CN108478807A - A kind of nucleic acid drug delivery system and its application - Google Patents
A kind of nucleic acid drug delivery system and its application Download PDFInfo
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
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Abstract
The invention discloses a kind of nucleic acid drug delivery system and its applications.The system is made of neutral base lipid carrier and metal salt, wherein the structure of neutral base lipid carrier is shown in formula I.In addition, the delivery system of the present invention further includes metal salt, it is demonstrated experimentally that the presence of metal ion can effectively improve the silencing activity of nucleic acid drug, realize nucleic acid drug in intracellular and internal effectively transhipment.The present invention is by D, L heteronuclear glycosides is modified, deoxyinosine modification, and the chemical modification methods such as the conjugated modification of peptide and phosphorylation modification are provided commonly for nucleic acid delivery systems research, advantage and rule of the composite modified mode for delivery of nucleic acids are fully excavated, and by the modification strategy in the research of nucleic acid drug.Studies have shown that the product obtained through the chemical modification is more suitable for this system, and have many advantages, such as that stable in physicochemical property, bioactivity are good, permeable membrane is good.A kind of nucleic acid drug delivery system of the present invention will have broad application prospects in field of gene.
Description
Technical field
The present invention relates to a kind of nucleic acid delivery systems, the nucleic acid delivery systems answering in nucleic acid molecules delivery is further related to
With.The invention belongs to biomedicine fields.
Background technology
Gene therapy provides new therapeutic modality for a variety of acquired and genetic disease, is only used for single-gene in early days
The treatment of hereditary disease, at present therapeutic domain expanded to the multigenic disease for seriously threatening human health, including angiocardiopathy,
Hereditary disease, malignant tumour, metabolic disease and infectious diseases (such as hepatitis B, AIDS).Gene therapy is according to importing
The difference of mode can be divided into ex vivo gene therapy and vivo gene therapy.Ex vivo gene therapy refers to first obtaining certain from the patient
Kind cell is cultivated, and is screened in vitro and re-enters patient's body after being expanded, which is injected in vivo
The limitation of mode and cell culture condition.Interior therapeutic method refers to then that exogenous target gene is direct by specific carrier
It imports in organism, used genomic medicine can be genetic fragment (including RNA or DNA), can also be complete genome, just
In large-scale production.Gene (nucleic acid) drug for vivo gene therapy must meet claimed below：(1) there is height to target sequence
The specificity and compatibility of degree；(2) stability in vivo with height, can resist the degradation of various nucleases；(3) have and wear
Saturating cell membrane reaches the ability of target area.Natural gene (nucleic acid) cannot be satisfied these requirements, it is therefore desirable to by certain skill
Art carries out it structural modification, and is conducted into biological cell, the biology of nucleic acid drug delivery system using nucleic acid delivery vector
Compatibility and high efficiency often become the key factor for the treatment of success or failure.Monotechnics transformation nucleic acid drug, which is difficult to reach above-mentioned, to be wanted
It asks, it is then effective settling mode that chemical modification contains equal various ways and act synergistically with carrier.
In biological study field, Plasmid DNA is the important tool of modern biology research, in research genome and its work(
The tool molecule in increasingly important role and gene therapy research can be played in the process.Realize the effective of Plasmid DNA
Delivering is the key that play its function.
Nucleic acid carrier can be divided into viral vectors and non-virus carrier.Viral vectors method has very high transfection efficiency, but has
The shortcomings of preparing anisotropic difficulty, cytotoxicity, immunogenicity, mutagens, shortage target cell positioning, limits its extensive use.It is non-
Viral delivery method is mostly to use artificial synthesized carrier, has preparation simple, performance controllable, hypotoxicity, hypoimmunity etc. excellent
Point has huge application potential in field of gene.
Cationic-liposome is the non-viral genoid carrier being most widely used, usually by cationic head, aliphatic
Tail portion and linking arm composition, are combined with nucleic acid by the coulomb force effect between positive and negative charge, can effectively contain nucleic acid.But
Cationic-liposome is there are many limiting factor is urgently to be resolved hurrily, such as its stronger cytotoxicity and haemocyanin binding ability, more by force
Immunogenicity and liver cumulative effect etc.；In addition, so that it is combined with nucleic acid closer for stronger electrical function, after cross-film
It is difficult to be released effectively (Biomaterials 2008,29,3477-3496), these defect high degrees limit cation lipid
Body further applying in clinical studies.The present inventor, which designs early period, has synthesized cationic-liposome (CLD), in lipid
Stable transfection can be realized under suitable ratio and play effective bioactivity for body, nucleic acid, but have under a high concentration condition
There is certain cytotoxicity.
The base acetamide glycerin ether molecule that the present invention constructs, also construct early period base triacetin ether molecule (in
State's patent 201310006506.9), these molecules have base property head, can pass through hydrogen bond action and electron cloud π-π
Effectively containing for nucleic acid is realized in sedimentation, in addition, we are optimized to containing method, add certain metal from
Son, and chemical modification method is combined, obtain the neutral nucleic acid drug encapsulation delivery system of high-efficiency low-toxicity.
Invention content
The purpose of the present invention is to provide a kind of nucleic acid drug delivery system of high-efficiency low-toxicity and its in delivering nucleic acid drug
In application.
In order to achieve the above object, present invention employs following technological means：
A kind of nucleic acid delivery systems of the present invention, are made of, wherein described neutral base lipid carrier and metal salt
The structural formula of neutral base lipid carrier is shown in formula I：
Wherein, wherein X is oxygen atom or nitrogen-atoms, and B is natural purine and pyrimidine bases, i.e. adenine, guanine, secondary
Xanthine, cytimidine, thymidine and uracil, preferably cytimidine -1- bases or thymidine -1- bases.
Wherein, it is preferred that the fatty long-chain-C of institute in Formulas I18H35Structure be
Wherein, it is preferred that the neutral base lipid carrier has structure as shown below：
Wherein, it is preferred that the metal salt is calcium salt or manganese salt, preferably CaCl2。
Further, the application the invention also provides the nucleic acid delivery systems in preparing nucleic acid delivery reagent.
Wherein, it is preferred that the nucleic acid includes oligonucleotide, aptamers, siRNA and Plasmid DNA.
Wherein, it is preferred that the nucleic acid is the L- heteronuclear glycosides modification using D, deoxyinosine modification, the conjugated modification of peptide with
And the nucleic acid analog that at least one of phosphorylation modification method of modifying is modified.
Compared to the prior art, the beneficial effects of the invention are as follows：
1, a kind of nucleic acid delivery systems of the invention, are made of neutral base lipid carrier and metal salt, wherein neutral
Base lipid carrier has base property head, using between the nucleoside base and nucleic acid base of carrier hydrogen bond and π-π act on,
It forms supramolecular structured merging and forms liposome, avoid nano grain surface from generating cation, realize nucleic acid is transfected into born of the same parents.This
Outside, metal ion is added in delivery system, it is demonstrated experimentally that the silence that the presence of metal ion can effectively improve nucleic acid drug is lived
Property, realize nucleic acid drug effectively intracellular transport and transhipment in vivo.
2, the present invention modifies D, L- heteronuclear glycosides, deoxyinosine modification, the nucleic acid such as the conjugated modification of peptide and phosphorylation modification
Chemical modification method is provided commonly for nucleic acid delivery systems research, has fully excavated advantage of the composite modified mode for delivery of nucleic acids
And rule, and by the modification strategy in the research of nucleic acid drug.The product obtained through the chemical modification method has reason
Change the advantages that property is stable, bioactivity is good, permeable membrane is good, can be widely applied in nucleic acid drug research.This delivery system
Plasmid DNA is can also be used for, to have wide gene studies field application prospect.
Description of the drawings
Fig. 1 is the synthetic route of base acetamide glycerin ether molecule DNTA；
Fig. 2 is the synthetic route of base acetamide glycerin ether molecule DNCA；
Fig. 3 is DNTA (c, d) and the scanning electron microscope of DNCA (a, b) liposome observes result；
Fig. 4 is Cenersen (20bp, 4 μM), DOCA and Cenersen mixtures (base ratio=1:1) the front and back CD of annealing
Spectrum；
Fig. 5 is Cenersen (22bp, 4 μM), DNCA and Cenersen mixtures (base ratio=5:1) the front and back CD of annealing
Spectrum；
Fig. 6 is that base lipid carrier contains the investigation of G3139 antiproliferative activities；
(A)A549cells；(B)A549/TXL cells(DNXA:7.5μM)；(C) contain G3139 pairs of NC and DNCA
Influence (the AONs of A549/TXL cell vigor：400nM)；
Fig. 7 is that acrylamide gel electrophoresis investigates contain efficiency of the DNCA to ss/ds-miR-122 (FAM-RNA samples are
The holes 20pmol/；DNCA=0.44nmol (N/P=1:1))；
Fig. 8 is that acrylamide gel electrophoresis investigation DNCA contains conjugated modification RNA intracellular stabilities；
RNA samples (20pmol) are added among brand-new cell pyrolysis liquid (300cell/ μ L, 1 μ L), under the conditions of 37 DEG C
It is incubated different time；N/P=5:1；
Fig. 9 is that DNCA, lipo contain the conjugated ss/dsRNA cellular uptake abilities of peptide；
(A) unmodified rna s enters born of the same parents' situation；(B) the conjugated modification RNAs of peptide enters born of the same parents' situation；
Figure 10 is the cell inhibitory effect effect that cck-8 detects that DNCA, lipo contain the conjugated ss/dsRNA of peptide；
(A) cytotoxicity of the nano particle to HEK293A；(B) increment inhibitory activity of the nano particle to HepG2.
Figure 11 is that gel electrophoresis investigation DNCA contains single stranded DNA encapsulation efficiency；
N/P=0 represents pure nucleic acid sequence, no DNCA carriers；
Figure 12 is AS1411 (26bp, 4 μM), DNTA and AS1411 mixtures (base ratio=3:1) the Tm values after annealing；
Figure 13 A for DNTA/DOTA/DNCA/DOCA, test by 72 hours cell viabilities on MCF-7 cells；
Figure 13 B are AS1411 (being contained by DNTA/DOTA/DNCA/DOCA) to A549, the anti-increasing of MCF-7 and K562 cells
Grow activity；
Cell viability is measured after measuring addition AS1411 using CCK-8 within 48 hours：The set for implementing three groups of different experiments is (each
In triplicate), and by each value it is expressed as average value ± standard deviation (1：Control；2：26μM DNCA；3：200nM AS1411；
4：200nM AS1411+26μM DNTA；5：200nM AS1411+26μMDOTA；6：200nM AS1411+26μM DNCA；7：
200nM AS1411+26μM DOCA)；
Figure 13 C are intakes of the AS1411 of FAM- labels in A549 cells；
Figure 14 is GenOpti intermediate ions and amino acid composition to A549 and A549/TXL cell inhibitory effect effects；
Solvent：PBS；Component：50μM；AS1411(A)：200nM；DNCA(D)：15μM；Ca-2(C):0.2g/L；Mn-2:
9.9*10-8g/L；
Figure 15 is CaCl2Transfection concentrations and annealing temperature are to A549/TXL cell inhibitory effect effects；
Figure 16 be base lipid carrier transfect pEGFP-N1 plasmids transfection efficiency (n=3) (+indicate serum transfection
For 24 hours ,-indicate to enrich blood clearly after serum-free transfects 4h)；
Figure 17 is the Toxicity test (48h, n=2) that base lipid carrier is used for pMB3 plasmid transfections.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.People in the art
Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are each fallen in protection scope of the present invention.
The synthesis of first part's base lipid carrier
The synthesis of embodiment 1 base triacetin ether-ether molecule DOTA and DOCA
Base lipid carrier DOTA and DOCA is CN201310006506.9 according to number of patent application, entitled
The Chinese patent Shen of " a kind of base triacetin ether-ether molecule, chemical synthesis process and its application in field of gene "
Method that please be recorded synthesizes.
The synthesis of embodiment 2 base acetamide glycerin ether ester molecule DNTA and DNCA
(1) synthesis of oleyl alcohol sulfonyloxy methyl ester
By oleyl alcohol (50g, 85%purity, 158mmol), Et3N (40mL, 286mmol) is added to the round-bottomed flask of 1L
In, DCM (500mL) is added, is placed on ice bath and is sufficiently stirred, temperature is made to be reduced to 0 DEG C.It is slowly added into first thereto by syringe
Sulfonic acid chloride (16mL, 206mmol), solution becomes cloudy.Ice bath is removed later, reaction solution is made slowly to return back to room temperature, continues to stir
12h.Water (250mL) is added to quench the reaction, organic phase is then detached by separatory funnel.Water phase is anti-with DCM (250mL × 2)
Extraction, is then combined with organic phase.Organic phase after merging uses 1N hydrochloric acid (250mL), 10%NaHCO successively3Aqueous solution (250mL) and
Saturated salt solution (250mL) washs, anhydrous Na2SO4It is dry.Organic phase evaporated under reduced pressure, residue are detached by silica gel column chromatography
(eluant, eluent:Petrol ether/ethyl acetate=20/1, Rf=0.3) pale yellowish oil liquid 44.3g, yield 81%, are obtained.1H
NMR(400MHz,CDCl3):δ=5.30-5.43 (m, 2H), 4.22 (t, J=6.6Hz, 2H), 3.00 (s, 3H), 1.90-2.10
(m, 4H), 1.70-1.80 (m, 2H), 1.20-1.40 (m, 22H), 0.88 (t, J=6.8Hz, 3H)；13C NMR(100MHz,
CDCl3):δ=130.2,129.9,70.3,37.5,32.0,29.90,29.83,29.66,29.46,29.29,29.26,
29.15,27.36,27.30,25.6,22.8,14.3；IR (neat) ν=2925.5,2854.5,1463.6,1355.9,
1175.4,974.8,947.8,831.7,721.6,528.8；MS(ESI-TOF+)for C19H38O3SNa[M+Na]+
found369.2315,calcd369.2434；Anal.calcdfor C19H38O3S:C 65.85,H 11.05,Found:C
65.63,H 10.98.
(2) synthesis of 1- triphenylmethoxies glyceryl alcohol
Glycerine (40g, 435mmol), triphenylchloromethane (30g, 107mmol), DMAP (300mg, 2.46mmol) are set
In dry 500mL round-bottomed flasks, THF (80mL) and Et is added3N (18mL), is stirred at room temperature 12h.It is added into reaction solution
Water (100mL) to quench the reaction, is then added ethyl acetate (150mL) and dilutes.After fully shaking, mixed liquor is transferred to liquid separation
In funnel, organic phase is detached.Water phase is extracted with ethyl acetate (100mL × 2), is then combined with organic phase.Organic phase after merging
Successively with saturation NaHCO3Aqueous solution (200mL), water (200mL) and saturated salt solution (200mL) washing, anhydrous Na2SO4It is dry.
After filtering, solvent evaporated obtains yellow oil.It is dissolved into toluene/n-hexane (200mL, v/v=1/1), at room temperature
It places for 24 hours, crystallizes out white solid 29g, yield 85%.1H NMR(400MHz,CDCl3):δ=7.38-7.48 (m, 6H),
7.20-7.35(m,9H),3.84(s,1H),3.63-3.71(m,1H),3.53-3.63(m,1H),3.20-3.28(m,2H),
2.74(brs,1H),2.35(brs,1H)；13C NMR(100MHz,CDCl3):δ=1438,1287,1280,127
3,871,713,651,644；IR (film, KBr)=33808,30581,2920.0,2866.8,1490.0,
1447.8,1081.5,1028.5,699.8；MS(EI)for C22H22O3[M]+found 334.5,calcd 334.2；
Anal.calcd for C22H22O3:C 79.02,H 6.63,Found:C 79.26,H 6.49.
(3) synthesis of two oleyl alcohol ethers of 1- trityl groups -2,3--glycerine
By 1- triphenylmethoxies-glycerine -2,3- glycol (8g, 23.1mmol), KOH (3.3g, 58.9mmol) and oleyl alcohol
It is dissolved in dry benzene (150mL) solution after being mixed to sulfonyloxy methyl ester (19.2g, 55.42mmol), equips water knockout drum, heating
To 80 DEG C, flow back 32 hours.Ethyl acetate 100mL and water 150mL is added thereto later, extraction detaches organic phase.Water phase is used
Ethyl acetate (150mL × 3) extracts, and merges organic phase, anhydrous Na2SO4It is dry, decompression silica gel column chromatography separation after solvent evaporated,
Obtain target product 6.1g, yield 31%.Separately obtain 1- triphenylmethoxy -3- oleyl alcohol ether-glycerine -2- alcohol 3.7g, yield
It is 27%.Target product is weak yellow liquid.1H NMR(400MHz,CDCl3):δ=7.40-7.50 (m, 6H), 7.18-7.32
(m,9H),5.26-5.43(m,4H),3.50-3.60(m,5H),3.35-3.45(m,2H),3.12-3.20(m,2H),1.92-
2.08 (m, 8H), 1.50-1.58 (m, 4H), 1.26 (brs, 44H), 0.88 (t, J=6.6Hz, 6H)；13C NMR(100MHz,
CDCl3):δ=144.31,130.07,130.00,128.90,127.85,127.02,86.64,78.45,71 .76,71.33,
70.84,63.73,32.77,32.06,30.28,29.94,29.93,29.85,29.82,29.72,29.68,29.65,
29.47,27.37,27.06,26.31,26.25,22.84,14.27；IR (film, KBr) ν=3004.4,2925.3,
2854.1,1742.6,1597.7,1490.7,1450.0,1220.6,1118.7,763.9,745.0,704.1,632.8cm-1；
MS(ESI-TOF+)for C58H90O3Na[M+Na]+found 857.9059,calcd 857.6782；Anal.calcd for
C58H90O3:C 83.39,H 10.86,Found:C 83.10,H 10.62.
(4) synthesis of bis- oleyl alcohol ethers of 1,2--glycerine -3- alcohol
Two oleyl alcohol ethers of 1- trityl groups -2,3--glycerine (8.34g, 10mmol) is taken to be suspended in methanol-tetrahydrofuran
In (100mL, v/v=1/1) mixed solution, concentrated hydrochloric acid (2mL, 12M) is added, stirs 2h at room temperature.TLC detections have found raw material
Through reacting completely.Ethyl acetate (50mL) and water (100mL) are added into residue for evaporated under reduced pressure solvent, and being detached after extraction has
Machine phase.Water phase is extracted with ethyl acetate (3 × 100mL), merges organic phase, anhydrous Na2SO4It is dry.It is filtered to remove drier, is subtracted
Solvent evaporated, silica gel column chromatography is pressed to detach (eluant, eluent：Petrol ether/ethyl acetate=20/1, Rf=0.2) target product, is obtained
3.7g, yield 62%.Pale yellowish oil liquid.1H NMR(400MHz,CDCl3):δ=5.30-5.45 (m, 4H), 3.40-3.75
(m,9H),2.18(s,1H),1.90-2.10(m,8H),1.55-1.65(m,4H),1.25-1.40(brs,44H),0.88(t,J
=6.4Hz, 6H)；13C NMR(100MHz,CDCl3):δ=130.10,129.97,78.39,72.00,71.07,70.54,
63.27,32.06,30.23,29.92,29.85,29.81,29.77,29.67,29.65,29.60,29.47,29.41,
27.36,26.25,22.83,14.25；IR (film, KBr) ν=3470.1,3004.4,2925.4,2854.0,1651.2,
1463.2,1376.2,1117.5,1041.3,968.0,721.9cm-1；MS(ESI-TOF+)for C39H76O3Na[M+Na]+
found 615.7213,calcd 615.5687；Anal.calcd for C39H76O3:C 78.99,H 12.92,Found:C
78.72,H 12.68.
(5) bis- oleyl alcohol ether -3- glyceryl alcohol methanesulfonates of 1,2-
Take triethylamine (0.54mL, 3.91mmol), 1,2-, bis- oleyl alcohol ethers-glycerine -3- alcohol (1.932g, 3.26mmol) in
In 25mL single port bottles, dry dichloromethane (5ml) is added, magnetic agitation makes it dissolve, and ice-water bath makes reaction system be cooled to 0
DEG C, mesyl chloride (0.3mL, 3.91mmol) is added dropwise, 0 DEG C the reaction was continued 2 hours.Reaction system is added to saturation
NaHCO3In (50mL) solution, organic phase, water phase CH are detached2Cl2(2 × 50mL) is extracted, and merges organic phase, anhydrous Na2SO4It is dry
It is dry overnight.It is filtered to remove drier, evaporated under reduced pressure solvent, residue detaches (eluant, eluent with silica gel column chromatography：Petroleum ether/methanol
=120/1) target product 2.02mg (yield 92.6%), is obtained.1H NMR(400MHz,CDCl3)δ5.37(s,4H),4.40
(d, J=10.9Hz, 1H), 4.27 (d, J=10.7,5.8Hz, 1H), 3.54 (d, J=29.7,27.4,20.6Hz, 8H), 3.06
(s, 3H), 2.03 (d, J=5.2Hz, 7H), 1.58 (s, 4H), 1.29 (d, J=10.6Hz, 44H), 0.90 (t, J=6.1Hz,
6H).13C NMR(101MHz,CDCl3)δ127.93,127.27,77.34,77.22,77.02,76.70,76.38,71.90,
70.83,69.08,37.40,32.62,31.92,29.95,29.78,29.71,29.59,29.54,29.52,29.33,
29.28,27.23,26.08,26.02,22.70,14.13.MS(EI)for C40H78O5S[M+Na]+found 693.36,
calcd 693.55；
(6) bis- oleyl alcohol ethers of 1,2--glycerine -3- nitrine
Dry compound 1 is taken, bis- oleyl alcohol ethers of 2--glycerine -3- methanesulfonates (970mg, 1.44mmol) is in 25mL single port
In bottle, dry DMF (8mL) solution is added, magnetic agitation makes it dissolve, and NaN is added3(188mg, 2.88mmol) argon gas is protected
Shield, reaction are warming up to 70 DEG C and react 15 hours.Reaction solution is cooled to room temperature, acetone (30mL) is added and dilutes, reaction solution silicon
Diatomaceous earth filters, and filtrate decompression is evaporated, and residue detaches (eluant, eluent with silica gel column chromatography：Petrol ether/ethyl acetate=200/1),
Obtain colorless oil target product 345.5mg (yield 56%).1H NMR(400MHz,CDCl3)δ5.37(s,4H),3.60–
3.36 (m, 9H), 2.31 (s, 1H), 2.07-1.95 (m, 8H), 1.59 (d, J=8.3Hz, 4H), 1.30 (d, J=10.8Hz,
44H),0.90(s,6H).13C NMR(101MHz,CDCl3) δ 129.89 (d, J=10.6Hz), 77.90 (s), 77.45-76.95
(m),76.95–76.86(m),76.70(s),71.79(s),70.66(s),70.11(s),52.08(s),32.62(s),
31.92 (s), 30.38-29.54 (m), 29.37 (t, J=11.7Hz), 27.22 (s), and 26.07 (d, J=7.5Hz), 22.70
(s),14.13(s).MS(EI)for C39H75N3O2[M+Na]+found 640.49,calcd 640.58.
(7) bis- oleyl alcohol ethers of 1,2--glycerine -3- amine
1,2-, bis- oleyl alcohol ethers-glycerine -3- nitrine (309mg, 0.5mmol) is added in dry THF (10ml), magnetic force
It stirs to dissolve, solution is cooled to 0 DEG C, LiAlH is added4(95mg, 2.5mmol) argon gas is protected, will be molten after reaction 45min
Liquid is warming up to room temperature, the reaction was continued 2.5h, and saturation Na is added2SO4Reaction, reaction solution diatomite mistake is quenched in (0.7mL) solution
Filter, filtrate decompression are evaporated, residue silicagel column column chromatography for separation (eluant, eluent：Methylene chloride/methanol=50/1), obtain target production
Object 233.5mg (yield 79%).Pale yellow oil.
1H NMR(400MHz,CDCl3):δ=5.30-5.45 (m, 4H), 3.40-3.75 (m, 9H), 2.18 (s, 1H),
1.90-2.10 (m, 8H), 1.55-1.65 (m, 4H), 1.25-1.40 (brs, 44H), 0.88 (t, J=6.4Hz, 6H)；13C NMR
(100MHz,CDCl3):δ=130.10,129.97,78.39,72.00,71.07,70.54,63.27,32.06,30.23,
29.92,29.85,29.81,29.77,29.67,29.65,29.60,29.47,29.41,27.36,26.25,22.83,
14.25；MS(ESI-TOF+)for C39H77NO2[M-H]-found 590.75,calcd 591.60；
(8) synthesis of (thymidine -1- bases)-acetic acid
Thymidine (10.0g, 79.3mmol) is suspended in H2O (150mL), be added thereto KOH aqueous solutions (50mL,
3.6M).After 10min is stirred at room temperature in the mixture, solution gradually becomes clarification.Then thereto be added monoxone (15.0g,
159mmol), reaction solution is heated to reflux 90min.After reaction solution is cooled to room temperature, it is acidified to pH=3 with concentrated hydrochloric acid, then at 4 DEG C
Under stand overnight, be precipitated white crystalline precipitate.The white crystalline precipitate, P is obtained by filtration2O5Vacuum drying, obtains target product
4.5g (yield 31%).1H NMR(400MHz,DMSO-d6):δ=13.11 (s, 1H), 11.34 (s, 1H), 7.50 (s, 1H),
4.37(s,2H),1.75(s,3H)；13C NMR(100MHz,DMSO-d6):δ=169.6,164.4,151.0,141.8,
108.4,48.4,11.9；IR (film, KBr) ν=3180.2,3076.2,3027.0,2962.3,2835.8,1737.7,
1708.4,1664.8,1631.9,1418.3,1356.3,1201.7,1147.0,829.8,566.9cm-1；MS(EI):m/z
(%):184.1(39)[M+],95.9(100)；Anal.Calcd for C7H8N2O4:C 45.66,H 4.38,N 15.21,
Found:C 45.59,H 4.40,N 15.25.
(9) synthesis of (thymidine -1- bases)-acetyl-(n-hydroxysuccinimide) -ester
(thymidine -1- bases)-acetic acid (3g, 16.3mmol) and dry DMF are added into dry 25mL eggplant-shape bottles
(30mL), stirring makes it completely dissolved.Then n-hydroxysuccinimide (2.38g, 21mmol) and N, N '-two are added thereto
Carbodicyclo hexylimide (DCC, 3.36g, 16.3mmol).It is stirred overnight at room temperature, a large amount of white precipitates is precipitated.It is heavy to be filtered to remove
It forms sediment, then filtrate decompression distillation redissolves residue in DMF (5mL).Anhydrous ether (30mL) is added thereto, is precipitated white
Color solid.The solid is obtained by filtration, is dried in vacuo, obtains target product 4.6g (yield 61%).White solid.1H NMR
(400MHz,DMSO-d6):δ=11.52 (s, 1H), 7.63 (s, 1H), 4.96 (s, 2H), 2.83 (brs, 4H), 1.77 (s,
3H)；13C NMR(100MHz,DMSO-d6):δ=169.8,165.0,164.2,150.7,140.8,109.3,46.4,25.5,
11.9；IR (film, KBr) ν=3154.4,3003.4,2830.5,1827.4,1785.4,1739.8,1697.2,1467.8,
1422.8,1382.8,1358.6,1213.7,1106.8,1065.1,793.9,651.3cm-1；MS(ESI-TOF+)for
C11H11N3O6Na[M+Na]+found 304.0489,calcd 304.0540；Anal.Calcd for C11H11N3O6:C
46.98,H 3.94,N 14.94,Found:C 46.75,H 3.96,N 14.95.
(10) 1,2, the synthesis of-two (oleyl)-glycerine -3- amine-(thymidine -1- bases)-acetonyl ester (DNTA)
The synthetic route of DNTA is as shown in Figure 1.
By (thymidine -1- bases)-acetyl-(n-hydroxysuccinimide) -ester (280mg, 1.0mmol), 1,2,-two
(oleyl)-glycerine -3- amine (709mg, 1.2mmol), DMPA (14.6mg, 0.1mmol), pyridine (0.4mL) and anhydrous DMF
(20mL) is mixed, and argon gas protection is stirred overnight at room temperature.Ethyl acetate (200mL) is added thereto to dilute, is transferred to separatory funnel
In, dilute hydrochloric acid (0.1M), saturation NaHCO are used successively3Aqueous solution, water, saturated salt solution rinse, and organic phase is dry with anhydrous sodium sulfate
It is dry.It is filtered to remove drier, filtrate decompression is evaporated, and residue detaches (eluant, eluent with silica gel column chromatography：Methylene chloride/methanol=
50/1) target product 537.5mg (yield 71%), is obtained.Pale yellow oil.1H NMR(400MHz,CDCl3) δ=8.12
(s, 1H), 7.09 (s, 1H), 6.43 (s, 1H), 5.38 (d, J=18.8Hz, 4H), 4.31 (s, 2H), 3.58 (s, 1H), 3.52-
3.42 (m, 6H), 2.05-1.94 (m, 10H), 1.60 (s, 4H), 1.29 (d, J=11.7Hz, 45H), 0.89 (d, J=6.7Hz,
6H).(ESI-MS)for C46H83N3O5[M-H]-found756.61,calcd.757.23.13C NMR(100MHz,CDCl3):δ
=167.54,163.69,150.61,140.18,130.13,129.98,111.38,76.35,7 2.03,70.89,69.97,
65.77,48.60,32.06,29.93,29.78,29.67,29.61,29.48,27.37,26.19,14.25,12.45；(ESI-
MS)for C46H83N3O5[M-H]-found 756.61,calcd.757.23.
(11) synthesis of (4-N- (hexichol methoxycarbonyl group)-cytimidine) -1- acetic acid
Cytimidine (10.3g, 90mmol, 1.0eq) is taken to be dissolved in DMF (90mL), addition potassium tert-butoxide (11.6g,
103.5mmol, 1.15eq), reaction system is heated to 100 DEG C later and is reacted 2 hours.Reaction system is cooled to 10 DEG C, point
2- benzyl acetate bromides (16.05mL, 101mmol, 1.12eq) are added dropwise dropwise within 30 minutes, are warming up to reaction system after being added dropwise
The reaction was continued 12 hours for room temperature, and acetic acid (5.9mL, 103.5mmol, 1.2eq) is added and is quenched instead, reaction solution is spin-dried for.Residue
It is resuspended in H2In O (100mL), filtered after continuing stirring 4 hours, H2O (4x 150mL) is washed, and cytimidine-is obtained after drying
1- benzylacetic acids 20.6g.It takes cytimidine -1- benzylacetic acids (20.6g, 82mmol, 1.0eq) to be dissolved in DMF (160mL), is added
N, N'- carbonyl dimidazoles (21.25g, 131.25mmol, 1.6eq).Methanol is added after the reaction was complete in TLC detections, continues to stir
1.5 hours, benzhydrol (19.65g, 106.5mmol, 1.3eq) is added.Reaction system is heated to 60 DEG C, in two batches later
Benzohydrol (2x1.825g, 9.9mmol, 0.12eq) is added in 1 hour in interval, and the reaction was continued 6 hours.Stop heating reaction 12
Hour, methanol (4.65g, 115mmol, 1.4eq) is added, reaction is quenched.Reaction solution is spin-dried for, residue continues to be tied again with ethyl alcohol
Crystalline substance, later methanol (100mL) be recrystallized to give (4-N- (hexichol methoxycarbonyl group)-cytimidine) -1- acetate 29.35g.Take (4-
N- (hexichol methoxycarbonyl group)-cytimidine) -1- acetate (29.35g, 62.5mmol, 1.0eq) is dissolved in acetonitrile:MeOH:H2O:
EtOH(2:2:1:1,350mL) in mixed solution system, heating makes compound dissolve, and is cooled to 0 DEG C later, and LiOH.H2O is added
The aqueous solution (196.8mL) of (25.5g, 0.61mol, 9.7eq).TLC detections find to be added after the reaction was complete citric acid (58.5g,
303.5,4.9eq reaction is quenched in aqueous solution (290mL)).Obtain (4-N- (hexichol methoxycarbonyl group)-cytimidine) -1- acetic acid
22.1g。1H NMR (400MHz, D6-DMSO, 25 DEG C) δ 8.03-8.01 (d, J=7.5,1H, C6), 7.46 (d, J=7.5,4H,
), Ph 7.38 (t, J=7.5,4H, Ph), 7.3 (d, J=7.5,2H, Ph), 6.96 (d, J=7.5,1H, C5),6.82(s,1H,
CH-(C6H5)2),4.50(s,2H,N-CH2-CO)；13C NMR(100MHz,D6-DMSO,25℃)δ169.9,163.5,155.5,
152.8,151.1,140.8,129.0,128.3,126.9,94.2,71.9,51.3；HRMS(ESI)calculated for
C20H17N3O5:(M+Na+):402.1060,found:402.1010.
(12) 1,2, the synthesis of-two (oleyl)-glycerine -3- amine-(cytimidine -1- bases)-acetonyl ester (DNCA)
The synthetic route of DNCA is as shown in Figure 2.
Into dry 25mL eggplant-shape bottles be added (4-N- (hexichol methoxycarbonyl group)-cytimidine) -1- acetic acid (3.29g,
10mmol) with dry DMF (30mL), stirring makes it completely dissolved.Then n-hydroxysuccinimide is added thereto
(14.73g, 13mmol) and N, N '-dicyclohexylcarbodiimides (DCC, 2.06g, 10mmol).It is stirred overnight at room temperature, is precipitated big
Measure white precipitate.It is filtered to remove precipitation, then filtrate decompression distillation redissolves residue in DMF (5mL).It is added thereto
White solid is precipitated in anhydrous ether (30mL).The solid is obtained by filtration, is dried in vacuo, obtains target product 2.62g (yields
55%).White solid.By (4-N- (hexichol methoxycarbonyl group)-cytimidine)-(n-hydroxysuccinimide) -ester (476mg,
1.0mmol), 1,2,-two (oleyl)-glycerine -3- amine (910mg, 1.2mmol), DMPA (14.6mg, 0.1mmol), pyridine
(0.4mL) and anhydrous DMF (20mL) mix, and argon gas protection is stirred overnight at room temperature.It is dilute that ethyl acetate (200mL) is added thereto
It releases, is transferred in separatory funnel, use dilute hydrochloric acid (0.1M), saturation NaHCO successively3Aqueous solution, water, saturated salt solution rinse, and have
Machine is mutually dried with anhydrous sodium sulfate.It is filtered to remove drier, filtrate decompression is evaporated, and residue detaches (elution with silica gel column chromatography
Agent：Methylene chloride/methanol=50/1), obtain target product 485.5mg (yield 51%).By 1,2 ,-two (oleyl)-glycerine-
3- amine-(4-N- (hexichol methoxycarbonyl group)-cytimidine)-acetonyl ester (476mg, 0.5mmol) is dissolved in the CH of 5%TFA2Cl2(20mL)
In, it is stirred at room temperature 0.5 hour.DCM (30mL) is added and water (30mL) extracts liquid separation, water phase is stripped with DCM (2x30mL),
Merge organic phase.Organic phase uses 10%NaHCO successively3(200mL), water (200mL) and saturated salt solution (200mL) washing, nothing
Aqueous sodium persulfate is dried overnight.Evaporated under reduced pressure organic solvent, silica gel chromatograph post separation (eluant, eluent：Methylene chloride/methanol=50/1),
Obtain target product 345mg (yield 93%) DNCA:1H NMR(400MHz,CDCl3) δ=7.44-7.29 (m, 4H), 5.80 (d,
J=3.8Hz, 1H), 5.37 (dd, J=12.0,7.4Hz, 4H), 4.45 (s, 2H), 3.59-3.40 (m, 7H), 2.11-1.91
(m, 8H), 1.55 (s, 4H), 1.44-1.10 (m, 45H), 0.90 (t, J=6.4Hz, 6H) (ESI-MS) for C45H82N4O4[M
+H]+found 743.50,[M+Na]+found 756.44.calcd.742.21.
The liposome preparation of embodiment 3, base acetamide glycerin ether
Base acetamide glycerin ether molecule has amphipathic structure, can be prepared into the supramolecular structures such as liposome.With
For DNTA, method for preparing lipidosome is：DNTA (1.18mg, 1.56 μm of ol) is taken to be dissolved in methanol (1mL) solution, vortex makes
It is fully dissolved, and the methanol solution (12.8 μ L) of DNTA is taken to be added in 200 μ L centrifuge tubes after centrifugation, and the whirlpools afterwards PBS (100 μ L) are added
Rotation (10s) makes it mix well.Place it in the annealing of PCR Programs.Program：95 DEG C are heated 10 minutes, and every 5 clock cools down 5 DEG C, directly
To 15 DEG C, 4 DEG C of preservations later.Annealing is completed to be placed on 40 DEG C of incubation 30min, is that methanol vapors away completely, obtains its liposome
Solution.
Granularity and its form：
It can be seen that neutral base lipid carrier molecule DNCA and DNTA can be in water under projection electron microscope (TEM)
The nano particle of stable uniform is formed in solution, DNTA molecules voluntarily assemble the liposomal particle size of formation on the left sides 150nm in water
The right side, DNCA carrier molecules voluntarily assemble the liposomal particle size of formation at 180nm or so (Fig. 3) in water.
The liposome preparation of embodiment 4, base triacetin ether
It is CN201310006506.9 according to number of patent application, entitled " a kind of base triacetin ether-ether molecule,
It is prepared by the method recorded in the Chinese patent application of its chemical synthesis process and its application in field of gene ".
Application of the second part base lipid carrier in nucleic acid drug delivering
The CD spectral investigations that embodiment 5, base lipid carrier are combined with nucleic acid
The present invention utilizes circular dichroism (CD) spectral investigation nucleoside base lipid carrier DOCA, DNCA and antisense nucleic acid
The combination of Cenersen (22bp, 4 μM), Cenersen are the antisense containing 22 bases for target with p53mRNA exons 1s 0
Nucleic acid sequence.By base lipid carrier DOCA, DNCA respectively with Cenersn mixing after annealings, compound is obtained.Annealing uses
PCR instrument, program are identical as the program of amplification of nucleic acid double-strand.Fig. 4 and Fig. 5 gives the CD light of oligonucleotide in aqueous solution
Spectrum.It can be seen that from the result and be mixed with base lipid carrier, there is no apparent variation occurs for CD spectrum.But
After annealing, CD spectrum have certain change.
The transfection efficiency that 6 base lipid carrier of embodiment contains oligonucleotides G3139 is investigated
The present invention has investigated the neutral base lipid carrier DNCA, DOCA, DNTA of synthesis, and DOTA contains G3139 activity effect
Fruit, G3139 are the antisense nucleic acid of a full thio-modification containing 18 bases, the first six codon of it and Bcl-2mRNA
Complementation, can be with efficient targeting drug resistant gene Bcl-2, and inhibits tumour growth.
1, method
By taking DNTA and DNCA as an example, the G3139 of FAM labels is template, has studied thymine alkali bases acetamide glycerin ether
Nucleic acid transfection ability.Specific coating is as follows with transfection process：
DNTA or DNCA liposomes and FAM-G3139 are configured to mixed solution with certain base ratio.96 DEG C are heated to,
Then it is decremented to 4 DEG C (annealing) step by step, is placed 2 days at 4 DEG C.Cell used in transfection experiment is A549 and its persister A549/TXL
Cell line.
(1) bed board：300,000 cells are added in six orifice plates, are 1.8mL per pore volume；
(2) after 18-24h, sample is diluted in appropriate opti-MEM, liquid-transfering gun blows and beats mixing, and 20 μ L are added per hole should
Mixed solution makes the final concentration of 100nM of FAM-G3139.It is not added with DNA and the hole of DNA is only added as two kinds of negative controls, addition
Transfection reagent Lipofectamine 2000-DNA compounds (method that transfection reagent is added with DNA groups is carried out by protocol)
For positive control；
(3) it is washed three times with 500 μ L PBS；
(4) after 4h, liquid in culture hole is sucked, after 250 μ L, 5% trypsin digestions are added, is placed in 1.5mLEP pipes,
It is added and fixes cell 15min containing 4% paraformaldehyde；
(5) it centrifuges, abandons supernatant, add 400 μ LPBS；
(6) flow cytomery fluorescence intensity is used.
2, result
Experimental result as shown in fig. 6, should the result shows that, in A549 and its persister A549/TXL cell lines, DNCA's
Delivery capability is shown about better than other three kinds of base lipid carriers under conditions of DNCA (7.5 μM)/G3139 (400nM)
35% Cytostatic to tumor cell effect.In addition, under the conditions of G3139=400nM, DNCA concentration is gradually carried from 7.5 μM
Height is to 15 μM, and tumors inhibition activity is without significantly improving.
Embodiment 7, DNCA contain the investigation of ssRNA efficiency
This experiment investigates DNCA using 20% native gel electrophoresis and contains efficiency to ss/ds-miR-122.N/P setting from
1:2–10:1, RNA sample carries out 6-FAM labels in 5 '-ends.
MiR-122 is high in normal liver organization to express and participates in adjusting the related physiology course of various kinds of cell metabolism,
But it is substantially reduced in Expression In Hepatocellular Carcinoma amount.Related research confirms that miR-122 is overexpressed in liver cancer cells can inhibit cancer
The proliferation of cell shifts, and infiltration increases its sensibility for drug.In addition, miR-122 can also target carcinoma of urinary bladder VEGFC
Gene, and signal path AKT downstream and mTOR generations are significantly affected, it finally plays and efficiently inhibits tumour growth and new green blood
The effect that pipe generates.Natural miR-122 has the duplex structure of Incomplete matching, with lagging strand after Ago2 protein bindings from
It goes, form activity RISC and plays physiological function.Since miR-122 is there are mismatch site, the sequence is caused easily to be known by ribozyme
It does not degrade；In addition, the both ends miR-122 thermodynamic differences are not obvious, therefore lagging strand has larger possibility to enter RISC
Lead to the generation of undershooting-effect.Therefore this experiment by way of structure of modification by miR-122 be designed to double-strand non-fully match,
Double-strand exactly matches and single-stranded three kinds of forms.Enter RISC possibility to reduce lagging strand, L- heteronuclear glycosides is carried out in its 5 '-end
(L-isoA) it modifies, which is CN201210020028.2 according to number of patent application, entitled " a kind of using different
Method that nucleosides is chemically modified siRNA sequence, and products thereof and application " Chinese patent application recorded in method into
Row, to eliminate chain activity.In addition, to improve miR-122 stability, double-strand 3 '-end carries out peptide and (3 '-KALLAL- is conjugated
5 ') it modifies, reduces the possibility that RNA is degraded by 3 '-excision enzymes, which is according to number of patent application
WO2016197264A1, entitled " a kind of modification of combination heteronuclear glycosides, terminal peptide be conjugated and the small interference of cationic-liposome
Method recorded in the patent application of RNA method of modifying and preparation " carries out.(table 1：MiR-122 (122) represents target
microRNA；MiR-122-mimic represents the analogies of miR-122, and lagging strand is exactly matched with leading chain；M represents mimic
Analogies；The conjugated modification (3 '-KALLAL) of P representative peptides；1AL5 ' the 1st, ends are represented to be modified with L-isoA；Pi represents 5 '-phosphoric acid
Change modification；As, which is represented, dominates chain；S represents lagging strand.Such as：“PP-122m-S1AL", indicate that 1 L- of joint lagging strand is conjugated in double peptides
The miR-122 analogies of isoA modifications.)
Table 1.miR122mimics sequences
The results are shown in Figure 7, should the experimental results showed that, ssRNA and its peptide conjugation product electrophoretic band are in N/P=5:1
It disappears substantially under part；However, even if double-stranded RNA is in N/P=10:Electrophoretic band is without significantly reducing under the conditions of 1.It these results suggest that
DNCA carriers it is poor can to contain ability with Selective recognition combination single stranded RNA structures to double-stranded RNA.
Embodiment 8.DNCA contains ssRNA intracellular study on the stability
Effective vehicle delivery and chemical modification strategy can play RNAs protective effects, reduce the identification degradation of ribozyme.
Therefore, the change that the conjugated modification ssRNA (by taking miR-122 as an example) of peptide is contained front and back intracellular stability by DNCA has been investigated in this experiment
Change.
The experimental results showed that (Fig. 8), single stranded RNA is in N/P=5:It can completely be contained, and add by DNCA under the conditions of 1
It is released from nano particle after cell pyrolysis liquid.In addition, unentrapped sequence 122-SS (single-stranded miR-122), Pep-122-
SS (the single-stranded miR-122 of the conjugated modifications of terminal peptide LALLAK) is degradable within 1h, illustrates that the conjugated modification of 3 '-peptides cannot
Improve the stability of ssRNA.DNCA/122-SS nanoparticles stables illustrate that DNCA is opposite with RNA combinations loose without significantly improving
It dissipates, effective ribozyme protective effect cannot be played.It is obviously carried however, DNCA/Pep-122-SS nano particle serum stabilities have
Height illustrates to be interacted between peptide fragment (LALLAK) and DNCA carriers, to change DNCA and RNA conjugates it
Between assembly model, improve the compactness extent of nano particle.
Embodiment 9, DNCA contain the investigation of ssRNA cross-film abilities
This experiment has investigated DNCA and has contained the conjugated modification RNA of peptide (by taking miR-122 as an example, method of modifying is with embodiment 8)
Cross-film ability.And select commercial transfection reagent Lipofectaimne 2000 (lipo) as a contrast, further illustrate DNCA+ peptides
Advantage of the conjugated modification strategy on delivering ssRNA.
The results are shown in Figure 9, which shows that carrier-free RNAs is unable to cross-film and enters cell.In addition it is transfected in serum-free
Under the conditions of, DNCA has higher transfection efficiency relative to lipo.In addition, DNCA is suitable for having transfects ssRNA under serum condition,
Its transfection efficiency is better than serum-free condition.Consistent with efficiencies are contained, DNCA transfections ssRNA efficiency is significantly higher than dsRNA, into
One step illustrates that DNCA can be with Selective recognition combination ssRNA.
Embodiment 10, DNCA contain ssRNA safeties and anti-tumour cell proliferative effect
This experiment has investigated DNCA and has contained ssRNA (by taking miR-122 as an example) for human embryonic kidney cells (HEK293A) cell line
Safety.
DNCA/pi-AS：Indicate the leading chain simulative of the miR-122 for the 5 '-phosphorylation modifications that DNCA is contained
DNCA/Pep-pi-AS：Indicate the miR- for the combination 5 '-phosphorylation modification and the conjugated modification of 3 ' peptides that DNCA is contained
122 leading chain simulative
Transfection method is the same as embodiment 6.
Experimental result is as shown in Figure 10, should the result shows that, DNCA/pi-AS and DNCA/Pep-pi-AS are dense in high dose
Without apparent cytotoxicity under degree.In contrast, lipo2000 contains identical sequence and shows significant dose-dependent effect,
When RNA concentration reaches 100nM, HEK293A cells dead is more than half.Result above absolutely proved DNCA relative to
Lipo2000 has better safety and lower cytotoxicity.In addition, for the Activity Results table of HepG2 cell lines
Bright, DNCA/pi-AS is relative to NC sequences without apparent active function, however DNCA/Pep-pi-AS is shown under the conditions of 200nM
Preferable Cytostatic to tumor cell effect, further illustrates the dominance of strategies.
Embodiment 11, DNCA contain efficiency to single stranded DNA
Single stranded DNA whether can be effectively contained for verification DNCA, this experimental system has investigated under the conditions of different N/P DNCA pairs
The packet of AS1411 ((5'-GGT GGT GGT GGT TGT GGTGGT GGT GG-3', 26-mer)), G3139 and N-G3139
Carry efficiency.Experimental result is as shown in figure 11, should the result shows that, in N/P=4-5, DNCA can effectively contain corresponding nucleic sequence
Row.Wherein G3139 contains that efficiency is slightly lower, this may be because full site PS modifications affect the phase interaction between DNCA- nucleic acid
With.
Embodiment 12DNCA/AS1411 compound Tm values measure
The stable composite degree that the present invention is formed using circular dichroism (CD) spectral investigation DNCA and AS1411 (20bp).
As a result as shown in figure 12, Tm values (the Tm values of AS1411 after the PBS buffer solutions annealing of AS1411 are we illustrated
It is 41 DEG C；AS1411/DNCA compound Tm values are 53 DEG C).DNCA is mixed with, after annealing process, DNCA/AS1411
The Tm values of compound are obviously changed, and 52 DEG C are increased to from 42 DEG C, display DNCA and AS1411 forms new two level knot
Structure is more stablized relative to AS1411 itself secondary structures formed.
The bio-compatibility of 13 base lipid carrier of embodiment is investigated
Base acetamide glycerin ether molecule will be as novel biomaterial, and realizes answering in terms of gene therapy
With itself must have the characteristics that low cytotoxicity.We are thin to four kinds of nucleoside base acetamide/triacetin ether compounds
Cellular toxicity is investigated, the results showed that these four compounds are all without apparent cytotoxicity.In 65 μM of concentration, nucleosides is added
For base lipid molecular compound after 72 hours, cells survival rate equal 90% or more shows it with good bio-compatibility.Knot
Fruit is as shown in FIG. 13A.
14 base lipid carrier of embodiment contains the anti-cck-8 proliferation activities of AS1411 and investigates
This experiment contains base lipid carrier by cck-8 proliferation inhibition tests the concentrations of nanoparticles ladder of AS1411
Degree-tumor cell proliferation rate is evaluated.The experimental results showed that unentrapped AS1411 and DNCA press down without apparent tumour
Effect processed；AS1411/DNCA systems show higher antiproliferative activity at low concentrations, and with the raising pair of concentration
A549 cellkilling capacities gradually increase, and illustrate that DNCA effectively delivers AS1411 and enters cell and play its active function.In addition,
A549-TXL cell inhibitions are remarkably reinforced in DNCA/AS1411, have reversing multiple medicine resistance of tumor cells effect.As a result
As shown in Figure 13 B.
The AS1411 cellular uptakes experiment that embodiment 15DNCA is contained
Experiment shows that the cellular uptake amount of DNCA/AS1411 is about 3 times of unentrapped AS1411 cellular uptake amounts, explanation
DNCA has good transfection efficiency for G-4 structures.As a result as shown in fig. 13 c.
Influence of 16 salt of the embodiment/amino acid to DNCA/AS1411 transfections
Find that certain ingredient has promotion DNCA to AS1411 transfections in GenOpti solution in experiment, to understand
DNCA and AS1411 concentration is respectively increased to 15 for influence of the GenOpti each components to DNCA/AS1411 transfections, this experiment
μM and 200nM, investigated the influence of salt component and salt/amino acid (50 μM) ingredient to AS1411 transfections respectively.
Experimental result is as shown in figure 14, should the experimental results showed that, CaCl2And MnCl2DNCA/AS1411 can be improved to tumour
Cell inhibitory effect effect, inhibition is without further increasing after increasing amino acid.Then concentration of metal ions is reduced to
GenOpti solution concentrations, CaCl2Keep high activity and MnCl2Active basic disappearance.Illustrate the CaCl in GenOpti2Component is
Improve the major reason of the efficiency of liposome transfection nucleic acid.
Embodiment 17CaCl2AS1411 transfection efficiencies are influenced to investigate
This experiment is to a series of AS1411 and its modification sequence (6L12D24I), oligonucleotides G3139, optimize formulation protocol
(CaCl is added2) carry out system thinking (table 2).
As a result as shown in figure 15, the experimental results showed that, CaCl2Nano particle has reason in DNCA contains AS1411 systems
Want inhibitory activity of rising in value；DNCA contain G3139 active effects be less than AS1411, wherein sequence 4,11,30 and 36 activity have compared with
Significantly improve (6L12D24I：6 L- heteronuclear glycosides of AS1411 are modified, 12 D- heteronuclear glycosides modifications, 24 deoxyinosine modifications).
2. scheme of table
18 neutral lipid carrier of embodiment is used for the transfection of pEGFP-N1 plasmids
Selection can with the pEGFP-N1 plasmids of enhanced green fluorescent protein, under the conditions of same preparation, investigate have serum and
(DNCA and DNTA are according to 1 by neutral lipid carrier DNCA, DNTA, DNXA under serum-free condition:1 mixing) transfection efficiency,
Transfection method is the same as embodiment 6.And it is compared with commercial transfection reagent LIPO.
As a result as shown in figure 16, the experimental results showed that, neutral lipid carrier DNCA, DNTA, DNXA (DNCA:DNTA=1:
1) more preferable to the transfection of plasmid in the case where there is serum condition, under serum-free condition, cell is in starvation, transfection effect
Rate is decreased obviously.And then the transfection efficiency in serum-free is very high by cationic transfection reagent LIPO, transfection efficiency is substantially when having serum
Degree declines.The result illustrates that, although compared with LIPO, neutral lipid carrier need to be improved to the transfection efficiency of plasmid, should
The compound that lipid carrier is formed with plasmid has preferable stability in serum, may be implemented to turn under serum condition
Dye, there may be advantages in terms of the following transfection in vivo.
19 neutral lipid carrier of embodiment is used for the Toxicity test of plasmid DNA transfection
Plasmid itself used is non-toxic in this experiment, for neutral lipid carrier in investigation/DNA compound toxicity sizes,
Therefore contrived experiment, under the conditions of common fat timber-used amount (1nmol lipid carriers contain 10ng plasmids), at the same compare DNCA, DNTA,
(DNCA and DNTA are according to 1 by DNXA:1 mixing) in transfected plasmids process to cell (HEK293A) toxicity itself, and be added one group
The experimental group of 5 times of dosage DNCA (5nmol fat materials contain 10ng plasmids) transfection equal amount plasmids.96 orifice plates, per hole 1nmol
It is 10 μM that DNCA, which contains siQuant plasmid pMB3 10ng, DNCA activities, has serum condition to transfect 48h.Control group is
LIPO, per 0.1 μ L of hole, serum-free acts on 4h, enrich blood it is clear after continue to cultivate 44h, by CCK-8 cell viability detection reagents, with
Neutral lipid vehicle group detects cell survival rate simultaneously.Transfection method is the same as embodiment 6.
As a result as shown in figure 17, should the result shows that, LIPO shows overt toxicity, and DNCA, DNXA are at usual amounts (10 μM)
Substantially non-toxic, DNTA is also apparent from cytotoxicity also very little, 5 times of DNCA dosages (50 μM) toxic effects.The result
Illustrate that neutral lipid carrier DNCA, DNTA is smaller as the toxicity of plasmid transfection reagent, there is widely applied foreground.On a small quantity
DNTA there is toxicity, may be related with its dissolubility, more muddy with the complex solution of plasmid, after culture medium is added,
The particle floated a little can be observed, insoluble larger particles are easy stimulation cell membrane and cause to damage to cell, generate acute poison
Property effect, if composite particles enter after cell to degrade in time and can also cause cumulative toxicity in addition.Neutral lipid carrier energy
It is transfected in the case where there is serum condition, cell normal proliferative is more advantageous to relative to serum-free environment, with cationic formulation LIPO
It compares, may have some superiority in terms of transfection in vivo.
The information for showing and being described in detail herein is enough to realize the above-mentioned purpose of the present invention, therefore the preferred implementation of the present invention
Scheme represents subject of the present invention, which is that the present invention covers extensively.The scope of the present invention is fully contemplated by other to ability
Obvious embodiment for field technique personnel, therefore, the scope of the present invention is not by appointing in addition to appended claims
What content is limited, wherein in addition to clearly stating other than, the singulative of element used does not imply that " one with uniquely ", and refers to
" one or more ".For persons skilled in the art, all well known above-mentioned preferred embodiments and additional implementation
Therefore structure, composition and the equivalent functionally of scheme section are incorporated herein by, and attempt by the right of the present invention
It is required that being covered.
Furthermore, it is not necessary that certain equipment or method express each problem solved by the invention, because they have all been wrapped
It includes within the claim of the present invention.In addition, no matter the present invention discloses all parts, ingredient or method step in the fact
Suddenly whether clearly described in the claims, they all do not contribute to the public.But those of ordinary skill in the art are come
It says, it is evident that, can be in shape under the premise of without departing substantially from the spirit and scope of the invention as illustrated in appended claims
Various changes and modification are made in formula, reagent and synthesis details.
Claims (7)
1. a kind of nucleic acid delivery systems, which is characterized in that be made of neutral base lipid carrier and metal salt, wherein described
Neutral base lipid carrier structural formula it is shown in formula I：
Wherein, wherein X is oxygen atom or nitrogen-atoms, and B is natural purine and pyrimidine bases, i.e. adenine, guanine, secondary Huang is fast
Purine, cytimidine, thymidine and uracil, preferably cytimidine -1- bases or thymidine -1- bases.
2. nucleic acid delivery systems according to claim 1, which is characterized in that the fatty long-chain-C of institute in Formulas I18H35Structure
For
3. nucleic acid delivery systems according to claim 1, which is characterized in that the neutral base lipid carrier have with
Structure shown in lower：
4. according to the nucleic acid delivery systems in claim 1, which is characterized in that the metal salt be calcium salt or manganese salt, preferably
For CaCl2。
5. application of the claim 1-4 any one of them nucleic acid delivery systems in preparing nucleic acid delivery reagent.
6. application according to claim 5, which is characterized in that the nucleic acid include oligonucleotide, aptamers,
SiRNA and Plasmid DNA.
7. application according to claim 6, which is characterized in that the nucleic acid is using D, the modification of L- heteronuclear glycosides, deoxidation
Inosine is modified, the nucleic acid analog that at least one of the conjugated modification of peptide and phosphorylation modification method of modifying are modified.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112826808A (en) * | 2021-01-18 | 2021-05-25 | 北京大学 | Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof |
| CN113373155A (en) * | 2021-06-23 | 2021-09-10 | 北京大学 | Comprehensive chemical modification method and product of G-four-chain aptamer and application of G-four-chain aptamer in resisting multidrug-resistant tumor cells |
| CN113797327A (en) * | 2021-09-24 | 2021-12-17 | 北京大学 | Nucleic acid drug delivery carrier for carrying mRNA and preparation method and application thereof |
| CN114246829A (en) * | 2021-12-31 | 2022-03-29 | 北京大学 | Method for encapsulating antisense nucleic acid medicament, medicinal preparation prepared by method and application of medicinal preparation in liver cancer treatment |
| CN114605338A (en) * | 2022-05-11 | 2022-06-10 | 深圳厚存纳米药业有限公司 | Nanoparticle and nucleic acid nanocomposite containing uracil derivative, and preparation method and application thereof |
| CN114933565A (en) * | 2022-05-12 | 2022-08-23 | 深圳厚存纳米药业有限公司 | Nucleobase derivative nanoparticles and composition thereof |
| CN116143707A (en) * | 2023-02-24 | 2023-05-23 | 山东大学 | Base ionizable lipid and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103242243A (en) * | 2013-01-08 | 2013-08-14 | 北京大学 | Base acetic acid glycerin ester ether molecule, chemical synthesis method of molecule, and application of molecule in field of gene therapy |
-
2018
- 2018-04-11 CN CN201810322098.0A patent/CN108478807B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103242243A (en) * | 2013-01-08 | 2013-08-14 | 北京大学 | Base acetic acid glycerin ester ether molecule, chemical synthesis method of molecule, and application of molecule in field of gene therapy |
Non-Patent Citations (2)
| Title |
|---|
| YUEJIE ZHU ETAL: "The Antitumor Activity of G-Quadruplex Aptamer AS1411 Encapsulated by Nucleobase Lipid DNCA", 《第十届全国化学生物学学术会议论文摘要集（墙报）》 * |
| 周雪雁等: "脂质体介导转染法原理及其研究近展", 《上海畜牧兽医通讯》 * |
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| CN112826808A (en) * | 2021-01-18 | 2021-05-25 | 北京大学 | Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof |
| CN113373155A (en) * | 2021-06-23 | 2021-09-10 | 北京大学 | Comprehensive chemical modification method and product of G-four-chain aptamer and application of G-four-chain aptamer in resisting multidrug-resistant tumor cells |
| CN113797327A (en) * | 2021-09-24 | 2021-12-17 | 北京大学 | Nucleic acid drug delivery carrier for carrying mRNA and preparation method and application thereof |
| CN114246829A (en) * | 2021-12-31 | 2022-03-29 | 北京大学 | Method for encapsulating antisense nucleic acid medicament, medicinal preparation prepared by method and application of medicinal preparation in liver cancer treatment |
| CN114605338A (en) * | 2022-05-11 | 2022-06-10 | 深圳厚存纳米药业有限公司 | Nanoparticle and nucleic acid nanocomposite containing uracil derivative, and preparation method and application thereof |
| CN114933565A (en) * | 2022-05-12 | 2022-08-23 | 深圳厚存纳米药业有限公司 | Nucleobase derivative nanoparticles and composition thereof |
| CN116143707A (en) * | 2023-02-24 | 2023-05-23 | 山东大学 | Base ionizable lipid and preparation method and application thereof |
| CN116143707B (en) * | 2023-02-24 | 2024-04-19 | 山东大学 | Base ionizable lipid and preparation method and application thereof |
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