TDNs-AS1411-Nucleic Acid Drug Nanocomposite Based Drug Delivery System and Preparation Method Thereof

Abstract

This invention discloses a method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system, which includes the following steps: binding AS1411 and nucleic acid drug to a tetrahedral DNA nanostructure respectively; selecting four DNA single strands that respectively carry AS1411 and nucleic acid drug; mixing the four DNA single strands; mixing the DNA single strands and the TM buffer uniformly; putting the mixture into a PCR apparatus; raising the temperature to 95 C. quickly and maintaining for 10 min; and next cooling down to 4 C. and maintaining for 20 min to obtain the TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system. This drug delivery system can directly act on cell nucleus and will not be degraded by lysosomal. The targeting specificity is good. The drug can take a good efficacy and the pertinency is high.

Claims

1. A method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system, comprising the following steps: binding AS1411 and nucleic acid drug to a tetrahedral DNA nanostructure respectively; selecting four DNA single strands that respectively carry AS1411 and nucleic acid drug; mixing the four DNA single strands; adding the DNA single strands to TM buffer; mixing the DNA single strands and the TM buffer uniformly; putting the mixture into a PCR apparatus; raising the temperature to 95 C. quickly and maintaining for 10 min; and cooling down to 4 C. and maintaining for 20 min to obtain the TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system.

2. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 1, wherein the TM buffer with pH value of 8.0, includes 5-10 mM Tris-HCl and 5-50 mM MgCl.sub.2.

3. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 2, wherein the TM buffer with pH value of 8.0, includes 10 mM Tris-HCl and 50 mM MgCl.sub.2.

4. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 1, wherein four DNA single strands respectively carrying AS1411 and nucleic acid drug are mixed with a mole ratio of 1:1:1:1.

5. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 1, wherein the volume ratio of four DNA single strands respectively carrying AS1411 and nucleic acid drug to the TM buffer is 1:1:1:1:96.

6. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 1, wherein the concentration of each single strand of the tetrahedral DNA nanostructure is 1 M.

7. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 1, wherein the nucleic acid drug is selected from the group consisting of CpG, antisense oligonucleotide, microRNA, and siRNA.

8. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 1, wherein the CpG is selected from the group consisting of Class-A CpG, Class-B CpG, Class-C CpG, and Class-P CpG.

9. The method for preparing TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system of claim 1, wherein the antisense oligonucleotide is selected from the group consisting of ISIS 8005, ISIS 1082, ISIS 2105, ISIS 2302, ISIS 3521, ISIS 5132, ISIS 2922, ISIS 1082, ISIS 11061, ISIS 12959, and ISIS 481464.

10. A TDNs-A51411-nucleic acid drug nanocomposite based drug delivery system prepared by a method comprising the following steps: binding AS1411 and nucleic acid drug to a tetrahedral DNA nanostructure respectively; selecting four DNA single strands that respectively carry AS1411 and nucleic acid drug; mixing the four DNA single strands; adding the DNA single strands to TM buffer; mixing the DNA single strands and the TM buffer uniformly; putting the mixture into a PCR apparatus; raising the temperature to 95 C. quickly and maintaining for 10 min; and cooling down to 4 C. and maintaining for 20 min to obtain the TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system.

11. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 10, wherein the TM buffer with pH value of 8.0, includes 5-10 mM Tris-HCl and 5-50 mM MgCl.sub.2.

12. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 11, wherein the TM buffer with pH value of 8.0, includes 10 mM Tris-HCl and 50 mM MgCl.sub.2.

13. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 10, wherein four DNA single strands respectively carrying AS1411 and nucleic acid drug are mixed with a mole ratio of 1:1:1:1.

14. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 10, wherein the volume ratio of four DNA single strands respectively carrying AS1411 and nucleic acid drug to the TM buffer is 1:1:1:1:96.

15. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 10, wherein the concentration of each single strand of the tetrahedral DNA nanostructure is 1 M.

16. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 10, wherein the nucleic acid drug is selected from the group consisting of CpG, antisense oligonucleotide, microRNA, and siRNA.

17. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 10, wherein the CpG is selected from the group consisting of Class-A CpG, Class-B CpG, Class-C CpG, and Class-P CpG.

18. The TDNs-AS1411-nucleic acid drug nanocomposite based drug of claim 10, wherein the antisense oligonucleotide is selected from the group consisting of ISIS 8005, ISIS 1082, ISIS 2105, ISIS 2302, ISIS 3521, ISIS 5132, ISIS 2922, ISIS 1082, ISIS 11061, ISIS 12959, and ISIS 481464.

Description

DETAILED DESCRIPTION OF THE INVENTION

Embodiment 1

[0031] A method to prepare TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system includes the following steps. AS1411 and CpG are bound to tetrahedral DNA nanostructure respectively. The tetrahedral DNA nanostructure includes four DNA single strands. The concentration of each DNA single strand is 1 M. There is a binding site in each single strand. There are four binding sites in total. Both the AS1411 and the CpG can bind to the 5 end of each single strand (S1, S2, S3, and S4) of the tetrahedral DNA nanostructure. Effective binding ways are as follows: (1) one of the strands combines with AS1411, and the other one of three strands combine with CpG; (2) one of the strands combines with AS1411, and the other two strands combine with CpG; (3) one of the strands combines with AS1411, and the remaining three strands combine with CpG; (4) two of the strands combines with AS1411, and the other one of three strands combine with CpG; (5) two strands combine with AS1411, and the remaining two strands combine with CpG; (6) three strands combine with AS1411, and the remaining one strand combines with CpG.

[0032] Four single strands (with a molar ratio of 1:1:1:1) obtained by the binding way (1) are mixed with the TM buffer (10 mM Tris-HCl, 50 mM MgCl.sub.2, pH8.0). The volume of each strand is 1 L. The volume of the TM buffer is 96 L. The total reaction system is 100 L. After well mixed, the mixture is put into a PCR apparatus. The temperature is quickly raised to 95 C. and maintained for 10 min. Next, the temperature is cooled down to 4 C. and maintained for 20 min, such that the TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system is obtained.

[0033] Methods of 6% non-denaturing polyacrylamide gel electrophoresis (PAGE), dynamic light scatter (DLS), atomic force microscope (AFM), etc. can be conducted to verify whether the TDNs-AS1411-nucleic acid drug nanocomposite based drug delivery system is successfully prepared.

[0034] The sequences of the four DNA single strands are respectively as below:

TABLE-US-00002 S1: (SEQIDNO:1) 5-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTG AGACGAACATTCCTAAGTCTGAA-3; S2: (SEQIDNO:2) 5-ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTAC ACGATTCAGACTTAGGAATGTTCG-3; S3: (SEQIDNO:3) 5-ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAAT CGACGGGAAGAGCATGCCCATCC-3; S4: (SEQIDNO:4) 5-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATA CGAGGATGGGCATGCTCTTCCCG-3.

[0035] AS1411 can bind to any single strand of tetrahedral DNA nanostructure at the 5 terminal of the DNA. The sequences after binding are as follows:

TABLE-US-00003 S1-AS1411: (SEQIDNO:5) 5-GGTGGTGGTGGTTGTGGTGGTGGTGGT-ATTTATCACCCGCCATAGT AGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3; S2-AS1411: (SEQIDNO:6) 5-GGTGGTGGTGGTTGTGGTGGTGGTGGT-ACATGCGAGGGTCCAATAC CGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG-3; S3-AS1411: (SEQIDNO:7) 5-GGTGGTGGTGGTTGTGGTGGTGGTGGT-ACTACTATGGCGGGTGATA AAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3; S4-AS1411: (SEQIDNO:8) 5-GGTGGTGGTGGTTGTGGTGGTGGTGGT-ACGGTATTGGACCCTCGCA TGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG-3;

[0036] CpG can also bind to any single strand of tetrahedral DNA nanostructure at the 5 terminal of the DNA. The sequences after binding are as follows:

TABLE-US-00004 CpG: (SEQIDNO:13) 5-TCCATGACGTTCCTGACG-3; S1-CpG: (SEQIDNO:9) 5-TCCATGACGTTCCTGACG-ATTTATCACCCGCCATAGTAGACGTATC ACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3; S2-CpG: (SEQIDNO:10) 5-TCCATGACGTTCCTGACG-ACATGCGAGGGTCCAATACCGACGATTA CAGCTTGCTACACGATTCAGACTTAGGAATGTTCG-3; S3-CpG: (SEQIDNO:11) 5-TCCATGACGTTCCTGACG-ACTACTATGGCGGGTGATAAAACGTGTA GCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3; S4-CpG: (SEQIDNO:12) 5-TCCATGACGTTCCTGACG-ACGGTATTGGACCCTCGCATGACTCAAC TGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG-3.

Embodiment 2

[0037] AS1411 and CpG bind to tetrahedral DNA nanostructure in the binding way (2). Next, the combined DNA is mixed with the TM buffer. The remaining operations are the same as those in Embodiment 1.

Embodiment 3

[0038] AS1411 and CpG bind to tetrahedral DNA nanostructure in the binding way (3). Next, the combined DNA is mixed with the TM buffer. The remaining operations are the same as those in Embodiment 1.

Embodiment 4

[0039] AS1411 and CpG bind to tetrahedral DNA nanostructure in the binding way (4). Next, the combined DNA is mixed with the TM buffer. The remaining operations are the same as those in Embodiment 1.

Embodiment 5

[0040] AS1411 and CpG bind to tetrahedral DNA nanostructure in the binding way (5). Next, the combined DNA is mixed with the TM buffer. The remaining operations are the same as those in Embodiment 1.

Embodiment 6

[0041] AS1411 and CpG bind to tetrahedral DNA nanostructure in the binding way (6). Next, the combined DNA is mixed with the TM buffer. The remaining operations are the same as those in Embodiment 1.

[0042] CpG is the well-known immunostimulation nucleic acid, the main function of which is to activate immune response of cells to treat diseases including infections, tumors, and allergies, etc. AS1411 can specifically recognize the over-expressed pyrenin receptor in the cancer cell membrane. Moreover, AS1411 can be delivered into the nucleus with the mediation of the pyrenin during shuttling in cells. There are four binding sites on tetrahedral DNA nanostructure in total. That is, several different CpGs and AS1411s can be bound respectively.

[0043] (1) When one binding site connects with CpG, and one binding site connects with AS1411, the prepared drug delivery system can enter the nucleus (compared with that the tetrahedral DNA nanostructures enters the nucleus alone) and has certain immunostimulatory effect, but the effect is relatively weak.

[0044] (2) When one binding site connects with CpG, and two binding sites connect with AS1411, the prepared drug delivery system enters the nucleus with an increased amount (compared with that the tetrahedral DNA nanostructures with one AS1411 enters the nucleus), and has certain immunostimulatory effect, but the effect is still relatively weak.

[0045] (3) When one binding site connects with CpG, and the remaining three binding sites connect with AS1411, the prepared drug delivery system enters the nucleus with a significantly increased amount (compared with that the tetrahedral DNA nanostructures enters the nucleus alone). However, since only one CpG is carried, there is certain immunostimulatory effect, but not so strong.

[0046] (4) When two binding sites connect with CpG, and one binding site connects with AS1411, the prepared drug delivery system can enter the nucleus (compared with that the tetrahedral DNA nanostructures enters the nucleus alone), and has certain immunostimulatory effect, and the effect is relatively strong.

[0047] (5) When two binding sites connect with CpG, and the other two connect with AS1411, the prepared drug delivery system enters the nucleus with significantly increased amount (compared with that the tetrahedral DNA nanostructures enters the nucleus alone). The amount of entering the nucleus goes beyond that of (4). Also, the achieved immunostimulatory effect is relatively strong.

[0048] (6) When three binding sites connect with CpG, and the other one connects with AS1411, the prepared drug delivery system enters the nucleus with a lower amount than those of (4) and (5). However, the immunostimulatory effect is stronger than that of (5).

[0049] After comprehensive consideration, method (5) is chosen. Because the amount of the drug delivery system entering nucleus is large, immunostimulatory effect is strong, and functioning time in nucleus is long.