonds. The water-soluble core molecule, namely compound 6.

3. Preparation of the final dendritic polycation compound (compound 7): Dissolve the water-soluble core molecule compound 6 containing disulfide bonds in dimethyl sulfoxide with a mass-volume ratio of 5-10 times, and then weigh the activated hydroxyl The linking agent used is 15-30 times the amount of the compound 6 substance, the linking agent is dissolved in dimethyl sulfoxide with a mass-volume ratio of 5-10 times, and the amount of the linking agent added is 1-2 times triethylamine, the two solutions were mixed under the protection of nitrogen and protected from light, and then stirred and reacted for 4-8 hours. Then add the reaction solution to a mixed solution with a volume ratio of 10-50 times diethyl ether: tetrahydrofuran = 3:1-5:1 volume ratio, let it stand at 0-5 degrees Celsius for 1-2 hours, and filter to obtain a precipitate that is an intermediate product .

Then the intermediate product obtained by filtration is dissolved in dimethyl sulfoxide with a mass volume ratio of 10-20 times, and then a certain amount of compound 4 is weighed, and the amount is 5-10 times the amount of the intermediate product substance, and it is dissolved In dimethyl sulfoxide with a mass-volume ratio of 10-20 times. After that, the solution of the intermediate product was slowly added dropwise to the solution of compound 4 for 2-4 hours, and then the reaction was continued for 12-18 hours. After the reaction was completed, the solution was dialyzed in the dialysis bag for 24-48 hours, and then frozen After drying, a powdery substance was obtained, namely the final product dendritic polycation material (Compound 7).

The reaction formula is as follows:

In the reaction formula, compound 1 is a known compound (Newkome GR Journal of Organic Chemistry , 1988 , V53(23): P5552-5554; Leonard NJ Journal of the American Chemical Society , 1949 , V71: P1762-1764); compound 2 is The reduction product of compound 1 and N-tert-butoxycarbonylethylenediamine condensation; compound 3 is a known compound (Newkome GR, Young JK, Baker GR, Potter R, Audoly LP, Cooper D., and Weis CD Macromolecules , 1993 , 26:2394-2396; Huang QR Langmuir , 2005 , V21(7):P2737-2742); compound 4 is the hydrolyzate of compound 2 and compound 3 after condensation, that is, the second generation AB type ₃ dendritic cationic polymer (AB ₃ -Dendeimer G2); compound 5 is eight-branched polyethylene glycol; compound 6 is a water-soluble core molecule containing disulfide bonds, and eight-branched polyethylene glycol (8arm -PEG-SS-PEG); compound 7 is the condensation product of compound 4 and compound 6, that is, the final dendritic polycation material (8arm-PEG-SS-PEG-Dendrimer G2, referred to as PSPG2s).

In addition, n in the reaction formula is the number of repeating units in each branch of eight-branched polyethylene glycol, m is the number of repeating units in straight-chain polyethylene glycol, and n and m are composed of polyethylene glycol Depends on the molecular weight of the raw material. HOAT in the reaction conditions means 1-hydroxy-7-azobenzotriazole, EDCI means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, TFA means trifluoro Acetic acid, CDI means N,N'-carbonyldiimidazole, R group in compound 7 means compound 4 group (Dendrimer G2).

The dendritic polycation compound of the present invention, that is, compound 7 is: AB ₃ type polyamide-amine type dendrimer-G2 (Dendrimer G2), AB ₂ type polyamide-amine type dendrimer-G2, G3, G4 (PAMAM G2, G3, G4).

The water-soluble dendritic inner core (compound 5) of the present invention is: multi-branched polyethylene glycol (3arm, 4arm, 8arm) with a molecular weight of 10000Da-40000Da.

The molecular weight of linear polyethylene glycol activated by unilateral N-hydroxysuccinimide of the present invention is MW 2000Da-5000Da.

The linking agent described in the present invention is: N,N'-carbonyldiimidazole (CDI), N,N'-disuccinimidyl carbonate (DSC), N-hydroxysuccinimide chloroformate, Any of 9-fluorenylmethyl 1-benzotriazolyl carbonate (FMOC-OBT).

The ratio of the amount of the linking agent used in the present invention to the amount of the substance corresponding to the water-soluble dendritic inner core is 10-30 times the amount.

The mass-to-volume ratio of the water-soluble dendritic core in the present invention to dimethyl sulfoxide is 3-5 times the amount.

The mass-to-volume ratio of the linking agent used in the present invention to dimethyl sulfoxide is 1-5 times the amount.

Catalyst of the present invention is triethylamine, and consumption is 2-4 times amount with the amount ratio of activator HOAT substance, is 1-2 times amount with the amount ratio of linker substance; The amount ratio is 3-5 times the amount.

The dosage ratio of cystamine dihydrochloride in the present invention to the substance corresponding to the water-soluble dendritic inner core is 15-30 times.

The ratio of the amount of linear water-soluble dendritic inner core (linear polyethylene glycol) (NHS-PEG-OH) in the present invention to the amount of the corresponding compound 5 is 5-10 times.

The ratio of the amount of the dendrimer compound (compound 4) used in the present invention to the amount of the substance corresponding to PEG (compound 6) is 5-10 times.

The third object of the present invention is to provide the application of the novel dendritic polycation material as a gene drug carrier, wherein the drug is a drug with hydrophobic properties.

The material of the present invention is used as a non-viral gene drug carrier. The carrier consists of a class of multi-branched water-soluble polymer (such as polyethylene glycol) as the core, and multiple low-generation (G2- G4) Dendritic cationic polymers (such as AB _3- type PAMAM synthesized in the present invention and commercialized AB _2- type PAMAM), forming a series of new dendritic cationic materials (Dendritic cationic polymer). This kind of material can not only efficiently transport genes and drugs, but also has low toxicity and degradability, and the synthesis method is simple and the cost is low. It is a kind of material with good application prospects.

In the preparation of the compound of the present invention, the simple and easy-to-obtain multi-branch water-soluble polymer and dendritic cationic polymer with low algebra are selected as raw materials, and the cost is relatively low; Harsh reaction conditions such as oxygen, the post-treatment generally adopts the separation method of extraction or dialysis, and the operation is simple.

The invention cleverly utilizes the respective characteristics of the water-soluble core and the dendritic cationic polymer, and achieves good effects through simple connection. First, because the surface of the dendritic cationic polymer has a large number of positive charges, which repel each other, the entire carrier material presents a radial shape in water, and the spherical dendritic cationic polymer is dispersed around the water-soluble core. The unique hydrophobic cavity inside the polymer itself can easily carry fat-soluble drugs; secondly, the positive charge on the outer layer of the dendritic cationic polymer can effectively combine with negatively charged genes (DNA or RNA). The charge is neutralized, and due to the lack of water solubility, it will be wrapped by a more water-soluble core structure to form nanoparticles that are close to neutrality and suitable for cell phagocytosis. Third, since the water-soluble core molecule and the dendritic cationic polymer are linked by disulfide bonds, the disulfide bonds are easily reduced and degraded in the cell, thereby releasing the encapsulated genes and drugs to make them work.

The present invention uses the characteristics of its own structure and the different hydrophilicity and hydrophobicity of each part of the compound, through the inversion and transposition of the inner and outer layer structures of the compound, to effectively carry the function of genes (DNA or RNA) and medicines, and at the same time form a compound with stable properties and a size of about 200nm of nanoparticles. The nanoparticle has a high gene delivery efficiency in the cell, and the drug can also be stably and continuously released in the cell, thereby improving the bioavailability of the gene and the drug. In addition, the delivery efficiency of traditional low-algebra dendritic cationic polymers for genes and drugs is very low, while high-algebra dendritic cationic polymers have a certain delivery efficiency, but due to their high molecular weight and cannot be degraded in vivo, cytotoxicity Very large, the present invention uses disulfide bonds to link the low-generation dendritic cationic polymer with low toxicity to the water-soluble inner core with good biocompatibility to form a high-molecular-weight polymer, which not only has a good gene and drug delivery efficiency, and can be degraded in cells, effectively alleviating the contradiction between the delivery efficiency and toxicity of dendritic cationic polymers.

Description of drawings

Figure 1 is a schematic diagram of the three-dimensional structure of a novel dendritic polycation compound.

Fig. 2 is the H NMR spectrum characterization diagram of AB ₃ -Dendrimer G2.

Fig. 3 is a high-resolution mass spectrogram of AB ₃ -Dendrimer G2.

Figure 4 is 8armPEG-SS-PEG-Dendrimer H NMR spectrum characterization of G2 and the products of the synthesis process.

Figure 5 is the H NMR spectrum characterization of four different ratios of PSPG2s.

Figure 6 is an agarose gel electrophoresis retardation test of materials carrying DNA and RNA.

Fig. 7 is the attenuated total reflection Fourier transform infrared spectrum of PSPG2s-6 and PSPG2s-6/Dox/DNA.

Fig. 8 is a surface X-ray photoelectron spectrum diagram of PSPG2s-6 and PSPG2s-6/Dox/DNA.

Fig. 9 is an electron micrograph of PSPG2s-6/DOX/DNA, wherein (a) is a transmission electron micrograph, and (b) is a scanning electron micrograph.

Figure 10 (a-h) is the particle size and potential diagram of PSPG2s-6, Dendrimer G2 and its drug loading and gene, where picture a is Dendrimer The particle size and potential of G2 and 4 different ratios of PSPG2s, Figure b is the particle size and potential of Dendrimer G2 and PSPG2s-6 carrying different proportions of doxorubicin, Figures c and d are the combination of Dendrimer G2 and 4 different proportions of PSPG2s The particle size and potential of DNA at different N/P ratios, Figure e and Figure f are the particle size and potential of Dendrimer G2 and PSPG2s-6 carrying doxorubicin, respectively, and DNA binding at different N/P ratios, Figure g Figure h and Figure h are the particle size and potential of Dendrimer G2, 2 different ratios of PSPG2s and PSPG2s-6 carrying 2 different ratios of doxorubicin and binding siRNA at different N/P ratios, respectively.

Figure 11 is the heparin interference experiment after Dendrimer G2 and PSPG2s-6 combined with DNA or combined with doxorubicin and DNA.

Figure 12 is the degradation experiment of PSPG2s-6 in PBS solutions with pH=7.4, 6.5 and 5.0.

Figure 13 shows the cytotoxicity of 6 materials under different concentration conditions for 4 hours.

Figure 14 shows the cytotoxicity of 6 materials under different concentration conditions for 24 hours.

Figure 15 shows the transfection results of 4 different ratios of PSPG2s and 2 different ratios of PPG2s carrying luciferase protein gene (Luciferase) on Hek293 cells.

Figure 16 shows the GFP silencing within 24 hours of 4 different ratios of PSPG2s and 2 different ratios of PPG2s carrying green fluorescent protein interfering RNA (GFP-siRNA) on human kidney epithelial cells (293T-GFP) expressing GFP Experimental results.

Figure 17 is the release result of doxorubicin from PSPG2s-6/Dox9% in PBS with pH=5.0 and 7.4.

Figure 18 is the TEM pictures of PSPG2s-6/Dox9% at 0h and 12h under the acidic condition of PH=5.0, and the white light photos of PSPG2s-6/Dox9% at 0h and 12h under the conditions of PH=5.0 and 7.4.

Fig. 19 is the in vivo fluorescence diagram of metabolism of Dox, siRNA and PSPG2s/Dox9%/siRNA in tumor sites of nude mice at different time points.

Fig. 20 is the relative intensity change curve of red and green fluorescence in Fig. 19 .

Figure 21 is a picture of tumor sections after Dox and PSPG2s-6/Dox9%/siRNA were metabolized in the tumor site of nude mice for 24 hours.

Figure 22 is PSPP3s/DNA with two access ratios (PSPP3s-4/6 respectively represent the molar ratio of 8arm-PEG and PAMAM G3 in the dendritic polycation compound PSPG2s is 4/6) under different N/P ratios Agarose gel electrophoresis retardation test carrying DNA.

Figure 23 is the transfection experiment of the luciferase reporter gene Luciferase under the conditions of PAMAM G3, PEI25KD, and PSPP3s-6 at the optimal N/P ratio, and the serum concentrations were 0%, 10%, 25%, and 50% respectively result.

Fig. 24 is the detection experiment result of lysosome escape ability of different materials.

Fig. 25 is the experimental results of the effect of different drug loading ratios on transfection.

Figure 26 shows 4PSPP4s/DNA with two access ratios (4PSPP4s-2/4 respectively represent the molar ratio of 4arm-PEG and PAMAM G4 in the dendritic polycation compound 4PSPP4s is 2/4) under different N/P ratios Agarose gel electrophoresis retardation test carrying DNA.

Figure 27 is an atomic force microscopy (AFM) image of 4PSPP4s (a) and 4PSPP4s/Gef/DNA (b).

Figure 28 is an experiment of the effect of buthionine sulfate imide (BSO) on the cytotoxicity of materials.

Fig. 29 is an experiment of the effect of buthionine sulfate (BSO) on material cell transfection.

Figure 30 shows the potential changes of PAMAM G4, 4PSPP4s-4 and 4PPP4s-4 in the heparin dissociation experiment after binding gefitinib and DNA.

Detailed ways

The present invention is further described in conjunction with the accompanying drawings and embodiments, but is not limited to the content disclosed in the embodiments.

Example 1 : Preparation of the carrier material 8armPEG-SS-PEG-Dendrimer G2 (PSPG2s) and carrying drugs and genes. (Dendrimer is AB3-Dendrimer G2, 8armPEG is 15000Da, NHS-PEG-OH is 2000Da, linker is CDI, drug is doxorubicin, gene is blank reporter gene DNA)

(1) Synthesis of AB ₃ -Dendeimer G2

Weigh 445.6 mg of compound 1 and dissolve it in 15 ml of dichloromethane, add 871 mg of 1-hydroxy-7-azobenzotriazole (HOAt) and 3 ml of triethylamine, stir at room temperature for 10 minutes, then add 2.1 g of N-tert-butoxycarbonylethylenediamine, then slowly add 4.05 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) at 0°C After the addition, keep the temperature at 0°C for 1 hour, and then react at room temperature for 4 hours. After the reaction was completed, 30 milliliters of ethyl acetate was added to the reaction solution, and 20 milliliters of the organic phase was washed three times with 0.4 mol/liter hydrochloric acid, three times with 20 milliliters of saturated sodium bicarbonate solution, and once with 20 milliliters of saturated sodium chloride solution. , and then dried with a small amount of anhydrous sodium sulfate, filtered and rotary evaporated to obtain a white solid.

703.8 mg of the white solid was weighed and dissolved in 10 ml of methanol, 356.7 mg of nickel chloride hexahydrate was added thereto, and then 189.4 mg of sodium borohydride was slowly added at 0°C. After the addition, react at 0°C for 0.3 hours, then at room temperature for 4 hours. After the reaction was completed, the pH value was adjusted to 5 with 1 mol/L hydrochloric acid solution, and then adjusted to 9 with saturated sodium bicarbonate solution. The methanol in the solution was rotary evaporated, then extracted three times with ethyl acetate, the organic phase was washed once with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and then concentrated, separated by column chromatography (n-hexane/ Ethyl acetate=1/1) to obtain a colorless oily substance, compound 2.

Weigh 337 mg of compound 2, 47 mg of compound 3 and 136 mg of HOAt and dissolve them in 10 ml of dichloromethane, add 0.5 ml of triethylamine, then slowly add 575 mg of EDCI at 0 degrees Celsius, and control 0 The reaction was carried out for 2 hours at 100°C, and then at room temperature for 36 hours. After the reaction was completed, 75 milliliters of ethyl acetate was added to the reaction solution, and the organic phase was washed twice with 50 milliliters of 0.4 mol/liter hydrochloric acid, twice with 50 milliliters of saturated sodium bicarbonate solution, and washed twice with 50 milliliters of saturated sodium chloride solution. Wash once, then dry with anhydrous sodium sulfate, filter and rotary evaporate, and separate the remaining residue by column chromatography (methanol/ethyl acetate=1/4) to obtain a white solid.

Weigh 460 mg of the white solid, dissolve it in a mixed solvent of 10 ml of trifluoroacetic acid and 10 ml of dichloromethane at 0°C, stir at room temperature for 12 hours, then use a dialysis bag with a molecular weight of 2000 to dialyze in water for 24 hours, freeze-dry A white powder was obtained, namely the second generation dendrimer 4 (AB ₃ -Dendeimer G2).

(2) Synthesis of 8armPEG-SS-PEG

Weigh 6 g of 15,000 Da 8armPEG and dissolve it in 18 mL of DMSO, weigh 19 g of N,N'-carbonyldiimidazole (CDI), dissolve it in 20 mL of DMSO, and add catalyst triethyl 1.6 ml of amine, 8 armPEG solution was added dropwise to the CDI solution under the protection of nitrogen and protected from light, and then the stirring reaction was continued for 3 hours. Then the reaction solution was added to a mixed solution with a volume of 2 liters of diethyl ether: tetrahydrofuran = 4:1 volume ratio, allowed to stand at 0°C for 1 hour, and then filtered to obtain a white powder.

Then weigh 3 grams of the white powder and dissolve it in 15 milliliters of dimethyl sulfoxide, weigh 1.35 grams of cystamine dihydrochloride and dissolve it in 5 milliliters of dimethyl sulfoxide, add 2.5 milliliters of triethylamine, and stir for a while until completely dissolved. Afterwards, the PEG solution was slowly added dropwise to the cystamine solution for 3 hours, and then continued to react for 3 hours. After the reaction was completed, the solution was dialyzed in a MW14000 dialysis bag for 24 hours, and then freeze-dried to obtain a white powder.

Then weigh 1.75 grams of the white powder and dissolve it in 10 milliliters of dimethyl sulfoxide, then weigh 2 grams of 2000 Da unilateral N-hydroxysuccinimide-activated linear polyethylene glycol (NHS-PEG-OH ) was dissolved in 10 ml of dimethyl sulfoxide, and the solution of white powder was added dropwise to the solution of NHS-PEG-OH, stirred and reacted for 2 hours, and then the reaction solution was added to 400 ml of diethyl ether: tetrahydrofuran = 4:1 volume In the mixed solution of the ratio, let it stand for 1 hour at 0 degrees Celsius, and then filter to obtain a white powder, which is 8armPEG-SS-PEG.

(3) 8armPEG-SS-PEG-Dendrimer Synthesis of G2 (PSPG2s)

Dissolve 3.4 g of 8armPEG-SS-PEG in 35 mL of DMSO, then weigh 500 mg of N,N'-carbonyldiimidazole (CDI) and dissolve it in 5 mL of DMSO, and add Catalyst III 400 microliters of ethylamine, the two solutions were mixed under the protection of nitrogen and protected from light, and then stirred and reacted for 6 hours. Then the reaction solution was added to 400 ml of a mixed solution of diethyl ether: tetrahydrofuran = 4:1 volume ratio, allowed to stand at 0°C for 2 hours, and then filtered to obtain a white powder.

Then weigh 1 gram of the white powder and dissolve it in 10 milliliters of dimethyl sulfoxide, then weigh 1.5 grams of AB ₃ -Dendrimer G2 dendrimers and dissolve it in 15 milliliters of dimethyl sulfoxide, then dissolve the white powder solution Slowly added dropwise to the solution of dendrimers, added dropwise for 3 hours, then continued to react for 12 hours. After the reaction was completed, the solution was dialyzed in a dialysis bag of MW14000 for 36 hours, and then freeze-dried to obtain a white powdery substance, namely The final product is the dendritic polycation material PSPG2s.

The reaction formula is the same as above, compound 7 is the structural formula of the final dendritic polycation compound, wherein the R group is compound 4, and the stereostructure of compound 7 is shown in Figure 1.

(4) Preparation of nanoparticles PSPG2s-6/Dox

Dissolve 4 mg of doxorubicin (Doxorubicin, Dox) in 1 ml of CH ₂ Cl ₂ , add 3 μl of triethylamine, and dissolve 36 mg of PSPG2s-6 in 10 ml of DMSO, then mix the two, and stir at room temperature overnight. Then the solution was dialyzed for 4 hours with a dialysis bag with a molecular weight of 14000, and freeze-dried to obtain PSPG2s-6/Dox.

(5) Preparation of nanoparticles PSPG2s-6/DNA and PSPG2s-6/Dox/DNA

Dissolve the blank gene DNA in water to make a 0.1 μg/μl solution, take 10 μl of this solution, and then add 10 μg/μl of PSPG2s-6 or PSPG2s-6/Dox containing 10 μg/μl of PSPG2s-6 Add an equal volume of the aqueous solution into the DNA solution, and let it stand at room temperature for 30 minutes to form nanoparticles PSPG2s-6/DNA or PSPG2s-6/Dox/DNA for subsequent use.

Example 2 : Characterization of materials

(1) Characterization of AB ₃ -Dendrimer G2

Figure 2 is the H NMR and C NMR spectra of AB ₃ -Dendrimer G2, and Figure 3 is the high-resolution mass spectrum (MALDI-TOF MS) spectrum of AB ₃ -Dendrimer G2, which is basically consistent with the theoretical molecular weight of 5604, indicating that AB The preparation method of ₃ -Dendrimer G2 is feasible.

(2) 8armPEG-SS-PEG-Dendrimer Synthetic characterization of G2

Figure 4 shows eight-branched polyethylene glycol (8arm-PEG), eight-branched polyethylene glycol (8arm-PEG-SS-PEG) linked by disulfide bonds to linear polyethylene glycol, and the second-generation AB type ₃ dendritic The H NMR spectrum of the polymer (AB ₃ -Dendrimer G2) and the final product dendritic polycation material (8armPEG-SS-PEG-Dendrimer G2, PSPG2s), the displacement of each inactive hydrogen atom in the figure is shown in the figure, and the final The proton nuclear magnetic resonance spectrum of the product PSPG2s shows that the polyethylene glycol and the dendritic polymer are a whole, which fully proves the feasibility of the preparation method provided. Figure 5 is the H NMR spectra of four different ratios of PSPG2s, in which the ratios of core 8armPEG-SS-PEG and Dendrimer G2 are approximately 1:2, 1:4, 1:6, 1:8, indicating that the synthetic The method can control the synthesis of different ratios of products.

Embodiment 3 : the ability of compound to carry gene

Figure 6(a) is Dendrimer G2/DNA and PSPG2s/DNA of four access ratios (PSPG2s-2/4/6/8 represent the ratio of 8arm-PEG and Dendrimer G2 in the dendritic polycation compound PSPG2s, respectively 2/4/6/8) Agarose gel electrophoresis retardation test carrying DNA under different N/P ratios. The figure shows that compounds such as PSPG2s can effectively carry DNA, and the efficiency is higher than that of Dendrimer G2 that exists alone. Figure (b) is the agarose gel electrophoresis retardation test of PSPG2s-2/4/6/8 carrying RNA under different N/P ratios. The figure shows that compounds such as PSPG2s can also effectively carry RNA.

Embodiment 4 : Structural characterization of compound-carrying genes and drugs

(1) Attenuated total reflection Fourier infrared experiment of PSPG2s-6 and PSPG2s-6/Dox/DNA

Figure 7 is the attenuated total reflection Fourier transform infrared spectrum of PSPG2s-6 and PSPG2s-6/Dox/DNA (PSPG2s-6 represents 8arm-PEG-SS-PEG-Dendrimer G2-6, that is, each 8armPEG accesses 6 Dendrimer G2 on average). As shown in the two bands in the figure, after the PSPG2s-6 material carried DOX and DNA, the peak area of Dendrimer G2 in the bands was greatly reduced, indicating that the structure was reversed when carrying DOX and DNA, and the original exposed The cationic Dendrimer of the circle combines negatively charged DNA and is encapsulated inside the nanosphere by PEG, which forms a protective layer on the outside.

(2) Surface X-ray photoelectron spectroscopy experiments of PSPG2s-6 and PSPG2s-6/Dox/DNA

Figure 8 is the high-resolution X-ray photoelectron energy spectrum of C1s (a, b) and O1s (c, d) of PSPG2s-6 and PSPG2s-6/Dox/DNA, and the attribution description of each peak is shown below the figure. In the peak division results corresponding to C1s in the figure, the comparison between a and b shows that after PSPG2s-6 carries DOX and DNA, the C=O peak originally belonging to Dendrimer G2 basically disappeared; while the peak corresponding to O1s In the results, most of the c-graphs belong to Dendrimer The peak of C=O in G2 is almost entirely the peak of C-O in PEG in d. The above evidences all indicate that after carrying DOX and DNA, the surface of PSPG2s-6 basically exhibits the properties of PEG, while Dendrimer G2, together with DOX and DNA carried, are wrapped inside the nanospheres.

Embodiment 5 : electron microscope experiment of material carrying gene and medicine

Figure 9 is the transmission electron micrograph (a) and scanning electron micrograph (b) of PSPG2s-6/DOX/DNA. The pictures of the two kinds of electron microscopes all show that the particle size of the nano-microsphere is about 200nm, which belongs to the range suitable for cell phagocytosis, which is conducive to its function as a gene drug carrier.

Embodiment 6 : Compound and the particle size and site characterization after carrying genes and drugs

In Figure 10, panel a is the particle size and potential of Dendrimer G2 and 4 different ratios of PSPG2s. Figure b is the particle size and potential of Dendrimer G2 and PSPG2s-6 carrying different proportions of doxorubicin. Figures c and d are the particle size and potential of Dendrimer G2 and four different ratios of PSPG2s bound to DNA at different N/P ratios, respectively. Panel e and panel f are the particle size and potential of DNA binding at different N/P ratios after Dendrimer G2 and PSPG2s-6 carry doxorubicin, respectively. Figures g and h respectively show the particle size and potential of Dendrimer G2, 2 different ratios of PSPG2s and PSPG2s-6 carrying 2 different ratios of doxorubicin and binding siRNA at different N/P ratios. Based on the above results, the following conclusions can be drawn: the particle size of PSPG2s and PSPG2s/DOX is stable at about 200nm after binding to DNA or siRNA, and at the same time, the surface charge has changed greatly, from obviously positive to close to neutral. with Dendrimer alone Comparing the potential changes of G2 and Dendrimer G2/DOX after binding DNA or siRNA, this once again proves that the material itself has reversed the inner and outer layers in structure. The positively charged Dendrimer is wrapped inside the nanospheres, while the surface is composed of PEG. shell structure.

Embodiment 7 : the heparin interference test of compound

Figure 11 is Dendrimer After G2 and PSPG2s-6 combined with DNA or combined with doxorubicin and DNA, the particle size changes in the heparin dissociation experiment, in which the role of DTT is to degrade the disulfide bond, that is, to break the link between PEG and Dendrimer. The results in the figure show that the nanospheres carrying the gene can effectively avoid the interference of external charges due to the protection of PEG, and the structure will no longer be stable without the protection of PEG.

Embodiment 8 : the degradation experiment of material

Figure 12 is the degradation experiment of PSPG2s-6 in PBS solutions with pH=7.4, 6.5 and 5.0. The results showed that the compound PSPG2s-6 could be degraded under acidic conditions, which could reduce its cytotoxicity when used.

Embodiment 9 : the cytotoxicity experiment of compound

Figure 13 and Figure 14 are 4 different ratios of PSPG2s and 2 different ratios of PPG2s (8arm-PEG-PEG-Dendrimer G2, indicating that there is no disulfide bond synthesized by using 1,4-butanediamine instead of cystamine dihydrochloride 4-hour and 24-hour cytotoxicity on Hek293 cells. The results showed that PSPG2s showed little cytotoxicity within 24 hours, while compounds without disulfide bonds showed some toxicity. Since the disulfide bond can be reduced in the cell, the compound is decomposed into small molecules with almost no cytotoxicity, so the overall toxicity is low, while the PPG2s without disulfide bond is more toxic, indicating that the use of disulfide bond is beneficial to reduce Cytotoxicity of compounds.

Embodiment 10 :

Figure 15 shows the transfection results of 4 different ratios of PSPG2s and 2 different ratios of PPG2s carrying luciferase protein gene (Luciferase) on Hek293 cells. The results show that the disulfide bonds play a crucial role in the process of releasing genes to function after the compounds enter the cells, and compounds such as PSPG2s have a good ability to deliver genes.

Embodiment 11 :

Figure 16: 4 different ratios of PSPG2s and 2 different ratios of PPG2s carrying green fluorescent protein interfering RNA (GFP-siRNA), GFP silencing within 24 hours on human kidney epithelial cells expressing GFP (293T-GFP) Experimental results. The results show that the disulfide bonds play a vital role in the process of releasing genes to make them function after the compound enters the cell, and compounds such as PSPG2s can also effectively carry siRNA into the cell to play a role.

Example 12:

Figure 17 is the release result of doxorubicin from PSPG2s-6/Dox9% in PBS with pH=5.0 and 7.4. The results show that the compound can effectively carry and release drugs, and the release efficiency under acidic conditions is much higher than that under neutral conditions, which is beneficial for the compounds to release drugs in the acidic environment of the tumor to achieve curative effect. Figure 18 is the TEM pictures of PSPG2s-6/Dox9% at 0h and 12h under the acidic conditions of PH=5.0, and the white light photos of PSPG2s-6/Dox9% at 0h and 12h under the conditions of PH=5.0 and 7.4, the above results It shows that the structure of PSPG2s-6/Dox9% nano-microspheres will be dispersed under acidic conditions, and the drug will be released, but the drug release will be inhibited under neutral conditions. This is because the solubility of compounds and drugs inside the nanospheres increases under acidic conditions, resulting in loosening and accelerating drug release.

Embodiment 13 :

Figure 19 is the in vivo fluorescence diagram of Dox, siRNA and PSPG2s/Dox9%/siRNA at different time points in nude mouse tumor site metabolism; Figure 20 is the relative intensity change curve of red and green fluorescence in Figure 19; Figure 21 is Dox and PSPG2s- Tumor slices after 6/Dox9%/siRNA metabolized in the tumor site of nude mice for 24 hours. The results show that the material can effectively carry and release genes and drugs in the body at the same time, and has a good slow-release effect at the tumor site.

Embodiment 14 :

Preparation of carrier material 8armPEG-SS-PEG-PAMAM G3 (PSPP3s) and carrying drugs and genes. (Dendrimer is PAMAM G3, 8armPEG is 20000Da, NHS-PEG-OH is 3500Da, linker is DSC, drug is paclitaxel (Taxol), gene is luciferase reporter gene Luciferase)

(1) Synthesis of 8armPEG-SS-PEG

Weigh 8g of 20000Da 8armPEG and dissolve in 40ml of DMSO, weigh 2g of N,N'-disuccinimidyl carbonate (DSC), dissolve in 6ml of DMSO, 2.2 ml of triethylamine catalyst was added, and the 8armPEG solution was added dropwise to the DSC solution under the protection of nitrogen and protected from light, and then the stirring reaction was continued for 5 hours. Then the reaction solution was added to a mixed solution with a volume of 1 liter of diethyl ether: tetrahydrofuran = 5:1 volume ratio, allowed to stand at 5 degrees Celsius for 3 hours, and then filtered to obtain a white powder.

Then weigh 4 grams of the white powder and dissolve it in 40 milliliters of dimethyl sulfoxide, weigh 1 gram of cystamine dihydrochloride and dissolve it in 10 milliliters of dimethyl sulfoxide, add 2.7 milliliters of triethylamine, and stir for a while until completely dissolved. Afterwards, the PEG solution was slowly added dropwise to the cystamine solution for 5 hours, and then continued to react for 5 hours. After the reaction was completed, the solution was dialyzed in a MW14000 dialysis bag for 48 hours, and then freeze-dried to obtain a white powder.

Then weigh 2.25 grams of the white powder and dissolve it in 20 milliliters of dimethyl sulfoxide, then weigh 2.8 grams of 3500 Da unilateral N-hydroxysuccinimide-activated linear polyethylene glycol (NHS-PEG-OH ) was dissolved in 20 ml of dimethyl sulfoxide, and the white powder solution was added dropwise to the NHS-PEG-OH solution, stirred and reacted for 5 hours, and then the reaction solution was added to 2 liters of diethyl ether: tetrahydrofuran = 3:1 volume In the mixed solution of the ratio, let it stand at 5 degrees Celsius for 3 hours, and then filter to obtain a white powder, which is 8armPEG-SS-PEG.

(2) 8armPEG-SS-PEG- Synthesis of PAMAM G3 (PSPP3s)

Dissolve 4.5 g of 8armPEG-SS-PEG in 25 mL of DMSO, then weigh 650 mg of N,N'-disuccinimidyl carbonate (DSC) and dissolve in 4 mL of DMSO , and 650 microliters of catalyst triethylamine were added, and the two solutions were mixed under the protection of nitrogen and protected from light, and then stirred and reacted for 8 hours. Afterwards, the reaction solution was added to 1.2 liters of a mixed solution with a volume ratio of diethyl ether: tetrahydrofuran = 3:1, allowed to stand at 5° C. for 1.5 hours, and then filtered to obtain a white powder.

Then take by weighing 1.3 grams of this white powder and dissolve it in 25 milliliters of dimethyl sulfoxide, then weigh 1.7 grams of PAMAM G3 dendrimers and dissolve it in 35 milliliters of dimethyl sulfoxide, then slowly add the solution of the white powder Into the solution of the dendritic polymer, add dropwise for 4 hours, then continue to react for 18 hours, after the reaction is completed, the solution is dialyzed in a dialysis bag of MW14000 for 48 hours, and then freeze-dried to obtain a white powdery substance, the final product branch polycationic compound PSPP3s.

(3) Preparation of nanoparticles PSPP3s-6/Taxol

4mg of paclitaxel (Paclitaxel, Taxol) was dissolved in _{1ml of CH2Cl2} , 3μl of triethylamine was added, 36mg of PSPP3s-6 was dissolved in 10ml of DMSO, the two were mixed, and stirred overnight at room temperature. Then the solution was dialyzed for 4 hours with a dialysis bag with a molecular weight of 14000, and freeze-dried to obtain PSPP3s-6/Taxol.

(4) Preparation of nanoparticles PSPP3s-6/DNA, PSPP3s-6/Taxol/DNA

Dissolve the luciferase reporter gene Luciferase in water to make a 0.1 μg/μl solution, take 10 μl of the solution, and then add 10 μg/μl of PSPP3s-6 or PSPP3s-6 containing 10 μg/μl of PSPP3s-6 Add an equal volume of 6/Taxol aqueous solution to the DNA solution and let it stand at room temperature for 30 minutes to form nanoparticles PSPP3s-6/DNA or PSPP3s-6/Taxol/DNA for subsequent use.

Embodiment 15 : the ability of compound to carry gene

Figure 22 is PSPP3s/DNA with two access ratios (PSPP3s-4/6 respectively represent the molar ratio of 8arm-PEG and PAMAM G3 in the dendritic polycation compound PSPG2s is 4/6) under different N/P ratios Agarose gel electrophoresis retardation test carrying DNA. The figure shows that compounds such as PSPP3s can efficiently carry DNA.

Example 16 : Effect of Serum Concentration on Transfection

Figure 23 is PAMAM G3, PEI25KD, PSPP3s-6 transfection experiment results of luciferase reporter gene Luciferase under the conditions of optimal N/P ratio and serum concentrations of 0%, 10%, 25%, and 50%, respectively. As shown in the figure, the transfection efficiencies of PAMAM G3 and PEI25KD showed a significant downward trend with the increase of serum concentration, while PSPP3s-6 was hardly affected. This is because the protein and other substances in the serum will affect the stability of the nanospheres, and the surface of the nanospheres formed by PSPP3s-6/DNA is a neutral material of PEG, which is hardly affected by the protein, which improves the stability of the microspheres. Stability, so that the transfection efficiency is not significantly affected. It is also worth mentioning that PAMAM The transfection efficiency of the G3-branched compound is basically the same as that of PEI25KD, while the transfection efficiency of our self-synthesized Dendrimer G2-branched PSPG2s-6 is 1.13 times that of PEI25KD (Example 10), which has better delivery ability.

Embodiment 17 : detection experiment of lysosome escape ability

Figure 24 is PAMAM G3, PEI25KD, PSPP3s-6 under the condition of the optimal N/P ratio, the transfection experiment results of the luciferase reporter gene Luciferase in the presence or absence of chloroquine and bafilomycin A1 respectively. The role of chloroquine is to promote lysosomal escape of endocytic materials, while the role of bafilomycin A1 is to inhibit lysosomal escape by inhibiting the normal operation of ion pumps. The experimental results show that PSPP3s-6 has a good ability to escape from lysosomes, and the promotion effect of chloroquine on it is not obvious under the same conditions, while the PAMAM G3 monomer has poor escape ability, which is obvious under the condition of chloroquine. On the other hand, it shows that the lysosome escape of PSPP3s-6 is due to its own proton sponge effect, which inhibits the function of the ion pump, which will greatly inhibit its escape ability.

Embodiment 18 :

Fig. 25 is the transfection experiment results of luciferase reporter gene Luciferase loaded with different proportions of paclitaxel (Taxol) in PSPP3s-6, and the drug loading rates are 0%, 9%, 20%, and 30%, respectively. The results show that with the increase of drug loading rate, the transfection efficiency will decrease to a certain extent. This is because the drug itself has a certain degree of lethality to cells, which inhibits the normal function of cells and indirectly leads to a decrease in transfection efficiency. .

Example 19 : Preparation of carrier material 4armPEG-SS-PEG-PAMAM G4 (4PSPP4s) and carrying drugs and genes. (Dendrimer is PAMAM G4, 8armPEG is 40000Da, NHS-PEG-OH is 5000Da, linker is FMOC-OBT, drug is Gefitinib (Gef), gene is luciferase reporter gene Luciferase)

(1) Synthesis of 4armPEG-SS-PEG

Weigh 10 g of 4armPEG of 40,000 Da and dissolve in 40 mL of DMSO, weigh 0.9 g of 9-fluorenylmethyl 1-benzotriazolyl carbonate (FMOC-OBT), and dissolve in 5 mL of DMSO sulfoxide, and 0.5 ml of catalyst triethylamine was added, and the solution of 8armPEG was added dropwise to the solution of FMOC-OBT under the protection of nitrogen and protected from light, and then the stirring reaction was continued for 4 hours. Then the reaction solution was added to a mixed solution with a volume of 2 liters of diethyl ether: tetrahydrofuran = 3:1 volume ratio, and stood at 0 degrees Celsius for 2 hours, and then filtered to obtain a powdery solid.

Then weigh 8 grams of the powder and dissolve it in 60 milliliters of dimethyl sulfoxide, weigh 0.7 grams of cystamine dihydrochloride and dissolve it in 5 milliliters of dimethyl sulfoxide, add 1.6 milliliters of triethylamine, stir for a while to completely dissolved. Afterwards, the PEG solution was slowly added dropwise to the cystamine solution for 4 hours, and then continued to react for 4 hours. After the reaction was completed, the solution was dialyzed in a MW14000 dialysis bag for 36 hours, and then freeze-dried to obtain a white powder.

Then weigh 4 grams of the white powder and dissolve it in 30 milliliters of dimethyl sulfoxide, then weigh 2.5 grams of unilateral N-hydroxysuccinimide-activated linear polyethylene glycol (NHS-PEG-OH ) was dissolved in 25 ml of dimethyl sulfoxide, and the white powder solution was added dropwise to the NHS-PEG-OH solution, stirred and reacted for 3.5 hours, and then the reaction solution was added to 2 liters of diethyl ether: tetrahydrofuran = 5:1 volume In the mixed solution of the ratio, let stand at 0 degrees Celsius for 2 hours, and then filter to obtain a white powder, which is 4armPEG-SS-PEG.

(2) 4armPEG-SS-PEG-PAMAM Synthesis of G4 (4PSPP4s)

Dissolve 3 g of 8armPEG-SS-PEG in 25 mL of dimethyl sulfoxide, then weigh 270 mg of 9-fluorenylmethyl 1-benzotriazolyl carbonate (FMOC-OBT) and dissolve in 2 mL of dimethyl sulfoxide In the base sulfoxide, 100 microliters of catalyst triethylamine was added, and the two solutions were mixed under the protection of nitrogen and protected from light, and then stirred and reacted for 4 hours. After that, the reaction solution was added to 1 liter of ether:tetrahydrofuran=5:1 volume ratio mixed solution, allowed to stand at 0°C for 1 hour, and then filtered to obtain a white powder.

Then take by weighing 1.8 grams of this white powder and dissolve it in 25 milliliters of dimethyl sulfoxide, then weigh 2.7 grams of PAMAM G4 dendrimers and dissolve it in 40 milliliters of dimethyl sulfoxide, then slowly add the solution of the white powder Into the solution of the dendritic polymer, add dropwise for 2 hours, then continue to react for 15 hours. After the reaction is completed, the solution is dialyzed in a dialysis bag of MW50000 for 24 hours, and then freeze-dried to obtain a white powdery substance, which is the final product branch Polycation-like compound 4PSPP4s.

(3) Preparation of nanoparticles 4PSPP4s-4/Gef

Dissolve 4 mg of Gefitinib (Gefitinib, Gef) in 1 ml of CH ₂ Cl ₂ , add 3 μl of triethylamine, and dissolve 36 mg of 4PSPP4s-4 in 10 ml of DMSO, then mix the two, and open at room temperature Stir overnight. Then the solution was dialyzed for 4 hours with a dialysis bag with a molecular weight of 14000, and freeze-dried to obtain 4PSPP4s-4/Gef.

(4) Preparation of nanoparticles 4PSPP4s-4/DNA and 4PSPP4s-4/Gef/DNA

The gene is the luciferase reporter gene Luciferase dissolved in water to make a 0.1 μg/μl solution, take 10 μl of the solution, and then add 10 μg/μl of 4PSPP4s-4 or 10 μg/μl of 4PSPP4s-4 Add an equal volume of 4PSPP4s-4/Gef aqueous solution to the DNA solution, and let it stand at room temperature for 30 minutes to form nanoparticles 4PSPP4s-4/DNA or 4PSPP4s-4/Gef/DNA for subsequent use.

Embodiment 20 : the ability of material carrying gene

Figure 26 shows 4PSPP4s/DNA with two access ratios (4PSPP4s-2/4 respectively represent the molar ratio of 4arm-PEG and PAMAM G4 in the dendritic polycation compound 4PSPP4s is 2/4) under different N/P ratios Agarose gel electrophoresis retardation test carrying DNA. The figure shows that materials such as 4PSPP4s can efficiently carry DNA.

Embodiment 21 :

Figure 27 is an atomic force microscopy (AFM) image of 4PSPP4s (a) and 4PSPP4s/Gef/DNA (b). The figure shows that after carrying genes and drugs, 4PSPP4s can form nano-microspheres with a particle size of about 200nm, which is suitable for cell phagocytosis and is conducive to its function as a gene drug carrier.

Example 22 : Effect of disulfide bonds on material cytotoxicity

Figure 28 is a study of the effect of disulfide bonds on the cytotoxicity of compounds, specifically 4PSPP4s-4 and 4PPP4s-4 (4arm-PEG-PEG-PAMAM G4, represents the dendritic polycation compound without disulfide bonds synthesized by using 1,4-butanediamine instead of cystamine dihydrochloride) in the presence or absence of butythionine sulfate imine (BSO) Cytotoxicity experiments. Buthionine sulfate imine is a specific inhibitor of glutathione (GSH) synthesis in the regulation of cell oxidation-reduction state. It can inhibit the degradation of disulfide bonds in the compound by inhibiting the synthesis of GSH. The experimental results showed that the degradation of the disulfide bond played a crucial role in the low toxicity of the compound. 4PSPP4s-4 without the presence of BSO had almost no toxicity in cells, while in the presence of BSO, the 4PSPP4s-4 Cytotoxicity was similar to that of 4PPP4s-4 without disulfide bonds.

Embodiment 23 : Effect of disulfide bond on compound cell transfection

Figure 29 is a study of the effect of disulfide bonds on the transfection of compounds, specifically the transfection of the luciferase reporter gene Luciferase in the presence or absence of buthionine sulfate imine (BSO) of 4PSPP4s-4 and 4PPP4s-4 experiment. Buthionine sulfate imine is a specific inhibitor of glutathione (GSH) synthesis in the regulation of cell oxidation-reduction state. It can inhibit the degradation of disulfide bonds in the compound by inhibiting the synthesis of GSH. The experimental results show that the degradation of disulfide bonds plays a crucial role in the transfection of the compound, because the degradation of disulfide bonds promotes the release of DNA, thereby effectively improving the transfection efficiency.

Embodiment 24 : the heparin interference test of compound

Figure 30 is PAMAM The potential changes of G4, 4PSPP4s-4 and 4PPP4s-4 in the heparin dissociation experiment after binding gefitinib and DNA, where the role of dithiothreitol (DTT) is to degrade the disulfide bond, ie Disconnect 4arm-PEG from PAMAM Link between G4. The results in the figure show that the potential of PAMAM G4 is affected by heparin, 4PSPP4s-4 and 4PPP4s-4 are basically not affected by heparin under the protection of PEG, and 4PSPP4s-4 is degraded by the disulfide bond under the action of DTT, PEG After leaving, it will also be affected by heparin. This shows that only when 4PSPP4s-4 carries genes and drugs to form nano-microspheres, due to the protection of its own PEG, it can effectively avoid the interference of external charges, and its structure will no longer be stable without the protection of PEG.

Claims (10)

1. a class of dendritic polycationic compounds, characterized in that, the structural formula is as follows: in: R is the second generation AB ₃ type dendritic cationic polymer, the structural formula is: n is the number of repeating units in each branch of eight-branch polyethylene glycol; m is the number of repeating units in linear polyethylene glycol. 2. the preparation method of a class of dendritic polycationic compounds according to claim 1, is characterized in that, realizes by following steps, is example with 8armPEG-SS-PEG-Dendrimer G2: (1) Preparation of Compound 4: Dissolve Compound 1 in dichloromethane, add the activator 1-hydroxy-7-azobenzotriazole and the catalyst triethylamine, stir at room temperature, and then add N-tert Butoxycarbonyl ethylenediamine, then slowly add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, continue the reaction after the addition, after the reaction, add Ethyl acetate, the organic phase was washed successively with hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution, then dried with anhydrous sodium sulfate, and rotary evaporated to obtain an intermediate product, which was dissolved in methanol, and six Nickel chloride hydrate, then slowly add sodium borohydride, continue the reaction after the addition, after the reaction, adjust the pH value to acidic with hydrochloric acid solution, then adjust the pH value to alkaline with saturated sodium bicarbonate solution, and add The methanol was rotary evaporated, then extracted with ethyl acetate, the organic phase was washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, then concentrated, and the compound 2 was obtained by column chromatography; Dissolve compound 2, compound 3 and 1-hydroxy-7-azobenzotriazole in dichloromethane, add catalyst triethylamine, then add 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride, continue to react after adding, after the reaction is completed, add ethyl acetate to the reaction solution, the organic phase is washed with hydrochloric acid, saturated sodium bicarbonate solution, saturated sodium chloride solution successively, and then use Dry over sodium sulfate, filter and rotary evaporate, the remaining residue is separated by column chromatography to obtain an intermediate product, then dissolve the obtained intermediate product in a mixed solvent of trifluoroacetic acid and dichloromethane, stir at room temperature, then dialyze with water, freeze-dry After obtaining compound 4; (2) The preparation method of compound 6: dissolving compound 5 in dimethyl sulfoxide, then dissolving the linker in dimethyl sulfoxide, and adding triethylamine, and protecting compound 5 from light under the protection of nitrogen The solution was added dropwise to the solution of the linking agent to continue the reaction, and then the reaction solution was added to the mixed solution of ether and tetrahydrofuran, left to stand, filtered to obtain the precipitate, that is, the intermediate product, and the intermediate product was dissolved in dimethyl sulfoxide, and then the cyst Dissolve amine dihydrochloride in dimethyl sulfoxide, add triethylamine, stir until completely dissolved, slowly add the solution of the intermediate product into the solution of cystamine, and continue the reaction. After the reaction is completed, the solution is watered Dialysis, freeze-drying to obtain the intermediate product, and then dissolve the intermediate product in dimethyl sulfoxide, and then dissolve the unilaterally activated linear polyethylene glycol in dimethyl sulfoxide, drop the solution of the intermediate product Add to the solution of polyethylene glycol, stir and react, then add the reaction solution to the mixed solution of diethyl ether and tetrahydrofuran, let it stand, and filter to obtain a white powder which is compound 6; (3) Preparation of the final product compound 7: dissolving compound 6 in dimethyl sulfoxide, then dissolving the linker in dimethyl sulfoxide, adding triethylamine, and protecting the two The solutions were mixed, stirred and reacted, the reaction solution was added into the mixed solution of diethyl ether and tetrahydrofuran, left to stand, filtered to obtain the precipitate, that is, the intermediate product, and then the intermediate product and compound 4 were dissolved in dimethyl sulfoxide respectively, and the intermediate product The solution was slowly added dropwise to the solution of Compound 4, and then the reaction was continued. After the reaction was completed, the solution was dialyzed with water, and then freeze-dried to obtain a powdery substance, the final product Compound 7; the reaction formula is as follows: In the reaction formula, n is the number of repeating units in each branch of eight-branch polyethylene glycol, and m is the number of repeating units in linear polyethylene glycol, and n and m are determined by the molecular weight of polyethylene glycol raw material Depends; HOAT in the reaction conditions means 1-hydroxyl-7-azobenzotriazole, EDCI means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, TFA represents trifluoroacetic acid, CDI represents N,N'-carbonyldiimidazole, and the R group in compound 7 represents compound 4. 3. the preparation method of a class of dendritic polycationic compounds according to claim 2, is characterized in that, described dendritic polycationic compounds, that is, compound 7 is: AB ₃ type polyamide-amine type dendritic macromolecule - G2, AB Type ₂ polyamidoamine dendrimers - G2, G3, G4. 4. the preparation method of a class of dendritic polycationic compounds according to claim 2, is characterized in that, described compound 5 is: 3arm, 4arm, 8arm multi-branch polyethylene glycol, and molecular weight is 10000Da-40000Da; The molecular weight of the described linear polyethylene glycol is MW 2000Da-5000Da. 5. the preparation method of a class of dendritic polycationic compounds according to claim 2, is characterized in that, described linking agent is: N, N'-carbonyldiimidazole, N, N'-disuccinimide Any of 1-benzotriazolyl carbonate, N-hydroxysuccinimide chloroformate, and 9-fluorenylmethyl 1-benzotriazolyl carbonate. 6. the preparation method of a class of dendritic polycationic compounds according to claim 2, is characterized in that, the ratio of the amount of substance of described connecting agent consumption and corresponding compound 5 is 10-30 times amount, and described The mass-volume ratio of compound 5 to dimethyl sulfoxide is 3-5 times. 7. The preparation method of a class of dendritic polycationic compounds according to claim 2, characterized in that, the mass volume ratio of the amount of the linking agent to dimethyl sulfoxide is 1-5 times the amount. 8. the preparation method of a class of dendritic polycationic compounds according to claim 2, is characterized in that, described catalyzer is triethylamine, and the amount ratio of consumption and activator HOAT substance is 2-4 times amount, and The amount ratio of the linker substance is 1-2 times, and that of cystamine dihydrochloride is 3-5 times. 9. the preparation method of a class of dendritic polycationic compounds according to claim 2, is characterized in that, described cystamine dihydrochloride consumption and the amount ratio of substance of corresponding compound 5 are 15-30 times amount; The amount of linear polyethylene glycol is 5-10 times the amount of the substance corresponding to compound 5; the amount of compound 4 is 5-10 times the amount of the substance corresponding to compound 6. 10. The use of a class of dendritic polycationic compounds as a gene drug carrier according to claim 1, wherein the drug is a drug with hydrophobic properties.