A thrombin-responsive dna nanomachine and its preparation method and application

Abstract

The invention provides a preparation method and application of a thrombin-responsive DNA nanomachine, wherein the nanomachine includes a DNA nanostructure, a thrombolytic drug (tPA) complex and a thrombin aptamer chain structure; The capture chain is precisely loaded with tPA molecular drugs, and the controllable DNA molecule "lock" chain is used. Adding the third thrombin aptamer chain can "lock" the DNA nanostructure, so that the tPA drug is entrapped in the DNA nanostructure . The DNA nanomachine disclosed in the present invention can be used as a nanoscale molecular machine for loading tPA thrombolytic drugs, has high thrombin responsiveness, can effectively respond to the thrombin environment at the thrombus site, and accurately releases thrombin to achieve thrombolysis. The treatment is effectively transported to the thrombus site, and it is a new type of thrombolytic machine with precise drug loading, controlled release and high thrombolytic effect.

Description

technical field

[0001] The invention belongs to the technical field of nanomedicine, and in particular relates to a thrombin-responsive DNA nanomachine and its preparation method and application, in particular to a thrombin-responsive DNA nanomachine and its preparation method and application. Background technique

[0002] Currently, the standard therapy for the treatment of ischemic stroke and other thrombosis-related diseases in the clinic is intravenous injection of tissue plasminogen activator tPA within 3 to 4.5 hours after the onset of symptoms. tPA can catalyze the conversion of plasminogen into plasmin, and after intravenous injection of tPA, it will rapidly decompose the fibrin network to achieve the purpose of thrombolysis. However, the current use of tPA therapy in thrombotic patients still has the disadvantages of high risk of bleeding complications, short cycle life, and low targeting. Therefore, the precise delivery of tPA to the thrombus site and the on-deman...

Examples

Embodiment 1

[0049] The preparation method of the DNA nanomachine of embodiment 1 thrombin response

[0050] In this embodiment, the DNA nanomachine preparation method comprises the following steps:

[0051] Step 1: Preparation of rectangular flat DNA nanostructures

[0052] The raw materials for preparation include: M13mp18 template strands shown in SEQ ID NO:1 and short DNA strands, the short DNA strands include: DNA staple strands with 156 sequences, 24 DNAs with sequences shown in SEQ ID NO:3-26 Capture strand, 12 kinds of DNA "lock" strands with sequences shown in SEQ ID NO:28-39, the above DNA strands were mixed in 1×TAE / Mg 2+ In the buffer, the total volume after mixing is 100 μL, the template strand concentration is 10 nM, the short DNA strands of each sequence are added in equal amounts and in excess, and the total concentration of short DNA strands is 80 nM;

[0053] Among them, the DNA staple strands of 156 sequences are selected from the "Supporting information" of the docume...

Embodiment 2

[0062] Example 2: Regulation of DNA nanomachines in response to thrombin environments in different concentration ranges

[0063]The DAN "lock" chain used in Example 1 is a sequence that is not completely complementary to the thrombin aptamer chain, and is a DAN "lock" chain that mismatches two bases. By modifying the DNA "lock" chain The number of matching bases can change its stability when it is complementary to the thrombin aptamer chain, thereby regulating the DNA nanomachine response to the thrombin concentration in different concentration ranges; it is confirmed by experiments that corresponding to Example 1, it is completely complementary and only The DAN "lock" chain with a mismatch of 1 base can also realize the response of DNA nanomachines to thrombin, and the mismatch of 1 base is the sixth in the repeated sequence 5'-AAAACACCAACC-3' in the "lock" chain A base is replaced by a T base; no mismatch base, that is, the sixth A base of the repeat sequence 5'-AAAACACCAACC...

experiment example 1

[0064] Experimental Example 1: Evaluation of Thrombus Targeting Effect of Tubular DNA Nanomachines

[0065] A certain dose of Cy5.5 fluorescent labeling, according to the tubular DNA nanomachines constructed under the condition of DNA "lock" strand mismatch of 2 bases as described in Example 1, was injected into C57 / BL6 mice through the tail vein After 20 min, the lung tissue was isolated and observed by IVIS in vivo fluorescence imaging to evaluate the thrombus targeting effect of the tubular DNA nanomachine. The result is as image 3 As shown, compared with the control group, that is, Cy5.5-labeled single-stranded DNA and fluorescently-labeled rectangular flat DNA nanostructures, the ratio of Cy5.5 / FITC fluorescence intensity in the tubular DNA nanomachine treatment group was higher than that in the rectangular flat DNA nanostructure treatment group. More than 2.1 times, it shows that the tubular DNA nanomachine provided by the present invention can protect the drug as a dr...

technical field

[0001] The invention belongs to the technical field of nanomedicine, and in particular relates to a thrombin-responsive DNA nanomachine and its preparation method and application, in particular to a thrombin-responsive DNA nanomachine and its preparation method and application. Background technique

[0002] Currently, the standard therapy for the treatment of ischemic stroke and other thrombosis-related diseases in the clinic is intravenous injection of tissue plasminogen activator tPA within 3 to 4.5 hours after the onset of symptoms. tPA can catalyze the conversion of plasminogen into plasmin, and after intravenous injection of tPA, it will rapidly decompose the fibrin network to achieve the purpose of thrombolysis. However, the current use of tPA therapy in thrombotic patients still has the disadvantages of high risk of bleeding complications, short cycle life, and low targeting. Therefore, the precise delivery of tPA to the thrombus site and the on-deman...

Examples

Embodiment 1

[0049] The preparation method of the DNA nanomachine of embodiment 1 thrombin response

[0050] In this embodiment, the DNA nanomachine preparation method comprises the following steps:

[0051] Step 1: Preparation of rectangular flat DNA nanostructures

[0052] The raw materials for preparation include: M13mp18 template strands shown in SEQ ID NO:1 and short DNA strands, the short DNA strands include: DNA staple strands with 156 sequences, 24 DNAs with sequences shown in SEQ ID NO:3-26 Capture strand, 12 kinds of DNA "lock" strands with sequences shown in SEQ ID NO:28-39, the above DNA strands were mixed in 1×TAE / Mg 2+ In the buffer, the total volume after mixing is 100 μL, the template strand concentration is 10 nM, the short DNA strands of each sequence are added in equal amounts and in excess, and the total concentration of short DNA strands is 80 nM;

[0053] Among them, the DNA staple strands of 156 sequences are selected from the "Supporting information" of the docume...

Embodiment 2

[0062] Example 2: Regulation of DNA nanomachines in response to thrombin environments in different concentration ranges

[0063]The DAN "lock" chain used in Example 1 is a sequence that is not completely complementary to the thrombin aptamer chain, and is a DAN "lock" chain that mismatches two bases. By modifying the DNA "lock" chain The number of matching bases can change its stability when it is complementary to the thrombin aptamer chain, thereby regulating the DNA nanomachine response to the thrombin concentration in different concentration ranges; it is confirmed by experiments that corresponding to Example 1, it is completely complementary and only The DAN "lock" chain with a mismatch of 1 base can also realize the response of DNA nanomachines to thrombin, and the mismatch of 1 base is the sixth in the repeated sequence 5'-AAAACACCAACC-3' in the "lock" chain A base is replaced by a T base; no mismatch base, that is, the sixth A base of the repeat sequence 5'-AAAACACCAACC...

experiment example 1

[0064] Experimental Example 1: Evaluation of Thrombus Targeting Effect of Tubular DNA Nanomachines

[0065] A certain dose of Cy5.5 fluorescent labeling, according to the tubular DNA nanomachines constructed under the condition of DNA "lock" strand mismatch of 2 bases as described in Example 1, was injected into C57 / BL6 mice through the tail vein After 20 min, the lung tissue was isolated and observed by IVIS in vivo fluorescence imaging to evaluate the thrombus targeting effect of the tubular DNA nanomachine. The result is as image 3 As shown, compared with the control group, that is, Cy5.5-labeled single-stranded DNA and fluorescently-labeled rectangular flat DNA nanostructures, the ratio of Cy5.5 / FITC fluorescence intensity in the tubular DNA nanomachine treatment group was higher than that in the rectangular flat DNA nanostructure treatment group. More than 2.1 times, it shows that the tubular DNA nanomachine provided by the present invention can protect the drug as a dr...

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