CN101899154A

CN101899154A - Derivative of novel PAMAM and application thereof

Info

Publication number: CN101899154A
Application number: CN 201010142282
Authority: CN
Inventors: 丁克; 吴东海; 周毅; 常少华; 高学飞; 杨淑伟
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2010-04-01
Filing date: 2010-04-01
Publication date: 2010-12-01

Abstract

The invention discloses a derivative of novel PAMAM and application thereof. The derivative has a phenylalanine coupled and modified PAMAM tree-like molecular structure, and the molecular structure of the derivative is as follows, wherein n is 50 to 60. The derivative of the PAMAM, serving as a transfection reagent, is applied to biological transfection; the PAMAM-Phe greatly enhances the gene transfer capability of the primary reagent PAMAM and remarkably reduces the toxicity of cells, the transfection capability of the PAMAM is not affected by serum, and a serum culture medium does not need to be changed before transfection so as to greatly simplify the steps of experiment operations; and the PAMAM-Phe also can be effectively used for siRNA transfection and in vivo transfection of genes, and displays potential application value in gene therapy.

Description

A kind of derivative of novel PAMAM and application thereof

Technical field

The present invention relates to biological technical field, relate to a kind of derivative and application thereof of novel PAMAM specifically.

Background technology

Gene therapy is a kind of approach of extremely effectively treating for the disease of heredity and the formation day after tomorrow, but transfer of genetic material to be remained a very big technical barrier in body, seek the bottleneck that safe, effective and specific gene transfection method remains restriction gene treatment widespread use.Gene delivery mainly contains two class carriers at present, and viral carrier and non-viral carrier.Although viral carrier all shows very high efficient in the gene delivery in vitro and in vivo, the permanent integration of their immunogenicity, virogene and operation loaded down with trivial details limited their application in gene therapy.Comparatively speaking, non-virus carrier system (also being the synthetic gene movement system), non-immunogenicity simple to operate because of it, gene delivery ability be big etc., and advantage more and more is subject to people's attention.But the transhipment of right and wrong virus also has the relatively low and bigger weakness of cytotoxicity of transfection efficiency.However, the polycation dendrimer still has very big magnetism, be because their texture ratio is clearer and more definite on the one hand, have a plurality of functional groups on these macromolecular surfaces on the other hand, obtain biological activity and the higher novel gene transport vehicle of biocompatibility thereby can carry out chemically modified further.

Daiamid (PAMAM) is one of dendrimer family of studying at most at present, studies have shown that it has a stronger gene delivery ability external.Owing to have a lot of positively charged primary amine groups on the dendritic surface of PAMAM, it can be compressed into the complex body form of a poly with electronegative DNA or RNA, thereby promotes gene to intracellular transport.The gene delivery ability of PAMAM and cytotoxicity are that the character with its surface functional group is closely related.The surface is electronegative or the neutral dendrimer is bigger than positively charged dendrimer toxicity.In addition, the gene delivery decisive role that the physicochemical property such as difficulty of DNA/RNA release also mediate PAMAM in the solvability of polymkeric substance-DNA/RNA complex body, the born of the same parents.In order further to increase the gene delivery ability and the biocompatibility thereof of PAMAM dendrimer, a variety of parts are connected on the primary amine group on PAMAM surface to change its surface properties.The gene delivery ability with remarkable increase that the derivative that obtains through these modifications has, the cytotoxicity that has descends, and also has some then improvement all to be arranged at this aspect two.

Summary of the invention

One of purpose of the present invention provides the derivative of a class novel PAMAM, and the derivative of this PAMAM has good transfection efficiency.

The derivative of one class novel PAMAM, this derivative is modified the PAMAM dendrimer for the phenylalanine coupling, and its molecular structure is:

Wherein, n is 50～60.

Another object of the present invention provides the application of the derivative of above-mentioned PAMAM.

Concrete technical scheme is as follows:

The derivative of PAMAM as transfection reagent in APPLICATIONS IN BIOLOGY.

Preferably, the described biological transfection cell transfecting that is plasmid DNA and microRNA; The described siRNA cell transfecting that is applied as; Or described being applied as is used for packaging virus.

Another object of the present invention provides a kind of biological transfection reagent.

Concrete technical scheme is as follows:

A kind of biological transfection reagent, its major ingredient is modified PAMAM dendrimer structure for the phenylalanine coupling, and molecular structure is as follows:

Wherein, n is 50～60.

The present invention adopts amino acid link coupled method, amino acid by relatively different sorts and number is to a series of modification reaction of the 4th generation PAMAM dendrimer, utilize HEK 293 cells and 3T3-L1 cell that the transfection ability of the numerous compounds of this class is screened, find that the surperficial derivative PAMAM-Phe that is connected with phenylalanine has remarkable enhanced gene delivery ability and biocompatibility.Further, find that this compound has stronger vivo gene turn-over capacity, and it can be used for the biological experiment operation of multiple widespread uses such as siRNA transhipment, virus packing.

The more not modified PAMAM of PAMAM-Phe of the present invention has significant advantage: first, its gene delivery ability strengthens greatly, both shown in the transfection to single plasmid, also show in the transfection of the transfection of siRNA and a plurality of genes, in addition, its gene delivery ability in vivo also strengthens greatly; The second, the gene transfection ability of PAMAM-Phe is not answered the existence of serum and is reduced, and therefore need not need change serum free medium before cell transfecting as the transfection of PAMAM mediation, has simplified operating process greatly; The 3rd, its biocompatibility significantly strengthens, and shows lower cytotoxicity, for road has been paved in its application in gene therapy; At last, our PAMAM-Phe that studies have shown that can be used for the packing of efficient transfection, retrovirus and the slow virus of siRNA as a kind of novel transfection reagent, and the feasibility of its widespread use in biological study is described.

Description of drawings

Fig. 1 amino acid coupled derivative PAMAM-Phe synthesizes synoptic diagram;

Fig. 2 PAMAM and PAMAM-Phe's ¹H NMR spectroscopic analysis figure;

19 kinds of PAMAM derivatives of table 1 synthetic table;

The preliminary screening synoptic diagram of Fig. 3 PAMAM amino acid coupled derivative (No. 7 is PAMAM-Phe, and No. 0 is PAMAM);

The quantitative synoptic diagram of Fig. 4 PAMAM-Phe mediation FUGW plasmid (containing the eGFP gene) transfection HEK 293 cell fluorescence intensities;

The quantitative synoptic diagram of Fig. 5 PAMAM-Phe mediation RSV-Luc plasmid (containing luciferase reporter gene) transfection HEK 293 cells;

Fig. 6 PAMAM-Phe mediation siRNA transfection HEK 293 cell synoptic diagram (A, the white light visual field; B, Lipofectamin 2000; C, PAMAM-Phe; D, PAMAM);

Fig. 7 PAMAM-Phe is applied to virus packing (A, C: utilize the slow virus and the retrovirus of Lipofectamin 2000 mediation packings to infect the 3T3-L1 cell respectively; B, D: utilize the slow virus and the retrovirus of PAMAM-Phe mediation packing to infect the 3T3-L1 cell respectively);

Fig. 8 PAMAM-Phe is to HEK 293 cytotoxic assay synoptic diagram;

GFP gene (FUGW plasmid) transhipment synoptic diagram (A, PAMAM-Phe transfection FUGW in Fig. 9 PAMAM-Phe mediation mouse gastrocnemius muscle; B, PAMAM transfection FUGW; C, PAMAM-Phe inject separately; D, FUGW plasmid are injected separately).

Embodiment

Chemosynthesis agents useful for same of the present invention is conventional commercial reagents, and the Bioexperiment material therefor is commerical prod.

PAMAM-Phe of the present invention; according to the state of the art; can be achieved through the following technical solutions: in organic solvent, carry out; with hydroxybenzotriazole HOBt; 2-(1H-benzotriazole)-N; N; N ', N '-tetramethyl-urea phosphoric acid salt HBTU, N; N-diisopropylethylamine DIPEA is a catalyzer; tert-butoxycarbonyl Boc is that the phenylalanine and amino PAMAM the 4th generation dendrimer of end of protecting group is raw material generation condensation reaction, and the back is refining with the anhydrous ether precipitator method, with the Boc protecting group on the trifluoroacetic acid deaminize acid amino; the anhydrous ether precipitator method are refining, the PAMAM dendrimer that the terminal amino acid that obtains purifying with 4 ℃ of pure water dialysis in semi-permeable membranes is modified.

The chemosynthesis of embodiment 1, PAMAM-Phe dendrimer and purifying:

As shown in Figure 1, the PAMAM G4 powder of getting 0.07mmol is dissolved in the 25ml dry DMF, the Boc-L-phenylalanine that adds 5.63mmol in proper order, the HBTU of 5.63mmol, the HOBt of 5.63mmol, 5.63mmol DIPEA, stirring at room 12h adds cold anhydrous diethyl ether and separates out precipitation, leaches precipitation, add 10ml dry DMF dissolving intermediate, add cold anhydrous diethyl ether once more and separate out precipitation and obtain thick intermediate product.

Get intermediate product and be dissolved in 22ml 90% trifluoroacetic acid, stirring at room 2h sloughs the Boc protecting group, adds a large amount of cold anhydrous diethyl ethers and separates out precipitation, leaches precipitation, adds 10ml dry DMF lysate, adds cold anhydrous diethyl ether once more and separates out than clean product.

Get product and be dissolved in the pure water, change 4 ℃ of dialysed overnight of dialysis tubing over to, lyophilize obtains white powder PAMAM-Phe product.(738mg, productive rate 47.6%, amino acid modified degree 84.4%). ¹H NMR spectrum (400MHz, D ₂O) Fig. 2, PAMAM-Phe: δ 2.47 (NCH ₂CH ₂CO-of PAMAM unit, 248H), 2.83 (CONHCH ₂CH ₂N-ofPAMAM unit and-NCH ₂CH ₂N-of PAMAM unit), 2.98 (CONHCH ₂CH ₂NH ₂Of PAMAM unit), 3.12 (NCH ₂CH ₂CO-of PAMAM unit and-CHCH ₂C ₆H ₅Of phenylalanine), 3.36 (CONHCH ₂CH ₂N-of PAMAM unit and-CONHCH ₂CH ₂NHCO-of PAMAM unit), 4.08 (HCCH ₂C ₆H ₅Of phenylalanine 54H), 7.27 (HCCH ₂C ₆H ₅Of phenylalanine 270H).

The preliminary screening of embodiment 2, PAMAM amino acid coupled derivative:

Utilize HEK 293 cells and FUGW plasmid that can the high expression level green fluorescent protein to be carrier, the outer-gene turn-over capacity of the 19 class PAMAM derivatives (table 1) that purifying is obtained is carried out preliminary evaluation.In 96 orifice plates, screen, every hole adds FUGW plasmid 0.1 μ g, the final concentration of PAMAM amino acid coupled derivative is 0.04 μ g/ μ l, in this ratio (N/P=40) FUGW and PAMAM amino acid coupled derivative are added to mixing among the 20 μ l PBS, room temperature leaves standstill 30min, join then in the nonresistant DMEM substratum and cultivate 4～6h, changing complete culture solution continues to cultivate, under the fluorescence inverted microscope, observe the egfp expression situation behind the 24h, find that phenylalanine coupled derivative (PAMA-Phe) has the strongest gene delivery ability (Fig. 3).

Tablel

Surface?modification?of?PAMAM?with?amino?acids

The physicochemical property analysis of embodiment 3, PAMA-Phe-DNA complex body:

Carry out dna gel retardance experiment, the PAMA-Phe-DNA complex body that will form by different N/P ratios (N/P) is (in the PAMA-Phe molecular structural formula, n is 50～60 these classes) analyze through 1% agarose gel electrophoresis, observe the migration situation of DNA band, find when N/P＞2, the migration of DNA in electric field is blocked fully, illustrates that PAMA-Phe dendrimer and dna molecular form very tight complex body.Further, utilize the particle diameter of the technical measurement complex body of dynamic light scattering, when N/P was 40, the complex body particle diameter of formation was 233nm, and this size helps the endocytosis of cell.

The in-vitro transfection ability of embodiment 4, PAMA-Phe is measured:

Detect the method for (Fig. 5) by fluorescence intensity quantitative (Fig. 4) and luciferase reporter gene (luciferase reporter gene), be determined at the in-vitro transfection ability under the situation of different N/P ratio, and to serum-free with there is the transfection effect under the serum environment to compare.In 96 orifice plates in N/P than be ratio every hole transfection 0.1 μ g FUGW plasmid of 40 to the HEK293 cell, renewed bright complete culture solution in four hours after the transfection, continue to be cultured to 24h after the transfection, under fluorescent microscope, observe fluorescence and fluorescence intensity.Similarly, in 96 orifice plates in N/P than being that ratio every hole transfection 0.05 μ g RSV-Luc plasmid of 40 is to the HEK293 cell, renew bright complete culture solution in four hours after the transfection, continue to be cultured to 48h after the transfection, measure the expression intensity of luciferase with the luciferase detection kit.The result shows that PAMA-Phe transfection ability compares remarkable enhancing with PAMAM, and the existence of serum does not have tangible negative interaction to its transfection effect;

Embodiment 5, PAMAM-Phe are in APPLICATIONS IN BIOLOGY:

Utilize PAMAM-Phe (in the PAMA-Phe molecular structural formula, n is 50～60 these classes) to come the siRNA of transfection fluorescent probe Cy3 mark, transfection efficiency is obviously wanted high a lot (Fig. 6) than PAMAM and business-like transfection reagent Lipofectamin 2000.In addition, and, also obtained good effect, can produce and obtain very high retrovirus of titre and slow virus with the cell transfecting in its mediation virus packets process of assembling.For retroviral packing, change the pMXs-GFP plasmid over to the Plat-E cell in the ratio of N/P40 with PAMAM-Phe; For the packing of slow virus, the Packing Mix (mixed by three kinds of packaging plasmids, the mass ratio of FUGW and Packing Mix is 1: 2) that FUGW plasmid and Invitrogen is bought with PAMAM-Phe changes the Plat-E cell in the ratio of N/P 40.Transfection was changed complete culture solution after four hours, collect the culture supernatant of 48h and 72h after the transfection respectively, with these two kinds of supernatants and 1: 1 mixing of fresh culture, each infecting mouse 3T3-L1 cell 12h, continue to cultivate 24h with fresh complete culture solution afterwards, under fluorescent microscope, observe the luciferase expression situation.

Embodiment 6, PAMAM-Phe cytotoxic assay:

In 96 orifice plate middle berth HEK, 293 cells, 8 * 10 ³Individual cells/well, use the PAMAM-Phe of different concns (in the PAMA-Phe molecular structural formula after one day, n is 50～60 these classes) and PAMAM processing HEK 293 cell 24h, add MTT (final concentration 1mg/ml) 37 ℃ and hatch 4h, every hole adds 150 μ l DMSO, measure the absorbancy of 570nm, the result shows that the cytotoxicity of (Fig. 7) PAMAM-Phe and PAMAM significantly reduce, and it is less to use the concentration cytotoxicity in its transfection.

The vivo gene transfection of embodiment 7, PAMA-Phe:

PAMA-Phe is (in the PAMA-Phe molecular structural formula, n is 50～60 these classes) form complex body with the FUGW plasmid according to the ratio of N/P 40, inject the dosage injection C57BL/6J mouse gastrocnemius muscle of 2 μ g plasmids with every gram body weight, get the injection areas frozen section behind the 48h, microscopically is observed the fluorescent protein expression situation, and the result shows that PAMA-Phe can mediate mouse vivo gene transhipment (Fig. 8) preferably.

More than be at the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of this case.

Claims

1. the derivative of a PAMAM is characterized in that, described derivative is modified PAMAM dendrimer structure for the phenylalanine coupling, and its molecular structure is as follows:

Wherein, n is 50～60.

The derivative of the described PAMAM of claim 1 as transfection reagent in APPLICATIONS IN BIOLOGY.

3. application according to claim 2 is characterized in that, described APPLICATIONS IN BIOLOGY is the cell transfecting of plasmid DNA and microRNA.

4. application according to claim 3 is characterized in that, the described cell transfecting that is applied as siRNA.

5. application according to claim 2 is characterized in that, described being applied as is used for packaging virus.

6. a biological transfection reagent is characterized in that, its major ingredient is modified PAMAM dendrimer structure for the phenylalanine coupling, and molecular structure is as follows:

Wherein, n is 50～60.