CN109568595A - Nucleic acid-drug conjugates, drug delivery system and its preparation method and application - Google Patents
Nucleic acid-drug conjugates, drug delivery system and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to biomedicine field, a kind of nucleic acid-drug conjugates based on phosphorothioate nucleic acid, drug delivery system and their preparation method are specifically disclosed.Nucleic acid-the drug conjugates are to be formed by the phosphorothioate group in phosphorothioate nucleic acid with the group reaction bonded modified on drug molecule, electrophilic reaction can occur with phosphorothioate group, and, by selecting different nucleic acid sequences including functional nucleic acid including, the nucleic acid-drug conjugates can self assembly be various forms of drug containing nano-carriers progress drug delivery.Compared with prior art, the present invention can reach by simple solid phase synthesis technique, and accurately control graft site and its assembling form of the drug molecule in nucleic acid backbone, and the method has universality to chemotherapeutics；The present invention can significantly improve the physicochemical property of chemotherapeutics and internal distribution property and promote its therapeutic effect, can also be achieved the combination therapy of gene therapy, chemotherapy.
Description
Technical field
The invention belongs to biomedicine field, in particular to a kind of nucleic acid-drug based on nucleic acid phosphorothioate
Conjugate, drug delivery system and its preparation method and application.
Background technique
Chemotherapy is one of the important means of oncotherapy, however since there are poorly water-solubles, non-target for most of chemotherapeutics
The defects of tropism, blood clearance height or even serious toxic side effect, lead to the lower (Nat.Rev.Cancer of its bioavilability
2006,6,789.) it, and is used for a long time and can generate drug resistance, certain limitation is brought to their clinical application.
In order to capture this problem, researchers, which devise, in the past few decades a series of based on polymer or inorganic is received
The Nano medication delivery system of rice corpuscles improves chemotherapeutics property and promotes its therapeutic effect, such as micella, vesica, lipid
Body, albumin nano granular, microvesicle etc. (Science 2004,303,1818.).Currently, to contain chemotherapeutics main for nano-carrier
Including two kinds of mode of physically trapping mode and chemical bonding, but since the former drugloading rate is lower and drug is easy leakage and causes poison
Side effect, therefore, drug conjugates strategy become another hot spot of drug conveying.Material for drug conjugates has very much,
Including polymer (J.Controlled Release 2016,222,116.), polypeptide (Adv.Drug Delivery
Rev.2017,110-111,112.), antibody (Trends Biotechnol.2017,35,466.) etc..However, being used to prepare medicine
The material of combination tends not to meet three biocompatibility, biodegradability and low immunogenicity features simultaneously, because
This limits its clinical Transformation Application.In addition, some drug conjugates reported at present, all have the following problems: drug is connect
Branch less, drug graft site and graft numbers are unable to control, graftable drug molecule is restricted.
In recent years, biocompatibility can be met simultaneously as a kind of natural boiomacromolecule due to nucleic acid, biology can
Three conditions of degradability and low immunogenicity, and there is some other specific characterization of molecules such as targeting, specific molecule to know
The exact controllability of other function and the nanostructure being self-assembly of has obtained it in biomedicine field more and more extensive
Concern.
Currently, carrying out medicine using nucleic acid nano structure (J.Polym.Sci.2017,35,1.) such as polyhedron and origami structure
The research of object conveying also emerges one after another, for example the characteristics of DNA double helical structure can be inserted using adriamycin (DOX) carries out DOX
Conveying, all achieve good antitumous effect in vivo and in vitro.But physics is carried out using this nucleic acid insertion means and is contained
Drug not only limits drug, and its internal stability need to be gone into seriously.
Nucleic acid and chemotherapeutics by the end modified functional group of nucleic acid sequence or are led in addition, there are also researchers
It crosses the polymer etc. connecting with DNA to be linked into DNA nanostructure, the conveying of Lai Shixian drug.However the former is due to by function
Group quantity limitation drugloading rate it is lower, and the latter due to containing polymer segment thus certain biocompatibility may be brought to ask
Topic, and both modes generally require more complicated synthesis process.
Thus, it needs to establish blanket a kind of pair of chemotherapeutics, drugloading rate and carries the novel of medicine site controllable precise
Biocompatible nucleic acid nano medicine-carried system.
Summary of the invention
The first object of the present invention is to provide a kind of nucleic acid-drug conjugates based on nucleic acid phosphorothioate, with
It realizes chemotherapeutics accurate controllable grafting and high-efficiency delivery, solves following present in current drug conjugates delivery system lack
Point: (1) three big requirements of the bio-compatible, degradable in vivo and low immunogenicity of carrier material cannot simultaneously be met；(2) it is difficult to
Accurate control drug graft site and drugloading rate；(3) graftable drug is limited, cannot achieve nucleic acid as carrier material
The universality of material, it is difficult to reduce the cost that nucleic acid carries medicine；(4) more complicated synthesis process is needed；(5) it cannot achieve multi-functional
The reasonable simple construction of medicine-carried system.
The second object of the present invention is to provide a kind of drug delivery system, and the drug delivery system is medicament-carried nano body
System, is self-assembly of by above-mentioned nucleic acid-drug conjugates.
The third object of the present invention is to provide above-mentioned nucleic acid-drug conjugates based on nucleic acid phosphorothioate
Preparation method.
The fourth object of the present invention is to provide the preparation method of above-mentioned drug delivery system.
The fifth object of the present invention is to provide a kind of to be made based on above nucleic acid-drug conjugates and drug delivery system
It is standby based on nucleic acid as the purposes in the tumor therapeutic agent of carrier.
The sixth object of the present invention is to provide a kind of drug comprising the drug that above-mentioned nucleic acid-drug conjugates are formed
Delivery system.
Technical scheme is as follows:
A kind of nucleic acid-drug conjugates based on phosphorothioate nucleic acid, the nucleic acid including phosphorothioate
Skeleton and the drug molecule being grafted in the nucleic acid backbone, the grafting pass through the thiophosphoric acid ester group in the nucleic acid backbone
Group carries out reacting realization with the group that electrophilic reaction can occurs with thiophosphate modified on the drug molecule.The nucleic acid
Skeleton and the drug molecule form functional nucleic acid-drug conjugate (i.e. nucleic acid-drug conjugates), the nucleic acid-drug
Conjugate is able to carry out self assembly and obtains medicament-carried nano system.
Preferably, in the nucleic acid backbone of the phosphorothioate, the site of thio-modification and quantity can be according to need
It adjusts and controls, thiophosphate is continuously modified in certain one end of nucleic acid sequence, and/or in the intermediate alkali of nucleic acid sequence
Basic sequence selective modification, modification mode are more modifications or mono-modified.
Preferably, the nucleic acid of phosphorothioate is prepared by solid phase synthesis process, it will be able to realize thio-modification
Site and quantity it is adjustable.
Preferably, the sequence of the oligonucleotides of the nucleic acid backbone of the phosphorothioate and segment type can be autonomous
Design, and can further assemble to obtain controllable DNA nanostructure, the nucleic acid-drug conjugates by molecular recognition
And its package assembly can be used as new drug delivery systems, and then prepare controllable drug delivery system.
Preferably, the nucleic acid backbone can carry out the thio-modification design of different loci and quantity according to different needs,
For example, for the nucleic acid that gel and tetrahedral structure assemble, because considering after modification steric effect and its to the influence of base pairing,
One thio-modification site is set every 2 to 3 bases in nucleic acid backbone；And the nucleic acid for being used for micella assembling considers micella group
Installation reason, it is intended to carry out continuous thio-modification in one end of nucleic acid sequence, and then prepare and contain phosphodiester bond and thio phosphorus
The block type nucleic acid of acid esters key.
Preferably, the drug molecule is introduced by simply esterification or acylation reaction parent occurs with thiophosphate
The group of electricity reaction.
Preferably, the drug molecule also introduces the response type chemical bond that can be broken, the response type being broken
Learning key can be disulfide bond, acylhydrazone key, ester bond etc., but be not limited to above-mentioned listed response type chemical bond.
Preferably, the modification on drug molecule and can with thiophosphate occur electrophilic reaction group be selected from
Under one or more: 1) functional groups brominated or containing iodine, such as the acetyl compound of iodo or bromo, the bromo- α of γ-, β-is no
It is saturated carbonyl, benzyl bromine or bromo maleimide；2) maleimide base group；3) aziridinyl sulfanilamide (SN) group, but be not limited to
It is upper several.
Preferably, the drug molecule is selected from anticancer drug such as taxol, camptothecine, cis-platinum, Docetaxel, benzenebutanoic acid
Mustargen, methopterin, Doxorubicin, cis-platinum prodrug etc., but it is not limited to above-mentioned several, cancer targeting drug molecule Lip river such as in distress is replaced
Buddhist nun, Imatinib, Gefitinib, Sorafenib etc., but be not limited to above-mentioned listed several.
Preferably, the drug molecule is the drug molecule of the bromo modification containing disulfide bond, such as can be by simple
Chemical reaction introduce carbonyl ethyl bromine, benzyl bromine structure, to realize the further of the nucleic acid backbone modified with phosphorothioate backbone
Reaction, and introducing disulfide bond can also realize the redox release of drug in the process.But it is not limited only to during this
Carbonyl ethyl bromine and benzyl bromine structure, other, which contain the compound that electrophilic reaction can occurs with thiophosphate, can introduce；And this
Structure is also not limited to introducing disulfide bond, and the key of other modes being broken can also be used to substitution disulfide bond, as esterase can be broken
Ester bond, illumination can be broken or the acid chemical bond that can be broken etc..
Preferably, the drug molecule being grafted in the nucleic acid backbone is functional drug molecule or fluorescent probe molecule, thin
Born of the same parents' targeted molecular.
Preferably, the drug molecule is anti-tumor drug.But drug molecule of the invention is not limited to antineoplastic
Object, treating the drug of other diseases or the drug molecule for imaging can also realize that nucleic acid is modified in this way；Some
In specific embodiment, the anti-tumor drug is camptothecine or taxol.But anti-tumor drug of the present invention also not office
It is limited to camptothecine and taxol.Other modifiable drugs be, for example, cis-platinum, Docetaxel, Chlorambucil, methopterin,
Doxorubicin etc..
In the present invention, the type of nucleic acid backbone and sequence are not limited.Deoxyribose core both may be selected in type
RNA sequence also may be selected in acid sequence；On sequence requirements, that is, non-functional common base sequence may be selected, including
The simple nucleic acid sequence being made of a kind of base and the complex nucleic acid sequence that can be used for the assembling of nucleic acid precision architecture；Also function may be selected
Can property nucleic acid sequence, the functional core acid sequence is selected from anti sense nucleotide sequence, nucleic acid aptamer sequence, nucleotide sequence, small
RNA interfering, mRNA, Microrna, long-chain non-coding RNA, children purpura nephritis, for gene editing guide RNA, in circular rna
One kind.
Preferably, the nucleic acid molecules after being grafted drug keep the property of its base pair complementarity, match it by this property
His functional core acid sequence assigns nucleic acid-drug conjugates drug delivery system targeting and imaging function, prepares multifunctionality
Nucleic acid-drug conjugates drug delivery system, wherein the functional nucleic acid for pairing is selected from aptamer, antisense nucleic acid
Sequence, the nucleic acid sequence of fluorescent molecule modification, the nucleic acid sequence of functional polypeptide modification, the nucleic acid sequence for targeting galactose modification
Deng one kind.
The present invention also provides a kind of drug delivery systems, to be self-assembly of by above-mentioned nucleic acid-drug conjugates
Medicament-carried nano system.
In the present invention, the form using assembly prepared by above-mentioned nucleic acid-drug conjugates is unrestricted, such as:
Simple nucleic acid-drug macromolecular prodrug can be prepared by design；The method of design may is that reduction thiophosphoric acid
The strong small molecule of the number or selection hydrophily of ester；
The DNA nanostructure that can be accurately assembled by designing preparation, such as carries medicine nucleic acid polyhedral structure, and be not limited to
DNA tetrahedron can also construct the DNA origami structure of different structure size；It is also not limited to DNA, RNA nanostructure can also be with
Selection.The method of design may is that selection particular sequence DNA or RNA sequence, by nucleic acid main chain specific position carry out
Thio-modification and drug are assembled by way of base pair complementarity after combining；
Design preparation DNA nanogel etc. can be passed through；The method of design may is that by select special sequence DNA or
RNA sequence selects the DNA or RNA sequence of particular sequence, by the specific position progress thio-modification in nucleic acid main chain, prepares Y
Type or i type assemble corpusculum, and further assembling preparation carries the nanogel of medicine；
The micelle-type spherical shape nucleic acid for carrying medicine can be prepared by design；The method of design, which may is that, selects common or function
Property nucleic acid sequence, be carried out continuously thio-modification at one end, prepare one end di-phosphate ester bond structure and one end thiophosphoric acid ester structure
Block type nucleic acid, thio-modification site carry out drug modification；Because the hydrophilic outer shell of micelle-type spherical shape nucleic acid maintains its alkali
The property of base complementary pairing can assign the function of the targeting of micelle-type spherical shape nucleic acid, imaging by this property；
Carry medicine nucleic acid hydrogel.
Preferably, the above-mentioned drug molecule for being used to prepare the DNA nanostructure accurately assembled can select as far as possible hydrophobicity it is weak,
The small molecule of molecular weight, and the quantity of nucleic acid backbone thio-modification can be reduced；And the drug molecule for being used for micella assembling may be selected
The strong molecule of hydrophobicity, and the quantity of nucleic acid backbone thio-modification can be increased.
It yet still another aspect, the present invention provides a kind of above-mentioned nucleic acid based on nucleic acid phosphorothioate-drug knots
The preparation method for closing object, mainly includes the following steps:
The first step, the nucleic acid molecules for preparing thio-modification；Preferably, the core of thio-modification is prepared by solid phase synthesis process
Acid molecule, site and the quantity that can be realized thio-modification are adjustable；
Drug molecule by chemical reaction method preparation containing the group that electrophilic reaction can occur with thiophosphate；?
In preferred embodiment, the drug molecule also contains the response type chemical bond that can be broken, and is broken it is highly preferred that described
Response type chemical bond is disulfide bond, and the group that electrophilic reaction occurs is bromo modification group, by chemical reaction method system
The drug molecule of the standby bromo modification containing disulfide bond, can be used one of following methods: under protection of argon gas, passing through three
Disulfide bond is accessed camptothecin drug molecule by phosgene, then by reacting with bromoacetyl bromide, obtains bromo camptothecin drug molecule；
Or, obtaining the carboxylic acid structure containing disulfide bond by the esterification of 4- bromomethyl benzyl alcohol and dithiodipropionic acid, then pass through
The esterification of carboxylic acid and Japanese yew alcoholic extract hydroxyl group obtains the taxol drug molecule of benzyl bromine structural modification；
Nucleic acid molecules made above do not have sequencing with the step of preparing drug molecule；
Second step prepares nucleic acid-drug conjugates, and the drug molecule of modification is dissolved in organic solvent, is then added suitable
The nucleic acid molecules of amount are reacted, the drug molecule in this reaction relative to thiophosphate be it is large excess of, reacted it
Extra small molecule is removed afterwards, and nucleic acid-drug conjugates can be obtained after dry.Reacted treatment process may is that addition
The aqueous solution (volume of aqueous solution can be determined by limited trials, be not described in detail herein) of certain volume, then passes through acetic acid second
The method of ester extraction or the method for ethanol precipitation remove extra small molecule, and nucleic acid-drug can be obtained after water phase is dry and combine
Object.
Preferably, in above-mentioned preparation method, by controlling nucleic acid molecules concentration, the ratio of drug molecule and thiophosphate
And whether can control the grafting efficiency of drug molecule in reaction solution containing salting liquid.
In some embodiments, organic solvent one kind chosen from the followings: reaction system used when grafting camptothecine
It is in the mixed system of dimethyl sulfoxide and phosphate buffer (volume ratio 4:1)；Being grafted reaction system used in taxol is two
Methyl sulfoxide.
In some embodiments, the temperature and time of the reaction is: the overnight or longer time is reacted at 50 DEG C -58 DEG C,
But reaction temperature and time are changeable, however it is not limited to this.
It yet still another aspect, the present invention provides above-mentioned nucleic acid-drug conjugates drug delivery system preparation method, choosing
From one of following methods:
It is prepared using direct dissolution method, nucleic acid-drug conjugates is directly dissolved in aqueous solution or salting liquid, preparation carries medicine core
Sour macromolecular prodrug；Or
Carry the preparation of medicine nucleic acid polyhedral structure or nucleic acid gel: selection can complementary pairing nucleic acid sequence, in feature position
Point carries out the nucleic acid-drug conjugates obtained after phosphorothioate according to above-mentioned method, by the nucleic acid sequence of complementary pairing
It is listed in TAE/Mg2+It mixes in solution, is prepared by the method for annealing in proportion；By taking camptothecine as an example: not homotactic by four kinds
Nucleic acid-camptothecine prodrug is in 1 × TAE/Mg2+It is mixed in solution buffer with equimolar amounts, at 90 DEG C after placement 5min rapidly
4 DEG C are cooled to get the DNA nanometer tetrahedron for carrying camptothecine is arrived；Or
The preparation of carrier micelle type spherical shape nucleic acid: one section of continuous phosphodiester bond of selection and one section of continuous phosphorothioate bond
The block type nucleic acid of modification, the nucleic acid-drug conjugates obtained according to above-mentioned method, prepares carrier micelle using dialysis,
Wherein, the hydrophilic outer shell without the nucleic acid block of phosphorothioate as micella, hydrophobic drug is as kernel；Specifically
Ground, by taking taxol as an example: nucleic acid-paclitaxel conjugates of obtained block structure are dissolved in dimethyl sulfoxide, be added etc.
After the moisture of volume in water dialysed overnight to get carry taxol micelle-type spherical shape nucleic acid；
Carry the preparation of medicine multifunctional sphere nucleic acid: selection is containing the core with continuous phosphodiester bond block complimentary mating portion
Acid sequence, there is this nucleic acid sequence fluorescent molecule to modify, targeting aptamers are modified, target polypeptide is modified, it is small numerator modified to target
Function, by passing through the targeting of method preparation collection, imaging, gene therapy and the chemotherapy of annealing for micella prepared in above-mentioned (3)
Multi-functional load medicine ball forming core acid.Specifically, such as selection contains and the phosphodiester bond block in this spherical nucleic acid
The functional core acid sequence (targeting or fluorescent decoration) of complimentary mating portion passes through the targeting of method preparation collection, the imaging, base of annealing
Because treating the multi-functional load medicine ball forming core acid with chemotherapy.
It yet still another aspect, being existed the present invention provides a kind of based on above-mentioned nucleic acid-drug conjugates and drug delivery system
Prepare based on gene therapy, chemotherapy combination therapy disease nucleic acid nano drug and chemotherapeutics in purposes.
It yet still another aspect, the present invention provides a kind of drugs comprising above-mentioned nucleic acid-drug conjugates or its formed
Drug delivery system.
Preferably, the drug is tumor therapeutic agent.
Benefit of the invention is that:
1, by being grafted drug molecule on the nucleic acid molecules of phosphorothioate, nucleic acid-drug conjugates are obtained, and
Drug delivery system is obtained by the self assembly of nucleic acid-drug conjugates, realizes the mesh delivered using nucleic acid drug
, it is successfully realized and carries out drug delivery using nucleic acid, meet bio-compatible, degradable in vivo and the low immunogene of carrier material
Property three big require；
2, the oligonucleotides that thio-modification is obtained by way of synthesis in solid state, without cumbersome chemical synthesis, and sulphur
The site of generation modification and quantity can according to need adjustment control, and the drugloading rate and load medicine site for the later period are realized and accurately set
The purpose of meter；
3, to graftable drug less-restrictive, universality of the nucleic acid as carrier material is realized；
4, synthesis process is simple；
5, required DNA nanostructure can be obtained by the sequence and segment type of autonomous Design oligonucleotides, into
And prepare controllable drug delivery system；
6, medicine-carried system uses the nucleic acid of biocompatibility for material, has lower immunogenicity to organism, lower
Metabolic burden has no toxic side effect；
7, this medicine-carried system can also be further introduced into targeting group and functional core acid sequence, prepare multi-functional nucleic acid
Medicine-carried system realizes purpose associated with gene therapy, chemotherapy；It can also be achieved the DNA nanostructure that tumor imaging is integrated；
8, the reasonable simple construction of multi-functional medicine-carried system is realized.
Certainly, it implements any of the products of the present invention and does not necessarily require achieving all the advantages described above at the same time.
Detailed description of the invention
Fig. 1 is the synthetic route of bromo camptothecine prodrug and bromo taxane molecule in embodiment 1 and embodiment 2；
Fig. 2 is prodrug compound 1 in embodiment 11H NMR spectra；
Fig. 3 is the LC-MS spectrogram of prodrug compound 1 in embodiment 1；
Fig. 4 is prodrug compound 2 in embodiment 11H NMR spectra；
Fig. 5 is the LC-MS spectrogram of prodrug compound 2 in embodiment 1；
Fig. 6 is the ultraviolet-visible light light-splitting luminosity spectrogram for the oligonucleotides prodrug that camplotheca acuminata is base-modified in embodiment 1；
Fig. 7 is the denaturing gel electrophoresis figure for the oligonucleotides prodrug that camplotheca acuminata is base-modified in embodiment 1；
Fig. 8 is the substance assistant laser desorpted ionized flight time for the oligonucleotides prodrug that camplotheca acuminata is base-modified in embodiment 1
Mass spectrogram；
Fig. 9 is DTDP-Bz-Br in embodiment 21H NMR spectra；
Figure 10 is PTX-Bz-Br in embodiment 21H NMR spectra；
Figure 11 is the mass spectrogram of PTX-Bz-Br in embodiment 2；
Figure 12 is DNA-b-PTX-g-DNA in embodiment 231P NMR spectra；
Figure 13 is the Matrix-assisted laser desorption ionization figure of DNA-b-PTX-g-DNA in embodiment 2；
Figure 14 is the agarose gel electrophoresis figure for the DNA tetrahedron origami structure that camplotheca acuminata is base-modified in embodiment 3；
Figure 15 is the hydrodynamic diameter datagram for the DNA tetrahedron origami structure that camplotheca acuminata is base-modified in embodiment 3；
Figure 16 is the atomic force microscopy for the DNA tetrahedron origami structure that camplotheca acuminata is base-modified in embodiment 3；
Figure 17 is the base-modified DNA tetrahedron origami structure of camplotheca acuminata in embodiment 3 to cancer cell toxicity assessment schematic diagram；
Figure 18 is the realization cancer cell-apoptosis schematic diagram for the DNA tetrahedron origami structure that camplotheca acuminata is base-modified in embodiment 3；
Figure 19 is the 20% deformable gel electrophoretogram of PTX-SNA in embodiment 4；
Figure 20 is 1% agarose gel electrophoresis figure and dynamic light scattering diagram of PTX-SNA in embodiment 4；
Figure 21 is the transmission electron microscope picture of PTX-SNA in embodiment 4；
Figure 22 is 1% agarose gel electrophoresis of the FAM/AS1411/PTX-SNA of multifunctional sphere nucleic acid in embodiment 4
Figure；
Figure 23 is the fine jade for the spherical nucleic acid AS1411/Bcl-2-PTX-SNA that the gene that targets and chemotherapy coexist in embodiment 4
Sepharose electrophoretogram；
Figure 24 is the extracorporeal anti-tumor and reversing tumor multiple medicine that the various micelle-type spherical shape nucleic acid of taxol are carried in embodiment 4
Drug resistance figure.
Specific embodiment
The present invention provide it is a kind of based on nucleic acid phosphorothioate backbone modification nucleic acid-drug conjugates, drug delivery system
System, their preparation method and application.
The invention belongs to biomedicine fields, specifically disclose a kind of nucleic acid-medicine based on phosphorothioate nucleic acid
Combination, drug delivery system and their preparation method.Nucleic acid-the drug conjugates are by nucleic acid thiophosphoric acid
Thiophosphate in ester skeleton with the drug molecule for the electrophilic reaction base group modification that can be reacted and being grafted
At, wherein the nucleic acid sequence including functional nucleic acid can be selected by different target, in addition, the nucleic acid-drug
Conjugate can self assembly be drug containing nano-carrier carry out drug delivery.Compared with prior art, thio phosphorus of the present invention
Acid esters nucleic acid backbone can reach by simple solid phase synthesis technique, carry out drug grafting by phosphorothioate group, can
Graft site and its assembly behavior of the accurate control drug molecule in nucleic acid backbone, the method has chemotherapeutics pervasive
Property.The present invention, as carrier, can significantly improve chemotherapeutic using bio-compatible and the water-soluble nucleic acid molecule that can be degraded in vivo
The physicochemical property of object and internal distribution property simultaneously promote its therapeutic effect, can also be achieved the combination therapy of gene therapy, chemotherapy, and
Avoid complicated synthesis and modification step.
Herein, the range indicated by " numerical value to another numerical value ", is that one kind avoids enumerating in the description
The summary representation of all numerical value in the range.Therefore, the record of a certain special value range, covers the numberical range
Interior any number and the relatively fractional value range defined by any number in the numberical range, as bright in the description
Text writes out any number as should be compared with fractional value range.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair
It is bright, rather than limit the scope of protection of the present invention.What those skilled in the art made according to the present invention in practical applications changes
Into and adjustment, still fall within protection scope of the present invention.
Embodiment 1
The synthesis of 1.1 bromo camptothecine prodrugs, step are shown in Fig. 1 (A) that synthesis is divided into two steps:
(1), the synthesis of the prodrug compound 1 of isotope of redox-sensitive: under argon gas protection, by camptothecine (1g) and triphosgene
(313mg) is dissolved in 150ml anhydrous methylene chloride, and 4-dimethylaminopyridine is slowly added dropwise, and (DMAP, 1.12g are dissolved in 20mL dichloro
Methane), it is stirred to react half an hour at room temperature, 2,2 '-two sulphur diethanols (4.43g) are added, room temperature reaction is overnight.
Mixed liquor is washed with 80mL 0.1M HCL solution after reaction, and layering discards supernatant, HCL solution cleans three times repeatedly
Afterwards, it is washed with 80mL saturation NaCl solution, is layered, discards supernatant, finally distill water washing with 80mL, layering discards supernatant, and uses
Anhydrous MgSO4Dry, crude product is isolated and purified using column chromatography gradient elution, and separating polar is methanol: methylene chloride
=1:100.
This step products yield 64.5%, the nuclear magnetic spectrogram of product are shown in Fig. 2, Spectra peak recognition are as follows:1H NMR(400MHz,
d6- DMSO) δ (ppm): 8.67 (s, 1H), 8.15 (d, J=8.6Hz, 1H), 8.11 (d, J=7.9Hz, 1H), 7.85 (ddd, J
=8.4,6.9,1.3Hz, 1H), 7.70 (dd, J=11.1,3.9Hz, 1H), 7.03 (s, 1H), 5.56-5.38 (m, 2H),
5.34–5.19(m,2H),4.38–4.19(m,2H),3.62–3.46(m,2H),3.02–2.88(m,2H),2.80–2.64(m,
2H),2.30–2.01(m,2H),0.98–0.84(m,3H).13C NMR(400MHz,d6-DMSO)δ(ppm):167.54,
156.93,153.29,152.62,148.33,146.69,145.22,132.00,130.86,130.19,129.45,128.94,
128.40,128.14,119.58,94.86,78.36,66.92,59.75,50.70,41.56,36.63,30.78,8.05.。
LC-MS liquid chromatography mass is combined the molecular weight and theoretical calculation for measuring compound 1, such as Fig. 3, ESI-MS m/z=(M+H+)
Calculated value 529.106, detected value m/z=(M+H+)529.103。
(2), compound 1 (330mg) and N, N- diisopropylethylamine the synthesis of bromination camptothecine prodrug compound 2: are taken
(88.8mg) is dissolved under protection of argon gas in the anhydrous methylene chloride of 150mL, stirring, be slowly added dropwise bromoacetyl bromide (126.1mg,
It is dissolved in super dry dichloromethane), reaction is stayed overnight at room temperature.After the reaction was completed, solvent is evaporated using Rotary Evaporators, column chromatography
The purifying of gradient elution progress product.Silica does filled column chromatography, and separating polar is methanol: methylene chloride=1:200.
This step products yield 80.7%, the nuclear magnetic spectrogram of product are shown in Fig. 4, Spectra peak recognition are as follows:1H NMR(400MHz,
CDCl3) δ (ppm): 8.41 (d, J=13.0Hz, 1H), 8.21 (d, J=8.5Hz, 1H), 7.91 (t, J=10.3Hz, 1H),
7.90-7.71 (m, 1H), 7.66 (ddd, J=8.0,7.0,1.0Hz, 1H), 7.33 (s, 1H), 5.48 (dt, J=27.5,
13.8Hz,2H),5.31–5.22(m,2H),4.48–4.20(m,4H),3.87–3.72(m,2H),3.01–2.80(m,4H),
2.38-2.04 (m, 2H), 0.99 (t, J=7.5Hz, 3H)13C NMR (400MHz, CDCl3) δ (ppm): 167.27,166.98,
157.24,153.44,152.18,148.71,146.40,145.57,131.35,130.82,129.51,128.50,128.25,
128.18,128.01,120.24,96.06,78.06,67.06,66.48,63.76,53.50,36.66,31.85,29.93,
25.66,7.67..The molecular weight that LC-MS liquid chromatography mass is combined detection compound 2 is m/z (M+H+)=649.031, with reason
By value m/z (M+H+) 649.530 consistent, as shown in Figure 5.
The synthesis of 1.2 oligonucleotides-camptothecin conjugates prodrug
The oligonucleotide sequence of four base complementrities is selected in this example 1.2, thio-modification site is among skeleton, sulphur
Substituted phosphate group number is TET-A, and TET-C:7 is a；TET-B, TET-D:8.The sequence of four oligonucleotides is as follows:
TET-A:5 '-ACATTC*CTAAG*TCTGAAACATTAC*AGCT*TGCT*ACACGAGAAGAGC*CGCC*
ATAGTA-3'；
TET-B:5’-TATCA*CCAG*GCAG*TTGACAGTGTAGC*AAGC*TGTA ATAGATGCG*AGGG*TCCA*
ATAC-3'；
TET-C:5 '-TCA ACTG*CCTG*GTGATA AAACGACAC*TACG*ACTA*TGGC*GGCT*CTTC-3 '；
TET-D:5’-TTCAG*ACTT*AGGA*ATGTGCTTCCC*ACGT*AGTG*TCGTTTGTA TTGG*ACCC*
TCGCAT-3'；
(* indicates phosphorothioate group decorating site).
It takes bromination camptothecine prodrug compound 2 to be dissolved in 80 μ L dimethyl sulphoxide solutions, 20 μ L thio-modification widow's cores is added
The phosphate buffer of thuja acid, oligonucleotides concentration are set 350 μM (ratio of phosphorothioate group and compound 2 is 1:50)
At 55 DEG C, concussion reaction 20h.After reaction, it is repeatedly extracted using ethyl acetate except excessive compound 2 in dereaction,
After being evaporated, it is redissolved in ultrapure water to get the oligonucleotides prodrug base-modified to camplotheca acuminata.4 kinds of base complementrities of acquisition are liked
Drug solns are by UV spectrophotometer measuring before setting base-modified oligonucleotides, in addition to the characteristic absorption of DNA at 260nm,
Occurs the characteristic absorption peak of camptothecin molecule at 365nm, as shown in Figure 6.
The camptothecin molecule of 2-6 different number, most of few nucleosides have been accessed in this step on every oligonucleotide chain
Acid linker has entered 4 camptothecin molecules, and TET-A and TET-C access quantity are less than TET-B and TET-D, and as shown in Figure 7 20%
The analysis of denaturing polyacrylamide (PAGE) detected through gel electrophoresis and substance assistant laser desorpted ionized flight time shown in Fig. 8
Mass Spectrometer Method.
The synthesis of 2 nucleic acid of example-taxol graft
The synthesis of the taxol drug (PTX-Bz-Br) of 2.1 benzyl bromines modification, step are shown in Fig. 1 (B) that synthesis is also classified into two
Step:
(1) 4- bromomethyl benzyl alcohol (500mg, 1 equivalent) and dithiodipropionic acid (DTDP, 2.6g, 5 equivalent) are dissolved in
The mixed solution (1/1, v/v) of super dry methylene chloride and tetrahydrofuran；Then DMAP (91mg, 0.3 equivalent) is added, stirs several
Dicyclohexylcarbodiimide (DCC, 615mg, 1.2 equivalent are dissolved in super dry methylene chloride) is added dropwise after minute, reacted at room temperature
After night, solvent is evaporated using Rotary Evaporators, passes through the isolated benzyl bromine structure containing disulfide bond of silica gel column chromatography
(DTPA-Bz-Br), eluant, eluent is petrol ether/ethyl acetate.Product nuclear-magnetism figure and ownership are shown in Fig. 9.
(2) taxol (500mg, 1 equivalent) and DTPA-Bz-Br (230mg, 1 equivalent) are dissolved in methylene chloride, then
It is added DMAP (71mg, 1 equivalent), (DCC, 145mg, 1.2 equivalent, are dissolved in dropwise addition dicyclohexylcarbodiimide after stirring a few minutes
In super dry methylene chloride), after room temperature reaction overnight, solvent is evaporated using Rotary Evaporators, is separated by silica gel column chromatography
The taxane molecule (PTX-Bz-Br) of benzyl bromine modification is obtained, eluant, eluent is petrol ether/ethyl acetate system.Product nuclear-magnetism figure is shown in
Figure 10, mass spectrogram are shown in Figure 11, [M+1] of resulting m/z+It is 1229.
The synthesis of 2.2 oligonucleotides-paclitaxel conjugates prodrug
It is block type DNA nucleic acid sequence employed in this example, is the DNA (26 of phosphodiester bond modification close to 5 ' ends
A base), it is the DNA (19 bases, 18 thiophosphoric acid diester linkages) of continuous phosphorothioate bond modification close to 3 ' ends.
Nucleic acid sequence is as follows:
POT26-PST18- T:5 '-TTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T*T*T*T*T*T*T*T*T*
T*T*T*T*T-3'；(* indicates phosphorothioate group decorating site).
Bcl-2-POT10-PST18- T:5 '-TCTCCCAGCGTGCGCCATTTTTTTTTTTT*T*T*T*
T*T*T*T*T*T*T*T*T*T*T*T*T*T*T-3；(* indicates phosphorothioate group decorating site).
Synthetic method are as follows: PTX-Bz-Br is dissolved in DMSO, after DNA is added, concussion reaction at 55 DEG C is placed in and stays overnight.So
It after water is added afterwards, is extracted with ethyl acetate except PTX-Bz-Br excessive in dereaction, is concentrated after being evaporated to get block type core is arrived
Acid-taxane molecule.It verifies it by nuclear magnetic resonance phosphorus spectrum and Matrix-assisted laser desorption ionization and successfully connects
Branch, is shown in Figure 12 and 13.
The DNA polyhedron of the conjugation camptothecine of example 3
The polyhedral self assembly of DNA of 3.1 conjugation camptothecines
By the oligonucleotides prodrug of four kinds of base complementrities of acquisition in 1 × TAE/Mg2+(40mM trishydroxymethylaminomethane,
2mM disodium ethylene diamine tetraacetate, 20mM acetic acid, tetra- acetate hydrate magnesium of 12.5mM, pH=7.4, second acid for adjusting pH value) in buffer
Equimolar amounts mixing, is warming up to 90 DEG C, 4 DEG C is rapidly cooled in 2min.It is detected by 2% agarose gel electrophoresis, gradually
Oligonucleotides prodrug not of the same race is added, nanostructure constantly becomes larger, and final assembling obtains polyhedral structure, compared to unmodified
DNA polyhedron, size increased, as shown in figure 14.Dynamic light scattering detection display, the polyhedral water of the DNA of drug modification
Partial size is closed in 24nm or so, relatively literalness DNA polyhedron partial size increases about 10nm, such as Figure 15.Figure 16 is aobvious for atomic force
The DNA polyhedron of the drug modification of micro mirror shooting, partial size is in 18nm or so.
The DNA polyhedron extracorporeal anti-tumor function of 3.2 conjugation camptothecines
The DNA polyhedron that drug modification is carried out by G 3139 produced above, can be effectively thin by tumour
Born of the same parents absorb and generate cancer cell fragmentation effect even more preferably close with raw medicine.
After the drug modification polyhedron prepared in example 3.1 and 116 cell of HCT are incubated for 72h altogether, detected by MTT thin
Born of the same parents' survival rate, as a result as shown in figure 17, the cell being incubated for altogether with the polyhedron of drug modification are increased with drug concentration, survival rate by
It gradually reducing, and under higher drug concentration, the polyhedron of drug modification is resulted in relative to the lower cell survival rate of raw medicine,
It proves the drug effective conveying of drug may be implemented in such a way that thio-modification oligonucleotides realizes drug conveying, and produces
Raw good tumor cytotoxicity effect.
The DNA polyhedron apoptosis in vitro effect of 3.3 conjugation camptothecines
The DNA polyhedron that drug modification is carried out by G 3139 of the invention, passes through cancer cell specific induction of apoptosis
Mode, which is realized, acts on tumor cytotoxicity.
After the drug modification polyhedron prepared in example 3.1 and 116 cell of HCT are incubated for 48h altogether, using Annexin
V-FITC/PI method detects apoptosis of tumor cells, and as a result as shown in figure 18, the cell being incubated for altogether with the polyhedron of drug modification can
To realize the effect of good cancer cell specific induction of apoptosis, and cause and apoptosis rate similar in raw medicine.Proof leads to drug
It crosses thio modified oligonucleotide and realizes that the mode of drug conveying may be implemented the quick release of drug, inducing apoptosis of tumour cell,
It is finally reached the purpose for the treatment of cancer.
The micelle-type spherical shape nucleic acid of the conjugation taxol of example 4
4.1 common nucleic acid block types carry the preparation (PTX-SNA) of the spherical nucleic acid of taxol
Select block type polyT base sequence (POT26-PST18- T), block type core is obtained according to the method in example 2.2
Acid-paclitaxel conjugates (PODNA-b-(PSDNA-g-PTX)), it is dissolved in DMSO that isometric water then is added, water-soluble
PTX-SNA can be obtained in dialysed overnight in liquid.Pass through the polyacrylamide gel electrophoresis of 20% denaturation, 1% Ago-Gel
Electrophoresis proves that it is assembled successfully.Its particle size and pattern are characterized by dynamic light scattering and transmission electron microscope.As a result see figure
19- Figure 21.
4.2 have preparation (AS1411/PTX-SNA, the FAM/ of the multi-functional carrier micelle of targeting and imaging function
AS1411/PTX-SNA)
By the PTX-SNA and polyA in 4.120-AS1411 and polyA20-FAM base sequence is proportionally blended in 1 ×
TAE/Mg2+, room temperature is then annealed to since 65 DEG C can be obtained the multi-functional carrier micelle with targeting and imaging function.It is logical
It crosses 1% Ago-Gel and characterizes its assembling, such as Figure 22.
Nucleic acid sequence is as follows:
PolyA20- FAM:5 '-AAAAAAAAAAAAAAAAAAAA-FAM-3 '；
PolyA20-AS1411:5 '-AAAAAAAAAAAAAAAAAAAAGGTGGTGGTGGTTGTGGTGGTGGTGG-3 '.
Preparation (the Bcl-2-PTX- of the 4.3 load medicine ball forming core acid without targeting or targeting coexisted with gene and chemotherapy
SNA, AS1411/Bcl-2-PTX-SNA) by the way that the di-phosphate ester key sequence in block D NA is changed to antisense nucleic acid Bcl-2 sequence
Column, can achieve the purpose of reverse multiple drug resistance of tumor.
Bcl-2- of the selection function nucleic acid (antisense nucleic acid) as blockPOT10-PST18- T base sequence, according to example
Method in 2.2 obtain containing functional nucleic acid sequence blocks type nucleic acid-paclitaxel conjugates (Bcl-2-b- (PSDNA-g-
PTX)), it is dissolved in DMSO that isometric water then is added, non-targeted gene can be obtained in dialysed overnight in aqueous solution
The Bcl-2-PTX-SNA coexisted with chemotherapy.By Bcl-2-b- (PSDNA-g-PTX) andPOT26-b-(PSDNA-g-PTX) in proportion altogether
With the resulting SNA that dialyses, with polyA20-AS1411 is proportionally blended in 1 × TAE/Mg2+, then it is annealed to since 65 DEG C
The AS1411/Bcl-2-PTX-SNA that gene with targeting can be obtained in room temperature and chemotherapy coexists.Pass through 0.5% agar
Sugared gel characterizes its assembling, such as Figure 23.
The extracorporeal anti-tumor of the micelle-type spherical shape nucleic acid of 4.4 conjugation taxols and reverse multiple drug resistance of tumor effect
After the various PTX-SNA and tumour cell that prepare in example 4.1-4.3 are incubated for 72h altogether, cell is detected by MTT
Survival rate, as a result as shown in figure 24.Wherein, MCF-7 and HeLa cell is Sensitive Tumor Cells, and L929 cell is normal cell,
For characterizing the antitumous effect of targeting AS1411/PTX-SNA；HeLa/PTX is taxol resistance cell, for characterizing
The drug resistant effect of AS1411/Bcl-2-PTX-SNA reversing tumor.The experimental results showed that the AS1411/ with targeted molecular modification
PTX-SNA to tumour cell have preferable target function, and tumor cytotoxicity effect be better than non-targeted PTX-SNA.
It, can be by lowering anti-apoptotic Bcl-2 egg for the targeting AS1411/Bcl-2-PTX-SNA that gene and chemotherapy coexist
White gene expression plays the purpose of reverse multiple drug resistance of tumor, realizes the purpose of good gene and chemotherapy combined application.
The above embodiment of the present invention has obtained medicine-carried system DNA tetrahedron origami structure first, and nanometer hydrated partial size is
20 nanometers.Extremely hydrophobic camptothecin drug molecule is accurately accurately grafted in oligonucleotides segment, camptothecine is improved
The dissolubility of drug forms water-soluble nano prodrug, and the DNA sequence dna by being pre-designed realizes polyhedral structure
Assembling, has reached the structural controllability of medicine-carried system.Secondly, carrier micelle type spherical nuclei are also obtained in the above embodiment of the present invention
Acid, hydration partial size are 68 nanometers；Taxol drug is precisely grafted on the phosphorothioate group of nucleic acid, utilizes di(2-ethylhexyl)phosphate
The hydrophobic property of the DNA structure and taxol of ester bond and phosphorothioate bond block type is prepared using drug as kernel, with di(2-ethylhexyl)phosphate
The nucleic acid block of ester bond is the micellar structure of shell, and prepared micelle medicine carrying amount is up to 53%.
Under the teaching of the present invention and the above embodiments, those skilled in the art are easy to it is envisioned that cited by the present invention
Or each raw material enumerated or its equivalent alterations, each processing method or its equivalent alterations can realize the present invention and each original
The parameter bound value of material and processing method, interval value can realize the present invention, embodiment numerous to list herein.
Claims (24)
1. a kind of nucleic acid-drug conjugates based on phosphorothioate nucleic acid, which is characterized in that repaired including thiophosphate
The nucleic acid backbone of decorations and the drug molecule being grafted in the nucleic acid backbone, the grafting is by thio in the nucleic acid backbone
The group that electrophilic reaction can occurs with thiophosphate modified in bound phosphate groups and the drug molecule carries out reacting realization.
2. nucleic acid-drug conjugates as described in claim 1, which is characterized in that the nucleic acid bone of the phosphorothioate
On frame, the site of thio-modification and quantity can be adjusted and be controlled as needed, and thiophosphate is in certain one end of nucleic acid sequence
It is continuously modified, and/or in the intermediate base sequence selective modification of nucleic acid sequence, modification mode is more modifications or singly repairs
Decorations.
3. nucleic acid-drug conjugates as claimed in claim 1 or 2, which is characterized in that prepare sulphur by solid phase synthesis process
The nucleic acid backbone of substituted phosphate modification.
4. nucleic acid-drug conjugates as claimed in claim 1 or 2, which is characterized in that the nucleic acid of the phosphorothioate
The sequence and segment type of the oligonucleotides of skeleton can autonomous Design, and further can assemble to obtain by molecular recognition can
The DNA nanostructure of control, the nucleic acid-drug conjugates and its package assembly can be used as new drug delivery systems.
5. nucleic acid-drug conjugates as claimed in claim 2, which is characterized in that assembled for gel and tetrahedral structure
A thio-modification site is arranged every 2 to 3 bases in nucleic acid backbone in nucleic acid；And it is used for the nucleic acid of micella assembling, in core
One end of acid sequence carries out continuous thio-modification, and then prepares the block type core containing phosphodiester bond and phosphorothioate bond
Acid.
6. nucleic acid-drug conjugates as described in claim 1, which is characterized in that the drug molecule is by being simply esterified
Or acylation reaction introduces the group that electrophilic reaction can occur with thiophosphate.
7. nucleic acid-drug conjugates as described in claim 1 or 6, which is characterized in that the drug molecule, which also introduces, to break
The response type chemical bond split.
8. nucleic acid-drug conjugates as described in claim 1, which is characterized in that the modification is on drug molecule and can
The group one or more selected from the following of electrophilic reaction: 1) functional groups brominated or containing iodine occur with thiophosphate；
2) maleimide base group；3) aziridinyl sulfanilamide (SN) group.
9. nucleic acid-drug conjugates as described in claim 1, which is characterized in that the drug molecule be selected from anticancer drug or
The one or more of cancer targeting drug molecule.
10. nucleic acid-drug conjugates as described in claim 1, which is characterized in that the drug being grafted in the nucleic acid backbone point
Son is functional drug molecule or fluorescent probe molecule, cell-targeting molecule.
11. nucleic acid-drug conjugates as described in claim 1, which is characterized in that the type of the nucleic acid backbone is selected from deoxidation
RNA sequence or RNA sequence；The sequence of the nucleic acid backbone, one or more chosen from the followings:
Non-functional common base sequence including a kind of be made of base simple nucleic acid sequence and can be used for nucleic acid and accurately tie
The complex nucleic acid sequence of structure assembling；
Functional core acid sequence, the functional core acid sequence are selected from anti sense nucleotide sequence, nucleic acid aptamer sequence, nuclease sequence
Column, mRNA, Microrna, long-chain non-coding RNA, children purpura nephritis, are used for gene editing guide RNA, ring-type at siRNA
One of RNA.
12. nucleic acid-drug conjugates as described in claim 1, which is characterized in that the nucleic acid molecules after grafting drug keep it
It is defeated to match other function nucleic acid sequence imparting nucleic acid-drug conjugates drug by this property for the property of base pair complementarity
System targeting and imaging function are sent, prepares multi-functional nucleic acid-drug conjugates drug delivery system, wherein for matching
Functional nucleic acid be selected from aptamer, anti sense nucleotide sequence, fluorescent molecule modification nucleic acid sequence, functional polypeptide modification
Nucleic acid sequence, target galactose modification nucleic acid sequence etc. one kind.
13. a kind of drug delivery system, which is characterized in that be combined by the nucleic acid-drug any in claim 1-12
The medicament-carried nano system that object is self-assembly of.
14. drug delivery system as claimed in claim 13, which is characterized in that according to selected drug molecule and nucleic acid sequence
Difference, different drug delivery systems can be prepared by distinct methods, and the drug delivery system is selected from simple chain
Nucleic acid-drug macromolecular drug, the DNA nanostructure accurately assembled, DNA nanogel, carrier micelle type spherical shape nucleic acid nano
Structure carries medicine nucleic acid polyhedral structure or carries medicine nucleic acid hydrogel.
15. drug delivery system as claimed in claim 14, which is characterized in that the above-mentioned DNA accurately assembled that is used to prepare receives
The drug molecule selection molecule that hydrophobicity is weak, molecular weight is small of rice structure, and/or reduce the quantity of nucleic acid backbone thio-modification；
And the molecule that the drug molecule for being used for micella assembling selects hydrophobicity strong, and/or the quantity of nucleic acid backbone thio-modification can be increased.
16. any nucleic acid-drug conjugates preparation method in a kind of claim 1-12 characterized by comprising
The first step, the nucleic acid molecules for preparing thio-modification；
Drug molecule by chemical reaction method preparation containing the group that electrophilic reaction can occur with thiophosphate；
Nucleic acid molecules made above do not have sequencing with the step of preparing drug molecule；
Second step prepares nucleic acid-drug conjugates, and the drug molecule of modification is dissolved in organic solvent, is then added suitable
Nucleic acid molecules are reacted, the drug molecule in this reaction relative to phosphorothioate group be it is large excess of, reacted it
Extra small molecule is removed afterwards, and nucleic acid-drug conjugates can be obtained after dry.
17. the preparation method of nucleic acid-drug conjugates as claimed in claim 16, which is characterized in that by synthesis in solid state side
Method prepares the nucleic acid molecules of thio-modification.
18. the preparation method of nucleic acid-drug conjugates as claimed in claim 16, which is characterized in that the drug molecule
Modification is one of with the following method: (1) drug point of the carbonyl ethyl bromine containing disulfide bond is prepared by two step esterifications
Son；Or (2) prepare the drug molecule of the benzyl bromine modification containing disulfide bond by two step esterifications.
19. the preparation method of nucleic acid-drug conjugates as claimed in claim 16, which is characterized in that the drug molecule is also
The response type chemical bond that can be broken is introduced, the response type chemical bond being broken is disulfide bond, described and thiophosphate
The group that electrophilic reaction occurs is bromo modification group；The medicine of the bromo modification containing disulfide bond is prepared by chemical reaction method
Object molecule, uses one of following methods: under protection of argon gas, disulfide bond being accessed camptothecin drug point by triphosgene
Son, then by reacting with bromoacetyl bromide, obtain bromo camptothecin drug molecule；Or, passing through 4- bromomethyl benzyl alcohol and two sulphur
The carboxylic acid structure containing disulfide bond is obtained for the esterification of dipropionic acid, then anti-by the esterification of 2 ' position hydroxyl of carboxylic acid and taxol
It should obtain the taxol drug molecule of benzyl bromine structural modification.
20. the preparation method of nucleic acid-drug conjugates as claimed in claim 16, which is characterized in that pass through control nucleic acid point
Whether divide in sub- concentration, the ratio of drug molecule and phosphorothioate group and reaction solution containing salting liquid to control drug
The grafting efficiency of son.
21. the preparation method of nucleic acid-drug conjugates as claimed in claim 16, which is characterized in that the organic solvent choosing
From following one kind: reaction system used is in the mixed system of dimethyl sulfoxide and phosphate buffer when grafting camptothecine,
Volume ratio 4:1；Being grafted reaction system used in taxol is dimethyl sulfoxide；The temperature and time of the reaction is: 50 DEG C-
55 DEG C of reactions, it is overnight or longer.
22. any nucleic acid-drug conjugates drug delivery system preparation method in a kind of claim 13-15,
It is characterized in that, selected from one of following methods:
(1) it carries the preparation of medicine nucleic acid molecule drug molecule: being prepared using direct dissolution method, nucleic acid-drug conjugates are directly molten
In aqueous solution or salting liquid, preparation carries medicine nucleic acid molecule prodrug；
(2) carry the preparation of medicine nucleic acid polyhedral structure or nucleic acid gel: selection can complementary pairing nucleic acid sequence, in feature site
Nucleic acid-drug conjugates are obtained according to the method any in claim 16-21 after progress phosphorothioate, it will be mutual
The nucleic acid sequence recruited pair is in TAE/Mg2+It mixes in solution, is prepared by the method for annealing in proportion；
(3) preparation of carrier micelle type spherical shape nucleic acid: one section of continuous phosphodiester bond of selection and one section of continuous phosphorothioate bond
The block type nucleic acid of modification obtains nucleic acid-drug conjugates according to the method any in claim 16-21, using saturating
Analysis method prepares carrier micelle, wherein the hydrophilic outer shell without the nucleic acid block of phosphorothioate as micella, hydrophobicity
Drug is as kernel；
(4) carry the preparation of medicine multifunctional sphere nucleic acid: selection is containing the core with continuous phosphodiester bond block complimentary mating portion
Acid sequence, there is this nucleic acid sequence fluorescent molecule to modify, targeting aptamers are modified, target polypeptide is modified, it is small numerator modified to target
Function, by passing through the targeting of method preparation collection, imaging, gene therapy and the chemotherapy of annealing for micella prepared in above-mentioned (3)
Multi-functional load medicine ball forming core acid.
23. described in any in any nucleic acid-drug conjugates and claim 13-16 in a kind of claim 1-12
Drug delivery system preparation based on gene therapy, chemotherapy combination therapy disease nucleic acid nano drug and chemotherapeutics in
Purposes.
24. a kind of drug, which is characterized in that including the nucleic acid-drug conjugates or right any in claim 1-12
It is required that any drug delivery system in 13-16.
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Cited By (8)
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| CN110075312A (en) * | 2019-04-25 | 2019-08-02 | 中国科学院上海应用物理研究所 | A kind of method and the thus obtained compound of single stranded DNA and hydrophobic drug covalent linkage |
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| CN110257053A (en) * | 2019-07-02 | 2019-09-20 | 上海交通大学 | A kind of customized frame nucleic acid nano illuminator of shape and its preparation method and application |
| CN111317826A (en) * | 2020-03-19 | 2020-06-23 | 上海交通大学 | Nucleic acid composite nano-drug constructed based on metal ion coordination self-assembly and preparation method and application thereof |
| CN111803517A (en) * | 2020-06-24 | 2020-10-23 | 上海交通大学 | Platinum drug conjugate based on nucleic acid material loading, platinum drug delivery system, and preparation method and application thereof |
| CN113633613A (en) * | 2021-07-20 | 2021-11-12 | 河南大学 | siRNA micelle, preparation method, composition and application thereof |
| CN114681619A (en) * | 2020-12-30 | 2022-07-01 | 上海交通大学医学院附属仁济医院 | Aptamer compound based on Biotin-SA and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110075312A (en) * | 2019-04-25 | 2019-08-02 | 中国科学院上海应用物理研究所 | A kind of method and the thus obtained compound of single stranded DNA and hydrophobic drug covalent linkage |
| CN110170056A (en) * | 2019-04-25 | 2019-08-27 | 中国科学院上海应用物理研究所 | A kind of method and the thus obtained compound of DNA nanostructure and hydrophobic drug covalent linkage |
| CN110257053A (en) * | 2019-07-02 | 2019-09-20 | 上海交通大学 | A kind of customized frame nucleic acid nano illuminator of shape and its preparation method and application |
| CN111317826A (en) * | 2020-03-19 | 2020-06-23 | 上海交通大学 | Nucleic acid composite nano-drug constructed based on metal ion coordination self-assembly and preparation method and application thereof |
| CN111803517A (en) * | 2020-06-24 | 2020-10-23 | 上海交通大学 | Platinum drug conjugate based on nucleic acid material loading, platinum drug delivery system, and preparation method and application thereof |
| CN114681619A (en) * | 2020-12-30 | 2022-07-01 | 上海交通大学医学院附属仁济医院 | Aptamer compound based on Biotin-SA and application thereof |
| CN114681619B (en) * | 2020-12-30 | 2023-08-29 | 上海交通大学医学院附属仁济医院 | Aptamer complex based on Biotin-SA and application thereof |
| CN113633613A (en) * | 2021-07-20 | 2021-11-12 | 河南大学 | siRNA micelle, preparation method, composition and application thereof |
| CN113633613B (en) * | 2021-07-20 | 2023-04-25 | 河南大学 | siRNA micelle, preparation method, composition and application thereof |
| WO2023246827A1 (en) * | 2022-06-21 | 2023-12-28 | 上海交通大学 | Micromolecular drug-oligonucleotide conjugate and use thereof |
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