CN103948938A - Thrombin-loaded DNA (deoxyribonucleic acid) self-assembly nano-structure, as well as preparation method and application thereof - Google Patents
Thrombin-loaded DNA (deoxyribonucleic acid) self-assembly nano-structure, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing a thrombin-loaded DNA (deoxyribonucleic acid) self-assembly nano-structure. The method comprises the following steps: self-assembling a single-stranded DNA molecule into a rectangular nano-structure by utilizing a DNA paper folding technology; immobilizing thrombin on the surface of the rectangular nano-structure, and immobilizing a 'lock' structure capable of answering vascular endothelial specific receptor of a tumor at two long edges of the rectangular nano-structure so as to close the thrombin inside the structure and generate response when thrombin is carried to a tumor blood vessel tissue. Therefore, target delivery and controllable release of the tumor blood vessel tissue by the thrombin can be realized; and the prepared thrombin-loaded DNA (deoxyribonucleic acid) self-assembly nano-structure can be used for intravenous injection, realizes targeted treatment of tumors, and has a wide clinical application prospect.
Description
Technical field
The present invention relates to nano-carrier technical field of pharmaceuticals, relate in particular to a kind of DNA self-assembled nano structures, its preparation method and application of loading thrombin.
Background technology
Growth, the metastasis of tumor vessel system and tumor are closely related, it is the main place that tumor tissues obtains nutrient substance and oxygen, also be the passage that tumor is drained metabolite, tumor cell escape, transfer, thereby tumor vessel become the good target spot of neoplasm targeted therapy.Target treatment to tumor blood vessel method mainly comprises two kinds of strategies: the treatment of the treatment that anti-new vessels generates and the confession of blocking-up tumor blood.Wherein blocking tumor blood is the blood vessel having existed by thromboembolism for therapy, makes for want of nutrition of tumor cell and oxygen hungry and dead.This Therapeutic Method for target spot be blood constituent, thereby be difficult for to produce drug resistance, applied widely.In addition, for clinical most of cancer patients, medicine for be all the tumor of vascularization.Therefore, blocking-up tumor blood presents good potential applicability in clinical practice for therapy.Yet, find up to now one " bottleneck " that safe and effective tumor vessel thromboembolism method remains this research field.
Thrombin (thrombin) claim again prothrombin a, is a kind of key enzyme of body blood coagulation system.It can directly act on the Fibrinogen in blood on the one hand, make it to change into fibrin, form fibrin gel, by receptor-mediated approach induced platelet, activate on the other hand, the platelet of activation is assembled, under the effect of fibrin gel net, form platelet thrombus, afterwards in blood other visible components add the formation that causes stablizing thrombosis.Therefore,, as a kind of effective coagulant, thrombin, in the situation that not needing other numerous thrombins to participate in, is induced the formation of thrombosis fast and efficiently.So, if thrombin is delivered in tumor vessel as coagulant drug targeting, can effectively block the confession of tumor blood undoubtedly, reach the object for the treatment of tumor.Yet the high procoagulant activity of thrombin self makes it be unsuitable for intravenous injection, moreover the half-life of thrombin in blood is shorter.Therefore, how to realize the intravenous injection of thrombin and to the targeted of tumor tissues, be the key technology challenge that is applied to oncotherapy.
DNA nanotechnology has potential application widely in emerging nanometer field, and wherein DNA paper folding art causes that as a kind of brand-new DNA self-assembling method people pay close attention to widely.DNA paper folding art (origami) is by a long DNA single chain (is called to scaffold chain, be generally genomic DNA) carry out base complementrity with a series of short dna strands (being called staple chain) through particular design, construct the controlled nano-pattern of various complexity or structure.
Summary of the invention
The object of the invention is to propose a kind ofly can realize the DNA self-assembled nano structures of thrombin to the loading thrombin of the targeted of tumor tissues blood vessel and controllable release, this nanostructured has controlled " lock " structure with specificly-response tumor vascular endothelium specific receptor and then discharges thrombin, be a kind of addressable, safety, efficient, there is the tumor vessel blocker of higher medical value.
For reaching this object, the present invention by the following technical solutions:
First aspect, the invention provides a kind of preparation method of loading the DNA self-assembled nano structures of thrombin, and this preparation method comprises the following steps:
(1) utilize DNA paper folding technology, single strand dna is self-assembled into rectangular nanostructured;
(2) thrombin is fixed on to the surface of step (1) gained rectangle nanostructured;
(3) the auxiliary chain 2 of the auxiliary chain that comprises aptamers that can specific recognition tumor vascular endothelium receptor 1 and the complementary series that comprises this aptamers is hybridized, and two in this crossbred free strands parts are matched and are individually fixed in two long limits of described rectangle nanostructured by base complementrity.
In above-mentioned preparation method, as preferably, in step (1), folding assisted by staple chain, is self-assembled into rectangular nanostructured by described single strand dna;
Preferably, described single strand dna is M13 phage genome DNA;
Described rectangular length-width ratio can be adjusted according to actual needs, and preferably, described rectangular length-width ratio is (1.5～2.5): 1, be preferably (1.5～2): 1,1.5:1 more preferably.
In the preferred embodiment of the invention, described rectangular length * wide=90nm * 60nm.
Further preferably, the self assembling process of step (1) is:
Described single strand dna and staple chain are pressed to 1:(6-10), the preferred mixed in molar ratio of 1:8, gained mixed solution is from 60-70 ℃, preferably 62-68 ℃, more preferably 65 ℃, take 0.8-1.2 ℃, preferably 1 ℃ be a gradient, be cooled to gradually 20-30 ℃, preferably 22-28 ℃, more preferably 25 ℃ anneal, obtain described rectangle nanostructured;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 6-8 hour, is preferably 7 hours.
In above-mentioned preparation method, as preferably, in step (2), the oligomerization dT that described thrombin is coupled to sulfhydrylation by coupling agent is upper, and gained complex is covalently bonded in the surface of described rectangle structure again by the oligomerization dT of sulfhydrylation;
Preferably, described coupling agent is 4-(N-maleimide methyl) cyclohexane extraction-1-carboxylic acid sulfonic group butanimide ester sodium salt;
Preferably, on the surface of described rectangle structure, establish 3-6, be preferably 4 thrombin binding sites.
Further preferably, step (2) comprising:
(1 ') mixes thrombin with coupling agent, in 20-30 ℃, preferably 22-28 ℃, more preferably react 40-90min, preferred 60min at 25-26 ℃, obtain the complex of thrombin and coupling agent;
(2 ') mixes step (1 ') gained complex with the oligomerization dT of sulfhydrylation, in 2-8 ℃, preferably 4-6 ℃, more preferably react 12-20 hour, preferably 14-18 hour, more preferably 16 hours at 4 ℃, obtain the complex of thrombin-coupling agent-oligomerization dT;
Preferably, the mol ratio of the oligomerization dT of step (1) gained complex and sulfhydrylation is 1:(12-20), be preferably 1:15;
(3 ') mixes step (2 ') gained complex with described rectangle nanostructured, gained mixture is from 40-50 ℃, preferably 42-48 ℃, more preferably 45 ℃, take 0.8-1.2 ℃, preferably 1 ℃ be a gradient, be cooled to gradually 20-30 ℃, preferably 22-28 ℃, more preferably 25 ℃ anneal, thereby thrombin is covalently bonded in rectangle nanostructured;
Preferably, the mol ratio of step (2 ') gained complex and described rectangle nanostructured is (8-12): 1, be preferably 10:1;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 2-4 hour, is preferably 3 hours.
In above-mentioned preparation method, as preferably, in step (3), described aptamers that can specific recognition tumor vascular endothelium receptor is DNA or RNA aptamers, is preferably AS-1411;
Preferably, the sequence of the auxiliary chain 1 that comprises described aptamers is as shown in SEQ ID NO.1, and the sequence of the auxiliary chain 2 of the complementary series that comprises this aptamers is as shown in SEQ ID NO.2:
SEQ?ID?NO.1：
5’-GTGCGCAAAGAGTTTACAAAATTAAAGTACGGTGTCTGGAAGAGGTCA-3’；
SEQ?ID?NO.2：
5’-GTGCGCAAAGAGTTTATTTTTGCGCAGAAAACGAGAATGAATGTTTAG-3’。
Further preferably, step (3) comprising:
By described auxiliary chain 1 and auxiliary chain 2 annealing hybridization, gained crossbred mixes with step (2) products therefrom, and from 35-40 ℃, preferably 37 ℃, take 1 ℃ as a gradient, be cooled to gradually 12-18 ℃, be preferably 15 ℃ and anneal, thereby two of described crossbred free strands parts are matched and are individually fixed in two long limits of described rectangle structure by base complementrity;
Preferably, the mol ratio of described crossbred and step (2) products therefrom is (18-22): 1, be preferably 20:1;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 3-5 hour, is preferably 4 hours.
As preferably, above-mentioned preparation method also comprises:
(4) fixing on the broadside of described rectangle nanostructured can the tumor vascular targeting chain of specific recognition, described targeting chain is preferably aptamers, polypeptide, antibody or the antibody fragment of tumor vascular endothelium specific receptor, more preferably AS-1411 aptamers;
Preferably, identical with on long limit of the kind of the aptamers on broadside and number;
Preferably, AS-1411 aptamers is mixed with step (3) products therefrom, from 35-40 ℃, preferably 37 ℃, take 0.8-1.2 ℃, preferably 1 ℃ be a gradient, be cooled to gradually 12-18 ℃, be preferably 15 ℃ and anneal, form the Self-assembled DNA nanostructured of described loading thrombin;
Preferably, the mol ratio of described AS-1411 aptamers and step (3) products therefrom is (3-8): 1, be preferably 5:1;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 3-5 hour, is preferably 4 hours.
The increase of step (4), can strengthen described DNA self-assembled nano structures to the identification of neoplasm vascularity and binding ability, is more conducive to targeting transportation and the controllable release of thrombin.
In above-mentioned preparation method, due to controlled, the predictability in the self assembly of DNA nanostructured, thereby can realize thrombin accurate space orientation in nanostructured.
Second aspect, the invention provides a kind of DNA self-assembled nano structures that loads thrombin, by preparation method described in first aspect, is made;
Preferably, described DNA self-assembled nano structures is tubulose, and base diameter is 16-22nm, be preferably 18-20nm, 19nm more preferably, high for 80-100nm, be preferably 85-95nm, 90nm more preferably;
Preferably, the inside of each DNA self-assembled nano structures is in conjunction with 3-6, preferred 4 thrombin protein moleculars.
The third aspect, thus the invention provides DNA self-assembled nano structures described in second aspect for the preparation of blocking-up tumor blood for the application suppressing in the medicine of tumor growth; Preferably, described tumor is breast carcinoma, melanoma, sarcoma, nonsmall-cell lung cancer and gastric cancer.
The present invention utilizes DNA paper folding technology, take longer single stranded DNA as main chain (being scaffold chain), complementary in the hybridization of ad-hoc location by the staple chain with excessive, makes it be self-assembled into rectangular DNA nanostructured; The surface in rectangle nanostructured by thrombin proteopexy again, subsequently, by the auxiliary chain 1 of the aptamers that comprises tumor vascular endothelium specific receptor and auxiliary chain 2 hybridization that comprises aptamers complementary series, and two free strands of crossbred are partly separately fixed to rectangular two long limits, thereby form " lock " structure, by rectangular nanostructured closure, it is tubulose, finally obtain a kind of inside and be mounted with thrombin, there is controlled " lock " to respond the tubulose DNA nanostructured of tumor vascular endothelium specific receptor simultaneously.
The targeted of DNA self-assembled nano structures and the mechanism of controllable release of loading thrombin of the present invention are: in the blood vessel transportation before arriving neoplasm vascularity, auxiliary chain 1 and auxiliary chain 2 formed " lock " structure is closed in thrombin the inside of tubular structure, has avoided thrombin to bring into play non-specific blood coagulation effect; When arriving neoplasm vascularity, the contained specificity aptamers of auxiliary chain 1 can respond tumor vascular endothelium specific receptor, thereby opens " lock " structure, discharges thrombin, and then has realized thrombin to the targeted of neoplasm vascularity and controllable release.
The DNA self-assembled nano structures of loading thrombin of the present invention can be used for intravenous injection, has realized the targeted therapy to tumor; And, due to DNA nanostructured be easy to modification, good biocompatibility, size uniform, in solution good stability, therefore there is wide medical prospect.
Accompanying drawing explanation
Fig. 1 is atomic force microscope (AFM) shape appearance figure of rectangle DNA paper folding structure prepared in embodiment 1, and scale is 200nm.
Fig. 2 is transmission electron microscope (TEM) shape appearance figure of rectangle DNA paper folding structure prepared in embodiment 1, and scale is 200nm.
Fig. 3 is the inhibitory action of the prepared tubulose DNA nanostructured of embodiment 1 to breast cancer tumour growth; Arrow represents administration time point.
The specific embodiment
Below by the specific embodiment, further illustrate technical scheme of the present invention.It will be appreciated by those skilled in the art that these embodiment are only for the present invention is described, the scope that it does not limit the present invention in any way.
Embodiment 1: the present invention loads the preparation of the DNA self-assembled nano structures of thrombin
Concrete steps are as follows:
(1) preparation of rectangle DNA paper folding structure
M13 phage genome DNA backbone and staple chain are mixed, and the ultimate density of main chain and staple chain is respectively 10nM and 80nM.Utilize grads PCR instrument slowly to anneal to mixture, annealing conditions is: from 65 ℃ to 25 ℃, every 1 ℃ is a gradient, and each gradient time of staying is 10 minutes, and whole annealing process is about 7 hours.
After cycle of annealing is complete, DNA paper folding structure sample is taken out, with 100kDa centrifugal column (MWCO) centrifugalize, remove excessive staple chain and catch chain.Centrifugal condition is: get 100 μ L samples, add 350 μ L1 * TAE-Mg buffer solution, under 4700 revs/min of conditions centrifugal 3 minutes, in centrifugal column, the volume of surplus solution was approximately 100 μ L, repeats twice of this process.The final sample of collecting is respectively used to 1% agarose gel electrophoresis analysis, and the observation of the paper folding structure and morphology under atomic force microscope and projection Electronic Speculum, and the paper folding structure and morphology figure under atomic force microscope and transmission electron microscope respectively as shown in Figure 1 and Figure 2.After testing, the length of gained rectangle DNA paper folding structure is 90nm, and wide is 60nm, and thickness is 2nm.
(2) prepare the complex of the oligomerization dT of thrombin-coupling agent-sulfhydrylation
After the sulfo-SMCC of the thrombin of 200 μ L3 μ M (purchased from the magnificent Science and Technology Ltd. of middle life) and 9 μ L15mM (thinking biochemical technology company limited purchased from Tianjin Skien) is mixed in room temperature reaction 1 hour, reaction finish after, by mixture with 30kDa centrifugal column under the condition of 7500 revs/min centrifugal 30 minutes to remove excessive sulfo-SMCC.The oligomerization dT that adds 15 times of excessive sulfhydrylations in the solution of collecting, mixture spends the night in 4 ℃ of reactions, the thrombin product of modifying with 30kDa centrifugal column centrifugal purification Oligo dT, purified product is identified by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 4%-12%.
(3) load the preparation of the tubulose Self-assembled DNA nano-particle of thrombin
By the rectangle DNA paper folding structure-solution after step (1) gained purification and step (2) gained thrombin complex according to the molar ratio mix homogeneously of 1:10 after, put into grads PCR instrument, slowly from 45 ℃, be down to 25 ℃, every 1 ℃ is a gradient, each gradient time of staying is 10 minutes, and whole annealing process required time is about 3 hours.
After cycle of annealing finishes, the thrombin complex that sample is modified to remove excessive Oligo dT with the centrifugalize of 100kDa centrifugal column.Then, to the crossbred that adds in the sample of collecting 20 times of excessive auxiliary chains 1 (sequence is as shown in SEQ ID NO.1) with auxiliary chain 2 (sequence is as shown in SEQ ID NO.2), and 5 times of excessive targeting chain AS-1411 aptamers, again putting into grads PCR instrument slowly anneals, annealing conditions is for to be down to 15 ℃ from 37 ℃, every 1 ℃ is a gradient, and each gradient time of staying is 10 minutes, and whole annealing process is approximately lasted 4 hours.With 100kDa centrifugal column centrifugalize PCR product, agarose gel electrophoresis purification reclaims the tubulose DNA nano-particle that the bag that obtains purifying is loaded with thrombin.
Embodiment 2: the present invention loads the antitumous effect of the DNA self-assembled nano structures of thrombin
The evaluation methodology of antitumous effect:
By 2.0 * 10
6individual's source MDA-MB-231 breast cancer cell in-situ inoculating is in the breast pad of BALB/c nude mice, grows into about 100mm to gross tumor volume
3.By the DNA self-assembling nanoparticles solution of the prepared loading thrombin of embodiment 1 according to every the about 1.5U unit of mice thrombin dosage to tumor-bearing mice tail vein injection, every 2 days, inject once and measure gross tumor volume size, inject after 6 times statistical analysis gross tumor volume situation of change.Gross tumor volume calculates according to following formula: volume=(d
2* D)/2, wherein d is the minimum diameter of tumor, D is maximum gauge.Injecting normal saline, free thrombin, blank DNA nano-particle, as negative control.Experimental result as shown in Figure 3.
By Fig. 3, can be obtained, compare with normal saline group, free thrombin group and blank DNA nano-particle group, the gross tumor volume of the DNA nano-particle group of loading thrombin of the present invention increases obviously slack-off, and its antitumous effect is obvious.
Applicant's statement, the present invention illustrates technical scheme of the present invention by above-described embodiment, but the present invention is not limited to above-described embodiment, does not mean that the present invention must rely on above-described embodiment and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention comprises the suitable improvement of the present invention's nucleotide sequence used, the selection of design parameter etc., within all dropping on protection scope of the present invention and open scope.
Claims (10)
1. a preparation method of loading the DNA self-assembled nano structures of thrombin, is characterized in that, comprises the following steps:
(1) utilize DNA paper folding technology, single strand dna is self-assembled into rectangular nanostructured;
(2) thrombin is fixed on to the surface of step (1) gained rectangle nanostructured;
(3) the auxiliary chain 2 of the auxiliary chain that comprises aptamers that can specific recognition tumor vascular endothelium receptor 1 and the complementary series that comprises this aptamers is hybridized, and two in this crossbred free strands parts are matched and are individually fixed in two long limits of described rectangle nanostructured by base complementrity.
2. preparation method according to claim 1, is characterized in that, in step (1), folding assisted by staple chain, is self-assembled into rectangular nanostructured by described single strand dna;
Preferably, described single strand dna is M13 phage genome DNA;
Preferably, described rectangular length-width ratio is (1.5～2.5): 1, be preferably (1.5～2): 1,1.5:1 more preferably.
3. preparation method according to claim 2, is characterized in that, the self assembling process of step (1) is:
Described single strand dna and staple chain are pressed to 1:(6-10), the preferred mixed in molar ratio of 1:8, gained mixed solution is from 60-70 ℃, preferably 62-68 ℃, more preferably 65 ℃, take 0.8-1.2 ℃, preferably 1 ℃ be a gradient, be cooled to gradually 20-30 ℃, preferably 22-28 ℃, more preferably 25 ℃ anneal, obtain described rectangle nanostructured;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 6-8 hour, is preferably 7 hours.
4. according to the preparation method described in claim 1-3 any one, it is characterized in that, in step (2), the oligomerization dT that described thrombin is coupled to sulfhydrylation by coupling agent is upper, and gained complex is covalently bonded in the surface of described rectangle structure again by the oligomerization dT of sulfhydrylation;
Preferably, described coupling agent is 4-(N-maleimide methyl) cyclohexane extraction-1-carboxylic acid sulfonic group butanimide ester sodium salt;
Preferably, on the surface of described rectangle structure, establish 3-6, be preferably 4 thrombin binding sites.
5. preparation method according to claim 4, is characterized in that, step (2) comprising:
(1 ') mixes thrombin with coupling agent, in 20-30 ℃, preferably 22-28 ℃, more preferably react 40-90min, preferred 60min at 25-26 ℃, obtain the complex of thrombin and coupling agent;
(2 ') mixes step (1 ') gained complex with the oligomerization dT of sulfhydrylation, in 2-8 ℃, preferably 4-6 ℃, more preferably react 12-20 hour, preferably 14-18 hour, more preferably 16 hours at 4 ℃, obtain the complex of thrombin-coupling agent-oligomerization dT;
Preferably, the mol ratio of the oligomerization dT of step (1) gained complex and sulfhydrylation is 1:(12-20), be preferably 1:15;
(3 ') mixes step (2 ') gained complex with described rectangle nanostructured, gained mixture is from 40-50 ℃, preferably 42-48 ℃, more preferably 45 ℃, take 0.8-1.2 ℃, preferably 1 ℃ be a gradient, be cooled to gradually 20-30 ℃, preferably 22-28 ℃, more preferably 25 ℃ anneal, thereby thrombin is covalently bonded in rectangle nanostructured;
Preferably, the mol ratio of step (2 ') gained complex and described rectangle nanostructured is (8-12): 1, be preferably 10:1;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 2-4 hour, is preferably 3 hours.
6. according to the preparation method described in claim 1-5, it is characterized in that, in step (3), described aptamers that can specific recognition tumor vascular endothelium receptor is DNA or RNA aptamers, is preferably AS-1411;
Preferably, the sequence of the auxiliary chain 1 that comprises described aptamers is as shown in SEQ ID NO.1, and the sequence of the auxiliary chain 2 of the complementary series that comprises this aptamers is as shown in SEQ ID NO.2.
7. preparation method according to claim 6, is characterized in that, step (3) comprising:
By described auxiliary chain 1 and auxiliary chain 2 annealing hybridization, gained crossbred mixes with step (2) products therefrom, and from 35-40 ℃, preferably 37 ℃, take 1 ℃ as a gradient, be cooled to gradually 12-18 ℃, be preferably 15 ℃ and anneal, thereby two of described crossbred free strands parts are matched and are individually fixed in two long limits of described rectangle structure by base complementrity;
Preferably, the mol ratio of described crossbred and step (2) products therefrom is (18-22): 1, be preferably 20:1;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 3-5 hour, is preferably 4 hours.
8. according to the preparation method described in claim 1-7, it is characterized in that, also comprise:
(4) fixing on the broadside of described rectangle nanostructured can the tumor vascular targeting chain of specific recognition, described targeting chain is preferably aptamers, polypeptide, antibody or the antibody fragment of tumor vascular endothelium specific receptor, more preferably AS-1411 aptamers;
Preferably, identical on the kind of the aptamers on broadside and number and long limit;
Preferably, AS-1411 aptamers is mixed with step (3) products therefrom, from 35-40 ℃, preferably 37 ℃, take 0.8-1.2 ℃, preferably 1 ℃ be a gradient, be cooled to gradually 12-18 ℃, be preferably 15 ℃ and anneal, form the Self-assembled DNA nanostructured of described loading thrombin;
Preferably, the mol ratio of described AS-1411 aptamers and step (3) products therefrom is (3-8): 1, be preferably 5:1;
Preferably, each gradient time of staying is 8-12 minute, is preferably 10 minutes, and whole annealing process is 3-5 hour, is preferably 4 hours.
9. a DNA self-assembled nano structures that loads thrombin, is characterized in that, by preparation method described in claim 1-8 any one, is made;
Preferably, described DNA self-assembled nano structures is tubulose, and base diameter is 16-22nm, be preferably 18-20nm, 19nm more preferably, high for 80-100nm, be preferably 85-95nm, 90nm more preferably;
Preferably, the inside of each DNA self-assembled nano structures is in conjunction with 3-6, preferred 4 thrombin protein moleculars.
10. thereby the application of DNA self-assembled nano structures as claimed in claim 9 in the medicine that supplies inhibition tumor growth for the preparation of blocking-up tumor blood; Preferably, described tumor is breast carcinoma, melanoma, sarcoma, nonsmall-cell lung cancer and gastric cancer.
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