CN105837767A - Cationic polymer gene vector and its preparation method and use - Google Patents
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Abstract
The invention discloses a cationic polymer gene vector. N-(3-aminopropyl)methacrylamide and N-(1, 3-dihydroxy-2-propyl)methacrylamide as monomers undergo a polymerization reaction to produce an N-(1, 3-dihydroxy-2-propyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide polymer through a reverse addition-fragmentation chain transfer polymerization method, and a guanidine group is coupled to the chain segment of N-(3-aminopropyl)methacrylamide to obtain an N-(1, 3-dihydroxy-2-propyl)methacrylamide-b-N-(3-guanidylpropyl)methacrylamide polymer. A ratio of the guanidinated N-(3-aminopropyl)methacrylamide chain segment to the polymer is in a range of 8.3-57%. The invention also provides a preparation method of the gene vector. The gene vector can carry a gene drug and has good biocompatibility and a large application prospect.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of cationic polymer gene vector and preparation method thereof and
Application.
Background technology
Cationic polymer is a novel non-viral gene vector of class, and it is unexistent that it has virus gene carrier
Good physicochemical properties, the molecular diversity of height, reduced immunogenicity, structure are prone to modify, carry base
Because of size and the advantage such as type is unrestricted[1].Polymine is that the sun being most widely used at present is from polymer
Genophore, the reported first such as nineteen ninety-five Boussif PEI can carry as cationic polymer gene vector
DNA molecular containing negative electricity forms PEI/DNA complex[2].This PEI/DNA complex enters cell
Lysosome after, the nitrogen-atoms on PEI strand is protonated, and makes lysosomal pH raise, and causes Cl ion
Interior stream, thus cause lysosome to rupture complex and escape out, DNA is expressed.And N-3-aminopropyl first
Base acrylamide (APMA) is the monomer with primary amine groups residue, and the homopolymer carrier of this monomer synthesis is at base
Higher transfection is shown because delivering in research[3]。
N-(1,3-dihydroxy-2-propyl group) Methacrylamide (DHPMA) is the monomer that a class hydrophilic is forged a good relationship,
Its homopolymer, as the hydrophilic segment of polymer support, makes the DHPMA positive charge to cationic polymer
In masking action and blood, the inhibitory action of proteinosis is higher, makes polymer have the advantages such as hypotoxicity[4,5]。
Additionally, DHPMA is as artificial polymer, molecular weight ranges is the widest, and the degree of freedom of selection is high[6].Polymer
Skeleton has hydrophilic segment so that it is has the molecular structure unfolded, and carries out the positive charge of itself suitably
Protection, improves carrier stability outside target cell, reduces cytotoxicity[7,8]。
1988, the trans-activator Tat albumen of the confirmer immune deficiency virus HIV-1 such as Green can be across
Film is transferred in Cytoplasm and nucleus, thus is greatly improved the transcription and replication level of genome, finds simultaneously
The homology heterogeneous structure territory of Drosophila Antennapedia foot transcription factor ANTP, herpes simplex virus type 1 VP22 transcribe
The factors etc. all have powerful cell membrane penetration capacity, and are little affected by the restriction of cell type[9-11].People
This kind of macromolecular substances that can carry is entered cell, there is the small peptide of cell membrane penetration capacity, referred to as cell-penetrating
Peptide (Cell Penetrating Peptides, CPPs), or Trojan Horse peptide (Trojan horse peptides).Grind
Studying carefully result to show, arginine can effectively transport permeates cell membranes, plays the part of very important during cell-penetrating
Role, research speculates that the characteristic group (guanidino group) in arginine is to confer to this type of Materials Cell film and penetrates energy
The funtion part of power.Wender etc. have synthesized a series of poly-guanidine base class peptide derivant, utilize block and the third ammonia
The substituted Tat similar polypeptide of acid finds, cation district is critically important for the cellular uptake ability of Tat polypeptide, can
Cation district can be because contribute to the interaction between Tat polypeptide and cell thus promote cellular uptake[12]。
Experiment finds that the cellular uptake efficiency of N-alkylated amine acetic acid polypeptide derivative is even high 20 times than Tat, this
Illustrate that guanidine radicals plays the effect of key in cellular uptake.
RNA interference (RNAi) has established solid as one of 21 century most important discovery for gene therapy
Basis, it is possible to effective triggering gene silencing is the key of RNA perturbation technique successful Application.RNA disturbs the beginning
In Dicer enzyme, the Dicer enzyme as RNaseIII family shears the little of dsRNA 19-21 nucleotide of generation
RNA interfering (siRNA) fragment[13-15].These siRNA are by forming RNA with Argonaute 2 albumen
Reticent effect complex and then selective reticent purpose mRNA[16-18].Although cell is cultivated and dynamic in vitro
In thing In vivo study, siRNA demonstrates the good inhibition to target gene, but at treatment human diseases
During, siRNA is faced with many obstacles at the aspect that is administered systemically, such as more weak cell membrane penetration capacity, nothing
Targeting, and unstable in physiological environment.Therefore one of siRNA drug development it is crucial that transmission system,
And how to strengthen siRNA stability in vivo and the ability of permeates cell membranes, and strengthen in disease treatment
It it is all the eager problem needing to solve to cell and tissue-targeting etc..Therefore except finding and screening the most special
Property siRNA drug molecule outside, targeting particular organization or the transmission system of target cell, just become development and
Implement another key point of RNA interference therapy.And preferably gene delivery vector is wanted to make therapeutic gene
Safe efficient, controlled and stable expression is obtained in target cell.Generally, the character according to carrier can be by
Genophore is divided into two classes, viral Gene Delivery Vectors (viral vector) and non-viral-based gene carrier (non-viral
vector).But, since people recognize that virus can invade host cell and by self nucleic acid integration to host
In nucleic acid, viral vector is just used for clinical experiment as a kind of effective nucleic acid carrier.Conventional viral vector,
Including adenovirus (adenovirus, AV) and retrovirus (retrovirus, RV).But, high
Production cost, enter internal after shortcomings (as without targeting, produced immunogenicity etc.) limit in fact
Border uses[19].It addition, the U.S. in 1999 occurs that the first causes the thing of patient death because using adenovirus vector
Part accelerates the exploitation for non-viral-based gene carrier equally[20].So, find novel carrier and overcome
The shortcoming of viral vector also plays the advantage of nucleic acid drug and just becomes extremely important.
Genophore based on macromolecular material is widely used for the transmission to siRNA molecule, the most a lot
Achievement enters clinical trial.High polymer gene carrier has the potentiality of preferable biocompatibility and targeted delivery,
Bioactive molecule stability in physiological environment can be effectively improved.High polymer gene carrier is after being administered systemically
EPR effect (Enhanced permeability and retention) can be produced, and then promote that it is at tumor tissues
Enrichment, therefore macromolecule carrier is the genomic medicine carrier of great potential[21].And present invention offer has
The macromolecule carrier of high-efficiency transfection ability, energy payload dewatering medicament also protects siRNA molecule, it is achieved target
While treatment, disturb destination gene expression, reach synergistic therapeutic effect.Therefore, the high score that the present invention provides
Sub-carrier has more preferable advantage and application prospect in cooperativing medicine-feeding system.
List of references
[1] Lares MR, Rossi JJ, Ouellet DL. RNAi and small interfering RNAs in human disease therapeutic
applications.Trends in biotechnology.2010;28:570-9.
[2] Boussif O, Lezoualc ' hF, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al.A versatile
Vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine.
Proceedings of the National Academy of Sciences.1995;92:7297-301.
[3] York AW, Zhang Y, Holley AC, Guo Y, Huang F, McCormick CL.Facile Synthesis of Multivalent
Folate-Block Copolymer Conjugates via Aqueous RAFT Polymerization:Targeted Delivery of siRNA
and Subsequent GeneBiomacromolecules.2009;10:936-43.
[4] Luo D, Haverstick K, Belcheva N, Han E, Saltzman WM.Poly (ethylene glycol)-conjugated
PAMAM dendrimer for biocompatible, high-efficiency DNA delivery.Macromolecules.
2002;35:3456-62.
[5] Kichler A, Chillon M, Leborgne C, Danos O, Frisch B.Intranasal gene delivery with a
polyethylenimine-PEG conjugate.J Control Release.2002;81:379-88.
[6] Itaka K, Yamauchi K, Harada A, Nakamura K, Kawaguchi H, Kataoka K.Polyion complex micelles
from plasmid DNA and poly(ethylene glycol)-poly(-lysine)block copolymer as serum-tolerable
Polyplex system:physicochemical properties of micelles relevant to gene transfection efficiency.
Biomaterials.2003;24:4495-506.
[7] Li SD, Huang L.Targeted delivery of antisense oligodeoxynucleotide and small interference RNA
into lung cancer cells.Mol Pharmaceut.2006;3:579-88.
[8] de Wolf HK, Snel CJ, Verbaan FJ, Schiffelers RM, Hennink WE, Storm G.Effect of cationic
carriers on the pharmacokinetics and tumor localization of nucleic acids after intravenous
administration.International Journal of Pharmaceutics.2007;331:167-75.
[9] Green M, Loewenstein PM.Autonomous functional domains of chemically synthesized human
immunodeficiency virus tat trans-activator protein.Cell.1988;55:1179-88.
[10] Derossi D, Joliot AH, Chassaing G, Prochiantz A.The third helix of the Antennapedia
homeodomain translocates through biological membranes.J Biol Chem.1994;269:10444-50.
[11] Elliott G, O ' Hare P.Intereellular trafficking and protein delivery by a herpesvirus structural protein.
Cell.1997;88:223-33.
[12] Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB.The design,
Synthesis, and evaluation of molecules that enable or enhance eellular uptake:peptoid molecular
transporters.Proceedings ofthe National Academy of Sciences.2000;97:13003-8.
[13] Bernstein E, Caudy AA, Hammond SM, Hannon GJ.Role for a bidentate ribonuclease in the
initiation step ofRNA interference.Nature.2001;409:363-6.
[14] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T.Duplexes of 21-nucleotide
RNAs mediate RNA interference in cultured mammalian cells.Nature.2001;411:494-8.
[15] Hammond SM, Bernstein E, Beach D, Hannon GJ.An RNA-directed nuclease mediates
post-transcriptional gene silencing in Drosophila cells.Nature.2000;404:293-6.
[16] Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ.Argonaute2, a link between
genetic and biochemical analyses of RNAi.Science Signaling.2001;293:1146.
[17] Song JJ, Smith SK, Hannon GJ, Joshua-Tor L.Crystal structure of Argonaute and its implications
for RISC slicer activity.Science.2004;305:1434-7.
[18] Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song J-J, et al.Argonaute2is the
catalytic engine of mammalian RNAi.Science Signaling.2004;305:1437.
[19] Collins SA, Guinn B-a, Harrison PT, Scallan MF, OSullivan GC, Tangney M.Viral vectors in
Cancer immunotherapy:which vector for which strategy?Current gene therapy.2008;8:66-78.
[20] Glover DJ, Lipps HJ, Jans DA.Towards safe, non-viral therapeutic gene expression inh umans.
Nature reviews Genetics.2005;6:299-310.
[21] Hussain AF, Kr ü ger HR, Kampmeier F, Weissbach T, Licha K, Kratz F, et al.Targeted Delivery of
Dendritic Polyglycerol-Doxorubicin Conjugates by scFv-SNAP Fusion Protein Suppresses EGFR+
Cancer Cell Growth.Biomacromolecules.2013;14:2510-20.
Summary of the invention
Goal of the invention: for solving problems of the prior art, the present invention provides a kind of cationic polymer base
Because of carrier.
The present invention also to solve the technical problem that the preparation side being to provide above-mentioned cationic polymer gene vector
Method and application.
Technical scheme: for realizing above-mentioned technical purpose, the present invention proposes a kind of cationic polymer gene vector,
This genophore is with N-3-aminopropyl methacrylamide and N-(1,3-dihydroxy-2-propyl group) Methacrylamide
For monomer, Invertible ideal method is used to synthesize block polymer N-(1,3-dihydroxy-2-third
Base) Methacrylamide-b-N-3-aminopropyl methacrylamide polymer, subsequently at N-3-aminopropyltriethoxy third
Coupling guanidino group on acrylamide segment, obtains N-(1,3-dihydroxy-2-propyl group) Methacrylamide-b-N-3-
Guanidine propyl methacrylamide polymer, wherein, guanidinated N-3-aminopropyl methacrylamide segment accounts for
The ratio of polymer is 8.3%~57%.
Preferably, the number-average molecular weight of described cationic polymer gene vector is 23700~45700g/mol.
The polymerization of poly-N-3-aminopropyl methacrylamide in the main chain of described cationic polymer gene vector
Degree is 10-90 for 10-90, N-(1,3-dihydroxy-2-propyl group) the Methacrylamide degree of polymerization.
Present invention further proposes the preparation method of above-mentioned cationic polymer gene vector, comprise the steps:
(1) preparation of poly-DHPMA polymer:
Take dithiobenzoic acid (4-cyanopentanoic acid) ester and ACVA be dissolved in Isosorbide-5-Nitrae-dioxane, obtain solution A,
Then the aqueous solution of solution A and DHPMA is joined in polymerization bottle, logical nitrogen 1~2h, polymerization bottle of sealing with wax,
Then polymerization bottle is put into and be preheated in the oil bath of 70~80 DEG C reaction 12~24h, polymerization bottle is taken out, cooling,
Terminate polyreaction, the product obtained joined in ice acetone and precipitate, centrifugal, repeatedly dissolve, precipitate some
Secondary, finally product is placed in vacuum drying oven 50~60 DEG C and is dried and to obtain product poly-DHPMA polymer, be designated as
PDHPMA;
(2) preparation of poly-DHPMA-b-APMA polymer:
PDHPMA and APMA monomer is dissolved in distilled water, obtains solution B, by solution B and ACVA
Dioxane solution mix homogeneously after be transferred in polymerization bottle, system with drying nitrogen ventilate 1~2h, seal with wax
Polymerization bottle, puts into polymerization bottle and is preheated in 70 DEG C~80 DEG C of oil baths react 12~24h, and reaction will be poly-after terminating
Conjunction bottle takes out, cooling, terminates polyreaction, and product joins in the acetone of pre-cooling and precipitates, centrifugal, the most molten
Solve, precipitation several times, then the product obtained is placed in vacuum drying oven 50~60 DEG C be dried to obtain poly-
DHPMA-b-APMA polymer, is designated as DA polymer;
(3) N-3-guanidine propyl methacrylamide-b-N-(1,3-dihydroxy-2-propyl group) methacrylamide polymer
Synthesis:
By soluble in water for DA polymer, it is subsequently added guanidinated reagent, preferably anti-with saturated sodium carbonate regulation
Answer pH value of solution to 8~10, react 12~24h under room temperature, product is placed in deionized water dialysis, preferably
Dialysis 24h, uses MWCO 8000, and every 2~4h change water, and lyophilization obtains product N-3-guanidine propyl group first
Base acrylamide-b-N-(1,3-dihydroxy-2-propyl group) methacrylamide polymer, is designated as
DHPMA-b-GPMA。
Preferably, in step (1), dithiobenzoic acid (4-cyanopentanoic acid) ester with the mol ratio of DHPMA is
1: 10~90, dithiobenzoic acid (4-cyanopentanoic acid) ester is 1: 1~4 with the mol ratio of ACVA, DHPMA's
The quality of the water in aqueous solution is 1~5 times of DHPMA mass.
In step (2), PDHPMA polymer is 1: 10~90 with the mole dosage ratio of APMA monomer,
The mole dosage ratio of PDHPMA with ACVA is 1: 1~4.
In step (3), described guanidinated reagent is 1-H-pyrazoles-1-amitraz hydrochloride, in DA polymer
APMA segment is 1: 1~10 with the mol ratio of 1-H-pyrazoles-1-amitraz hydrochloride, the consumption of the most guanidinated reagent
For primary amino radical mole in DA polymer segment 2~10 times.
The invention allows for above-mentioned cationic polymer gene vector to carry at medicine as biological carrier materials
In application.
On the other hand, the present invention proposes a kind of cationic polymer carrier loading genomic medicine, and it is to pass through
Genomic medicine is supported on above-mentioned cationic polymer gene vector and obtains by electrostatic attraction, wherein, and described base
Because medicine is siRNA, shRNA, plasmid DNA etc..
Antitumor drug prepared by above-mentioned load genomic medicine, the especially cationic polymer carrier of dewatering medicament
In application equally within protection scope of the present invention.
Beneficial effect: compared with prior art, present invention have the advantage that
(1) polymer support prepared by the present invention can load genomic medicine in a large number, has preferable dewatering medicament
Carry and genomic medicine binding ability;
(2) polymer support prepared by the present invention has preferable permeates cell membranes ability;
(3) polymer prepared by the present invention has relatively small molecular weight distribution and reacts controlled;
(4) there is higher transfection effect than " goldstandard " PEI after polymer prepared by the present invention carries siRNA
Rate.
Accompanying drawing explanation
Fig. 1 is the synthetic route of DA polymer;
Fig. 2 is DG polymer1H NMR spectra;
Fig. 3 is that DG/siRNA complex is at the electrophoresis retardance figure that N/P is when 1~32;A figure is
DG1/siRNA-NC complex, B figure is DG2/siRNA-NC complex, and C figure is DG3/siRNA-NC
Complex;
In Fig. 4, A is the grain-size graph of DA3/siRNA-NC complex and series DG/siRNA-NC complex;
B is the surface potential figure of DA3/siRNA-NC complex and series DG/siRNA-NC complex;C is
DA3/siRNA-NC complex (on) and DG3/siRNA-NC complex (under) scanning electron microscope (SEM) photograph;
In Fig. 5, A is P-gp mrna expression amount schematic diagram;B be DG3/siRNA-NC group (1),
PEI/siRNA-2 group (2), DA3/siRNA-2 complex group (3) and the P-gp of DG3/siRNA-2 group (4)
Expressing quantity, * shows that each group has significant difference (p < 0.05, n=3) with blank group;DG polymer with
The compound ratio of siRNA is 12;
In Fig. 6, A be mtt assay detection hatched altogether DOX, DG/siRNA-3 complex and
DG/siRNA-3 complex and the DOX mixture IC to MCF-7/ADR cell50Value (n=3);B is stream
DOX, DG/siRNA-3 complex and DG/siRNA-3 complex and DOX have been hatched in the detection of formula cell art altogether
Mixture is (upper: DOX group;In: DG/siRNA-3 complex group;Under: DG/siRNA-3 complex with
DOX mixture group;DG polymer is 12 with the compound ratio of siRNA);
The abbreviation implication occurred in description is as shown in table 1 below:
Table 1
Detailed description of the invention
The present invention with N-3-aminopropyl methacrylamide and N-(1,3-dihydroxy-2-propyl group) Methacrylamide is
Monomer, uses Invertible ideal method to synthesize block polymer N-3-aminopropyltriethoxy propylene
Amide-b-N-(1,3-dihydroxy-2-propyl group) methacrylamide polymer, subsequently at N-3-aminopropyltriethoxy propylene
Coupling guanidino group on amide segment, obtains N-3-guanidine propyl methacrylamide-b-N-(1,3-dihydroxy-2-third
Base) methacrylamide polymer polymer, obtain the polymer support with relatively high transfection efficiency.Subsequently
Determined the molecular weight of polymer by gel exclusion chromatography, describe this in detail below by specific embodiment
Bright.
The preparation method of the polymer support of embodiment 1 genomic medicine.
(1) preparation of poly-DHPMA polymer:
DHPMA (1.0430g, 7.0mmol) is dissolved in 1.0ml distilled water, after join 5ml be polymerized bottle
In, dithiobenzoic acid (4-cyanopentanoic acid) ester (0.0336g, 0.12mmol), ACVA (0.0167g, 0.06mmol)
It is dissolved in 0.5mL Isosorbide-5-Nitrae-dioxane, adds it to be polymerized in bottle.System drying nitrogen is ventilated 1h,
Seal with wax polymerization bottle.Polymerization bottle is put into and is preheated in 70 DEG C of oil baths react 24h.Polymerization pipe is taken out, puts
Cool down in 0 DEG C of cold pump, terminate polyreaction, obtain product.Product is joined in ice acetone and precipitate, centrifugal,
Dissolution/precipitation three times repeatedly, product is placed in 50 DEG C of vacuum drying oven and is dried to obtain product poly-DHPMA polymer
(PDHPMA) 0.0362g (productivity 72%).
(2) preparation of poly-DA polymer:
APMA (0.2143g, 1.2mmol), PDHPMA (0.1812g, 0.02mmol) are dissolved in distilled water
In, ACVA (0.0028g, 0.01mmol) is dissolved in 0.5mL Isosorbide-5-Nitrae-dioxane, after the two mix homogeneously
It is transferred in 5mL polymerization bottle.System drying nitrogen is ventilated 1h, polymerization bottle of sealing with wax.By polymerization bottle
Put into and be preheated in 70 DEG C of oil baths react 24h.Polymerization pipe is taken out after terminating by reaction, is placed in 0 DEG C of cold pump cold
But, polyreaction is terminated.Product joins in the acetone of pre-cooling and precipitates, centrifugal, dissolution/precipitation three times repeatedly.
Product is placed in 50 DEG C of vacuum drying oven and is dried to obtain product poly-DA polymer 0.149g (productivity 64%).
(3) N-3-guanidine propyl methacrylamide-b-N-(1,3-dihydroxy-2-propyl group) Methacrylamide (DG)
The synthesis of polymer:
By soluble in water for DA polymer, making primary amine groups concentration is 12mM, adds excessive guanidinated reagent 1-H-
Pyrazoles-1-amitraz hydrochloride so that it is concentration is 65mM, saturated sodium carbonate solution regulation reaction solution pH is extremely
9.0, react 24h under room temperature.Product is placed in deionized water dialysis 24h (MWCO 8000, every 2~4h
Change time water), lyophilization product DG polymer 0.087g (productivity 83%).
Table 2 is the polymer composition of polymer DA1, DA2, DA3 of the different segment ratios of preparation and puts down
Average molecular weight.In the text, DA1, DA2, DA3 have identical implication.
2 three kinds of polymer compositions of table and molecular weight
Title | Polymer | The DHPMA degree of polymerization | The APMA degree of polymerization | Mw | PDI |
DA1 | DHPMA-b-APMA | 90 | 10 | 23700 | 1.51 |
DA2 | DHPMA-b-APMA | 60 | 40 | 31500 | 1.45 |
DA3 | DHPMA-b-APMA | 40 | 60 | 45700 | 1.54 |
The preparation of embodiment 2 DHPMA-b-GPMA/siRNA complex and sign.
(1) preparation of DHPMA-b-GPMA/siRNA complex:
Take quantitative siRNA storing solution and be dissolved in without in enzyme water, by the mole of amino nitrogen in DG polymer with
In siRNA (siRNA 1, siRNA 2, siRNA 3 and siRNA-NC), the mole of phosphate radical is than preparation
DHPMA-b-GPMA/siRNA complex, i.e. taking DG1, DG2 and DG3 polymer by N/P is 1: 1,
1: 4,1: 8 equal proportion adds siRNA (appointing in siRNA-1, siRNA-2, siRNA-3, siRNA-NC
Meaning one) storing solution mixed room temperature hatches 30min, and prepare series DHPMA-b-GPMA/siRNA and be combined
Thing (i.e. DG1, DG2, DG3 and siRNA-1, siRNA-2, siRNA-3, siRNA-NC any two
The complex of person), subsequently by ultrasonic for complex solution and cross 0.22 μm filter membrane be stored in 4 DEG C standby.
(2) agarose gel electrophoresis of DHPMA-b-GPMA/siRNA complex
By charge ratio 1: 1,1: 4,1: 8,1: 16 and 1: 32 mixing DG polymer and siRNA, incubated at room
30min obtains DG1/siRNA-NC, DG2/siRNA-NC and DG3/siRNA-NC complex.Take 1uL
Sample-loading buffer (EDTA 30mM, glycerol 36%, xylene blue 0.035%, bromophenol blue 0.05%,
TaKaRa) add in 1% agarose gel after mixing from the complex of above-mentioned different N/P, with 100V voltage,
Electrophoresis 15min in tbe buffer solution.DNA electrophoresis situation is observed subsequently under the ultraviolet light of 303nm
And take pictures.Result as it is shown on figure 3, when charge ratio is 8 DG3 polymer siRNA-NC can be coated with,
SiRNA-NC can be coated with by DG1 and DG2 when N/P is 32 and 16 simultaneously.Result shows, along with
The increase of molar ratio shared by APMA segment guanidinated in DG polymer, DG polymer is to siRNA's
Absorbability gradually strengthens.When N/P is 8, DG3 polymer can block siRNA completely and moves to positive pole,
Show that the DG3 polymer support of the present invention has higher gene carrying capacity.
(3) particle diameter of DHPMA-b-APMA/siRNA and DHPMA-b-GPMA/siRNA complex, Zeta
Current potential and scanning electron microscope analysis.
By the DA3/siRNA-NC of preparation and three kinds of DG/siRNA-NC complex (DG1/siRNA-NC,
DG2/siRNA-NC and DG3/siRNA-NC complex) at PBS (pH 7.2) solution, be placed in Malvern
In Zetasizer nano ZS laser particle analyzer, at room temperature detect particle diameter and the Zeta potential of four kinds of complex,
Result is as shown in table 3 and accompanying drawing 4.
Table 3
The relatively DG/siRNA complex particle diameter of different GPMA segment ratios understands, under identical N/P,
Along with GPMA segment ratio in the polymer increases, the mean diameter of DG/siRNA complex is the lowest,
SiRNA is under different N/P, and the mean diameter of the DG3/siRNA complex after guanidinated is less than non-guanidine radicals
The DA3/siRNA complex changed, shows that in polymer, GPMA segment proportion is to load and compression
SiRNA has decisive role, and the polymer after guanidinated further increases the compression energy to siRNA
Power.Document shows, particle diameter less than 300nm particle all can with cross-film enter cell, and the DG2 prepared,
When DG3 polymer and siRNA are 8 with charge ratio, the particle diameter of complex is less than 300nm (such as accompanying drawing 3), with
N/P to increase, polymer is the most abundant to the compression of siRNA, when N/P is 32, and all GD/siRNA
The mean diameter of complex all drops to below 300nm, DG2, DG3 polymer and siRNA complex grain
Footpath is further reduced to about 150nm.
Under different charge ratios, DA3/siRNA is the most attached with the surface zeta potential potential value of series DG/siRNA complex
Shown in Fig. 4.Result shows, the GD/siRNA complex of different GPMA segments zeta when N/P is more than 1
Current potential all show on the occasion of, show that electronegative siRNA molecule can be wrapped in the composite under this ranges of charge.
Along with N/P increases, i.e. the amount of polymer increases, and the positive charge density of complex is gradually increased so that zeta
Current potential gradually rises.Wherein, the DG3/siRNA complex after guanidine radicals goes out than the most guanidinated compound features
Strong electropositive.
Take each 10 μ L of DA3/siRNA and DG3/siRNA complex solution to drip on dry silicon chip, use
Filter paper sucks surplus liquid and the most naturally dries.Then under scanning electron microscope (ZEISS Ultra plus)
Observe pattern and the particle diameter distribution of two kinds of complex.Result shows, two kinds of complex forms become nearly ball-type, particle diameter
Distribution comparison rule, diameter is between 150~200nm.
Embodiment 3 in-vitro transfection is tested.
DA3 and DG3 and three kinds of P-gp siRNA of Example 1 preparation are 16 mixing by N/P and hatch
Standby, 6 orifice plates are inoculated (1 × 106Individual/hole) it is in the MCF-7/ADR cell of exponential phase,
After cell confluency is in 60~70%, changes serum-free antibiotic-free or has the culture medium of serum antibiotic-free.With
After above-mentioned DA3/siRNA and DG3/siRNA complex is separately added in different hole (n=3), co-culture
Change the fresh serum free culture system liquid that has after 4h to continue to cultivate to 48h by real-time quantitative PCR and Western
Blot detects P-gp expression.As shown in Figure 5, experiment shows, DA3/siRNA-2, DG3/siRNA-2
Reticent P-gp mrna expression (p < 0.05) notable with DA3/siRNA-3, DG3/siRNA-3 complex all energy,
Wherein DG3/siRNA-2 and DG3/siRNA-3 group is higher to P-gp mRNA silence rate, higher than sympathizing with mutually
PEI group under condition.Western blot test result indicate that DG3/siRNA-2 and DG3/siRNA-3 complex
Can effectively suppress the expression of P-gp albumen.
The reverse MCF-7/ADR drug resistance of embodiment 4 PECL3 and B-PECL3/siRNA complex.
Take the logarithm the human breast cancer cell (MCF-7/ADR) of trophophase, with 1 × 104Individual/hole adds 96 orifice plates
In, every hole adds 150 μ L culture medium, at 37 DEG C, 5%CO2Lower cultivation is to cell confluency 70~80%
Rear replacing fresh culture, every hole is separately added into DOX (0.001 μM~1mM), DG3/siRNA-3 in proportion
The mixed solution of complex (100nM siRNA-3) and DG3/siRNA-3 complex and DOX is (containing 0.001
μM~1mM DOX, 100nM siRNA-3) continue cultivate 72h.Every hole adds 20 μ L (5mg/mL) subsequently
MTT solution continue cultivate 4h, carefully go all culture medium and add 150 μ L dimethyl sulfoxide (DMSO)
The 10min that vibrates on rearmounted shaker makes blue first a ceremonial jade-ladle, used in libation fully dissolve, and detects every hole at 490nm in microplate reader
Absorbance at wavelength.Separately take the logarithm the human breast cancer cell (MCF-7/ADR) of trophophase, with 1 × 106Individual
/ hole adds in 6 orifice plates, and every hole adds 2mL culture medium, at 37 DEG C, 5%CO2Lower cultivation is melted to cell
Replacing fresh culture after conjunction rate 70~80%, every hole addition DOX (0.001 μM~1mM),
The mixing of DG3/siRNA-3 complex (100nM siRNA-3) and DG3/siRNA-3 complex and DOX
Solution (containing 0.001 μM~1mM DOX, 100nM siRNA-3) continues to cultivate 48h.Subsequently, pancreatin
Digestion is collected cell and detects apoptosis rate with flow cytometer after 7AAD/APC apoptosis reagent dyeing.
As shown in Figure 6, DG3/siRNA-3 complex does not affect P-gp siRNA and reverses MCF-7/ADR result
The drug resistance of cell, wherein the drug resistance of DOX group MCF-7/ADR cell be 32.58 μMs and
The drug resistance of DG3/siRNA-3 Yu DOX mixed solution group MCF-7/ADR cell significantly reduces to 4.43 μMs,
Show that DG3 polymer can carry siRNA and to tumor cell and play synergism.
In sum, the present invention proposes gene drug carriers N-(1, the 3-dihydroxy of a kind of novel tumor targeting
-2-propyl group) Methacrylamide-b-N-3-guanidine propyl methacrylamide polymer, it can lead in polar solvent
Cross electrostatic attraction load genomic medicine.The surface of this carrier is N-(1,3-dihydroxy-2-propyl group) methacryl
Amine hydrophilic layer, inside is the N-3-guanidine propyl methacrylamide structure containing positive electricity.This genomic medicine
Carrier can increase therapeutic gene long circulating time, protection therapeutic gene not by internal enzymatic degradation and target tumor group
Knit.The polymer of good biocompatibility that the present invention provides, can in aqueous can be with genomic medicine self assembly shape
Become nano-particle, there is good stability, and preparation method is simple, repeatable high.Polymer reaction bar
Part is the gentleest, and monomer material is cheap and easy to get, and synthesis and the simple productivity of post-processing approach are higher, itself and gene medicine
The complex preparation method that thing is formed is simple, can load genomic medicine in a large number, and the guanidino group of grafting can efficiently be taken
Band genomic medicine and transfection efficiency are high, and have good biocompatibility, have the spies such as good stability simultaneously
Point.This kind of portability genomic medicine and there is the genophore of good biocompatibility have good in disease treatment
Good application prospect.
Genomic medicine, N-(1,3-dihydroxy-2-propyl group) methyl is combined by N-3-aminopropyl methacrylamide
Acrylamide segment forms hydrophilic layer.Experiment finds N-(1,3-dihydroxy-2-propyl group) Methacrylamide segment tool
Best hydrophilic, N-3-aminopropyl methacrylamide segment is had to have preferable genomic medicine load capacity,
Its reason is to have more primary amino radical on segment, improves its ability combining genomic medicine, but N-3-
On aminopropyl methacrylamide segment, primary amino radical its toxicity the highest is the biggest, and N-(1,3-dihydroxy-2-propyl group)
The outer layer that Methacrylamide segment is formed can reduce the cytotoxicity that internal primary amino radical brings.
The complex that three kinds of polymer (DG1, DG2 and DG3) prepared by the present invention are formed with siRNA
Particle diameter and form result show, three kinds of complex size all can meet the requirement being phagocytized by cells.
The outer transfection experiment of DG3/siRNA composite body prepared by the present invention is by transfection P glycoprotein (P-gp)
SiRNA checking carrier transfection efficiency.By, compared with " goldstandard " PEI, illustrating that DG3/siRNA has
There is higher transfection efficiency.
Breast carcinoma (MCF-7/ADR) drug resistance is reversed outside DG3/siRNA-3 composite body prepared by the present invention
Being shown by cytotoxicity experiment, by compared with DOX, it is aobvious that DG3/siRNA-3 Yu DOX hatches group altogether
(the p < 0.05) write has reversed the drug resistance of MCF-7/ADR cell.
Claims (10)
1. a cationic polymer gene vector, it is characterised in that this genophore is with N-3-aminopropyl first
Base acrylamide and N-(1,3-dihydroxy-2-propyl group) Methacrylamide are monomer, use reversible addition fracture chain
Transfer polymerization method has synthesized block polymer N-(1,3-dihydroxy-2-propyl group) Methacrylamide-b-N-3-ammonia third
Butylmethacrylamide polymer, subsequently coupling guanidino group on N-3-aminopropyl methacrylamide segment,
Obtain N-(1,3-dihydroxy-2-propyl group) Methacrylamide-b-N-3-guanidine propyl methacrylamide polymer poly
Compound, wherein, it is 8.3%~57% that guanidinated N-3-aminopropyl methacrylamide segment accounts for the ratio of polymer.
Cationic polymer gene vector the most according to claim 1, it is characterised in that described sun
The number-average molecular weight of ionomer genophore is 23700~45700g/mol.
Cationic polymer gene vector the most according to claim 1, it is characterised in that described sun
In the main chain of ionomer genophore, the degree of polymerization of poly-N-3-aminopropyl methacrylamide is 10-90,
N-(1,3-dihydroxy-2-propyl group) the Methacrylamide degree of polymerization is 10-90.
4. the preparation method of the cationic polymer gene vector described in claim 1, it is characterised in that bag
Include following steps:
(1) preparation of DHPMA polymer:
Take dithiobenzoic acid (4-cyanopentanoic acid) ester and ACVA be dissolved in Isosorbide-5-Nitrae-dioxane, obtain solution A,
Then the aqueous solution of solution A and DHPMA is joined in polymerization bottle, logical nitrogen 1~2h, polymerization bottle of sealing with wax,
Then polymerization bottle is put into and be preheated in the oil bath of 70~80 DEG C reaction 12~24h, polymerization bottle is taken out, cooling,
Terminate polyreaction, the product obtained joined in ice acetone and precipitate, centrifugal, repeatedly dissolve, precipitate some
Secondary, finally product is placed in vacuum drying oven 50~60 DEG C and is dried and to obtain product DHPMA polymer, be designated as
PDHPMA;
(2) preparation of DHPMA-b-APMA polymer:
PDHPMA and APMA monomer is dissolved in distilled water, obtains solution B, by solution B and ACVA
Dioxane solution mix homogeneously after be transferred in polymerization bottle, system with drying nitrogen ventilate 1~2h, seal with wax
Polymerization bottle, puts into polymerization bottle and is preheated in 70 DEG C~80 DEG C of oil baths react 12~24h, and reaction will be poly-after terminating
Conjunction bottle takes out, cooling, terminates polyreaction, and product joins in the acetone of pre-cooling and precipitates, centrifugal, the most molten
Solve, precipitation several times, then the product obtained is placed in vacuum drying oven 50~60 DEG C be dried to obtain
DHPMA-b-APMA polymer, is designated as DA polymer;
(3) synthesis of guanidinated DHPMA-b-APMA polymer:
By soluble in water for DA polymer, it is subsequently added guanidinated reagent, regulates reaction solution pH to 8~10,
Reacting 12~24h under room temperature, product is placed in deionized water dialysis, lyophilization obtains product N-3-guanidine third
Butylmethacrylamide-b-N-(1,3-dihydroxy-2-propyl group) methacrylamide polymer, is designated as
DHPMA-b-GPMA or DG.
Preparation method the most according to claim 4, it is characterised in that in step (1), dithio benzene
Formic acid (4-cyanopentanoic acid) ester is 1: 10~90 with the mol ratio of DHPMA, dithiobenzoic acid (4-cyanopentanoic acid)
Ester is 1: 1~4 with the mol ratio of ACVA, and the quality of the water in the aqueous solution of DHPMA is DHPMA mass
1~5 times.
Preparation method the most according to claim 4, it is characterised in that in step (2), PDHPMA
Polymer is 1: 10~90 with the mole dosage ratio of APMA monomer, the mole dosage of PDHPMA Yu ACVA
Ratio is 1: 1~4.
Preparation method the most according to claim 4, it is characterised in that in step (3), described guanidine
Base reagent is 1-H-pyrazoles-1-amitraz hydrochloride, APMA segment and 1-H-pyrazoles-1-first in DA polymer
The mol ratio of amidine hydrochlorate is 1: 1~10.
8. the cationic polymer gene vector described in claim 1 carries at medicine as biological carrier materials
In application.
9. the cationic polymer carrier loading genomic medicine, it is characterised in that by by genomic medicine
It is supported on the cationic polymer gene vector described in claim 1 by electrostatic attraction and obtains, wherein, institute
The genomic medicine stated is siRNA, shRNA, plasmid DNA etc..
10. the cationic polymer carrier of the load genomic medicine described in claim 9 is preparing antitumor drug
In application.
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