WO2008058457A1

WO2008058457A1 - A biodegradable crosslinked polyethyleneimine and its uses

Info

Publication number: WO2008058457A1
Application number: PCT/CN2007/003194
Authority: WO
Inventors: Wei Dong; Yunzhen Wei
Original assignee: Wei Dong; Yunzhen Wei
Priority date: 2006-11-16
Filing date: 2007-11-12
Publication date: 2008-05-22
Also published as: CN1970591A; CN100549044C

Abstract

A biodegradable crosslinked polyethyleneimine is obtained by reacting a linear or branched polyethyleneimine with molecular weight of 200-100000Da with a crosslinker having biodegradable ester bonds, wherein the crosslinker has molecular weight of 50-50000Da and is selected from glycidyl esters of diacid , glycidyl esters of (meth)acrylic, polyol, polyol multi-(meth)acrylates, or their mixtures. The crosslinked polyethyleneimine can be conjugated with a targeting molecule to obtain a targeting crosslinked polyethyleneimine. A method of cell transfection is accomplished by contacting a cell with the compositon comprising the crosslinked polyethyleneimine and a nucleic acid. The polyethyleneimine is used in the gene transfection reagent, the delivery system of gene therapy drug, and the carrier of drug and bioactivator such as protein and polypeptide and the like.

Description

Biodegradable crosslinked polyethyleneimine and its application

The present invention belongs to the field of biotechnology, and in particular to a delivery vector and method for a biological active substance which can be used for various cells and tissues or organs in vitro, and is a drug, a protein, a polypeptide, and particularly a DNA. RNA, and expression plasmids encoding therapeutic genes, are transferred into cells or tissues and organs. The invention also relates to the preparation and use of such delivery vehicles. Another object of the present invention is to provide a composition and method for gene delivery wherein the gene vector is biodegradable and biocompatible. technical background

Gene therapy refers to the transfer of foreign genes into the interior of cells, by restoring or adding gene expression to correct the structural or functional disorder of human genes, preventing the development of lesions, killing diseased cells, or inhibiting the genetic material of foreign pathogens. Copy, so as to achieve the purpose of treatment. Gene therapy is currently considered to be a very promising treatment, not only for the treatment of genetically deficient diseases, but also for the development or prevention of chronic diseases such as cancer, cardiovascular disease and rheumatoid arthritis. Gene therapy involves three important aspects, namely the gene of interest, the transgenic vector and the target cell. A safe and efficient gene delivery system is the core of successful gene therapy. The gene therapy vectors mainly include viral vectors and non-viral vectors. Although the transfection efficiency of the viral vector is high, the viral vector has some serious disadvantages, such as limited packaging capacity, complicated preparation, and immunogenicity (the body cannot be used repeatedly; And potential safety hazards. Synthetic non-viral vectors have received great attention [1]. Non-viral vectors have the advantages of high safety, low immunogenicity, and easy manipulation of DNA. Therefore, people have recently paid more and more attention to synthetic non-viral vectors. Research [2]. A variety of non-viral vectors have been synthesized, of which polymer gene vectors occupy an important position. The most studied polymer gene therapy systems are cationic polymer type carriers, non-polycondensed polymer systems, biodegradable polymer systems, multi-complex liposome systems, heat sensitive polymer systems and polymeric micelles. System, etc. Although in the early days of non-viral vector research, people's main interest was on liposomes, in recent years, studies have found that cationic liposome gene vectors are found to be highly cytotoxic in repeated use. Moreover, there are potential anti-inflammatory manifestations in the body, and lymphocytes are cultured in vivo for a long time, and liposomes are highly toxic. Recent studies have also shown that it also has some structural shortcomings. For example: The hydrophobic group of a liposome determines its shape, size, stability in the aqueous phase, and interactions with other liposomes, cell membranes, DNA, and the like. This ultimately affects the transfection efficiency of liposome polymers [3, 4]. It is precisely because of the difficulty in controlling the size of liposomes that its properties become unstable with time [5]. In addition, liposome complexes often require some adjuvants in order to improve their transfection efficiency [6]. In conclusion, liposomes, as gene-transducing media, have low transduction efficiency in humans and lack effective chromosomal integration mechanisms. Liposomes, especially cationic liposomes, have certain potential as gene vectors, but the gene expression efficiency is low, and instability in the body has been a problem that plagues the medical community. Due to viral vectors as well The limitations of cationic liposomes have led to an increasing interest in cationic polymers as gene carriers. The more studied cationic transgenic vectors are polylysine (PLL), polyethyleneimine (PEI) and polyamide-amine dendrimer (EDA-PMAM) with ethylenediamine as the core, in which dendrimer The use of polymers and polyethyleneimine (PEI) has received particular attention. Dendrimers rich in amine groups (Dendrimer) and branched polyethyleneimine (PEI) are high-frequency materials that have attracted attention in recent years. Under normal physiological conditions, positive charges will be caused by amine protonation. Interacts with negatively charged DNA to form nano-sized supramolecular complexes, and the unique structure of dendrimers and branches makes the complexes formed with DNA extremely easy to enter cells, thereby transferring genes. If they are modified, such as linking specific proteins, receptors, carrying magnetism, etc., gene transfer can be targeted. In the experiment, by controlling the ratio of their interaction with DNA, controlling the size of the complex with DNA, introducing groups with affinity for the cell membrane, and adjusting their hydrophilic-lipophilic balance values, the gene transfer efficiency can be significantly improved. . However, the dendrimer synthesis method is relatively complicated, and each step of the reaction needs to be accurately controlled. As the molecular weight increases in the synthesis, the separation and purification of each generation of samples becomes more and more difficult. PEI has a variety of structural and molecular weight products to choose from. Various linear, branched and ultra-high-branched PEI synthesis techniques and processes are mature, so PEI can be easily designed and synthesized based on the structure. A series of novel gene vectors.

Compared with viral vectors, the previous gene vectors have lower transfection efficiency and generally have greater cytotoxicity, which may be due to their lack of biodegradability. Therefore, in recent years, people have begun to study biodegradable carriers [ 7-12], the ideal gene carrier should be low toxicity or non-toxic at the dose of high-efficiency transfection, and biodegradability is very important in order to avoid toxic side effects when applied in vivo. However, the currently studied degradable cationic polymers have only met with limited success [13-15]. Polyethylenimine is one of the most transfected non-viral vectors to date, and its transfection efficiency is comparable to that of viral vectors on some cells, so it is often used as a new gene transfection vector evaluator. The new design is compared to the newly developed gene transfection vector. · However, its high transfection efficiency is often accompanied by greater cytotoxicity. Generally, polyethyleneimine below 2000Da is not cytotoxic, but there is no transfection efficiency [16, 17]. 25 kDa polyethyleneimine has high transfection efficiency, but has greater cytotoxicity [18]. . Therefore, many studies have focused on chemically modifying them to couple various glycosyl groups, cyclodextrins, etc., thereby reducing cytotoxicity. Recently, many studies have used various cross-linking agents to crosslink small molecule polyethyleneimine with no toxic side effects, thereby obtaining highly effective and low toxicity biodegradable polyethyleneimine [9,10,15]. However, like most cationic polymers and polyethyleneimine, these crosslinked polyethyleneimines tend to aggregate into larger particles after forming a complex with DNA under physiological conditions [19-22]. Daniel G. [23] The combination of various diacrylates with various small molecular amines into thousands of polymers. It was found that hydroxyl-containing amine monomers crosslink with hydrophobic diacrylate. The product obtained by crosslinking the agent has the highest transfection efficiency. Zhong Z's research found that the addition of hydrophobic fragments to the polymer enhances its hydrophobic interaction with the cell membrane, thereby increasing its affinity for the cell membrane, but this hydrophobic interaction with the cell membrane also impairs the cell membrane [24]. . Therefore, it is very important to reasonably regulate the hydrophilic-lipophilic balance of the synthesized polymer. Summary of the invention

After our analysis, we believe that there are many glycosyl acceptors on the surface of the cell membrane, and there are many hydroxyl groups in the sugar molecule. Similarly, the introduction of hydroxyl groups into the polymer may increase its affinity for the cell membrane, and the hydrophobicity of the cross-linking agent is also promoted. The hydrophobic interaction of the polymer with the cell membrane increases the transfection efficiency. We use 200-100OOODa linear, branched and ultra-branched polyethyleneimine and various diacid glycidyl esters, acrylic acid / glycidyl methacrylate for crosslinking, due to the ring in the crosslinker The oxy group forms a hydroxyl group during the crosslinking reaction, and the aliphatic chain in the crosslinking agent is hydrophobic, and the reaction conditions of the double bond and the epoxy bond are similar, so that the hydroxyl group and the hydrophobic group can be simultaneously introduced into the branched polyethyleneimine. The fatty chain thus improves the solubility of polyethyleneimine under physiological conditions. Since the small molecule polyethyleneimine is rich in various amine groups, this crosslinking is more likely to improve the transfection efficiency of the product than Daniel G.'s research work. The above cross-linking method can be used in combination with various polyhydric alcohols (polyhydric alcohols) of acrylic acid/methacrylic acid polyester to adjust the hydrophilic-lipophilic balance of the synthesized polymer to prepare a highly efficient and low-toxic gene carrier. Since these highly efficient and low-toxicity gene vectors have hydroxyl and amine groups, it is very easy to couple various targeting molecules. These studies have not been reported yet.

Accordingly, it is an object of the present invention to provide a biodegradable crosslinked polyethyleneimine, and a method of synthesis and use thereof.

The singularity of the polyethyleneimine to the crosslinker is 0.1. 1-5 : 1 ; said polyethyleneimine has a molecular weight of 200 to 100,000 Da, preferably 600 to 20,000 Da; said crosslinking agent contains an ester bond which is degradable under physiological conditions, and has a molecular weight of 50 to 50,000 Da , preferably 100-20000 Da.

The cross-linked polyethyleneimine can transfer drugs or various biological actives such as various proteins, polypeptides, especially DNA, RNA, and expression plasmids encoding therapeutic genes into various cells and various tissues or organs in vivo. . Although the small molecule (200-2000 Da;) of polyethyleneimine is not toxic to cells or tissues, it is also substantially free of transfection performance. These various amine-rich small molecule polyethyleneimine are crosslinked by various crosslinkers containing biodegradable bonds to obtain higher molecular weight polyethyleneimine. This synthetic product has high gene transfer performance due to its structure and suitable molecular weight of polyethyleneimine. At the same time, it has no cytotoxicity and can be degraded into non-cytotoxic small molecule polyphores under physiological conditions after entering cells. Ethyleneimine, so it is very toxic in its use.

The above-mentioned crosslinking agent containing a degradable ester bond under physiological conditions includes various glycidyl esters of diacids, acrylic acid/glycidyl methacrylate or acrylic acid/methyl groups of various polyols or polyhydric alcohols. A complex of one or more of the polyacrylates of acrylic acid.

Crosslinking of small molecular weight (200-20000Da) linear or branched polyethyleneimine with various diacid glycidyl esters or glycidyl acrylates can introduce hydroxyl groups into the polyethyleneimine structure, thereby improving physiological conditions Lower solubility and improved affinity for cell membranes. Crosslinking of small molecular weight (200-20000Da) linear or branched polyethyleneimine with various polyols or polyhydric alcohols of acrylic acid/methacrylic acid can introduce appropriate hydrophobicity in the polyethyleneimine structure Hydrophilic structure, thereby improving the cross-linking product to fine Hydrophobic interaction of the membrane. If a polyethyleneimine is reacted with a polyacrylic acid/methacrylic acid polyester of a polyol (for example, pentaerythritol triacrylate or pentaerythritol tetraacrylate), the degree of branching of the crosslinked product can be increased, thereby adjusting the degradation rate and realizing a drug or organism. Controlled release of active substances. The reaction of the polyethyleneimine with the complex of the above three cross-linking agents can further adjust the hydrophilic-lipophilic balance value and the degree of branching of the product, thereby synthesizing gene-transfecting vectors of various structures.

The polyol/polymethacrylate polyacrylate of the above-mentioned polyol or polyhydric alcohol comprises: ethoxylated 1,6-hexanediol diacrylate (E0-HDM), tripropylene glycol diacrylate (TPGDA) ), dipropylene glycol diacrylate (DPGDA), propylene glycol diacrylate (PGDA), neopentyl glycol diacrylate (NPGDA), propoxylated (2) neopentyl glycol diacrylate (P0-NPGDA), Ethylene glycol diacrylate (EGDA), diethylene glycol diacrylate (DEGDA), triethylene glycol diacrylate (TEGDA), tetraethylene glycol diacrylate (TEGDA), 1, 6-hexanediol Diacrylate (HDDA), 1, 4-butanediol diacrylate (BDDA), pentaerythritol tetraacrylate, pentaerythritol triacrylate, dipentaerythritol pentaacrylate, polyethylene glycol (200) diacrylate [PEG ( 200) DA], polyethylene glycol (400) diacrylate [PEG (400) M], neopentyl glycol dimethacrylate (NPGDMA), trimethylolpropane trimethacrylate (TMPTMA), Tripropylene glycol dimethacrylate (TPGDMA), dipropylene glycol dimethacrylate (DPGDMA), propylene glycol dimethacrylate (PGDMA), ethylene glycol dimethacrylate (GDMA), diethylene glycol dimethacrylate (DEGDMA), triethylene glycol dimethacrylate (TRGDMA) ), tetraethylene glycol dimethacrylate (TEGDMA), pentaerythritol trimethacrylate (PETMA), pentaerythritol tetramethacrylate, 1,6-hexanediol dimethacrylate (HDDMA), 1, 4-butanediol dimethacrylate, glycidyl methacrylate (GMA), glycidyl acrylate (GA), polyethylene glycol (200) dimethacrylate, polyethylene glycol (400) II Methacrylate, 1, 3-butanediol dimethacrylate, diethylene glycol diacrylate.

The present invention also provides a derivative of the above biodegradable crosslinked polyethyleneimine, which is a biodegradable crosslinked polyethyleneimine, further glycosylated, polyglycolized, acylated or alkyl Thereby, various derivatives of crosslinked polyethyleneimine are obtained.

The present invention also discloses various targeted crosslinked polyethyleneimine based on the above biodegradable crosslinked polyethyleneimine and derivatives thereof.

The non-viral gene carrier based on the polymer material is not only safe, but also has a large number of characteristics that the reactive functional group is easily modified, and can impart a targeted delivery ability between the gene carrier cells through a specific modification reaction. Since the synthesized product contains an amine group and a hydroxyl group, these groups can be coupled to the targeting ligand either directly or via a suitable tether, which is a polyethylene glycol (P EG) chain, polysuccinic acid. , polysebacic acid (PSA), poly-L-glutamic acid, oligosaccharides, amino acid chains, or any other suitable linker. There may be multiple types of linkers in a particular polymer or polymerization reaction. Preferably, it represents a linear or branched polymer composed of carbon and oxygen, and suitably may also contain a cyclic, star-shaped or dendritic structure, such as, for example, a linear PEG residue, a multi-side grafted PEG, a star-shaped PEG, but preferably linear and multi-side grafting and star-shaped PEG. The latter is commercially available from Aldrich, Fluka, Sigma and Nectar (shearwater). The targeting molecule can be a ligand that targets a specific interaction and absorbs into the target organ tissue or cell, such as transferrin, asialoglycoprotein (ASGP), antibody/antibody fragment, low density lipoprotein, leukocyte , GM-CSF, G-CSF, M-CSF, stem cell growth factor, erythropoietin (EPO), epidermal growth factor (EGF), insulin, folic acid, lactose, galactose, asialo-serum mucin Mannose, mannose-6-phosphate, N-hexanoyl lactosamine, thrombomodulin, fusion agent, hemagglutinin HA2 and nuclear localization signals (NLS).

The crosslinked polyethyleneimine of the present invention is capable of efficiently transmitting hepatocytes or liver tissues by endocytosis of a selected nucleic acid mediated by a galactosyl receptor on the surface of hepatocytes. Gene delivery to other tissues can be performed by coupling corresponding targeting molecules, such as mannose-6-phosphate (directed monocytes), mannose (directed macrophages and certain B-cells), N-acetyl Lactosamine (directed T-cell), galactose (directed melanoma cells), glucose (directed colon cancer cells) and thrombomodulin (directed mouse lung endothelial cells) and the like.

In gene delivery systems, particle size optimization is critical because particle size often controls transfection efficiency, cytotoxicity, and tissue orientation in vivo. See Haensler, J. and Szoka, FC, Jr. Bioconjugate Chem. 1993, 4, 372-379. Generally, the size of the gene delivery particles should not exceed the size of the virus, thereby enabling the gene delivery particles to effectively penetrate into the tissue. In the present invention, the size of the particles can be easily changed by using different combinations of the crosslinking agent and the polymer. The size and structure of a single polymer determines the number of aggregations, i.e., the number of individual copolymers that aggregate to form micelles. Thus, the size and structure portion control the particle size of the complex formed by the polymer and the nucleic acid, i.e., the micelle. The particle size can in turn be further controlled by the conditions and methods of preparing the particles. The particle size and charge density of the complex formed by the polymer and the nucleic acid can be easily controlled by changing the chemical composition and structure, molecular weight and charge density of the synthesized cationic polymer. An advantage of the present invention is that the particle size and charge density of the complex formed by the synthesized gene vector and DNA are easily controlled. Since the particle size of the transfection complex has a great influence on transfection efficiency, cytotoxicity and distribution within the composite object and tissue targeting, in order to effectively enter the cell or penetrate the tissue, the particles of the transfection complex are generally The diameter is smaller than the size of the virus particles. The present invention adjusts the size of the particle size of the composite by controlling the hydrophilic-lipophilic balance of the synthetic polymer by crosslinking the small molecule PEI with a combination of various crosslinking agents. Specifically, after optimizing the composition of the synthesized gene carrier and the ratio of the complex formed with the DNA, the formed complex has a particle size ranging from 20 to 200 nm. It is reported that particles of different sizes will accumulate in different organs of the body when injected into the body. For example, particles less than 150 nanometers can be administered through the membrane opening of the sinusoidal canal of the liver endothelium, and remain in tissues such as the spleen, bone marrow, and tumor. By using the novel cationic polymer produced by the invention and various plasmids or oligonucleotides, the particle size of the microparticles formed is completely controllable, has excellent dispersion, and thus has good tissue or Organ targeting. It is generally accepted in the industry that after microparticles, nanospheres and microspheres are injected into the body, their relative distribution ratio in different organs depends on their particle size. For example, for particles with a particle size below 150 ran, after systemic injection, it can pass through the membrane opening of the sinusoidal tubules of the liver's endothelium, which in turn can reach the pancreas, bone marrow, and tumor tissue. For particles with a particle size between 100 nm and 200 nm, intravenous, arterial or intraperitoneal injections are usually easily phagocytosed by macrophages from the reticuloendothelial tissue in the bloodstream. The biodegradable, biocompatible crosslinked polyethyleneimine and its derivatives obtained according to the present invention are non-immunogenic and non-toxic. The preferred polymers disclosed herein degrade into non-toxic small molecules that can be excreted by the kidney, which are inert during the desired gene expression. Degradation is carried out by a simple hydrolysis reaction. When the polymer backbone contains ester linkages, degradation by simple hydrolysis predominates. The length of the degradation period can be changed by using different kinds and molecular weight crosslinking agents. Therefore, biodegradable polymers can be used for gene delivery to address the toxicity problems associated with polycationic gene vectors. It is well known that most polycationic gene carriers have severe cytotoxicity and can cause serious consequences if they persist in the body for a long time. Therefore, a preferred gene vector should be capable of degrading into a non-toxic product upon completion of the action. The present invention utilizes biodegradable crosslinked polyethyleneimine containing ester linkages which have a safe, biocompatible degradation pathway. Such polymers facilitate the preparation of formulations for injectable, sustained and continuous release of encapsulated drugs. The highly branched molecular structure of the present invention can further reduce cytotoxicity because branched cations such as dendritic polyamidoamines have lower cytotoxicity than linear polycations. See Haensler, J. and Szoka, FC, Jr. Bioconjugate Chem. 1993, 4, 372-379. Thus, advantageous components and structures of the polymers of the present invention will be desirable due to reduced cytotoxicity. DRAWINGS

Figure 1 shows the degradation of cross-linked PEI in PBS (140 mM NaCl, 2.7 mM KC1, 10 mM Na ₂ HP0 ₄ , 1.8 mM KH ₂ P0 ₄ , pH 7.4). The synthesized crosslinked PEI was dissolved in PBS, allowed to stand at 37 ° C for a while, and the molecular weight was measured by a capillary viscosity method and the molecular weight of the polymer was plotted against time.

Figure 2 shows agarose gel electrophoresis of cross-linked PEI/DNA complexes. The results show that the synthesized cross-linked PEI has the same ability to bind DNA as PEI 25 kDa. Lane 1 : DNA only; from lane 2 to 7, the polymer:DNA ratio (w/w) is 0.1 :1 (lane 2), 0.2:1 (lane 3), 0.3:1 (lane 4), 0.35:1 (lane 5), 0.4:1 (lane 6) and 0.45:1 (lane 7). (A) Cross-linked PEI (B) 25 kDa PEL

Figure 3 shows that when the complex formed by cross-linking PEI and DNA in PBS is diluted with serum-containing complete medium, the complex can be stabilized against salt-induced aggregation.

Figure 4 is a comparison of the toxicity of cross-linked PEI with 25 kDa PEI on cells. The results show that the cytotoxicity of the synthesized cross-linked PEI is significantly less than 25 kDa PEI.

Figure 5 is a graph showing the percentage of survival of HEK293 cells after gene transfection of various transfection reagents under optimal transfection conditions. Figure 6 is a fluorescent photograph of cross-linked PEI-mediated GFP plasmid transfection in various cells for 24 hours. White light photo and superimposed photo _P

Figure 7 shows the transfection efficiency of the synthesized cross-linked PEI after 24 hours of transfection of different cells by flow cytometry.

Figure 8 is a comparison of the transfection efficiency of CLPEI and Gal-PEI for different cells. Figure 9 is a comparison of the transfection efficiency of CLPEI and FOL-PEI for different cells.

Figure 10 is a comparison of transfection efficiency of transfected PEI with various commercial transfection reagents and 25 kDa PEI, respectively, in NIH 3T3 cells under respective optimized transfection conditions.

Figure 11 RT-PCR analysis of cross-linking PEI-mediated EGFR R A interference plasmid affects EGFR mRNA levels. Where 1: PBS/PBS; 2: CLPEI/PBS; 3: CLPEI/interference plasmid; 4: CLPEI/control interference plasmid

Figure 12 Western blotting analysis of cross-linked PEI-mediated Erk RNA interference plasmid affects Erk protein expression.

Wherein: A549; 2: A549 + siRNA; 3: Hela; 4: Hela + siRNA

Figure 13 Western blotting analysis of cross-linked PEI-mediated PTEN RNA interference fragment affects PTEN protein expression. 1 : CLPEI/chemically synthesized interference fragment control; 2: CLPEI/chemically synthesized interference fragment 3: CLPEI/chemically synthesized interference fragment Control; 4: CLPEI/chemically synthesized interference fragment. Among them, 1 and 2 are A549 cells, and 3 and 4 are Hda cells.

Figure 14 is a photomicrograph of a cross-linked PEI-mediated GFP transfected for three days in the muscle site of C57 mice, a white light photograph, and a superimposed photograph of both.

Figure 15 is a photomicrograph and white light photograph of cross-linked PEI-mediated GFP transfected three days after C57 mouse B16F10 tumor site. detailed description

The present invention will be more fully understood from the following description of the preferred embodiments of the invention.

Polyethylenimine can be prepared in a well-known manner or commercially available according to its own needs, from the BASF trade name Lupasol or the name polyethyleneimine or ethyleneimine polymer, with different molecular weights from 200 to 2 000 00 g / mol (from Aldrich, sigma, Fluka, Polysciences or directly from BASF). Preference is given to polyethyleneimine having a molecular weight of from 400 to 20,000 g/mol, particularly preferably from 400 to 5,000 g/mol of polyethyleneimine.

Various cross-linking agents can be obtained commercially according to their needs (from Aldrich, sigma, Fluka, or directly from the manufacturer). If necessary, some cross-linking agents can also be prepared by literature methods, such as adipic acid. The diglycidyl ester (DA) can be synthesized by literature methods (Zondler, H. Helv. Chim. Acta 1977, 60, 1845-1860). Synthesis and properties of the crosslinked PEI of Example 1.

1 g of PEI (Mw: 2000 Daltons) is dissolved in 3 ml of freshly distilled dichloromethane, then a suitable ratio of diglycidyl adipate and hexanediol diacrylate is added at 40 ° C for a suitable period of time. The solution gradually turns yellow. The solution was then transferred to a spectra / Por Mwco 10,000 membrane and dialyzed against double distilled water for 4 days at 4 °C. It was then lyophilized to give a pale solid (CLPEI). Store at _70 °C. Various crosslinked PEI polymers can be obtained using different crosslinkers and PEI combinations (Table 1). The reaction combination is shown in Formula 1, and the obtained polymer structure is shown in Formula 2. The product was subjected to HNMR (varian 300 MHz, D ₂ O ) analysis, and it was found that there was no absorption peak of double bond hydrogen (5.5-6.0 ppm CH ₂ =CH-) in the starting material, but there was 3.4 'ppm (HO-CH2). ), an absorption peak of 2.95 ppm (PEI-NH-CH2-) and 2.69 ppm (-CH ₂ -C=0).

Equation 1. Possible cross-linking reaction

2. Possible structure of crosslinked polyethyleneimine Crosslinking PEI (CLPEI) molecular weight determination and degradability experiment

A PEI standard of known molecular weight or a synthetic crosslinked PEI sample was formulated into a series of concentrations of PBS solution at a pH of 7.4. Record the time at which the solution of each sample flows through the capillary viscometer at atmospheric pressure. The intrinsic viscosity of the solution is η _χύι = \ηη _κ \Ιο, where η _κ ι Relative viscosity ^rei = oiution/ oivent, The ratio of the time the polymer solution exits the capillary to the time the solvent flows out of the capillary, and C is the concentration of the polymer. The molecular weight of the synthesized polymer is calculated by the following formula To, ? /i _n h= 3⁄4f, where M is the molecular weight, and a is the Mark-Houwink parameter, and the value of a can be calculated from the PEI standard of known molecular weight using the above formula. The synthesized crosslinked PEI was dissolved in PBS, allowed to stand at 37 ° C for a while, and the molecular weight was measured as described above and the molecular weight of the polymer was plotted against time. The molecular S of the obtained polymer and its degradability results are shown in Fig. 1. The results showed that the molecular weight of the polymer was 13,000, and after about 50 hours, it was degraded into a small molecular material having a molecular weight of 2000. Table 1 Properties of various synthetic crosslinked polyethyleneimine

Raw material product properties

PEI crosslinker water soluble HEK 293 (GFP%)

PEI 600 ethylene glycol diacrylate (EGDA) dissolution 40

1,4-butanediol diacrylate (BDDA) dissolution 30

Pentaerythritol triacrylate dissolution <10

Glycol dimethacrylate (GDMA) dissolves 55

Triethylene glycol dimethacrylate (TRGDMA) dissolution <10

Polyethylene glycol (400) dimethacrylate [PEG(400)DMA] Dissolved <10

Adipic acid diglycidyl ester (DA) dissolved <10

Glycidyl methacrylate (GMA) dissolved 10

GDMA and GMA dissolve <10

HDDA and GMA dissolve <10

BDDA and GMA dissolve <10

PEI 2000 ethylene glycol diacrylate (EGDA) dissolution 40

Propylene glycol diacrylate (PGDA) dissolves 40

1,4-butanediol diacrylate (BDDA) dissolution 50

1,6-hexanediol diacrylate (HDDA) slightly soluble 40

Diethylene glycol diacrylate (DEGDA) dissolution 40

Pentaerythritol triacrylate Dissolution 30

Diethylene glycol dimethacrylate (DEGDMA) dissolution 40

Triethylene glycol dimethacrylate (TRGDMA) dissolution 60

Polyethylene glycol (400) dimethacrylate [PEG(400)DMA] Dissolve 40

Adipic acid diglycidyl ester (DA) dissolves 30 Glycidyl methacrylate (GMA) dissolves 50

EGDA and GMA dissolve 60

HDDA and GMA slightly soluble -

BDDA and GMA slightly soluble -

PEI 3000 ethylene glycol diacrylate (EGDA) dissolution 40

Propylene glycol diacrylate (PGDA) dissolves 40

1,4-butanediol diacrylate (BDDA) slightly soluble 60

1,6-hexanediol diacrylate (HDDA) insoluble - diethylene glycol diacrylate (DEGDA) dissolution 40

Pentaerythritol triacrylate Dissolution 30

Diethylene glycol dimethacrylate (DEGDMA) dissolution 50

Triethylene glycol dimethacrylate (TRGDMA) dissolves 70

Polyethylene glycol (400) dimethacrylate [PEG(400)DMA] Dissolve 40

Adipic acid diglycidyl ester (DA) dissolved 40

Glycidyl methacrylate (GM A) dissolves 60

EGDA and GMA dissolve 80

HDDA and GMA are insoluble ―

BDDA and GMA are poorly soluble - Example 2 Cross-linking PEI lactose and folic acid modification

Crosslinking PEI is activated by EDC ( l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide ) and the amino group is linked to lactobionic acid by an amide bond. The crosslinked PEI synthesized in 200 mg of Example 1 was dissolved in 6 ml of 10 mM TEMED/HCl buffer. Then 180 mg of EDC was added and stirred at 25 ° C for 24 hours. Then, an appropriate amount of lactobionic acid was added, and the mixture was stirred at 25 ° C for 72 hours. The resulting product was dialyzed against double distilled water for 4 days and lyophilized to give a galactosyl modified crosslinked PEI (Gal-PEI).

10 mg of folic acid and appropriate amount of DCC were dissolved in freshly distilled dimethyl sulfoxide in DMSO for a period of time, and then 200 mg of PEI synthesized in Example 1 was added. After reacting for 12 hours with stirring, it was dialyzed against double distilled water for 4 days under 4 Torr. The water is then lyophilized to obtain a folic acid modified crosslinked PEI (Fol-PEI). Example 3 Crosslinking Preparation and Characterization of PEI and pEGFP-Cl Complexes

Preparation of complex The biodegradable cross-linked PEI or PEI 25kDa synthesized in Example 1 was dissolved in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HP0 ₄ , 1.8 mM KH ₂ P0 ₄ , pH 7.4), respectively. Formulated as a stock solution of 1 mg/ml. The plasmid DNA was diluted to lmg / ml stock solution with ddH ₂ 0. Prepare a complex of PEI and DNA of cross-linked PEI or PEI 25kDa according to a certain PEI/DNA (mass ratio), for example, to prepare a complex of cross-linked PEI/DNA=2:1, the specific process is as follows: Take Ιμΐ plasmid DNA solution (lmg/ml) Dissolve in 50 μM PBS, mix gently, then take 2 μM cross-linking ΡΕΓ dissolved in 50 μl PBS, mix gently, then mix the two, shake for 10 seconds, let stand at room temperature 10 — 15 minutes, which can be used to characterize its performance and cell transfection experiments.

Electrophoresis block experiment

Cross-linking ΡΕΙ and ΡΕΙ 25 kDa and DNA were prepared in a series of cross-linked enthalpy to DNA mass ratios (0:1, 0.1:1, 0.2:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1) The complex, containing 2.5 (Ο.ΐμ^μΐ) of plasmid DNA (pEGFP-Cl) and a certain amount of cross-linked polymer in PBS, total volume 50 μl, this solution was allowed to stand at room temperature for 10-15 minutes. ΙΟ μΙ was mixed with 2 μl of the loading buffer, and then 0.8% agarose (containing 0.5 g/ml ethidium bromide) gel was applied, and the electrophoresis was carried out at a voltage of 5 V/cm. As shown in Fig. 2, cross-linked PEI has the same DNA binding ability as PEI 25 kDa, and they completely encapsulate DNA molecules at PEI/DNA=0.35:1, so that DNA molecules cannot migrate in electrophoresis.

Particle size analysis of composites

The particle size of the composite was determined by light scattering at 25 ° C using the Brookhaven 90 PLUS particle size analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA). Wavelength 660 nm, constant angle 90. On the basis of gravimetric analysis, assuming that the crosslinked PEI/DNA complex is a lognormal distribution, its size is expressed as the effective diameter. The composite was prepared in the same manner as above except that the total volume of the complex was increased to an equal ratio of 1 ml. Specifically: 10 μ _§ ϋΝΑ dissolved in 500 μl of PBS, appropriate amount (according to PEI / DNA mass ratio) of cross-linked PEI dissolved in 500 μΐ PBS, then mix the two, shake at room temperature for 10 seconds, static After 45 minutes, a light scattering experiment was performed. The results are expressed as mean particle size ± SD, n = 3. In order to study the effect of serum on the stability of the complex, the comparative experiment was as follows: Ιμΐ plasmid DNA solution (1mg/ml) was dissolved in 50 μl ΡΒβ, and the appropriate amount (according to the PEI DNA mass ratio requirement) of cross-linked PEI was dissolved. Into 50 l PBS, mix the two, shake for 10 seconds, let stand for 10-15 minutes at room temperature, add 0.9 ml of DMEM medium containing 10% newborn calf serum, and perform light scattering experiment after 30 minutes. The results are expressed as mean particle size ± SD, n = 3. As shown in Fig. 3, the complex of cross-linked PEI and DNA without complete serum-containing medium was aggregated after 45 minutes, and the particle size reached about 400 nm, which significantly inhibited cross-linking after adding complete medium. The aggregation of the complex formed by PEI and DNA has a particle size of only 150 nm or less under the same conditions and can be maintained for several hours.

Cytotoxicity test

The cytotoxicity of the crosslinked polymer was determined by tetrazolium salt (MTT) colorimetry and compared to PEI 25 kDa. 3T3 and 293T cells were cultured in DMEM containing 10% newborn calf serum (containing 100 U/ml penicillin and 100 g/ml streptomycin) and placed in Grow in 37 ° C, 5% C0 ₂ incubator. After at logarithmic growth phase 3T3 and 293T cells, containing 0.02% EDTA and 0.25% trypsin digestion solution per well digested lxlO ⁴ cells were seeded in 96-well plates, each well volume of 100 μΐ. The plates were incubated overnight at 37 ° C in a 5% CO ₂ incubator. The medium was removed, washed with 1 x PBS, and different doses of synthetic polymer or PEI 25 kDa and serum-free medium were added to each well for 5 hours. The medium was removed, washed in 1 x PBS, and 100 μL of complete medium was added to each well for 24 hours. Then 20 l (5 mg/ml) MTT solution was added to each well and reacted at 37 ° C for 4 hours. The culture supernatant in the wells was carefully discarded, 100 μM dimethyl sulfoxide (DMSO) was added to each well, and incubated for 30 minutes at room temperature. After shaking, the absorbance at 570 nm of each well was measured by a microplate reader (Bio-RAD, Microplate Reader 3550).

Cell viability (%) = (OD _57t) sample / 00 _{57 ()} control) xl00

The results are shown in Figures 4 and 5. Figure 4 shows that for the two cell lines 293T and 3T3, the synthesized cross-linked PEI is much less toxic than the 25 kDa PEI cell, and under optimal transfection conditions (2 g/ml) ), cross-linked PEI has little cytotoxicity and cell survival rate of more than 95%. Figure 5 shows that the synthesized cross-linked PEI cells are also less toxic than other commercial transfection reagents. Example 4 Crosslinking Determination of PEI Transfection Performance

Cell transfection experiment

Various cell cultures were grown in DMEM or RPMI1640 medium (containing 100 U/ml penicillin and 100 g ml streptomycin) containing 10% newborn calf serum and placed in an incubator at 37 ° C, 5 % €0 ₂ . The specific steps of cross-linking PEI or PEI 25kDa and DNA complex transfected cells are as follows: 24 hours before transfection, cells in logarithmic growth phase are digested with 0.02% EDTA and 0.25% trypsin digest. Holes lxlO ⁵ cells were seeded in 24-well plates, 0.5 ml of complete medium was added to each well, and the plates were transferred to an incubator for 24 hours. When the cell confluence reaches 70-80%, the culture solution is removed, and the transfection complex is prepared by the method described above, specifically: a solution of Ιμΐ plasmid DNA (1 mg/ml) is dissolved in 50 μM PBS, and gently mixed. Then take 2 μΐ of cross-linked PEI dissolved in 50μ1 PBS, mix gently, then mix the two, shake for 10 seconds, let stand for 10-15 minutes at room temperature, then add 900 μΐ DMEM containing 10% newborn calf serum. After the liquid was mixed, a total of 1000 μΐ of the transfection solution was obtained, and then added to one well. The cells were cultured at 37 ° C for 24 hours, and observed under a fluorescence microscope and photographed. The results are shown in Figure 6. The synthesized cross-linked PEI has high transfection efficiency for the three cell lines 293T, A549 and B16F10, and the transfection efficiency of the two cell lines 293T and B16F10 is over 90%. The transfection efficiency of A549 cells was nearly 70%.

Expression of EGFP and analysis by flow cytometry

The above-mentioned transfection expression system was cultured for 24 hours after transfection, and the expression of EGFP was analyzed by flow cytometry (FACS Calibur). Specifically: an argon laser was used for analysis at 488 nm, and cells that were not transfected were used as background. Cells transfected were digested with 0.02% EDTA and 0.25% trypsin, centrifuged and resuspended in PBS. Medium, then flow cytometry analysis. Randomly collect 10,000 cells, calculate the proportion of cells in which EGFP is expressed, and use the data. CellQuest (Becton Dickinson) treatment, CLPEI transfection results of various cell lines are shown in Figure 7, the transfection efficiency of the selected 9 kinds of cells except C6 cell line is only about 30%, and other cell lines are More than 50%.

In order to observe the selectivity of Gal-PEI for the asialo-glycoprotein (ASGP) cell line on liver-derived cell membranes, Gal-PEI and CLPEI were used to transfect SMMC-7721 hepatoma cell line with ASGR and not, respectively. Hela cell line with this receptor. The results showed that the Gal-PEI obtained by the modified galactosyl group of CLPEI had certain selectivity to the cell line with ASGR receptor, and under the condition of low cross-linked PEI to DNA weight ratio, this choice Sex is more obvious (Figure 8).

To observe the selectivity of Fol-PEI to the cells of the folate receptor on the cell membrane, Fol-PEI and CLPEI were used to transfect KB cells expressing the folate receptor and A549 cells without the receptor, respectively. The results showed that the Fol-PEI obtained by the modification of CLPEI by folic acid had certain selectivity to the cell line with folate receptor, and this selectivity was more under the condition of low cross-linked PEI to DNA weight ratio. Obvious (Figure 9).

To compare the transfection efficiency of various commercial transfection reagents with our synthetic cross-linked PEI for NIH 3T3 cells, various transfection reagents were transfected into pEGFP-Cl plasmids to NIH 3T3 under optimal transfection conditions. In the cells, the transfection efficiency was analyzed by flow cytometry. As shown in Fig. 10, the transfection efficiency of the synthesized cross-linked PEI to the 3T3 cell line was significantly higher than that of several other commercial transfection reagents. Example 5 Cross-linked polyethyleneimine (CLPEI) mediated RNAi interference assay

1. Cross-linking PEI-mediated RNAi study of epidermal growth factor receptor (EGFR)

B16F10 cells were cultured in DMEM containing 10% newborn calf serum (containing 100 U/ml penicillin and 100 g/ml streptomycin) and placed in an incubator at 37 ° C, 5 % C0 ₂ . The successfully constructed murine EGFR interference vector pBSU6-EGFR was transfected into cells as in Example 4. After 48 hours, the cells were harvested and total mR A was extracted with Trizol (Invitrogen), and the mRNA level of EGFR after interference was analyzed by RT-PCR, and GAPDH was set as an internal reference. As a result, as shown in Figure 11, the synthesized cross-linked PEI successfully mediates the entry of EGFR interference plasmid into the cells, resulting in a significant down-regulation of EGFR mRNA levels.

2. Cross-linking RNAi study of PEI-mediated extracellular-signal regulated kinase (ERK)

A549 and Hela cells were cultured in DMEM containing 10% newborn calf serum (containing 100 U/ml penicillin and 100 g/inl streptomycin) and placed in an incubator at 37 ° C, 5 % C0 ₂ . The successfully constructed Erk interference vector pBSU6-ERKl/2 was transfected into cells in the same manner as in Example 4. After 48 hours, the cells were collected and the total protein was extracted. Western blot was used to analyze the changes in the protein level of Erk after the interference, and Tubulin was set as the internal reference. As a result, as shown in Figure 12, the synthesized cross-linked PEI successfully mediates the entry of the Erk interference plasmid into the cells, resulting in a significant down-regulation of the protein level of Erk.

3. Cross-linked PEI-mediated PTEN (phosphatase and tensin homologue deleted on chromosome ten) RNAi study A549 and Hela cells were cultured in DMEM containing 10% newborn calf serum (containing 100 U/ml penicillin and 100 g/ml streptomycin) and placed in an incubator at 37 ° C, 5 % C0 ₂ . The chemically synthesized small interfering R As mixture (siRNA/siABTM Assay Kit, Upstate Catalog # 60-036) was transfected into cells according to a similar method as in Example 4. After 48 hours, the cells were collected and the total protein was extracted. The protein level of PTEN after interference was analyzed by Western-blot analysis, and Beta-actin was used as an internal reference. As a result, as shown in Figure 13, the synthesized cross-linked PEI successfully mediates the entry of PTEN-interfering fragments into cells, resulting in a significant down-regulation of PTEN protein levels. Example 6 In vivo transfection experiment of cross-linked PEI

Expression of EGFP in muscle of C57 mice

10 pEGFP-Cl DNA (Clontech) ddH ₂ 0 solution (1 mg / ml) and cross-linked PEI in PBS (1 mg / ml) mixed 1: 2, to prepare a total volume of 50 μ ΐ transfection complex, After standing for 15 minutes at room temperature, eight-week-old C57 mice (Shanghai Experimental Animal Center) cut leg hair on the left and right hind legs, and each of the left and right hind legs were injected with 50 ^ PBS and 50 μΐ of the above transfection complex. . Four groups of 6 mice in each group were set up and killed on the first day, the third day, the fifth day, and the seventh day after administration, and then frozen sections were prepared, and the expression of EGFP was observed under a fluorescence microscope. Happening. The results are shown in Figure 14. The superposition of white light and fluorescence indicates a transfection efficiency of 90%.

Expression of EGFP in C57 mouse tumors

C57BL/6J mice (6 to 8 weeks) were subcutaneously inoculated with B16F10 cells ⁵ ×10 5/50 μ1, and animals were randomly divided into tumors with a diameter of 50 mm ³ or so. In the transfection group and the blank control group, 50 μΐ of the cross-linked PEI-plasmid complex was injected into the tumor (the amount of the injected plasmid was 10 μ _§ /50 μ1), and the control group was intratumorally injected with an equal volume of 0.9% physiological saline, the third day. The mice were sacrificed, the subcutaneous tumors were removed, frozen in a cryostat, and sections were frozen. The expression of EGFP was observed under a fluorescence microscope. The results are shown in Figure 15, indicating that the transfection efficiency reached nearly 90%. The experiment of the above embodiment is repeated with PEI20000 and PEI10000 as raw materials, and the results show that the object of the present invention can also be achieved. It should be understood that after reading the above contents of the present invention, those skilled in the art can make various modifications to the present invention. And equivalents, such equivalents also fall within the scope defined by the claims of the present application. References to which the present invention relates:

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Claims

Rights request

A biodegradable crosslinked polyethyleneimine comprising a linear or branched polyethyleneimine having a molecular weight of from 200 to 100,000 Da and a degradable ester having a molecular weight of from 50 to 50,000 Da under physiological conditions. The molar ratio of the polyethyleneimine to the crosslinking agent is 0. 1: 1-5: 1.

2. The crosslinked polyethyleneimine according to claim 1, wherein: said crosslinking agent is a glycidyl ester of various diacids, acrylic acid/glycidyl methacrylate or various polyols or A complex of one or more of a polyhydric alcoholic acrylic acid/methacrylic acid polyester.

3. The biodegradable crosslinked polyethyleneimine according to claim 1, which may be further glycosylated, polyglycolized, acylated or alkylated to obtain various crosslinked polyethyleneimine. Derivatives.

4. The crosslinked polyethyleneimine and derivative thereof according to any one of claims 1 to 3, further coupled to various targeting molecules to obtain various targeted crosslinked polyethyleneimine; said target The molecular molecules are: transferrin, asialoglycoprotein, antibody/antibody fragment, low density lipoprotein, interleukin, GM-CSF, G-CSF, M-CSF, stem cell growth factor, erythropoietin, epidermis Growth factor, insulin, folic acid, lactose, galactose, asialo serum mucin, mannose, mannose-6-phosphate, N-hexanoyl lactosamine, thrombomodulin, fusion agent, hemagglutinin HA2 or nuclear Positioning signal.

A composition comprising the crosslinked polyethyleneimine of claim 1 and a nucleic acid.

6. A composition according to claim 5, wherein said nucleic acid is a DNA, a therapeutic gene, an RNA, a catalytically active nucleic acid, an antisense oligonucleotide or a modified nucleic acid.

A composition comprising the targeted cross-linked polyethyleneimine and nucleic acid of claim 4.

A method of transfecting a cell with a nucleic acid, which comprises the step of contacting the cell with the composition of the crosslinked polyethyleneimine of claim 1 and a nucleic acid.

9. A kit comprising the crosslinked polyethyleneimine of claim 1 and instructions for directing the binding of the above substances and nucleic acids to nucleic acid transfected cells.

10. The cross-linked polyethyleneimine according to claims 1-4, which is used in vitro as a gene transfection reagent and a drug delivery system for gene therapy in vivo, and can also be used as a bioactive agent such as a drug, a protein or a polypeptide. Transmission carrier.