CN112451666B - Nucleic acid-drug-loaded nano material for improving tumor permeability by VEGF response and preparation method and application thereof - Google Patents
Nucleic acid-drug-loaded nano material for improving tumor permeability by VEGF response and preparation method and application thereof Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a nucleic acid-drug-loaded nano material for improving tumor permeability by VEGF response, which comprises a drug-loaded nano particle inner core and a nucleic acid shell, wherein the nucleic acid shell comprises DNA1、DNA2、DNA3And DNA4，DNA3Contains a V7T1 aptamer specifically recognizing VEGF protein. Due to DNA3Contains VEGF aptamer, can specifically identify VEGF protein over-expressed in tumor microenvironment, and makes DNA3And DNA4Falling off from the nucleic acid-carrying nanoparticles, DNA2/DNA1The modified albumin drug-loaded nanoparticles have small particle size and good tumor permeability; and the whole nucleic acid-drug-loaded nano material mainly comprises nucleic acid and protein, and has good biocompatibility. The invention also discloses a preparation method and application of the nucleic acid-drug-loaded nano material, and the preparation method is simple to operate and low in cost.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a nucleic acid-albumin nano drug-loaded material for improving tumor permeability by VEGF response, and a preparation method and application thereof.
Background
The target drug delivery nano material is widely applied to the treatment of solid tumors so as to improve the accuracy of cancer diagnosis and the effectiveness of treatment. Generally, the 50-100 nm nanometer material can prolong blood circulation and effectively gather and stay at a tumor tissue part (EPR effect) through passive targeting. However, solid tumor microenvironments also include dense extracellular matrix, high cell packing density, increased interstitial pressure and slow interstitial flow rate. These factors result in the difficulty of penetration of the large-sized nanomaterial into the deep tissues of the tumor, thereby significantly reducing the therapeutic effect of solid tumors. Conversely, small size nanomaterials (< 20nm) can diffuse into the deep layers of the tumor, but are easily cleared by the liver and kidney and do not effectively aggregate at the tumor site.
Therefore, the nano drug delivery system with micro-environment response is developed, the nano drug delivery system keeps larger particle size before entering a tumor part, and the particle size can be reduced after being accumulated to the tumor part, so that the penetration of the tumor part is enhanced, the tumor effect is improved, and the nano drug delivery system has very important significance to the field.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects and defects in the background technology, providing a nucleic acid-albumin nano drug-loaded material which keeps larger particle size before entering a tumor part and can reduce the particle size after accumulating to the tumor part so as to respond to VEGF (vascular endothelial growth factor) and improve the tumor permeability, and a preparation method and application thereof.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a nucleic acid-medicine carrying nano material for improving tumor permeability by VEGF response comprises medicine carrying nano particlesAn inner core and a nucleic acid outer shell comprising DNA1、DNA2、DNA3And DNA4The DNA1The 5' end of (A) is modified with carboxyl, the DNA1A part of bases of 5' end of (3) and DNA2Part of bases at the 3' end of (3) are bound to and incorporated into DNA1Are paired in the 3' direction to form a double strand, and the DNA2The remaining part of the bases of the 5' end of (3) and DNA3Part of bases at the 3' end of (3) are bound to and incorporated into DNA2The 3' end directions of the two strands are paired to form a double strand; the DNA3A part of bases of 5' end of (3) and DNA4Part of bases at the 3' end of (3) are bound to and incorporated into DNA3The 3' end directions of the two strands are paired to form a double strand; the DNA4The remaining part of bases of the 5' end of (A) and the next DNA3Part of bases at the 3' end of (3) are bound to and incorporated into DNA4Are paired in the 3' direction to form a double strand, and the DNA3Contains a V7T1 aptamer specifically recognizing VEGF protein, and the nucleotide sequence of the V7T1 aptamer is 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3' (shown as SEQ ID NO: 1).
Preferably, the drug-loaded nanoparticles are any one or more of albumin nanoparticles, hemoglobin nanoparticles and lactoferrin nanoparticles; compared with lactoferrin and hemoglobin, the albumin nanoparticles have lower cost.
Preferably, the DNA15'-TTTGGGTTAGGGTTAGGGTTAGCG-3' (shown as SEQ ID NO: 2), the DNA25'-CACATTTTTTTTCCCTAACCCTAACCCTAACCC-3' (shown as SEQ ID NO: 3), the DNA35'-AAAAAAAATGTGGGGGTGGACGGGCCGGGTAGAAAAAAAA-3' (shown as SEQ ID NO: 4), the DNA4Is 5'-CACATTTTTTTTTTTTTTTTCTAC-3' (shown as SEQ ID NO: 5). Compared with the nanoparticle system constructed by RNA, the nanoparticle system constructed by DNA has lower cost.
DNA1The carboxyl modified by the 5' end can be connected with the drug-loaded nanoparticle inner core, and DNA1、DNA2、DNA3And DNA4The four nucleic acid single strands form double strands in pairs according to the base complementary pairing principle. DNA1The fourth of 5' end of (3)Single base and DNA2The first base at the 3' end of (3) is bound to and incorporated into DNA1The 3' end of the base pair form 21 pairs of bases; DNA2The first base of the 5' end of (3) and DNA3Binds to the twenty-ninth base at the 3' end of the DNA and adds to the DNA2The 3' end of the base pair is paired to form 12 pairs of bases; DNA3Twenty-ninth base and DNA of 5' end4The first base at the 3' end of (A) is bound to and added to DNA3The 3' end of the base pair is paired to form 12 pairs of bases; DNA4The first base of the 5' end of (A) and the next DNA3Binds to the twenty-ninth base at the 3' end of the DNA and adds to the DNA4The 3' -terminal pair of (A) form 12 bases in total.
Preferably, the particle size of the nucleic acid-drug-loaded nano material is 70-80 nm.
Due to their good sequence programmability, nucleic acid-based composite nanomaterials have been widely used as tumor therapeutics. The albumin nanoparticle surface contains a large amount of amino and carboxyl functional groups, and nucleic acid can be surface modified. The albumin nanoparticles have good biocompatibility and biodegradability, have the advantages of high in-vivo and storage stability, no antigen and the like, and are a good drug carrier. In particular, the albumin nanoparticles can encapsulate a photosensitizer to obtain a nano drug delivery system with the size less than 20nm, and the tumor penetration is greatly enhanced. And the number of outer-layer nucleic acid layers of the albumin drug-loaded nanoparticles is increased through base complementary pairing, so that the size of the nano drug-loaded system can be increased.
VEGF is a multifunctional angiogenic cytokine expressed and secreted at high levels by tumors. In a normal tumor microenvironment, the secretion level of VEGF has obvious difference, and the VEGF can be used as an effective tumor microenvironment response target. The V7T1 aptamer is a DNA single strand capable of specifically recognizing VEGF protein, and has a sequence 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3' (shown as SEQ ID NO: 1). When a large amount of VEGF protein exists, the VEGF protein can be folded into a unique G-quadruplex structure to be combined with the VEGF protein, and the VEGF responsiveness is good. A plurality of layers of nucleic acid single chains containing V7T1 aptamer are connected outside the albumin drug-loaded nanoparticle through base complementary pairing to obtain a large-size drug-loaded nanoparticle material, which can be effectively accumulated on a tumor part. Then, under the stimulation of VEGF in a tumor microenvironment, the V7T1 aptamer in the system can recognize VEGF protein and fall off from the albumin drug-loaded nanoparticles, so that the purpose of reducing the particle size from large to small is achieved, and the tumor penetration is increased, thereby achieving the optimal solid tumor treatment effect. Therefore, the nucleic acid-albumin nano drug-loaded material for improving tumor permeability by VEGF response has great significance and value.
Based on a general inventive concept, the invention also provides a preparation method of the nucleic acid-drug-loaded nano material for improving tumor permeability by VEGF response, which comprises the following steps:
(1) encapsulating the carboxyl-containing hydrophobic anti-tumor drug into drug-loaded nanoparticles through amidation reaction and hydrophobic interaction to obtain drug-loaded nanoparticles encapsulating the anti-tumor drug;
(2) connecting carboxyl modified DNA in the drug-loaded nanoparticle solution obtained after the step (1) through amidation reaction1Obtaining DNA1Modified drug-loaded nanoparticle solution;
(3) DNA obtained after said step (2)1DNA is connected in the modified drug-carrying nano-particle solution by the base complementary pairing principle2Obtaining DNA2/DNA1Modified drug-loaded nanoparticle solution;
(4) DNA obtained after said step (3)2/DNA1Adding DNA into the modified drug-loaded nanoparticle solution3And DNA4And (3) incubating, and finally performing ultrafiltration and purification to obtain the nucleic acid-drug-loaded nano material.
In the preparation method, preferably, the drug-loaded nanoparticles are albumin nanoparticles, and the specific operation of the step (1) comprises the following steps: dissolving a hydrophobic antitumor drug containing carboxyl in dimethyl sulfoxide, adding a carboxyl activating agent, stirring for reaction, then adding the mixture into an albumin PBS buffer solution, stirring for reaction, and performing centrifugal ultrafiltration to obtain the drug-loaded nanoparticles coated with the antitumor drug.
More preferably, the anti-tumor drug is photosensitizer chlorin e6, and the photosensitizer chlorin e6 contains carboxyl and hydrophobic property; the albumin is human serum albumin, bovine serum albumin or ovalbumin; the carboxyl activating agents include NHS and edc.
More preferably, the specific operation of step (2) comprises the following steps: DNA modified at the 5' end by carboxyl groups1(5’-COOH-DNA1) Adding a carboxyl activating agent into the aqueous solution, stirring for reaction, adding the drug-loaded nanoparticles obtained in the step (1), continuing stirring for reaction at 15-30 ℃ for 4-6h, and performing ultrafiltration purification to obtain DNA1Modified drug-loaded nanoparticle solution; the carboxyl activating agents include NHS and edc.
More preferably, the specific operation of step (3) includes the following steps: DNA obtained after said step (2)1Adding DNA into the modified drug-loaded nanoparticle solution2Incubating at 35-37 deg.C, ultrafiltering and purifying to obtain DNA2/DNA1Modified drug-loaded nanoparticle solution; the DNA2/DNA1The particle size of the modified drug-loaded nanoparticles is 10-20 nm.
More preferably, the specific operation of step (4) includes the following steps: subjecting the DNA obtained after the step (3) to2/DNA1Adding DNA into the modified drug-loaded nanoparticle solution3And DNA4Wherein the DNA2、DNA3And DNA4The molar ratio of the nucleic acid to the drug-loaded nano-material is 1:4-6:4-6, incubation is carried out at the temperature of 35-37 ℃, and the nucleic acid-drug-loaded nano-material is obtained after ultrafiltration and purification; the particle size of the nucleic acid-drug-loaded nano material is 70-80 nm.
Based on a general inventive concept, the invention also provides an application of the nucleic acid-drug-loaded nano material in preparation of targeted antitumor drugs.
In the above application, preferably, the targeted antitumor drug is composed of an albumin nanoparticle inner core and a nucleic acid shell, wherein the albumin nanoparticle inner core is loaded with a photosensitizer chlorin e6, and the nucleic acid shell is composed of DNA1、DNA2、DNA3And DNA4Composition of, the DNA3Contains a V7T1 aptamer specifically recognizing VEGF protein, and the nucleotide sequence of the V7T1 aptamer is 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3' (shown as SEQ ID NO: 1).
Compared with the prior art, the invention has the beneficial effects that:
1. the nucleic acid-drug-loaded nano material can respond to VEGF protein secreted by a high level in a tumor microenvironment to realize size reduction and improve tumor permeability, mainly because DNA3Contains VEGF nucleic acid aptamer sequence, can specifically identify VEGF protein over-expressed in tumor microenvironment, and makes DNA3And DNA4Falling off from the nucleic acid-drug-loaded nanoparticles to obtain DNA2/DNA1Modified drug-loaded nanoparticles, DNA2/DNA1The modified albumin drug-loaded nanoparticles have small particle size and good tumor permeability; and the whole nucleic acid-drug-loaded nano material mainly comprises nucleic acid and protein, and has good biocompatibility.
2. The preparation method of the invention has simple operation and low cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is BCD in example 11,2An atomic AFM map of (a);
FIG. 2 is BCD in example 11,2,3,4An AFM map of (a);
FIG. 3 is BCD of example 11,2，BCD1,2,3,4And control product BCD1,2,3’,4Distribution in a549 tumor sphere.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1:
a nucleic acid-albumin drug-loaded nano material with VEGF response for improving tumor permeability comprises an albumin drug-loaded nano particle inner core and a nucleic acid outer shell, wherein the nucleic acid outer shell comprises DNA1、DNA2、DNA3And DNA4The DNA15'-TTTGGGTTAGGGTTAGGGTTAGCG-3' (shown as SEQ ID NO: 2), the DNA25'-CACATTTTTTTTCCCTAACCCTAACCCTAACCC-3' (shown as SEQ ID NO: 3), the DNA35'-AAAAAAAATGTGGGGGTGGACGGGCCGGGTAGAAAAAAAA-3' (shown as SEQ ID NO: 4), the DNA45'-CACATTTTTTTTTTTTTTTTCTAC-3' (shown as SEQ ID NO: 5), wherein the DNA3Contains a V7T1 aptamer specifically recognizing VEGF protein, and the nucleotide sequence of the V7T1 aptamer is 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3' (shown as SEQ ID NO: 1).
The preparation method of the nucleic acid-drug-loaded nanomaterial specifically comprises the following steps:
(1) preparation of albumin drug-loaded nanoparticles (BC): dissolving 0.8mg of photosensitizer chlorin e6(Ce6), 0.18mg of NHS and 0.28mg of EDC.HCl in 200 mu L of dimethyl sulfoxide, taking the NHS and the EDC.HCl as Ce6 carboxyl activators, and stirring at room temperature for 2 hours; adding the activated Ce6 solution into 10mL of 2mg/mL bovine serum albumin PBS buffer solution (pH 7.4), stirring overnight at room temperature, then centrifuging at 12000rpm for 8min, discarding the precipitate, and then performing ultrafiltration centrifugal purification with the molecular weight cutoff of 10kDa to obtain the albumin drug-loaded nanoparticle solution. It is conceivable that: wherein the bovine serum albumin can be replaced by albumin of human serum albumin, ovalbumin and other similar substances.
(2) Preparation of DNA1Modified albumin drug-loaded nanoparticles (BCD)1): take 8mg NHS and 10mg EDCL was dissolved in 200. mu. L, pH of 6.0 MES buffer and 30. mu.L of 100. mu.M 5' -carboxyl-modified DNA was added1(5’-COOH-DNA1) Stirring the aqueous solution at room temperature for 30 min; adding PBS (pH 7.4) to adjust pH to 7.2, adding 30 μ L of 25mg/mL albumin drug-loaded nanoparticle solution, stirring at room temperature for 4h, and performing ultrafiltration centrifugal purification with molecular weight cutoff of 10kDa to obtain DNA1Modified albumin drug-loaded nanoparticle solution.
(3) Preparation of DNA2/DNA1Modified albumin drug-loaded nanoparticles (BCD)1,2): 1OD Single-stranded DNA2Dissolving in 31 μ L PBS (pH 7.4) buffer solution, denaturing at 95 deg.C for 10min, and placing on ice for 10 min; taking 10 mu LDNA2And DNA1Incubating the modified albumin drug-loaded nanoparticle solution for 2h at 37 ℃, and performing ultrafiltration centrifugal purification by using the molecular weight cutoff of 50kDa to obtain DNA2/DNA1Modified albumin drug-loaded nanoparticle solution (DNA, shown in FIG. 1)2/DNA1The particle size of the modified albumin drug-loaded nanoparticles is 10-20 nm).
(4) Preparation of VEGF-responsive nucleic acid-albumin drug-loaded nanomaterial (BCD) for improving tumor permeability1,2,3,4): separately, 1OD single-stranded DNAs3(containing a V7T1 aptamer specifically recognizing VEGF protein, the nucleotide sequence of the V7T1 aptamer is shown as SEQ ID NO: 1) and DNA4Dissolving in 19.7 μ L and 44.7 μ L PBS (pH 7.4) buffer solution, denaturing at 95 deg.C for 10min, and placing on ice for 10 min; adding to DNA2/DNA1Modified albumin drug-loaded nanoparticle solution, DNA2、DNA3And DNA4Is 1:5:5, incubated for 2h at 37 ℃, and subjected to ultrafiltration centrifugal purification with molecular weight cutoff of 100kDa to obtain a target product DNA1,2,3,4Modified albumin nano drug-loaded material (shown in figure 2, DNA)1,2,3,4The particle size of the modified albumin nano drug-loaded material is 70-80 nm).
(5) Preparation of non-VEGF-responsive nucleic acid-albumin Nanocarrier System (BCD)1,2,3’,4): separately, 1OD single-stranded DNAs3’(containing VEGF aptamer control sequence) and DNA4Dissolving in 19.7. mu.L and 44.7. mu.L PBS (pH 7.4) buffer solution, denaturing at 95 deg.C for 10min, and placing on ice10 min; adding to DNA2/DNA1Modified albumin drug-loaded nanoparticle solution, DNA2、DNA3’And DNA4Is 1:5:5, incubated at 37 ℃ for 2h, and subjected to ultrafiltration centrifugal purification with molecular weight cutoff of 100kDa to obtain a control product DNA1,2,3’,4Modified nucleic acid-albumin drug-loaded nanomaterial;
DNA3’5'-AAAAAAAATGTGGGGTTGGACGGGCCGTGTAGAAAAAAAA-3' (shown in SEQ ID NO: 6), which contains VEGF aptamer control sequence 5'-TGTGGGGTTGGACGGGCCGTGTAGA-3' (shown in SEQ ID NO: 7), and does not specifically bind VEGF protein.
In order to verify the nucleic acid-albumin drug-loaded nano material BCD1,2、BCD1,2,3,4And BCD1,2,3’,4The effect of (1) in this example, the in vitro tumor sphere experiment was performed to investigate the tumor penetration ability of several nucleic acid-albumin nano drug-loaded materials, which specifically includes the following steps:
(1) constructing an in vitro tumor sphere model: pre-paving 2% agarose gel on a 96-hole cell culture plate to obtain a low-adsorption 96-hole cell culture plate; inoculating A549 cells into a 96-well cell culture plate with low adsorption at a cell density of 3000/well, wherein the volume of cell suspension added to each well is 200 mu L; the inoculated cells were incubated at 37 ℃ with 5% CO2The culture is carried out in the incubator, and semiquantitative liquid change is carried out every 3 days; after 7 days of cell inoculation, the formation condition of the tumor spheres is observed under a microscope, and the tumor spheres with round and compact shapes and uniform sizes are selected for experiments.
(2) The research on the tumor penetration capability of the nucleic acid-albumin nano drug delivery system comprises the following steps: adding BCD into the wells containing tumor balls respectively1,2、BCD1,2,3,4And BCD1,2,3’,4Wherein the concentration of Ce6 is 1 μ M, placing into an incubator to continue culturing for 4 h; after incubation, the culture medium was discarded, washed 3 times with pre-cooled PBS, and photographed in high content confocal mode, as shown in FIG. 3.
The results show that after 4h incubation, the small size of BCD1,2The vast majority of the groups gather in the inner layer of the tumor sphere, the large size of the BCD containing VEGF aptamer1,2,3,4The group is more evenly dispersed in the tumor ballComparison with BCD containing VEGF aptamer control sequence1,2,3’,4The group can more easily enter the interior of the tumor sphere, which indicates that the nucleic acid-albumin drug-loaded nano material BCD of the invention is prepared1,2,3,4VEGF protein which can respond to the over-expression of a tumor microenvironment becomes small, so that tumor penetration is realized, and the VEGF protein is expected to be further prepared into a targeted antitumor drug.
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Claims (10)
1. The VEGF-responsive nucleic acid-drug-loaded nanomaterial capable of improving tumor permeability is characterized by comprising a drug-loaded nanoparticle inner core and a nucleic acid shell, wherein the nucleic acid shell comprises DNA1、DNA2、DNA3And DNA4The DNA1The 5' end of (A) is modified with carboxyl, the DNA1A part of bases of 5' end of (3) and DNA2Part of bases at the 3' end of (3) are bound to and incorporated into DNA1Are paired in the 3' direction to form a double strand, and the DNA2The remaining part of the bases of the 5' end of (3) and DNA3Part of bases at the 3' end of (3) are bound to and incorporated into DNA2The 3' end directions of the two strands are paired to form a double strand; the DNA3A part of bases of 5' end of (3) and DNA4Part of bases at the 3' end of (3) are bound to and incorporated into DNA3The 3' end directions of the two strands are paired to form a double strand; the DNA4The remaining part of bases of the 5' end of (A) and the next DNA3Part of bases at the 3' end of (3) are bound to and incorporated into DNA4Are paired in the 3' direction to form a double strand, and the DNA3Contains a V7T1 aptamer which specifically recognizes VEGF protein, and the nucleotide sequence of the V7T1 aptamerColumn is 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3'.
2. The nucleic acid-drug-loaded nanomaterial according to claim 1, wherein the drug-loaded nanoparticles are any one or more of albumin nanoparticles, hemoglobin nanoparticles and lactoferrin nanoparticles; the DNA15'-TTTGGGTTAGGGTTAGGGTTAGCG-3', the DNA25'-CACATTTTTTTTCCCTAACCCTAACCCTAACCC-3', the DNA35'-AAAAAAAATGTGGGGGTGGACGGGCCGGGTAGAAAAAAAA-3', the DNA4Is 5'-CACATTTTTTTTTTTTTTTTCTAC-3'.
3. The nucleic acid-drug-loaded nanomaterial according to claim 1 or 2, wherein the particle size of the nucleic acid-drug-loaded nanomaterial is 70-80 nm.
4. A method for preparing a nucleic acid-drug loaded nanomaterial with VEGF enhancing response to tumor permeability according to any one of claims 1 to 3, comprising the steps of:
(1) encapsulating the carboxyl-containing hydrophobic anti-tumor drug into drug-loaded nanoparticles through amidation reaction and hydrophobic interaction to obtain drug-loaded nanoparticles encapsulating the anti-tumor drug;
(2) connecting carboxyl modified DNA in the drug-loaded nanoparticle solution obtained after the step (1) through amidation reaction1Obtaining DNA1Modified drug-loaded nanoparticle solution;
(3) DNA obtained after said step (2)1DNA is connected in the modified drug-carrying nano-particle solution by the base complementary pairing principle2Obtaining DNA2/DNA1Modified drug-loaded nanoparticle solution;
(4) DNA obtained after said step (3)2/DNA1Adding DNA into the modified drug-loaded nanoparticle solution3And DNA4And (3) incubating, and finally performing ultrafiltration and purification to obtain the nucleic acid-drug-loaded nano material.
5. The preparation method according to claim 4, wherein the drug-loaded nanoparticles are albumin nanoparticles, and the specific operation of the step (1) comprises the following steps: dissolving a hydrophobic antitumor drug containing carboxyl in dimethyl sulfoxide, adding a carboxyl activating agent, stirring for reaction, then adding the mixture into an albumin PBS buffer solution, stirring for reaction, and performing centrifugal ultrafiltration to obtain the drug-loaded nanoparticles coated with the antitumor drug.
6. The method according to claim 5, wherein the antitumor drug is a photosensitizer chlorin e 6; the albumin is human serum albumin, bovine serum albumin or ovalbumin; the carboxyl activating agents include NHS and edc.
7. The method for preparing a composite material according to any one of claims 4 to 6, wherein the specific operation of the step (2) comprises the steps of: DNA modified at the 5' end by carboxyl groups1Adding a carboxyl activating agent into the aqueous solution, stirring for reaction, adding the drug-loaded nanoparticles obtained in the step (1), continuously stirring for reaction at 15-30 ℃ for 4-6h, and performing ultrafiltration purification to obtain DNA1Modified drug-loaded nanoparticle solution; the carboxyl activating agents include NHS and edc.
8. The method for preparing a composite material according to any one of claims 4 to 6, wherein the specific operation of the step (3) comprises the steps of: DNA obtained after said step (2)1Adding DNA into the modified drug-loaded nanoparticle solution2Incubating at 35-37 deg.C, ultrafiltering and purifying to obtain DNA2/DNA1Modified drug-loaded nanoparticle solution; the DNA2/DNA1The particle size of the modified drug-loaded nanoparticles is 10-20 nm.
9. The method for preparing a composite material according to any one of claims 4 to 6, wherein the specific operation of the step (4) comprises the steps of: subjecting the DNA obtained after the step (3) to2/DNA1Adding DNA into the modified drug-loaded nanoparticle solution3And DNA4Wherein the DNA2、DNA3And DNA4The molar ratio of the nucleic acid to the drug-loaded nano-material is 1:4-6:4-6, incubation is carried out at the temperature of 35-37 ℃, and the nucleic acid-drug-loaded nano-material is obtained after ultrafiltration and purification; the particle size of the nucleic acid-drug-loaded nano material is 70-80 nm.
10. The application of the nucleic acid-drug-loaded nano material prepared by the preparation method of any one of claims 1-3 or any one of claims 4-9 in preparing targeted antitumor drugs.
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