CN104017828B - A kind of cationic polymer that fluorine-containing aliphatic chain is modified and its purposes as genophore - Google Patents

Abstract

The present invention provides fluorine-containing aliphatic chain modification and is improving the application in cationic polymer gene transfection efficiency.The present invention provides a kind of cationic polymer that fluorine-containing aliphatic chain is modified and its purposes as non-viral gene vector.The fluorine-containing aliphatic chain is covalently attached on cationic polymer surface；The cationic polymer includes L-PEI, branched polyethylene imine, linear polylysine, polylysine tree form modification, polypropyleneimine tree form modification, polyamide-amide tree form modification and its derivative；The gene includes the nucleic acid of DNA, siRNA, shRNA, microRNA and modification.The cationic polymer small toxicity of fluorine-containing aliphatic chain modification provided by the invention, it can realize that high efficiency gene transfects under the conditions of low nucleic acid and material utilization amount, the efficiency gene transfection in various kinds of cell is significantly better than the commercialization transfection reagent such as Lipofectamine2000.

Description

The cationic polymer of a kind of fluorine-containing aliphatic chain modification and its as genophore Purposes

Technical field

The present invention relates to polymer chemistry and technical field of biological materials, and in particular to based on fluoride modification Gene transfer vector of tree form modification and its preparation method and application.

Background technique

Gene transfection, which refers to the process of, to be imported cell or tissue for foreign gene and expresses.This technology is widely used in section Learn research and disease treatment.In this process, Gene transfer vector plays critically important role.In general, gene turns Dye carrier is broadly divided into two classes: viral vector and non-viral vector.It can be obtained using virus transfection method long-term efficient The cell or tissue of expression alien gene, but there are the safety risks such as immunogenicity and genetoxic for viral vector.At present Efficiently, low toxicity, easy-operating non-viral gene transfection method receive the extensive concern of people.Cationic polymer includes poly- second Alkene imines (PEI), poly arginine, L- polylysine (PLL), the non-viral transfections such as liposome carrier and its derivative obtain Extensive research.Although such methods provide comparatively safe gene rotaring transfecting mode, relatively low transfection efficiency and Higher cytotoxicity limits its extensive use on clinical medicine.

Polyamide-amide tree form modification (polyamidoamine, PAMAM) is that Tomalia D.A. was reported for the first time in 1985 The tree form modification of the elliposoidal or spherical structure with geometry branch in road.The cationic polymer is in gene transfection It is widely studied and applied, has obtained the extensive concern of people.1993, Meijer etc. was synthesized poly- with export-oriented divergent method Propyleneimine (Polypropyleneimine, PPI) tree form modification.Extensive synthesis and commercialization are realized, for the first time for tree The high molecular extensive research of shape and application are laid a good foundation.The special construction of tree form modification makes it easier to combine gene plasmid piece Section, internal tertiary amine group can play the role of " proton sponge ", it promoted to contain the escape of body in the cell, improve purpose The stability of gene in the cell.In recent years, by carrying out chemistry in polyamide-amide and polypropyleneimine tree form modification surface It is one of main path that modification, which obtains new Gene transfer vector,.Such as in surface amino groups be covalently attached cyclodextrin, amino acid, The modification groups such as PEG can obtain relatively good gene transfection (Il-Doo Kim et al.2010；Yuya Hayashi et al.2012；Quan Yuan et al.2010).Another kind of modification protocols are in the tree-like high score of polyamide-amide The groups such as modified biological element, glycosyl, polypeptide fragment and protein in sub- surface amino groups can make carrier have targeting transfection (Zhang Q et al.2010, Yuya Hayashi et al.2012；Shixian Huang et al.2011；Arnaud E.Felber et al.2011).In addition, connecting hydrophobic molecule such as lauric acid, myristic acid, palm on tree form modification surface Acid, oleic acid, cholesterol etc. can improve its transfection efficiency (Jose L.Santos et al.2010 to a certain extent；Julia Morales-Sanfrutos et al.2011).Currently, the gene based on the tree form modifications such as polyamide-amide is transfected and is carried BodyCommercial applications have had successfully been obtained.Polylysine tree form modification (poly-_L-lysine Dendrimer) equally it is a kind of polycation tree form modification polymer, becomes a kind of in recent years and be widely studied and applied Genophore.Polylysine tree form modification can effectively combine DNA molecular, and effective protection DNA is not degraded by nuclease, Realize gene transfection.In fact polylysine is the polypeptide being polymerized by lysine as repetitive unit, and can be dropped Solution, therefore this is conducive to the research and application of polylysine in vivo.However polylysine carries gene and enters cell and express Efficiency it is not efficient, this may be to release since polylysine not can induce or DNA is promoted to contain the escape in body in the cell Clearance is.In addition, transfection process often will receive the shadow of cytotoxicity and immunogenicity since polylysine is polypeptide structure It rings.Based on this, polylysine surface modification functional group come improve its function and structure have been obtained extensive research and Using.Such as polyethylene glycol, the block polymers such as polycaprolactone (Polycaprolactone, PCL), and repaired on other timbering materials Polylysine is adornd, modifies polylysine such as on nano silicon particles to enhance gene transfection (Xin Zhang et al.2010).Generally speaking, the relatively low transfection efficiency of the Gene transfer vector based on these tree form modifications, high cell toxicant Property and high material cost be still restrict these materials in the widely applied principal element of field of biomedicine.

Polyethyleneimine (PEI) is a kind of common cationic polymer, including L-PEI and the poly- second of branching Alkene imines.Polyethyleneimine has widely been proved to be a kind of polycationic gene transfection carrier.Polyethyleneimine can be steady Surely it combines DNA to enter cell, and can be escaped by " proton pump " mechanism from cellular inclusion, released dna molecule reaches gene and turns The purpose of dye.But the high toxicity of polyethyleneimine and transfection efficiency deficiency are always the root that it is denounced, therefore many bases It is modified in the amine-modified Gene transfer vector of polyethyleneimine, including PEG, the transformation of the modification methods such as carbamate mannose-modified Gene transfer vector (Chunxi Liu et al.2013；Wei Cheng et al.2013).These methods can be from certain journey It solves the above problems on degree, but still cannot achieve efficient, less toxic gene transfection.

In order to enable cationic polymer obtains better transfection and biocompatibility, the present invention are prepared for being based on The cationic polymer gene transfection carrier of fluorine-containing aliphatic chain modification.Fluorine is a kind of unique element, fluorochemical tool There is very special performance, such as: (1) fluorine-containing aliphatic chain was not only hydrophobic, but also lipophobic, and all had in polarity and nonpolar environment Have the tendency that mutually separating, therefore it is presumed that the fluorine-containing aliphatic chain of tree form modification surface modification can be enhanced tree form modification with The affinity of cell membrane makes it easier to be also beneficial to its endosome escape in the cell by cell membrane；(2) fluorine-containing rouge Fat chain has stronger chemistry and biologically inert, and the compound that the cationic polymer and nucleic acid of this kind of material modification are formed can It is effective against the combination of haemocyanin and lipid molecule, therefore the cationic polymer of fluorine-containing aliphatic chain modification is carried as gene There is good serum stability when body, can realize that high efficiency gene transfects under the conditions of high serum-concentration, there is internal gene The potential application of transfection.(3) due to the high-affinity between fluorine-containing aliphatic chain and fluorine-containing aliphatic chain, point of fluorine-containing aliphatic chain modification Son has good self assembly behavior (V.Percec et al.2003；Jason M.Criscione et al.2009), it is this kind of Molecule can form aggregate structure under extremely low concentration conditions.Donald A.Tomalia is specially in Nature It makes comments on Materials and points out that the presence of fluorine can cause self assembly behavior great difference (Donald occur A.Tomalia, 2003), for example the self assembly behavior of carbon fluorine chain molecule and hydrocarbon chain molecule is entirely different, and this species diversity is not It is as caused by hydrophobicity.This feature of fluorine-containing aliphatic chain is conducive to the material that it is modified to be made in gene transfection process High-efficiency transfection can be thus achieved with less carrier dosage or N/P ratio, so that the cost of gene transfection is greatly saved, drop The cytotoxicity of low transfection Materials.Due to these design features, the present invention is proposed, a variety of cations of fluorine-containing aliphatic chain modification are poly- Closing object can be used for high efficiency gene transfection.Transfection efficiency in various kinds of cell can achieve 90% or more, be much higher than a variety of quotient The transfection reagent of industry is as such asLipofectamine2000；And it can be in the condition for being 1 close to N/P ratio Under (close to electroneutral) realize high efficiency gene transfect (Wang M et al.Nat Commun2014；5:3053；Liu H et al.Biomaterials；201435,5407-5413).In particular, it should be pointed out that although modification containing fluorination can improve tree-like height The hydrophobicity (Mandeep Singh Bakshi et al.2005) of molecule, and hydrophobic modification can improve to a certain extent The efficiency gene transfection (Jose L.Santos et al.2010) of cationic polymer, but what is proposed in the present invention this contains The hydrophobicity that there is the cationic polymer of fluorine aliphatic chain modification high gene transfection to be not based on fluorine-containing aliphatic chain. After if the fluorine in the cationic polymer that fluorine-containing aliphatic chain is modified is substituted for hydrogen by we, discovery common fats chain modification Cationic polymer is not improved efficiency gene transfection under equal conditions, " negative " work played to a certain extent instead With (Figure S13, Wang M et al.Nat Commun2014；5:3053), in addition, the cation of fluorine-containing aliphatic chain modification The transfection efficiency of polymer is also much higher than the common fats chain gene transfection Materials of other reported in literature.These results show that containing The modification of fluorine aliphatic chain has in terms of improving cationic polymer gene transfection efficiency to have an unexpected effect.Further research Fluorination modification is also indicated that in the cellular uptake for improving cationic polymer and nucleic acid complexes, endosome is escaped, nucleic acid release, with And multiple processes such as seroresistance play the role of vital (Wang M et al.Nat Commun2014；5: 3053), these Mechanism Studies also show fluorine-containing aliphatic chain modification and are different from the aliphatic chain modification of conventional method proposition and dredge Water regulatory mechanism.

The cationic polymer synthesis technology of fluorine-containing aliphatic chain modification proposed by the present invention is very mature and operates simply, Combined coefficient is high, and the period is short, and the Gene transfer vector of high yield can be quickly obtained without cumbersome purification step, easy Synthetic method is provided for commercialized good basis.The cationic polymer of this kind of partially fluorinated object surface modification has height The advantages that imitating transfection, low cytotoxicity, synthetic method simple and high yield, having can develop as a new class of gene transfection material Expect and there are the potentiality being commercialized.

Summary of the invention

The present invention improves the deficiency of prior art cationic polymer gene transfection material, and the present invention innovatively utilizes fluorine-containing Cationic polymer such as L-PEI, branched polyethylene imine, the linear polylysine, polyamide-of aliphatic chain modification Amine tree form modification, polypropyleneimine tree form modification and polylysine tree form modification are transfected for gene.With fluorine-containing rouge The cationic polymer of fat chain modification is combined nucleic acid and transfects for cytogene.The present invention is efficient, safe, in low nitrogen phosphorus Than high transfection efficiency can be obtained under conditions of (N/P).

The present invention provides one kind fluorine-containing aliphatic chain modification as shown in formula (II) and is improving cationic polymer gene transfection Application in efficiency.The present invention provides a kind of cationic polymer of fluorine-containing aliphatic chain modification and its as non-viral gene vector Purposes；The fluorine-containing aliphatic chain is covalently attached on cationic polymer surface；Polymer is that the cation is poly- in formula (II) Close object；The gene is the nucleic acid of DNA, siRNA, shRNA, microRNA and modification.The fluorine-containing aliphatic chain includes such as right It is required that formula (I)Shown in functional fluoropolymer group；Wherein X is the reactive group of fluorine-containing aliphatic chain, such as halogen (-Cl、-Br、-I)、-COOH、(R is-CF₃、- CF₂CF₃、-CF₂CF₂CF₃、-CF₂CF₂CF₂CF₃) etc. can be chemically reacted with the amino in cationic polymer；A is 0-3 Integer, b be 0-8 integer, Y be-CH₂F、-CHF₂Or-CF₃；The X includes trifluoroacetic anhydride, pentafluoropropionic anhydride, seven fluorine fourths Acid anhydrides, 2,2,3,3,4,4,4- seven fluorine butyl propyleneglycol acid esters, 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide, nine fluorine Butyl semi-annular jade pendant acyl acid anhydride and its derivative.

In formula (II), Z is-NH- ,-N=C- ,-NHCH₂CH(OH)-、-NHCH₂CH(OH)CH₂O-、 Group；Y is-CH₂F、-CHF₂Or-CF₃Group.C is containing for tree form modification surface covalent linkage The quantity of fluorine compounds modification group, c are the integer of 1-256, and a is the integer of 0-3, and b is the integer of 0-8；Polymer is such as formula (VI) L-PEI shown in (linear polyethyleneimine polymer) is propped up as shown in formula (VIII) Change polyethyleneimine (branched polyethyleneimine polymer), the linear polylysine as shown in formula (VII) (linear poly-_L- lysine), the polyamide-amide tree form modification (polyamidoamine as shown in formula (III) Dendrimer), the polypropyleneimine tree form modification as shown in formula (IV) (poly (propyleneimine) dendrimer) Or the polylysine tree form modification (Poly- as shown in formula (V)_L-lysine dendrimer)；

In formula (VI), formula (VII), formula (VIII), the integer that x and y are 0 or more；Formula (III), formula (IV), in formula (V), M For the core of tree form modification, composition includes ammonia, ethylenediamine, butanediamine, pentanediamine, hexamethylene diamine, octamethylenediamine, certain herbaceous plants with big flowers diamines, or 1,12- dodecamethylene diamine；N is the integer of 1-10；M is the integer of 2-4；

One of the specific example of formula (II) is, when Polymer is polyamide-amide tree form modification, n=6, m=2, Z isY is-CF₃, a=0, b=3, when c=32, the structure of the Gene transfer vector is as follows:

Wherein, G5 is the 5th polyamide-amine tree form modification.

In the present invention, it is by the cationic polymer such as L-PEI, branched polyethylene imine, linear poly- relies Propylhomoserin, polyamide-amide tree form modification, polypropyleneimine tree form modification or polylysine tree form modification and fluorochemical It is covalently attached, is modified on the cationic polymer surface, constitute a kind of cation of novel fluorine-containing aliphatic chain modification Polymer gene transfection carrier；The L-PEI, branched polyethylene imine are using aziridine as monomer polymerization shape At cationic polymer, the end groups of both cationic polymers is primaquine group；Polyamide-amide tree form modification is The tree form modification synthesized using ethylenediamine and methyl acrylate as monomer, the end group of the polyamide-amide tree form modification For primaquine group, n is the integer of 1-10；M is the integer of 2-4.The polypropyleneimine is using butanediamine as core and propane diamine As the tree form modification of monomer synthesis, the end group of the polypropyleneimine tree form modification is primaquine group, and n is 1-10 Integer；M is the integer of 2-4.The linear polylysine is the cationic polymer synthesized by monomer of lysine, the cation The end group of polymer is primaquine group；Polylysine tree form modification is the tree-like high score synthesized by monomer of lysine Son, the polylysine tree form modification end group are primaquine group, and n is the integer of 1-10；M is the integer of 2-4.

In the present invention, the cationic polymer is cationic tree form modification (cationic dendrimer), more Body for example, cationic polymer be polyamide-amide tree form modification (polyamidoamine dendrimer), structure is such as Shown in formula (III)；Or the cationic polymer is polypropyleneimine tree form modification (poly (propyleneimine) Dendrimer) shown in its structure such as formula (IV)；Or the cationic polymer is polylysine tree form modification (Poly-_L- Lysine dendrimer), shown in structure such as formula (V).

In the present invention, the cationic polymer is linear cationic polymer (linear cationic polymer), More specifically for example, cationic polymer is L-PEI (linear polyethyleneimine), structure is such as Shown in formula (VI), alternatively, cationic polymer is linear polylysine (linear poly-_L- lysine), structure such as formula (VII) shown in.

In the present invention, the cationic polymer is branched cationic polymer (branched cationic Polymer), more specifically for example, cationic polymer is the polyethyleneimine (branched of branching Polyethyleneimine), shown in structure such as formula (VIII).

In the present invention, " fluorine-containing aliphatic chain " refers to fluorine-containing alkyl and its derivative, such as trifluoroacetic anhydride, five fluorine propionic acid Acid anhydride, heptafluorobutyric anhydride, 2,2,3,3,4,4,4- seven fluorine butyl propyleneglycol acid esters, 3- (1H, 1H, 5H octafluoro amoxy) -1,2- epoxy Propylene, nona-fluoro butyl group sulphonic acid anhydride etc. and its derivative；The fluorine-containing aliphatic chain is with the active group that can be reacted with amino Fluorine compound.Shown in structure example such as formula (IX):

Wherein, X be halogen (- Cl ,-Br ,-I) ,-COOH,Etc. the active group that can be reacted with primary amino group, a 0- 3 integer, b are the integer of 0-8.

The present invention proposes that a kind of compound includes the cationic polymer and gene of fluorine-containing aliphatic chain modification, the gene Nucleic acid and its purposes in cell transfecting including DNA, siRNA, shRNA, microRNA and modification.

Using green fluorescence protein gene and luciferase gene as reporter gene, prepared by the method fluorine-containing fat The cationic polymer of chain modification conveys gene reported above as genophore, test show the invention has the following advantages that The present invention keeps preferable biocompatibility (to survive in a variety of transfection cells while keeping high efficiency gene transfection 90%) rate is greater than.It is found by gene transfection experiments, the cationic polymer transfection luciferase of fluorine-containing aliphatic chain modification and green The efficiency of color fluorescence protein gene is much higher than unmodified cationic polymer, and the transfection efficiency of some materials is far more than commercialization Transfection reagent such asAnd Lipofectamine2000；The cationic polymer of fluorine-containing aliphatic chain modification simultaneously can be with High efficiency gene transfection is realized under the conditions of low N/P ratio or low carrier dosage.Genophore proposed by the present invention has both efficient, low It is malicious, cheap, synthesis it is simple the advantages that.

Detailed description of the invention

Fig. 1 is the conjunction of the cationic polymer gene transfection carrier of fluorine-containing aliphatic chain modification prepared in embodiment 1-14 At route and genophore structure chart.

Fig. 2 is that the branching of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification prepared in embodiment 1 is poly- The one-dimensional nuclear magnetic resonance map (1H NMR) of the transfection carrier of aziridine.

Fig. 3 is that the branching of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification prepared in embodiment 1 is poly- The transfection carrier of aziridine and transfection efficiency comparison diagram of other transfection carriers on HeLa cell.

Fig. 4 is embodiment 2,3 and 4 gained Gene transfer vector G3-PPI-F7-9, G4-PPI-F7-16 and G5-PPI- The dynamic light scattering detection data figure for the compound that F7-26 and DNA is formed.

Fig. 5 is Gene transfer vector G3-PPI-F7-9 obtained in embodiment 2 and other transfection carriers in HEK293 cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Fig. 6 is Gene transfer vector G3-PPI-F7-9 obtained in embodiment 2 and other transfection carriers in HEK293 cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Fig. 7 is Gene transfer vector G3-PPI-F7-9 obtained in embodiment 2 and other transfection carriers in HeLa cell Transfect the transfection picture comparison diagram of green fluorescence protein gene.

Fig. 8 is Gene transfer vector G3-PPI-F7-9 obtained in embodiment 2 and other transfection carriers in HeLa cell Transfect the transfection efficiency streaming comparative result figure of green fluorescence protein gene.

Fig. 9 is that Gene transfer vector obtained in embodiment 2-4 and other transfection carriers compare the toxicity of HEK29 cell Figure.

Figure 10 is that Gene transfer vector obtained in embodiment 2-4 and other transfection carriers compare the toxicity of HeLa cell Figure.

Figure 11 is that Gene transfer vector G4-PPI-F7-16 obtained in embodiment 3 and other transfection carriers are thin in HEK293 The transfection picture comparison diagram of born of the same parents' transfer green colouring fluorescence protein gene.

Figure 12 is that Gene transfer vector G4-PPI-F7-16 obtained in embodiment 3 and other transfection carriers are thin in HEK293 The transfection efficiency streaming comparative result figure of born of the same parents' transfer green colouring fluorescence protein gene.

Figure 13 is Gene transfer vector G4-PPI-F7-16 obtained in embodiment 3 and other transfection carriers in HeLa cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Figure 14 is Gene transfer vector G4-PPI-F7-16 obtained in embodiment 3 and other transfection carriers in HeLa cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 15 is that Gene transfer vector G5-PPI-F7-26 obtained in embodiment 4 and other transfection carriers are thin in HEK293 The transfection picture comparison diagram of born of the same parents' transfer green colouring fluorescence protein gene.

Figure 16 is that Gene transfer vector G5-PPI-F7-26 obtained in embodiment 4 and other transfection carriers are thin in HEK293 The transfection efficiency streaming comparative result figure of born of the same parents' transfer green colouring fluorescence protein gene.

Figure 17 is Gene transfer vector G5-PPI-F7-26 obtained in embodiment 4 and other transfection carriers in HeLa cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Figure 18 is Gene transfer vector G5-PPI-F7-26 obtained in embodiment 4 and other transfection carriers in HeLa cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 19 is that the first generation that surface hyptafluorobutyric acid obtained in embodiment 5-6 is modified and the second polyamide-amine are tree-like High molecular Gene transfer vector one-dimensional nuclear magnetic resonance map (¹H NMR)。

Figure 20 is Gene transfer vector G1-F7-5 obtained in embodiment 5 and other transfection carriers in HEK293 cell Transfect the transfection picture comparison diagram of green fluorescence protein gene.

Figure 21 is Gene transfer vector G1-F7-5 obtained in embodiment 5 and other transfection carriers in HEK293 cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 22 is Gene transfer vector G2-F7-11 obtained in embodiment 6 and other transfection carriers in HEK293 cell Transfect the transfection picture comparison diagram of green fluorescence protein gene.

Figure 23 is Gene transfer vector G2-F7-11 obtained in embodiment 6 and other transfection carriers in HEK293 cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 24 is Gene transfer vector D-PLL-F7-50% obtained in embodiment 7 and other transfection carriers in HEK293 The efficiency comparative of cell transfer green colouring fluorescence protein gene schemes.

Figure 25 is embodiment 8, the 5th polyamide-amine tree of fluorine-containing aliphatic chain modification obtained in 10,11,12,13 The high molecular Gene transfer vector of shape one-dimensional nuclear magnetic resonance map (¹H NMR)。

Figure 26 is embodiment 8, the 5th polyamide-amine tree of fluorine-containing aliphatic chain modification obtained in 10,11,12,13 The ninhydrin detection data and nuclear magnetic resonance meter of the high molecular Gene transfer vector of shape count according to comparison diagram.

Figure 27 is Gene transfer vector G5-F9-32 obtained in embodiment 8 and other transfection carriers in HEK293 cell Transfect the transfection picture comparison diagram of green fluorescence protein gene.

Figure 28 is Gene transfer vector G5-F9-32 obtained in embodiment 8 and other transfection carriers in HEK293 cell Transfect the transfection efficiency streaming comparative result figure of green fluorescence protein gene.

Figure 29 is G5-O-F8 prepared in embodiment 9₈₀Prepared surface modification 3- (penta oxygen of 1H, 1H, 5H octafluoro Base) -1,2- propylene oxide the 5th polyamide-amine Gene transfer vector one-dimensional nuclear magnetic resonance map (¹H NMR)。

Figure 30 is Gene transfer vector G5-O-F8 prepared in embodiment 9₈₀With other transfection carriers in HEK293 cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Figure 31 is Gene transfer vector G5-F3-26 obtained in embodiment 10 and other transfection carriers in HEK293 cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Figure 32 is Gene transfer vector G5-F3-26 obtained in embodiment 10 and other transfection carriers in HEK293 cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 33 is Gene transfer vector G5-F5-36 obtained in embodiment 11 and other transfection carriers in HEK293 cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Figure 34 is Gene transfer vector G5-F5-36 obtained in embodiment 11 and other transfection carriers in HEK293 cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 35 is what Gene transfer vector G5-F7-49 and G5-F7-68 obtained in embodiment 12-13 and DNA was formed The compound that the agarose gel electrophoresis figure (a) and Gene transfer vector G5 and G5-F7-68 and DNA of compound are formed exists Agarose gel electrophoresis figure (b) under the competitive binding of heparin sodium.

Figure 36 is what Gene transfer vector G5-F7-49 and G5-F7-68 obtained in embodiment 12-13 and DNA was formed The dynamic light scattering detection data figure of compound.

Figure 37 is what Gene transfer vector G5-F7-49 and G5-F7-68 obtained in embodiment 12-13 and DNA was formed The zeta potential detection datagram of compound.

Figure 38 is Gene transfer vector G5-F7-49 obtained in embodiment 12 and other transfection carriers in HEK293 cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Figure 39 is Gene transfer vector G5-F7-49 obtained in embodiment 12 and other transfection carriers in HEK293 cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 40 is Gene transfer vector G5-F7-49 obtained in embodiment 12 and other transfection carriers in HeLa cell Transfect the transfection picture comparison diagram of green fluorescence protein gene.

Figure 41 is Gene transfer vector G5-F7-49 obtained in embodiment 12 and other transfection carriers in HeLa cell Transfect the transfection efficiency streaming comparative result figure of green fluorescence protein gene.

Figure 42 is that Gene transfer vector G5-F7-49 obtained in embodiment 12-13 and the other transfection carriers of G5-F7-68 exist (a) HEK293 (b) transfects the transfection efficiency comparison diagram of luciferase gene in HeLa cell.

Figure 43 is Gene transfer vector G5-F7-68 obtained in embodiment 13 and other transfection carriers in HEK293, cell The transfection picture comparison diagram of transfer green colouring fluorescence protein gene.

Figure 44 is Gene transfer vector G5-F7-68 obtained in embodiment 13 and other transfection carriers in HEK293 cell The transfection efficiency streaming comparative result figure of transfer green colouring fluorescence protein gene.

Figure 45 is Gene transfer vector G5-F7-68 obtained in embodiment 13 and other transfection carriers in HeLa cell Transfect the transfection picture comparison diagram of green fluorescence protein gene.

Figure 46 is Gene transfer vector G5-F7-68 obtained in embodiment 13 and other transfection carriers in HeLa cell Transfect the transfection efficiency streaming comparative result figure of green fluorescence protein gene.

Figure 47 is Gene transfer vector G5-F7-68 obtained in embodiment 13 and other transfection carriers in HeLa cell In the transfection efficiency comparison diagram of the culture medium transfer green colouring fluorescence protein gene of different serum contents.

Figure 48 is Gene transfer vector G5-F7-49 obtained in embodiment 12-13 and the other transfection carriers pair of G5-F7-68 (a) HEK293, (b) the toxicity comparison diagram of HeLa cell.

Figure 49 is that Gene transfer vector G5-F7-49 obtained in embodiment 12-13 and the other transfection carriers of G5-F7-68 exist It is formed after compound with DNA to (a) HEK293, (b) the toxicity comparison diagram of HeLa cell.

Figure 50 is that Gene transfer vector G5-F7-68 obtained in embodiment 13 transfects siRNA's in HeLa-Luc cell Efficiency chart.

Figure 51 is the base of the 5th polyamide-amine tree form modification of Butyrylation modification in surface prepared in embodiment 15 Because of the synthetic route chart of transfection carrier.

Figure 52 is the gene of the 5th polyamide-amine tree form modification of the modification of surface Butyrylation obtained in embodiment 15 Transfection carrier one-dimensional nuclear magnetic resonance map (¹H NMR)。

Figure 53 is that Gene transfer vector G5-H7-69, G5-F7-68 obtained in embodiment 15 and other transfection carriers exist The transfection picture comparison diagram of HEK293 cell and HeLa cell transfer green colouring fluorescence protein gene.

Figure 54 is the Gene transfer vector and other turns of the linear polylysine of the resulting hyptafluorobutyric acid of embodiment 16 modification Dye carrier transfects the transfection picture comparison diagram of green fluorescence protein gene on HEK293 cell.

Figure 55 is obtained Gene transfer vector G4-F7-27 in embodiment 17 and other transfection carriers in HeLa cell The transfection efficiency comparison diagram of transfer green colouring fluorescence protein gene.

Figure 56 is the forth generation polyamide-of the diamino dodecane core for preparing hyptafluorobutyric acid modification prepared in embodiment 18 The synthetic route chart of the Gene transfer vector of amine tree form modification.

Figure 57 is obtained Gene transfer vector C12G4-HFA in embodiment 18₂₃It is thin in HeLa with other transfection carriers The transfection efficiency comparison diagram of born of the same parents' transfer green colouring fluorescence protein gene.

Figure 58 is that the polypropyleneimine tree form modification Gene transfer vector of fluorine-containing aliphatic chain modification in embodiment 19 exists The transfection efficiency comparison diagram of HeLa cell transfer green colouring fluorescin base

Figure 59 is the first generation and the second polyamide-amine tree form modification gene of fluorine-containing aliphatic chain modification in embodiment 20 Transfection efficiency comparison diagram of the transfection carrier in HeLa cell transfer green colouring fluorescin base.

Figure 60 is the 5th polyamide-amine tree form modification Gene transfer vector of fluorine-containing aliphatic chain modification in embodiment 21 In the transfection efficiency comparison diagram of HeLa cell transfer green colouring fluorescin base.

Figure 61 is the branched polyethylene imine Gene transfer vector of fluorine-containing aliphatic chain modification in embodiment 22 in HeLa cell The transfection efficiency comparison diagram of transfer green colouring fluorescin base.

Figure 62 is the Gene transfer vector of the polylysine of fluorine-containing aliphatic chain modification in embodiment 23 in HeLa cell transfer The transfection efficiency comparison diagram of green colouring fluorescin base.

Figure 63 is that the Gene transfer vector of the polyamide-amide tree form modification of fluorine-containing aliphatic chain modification in embodiment 24 exists The transfection efficiency comparison diagram of HeLa cell transfer green colouring fluorescin base.

Specific embodiment

In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail, protection content of the invention It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change Change and advantage is all included in the present invention, and using appended claims as protection scope.Implement process of the invention, Condition, reagent, experimental method etc. are among the general principles and common general knowledge in the art in addition to what is specifically mentioned below, There are no special restrictions to content by the present invention.

Embodiment 1: the branched polyethylene imine of preparation 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification, Wherein the molar ratio example of branched polyethylene imine and 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide is 1: 133.

Synthetic method: take 50 milligrams of branched polyethylene imine macromolecule in 2 milliliters of aqueous solutions, to this dissolved with average mark It is slowly added dropwise in the high molecular aqueous solution of branched polyethylene imine that son amount is 25000Da containing 3- (penta oxygen of 1H, 1H, 5H octafluoro Base) -1,2- propylene oxide 2 milliliters of ethanol solutions, continue the reaction solution to be stirred to react 48 at room temperature after being added dropwise to complete Hour, by dialysing and being freeze-dried, the branched polyethylene imine that the surface fluorination that appearance is white powder is modified can be obtained Macromolecule (the branching PEI of fluorination modification).

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is the branched polyethylene imine of average molecular weight 25000Da, and Z is-NHCH₂CH(OH) CH₂O-, a=1, b=3, c=133, Y CHF₂。

With nuclear-magnetism characterize detection fluorination modification branching PEI Gene transfer vector: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) inspection Survey the joint efficiency of the PEI of the prepared Gene transfer vector fluorination modification of characterization.

The branching PEI of prepared Gene transfer vector fluorination modification is dissolved in D with certain concentration₂O, concentration about 5 Mg/ml carries out nuclear magnetic resonance spectroscopy.According to¹H NMR spectra, as shown in Fig. 2, calculating the efficiency of octafluoro amoxy group.

Experimental result: as shown in Fig. 2, each branching PEI is averagely connected to 133 octafluoro amoxy groups.Product name For PEI-O-F8₁₃₃。

PEI-O-F8₁₃₃Efficiency gene transfection: by PEI-O-F8 prepared in the present embodiment₁₃₃And green fluorescent protein Plasmid DNA forms compound at room temperature, is then transfected in HeLa cell, by the expression for detecting green fluorescent protein It measures to assess the efficiency gene transfection of carrier.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA and PEI-O-F8₁₃₃It is mixed with N/P ratio to be added in the culture medium containing 10% serum after 2 mixing, volume is 100 micro- It rises, 150 microlitres of culture mediums for containing 10% serum is added after standing half an hour.Culture medium containing compound is incubated with cell 500 microlitres of culture medium for containing 10% serum are added after 6 hours.48 hours post-processing cells.With commercialization reagent Lipofectamine2000 is to the transfection efficiency of HeLa cell as control.

Experimental result: Gene transfer vector PEI-O-F8 as shown in Figure 3₁₃₃With other transfection carriers in HeLa cell Transfect the transfection efficiency comparison diagram of green fluorescence protein gene, wherein N/P is N/P ratio.Fig. 3 shows PEI-O-F8₁₃₃In N/P Successfully by green fluorescent protein transfection HeLa cell when being 2, transfection efficiency and Lipofectamine2000 transfection HeLa cell Green fluorescent protein transfection efficiency is suitable.

Embodiment 2: prepare hyptafluorobutyric acid modification third generation polypropyleneimine tree form modification, wherein tree form modification with The molar ratio of heptafluorobutyric anhydride is 1: 10.

Synthetic method: 70.5 milligrams of third generation polypropyleneimine tree form modification is taken to be dissolved in 2 ml methanol solution, to this Dissolved with 69.94 microlitres of triethylamines are added dropwise in the methanol solution of dendriform molecule, then it is added dropwise dropwise into the solution micro- dissolved with 171.5 Gram 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, The product that appearance is white powder can be obtained.

The connection quantity of hyptafluorobutyric acid in detection synthetic product is characterized with ninhydrin method.

Specific implementation method: it is 0,5,10,15,20,25,30,35,40 that volume is separately added into 1.5 milliliters of EP pipe Microlitre 0.2 mg/ml G3PPI dendriform molecular solution.Then, it is slow that 100 microlitres of sodium acetate is added in each pipe Fliud flushing (0.2 mol/L, pH=5.4) and 100 microlitres of ninhydrin solution add to 300 microlitres of final volume with distilled water, Then it is heated 10 minutes in boiling water.With ultraviolet-uisible spectrophotometer in 570 nanometers colorimetrics, the absorption of each sample is measured. Using the concentration of G3PPI tree form modification as abscissa, sample is absorbed as ordinate at 570 nanometers, makees standard curve.

14.11 grams of sodium acetate is dissolved in 86 milliliters of distilled water, obtains the aqueous sodium acetate solution of 2 mol/Ls, so The acetic acid of 14 milliliters of 2 mol/Ls is added afterwards, adjusting pH value makes pH=5.4.By 85 milligrams of hydrindantin and 15 milligrams Ninhydrin is dissolved in 10 milliliters of ethylene glycol monomethyl ether solution.75 microlitres of sample and 25 micro- is added in 1.5 milliliters of EP pipe 100 microlitres of hydrindantin solution is added in the distilled water and 100 microlitres of sodium acetate buffer risen after mixing.EP is managed It is heated 10 minutes in boiling water, 300 microlitre of 60: 40 ethanol water is added in every pipe after cooling.Use ultraviolet-uisible spectrophotometer In 570 nanometers colorimetrics, the absorption of each sample is measured.Referring to the standard curve made with G3PPI tree form modification, calculate The quantity of surface primary amino group, and then the efficiency that tree form modification surface hyptafluorobutyric acid is modified is gone out with the form calculus of residual quantity.

Experimental result: average each tree form modification surface is connected to 9 hyptafluorobutyric acid groups.The product is named as G3- PPI-F7-9.Its structure is as shown below:

Wherein, G3PPI is third generation polypropyleneimine tree form modification, structural formula are as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer be third generation polypropyleneimine tree form modification, centronucleus M be butanediamine, n=3, M=2；Z is amido bond, a=0, b=2, c=9, Y CF₃。

The detection of Gene transfer vector G3-PPI-F7-9 dynamic light scattering size: by base prepared in the present embodiment Because transfection reagent G3-PPI-F7-9 and green fluorescent protein plasmid DNA (pEGFP-1) form compound at room temperature, by dynamic The ability and feature of the compound DNA of state scattering measuring gene transfection agent G3-PPI-F7-9.

Method particularly includes: by prepared in the present embodiment gene transfection agent G3-PPI-F7-9 and green fluorescence egg White Plasmid DNA (pEGFP-1) presses different N/P ratios (respectively 0: 1,0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) at room temperature mixing At aqueous solution, wherein DNA content is 1.6 microgram/1.5 milliliter.It is incubated for 30 minutes, detects dynamic light scattering size.

Experimental result: as shown in Fig. 4 (a), the size of naked DNA molecule in the solution is in 1500 rans, and DNA molecular It is capable of forming compound with Gene transfer vector G3-PPI-F7-9, is 1: 1 formation size on 300 nanometers of left sides being greater than N/P ratio Compound right or below.For this explanation under conditions of low N/P ratio, Gene transfer vector G3-PPI-F7-9 can be glimmering with green Photoprotein Plasmid DNA (pEGFP-1) forms the compound of 300 rans.

The efficiency gene transfection of Gene transfer vector G3-PPI-F7-9: gene prepared in the present embodiment is transfected into examination Agent G3-PPI-F7-9 and green fluorescent protein plasmid DNA form compound at room temperature, then thin in HEK293 cell and HeLa It is transfected in born of the same parents, the gene transfection effect of carrier is assessed with the expression quantity by Flow cytometry green fluorescent protein Rate.

Method particularly includes: HEK293 cell and HeLa cell culture are incubated for 24 hours in 24 orifice plates, 0.8 microgram is green The training containing 10% serum is added to after the ratio mixing that color fluorescin plasmid DNA and G3-PPI-F7-9 is 1.6: 1 with N/P ratio It supports and is mixed in base, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.It will contain compound 500 microlitres of culture medium containing 10% serum are added in the culture medium and cell of object after being incubated with 6 hours.After transfection 48 hours, place HeLa cell and HEK293 cell are managed, is digested with pancreatin, and is resuspended with 300 microlitres of PBS buffer solution, is examined immediately with flow cytometer Survey the cell percentages of Successful transfection green fluorescent protein.Third generation polypropyleneimine tree form modification is as negative control, quotient The transfection reagent Lipofectamine2000 of industry is as positive control.

Experimental result: such as Fig. 5, Gene transfer vector G3-PPI-F7-9 shown in 6 is thin in HEK293 with other transfection carriers Born of the same parents and such as Fig. 7, Gene transfer vector G3-PPI-F7-9 shown in 8 is glimmering in HeLa cell transfer green colouring with other transfection carriers The transfection efficiency comparison diagram of aequorin, N/P N/P ratio.Fig. 6 and Fig. 8 shows that with G3-PPI-F7-9 be carrier, and green is glimmering Aequorin is reporter gene, the efficiency (82.77%) of transfected HEK 293 and HeLa cell when N/P ratio 1.6: 1 Efficiency (67.06%) considerably beyond third generation polypropyleneimine tree form modification in optimal conditions N/P ratio be 10: 1 when turn The efficiency (0.22%) of HEK293 cell and the efficiency (0.15%) of transfection HeLa cell are contaminated, and is more than The efficiency (71.116%) of the transfected HEK 293 of Lipofectamine2000 with optimal conditions.But in HeLa cell On, the transfection efficiency of G3-PPI-F7-9 is not so good as Lipofectaming2000 transfection efficiency (73.66%).At the same time, carrier G3-PPI-F7-9 is that (fluorescence in HEK293 cell is strong for 1.6: 1 transfection green fluorescent protein average fluorescent strengths than phosphorus in nitrogen Degree is 4204.95, and the fluorescence intensity in HeLa cell is 716.09) to have also exceeded third generation polypropyleneimine tree form modification (fluorescence in HEK293 cell is strong for transfection green fluorescent protein average fluorescent strength when N/P ratio is 10: 1 in optimal conditions Degree is 3.81, fluorescence intensity in HeLa cell be more than commercialized transfection reagent Lipofectamine2000 2.626) Transfecting green fluorescent protein average fluorescent strength, (average fluorescent strength of Lipofectamine2000 is in HEK293 cell 2827.67, in HeLa cell 666.35) average fluorescent strength of Lipofectamine2000 is.

The cytotoxicity of Gene transfer vector G3-PPI-F7-9: Gene transfer vector G3-PPI-F7-9 is to cell for research Activity influence.

Specific experiment method is: HEK293 or HeLa cell, cell density 10 are incubated in 96 orifice plates⁴Cells/well, training Culture medium is removed after supporting 12 hours, 100 microlitres of fresh cultures for containing a certain amount of G3-PPI-F7-9 (transfection conditions) are added It (containing 10% serum), cultivates 48 hours.Cell activity is detected by mtt assay.

Experimental result: (1) Gene transfer vector G3-PPI-F7-9 as shown in Figure 9 and other transfection carriers are to HEK293 The toxicity comparison diagram of cell.Fig. 9 shows under Cell Transfection Conditions, Gene transfer vector G3-PPI-F7-9 and the third generation poly- third Alkene imines tree form modification all has a lower cytotoxicity, and cell survival rate is 90% or more, all table in HEK293 cell Reveal lower cytotoxicity.

(2) Gene transfer vector G3-PPI-F7-9 as shown in Figure 10 is with other transfection carriers to the toxicity of HeLa cell Comparison diagram, Figure 10 show that G3-PPI-F7-9 has lower cytotoxicity, and cell survival rate is 90% or more, illustrates the present invention Gene transfer vector biological safety with higher.

Embodiment 3: prepare hyptafluorobutyric acid modification forth generation polypropyleneimine tree form modification, wherein tree form modification with The molar ratio of heptafluorobutyric anhydride is 1: 16.

Synthetic method: 53.4 milligrams of forth generation polypropyleneimine tree form modification is taken to be dissolved in 2 ml methanol solution, to this It is added dropwise dropwise dissolved with 40.7 microlitres of triethylamines of dropwise addition in the methanol solution of dendriform molecule, then into the solution dissolved with 99.7 milligrams 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react at room temperature 12 hours after being added dropwise to complete, i.e., The forth generation polypropyleneimine tree form modification material that the surface hyptafluorobutyric acid that appearance is white powder is modified can be obtained.

The Gene transfer vector of detection the present embodiment synthesis is characterized with ninhydrin method: prepared by ninhydrin method characterization detection Gene transfer vector hyptafluorobutyric acid modification efficiency.

Specific implementation method: reference implementation example 2.

Experimental result: average each tree form modification surface is connected to 16 hyptafluorobutyric acid groups.It names in the present embodiment The material of synthesis is G4-PPI-F7-16.Its structure is as shown below:

Wherein, G4PPI is forth generation polypropyleneimine tree form modification, structural formula are as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polypropyleneimine tree form modification, wherein centronucleus M is butanediamine, n=4, m= 2；Z is amido bond, a=0, b=2, c=16, Y CF₃。

The detection of Gene transfer vector G4-PPI-F7-16 dynamic light scattering size: by base prepared in the present embodiment Because transfection reagent G4-PPI-F7-16 and green fluorescent protein plasmid DNA (pEGFP-1) form compound at room temperature, by dynamic The ability and feature of the compound DNA of state scattering measuring gene transfection agent G4-PPI-F7-16.

Method particularly includes: by prepared in the present embodiment gene transfection agent G4-PPI-F7-16 and green fluorescence egg White Plasmid DNA (pEGFP-1) presses different N/P ratios (respectively 0: 1,0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) at room temperature mixing At aqueous solution, wherein DNA content is 1.6 microgram/1.5 milliliter.It is incubated for 30 minutes, detects dynamic light scattering size.

Experimental result: as shown in Fig. 4 (b), the size of naked DNA molecule in the solution is in 1500 rans, and DNA molecular It is capable of forming compound with Gene transfer vector G4-PPI-F7-16, is received for 0.5: 1 to 8: 1 formation size 300 in N/P ratio Rice or so or compound below.For this explanation under conditions of low N/P ratio, Gene transfer vector G4-PPI-F7-16 can be with Green fluorescent protein plasmid DNA (pEGFP-1) forms the compound of 300 rans.

The efficiency gene transfection of Gene transfer vector G4-PPI-F7-16: gene prepared in the present embodiment is transfected into examination Agent G4-PPI-F7-16 and green fluorescent protein plasmid DNA form compound at room temperature, then in HEK293 cell and HeLa It is transfected in cell, the gene transfection effect of carrier is assessed with the expression quantity by Flow cytometry green fluorescent protein Rate.

Method particularly includes: HEK293 cell and HeLa cell culture are incubated for 24 hours in 24 orifice plates, 0.8 microgram is green The training containing 10% serum is added to after the ratio mixing that color fluorescin plasmid DNA and G4-PPI-F7-16 is 2: 1 with N/P ratio It supports and is mixed in base, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.It will contain compound 500 microlitres of culture medium containing 10% serum are added in the culture medium and cell of object after being incubated with 6 hours.After transfection 48 hours, place HeLa cell and HEK293 cell are managed, is digested with pancreatin, and is resuspended with 300 microlitres of PBS buffer solution, is examined immediately with flow cytometer Survey the cell percentages of Successful transfection green fluorescent protein.Forth generation polypropyleneimine tree form modification is as negative control, quotient The transfection reagent Lipofectamine2000 of industry is as positive control.

Experimental result: such as Figure 11, Gene transfer vector G4-PPI-F7-16 shown in 12 and other transfection carriers exist HEK293 cell and Figure 13, Gene transfer vector G4-PPI-F7-16 shown in 14 and other transfection carriers are in HeLa cell transfer The transfection efficiency comparison diagram of green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 12 and Figure 14 shows to use G4-PPI- F7-16 is carrier, and green fluorescence protein gene is reporter gene, the efficiency of transfected HEK 293 when N/P ratio 2: 1 (86.58%) and the efficiency of HeLa cell (49.04%) considerably beyond forth generation polypropyleneimine tree form modification in optimal item The efficiency (1.326%) of transfected HEK 293 when N/P ratio is 10: 1 under part and the transfection efficiency on HeLa cell And the transfected HEK 293 efficiency more than Lipofectamine2000 with optimal conditions (2.446%), (71.116%), the transfection HeLa cell efficiency (73.66%) not as good as Lipofectamine2000 with optimal conditions.With this Meanwhile carrier G4-PPI-F7-16 is 2: 1 transfection green fluorescent protein average fluorescent strengths (in HEK293 cell than phosphorus in nitrogen Fluorescence intensity be 3975, fluorescence intensity in HeLa cell is that 445.67) to have also exceeded forth generation polypropyleneimine tree-like Macromolecule transfects green fluorescent protein average fluorescent strength (in HEK293 cell when N/P ratio is 10: 1 in optimal conditions Fluorescence intensity is 32,9.643) fluorescence intensity in HeLa cell is.Compared with Lipofectamine2000, carrier G4- PPI-F7-16 is more than that Lipofectamine2000 is optimizing in HEK293 cell transfecting green fluorescent protein average fluorescent strength Under the conditions of average fluorescent strength (2827.67), but on HeLa cell transfect green fluorescent protein average fluorescent strength not Such as the average fluorescent strength (666.35) of Lipofectamine2000 with optimal conditions.

The cytotoxicity of Gene transfer vector G4-PPI-F7-16: Gene transfer vector G4-PPI-F7-16 is to cell for research Activity influence.

Specific experiment method is: HEK293 or HeLa cell, cell density 10 are incubated in 96 orifice plates⁴Cells/well, training Culture medium is removed after supporting 12 hours, 100 microlitres of fresh cultures for containing a certain amount of G4-PPI-F7-16 (transfection concentrations) are added It (containing 10% serum), cultivates 48 hours.Cell activity is detected by mtt assay.

Experimental result: (1) Gene transfer vector G4-PPI-F7-16 as shown in Figure 9 and other transfection carriers are to HEK293 The toxicity comparison diagram of cell.Fig. 9 shows under Cell Transfection Conditions, Gene transfer vector G4-PPI-F7-16 and forth generation poly- third Alkene imines tree form modification all has a lower cytotoxicity, and cell survival rate is 90% or so, all table in HEK293 cell Reveal lower cytotoxicity.

(2) Gene transfer vector G4-PPI-F7-16 as shown in Figure 10 is with other transfection carriers to the toxicity of HeLa cell Comparison diagram, Figure 10 show that G4-PPI-F7-16 has lower cytotoxicity, and cell survival rate is 80% or more, illustrates the present invention Gene transfer vector biological safety with higher.

Embodiment 4: prepare hyptafluorobutyric acid modification the 5th generation polypropyleneimine tree form modification, wherein tree form modification with The molar ratio of fluorine-containing aliphatic chain is 1: 32.

Synthetic method: taking 67 milligrams of the 5th generation polypropyleneimine tree form modification to be dissolved in 2 ml methanol solution, molten to this There are 102 microlitres of triethylamines of dropwise addition in the methanol solution of dendriform molecule, then is added dropwise dropwise into the solution dissolved with 250.3 micrograms 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete Obtain the 5th generation polypropyleneimine tree form modification material that the surface hyptafluorobutyric acid that appearance is white powder is modified.

The Gene transfer vector of detection embodiment synthesis is characterized with ninhydrin method: prepared with ninhydrin method characterization detection The hyptafluorobutyric acid of the carrier of Gene transfer vector embodiment synthesis modifies efficiency.

Specific implementation method: reference implementation example 2.

Experimental result: average each tree form modification surface is connected to 26 hyptafluorobutyric acid groups.It names in the present embodiment The material of synthesis is G5-PPI-F7-26.Its structure is as shown below:

Wherein, G5PPI is the 5th generation polypropyleneimine tree form modification, structural formula are as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polypropyleneimine tree form modification, wherein centronucleus M is butanediamine, n=5, m= 2；Z is amido bond, a=0, b=2, c=26, Y CF₃。

The detection of Gene transfer vector G5-PPI-F7-26 dynamic light scattering size: by base prepared in the present embodiment Because transfection reagent G5-PPI-F7-26 and green fluorescent protein plasmid DNA (pEGFP-1) form compound at room temperature, by dynamic The ability and feature of the compound DNA of state scattering measuring gene transfection agent G5-PPI-F7-26.

Method particularly includes: by prepared in the present embodiment gene transfection agent G5-PPI-F7-26 and green fluorescence egg White Plasmid DNA (pEGFP-1) presses different N/P ratios (respectively 0: 1,0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) at room temperature mixing At aqueous solution, wherein DNA content is 1.6 microgram/1.5 milliliter.It is incubated for 30 minutes, detects dynamic light scattering size.

Experimental result: as shown in Fig. 4 (c), the size of naked DNA molecule in the solution is in 1500 rans, and DNA molecular It is capable of forming compound with Gene transfer vector G5-PPI-F7-26, is 1: 1 to 8: 1 formation size at 300 nanometers in N/P ratio Left and right or compound below.This explanation under conditions of low N/P ratio, Gene transfer vector G5-PPI-F7-26 can with it is green Color fluorescin plasmid DNA (pEGFP-1) forms the compound of 300 rans.

The efficiency gene transfection of Gene transfer vector G5-PPI-F7-26: gene prepared in the present embodiment is transfected into examination Agent G5-PPI-F7-26 and green fluorescent protein plasmid DNA form compound at room temperature, then in HEK293 cell and HeLa It is transfected in cell, the gene transfection effect of carrier is assessed with the expression quantity by Flow cytometry green fluorescent protein Rate.

Method particularly includes: HEK293 cell and HeLa cell culture are incubated for 24 hours in 24 orifice plates, 0.8 microgram is green It is added to after the ratio mixing that color fluorescin plasmid DNA and G5-PPI-F7-26 is 1.8: 1 with N/P ratio containing 10% serum It is mixed in culture medium, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.It will be containing multiple 500 microlitres of culture medium containing 10% serum are added in the culture medium and cell for closing object after being incubated with 6 hours.After transfection 48 hours, HeLa cell and HEK293 cell are handled, is digested with pancreatin, and is resuspended with 300 microlitres of PBS buffer solution, uses flow cytometer immediately Detect the cell percentages of Successful transfection green fluorescent protein.5th generation polypropyleneimine tree form modification as negative control, Commercialized transfection reagent Lipofectamine2000 is as positive control.

Experimental result: such as Figure 15, Gene transfer vector G5-PPI-F7-26 shown in 16 and other transfection carriers exist HEK293 cell and Figure 17, Gene transfer vector G5-PPI-F7-26 shown in 18 and other transfection carriers are in HeLa cell transfer The transfection efficiency comparison diagram of green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 16 and Figure 18 shows to use G5-PPI- F7-26 is carrier, and green fluorescence protein gene is reporter gene, the efficiency of transfected HEK 293 when N/P ratio 1.8: 1 (87.4%) and the efficiency of HeLa cell (68.6%) considerably beyond the 5th generation polypropyleneimine tree form modification N/P ratio be 8 : the efficiency (2.43%) in the efficiency (2.08%) and transfection HeLa cell of transfected HEK 293 when 1, and be more than The transfected HEK 293 efficiency (71.116%) of Lipofectamine2000 with optimal conditions, is not so good as The transfection HeLa cell efficiency (73.66%) of Lipofectamine2000 with optimal conditions.At the same time, carrier G5-PPI- F7-26 is that (fluorescence intensity in HEK293 cell is 1.8: 1 transfection green fluorescent protein average fluorescent strengths than phosphorus in nitrogen 4585, the fluorescence intensity in HeLa cell is 847) to have also exceeded the 5th generation polypropyleneimine tree form modification in optimal conditions When lower N/P ratio is 8: 1 transfect green fluorescent protein average fluorescent strength (fluorescence intensity in HEK293 cell is 35.87, Fluorescence intensity in HeLa cell is glimmering 6.88), to be more than commercialized transfection reagent Lipofectamine2000 transfection green Photoprotein average fluorescent strength (average fluorescent strength of Lipofectamine2000 is 2827.67 in HEK293 cell, In HeLa cell the average fluorescent strength of Lipofectamine2000 be for 666.35).

The cytotoxicity and G5-PPI-F7-26 of Gene transfer vector G5-PPI-F7-26: research Gene transfer vector Activity influence of the G5-PPI-F7-26 to cell.

Specific experiment method is: HEK293 or HeLa cell, cell density 10 are incubated in 96 orifice plates⁴Cells/well is left The right side, culture removed culture medium after 12~16 hours, were added 100 microlitres and contained the new of a certain amount of G5-PPI-F7-26 (transfection concentrations) Fresh culture medium (containing 10% serum), cultivates 48 hours.Cell activity is detected by mtt assay.

Experimental result: (1) Gene transfer vector G5-PPI-F7-26 as shown in Figure 9 and other transfection carriers are to HEK293 The toxicity comparison diagram of cell.Fig. 9 shows under Cell Transfection Conditions, Gene transfer vector G5-PPI-F7-26 and the 5th generation poly- third Alkene imines tree form modification all has a lower cytotoxicity, and cell survival rate is 90% or so, all table in HEK293 cell Reveal lower cytotoxicity.

(2) Gene transfer vector G5-PPI-F7-26 as shown in Figure 10 and other transfection carriers are in the poison to HeLa cell Property comparison diagram (concentration that used concentration is equal to transfection experiment), Figure 10 show G5-PPI-F7-26 have lower cell Toxicity, cell survival rate are 80% or more, illustrate Gene transfer vector of the present invention biological safety with higher.

Embodiment 5: prepare hyptafluorobutyric acid modification the first polyamide-amine tree form modification, wherein tree form modification with The molar ratio of heptafluorobutyric anhydride is 1: 5.

Synthetic method: 42.7 milligrams of the first polyamide-amine tree form modification is taken to be dissolved in 2 ml methanol solution, to this It is added dropwise dropwise dissolved with 25 microlitres of triethylamines of dropwise addition in the methanol solution of dendriform molecule, then into the solution dissolved with 36.6 microlitres 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react 24 hours at room temperature after being added dropwise to complete Obtain the first polyamide-amine tree form modification material that the surface hyptafluorobutyric acid that appearance is white powder is modified.

With NMR method characterize detection the present embodiment synthesis Gene transfer vector: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) detection The modification efficiency of the hyptafluorobutyric acid of the prepared Gene transfer vector of characterization.

Method particularly includes: the Gene transfer vector synthesized in 5 milligrams of the present embodiment is taken, 2 ml methanol solution are dissolved in, to this Dissolved with 12 microlitres of triethylamines of dropwise addition in the methanol solution of dendriform molecule, then the second dissolved with 22 microlitres is added dropwise dropwise into the solution 1 milliliter of acid anhydrides methanol solution, the reaction solution is continued to be stirred to react 24 hours at room temperature after being added dropwise to complete, obtains complete second First polyamide-amine tree form modification material of acylated hyptafluorobutyric acid modification.

The Gene transfer vector of prepared acetylation is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance Analysis.According to¹H NMR spectra calculates acetylated ratio as shown in figure 19, with the tree-like high score of the form calculus of residual quantity The efficiency of sublist face hyptafluorobutyric acid modification, the practical connection number for obtaining hyptafluorobutyric acid is 5.The material synthesized in name the present embodiment For G1-F7-5.Its structure is as shown below:

Wherein, G1 is the first polyamide-amine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=2, m= 2；Z is amido bond, a=0, b=2, c=5, Y CF₃。

The efficiency gene transfection of Gene transfer vector G1-F7-5: by gene transfection agent G1- prepared in the present embodiment F7-5 and green fluorescent protein plasmid DNA form compound at room temperature, are then transfected in HEK293 cell, to pass through The expression quantity of Flow cytometry green fluorescent protein assesses the efficiency gene transfection of carrier.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein The ratio that Plasmid DNA and G1-F7-5 are 60 with N/P ratio is added in the culture medium containing 10% serum after mixing and mixes, and volume is 100 microlitres, 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Culture medium containing compound and cell one It rises and 500 microlitres of culture medium that contain 10% serum is added after being incubated for 6 hours.48 hours post-processing cells, are digested with pancreatin, are used in combination 300 microlitres of PBS buffer solution are resuspended, and use the cell percentages of flow cytomery Successful transfection green fluorescent protein immediately.The One polyamide-amine tree form modification is commercialized transfection reagent Lipofectamine2000 as positive right as negative control According to.

Experimental result: Gene transfer vector G1-F7-5 as shown in Figure 20, Figure 21 is thin in HEK293 with other transfection carriers The transfection efficiency comparison diagram of born of the same parents' transfer green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 20, Figure 21 show with G1- F7-5 is genophore, and green fluorescence protein gene is reporter gene, the effect of transfected HEK 293 when N/P ratio is 60: 1 The transfection efficiency (58.28%) of rate (60.20%) and commercialization reagent Lipofectamine2000 quite, are much higher than the first generation Transfection efficiency (0.18%) of the polyamide-amide tree form modification when N/P ratio is 200: 1.As shown in Figure 20, Figure 21, gene turns It contaminates carrier G1-F7-5 transfection green fluorescent protein average fluorescent strength (1803) when N/P ratio is 60: 1 and is slightly above commercialized examination The green fluorescent protein average fluorescent strength (1496) of agent Lipofectamine2000, it is tree-like far more than the first polyamide-amine Green fluorescent protein average fluorescent strength (6.59) of the macromolecule when N/P ratio is 200: 1.Therefore this material can be improved Gene transfection of the dendrimer in HEK293 cell.

Embodiment 6: prepare hyptafluorobutyric acid modification the second polyamide-amine tree form modification, wherein tree form modification with The molar ratio of heptafluorobutyric anhydride is 1: 12.

Preparation synthetic method: taking 88.5 milligrams of the second polyamide-amine tree form modification to be dissolved in 2 ml methanol solution, 55 microlitres of triethylamines are added dropwise into the methanol solution dissolved with dendriform molecule, then are added dropwise dropwise into the solution dissolved with 80 microlitres 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react at room temperature 24 hours after being added dropwise to complete, i.e., The first polyamide-amine tree form modification material that the surface hyptafluorobutyric acid that appearance is white powder is modified can be obtained.

With NMR method characterize detection the present embodiment synthesis Gene transfer vector: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) detection The efficiency of the hyptafluorobutyric acid modification of the prepared Gene transfer vector of characterization.

Method particularly includes: the Gene transfer vector synthesized in 10 milligrams of the present embodiment is taken, 2 ml methanol solution are dissolved in, to 13.5 microlitres of triethylamines are added dropwise in the methanol solution dissolved with dendriform molecule, then are added dropwise dropwise into the solution dissolved with 25 microlitres 1 milliliter of acetic anhydride methanol solution, the reaction solution is continued to be stirred to react 24 hours at room temperature after being added dropwise to complete, has been obtained Second polyamide-amine tree form modification material of full acetylated hyptafluorobutyric acid modification.

The Gene transfer vector of prepared acetylation is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance Analysis.According to¹H NMR spectra calculates acetylated ratio as shown in figure 19, with the tree-like high score of the form calculus of residual quantity The efficiency of sublist face hyptafluorobutyric acid modification, the practical connection number for obtaining hyptafluorobutyric acid is 11.The material synthesized in name the present embodiment Material is G2-F7-11.Its structure is as shown below:

Wherein, G2 is the second polyamide-amine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=3, m= 2；Z is amido bond, a=0, b=2, c=11, Y CF₃。

The efficiency gene transfection of Gene transfer vector G2-F7-11: by gene transfection agent prepared in the present embodiment G2-F7-11 and green fluorescent protein plasmid DNA form compound at room temperature, are then transfected in HEK293 cell, with The efficiency gene transfection of carrier is assessed by the expression quantity of Flow cytometry green fluorescent protein.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein The ratio that Plasmid DNA and G2-F7-11 are 3 with N/P ratio is added in the culture medium containing 10% serum after mixing and mixes, and volume is 100 microlitres, 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Culture medium containing compound and cell one It rises and 500 microlitres of culture medium that contain 10% serum is added after being incubated for 6 hours.48 hours post-processing cells, are digested with pancreatin, are used in combination 300 microlitres of PBS buffer solution are resuspended, and use the cell percentages of flow cytomery Successful transfection green fluorescent protein immediately.The One polyamide-amine tree form modification is commercialized transfection reagent Lipofectamine2000 as positive right as negative control According to.

Experimental result: the Gene transfer vector G2-F7-11 as shown in Figure 22, Figure 23 and other transfection carriers are in HEK293 The transfection efficiency comparison diagram of cell transfer green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 22, Figure 23 show with G2-F7-11 is genophore, and green fluorescence protein gene is reporter gene, the transfected HEK 293 when N/P ratio is 3: 1 The transfection efficiency (58.28%) of efficiency (58.38%) and commercialization reagent Lipofectamine2000 quite, are much higher than second Transfection efficiency (0.08%) of the polyamide-amine tree form modification when N/P ratio is 40: 1.As shown in figure 23, gene transfection carries Body G2-F7-11 transfection green fluorescent protein average fluorescent strength (1605) when N/P ratio is 3: 1 is slightly above commercialization reagent The green fluorescent protein average fluorescent strength (1496) of Lipofectamine2000, far more than the tree-like height of the second polyamide-amine Green fluorescent protein average fluorescent strength (3.91) of the molecule when N/P ratio is 40: 1.Therefore this material can be improved tree-like Gene transfection of the molecule in HEK293 cell.

Embodiment 7: the polylysine tree form modification of hyptafluorobutyric acid modification is prepared, wherein tree form modification surface primary amine groups The molar ratio of group and heptafluorobutyric anhydride is 1: 0.5.

Synthetic method: taking 100 milligrams of polylysine tree form modification, is dissolved in 4 ml methanol solution, to this dissolved with 65.33 microlitres of triethylamines are added dropwise in the methanol solution of dendriform molecule, then are added dropwise dropwise into the solution dissolved with 96.9 microlitres 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, be passed through The polylysine tree form modification material that the hyptafluorobutyric acid that appearance is pale yellow powder is modified can be obtained in dialysis and freeze-drying And it is named as D-PLL-F7-50%.Its structure is as shown below:

Wherein, D-PLL is polylysine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polylysine tree form modification, wherein centronucleus M is pentanediamine, n=5, m=2； Z is amido bond, a=0, b=2, the polylysine tree form modification surface primary amine number that c is 50%, Y CF₃。

The efficiency gene transfection of Gene transfer vector D-PLL-F7-50%: gene prepared in the present embodiment is transfected Reagent D-PLL-F7-50% and green fluorescent protein plasmid DNA form compound at room temperature, then in HEK293 cell into Row transfection assesses the efficiency gene transfection of carrier by detecting the expression quantity of green fluorescent protein.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein Plasmid DNA is added in the culture medium containing 10% serum after being mixed with D-PLL-F7-50% with N/P ratio for 22 and is mixed, and volume is 100 microlitres, 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Culture medium containing compound and cell one It rises and 500 microlitres of culture medium that contain 10% serum is added after being incubated for 6 hours.24 hours post-processing cells.BPEI25kD is as control.

Experimental result: Gene transfer vector D-PLL-F7-50% and other transfection carriers are in HEK293 as of fig. 24 The transfection efficiency comparison diagram of cell transfer green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 24 shows D-PLL-F7- 50% when N/P is 22 successfully by green fluorescent protein transfected HEK 293, transfection efficiency (8.22%), which is higher than, to be not decorated The green fluorescent protein of the polylysine tree form modification (D-PLL) of fluoro-containing group transfected HEK 293 when N/P is 32 turns It contaminates efficiency (1.99%).Meanwhile Figure 24 shows D-PLL-F7-50% transfected HEK 293 green fluorescence egg when N/P is 20 White average fluorescent strength is 135.21, and also above D-PLL, when N/P is 32, transfected HEK 293 green fluorescent protein is average glimmering Luminous intensity (30.46).

Embodiment 8: the 5th polyamide-amine tree form modification of preparation nona-fluoro butyl group semi-annular jade pendant acyl acid anhydride modification, wherein tree-like height The molar ratio of molecule and nona-fluoro butyl group semi-annular jade pendant acyl acid anhydride is 1: 32.

Synthetic method: taking 50 milligrams of the 5th polyamide-amine tree form modification to be dissolved in 2 ml methanol solution, molten to this There are 9.25 microlitres of triethylamines of dropwise addition in the methanol solution of dendriform molecule, then is added dropwise dropwise into the solution dissolved with 18.27 microlitres 1 milliliter of methanol solution of nona-fluoro butyl group semi-annular jade pendant acyl acid anhydride, continue the reaction solution to be stirred to react 12 at room temperature after being added dropwise to complete small When, the 5th polyamide-amine tree form modification material that the nona-fluoro butyl group semi-annular jade pendant acyl acid anhydride that appearance is white powder is modified can be obtained.

The Gene transfer vector of detection the present embodiment synthesis is characterized with ninhydrin detection method: being characterized and is examined with ninhydrin detection method Survey the acylated efficiency of nona-fluoro butyl group semi-annular jade pendant of prepared Gene transfer vector.

Specific implementation method: reference implementation example 2.

With NMR method characterize detection the present embodiment synthesis Gene transfer vector: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) detection The acylated efficiency of the nona-fluoro butyl group semi-annular jade pendant of the prepared Gene transfer vector of characterization.

Method particularly includes: the Gene transfer vector synthesized in 5 milligrams of the present embodiment is taken, 2 ml methanol solution are dissolved in, to this Dissolved with 41 microlitres of triethylamines are added dropwise respectively in the methanol solution of dendriform molecule, then it is added dropwise dropwise into the solution dissolved with 23 microlitres 1 milliliter of acetic anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, obtains table The 5th acylated polyamide-amine tree form modification material of the nona-fluoro butyl group semi-annular jade pendant of the complete acetylation in face.

The Gene transfer vector of prepared acetylation is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance Analysis.According to¹H NMR spectra calculates acetylated ratio as shown in figure 25, with the tree-like high score of the form calculus of residual quantity The acylated efficiency of sublist face nona-fluoro butyl group semi-annular jade pendant.As shown in figure 25, the result that obtains and with ninhydrin detect in UV, visible light be divided The result calculating that spectrophotometry obtains is compared.

Experimental result: as shown in figure 26, the result of two methods measurement is relatively coincide, average each tree form modification surface It is connected to 32 nona-fluoro butyl group semi-annular jade pendant acylate groups.The material synthesized in name the present embodiment is G5-F9-32.Its structure such as following figure It is shown:

Wherein, G5 is the 5th polyamide-amine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, and centronucleus M is ethylenediamine, n=6, m=2；Z For amido bond, a=0, b=2, c=32, Y CF₃。

The efficiency gene transfection of Gene transfer vector G5-F9-32: by gene transfection agent prepared in the present embodiment G5-F9-32 and green fluorescent protein plasmid DNA form compound at room temperature, are then transfected in HEK293 cell, lead to The expression quantity of detection green fluorescent protein is crossed to assess the efficiency gene transfection of carrier.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein Plasmid DNA is added in the culture medium containing 10% serum after being mixed with G5-F9-32 with N/P ratio for 8 and is mixed, and volume is 100 micro- It rises, 150 microlitres of culture mediums for containing 10% serum is added after standing half an hour.Culture medium containing compound is incubated with cell 500 microlitres of culture medium for containing 10% serum are added after 6 hours.48 hours post-processing cells, are digested with pancreatin, and with 300 microlitres PBS buffer solution is resuspended, and uses the cell percentages of flow cytomery Successful transfection green fluorescent protein immediately.5th generation polyamides Amine-amine tree form modification is as negative control, and bPEI25kD is as positive control.

Experimental result: the Gene transfer vector G5-F9-32 as shown in Figure 27, Figure 28 and other transfection carriers are in HEK293 The transfection efficiency comparison diagram of cell transfer green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 27, Figure 28 show G5- The cell percentages of F9-32 Successful transfection green fluorescent protein are suitable with commercialization reagent bPEI25kD, and are higher than for the 5th generation Polyamide-amide tree form modification.It is carrier with G5-F9-32, green fluorescence protein gene is reporter gene, when N/P ratio 8: 1 The efficiency (12.89%) of transfected HEK 293 not as good as commercialization reagent bPEI25kD N/P ratio is 8: 1 with optimal conditions when Transfection efficiency (32.24%), but be apparently higher than the 5th polyamide-amine tree form modification nitrogen phosphorus under respective optimal conditions Transfection efficiency (2.89%) when than being 8: 1.It is flat to be that carrier transfects green fluorescent protein when N/P ratio is 8: 1 with G5-F9-32 Equal fluorescence intensity (272.83) not as good as commercialization reagent bPEI25kD N/P ratio is 8: 1 with optimal conditions when green fluorescence egg White average fluorescent strength (1334.35), but N/P ratio is in optimal conditions more than the 5th polyamide-amine tree form modification Green fluorescent protein average fluorescent strength (205.12) when 8: 1.Therefore this material can be improved dendrimer in HEK293 Gene transfection in cell.

Embodiment 9: the 5th polyamide-amine of preparation 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification Tree form modification, wherein the molar ratio of tree form modification and 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide is 1 ∶80。

Synthetic method: it takes in 50 milligrams 2 milliliters of aqueous solutions of daiamid tree form modification, to this dissolved with daiamid tree 2 milliliters of ethyl alcohol containing 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide are slowly added dropwise in the high molecular aqueous solution of shape The reaction solution is continued to be stirred to react 48 hours at room temperature, by dialysing and being freeze-dried by solution after being added dropwise to complete Obtain the daiamid tree form modification that the surface fluorination that appearance is white powder is modified.

With nuclear-magnetism characterize detection the present embodiment synthesis Gene transfer vector: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) detection table The joint efficiency of the prepared Gene transfer vector of sign.

Prepared Gene transfer vector is dissolved in D with certain concentration₂O, about 5 mg/ml of concentration carry out nuclear-magnetism Resonance analyzing.According to¹HNMR map calculates the efficiency of octafluoro amoxy group as shown in figure 29.

Experimental result: as shown in figure 29, average each tree form modification surface has been successfully connected 80 octafluoro amoxy bases Group.The material synthesized in name the present embodiment is G5-O-F8₈₀.Its structure is as shown below:

Wherein, G5 is the 5th polyamide-amine tree form modification, structural formula are as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=6, m= 2；Z is-NHCH₂CH(OH)CH₂O-, a=1, b=3, c=80, Y CHF₂。

Gene transfer vector G5-O-F8₈₀Efficiency gene transfection: by gene transfection agent prepared in the present embodiment G5-O-F8₈₀Compound is formed at room temperature with green fluorescent protein plasmid DNA, is then transfected in HEK293 cell, is led to The expression quantity of detection green fluorescent protein is crossed to assess the efficiency gene transfection of carrier.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein Plasmid DNA and G5-O-F8₈₀It is mixed with N/P ratio to be added in the culture medium containing 10% serum after 1.5 mixing, volume 100 Microlitre, 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Culture medium containing compound is incubated together with cell 500 microlitres of culture medium for containing 10% serum are added after educating 6 hours.24 hours post-processing cells.It is tree-like with the 5th polyamide-amine Macromolecule is under conditions of N/P is 8 to the transfection efficiency of HEK293 cell as control.

Experimental result: Gene transfer vector G5-O-F8 as shown in figure 30₈₀With other transfection carriers in HEK293 cell Transfect the transfection efficiency comparison diagram of green fluorescence protein gene, wherein N/P is N/P ratio.Figure 48 shows G5-O-F8₈₀It is in N/P Successfully by green fluorescent protein transfected HEK 293 when 1.5, transfection efficiency is higher than the 5th polyamide-amine tree form modification and exists The green fluorescent protein transfection efficiency of transfected HEK 293 when N/P is 8.

Embodiment 10: prepare trifluoroacetic acid modification the 5th polyamide-amine tree form modification, wherein tree form modification with The molar ratio of trifluoroacetic anhydride is 1: 32.

Synthetic method: taking 50 milligrams of the 5th polyamide-amine tree form modification, is dissolved in 2 ml methanol solution, to 9.25 microlitres of triethylamines are slowly added dropwise in methanol solution dissolved with tree form modification in this, then be added dropwise dropwise into the solution dissolved with 1 milliliter of 7.68 microlitres of trifluoroacetic anhydride methanol solution, the reaction mixture after being added dropwise to complete continued to stir at room temperature The 5th tree-like high score of polyamide-amine that the surface trifluoroacetic acid that appearance is white powder is modified can be obtained in reaction 12 hours Sub- material.

The Gene transfer vector of detection the present embodiment synthesis is characterized with ninhydrin detection method: being characterized and is examined with ninhydrin detection method The trifluoroacetic acid for surveying prepared Gene transfer vector modifies efficiency.

Specific implementation method: reference implementation example 2.

Characterize the Gene transfer vector that synthesizes in detection the present embodiment with NMR method: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) inspection The trifluoroacetic acid for surveying the prepared Gene transfer vector of characterization modifies efficiency.

Method particularly includes: the Gene transfer vector synthesized in 5 milligrams of the present embodiment is taken, 2 ml methanol solution are dissolved in, to this It is slowly added dropwise dropwise dissolved with 23 dissolved with 41 microlitres of triethylamines are added dropwise respectively in the methanol solution of dendriform molecule, then into the solution Microlitre 1 milliliter of acetic anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, is obtained The 5th polyamide-amine tree form modification material modified to the trifluoroacetic acid of the complete acetylation in surface, and it is named as Ac-G5- F3-26。

The Gene transfer vector of prepared acetylation is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance Analysis.According to¹H NMR spectra calculates acetylated ratio as shown in figure 25, with the tree-like high score of the form calculus of residual quantity Sublist face trifluoroacetic acid modifies efficiency.As shown in figure 26, the result that obtains and light is divided with UV, visible light in ninhydrin detection method The result calculating that degree meter colorimetric method obtains is compared.

Experimental result: as shown in figure 26, the result of two methods measurement is relatively coincide.It is compared according to calculating, it is average each Tree form modification surface is connected to 26 trifluoroacetic acid groups.The product for naming the present embodiment synthesis is G5-F3-26.Its structure It is as shown below:

Wherein, G5 is the 5th polyamide-amine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=6, m= 2；Z is amido bond, a=0, b=0, c=26, Y CF₃。

The efficiency gene transfection of Gene transfer vector G5-F3-26: by gene transfection agent prepared in the present embodiment G5-F3-26 and green fluorescent protein plasmid DNA form compound at room temperature, are then transfected in HEK293 cell, with The efficiency gene transfection of carrier is assessed by the expression quantity of Flow cytometry green fluorescent protein.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein The ratio that Plasmid DNA and G5-F3-26 are 14 with N/P ratio is added in the culture medium containing 10% serum after mixing and mixes, volume It is 100 microlitres, 150 microlitres of culture mediums for containing 10% serum is added after standing half an hour.Culture medium and cell containing compound 500 microlitres of culture medium for containing 10% serum are added after being incubated with 6 hours.48 hours post-processing cells, are digested with pancreatin, are used in combination 300 microlitres of PBS buffer solution are resuspended, and use the cell percentages of flow cytomery Successful transfection green fluorescent protein immediately.The Five polyamide-amine tree form modifications are as negative control, and bPEI25kD is as positive control.

Experimental result: the Gene transfer vector G5-F3-26 as shown in Figure 31, Figure 32 and other transfection carriers are in HEK293 The transfection efficiency comparison diagram of cell transfer green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 31, Figure 32 show with G5-F3-26 is genophore, and green fluorescence protein gene is reporter gene, the transfected HEK 293 when N/P ratio is 14: 1 Efficiency (14.17%) not as good as commercialization reagent bPEI25kD with optimal conditions N/P ratio be 8: 1 when transfection efficiency (32.24%) effect, but this result be higher than the 5th polyamide-amine tree form modification in optimal conditions N/P ratio be 8: Transfection efficiency (2.89%) when 1.As shown in Figure 31, Figure 32, Gene transfer vector G5-F3-26 turns when N/P ratio is 14: 1 Not as good as commercialization reagent bPEI25kD, N/P ratio is 8 to green colouring fluorescin average fluorescent strength (363.59) with optimal conditions : the green fluorescent protein average fluorescent strength (1334.35) when 1, but more than the 5th polyamide-amine tree form modification most Green fluorescent protein average fluorescent strength (205.12) when N/P ratio is 8: 1 under the conditions of excellent.Therefore this material can be improved Gene transfection of the dendrimer in HEK293 cell.

Embodiment 11: preparation five fluorine propionic acid modification the 5th polyamide-amine tree form modification, wherein tree form modification with The molar ratio of pentafluoropropionic anhydride is 1: 40.

Synthetic method: taking 50 milligrams of the 5th polyamide-amine tree form modification, it is dissolved in 2 ml methanol solution, to 11.56 microlitres of triethylamines are added dropwise in the methanol solution dissolved with dendriform molecule, then are added dropwise dropwise into the solution dissolved with 13.69 Microlitre 1 milliliter of pentafluoropropionic anhydride methanol solution, continue the reaction solution to be stirred to react 12 at room temperature after being added dropwise to complete small When, the 5th polyamide-amine tree form modification material that the five fluorine propionic acid of surface that appearance is white powder is modified can be obtained.

The Gene transfer vector synthesized in detection the present embodiment is characterized with ninhydrin detection method: being characterized and is detected with ninhydrin method Five fluorine propionic acid of prepared Gene transfer vector modify efficiency.

Specific implementation method: reference implementation example 2.

Characterize the Gene transfer vector that synthesizes in detection the present embodiment with NMR method: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) inspection The five fluorine propionic acid for surveying the prepared Gene transfer vector of characterization modify efficiency.

Method particularly includes: the Gene transfer vector synthesized in 5 milligrams of the present embodiment is taken, 2 ml methanol solution are dissolved in, to this Dissolved with 41 microlitres of triethylamines are added dropwise respectively in the methanol solution of dendriform molecule, then it is added dropwise dropwise into the solution dissolved with 23 microlitres 1 milliliter of acetic anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, obtains table 5th polyamide-amine tree form modification material of the five fluorine propionic acid modification of the complete acetylation in face.

The Gene transfer vector of prepared acetylation is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance Analysis.According to¹H NMR spectra calculates acetylated ratio as shown in figure 25, with the tree-like high score of the form calculus of residual quantity The efficiency of five fluoric acid of sublist face modification.As shown in figure 26, the result that obtains and light is divided with UV, visible light in ninhydrin detection method The result that degree meter colorimetric method obtains compares.

Experimental result: as shown in figure 26, the result of two methods measurement is relatively coincide, and is compared according to calculating, average each Tree form modification surface is connected to 36 five fluorine propionic acid groups.The product synthesized in name the present embodiment is G5-F5-36.It is tied Structure is as shown below:

Wherein, G5 is the 5th polyamide-amine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=6, m= 2；Z is amido bond, a=0, b=1, c=36, Y CF₃。

The efficiency gene transfection of Gene transfer vector G5-F5-36: by gene transfection agent prepared in the present embodiment G5-F5-36 and green fluorescent protein plasmid DNA form gene composite at room temperature, are then turned in HEK293 cell Dye assesses the efficiency gene transfection of carrier by detecting the expression quantity of green fluorescent protein.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein Plasmid DNA is added in the culture medium containing 10% serum after being mixed with G5-F5-36 with N/P ratio for 8 and is mixed, and volume is 100 micro- It rises, 150 microlitres of culture mediums for containing 10% serum is added after standing half an hour.Culture medium containing compound is incubated with cell 500 microlitres of culture medium for containing 10% serum are added after 6 hours.48 hours post-processing cells, are digested with pancreatin, and with 300 microlitres PBS buffer solution is resuspended, and uses the cell percentages of flow cytomery Successful transfection green fluorescent protein immediately.5th generation polyamides Amine-amine tree form modification is as negative control, and bPEI25kD is as positive control.

Experimental result: the Gene transfer vector G5-F5-36 as shown in Figure 33, Figure 34 and other transfection carriers are in HEK293 The transfection efficiency comparison diagram of cell transfer green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 33, Figure 34 show to use G5-F5-36 is carrier, and green fluorescence protein gene is reporter gene, the efficiency of transfected HEK 293 when N/P ratio 8: 1 (12.13%) compared with the transfection efficiency (32.24%) when N/P ratio is 8: 1 to commercialization reagent bPEI25kD with optimal conditions Be not above the effect of bPEI25kD, but be above the 5th polyamide-amine tree form modification in optimal conditions N/P ratio be 8 : the transfection efficiency (2.89%) when 1.As shown in Figure 33, Figure 34, Gene transfer vector G5-F5-36 turns when N/P ratio is 8: 1 Green colouring fluorescin average fluorescent strength (266.52) is not above commercialization reagent bPEI25kD nitrogen phosphorus with optimal conditions Green fluorescent protein average fluorescent strength (1334.35) when than being 8: 1, but more than the 5th tree-like high score of polyamide-amine Green fluorescent protein average fluorescent strength (205.12) when sub N/P ratio in optimal conditions is 8: 1.Therefore this material energy Enough improve gene transfection of the dendrimer in HEK293 cell.

Embodiment 12: prepare hyptafluorobutyric acid modification the 5th polyamide-amine tree form modification, wherein tree form modification with The molar ratio of heptafluorobutyric anhydride is 1: 50.

Synthetic method: taking 50 milligrams of the 5th polyamide-amine tree form modification to be dissolved in 2 ml methanol solution, molten to this There are 14.45 microlitres of triethylamines of dropwise addition in the methanol solution of dendriform molecule, then is added dropwise dropwise into the solution dissolved with 21.25 microlitres 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react at room temperature 12 hours after being added dropwise to complete, i.e., The 5th polyamide-amine tree form modification material that the surface hyptafluorobutyric acid that appearance is white powder is modified can be obtained.

The Gene transfer vector synthesized in detection the present embodiment is characterized with ninhydrin method: made with ninhydrin method characterization detection The efficiency of the hyptafluorobutyric acid modification of standby Gene transfer vector.

Specific implementation method: reference implementation example 2.

Characterize the Gene transfer vector that synthesizes in detection the present embodiment with NMR method: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) inspection Survey the efficiency of the hyptafluorobutyric acid modification of the prepared Gene transfer vector of characterization.

Method particularly includes: the Gene transfer vector synthesized in 5 milligrams of the present embodiment is taken, 2 ml methanol solution are dissolved in, to this Dissolved with 41 microlitres of triethylamines are added dropwise respectively in the methanol solution of dendriform molecule, then it is added dropwise dropwise into the solution dissolved with 23 microlitres 1 milliliter of acetic anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, has been obtained 5th polyamide-amine tree form modification material of full acetylated hyptafluorobutyric acid modification.

The Gene transfer vector of prepared acetylation is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance Analysis.According to¹H NMR spectra calculates acetylated ratio as shown in figure 25, with the tree-like high score of the form calculus of residual quantity The efficiency of sublist face hyptafluorobutyric acid modification.As shown in figure 26, the result that obtains and with ninhydrin detect in UV, visible light be divided light The result that degree meter colorimetric method obtains compares.

Experimental result: as shown in figure 26, the result of two methods measurement is relatively coincide.It is compared according to calculating, it is average each Tree form modification surface is connected to 49 hyptafluorobutyric acid groups.The product synthesized in name the present embodiment is G5-F7-49.It is tied Structure is as shown below:

Wherein, G5 is the 5th polyamide-amine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=6, m= 2；Z is amido bond, a=0, b=2, c=49, Y CF₃。

The stability of Gene transfer vector G5-F7-49 and DNA compound: gene prepared in the present embodiment is turned Dye carrier G5-F7-49 and green fluorescent protein plasmid DNA (pEGFP-1) forms gene composite at room temperature, passes through agarose The ability of the compound DNA of detected through gel electrophoresis gene transfection agent G5-F7-49.

Method particularly includes: by gene transfection agent G5-F7-49 from green fluorescent protein plasmid DNA (pEGFP-1) by different N/P ratio (respectively 0: 1,0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) mixing at room temperature, be incubated for 30 minutes, then use DNA loading Sample is finally carried out agarose gel electrophoresis experiment by buffer dilution under 90 volts of voltage, and experimental period is 50 minutes, fine jade The concentration of sepharose is 1%.Using individual green fluorescent protein plasmid DNA (pEGFP-1) as control in experiment.Experiment knot Fruit: the agarose gel electrophoresis figure for the compound that Gene transfer vector G5-F7-49 and DNA as shown in figure 35 is formed, wherein N/P is N/P ratio.Figure 35 shows to be formed when gene transfection agent G5-F7-49 is mixed with DNA with 0.5: 1 or more N/P ratio steady Fixed compound.

The detection of Gene transfer vector G5-F7-49 dynamic light scattering size and zeta potential: will institute in the present embodiment The gene transfection agent G5-F7-49 and green fluorescent protein plasmid DNA (pEGFP-1) of preparation form compound at room temperature, lead to Cross the ability and feature of dynamic light scattering and the compound DNA of zeta potential detection gene transfection agent G5-F7-49.Specific method Are as follows: gene transfection agent G5-F7-49 and green fluorescent protein plasmid DNA (pEGFP-1) prepared in the present embodiment is pressed Different N/P ratios (respectively 0: 1,0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) being at room temperature mixed into aqueous solution, wherein DNA content For 1.6 microgram/1.5 milliliter.It is incubated for 30 minutes, detects dynamic light scattering size and zeta potential.As shown in figure 36, naked DNA The size of molecule in the solution in 1200 rans, and DNA molecular be capable of forming with Gene transfer vector G5-F7-49 it is compound Object forms size in 200 rans or compound below under the conditions of 0.5: 1 to 8: 1 N/P ratio.As shown in figure 37, When N/P ratio is 0.5: 1, the potential in the aqueous solution of compound is negative, when N/P ratio is equal to or more than 1, the water of compound Potential in solution is positive.For this explanation under conditions of low N/P ratio, Gene transfer vector G5-F7-49 can be with green fluorescence Albumen Plasmid DNA (pEGFP-1) forms the compound of 200 rans.

The efficiency gene transfection of Gene transfer vector G5-F7-49: by Gene transfer vector G5-F7-49 and luciferase or Green fluorescent protein plasmid DNA forms compound at room temperature, is then transfected in HEK293 cell or HeLa cell, Expression quantity by detecting green fluorescent protein assesses the efficiency gene transfection of carrier.

Method particularly includes: HEK293 cell or HeLa cell culture are incubated for 24 hours in 24 orifice plates, by 0.8 microgram Luciferase or green fluorescent protein plasmid DNA are added to the training containing 10% serum after mixing with G5-F7-49 with N/P ratio for 7 It supports and is mixed in base, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Contain compound Culture medium and cell be incubated with 6 hours after be added and contain 500 microlitres of culture medium of 10% serum.48 hours post-processing cells, It is digested with pancreatin, the detection method of luciferase expression amount is referring to the operation instruction for going out manufacturer (Promega company) offer.With Five polyamide-amine tree form modifications are as negative control, and Lipofectamine2000 is as positive control.In green fluorescence In the transfection experiment of albumen plasmid, using the efficiency of flow cytometric analysis transfection green fluorescent protein.Method particularly includes: After Transfection of Enhanced Green Fluorescent 24 hours (HEK293 cell) or 48 hours (HeLa cell), with pancreatin by cell dissociation, And be resuspended with 300 microlitres of PBS buffer solution, immediately use the cell hundred of flow cytomery Successful transfection green fluorescent protein Divide ratio.Using the 5th polyamide-amine tree form modification as negative control, Lipofectamine2000 is as positive control.

Experimental result: (1) the Gene transfer vector G5-F7-49 as shown in Figure 38, Figure 39, Figure 40, Figure 41 and other transfections The transfection efficiency comparison diagram of carrier transfection green fluorescence protein gene, wherein N/P is N/P ratio.Figure 38, Figure 39 show to use G5- F7-49 is carrier, and green fluorescence protein gene is reporter gene, the efficiency of transfected HEK 293 when N/P ratio 7: 1 (49.83%) transfection efficiency (32.24%) when N/P ratio is 10: 1 with optimal conditions higher than bPEI25kD, although cannot surpass The transfection efficiency (70.84%) of commercialization transfection reagent Lipofectamine2000 is crossed, but is much higher than the 5th polyamide-amine Tree form modification with optimal conditions N/P ratio be 8: 1 when transfection efficiency (2.89%).As shown in Figure 38, Figure 39, Gene transfer vector G5-F7-49 transfects green fluorescent protein average fluorescent strength when N/P ratio is 7: 1 in HEK293 cell (1341.79) green fluorescent protein mean fluorecence when N/P ratio is 8: 1 with optimal conditions with commercialization reagent bPEI25kD Intensity (1334.35) and Lipofectamine2000 (1237.33) quite, far more than the 5th tree-like high score of polyamide-amine Green fluorescent protein average fluorescent strength (205.12) when sub N/P ratio in optimal conditions is 8: 1.Such as Figure 40, Figure 41, In HeLa cell, for G5-F7-49 when N/P ratio is 7, the cell percentages (32.1%) of Successful transfection green fluorescent protein are too late The transfection efficiency of commercialization reagent Lipofectamine2000 (64.2%), but it is much higher than the 5th tree-like high score of polyamide-amine Transfection efficiency (3.78%) when sub N/P ratio with optimal conditions is 8: 1.Using G5-F7-49 as carrier, when N/P ratio is 7, turn Green colouring fluorescin average fluorescent strength (330) and commercialization reagent Lipofectamine2000 (331) quite, far more than 5th polyamide-amine tree form modification in optimal conditions N/P ratio be 8: 1 when green fluorescent protein average fluorescent strength (28.295).This result illustrates that the Gene transfer vector G5-F7-49 of the present embodiment can be improved the 5th tree-like height of polyamide-amine The efficiency gene transfection of molecule, the commercialization reagent that can match in excellence or beauty Lipofectamine2000.

(2) Gene transfer vector G5-F7-49 as shown in figure 42 and other transfection carriers are in HEK293 (a), HeLa (b) The transfection efficiency comparison diagram of luciferase gene is transfected in cell, wherein N/P is N/P ratio.Figure 42 (a) shows thin in HEK293 In born of the same parents, the transfection efficiency of G5-F7-49 is suitable with the efficiency of transfection reagent Lipofectamine2000 of the marketization, and is much higher than The efficiency of 5th polyamide-amine tree form modification.In HeLa cell, the transfection efficiency of G5-F7-49 is more than turning for the marketization Transfection reagent Lipofectamine2000, and it is much higher than the efficiency of the 5th polyamide-amine tree form modification.It is with G5-F7-49 Carrier, luciferase gene are reporter gene, when N/P ratio is 7 the transfection efficiency of transfected HEK 293 with Lipofectamine2000 efficiency is suitable, and is much higher than the 5th polyamide-amine tree form modification N/P ratio with optimal conditions Transfection efficiency when being 8: 1.It is carrier with G5-F7-49 when Figure 42 (b) shows that N/P ratio is 6,7,8, transfection HeLa cell Transfection efficiency is more than Lipofectamine2000 efficiency, and much higher than the 5th polyamide-amine tree form modification in optimal conditions Transfection efficiency when lower N/P ratio is 8: 1.Two above result illustrates that the Gene transfer vector of the present embodiment synthesis can be improved Gene transfection of the dendrimer in HEK293, HeLa cell, can match in excellence or beauty even more than commercialization reagent Lipofectamine2000。

The cytotoxicity and G5-F7-49 and DNA of Gene transfer vector G5-F7-49 is formed after compound to cell activity Influence: the activity influence for the complexes upon cell that research Gene transfer vector G5-F7-49 and itself and DNA are formed.

Specific experiment method is: HEK293 or HeLa cell, cell density 10 are incubated in 96 orifice plates⁴Cells/well, training Culture medium is removed after supporting 12 hours, is added 100 microlitres containing the fresh of a certain amount of G5-F7-49 or G5-F7-49/DNA compound Culture medium (contains 10% serum), cultivates 48 hours.Cell activity is detected by mtt assay.

Experimental result: (1) Gene transfer vector G5-F7-49 as shown in figure 48 and other transfection carriers are to (a) HEK293, (b) the toxicity comparison diagram of HeLa cell.Figure 48 shows under Cell Transfection Conditions, Gene transfer vector G5-F7-49 There is lower cytotoxicity, cell survival relative to Lipofectmine2000 and the 5th polyamide-amine tree form modification Rate is 90% or more, all shows lower cytotoxicity in HeLa cell and HEK293 cell.

(2) Gene transfer vector G5-F7-49 as shown in figure 49 and other transfection carriers are after forming compound with DNA To the toxicity comparison diagram of HEK293 (a), HeLa (b) cell, Figure 49 shows not produce after G5-F7-49 and DNA forms compound Raw additional cytotoxicity, cell survival rate are 90% or more, illustrate Gene transfer vector of the present invention biology peace with higher Quan Xing.

Embodiment 13: prepare hyptafluorobutyric acid modification the 5th polyamide-amine tree form modification, wherein tree form modification with The molar ratio of heptafluorobutyric anhydride is 1: 70.

Synthetic method: taking 50 milligrams of the 5th polyamide-amine tree form modification to be dissolved in 2 ml methanol solution, molten to this 20.3 microlitres of triethylamines are slowly added dropwise in the methanol solution for having dendriform molecule, then into the solution slowly be added dropwise dropwise dissolved with 1 milliliter of 29.74 microlitres of heptafluorobutyric anhydride methanol solution, the reaction solution after being added dropwise to complete continued to be stirred to react at room temperature 12 hours, the 5th polyamide-amine tree form modification material that the surface hyptafluorobutyric acid that appearance is white powder is modified can be obtained Material.

The Gene transfer vector synthesized in detection the present embodiment is characterized with ninhydrin detection method: with ninhydrin detection method characterization The hyptafluorobutyric acid of the prepared Gene transfer vector of detection modifies efficiency.

Specific implementation method: reference implementation example 2.

Characterize the Gene transfer vector that synthesizes in detection the present embodiment with NMR method: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) inspection The hyptafluorobutyric acid for surveying the prepared Gene transfer vector of characterization modifies efficiency.

Method particularly includes: the Gene transfer vector synthesized in 5 milligrams of the present embodiment is taken, 2 ml methanol solution are dissolved in, to this Dissolved with 41 microlitres of triethylamines are added dropwise respectively in the methanol solution of dendriform molecule, then it is added dropwise dropwise into the solution dissolved with 23 microlitres 1 milliliter of acetic anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, obtains table 5th polyamide-amine tree form modification material of the hyptafluorobutyric acid modification of the complete acetylation in face.

The Gene transfer vector of prepared acetylation is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance Analysis.According to¹H NMR spectra calculates acetylated ratio as shown in figure 25, with the tree-like high score of the form calculus of residual quantity The efficiency of sublist face hyptafluorobutyric acid modification.As shown in figure 26, the result that obtains and with ninhydrin detect in UV, visible light be divided light The result calculating that degree meter colorimetric method obtains is compared.

Experimental result: as shown in figure 26, the result of two methods measurement is relatively coincide.It is compared according to calculating, it is average each Tree form modification surface is connected to the modification group of 68 hyptafluorobutyric acids.The product synthesized in name the present embodiment is G5-F7- 68.Its structure is as shown below:

Wherein, G5 is the 5th polyamide-amine tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=6, m= 2；Z is amido bond, a=0, b=2, c=68, Y CF₃。

The stability of Gene transfer vector G5-F7-68 and DNA compound: gene prepared in the present embodiment is turned Transfection reagent G5-F7-68 and green fluorescent protein plasmid DNA (pEGFP-1) form compound at room temperature, pass through Ago-Gel The ability of the compound DNA of electrophoresis detection gene transfection agent G5-F7-68.

Method particularly includes: by gene transfection agent G5-F7-68 from green fluorescent protein plasmid DNA (pEGFP-1) by different N/P ratio (respectively 0: 1,0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) mixing at room temperature, be incubated for 30 minutes, then use DNA loading Sample is finally carried out agarose gel electrophoresis experiment by buffer dilution under 90 volts of voltage, and experimental period is 50 minutes, fine jade The concentration of sepharose is 1%.Using individual green fluorescent protein plasmid DNA (pEGFP-1) as control in experiment.By poly- Anion competition law further analyzes the stability of G5-F7-68/DNA compound: by gene transfection agent G5-F7-68 and green Fluorescin plasmid DNA (pEGFP-1) is mixed at room temperature with the ratio that N/P ratio is 4, is incubated for 30 minutes.G5PAMAM is tree-like Macromolecule and green fluorescent protein plasmid DNA (pEGFP-1) are mixed at room temperature with the ratio that N/P ratio is 8, are incubated for 30 minutes.Point It is not answered with the G5-F7-68/DNA that charge is 4 with N/P ratio than the proportional arrangement heparin sodium for 0,1/0.4,1/0.8,1/2,1/4 Close object mixed solution, be incubated at room temperature 10 minutes, in experiment using individual green fluorescent protein plasmid DNA (pEGFP-1) as Control.Sample is finally carried out to agarose gel electrophoresis experiment under 90 volts of voltage, experimental period is 50 minutes, and agarose is solidifying The concentration of glue is 1%.

Experimental result: (1) fine jade for the compound that the Gene transfer vector G5-F7-68 as shown in Figure 35 (a) and DNA is formed Sepharose electrophoretogram, wherein N/P is N/P ratio.Figure 35 (a) show gene transfection agent G5-F7-68 and DNA with 0.5: 1 with On N/P ratio mixing when form stable compound.

(2) as shown in Figure 35 (b), the compound that Gene transfer vector G5-F7-68 and DNA are formed is in lower charge ratio Heparin competition is lower to compare G5PAMAM tree form modification elder generation released dna, and Gene transfer vector G5-F7-68 and DNA is formed compound Object structure is more loose with respect to G5PAMAM tree form modification.It is not only because Gene transfer vector G5- in the sample of configuration The charge density of F7-68 and DNA is less than G5PAMAM tree form modification, also because the property of its hydrophobic lipophobic causes releasing for DNA It puts.These results suggest that this illustrates that Gene transfer vector G5-F7-68 can be effectively combined Plasmid DNA, and relative to G5PAMAM tree form modification is easier to released dna molecule, is conducive to release and expression of the DNA in guarantor in cell transfecting.

The detection of Gene transfer vector G5-F7-68 dynamic light scattering size and zeta potential: will institute in the present embodiment The gene transfection agent G5-F7-68 and green fluorescent protein plasmid DNA (pEGFP-1) of preparation form compound at room temperature, lead to Cross the ability and feature of dynamic light scattering and the compound DNA of zeta potential detection gene transfection agent G5-F7-68.

Method particularly includes: by prepared in the present embodiment gene transfection agent G5-F7-68 and egfp Grain DNA (pEGFP-1) presses different N/P ratios (respectively 0: 1,0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) being at room temperature mixed into water Solution, wherein DNA content is 1.6 microgram/1.5 milliliter.It is incubated for 30 minutes, detects dynamic light scattering size and zeta potential.

Experimental result: as shown in figure 36, the size of naked DNA molecule in the solution in 1200 rans, and DNA molecular with Gene transfer vector G5-F7-68 is capable of forming compound, forms size under the conditions of 0.5: 1 to 8: 1 N/P ratio and receives 200 Rice or so or compound below.As shown in figure 37, when N/P ratio is 0.5: 1, the potential in the aqueous solution of compound is negative, When N/P ratio is equal to or more than 1, the potential in the aqueous solution of compound is positive.This explanation is under conditions of low N/P ratio, base Because transfection carrier G5-F7-68 can form the compound of 200 rans with green fluorescent protein plasmid DNA (pEGFP-1).

The efficiency gene transfection of Gene transfer vector G5-F7-68: by Gene transfer vector G5-F7-68 and green fluorescence egg White Plasmid DNA forms compound at room temperature, is then transfected in HEK293 cell and HeLa cell, green by detecting The efficiency gene transfection of the expression quantity assessment carrier of color fluorescin.

Method particularly includes: HEK293 cell or HeLa cell culture are incubated for 24 hours in 24 orifice plates, by 0.8 microgram Luciferase or green fluorescent protein plasmid DNA and G5-F7-68 are added to after being mixed with N/P ratio for 2,2.5 and 3 containing 10% blood It is mixed in clear culture medium, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Contain 500 microlitres of culture medium containing 10% serum are added in the culture medium and cell of compound after being incubated with 6 hours.It post-processes within 48 hours Cell is digested with pancreatin, and the detection method of luciferase expression amount is said referring to the use that manufacturer (Promega company) provides is gone out It is bright.Using the 5th polyamide-amine tree form modification as negative control, Lipofectamine2000 is as positive control.Green In the transfection experiment of color fluorescin plasmid, using the efficiency of flow cytometric analysis transfection green fluorescent protein.Specifically Method are as follows:, will be thin with pancreatin after Transfection of Enhanced Green Fluorescent 24 hours (HEK293 cell) or 48 hours (HeLa cell) Born of the same parents' digestion, and be resuspended with 300 microlitres of PBS buffer solution, immediately use flow cytomery Successful transfection green fluorescent protein Cell percentages.Using the 5th polyamide-amine tree form modification as negative control, Lipofectamine2000 is as sun Property control.

Experimental result: (1) the Gene transfer vector G5-F7-68 as shown in Figure 43, Figure 44, Figure 45, Figure 46 and other transfections The transfection efficiency comparison diagram of carrier transfection green fluorescence protein gene, wherein N/P is N/P ratio.Figure 43, Figure 44 show G5-F7- The cell percentages of 68 Successful transfection green fluorescent proteins be higher than commercialization reagent bPEI25kD, Lipofectamine2000 with And the 5th polyamide-amine tree form modification.It is carrier with G5-F7-68, green fluorescence protein gene is reporter gene, nitrogen phosphorus When than being 2: 1 the efficiency (90.97%) of transfected HEK 293 be higher than bPEI25kD with optimal conditions N/P ratio be 8: 1 when Transfection efficiency (32.24%), and the transfection efficiency (70.84%) of commercialization transfection reagent Lipofectamine2000, are much higher than 5th polyamide-amine tree form modification with optimal conditions N/P ratio be 8: 1 when transfection efficiency (2.89%).Such as Figure 43, figure Shown in 44, Gene transfer vector G5-F7-68 transfects green fluorescent protein average fluorescent strength when N/P ratio is 2: 1 It (4223.12) is more than that green fluorescent protein of commercialization reagent bPEI25kD when N/P ratio is 8: 1 with optimal conditions is average glimmering Luminous intensity (1334.35) and Lipofectamine2000 (1237.33), while more than the 5th tree-like high score of polyamide-amine Green fluorescent protein average fluorescent strength (205.12) when sub N/P ratio in optimal conditions is 8: 1.Such as Figure 45, Figure 46, with G5-F7-68 is carrier, and in HeLa cell, G5-F7-68 is when N/P ratio is 2, the cell of Successful transfection green fluorescent protein Percentage (89.56%) is more than commercialization reagent Lipofectamine2000 (64.2%), is much higher than the 5th polyamide-amine tree Shape macromolecule with optimal conditions N/P ratio be 8: 1 when transfection efficiency (3.78%).Using G5-F7-68 as carrier, N/P ratio is When 2, transfection green fluorescent protein average fluorescent strength (662) is the two of commercialization reagent Lipofectamine2000 (331) Times, green fluorescent protein when N/P ratio is 8: 1 in optimal conditions far more than the 5th polyamide-amine tree form modification is average Fluorescence intensity (28.295).This result illustrates that the Gene transfer vector G5-F7-68 of the present embodiment can be improved the 5th generation polyamide- The efficiency gene transfection of amine tree form modification, effect can be more than commercialization reagent Lipofectamine2000.(2) such as Figure 42 Shown in Gene transfer vector G5-F7-68 and other transfection carriers transfect luciferase in HEK293 (a), HeLa (b) cell The transfection efficiency comparison diagram of gene, wherein N/P is N/P ratio.Figure 42 (a) shows that the transfection efficiency of G5-F7-68 is more than the marketization Transfection reagent Lipofectamine2000 efficiency, and be much higher than the 5th polyamide-amine tree form modification efficiency.With G5-F7-68 is carrier, and luciferase gene is reporter gene, and the transfection efficiency of transfected HEK 293 is more than when N/P ratio is 2 The efficiency of the transfection reagent Lipofectamine2000 of the marketization, and much higher than the 5th polyamide-amine tree form modification excellent Transfection efficiency when N/P ratio is 8: 1 under the conditions of change.Figure 42 (b) shows in HeLa cell, transfects by gene of G5-F7-68 The transfection efficiency of carrier is more than the transfection reagent Lipofectamine2000 of the marketization, and is much higher than the 5th polyamide-amine tree The high molecular efficiency of shape.It is carrier with G5-F7-68, luciferase gene is reporter gene, when N/P ratio 2,2.5,3, transfection The transfection efficiency of HeLa cell is more than Lipofectamine2000 efficiency, and is much higher than the 5th polyamide-amine tree form modification Transfection efficiency when N/P ratio is 8: 1 with optimal conditions.These results suggest that the Gene transfer vector energy of the present embodiment synthesis Gene transfection of the dendrimer in HEK293, HeLa cell is enough improved, can be more than commercialization reagent Lipofectamine2000。

Antiserum behavior of the Gene transfer vector G5-F7-68 in transfection process: research Gene transfer vector G5-F7-68 Transfection efficiency of the compound formed with DNA under conditions of higher serum.

Specific implementation method are as follows: by Hela cell inoculation in 24 porocyte culture plates, be incubated for 24 hours, by 0.8 microgram Fluorescin plasmid DNA and G5-F7-68 with N/P ratio be 2 under conditions of, respectively using different serum-concentrations (0%, 10%, 30%, 50%) DMEM culture medium and green fluorescence protein gene carries out compound, and volume is 100 microlitres.Compound 30 minutes it Afterwards, 150 microlitres of culture mediums for containing different serum-concentrations (0%, 10%, 30%, 50%) serum are separately added into after standing half an hour. 500 microlitres of culture medium containing 10% serum are added in culture medium containing compound and cell after being incubated with 6 hours.After 48 hours Handle cell.Using the efficiency of flow cytometric analysis transfection green fluorescent protein.Method particularly includes: green fluorescent protein After gene transfects 48 hours, with pancreatin by cell dissociation, and it is resuspended with 300 microlitres of PBS buffer solution, immediately uses fluidic cell The cell percentages of instrument detection Successful transfection green fluorescent protein.With the 5th polyamide-amine tree form modification, as negative right According to Lipofectamine2000 is as positive control.

Experimental result: as shown in figure 47, Gene transfer vector G5-F7-68 and Lipofectamine2000 is containing 10% blood Cell transfecting efficiency improves a lot with respect to serum free medium in clear culture medium, but works as serum-concentration and increase again, until 30% and 50%, the transfection efficiency of commercialization gene transfection agent Lipofectamine2000 declines rapidly (to be trained containing 50% serum Support base, transfection efficiency 3.90%, green fluorescent protein average fluorescent strength be 46.35), and the gene prepared in embodiment turn Dye carrier G5-F7-68 still can keep relatively high efficiency gene transfection (to contain under conditions of containing high concentration serum 674.29) 50% blood serum medium, transfection efficiency 55.41%, green fluorescent protein average fluorescent strength are, it was demonstrated that this implementation The polyamide-amide tree form modification Gene transfer vector G5-F7-68 of synthesized surface modification hyptafluorobutyric acid modification has in example Certain antiserum transfection.

The cytotoxicity and G5-F7-68 and DNA of Gene transfer vector G5-F7-68 is formed after compound to cell activity Influence: the activity influence for the complexes upon cell that research Gene transfer vector G5-F7-68 and itself and DNA are formed.

Specific experiment method is: HEK293 or HeLa cell, cell density 10 are incubated in 96 orifice plates⁴Cells/well, training Culture medium is removed after supporting 12 hours, is added 100 microlitres containing the fresh of a certain amount of G5-F7-68 or G5-F7-68/DNA compound Culture medium (contains 10% serum), cultivates 48 hours.Cell activity is detected by mtt assay.

Experimental result: (1) Gene transfer vector G5-F7-68 as shown in figure 48 and other transfection carriers are to HEK293 (a), the toxicity comparison diagram of HeLa (b) cell.Figure 48 shows that under Cell Transfection Conditions, Gene transfer vector G5-F7-68 is opposite There is lower cytotoxicity in Lipofectmine2000 and the 5th polyamide-amine tree form modification, cell survival rate is 90% or more, lower cytotoxicity is all shown in HeLa cell and HEK293 cell.

(2) Gene transfer vector G5-F7-68 as shown in figure 49 and other transfection carriers are after forming compound with DNA To the toxicity comparison diagram of HEK293 (a), HeLa (b) cell, Figure 49 shows not produce after G5-F7-68 and DNA forms compound Raw additional cytotoxicity, cell survival rate are 90% or more, illustrate Gene transfer vector of the present invention biology peace with higher Quan Xing.

Embodiment 14: the 5th polyamide-amine tree form modification of prepared hyptafluorobutyric acid modification turns in embodiment 13 Contaminate the Efficiency testing of siRNA.

By taking the material for the 5th polyamide-amine tree form modification that hyptafluorobutyric acid in embodiment 13 is modified as an example, structure As described in Example 13.

Gene transfer vector G5-F7-68 transfects the efficiency of siRNA-Luc in HeLa-Luc cell: gene being transfected and is carried Body G5-F7-68 and siRNA form compound at room temperature, are then transfected in HeLa-Luc cell, by detecting fluorescence The transfection siRNA efficiency of the relative expression quantity assessment carrier of plain enzyme gene gene.

Specific method: the HeLa-Luc cell culture for surely earning luciferase gene is incubated for 24 hours in 24 orifice plates, will 200 nanomoles/liter siRNA-Luc (siRNA sequence: positive-sense strand: 5 '-CCCUAUUCUCCUUCUUCGCdTdT-3 ') from it is different It is added to after G5-F7-68 (0.46 micromoles per liter, the 0.62 micromoles per liter and 0.93 micromoles per liter) mixing of concentration containing no blood It is mixed in clear culture medium, volume is 100 microlitres, and the culture medium of 150 microlitres of serum-frees is added after standing half an hour.Containing compound 500 microlitres of culture medium containing 10% serum are added in the culture medium and cell of object after being incubated with 6 hours.After 24 or 48 hours Manage cell.The detection method of luciferase expression amount is referring to the operation instruction for going out manufacturer (Promega company) offer.

Experimental result: Gene transfer vector G5-F7-68 as shown in figure 50 transfects siRNA's in HeLa-Luc cell Transfection efficiency comparison diagram, wherein abscissa is the concentration of transfection carrier G5-F7-68 in transfection experiment, and the concentration of siRNA is 200 Nanomole/liter.Figure 50 shows G5-F7-68 Successful transfection siRNA-Luc, can be with when concentration is 0.46 micromoles per liter By luciferase gene successful knockout to 22.8%.Illustrate that Gene transfer vector G5-F7-68 has high-efficiency transfection siRNA molecule Ability.

Embodiment 15: the 5th polyamide-amine tree form modification of butyric acid modification is prepared, wherein tree form modification and butyric acid The molar ratio of acid anhydride is 1: 69.

Preparation synthetic method: 50 milligrams of the 5th polyamide-amine tree form modification is taken, it is molten to be dissolved in 2 ml methanols 20 microlitres of triethylamines are slowly added dropwise into the methanol solution dissolved with tree form modification for liquid, then are added dropwise dropwise into the solution molten There is 1 milliliter of butyric anhydride methanol solution of 19.6 microlitres, the reaction mixture is continued into stirring instead at room temperature after being added dropwise to complete It answers 12 hours, the 5th polyamide-amine tree form modification material that the surface butyric acid that appearance is white powder is modified can be obtained. Synthetic route is as shown in figure 50.

Characterize the Gene transfer vector that synthesizes in detection the present embodiment with NMR method: with one-dimensional nucleus magnetic hydrogen spectrum (¹H NMR) inspection Survey the Butyrylation efficiency of the prepared Gene transfer vector of characterization.

Prepared Gene transfer vector is dissolved in D₂O, about 5 mg/ml of concentration carry out nuclear magnetic resonance spectroscopy.Root According to¹H NMR spectra calculates the efficiency of tree form modification surface Butyrylation as shown in figure 52.It is compared according to calculating, it is average each Tree form modification surface is connected to the group of 69 butyric acid modification.The product synthesized in name the present embodiment is G5-H7-69.Its Structure is as shown below:

Wherein, G5 is the 5th polyamide-amine tree form modification, and structural formula is as follows:

The efficiency gene transfection of Gene transfer vector G5-H7-69: by Gene transfer vector G5-H7-69 and green fluorescence egg White Plasmid DNA forms compound at room temperature, is then transfected in HEK293 cell and HeLa cell, green by detecting The efficiency gene transfection of the expression quantity assessment carrier of color fluorescin.

Method particularly includes: HEK293 cell or HeLa cell culture are incubated for 24 hours in 24 orifice plates, by 0.8 microgram Green fluorescent protein plasmid DNA is added in the culture medium containing 10% serum after being mixed with G5-H7-69 with N/P ratio for 2 and is mixed, Volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Culture medium containing compound with 500 microlitres of culture medium containing 10% serum are added in cell after being incubated with 6 hours.Transfection of Enhanced Green Fluorescent 24 hours (HEK293 cell) or after 48 hours (HeLa cell), in fluorescence microscopy microscopic observation transfection efficiency.With the 5th polyamide-amine Tree form modification is as control.

Experimental result: Gene transfer vector G5-H7-69 as shown in figure 50 and other transfection carriers transfect green fluorescence egg The transfection efficiency comparison diagram of white gene, wherein N/P is N/P ratio.Figure 53 shows that with G5-H7-69 be carrier, green fluorescent protein Gene is reporter gene, and the efficiency of transfected HEK 293 and HeLa cell is not so good as the 5th polyamide-amine when N/P ratio 2: 1 Tree form modification with optimal conditions N/P ratio be 8: 1 when transfection efficiency.And G5-F7-68 is in HEK293 and HeLa in figure Transfection efficiency in cell is significantly larger than G5-H7-69 and G5.

This comparative example explanation, the 5th polyamide-amine tree form modification modify hydrophobic butyric acid group it Afterwards, the gene transfection abilities in cell are not enhanced, i.e., are not centainly to modify hydrophobic group to increase Add transfection efficiency, fluorine element plays the role of vital in gene transfection process.Meanwhile by comparing above-mentioned each implementation From the point of view of example, the special nature based on fluoride can make turning for tree form modification in tree form modification surface modification fluoro-containing group Dye efficiency is largely increased, and can be more than commercialization reagent, and can obtain good biocompatibility simultaneously, this is before Document or disclosed patent in be unprecedented.Therefore, the tree form modification of the modification of fluoride obtained in this patent It is a kind of Gene transfer vector novel, with wide application prospect.

Embodiment 16: the linear polylysine of hyptafluorobutyric acid modification is prepared, wherein linear polylysine surface primary amine group Molar ratio with heptafluorobutyric anhydride is 1: 0.4.

Synthetic method: taking 100 milligrams of polylysine tree form modification, is dissolved in 4 ml methanol solution, to this dissolved with 52.27 microlitres of triethylamines are added dropwise in the methanol solution of dendriform molecule, then are added dropwise dropwise into the solution dissolved with 128.14 milligrams 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete, pass through Dialysis and freeze-drying are crossed, the linear polylysine material based on hyptafluorobutyric acid modification that appearance is pale yellow powder can be obtained And it is named as L-PLL-F7-40%.Its structure is as shown below:

Wherein, L-PLL is linear polylysine, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is linear polylysine, average molecular weight 20000-40000Da；Z is amide Key, a=0, b=2, the linear polylysine surface primary amine number that c is 40%, Y CF₃。

The efficiency gene transfection of Gene transfer vector L-PLL-F7-40%: gene prepared in the present embodiment is transfected Reagent L-PLL-F7-40% and green fluorescent protein plasmid DNA form compound at room temperature, then in HEK293 cell into Row transfection assesses the efficiency gene transfection of carrier by detecting the expression quantity of green fluorescent protein.

Method particularly includes: HEK293 cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram green fluorescent protein Plasmid DNA is added in the culture medium containing 10% serum after being mixed with L-PLL-F7-40% with N/P ratio for 22 and is mixed, and volume is 100 microlitres, 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.Culture medium containing compound and cell one It rises and 500 microlitres of culture medium that contain 10% serum is added after being incubated for 6 hours.24 hours post-processing cells.BPEI25kD is as control.

Experimental result: Gene transfer vector L-PLL-F7-40% as shown in figure 50 and other transfection carriers are in HEK293 The transfection efficiency comparison diagram of cell transfer green colouring fluorescence protein gene, wherein N/P is N/P ratio.Figure 54 shows L-PLL-F7- 40% when N/P is 16 successfully by green fluorescent protein transfected HEK 293, transfection efficiency, which is higher than, is not decorated fluoro-containing group Linear polylysine (L-PLL) N/P be 24 when transfected HEK 293 green fluorescent protein transfection efficiency.

Embodiment 17: prepare hyptafluorobutyric acid modification forth generation polyamide-amide tree form modification, wherein tree form modification with The molar ratio example of heptafluorobutyric anhydride is 1: 32.

Synthetic method: taking 50 milligrams of forth generation polyamide-amide tree form modification to be dissolved in 2 ml methanol solution, molten to this There are 9.25 microlitres of triethylamines of dropwise addition in the methanol solution of dendriform molecule, then is added dropwise dropwise into the solution dissolved with 13.6 microlitres 1 milliliter of heptafluorobutyric anhydride methanol solution, the reaction solution is continued to be stirred to react 12 hours at room temperature after being added dropwise to complete Obtain the forth generation polyamide-amide tree form modification material based on the modification of surface hyptafluorobutyric acid that appearance is white powder.

The Gene transfer vector synthesized in detection the present embodiment is characterized with ninhydrin method: made with ninhydrin method characterization detection The efficiency of the hyptafluorobutyric acid modification of standby Gene transfer vector.

Specific implementation method: reference implementation example 2.

Experimental result: comparing according to calculating, and average each tree form modification surface is connected to 27 hyptafluorobutyric acid groups.Life The material synthesized in name the present embodiment is G4-F7-27.Its structure is as shown below:

Wherein, G4 is forth generation polyamide-amide tree form modification, and structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is ethylenediamine, n=5, m= 2；Z is amido bond, a=0, b=2, c=27, Y CF₃。

The efficiency gene transfection of Gene transfer vector G4-F7-27: by Gene transfer vector G4-F7-27 and green fluorescence egg White Plasmid DNA forms compound at room temperature, is then transfected in HeLa cell, by the table for detecting green fluorescent protein Up to the efficiency gene transfection of amount assessment carrier.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA is added in the culture medium containing 10% serum after being mixed with G4-F7-27 with N/P ratio for 4 and is mixed, and volume is 100 microlitres, 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.It is small that culture medium containing compound with cell is incubated with 6 When after be added and contain 500 microlitres of culture medium of 10% serum.48 hours post-processing cells, are transfected using flow cytometric analysis The efficiency of green fluorescent protein.Using forth generation polyamide-amide tree form modification as negative control.Method particularly includes: green is glimmering After aequorin transfects 48 hours (HeLa cell), with pancreatin by cell dissociation, and it is resuspended with 300 microlitres of PBS buffer solution, with Use the cell percentages of flow cytomery Successful transfection green fluorescent protein immediately afterwards.It is tree-like with forth generation polyamide-amide Macromolecule is as negative control.

Experimental result: Gene transfer vector G4-F7-27 as shown in figure 50 and other transfection carriers transfect green fluorescence egg The transfection efficiency comparison diagram of white gene, wherein N/P is N/P ratio.Figure 55 shows that with G4-F7-27 be carrier, green fluorescence egg White gene is reporter gene, and the efficiency (92.4) of transfection HeLa cell is much higher than forth generation polyamide-amide tree when N/P ratio 4: 1 Shape macromolecule with optimal conditions N/P ratio be 8: 1 when transfection efficiency (0.98%).As shown in figure 55, the base in HeLa cell Because transfection carrier G4-F7-27 N/P ratio be 4: 1 when transfect green fluorescent protein average fluorescent strength (2227.89) far more than Forth generation polyamide-amide tree form modification in optimal conditions N/P ratio be 8: 1 when green fluorescent protein average fluorescent strength (16.53).This result illustrates that the Gene transfer vector G4-F7-27 of the present embodiment can greatly improve forth generation polyamide-amide tree The high molecular efficiency gene transfection of shape.

Embodiment 18: preparing the forth generation polyamide-amide tree form modification of the diamino dodecane core of hyptafluorobutyric acid modification, The molar ratio of middle tree form modification and heptafluorobutyric anhydride is 1: 32.

Synthetic method: the forth generation polyamide-amide tree form modification of 50 milligrams of diamino dodecane core is taken to be dissolved in 2 milliliters of first 37.3 microlitres of triethylamines are added dropwise into the methanol solution dissolved with dendriform molecule, then are added dropwise dropwise into the solution for alcoholic solution Dissolved with 1 milliliter of heptafluorobutyric anhydride methanol solution of 91.4 milligrams, the reaction solution is continued to stir at room temperature after being added dropwise to complete The forth generation for the diamino dodecane core based on the modification of surface hyptafluorobutyric acid that appearance is white powder can be obtained in reaction 12 hours Polyamide-amide tree form modification material.It is as shown in figure 50 to react synthetic route.

The Gene transfer vector synthesized in detection the present embodiment is characterized with ninhydrin detection method: with ninhydrin detection method characterization The efficiency of the hyptafluorobutyric acid modification of the prepared Gene transfer vector of detection.

Experimental result: average each tree form modification surface has been successfully connected 23 hyptafluorobutyric acid groups.Name the present embodiment The product of middle synthesis is C12G4-HFA₂₃.Its structure is as shown below:

Wherein, it is the forth generation polyamide-amide tree form modification of diamino dodecane core, structural formula is as follows:

In the present embodiment, the cationic polymer of the fluorine-containing aliphatic chain modification, structure such as claim 1 Chinese style (II) shown in, the cationic polymer is polyamide-amide tree form modification, wherein centronucleus M is 1,12- dodecamethylene diamine, N=5, m=2；Z is amido bond, a=0, b=2, c=23, Y CF₃。

Gene transfer vector C12G4-HFA₂₃Efficiency gene transfection: by C12G4-HFA prepared in the present embodiment₂₃ Compound is formed at room temperature with green fluorescent protein plasmid DNA, is then transfected in HeLa cell, and detection green is passed through The expression quantity of fluorescin assesses the efficiency gene transfection of carrier.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 1.6 microgram egfps Grain DNA and C12G4-HFA₂₃It is mixed with N/P ratio to be added in the culture medium containing 10% serum after 1 mixing, volume is 100 micro- It rises, 150 microlitres of culture mediums for containing 10% serum is added after standing half an hour.Culture medium containing compound is incubated with cell 500 microlitres of culture medium for containing 10% serum are added after 6 hours.48 hours post-processing cells.With commercialization reagent Lipofectamine2000 is to the transfection efficiency of HeLa cell as control.

Experimental result: Gene transfer vector C12G4-HFA as shown in figure 50₂₃With other transfection carriers in HeLa cell Transfect the transfection efficiency comparison diagram of green fluorescence protein gene, wherein N/P is N/P ratio.Figure 57 shows C12G4-HFA₂₃In N/P Successfully by green fluorescent protein transfection HeLa cell when being 1, transfection efficiency and Lipofectamine2000 transfection HeLa cell Green fluorescent protein transfection efficiency is suitable.

Embodiment 19: the efficiency gene transfection of the polypropyleneimine tree form modification based on hyptafluorobutyric acid modification.

By modified based on hyptafluorobutyric acid third and fourth, for the materials of five generation polypropyleneimine tree form modifications, wherein The cationic polymeric structures of the fluorine-containing aliphatic chain modification are as shown in formula (II) in claim 1, the cationic polymer For polypropyleneimine tree form modification, centronucleus M is butanediamine；Work as n=3, when m=2, a=0, b=2, c=9 or 12, Y are CF₃；Work as n=4, when m=2, a=0, b=2, c=16 or 20, Y CF₃；Work as n=5, when m=2, a=0, b=2, c=16 or 32, Y CF₃。

The gene transfection Materials of polypropyleneimine tree form modification based on hyptafluorobutyric acid modification are respectively designated as G3-PPI- F7-9, G3-PPI-F7-12, G4-PPI-F7-16, G5-PPI-F7-20, G5-PPI-F7-26 and G5-PPI-F7-35.

Detect G3-PPI-F7-9, G3-PPI-F7-12, G4-PPI-F7-16, G5-PPI-F7-20, G5-PPI-F7-26 and The efficiency gene transfection of G5-PPI-F7-35: by G3-PPI-F7-9, G3-PPI-F7-12, G4-PPI-F7-16, G5-PPI-F7- 20, G5-PPI-F7-26 and G5-PPI-F7-35 and green fluorescent protein plasmid DNA form compound at room temperature, then exist It is transfected in HeLa cell.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA and G3-PPI-F7-9, G3-PPI-F7-12, G4-PPI-F7-16, G5-PPI-F7-20, G5-PPI-F7-26 and G5- It is added to after the ratio mixing that PPI-F7-35 is respectively 1.6,2,2,1.5,1.8 and 1.7 with best N/P ratio containing 10% serum It is mixed in culture medium, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.It will be containing multiple 500 microlitres of culture medium containing 10% serum are added in the culture medium and cell for closing object after being incubated with 6 hours.After transfection 48 hours, In its transfection efficiency of fluorescence microscopy microscopic observation.The G3 being not decorated, G4, G5PPI tree form modification is as control.

Experimental result: the polypropyleneimine tree form modification Gene transfer vector of hyptafluorobutyric acid modification as shown in figure 50 exists The transfection efficiency comparison diagram of HeLa cell transfer green colouring fluorescence protein gene, N/P is the best of each Gene transfer vector in fact N/P ratio.Figure 58 show third and fourth, five generation polypropyleneimine tree form modifications after modifying fluorine-containing aliphatic chain, in HeLa cell The efficiency of upper transfection green fluorescence protein gene significantly improves.

Embodiment 20: the gene transfection of the polyamide-amide tree form modification of the first generation and second generation of hyptafluorobutyric acid modification Efficiency.

By taking the material of the polyamide-amide tree form modification of the first generation of hyptafluorobutyric acid modification and the second generation as an example, wherein institute The cationic polymeric structures of fluorine-containing aliphatic chain modification are stated as shown in formula (II) in claim 1, the cationic polymer is Polyamide-amide tree form modification, centronucleus M are ethylenediamine；Work as n=2, when m=2, a=0, b=2, the integer of c=4-7, Y For CF₃；Work as n=3, when m=2, a=0, b=2, the integer of c=8-11, Y CF₃。

The first generation of hyptafluorobutyric acid modification and the gene transfection Materials of the second polyamide-amine tree form modification are named respectively For G1-F7₄, G1-F7₅, G1-F7₆, G1-F7₇, G2-F7₈, G2-F7₉, G2-F7₁₁。

Detect G1-F7₄, G1-F7₅, G1-F7₆, G1-F7₇, G2-F7₈, G2-F7₉, G2-F7₁₁Efficiency gene transfection: will G1-F7₄, G1-F7₅, G1-F7₆, G1-F7₇, G2-F7₈, G2-F7₉, G2-F7₁₁With green fluorescent protein plasmid DNA shape at room temperature At compound, then transfected in HeLa cell.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA and G1-F7₄, G1-F7₅, G1-F7₆, G1-F7₇, G2-F7₈, G2-F7₉, G2-F7₁₁It is added to after mixing containing 10% serum It is mixed in culture medium, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.It will be containing multiple 500 microlitres of culture medium containing 10% serum are added in the culture medium and cell for closing object after being incubated with 6 hours.After transfection 48 hours, In its transfection efficiency of fluorescence microscopy microscopic observation.The first generation and the second polyamide-amine tree form modification conduct being not decorated Control.

Experimental result: the first generation of hyptafluorobutyric acid modification as shown in figure 50 and the polyamide-amide tree form modification of the second generation Gene transfer vector HeLa cell transfer green colouring fluorescence protein gene transfection efficiency comparison diagram.Figure 59 shows first The polyamide-amide tree form modification of generation and the second generation transfects green fluorescence egg after modifying fluorine-containing aliphatic chain on HeLa cell The efficiency of white gene significantly improves.

The polyamide-amide tree form modification of embodiment 21:3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification Efficiency gene transfection.

With the material of the polyamide-amide tree form modification of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification For, wherein the cationic polymeric structures of the fluorine-containing aliphatic chain modification are as shown in formula (II) in claim 1, the sun Ionomer is polyamide-amide tree form modification, and centronucleus M is ethylenediamine；Work as n=6, when m=2, Z is-NHCH₂CH (OH)CH₂The integer of O-, a=1, b=3, c=32-96, Y CHF₂。

The polyamide-amide tree form modification of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification is named respectively For G5-O-F8₃₂, G5-O-F8₄₈, G5-O-F8₆₄, G5-O-F8₇₂, G5-O-F8₈₀, G5-O-F8₈₈, G5-O-F8₉₆。

Detect G5-O-F8₃₂, G5-O-F8₄₈, G5-O-F8₆₄, G5-O-F8₇₂, G5-O-F8₈₀, G5-O-F8₈₈, G5-O-F8₉₆ Efficiency gene transfection: by G5-O-F8₃₂, G5-O-F8₄₈, G5-O-F8₆₄, G5-O-F8₇₂, G5-O-F8₈₀, G5-O-F8₈₈, G5- O-F8₉₆Compound is formed at room temperature with green fluorescent protein plasmid DNA, is then transfected in HeLa cell.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA and G5-O-F8₃₂, G5-O-F8₄₈, G5-O-F8₆₄, G5-O-F8₇₂, G5-O-F8₈₀, G5-O-F8₈₈, G5-O-F8₉₆After mixing It is added in the culture medium containing 10% serum and mixes, volume is 100 microlitres, is added 150 microlitres after standing half an hour and contains 10% blood Clear culture medium.The culture medium 500 for containing 10% serum is added after culture medium containing compound and cell are incubated with 6 hours Microlitre.After transfection 48 hours, in its transfection efficiency of fluorescence microscopy microscopic observation.The 5th polyamide-amine being not decorated is tree-like Macromolecule is as control.

Experimental result: the polyamide-amide of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification as shown in figure 50 Transfection efficiency comparison diagram of the tree form modification Gene transfer vector in HeLa cell transfer green colouring fluorescence protein gene.Figure 60 table Bright, the 5th polyamide-amine tree form modification transfects green fluorescent protein base after modifying fluorine-containing aliphatic chain on HeLa cell The efficiency of cause significantly improves.

The base of the branched polyethylene imine of embodiment 22:3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification Because of transfection efficiency.

By taking the material of the branched polyethylene imine of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification as an example, Wherein, for the cationic polymeric structures of the fluorine-containing aliphatic chain modification as shown in formula (II) in claim 1, the cation is poly- Conjunction object is branched polyethylene imine, average molecular weight 25000Da；Z is-NHCH₂CH(OH)CH₂O-, a=1, b=3, c= The integer of 36-133, Y CHF₂。

The branched polyethylene imine gene transfection Materials point of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification PEI-O-F8 is not named as it₃₆, PEI-O-F8₇₈, PEI-O-F8₁₃₃。

Detect PEI-O-F8₃₆, PEI-O-F8₇₈, PEI-O-F8₁₃₃Efficiency gene transfection: by PEI-O-F8₃₆, PEI-O- F8₇₈, PEI-O-F8₁₃₃Compound is formed at room temperature with green fluorescent protein plasmid DNA, is then turned in HeLa cell Dye.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA and PEI-O-F8₃₆, PEI-O-F8₇₈, PEI-O-F8₁₃₃It is added in the culture medium containing 10% serum and mixes after mixing, body Product is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.By the culture medium containing compound with 500 microlitres of culture medium containing 10% serum are added in cell after being incubated with 6 hours.After transfection 48 hours, under fluorescence microscope Observe its transfection efficiency.The branched polyethylene imine being not decorated is as control.

Experimental result: the poly- second of branching of 3- (1H, 1H, 5H octafluoro amoxy) -1,2- propylene oxide modification as shown in Figure 61 Transfection efficiency comparison diagram of the alkene imines Gene transfer vector in HeLa cell transfer green colouring fluorescence protein gene.Figure 61 shows After modifying fluorine-containing aliphatic chain, the efficiency that green fluorescence protein gene is transfected on HeLa cell obviously mentions branched polyethylene imine It is high.

Embodiment 23: the efficiency gene transfection of the polylysine of hyptafluorobutyric acid modification.

By taking the polylysine material of hyptafluorobutyric acid modification as an example, wherein the cationic polymerization of the fluorine-containing aliphatic chain modification For object structure as shown in formula (II) in claim 1, the cationic polymer is linear polylysine or the tree-like height of polylysine Molecule, average molecular weight 20000-40000Da；A=0, b=2, c are 20-50% polylysine surface primary amine number, and Y is CF₃。

The polylysine gene transfection Materials of hyptafluorobutyric acid rest are respectively designated as L-PLL-F7-20%, L-PLL-F7- 30%, L-PLL-F7-40%, L-PLL-F7-50%, D-PLL-F7-20%, D-PLL-F7-30%, D-PLL-F7-40%, D- PLL-F7-50%.

Detect L-PLL-F7-20%, L-PLL-F7-30%, L-PLL-F7-40%, L-PLL-F7-50%, D-PLL-F7- The efficiency gene transfection of 20%, D-PLL-F7-30%, D-PLL-F7-40%, D-PLL-F7-50%: by L-PLL-F7-20%, L-PLL-F7-30%, L-PLL-F7-40%, L-PLL-F7-50%, D-PLL-F7-20%, D-PLL-F7-30%, D-PLL- F7-40%, D-PLL-F7-50% and green fluorescent protein plasmid DNA form compound at room temperature, then in HeLa cell It is transfected.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA and L-PLL-F7-20%, L-PLL-F7-30%, L-PLL-F7-40%, L-PLL-F7-50%, D-PLL-F7-20%, It is added in the culture medium containing 10% serum and mixes after D-PLL-F7-30%, D-PLL-F7-40%, D-PLL-F7-50% mixing Even, volume is 100 microlitres, and 150 microlitres of culture mediums for containing 10% serum are added after standing half an hour.By the culture containing compound 500 microlitres of culture medium containing 10% serum are added in base and cell after being incubated with 6 hours.After transfection 48 hours, in fluorescence microscopy Its transfection efficiency under the microscope.The linear polylysine and polylysine tree form modification being not decorated are as control.

Experimental result: the Gene transfer vector of the polylysine of hyptafluorobutyric acid modification as shown in Figure 62 is in HeLa cell transfer The transfection efficiency comparison diagram of green colouring fluorescence protein gene.Figure 62 shows linear polylysine and polylysine tree form modification After modifying fluorine-containing aliphatic chain, the efficiency that green fluorescence protein gene is transfected on HeLa cell is significantly improved.

Embodiment 24: the efficiency gene transfection of the polyamide-amide tree form modification of hyptafluorobutyric acid modification.

By taking the polyamide-amide tree form modification of hyptafluorobutyric acid modification as an example, wherein the sun of the fluorine-containing aliphatic chain modification from For sub- polymer architecture as shown in formula (II) in claim 1, the cationic polymer is polyamide-amide tree form modification, Centronucleus M is ethylenediamine, n=6, m=2；A=0, b=2, c=40-89, Y CF₃。

Polyamide-amide tree form modification based on the modification of seven fluoric acids is respectively designated as G5-F7-40, G5-F7-44, G5-F7- 49, G5-F7-59, G5-F7-64, G5-F7-68, G5-F7-74, G5-F7-76, G5-F7-81, G5-F7-89.

G5-F7-40, G5-F7-44, G5-F7-49, G5-F7-59, G5-F7-64, G5-F7-68, G5-F7-74 are detected, The efficiency gene transfection of G5-F7-76, G5-F7-81, G5-F7-89: by G5-F7-40, G5-F7-44, G5-F7-49, G5-F7- 59, G5-F7-64, G5-F7-68, G5-F7-74, G5-F7-76, G5-F7-81, G5-F7-89 and green fluorescent protein plasmid DNA Compound is formed at room temperature, is then transfected in HeLa cell.

Method particularly includes: HeLa cell culture is incubated for 24 hours in 24 orifice plates, by 0.8 microgram egfp Grain DNA and G5-F7-40, G5-F7-44, G5-F7-49, G5-F7-59, G5-F7-64, G5-F7-68, G5-F7-74, G5-F7- It is added in the culture medium containing 10% serum and mixes after 76, G5-F7-81, G5-F7-89 mixing, volume is 100 microlitres, stands half 150 microlitres of culture mediums for containing 10% serum are added after hour.After culture medium containing compound and cell are incubated with 6 hours 500 microlitres of culture medium for containing 10% serum are added.After transfection 48 hours, in its transfection efficiency of fluorescence microscopy microscopic observation.No 5th polyamide-amine tree form modification of modification is as control.

Experimental result: the Gene transfer vector of the polyamide-amide tree form modification of hyptafluorobutyric acid modification as shown in Figure 63 exists The transfection efficiency comparison diagram of HeLa cell transfer green colouring fluorescin.Figure 63 shows the 5th polyamide-amine tree form modification After modifying fluorine-containing aliphatic chain, the efficiency that green fluorescence protein gene is transfected on HeLa cell is significantly improved.

Above-described embodiment is in the art general its object is to allow simply to illustrate that technical concepts and features of the invention Logical technical staff cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all Changes or modifications equivalent made by the essence of content according to the present invention, should be covered by the scope of protection of the present invention.

Claims (7)

1. a kind of fluorine-containing aliphatic chain modification is improving the application in cationic polymer gene transfection efficiency, which is characterized in that institute The cationic polymer of fluorine-containing aliphatic chain modification is stated, its structure of structure is as follows:

The integer that a is 32,64, the gene includes the nucleic acid of DNA, siRNA, shRNA, microRNA and modification；G5 is the 5th Polyamide-amine tree form modification, centronucleus are ethylenediamine, and end group is 128 primary amine groups, and structural formula is as follows:

2. a kind of fluorine-containing aliphatic chain modification is improving the application in cationic polymer gene transfection efficiency, which is characterized in that institute The cationic polymer of fluorine-containing aliphatic chain modification is stated, its structure of structure is as follows:

The integer that a is 32, the gene includes the nucleic acid of DNA, siRNA, shRNA, microRNA and modification；G5 is poly- for the 5th generation Amide-amine tree form modification, centronucleus are ethylenediamine, and end group is 128 primary amine groups, and structural formula is as follows:

3. a kind of fluorine-containing aliphatic chain modification is improving the application in cationic polymer gene transfection efficiency, which is characterized in that institute The cationic polymer of fluorine-containing aliphatic chain modification is stated, its structure of structure is as follows:

The integer that a is 32, the gene includes the nucleic acid of DNA, siRNA, shRNA, microRNA and modification；G5 is poly- for the 5th generation Amide-amine tree form modification, centronucleus are ethylenediamine, and end group is 128 primary amine groups, and structural formula is as follows:

4. the cationic polymer of fluorine-containing aliphatic chain modification described in -3 any one is transfected as gene according to claim 1 The purposes of carrier, which is characterized in that the gene includes the nucleic acid of DNA, siRNA, shRNA, microRNA and modification.

5. the cationic polymer after modification described in -3 any one is preparing gene transfection agent box according to claim 1 In purposes, which is characterized in that the gene includes the nucleic acid of DNA, siRNA, shRNA, microRNA and modification.

6. a kind of compound, which is characterized in that comprising fluorine-containing aliphatic chain described in claim any one of 1-3 modification sun from Sub- polymer and gene, the gene include the nucleic acid of DNA, siRNA, shRNA, microRNA and modification.

7. application of the compound according to claim 6 in cell transfecting.