CN117919157A - Use of tetrahedral framework nucleic acids for preparing vectors for transnasal administration to the brain - Google Patents
Use of tetrahedral framework nucleic acids for preparing vectors for transnasal administration to the brain Download PDFInfo
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Abstract
The invention provides application of tetrahedral framework nucleic acid in preparing a carrier for transnasal administration, and belongs to the field of biomedical materials. The tetrahedral framework nucleic acid is formed by base complementary pairing of nucleotide sequences shown in SEQ ID NO. 1-4. According to the invention, the framework nucleic acid is used as a drug carrier for realizing drug delivery from nose to brain for the first time, and the drug delivery mode can realize noninvasive drug delivery to brain tissues, so that the occurrence rate of invasive drug delivery complications is reduced. The research result of the invention shows that the tetrahedral framework nucleic acid can still maintain the complete tetrahedral structure after entering the brain through the nose, and the tetrahedral framework nucleic acid administration system can also protect the integrity of the medicine after carrying the medicine, thereby reducing the loss of the medicine before entering brain tissues. Because of the way of transnasal administration, non-invasive administration can be realized, the compliance of patients is high, and the method has obvious advantages in treating chronic diseases in nerve and mental systems due to non-invasive operation.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to application of tetrahedral framework nucleic acid in preparation of a carrier for transnasal administration.
Background
Administration of brain diseases presents a number of pain points and challenges, particularly for the delivery of drugs to the central nervous system. With the continuing progress in clinical therapy in recent years, how to efficiently and accurately deliver drugs into brain tissue has become a central subject of interest to many researchers. Currently, the process of treating brain diseases is often significantly limited by the blood brain barrier. The blood brain barrier is a highly selective barrier, which serves mainly to protect the brain and ensure that harmful substances cannot enter, but this also causes the problem that most drugs have difficulty reaching the brain. The blood brain barrier is effective in preventing most drugs from entering the brain. This presents a great challenge for drug administration. To bypass the blood brain barrier, researchers have developed methods of administering drugs directly to the brain or cerebrospinal fluid, such as epidural injections and spinal injections. While these methods are capable of delivering drugs directly into the brain or cerebrospinal fluid, their use is still limited due to their invasive nature, possible complications and patient acceptance.
Nasal administration has received extensive attention and research over the last decades as a novel form of administration. The principle of this method is relatively simple: the drug is delivered directly to the brain through the nasal cavity, using its specific link with the brain. Notably, nasal administration has significant advantages over other methods. For example, there is a direct neurological connection of the olfactory epithelium in the nasal cavity to the brain, which allows the drug to enter the brain efficiently via the "nose-brain" route. In addition, the method is simple, convenient, painless and low in risk, and can bypass the first pass effect of the liver, so that the rapid absorption and continuous effect of the medicine are ensured. Although nasal administration presents great therapeutic and application potential, it presents a number of challenges in practical use. For example, current research has mainly employed various types of nano-delivery systems, such as liposomes, polypeptides, and high molecular polymers, to enhance the bioavailability of drugs. However, these systems also have problems such as insufficient stability and uncontrollable drug release rates.
Recent studies began to investigate tetrahedral framework nucleic acids as delivery vehicles for drugs. The novel carrier is formed by independently assembling four nucleic acid single chains through specific base pairing, has excellent biocompatibility and stability, can be custom-designed, and loads different drug molecules according to requirements. However, it is not known whether tetrahedral framework nucleic acids can achieve stable nasal administration, ensuring stable drug release.
Disclosure of Invention
The object of the present invention is to provide the use of tetrahedral framework nucleic acids for the preparation of vectors for nasal administration into the brain.
The present invention provides the use of a tetrahedral framework nucleic acid formed by base complementary pairing of the nucleotide sequences shown in SEQ ID NO.1 to 4 for the preparation of a vector for nasal administration into the brain.
Further, the preparation method of the tetrahedral framework nucleic acid comprises the following steps: after dissolving four DNA single strands in TM buffer solution in equimolar manner, the mixture is maintained at 85-105 ℃ for 5-15min, and then maintained at 2-8 ℃ for 10-30min.
Further, after four DNA single strands were equimolar dissolved in TM buffer, they were maintained at 95℃for 10min and then at 4℃for 20min.
Further, the concentration of Tris-HCl in the TM buffer is 10mM, the concentration of MgCl 2 is 50mM, and the pH is 8.0; the concentration of the four DNA single strands was 1. Mu.M.
Further, the carrier for transnasal administration is a carrier for transnasal administration through the olfactory bulb and trigeminal nerve.
The invention also provides application of the tetrahedral framework nucleic acid in preparing medicines for transnasal administration, wherein the medicines are obtained by taking tetrahedral framework nucleic acid formed by base complementary pairing of nucleotide sequences shown in SEQ ID NO. 1-4 as a carrier and carrying the medicines.
Further, the preparation method of the tetrahedral framework nucleic acid comprises the following steps: after four DNA single strands are equimolar dissolved in TM buffer, the mixture is maintained for 5 to 15 minutes at the temperature of between 85 and 105 ℃ and then maintained for 10 to 30 minutes at the temperature of between 2 and 8 ℃;
preferably, after equimolar dissolution of the four DNA strands in TM buffer, they are maintained at 95℃for 10min and then at 4℃for 20min.
Further, the concentration of Tris-HCl in the TM buffer is 10mM, the concentration of MgCl 2 is 50mM, and the pH is 8.0; the concentration of the four DNA single strands was 1. Mu.M.
Further, the medicament is a medicament for treating brain diseases.
Further, the medicine is a medicine for treating sepsis encephalitis, parkinson's disease, alzheimer's disease, cerebral ischemia and epilepsy.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention uses nucleic acid as a small molecule drug carrier for research of nasal administration to brain for the first time, can be used for treating brain diseases including but not limited to Parkinson's disease, alzheimer's disease, cerebral ischemia, epilepsy and the like, widens the research of the brain tissue administration field, and provides a new thought.
(2) The drug carrying system only uses nucleic acid and other effective drugs as raw materials, has low synthesis cost and high biological safety, and in vivo and in vitro experiments prove that the pharmacological action of the drug carrying system provides a feasible solution for treating brain tissue diseases.
(3) Reducing systemic side effects: the direct delivery of the drug to the brain can reduce its side effects on other body parts, thereby improving the safety of the treatment.
(4) Acute treatment and rescue: because nasal administration allows for rapid administration, it has potential in the emergency treatment of acute brain injury or stroke. Meanwhile, because the intranasal administration has small wound and high patient compliance, the chronic disease can be managed. In addition, it can also transport neurotrophic substances to prevent brain diseases.
(5) The drug delivery system provided by the invention has the earliest verification that the frame nucleic acid is taken as the drug delivery system for nasal administration, and because the tetrahedron has very good editability, the frame nucleic acid delivery system can be used for carrying various effective drugs (such as polypeptide or RNA molecules), and the accurate targeting can be realized by adding the aptamer, so that the treatment of various brain tissue diseases is realized.
The invention takes the framework nucleic acid as a drug carrier to realize the drug delivery from nose to brain for the first time, no study exists at present on using the framework nucleic acid or the framework nucleic acid carrier for the drug delivery from nose to brain, and the drug delivery mode can bypass the obstruction of the blood brain barrier to realize the brain tissue delivery of the drug. The nasal-to-brain framed nucleic acid drug delivery route allows for noninvasive delivery of drugs to brain tissue, reducing the incidence of invasive drug delivery complications. The research result of the invention shows that the tetrahedral framework nucleic acid can still maintain the complete tetrahedral structure after entering the brain through nose, and the tetrahedral framework nucleic acid administration system can also protect the integrity of the medicine after carrying the medicine (such as polypeptide) and reduce the loss of the medicine before entering brain tissue. Because of the way of transnasal administration, non-invasive administration can be realized, the compliance of patients is high, and the method has obvious advantages in treating chronic diseases in nerve and mental systems due to non-invasive operation.
The invention utilizes the advantages of the size, biological safety, noninvasive administration and the like of the tetrahedral framework nucleic acid, evaluates the brain targeting potential of the tetrahedral framework nucleic acid through living body imaging and confocal cutting, and explores the main way of entering the brain through the nose. The tetrahedron framework nucleic acid has the characteristics of wide nasal cavity distribution, strong nasal mucosa permeability, strong nasal adhesion capability, high biological safety and the like. The tetrahedron framework nucleic acid is used for carrying small molecular polypeptide, and administration is carried out from nose to brain, so that the treatment of the sepsis encephalitis mice is realized, and the bioavailability value of the administration system is verified. The method provides an innovative scheme for nasal administration and is expected to be widely popularized and applied in future clinical application.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 shows the result of a tFNA PAGE gel electrophoresis.
FIG. 2 shows the transmission electron microscope results of tFNA.
FIG. 3 shows the dynamic light scattering results for tFNA: the left plot shows the Zeta potential of tFNA and the right plot shows the particle size of tFNA.
FIG. 4 shows the results of tFNA atomic force microscopy.
FIG. 5 is a graph showing the distribution of tFNA with Cy5 fluorescence into brain tissue 2 hours after drug instillation.
FIG. 6 is a view of the results of in vivo imaging system with and without cutting the olfactory nerve of the mice.
FIG. 7 shows immunofluorescent staining results of olfactory bulb tissue with and without cutting the olfactory nerve of mice.
Fig. 8 is a diagram showing the transmission of a tetrahedron along a trigeminal neuron by immunofluorescence co-localization of the trigeminal nerve.
FIG. 9 is a view of the results of observation of a nasal cavity Bio-Rad imaging system for tFNA drops into the nasal cavity of a mouse at different times.
FIG. 10 shows the result of tFNA penetration of artificial mucus.
FIG. 11 is the integrity results after tFNA penetration of artificial mucus: the left panel shows tFNA integrity results from agarose gel electrophoresis and the right panel shows fluorescence intensity statistics.
Fig. 12 shows tFNA transdermal efficiency test results: the left panel shows nasal mucosa results and the right panel shows dialysis membrane results.
FIG. 13 shows immunofluorescent staining of brain tissue after 1, 2, 3, 6, and 9 hours of drug instillation.
FIG. 14 shows the effect of different concentrations tFNA on cell activity.
Fig. 15 is a section of HE nasal mucosa showing tFNA nasal delivery safety.
FIG. 16 shows the results of HE staining of the vital organs.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
Example 1 Synthesis and characterization of tetrahedral framework nucleic acids
1. Synthesis of tetrahedral framework nucleic acids
Four DNA strands (S1, S2, S3, S4) shown in Table 1 were added to a TM buffer (10 mM Tris-HCl and 50mM MgCl 2 in water at an equimolar ratio and the pH was adjusted to 8.0 with hydrochloric acid) and the final concentration of the four DNA strands was 1. Mu.M, heated at 95℃for 10 minutes to allow the DNA strands to spread and reform into a tetrahedron structure, and then rapidly cooled to 4℃and maintained for 20 minutes to allow the DNA to recombine, giving a tetrahedron-framed nucleic acid designated tFNA.
TABLE 1 sequence of four DNA single strands
2. Characterization of tetrahedral framework nucleic acids
The morphology information of the tetrahedral framework nucleic acid is obtained through PAGE gel electrophoresis, transmission electron microscopy, dynamic light scattering and atomic force microscopy.
(1) PAGE gel electrophoresis: the successful synthesis was checked by PAGE tFNA. After the glass plate was leak-tested, the 8% PAGE gel solution was added to the loading well and the gel was allowed to solidify after insertion into a comb. tFNA and a loading buffer solution are added into a loading tank after being uniformly mixed, and electrophoresis is completed in a 1x TAE solution environment. The Gel was stained in a dye solution of 1x Gel Red for 20min and finally the nucleic acid Gel was developed under a chemiluminescent imager. FIG. 1 shows the result of a tFNA PAGE gel electrophoresis, which demonstrates the successful synthesis of tetrahedra.
(2) Transmission electron microscope: after 1. Mu.M tFNA was diluted to 100nM with TM buffer, a small sample was slowly dropped onto the copper mesh and counterstained for 3min, the morphology of tFNA was determined by transmission electron microscopy. FIG. 2 is a transmission electron microscope result of tFNA, which illustrates that tetrahedra are uniformly distributed in a medium.
(3) Dynamic light scattering: the synthesized tFNA was diluted to 125nM with TM buffer, 1mL of the sample was added to a potentiometric dish and a particle size dish, respectively, and both were placed in a potentiometric particle sizer for detection and analysis of the data. FIG. 3 shows the dynamic light scattering results of tFNA, which illustrate that the tetrahedra have a particle size of 11.61nm and are negatively charged.
(4) Atomic force microscope: after diluting the synthesized tFNA with TM buffer to 10nM, 20. Mu.L of the sample was removed onto freshly cut mica plates. The droplets were left on the mica surface for 15 minutes of adsorption. The morphology of tFNA was measured by AFM using tapping mode. FIG. 4 shows the results of an atomic force microscope of tFNA, which shows that tetrahedra were successfully synthesized, with a size of about 10-20 nm.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 investigation of tetrahedral framework nucleic acids of the invention from nose to brain
1. Experimental method
Attachment of a fluorophore to the tetrahedral framework nucleic acid (Cy 5 fluorophore attached to the S1 single strand in Table 1, followed by preparation of the fluorophore attached tFNA as described in example 1). Experiments are carried out by adopting Balb/c mice, the tFNA prepared is diluted to a concentration of 1 mu M by using TM buffer solution, then the diluted solution is dripped into the nasal cavity of the mice, the dripped dosage is 25 mu L, and the fluorescent distribution situation of the brain is observed by using a living animal imaging system after 1, 2, 3 and 6 hours of dripping the medicines, so that the distribution situation of tFNA in brain tissues is verified. Mice were then sacrificed, brain tissues were removed, the collected brain tissues were fixed with paraformaldehyde, sectioned with a frozen microtome, immunofluorescent stained, and the distribution of tFNA in the brain tissues was observed under a confocal microscope.
2. Experimental results
After observing that the fluorescence intensity is highest after dropping the medicine for 2 hours, fig. 5 shows the immunofluorescence staining result when the fluorescence intensity is highest, and fig. 5 shows that tFNA of the invention is distributed in most areas of brain tissues, so that tFNA nasal-to-brain delivery is realized.
Test example 2 nasal route to brain of tetrahedral framework nucleic acids of the invention
1. Experimental method
Attachment of a fluorophore to the tetrahedral framework nucleic acid (Cy 5 fluorophore attached to the S1 single strand in Table 1, followed by preparation of the fluorophore attached tFNA as described in example 1). Experiments were performed using Balb/c mice. The 6-8 week Balb/c mice were divided into two groups, one group was not treated, the other group was cut off with a blade in front of the olfactory bulb in order to isolate the olfactory nerve (the skin between the two eyeballs of the mice was gently cut off with a No. 12 blade, and after the nasal-brain junction was determined, the front of the olfactory bulb was cut off with a blade). The tFNA thus prepared was diluted to a concentration of 1. Mu.M with TM buffer within 2 hours of the treatment, and then instilled into the nasal cavity of a mouse at a dose of 25. Mu.L, and after instilling the drug for 2 hours, the distribution of fluorescence in the brain was observed with a small animal living imaging system, confirming the distribution of tFNA in brain tissue. Mice were then sacrificed, brain tissues were removed, the collected brain tissues were fixed with paraformaldehyde, sectioned with a frozen microtome, immunofluorescent stained, and the distribution of tFNA in the brain tissues was observed under a confocal microscope.
2. Experimental results
After the mouse olfactory nerve pathway was cut off, the fluorescence intensity in the olfactory bulb was observed to be significantly weakened using a small animal living body imaging system (fig. 6), and the fluorescence intensity in the olfactory bulb was observed to be significantly weakened by immunofluorescent staining (fig. 7). The results indicate that the olfactory bulb pathway may be one of the main pathways for tetrahedral nasal entry into the brain.
In test example 1, strong fluorescence of the material was observed in the trigeminal nerve (fig. 8), a cross section of the trigeminal nerve was made, and the degree of fusion with tFNA fluorescence was observed using the labeled myelin sheath and the labeled neuron, which indicated that tFNA was distributed in the neuronal tissue of the trigeminal nerve and transported along the axon of the neuron (MBP is myelin sheath, MAP is neuronal marker, cy5 and MAP overlap).
The above results demonstrate that the nasal route of the present invention tFNA into the brain is olfactory bulb and trigeminal nerve delivery.
Test example 3 distribution and Structure of tetrahedral framework nucleic acids of the invention from nose to brain
1. Experimental method
(1) Distribution experiment in nasal cavity
Attachment of a fluorophore to the tetrahedral framework nucleic acid (Cy 5 fluorophore attached to the S1 single strand in Table 1, followed by preparation of the fluorophore attached tFNA as described in example 1). Experiments were performed using Balb/c mice. The tFNA prepared was diluted to a concentration of 1. Mu.M with TM buffer, then instilled into the nasal cavity of the mouse at a dose of 25. Mu.L, and after instilling the drugs for 2,4, 6 and 8 hours, the mouse was sacrificed to obtain materials (the nasal cavity is obtained, fur and muscle tissues are not needed), and the fluorescence distribution of the nasal cavity was observed with a Bio-Rad imaging system to confirm the distribution of tetrahedral framework nucleic acid in the nasal cavity tissues.
(2) Mucus penetration experiment
Attachment of a fluorophore to the tetrahedral framework nucleic acid (Cy 5 fluorophore attached to the S1 single strand in Table 1, followed by preparation of the fluorophore attached tFNA as described in example 1).
An artificial mucus was prepared by adding sterile yolk emulsion (25. Mu.L), mucin (25 mg), DTPA aqueous solution (30. Mu.L at 1 mg/mL), deoxyribonucleic acid (20 mg), sodium chloride (25 mg), potassium chloride (11 mg) and RPMI-1640 medium (100. Mu.L) to 5 mL of water according to the following formulation, and stirring. And (3) sucking 300 mu L of artificial mucus, dripping the artificial mucus on the surface of the confocal small dish, and uniformly spreading the artificial mucus. tFNA. Mu.L (concentration: 1000nM, solvent: TM buffer) of Cy5 fluorescent group was added dropwise to the surface of the mucus, and the mixture was observed under a confocal laser microscope.
TFNA after penetration of the artificial mucus, it was checked by PAGE whether tFNA remained intact. After the glass plate was leak-tested, the 8% PAGE gel solution was added to the loading well and the gel was allowed to solidify after insertion into a comb. tFNA and a loading buffer solution are added into a loading tank after being uniformly mixed, and electrophoresis is completed in a 1x TAE solution environment. The Gel was stained in a dye solution of 1x Gel Red for 20min and finally the nucleic acid Gel was developed under a chemiluminescent imager.
(3) Diffusion experiments
The preparation method of the SD rat nasal mucosa comprises the following steps: healthy SD rats were prepared for 6-8 weeks, after sacrifice, nasal mucosa in nasal cavity was carefully peeled off using ophthalmic scissors and ophthalmic forceps, during which time physiological saline was continuously used to maintain the nasal mucosa moist.
The diffusion of tFNA on the dialysis membrane (dialysis membrane parameter 10 kilokda) and on the nasal mucosa of SD rats was evaluated by a modified Franz diffusion cell using phosphate buffered solution (PBS, pH 6.6± 0.2,37 ±0.5 ℃, continuous stirring) as diffusion medium. At a specific time, 5 μl of medium was withdrawn from the receptor chamber and replaced with fresh PBS of the same volume. The drug concentration is measured by a spectrophotometer (1700,Tokyo, japan) at 260 nm.
(4) Verification of integrity experiments
Attachment of a fluorophore to the tetrahedral framework nucleic acid (Cy 3-BHQ2 fluorophore attached to the S2 single strand in Table 1, followed by preparation of the attached fluorophore tFNA as described in example 1). Experiments were performed using Balb/c mice. The tFNA prepared is diluted to the concentration of 1 mu M by using TM buffer solution, then is dripped into the nasal cavity of a mouse, the dripping dosage is 25 mu L, and brain tissues are obtained after medicines are dripped for 1,2, 3, 6 and 9 hours respectively. The collected brain tissue is fixed by using paraformaldehyde, the brain tissue is sliced by using a frozen microtome, and distribution of tetrahedral framework nucleic acid materials in the brain tissue is observed under a confocal microscope after immunofluorescence staining.
2. Experimental results
(1) After nasal administration, the mice were harvested after 2, 4,6 and 8 hours, the nasal cavities were collected, and the distribution of tFNA in the nasal cavities was observed, which indicated (fig. 9), that the residence time of tFNA in the nasal cavities was long, the distribution range was wide, and tFNA distribution in the olfactory region of the nasal cavities could be maintained for at least 8 hours.
(2) Artificial mucus was prepared and observed tFNA for its ability to penetrate mucus, and the results indicate that tFNA was able to penetrate mucus rapidly (within 5 minutes) and accumulate over time. The results of the PAGE gel electrophoresis show (FIG. 11) that tFNA remained intact after penetration of the gel.
(3) The SD rat nasal mucosa or dialysis membrane was placed therein using Franz's diffusion cells, and the results indicated (fig. 12) tFNA were able to penetrate the SD rat nasal mucosa or dialysis membrane, indicating that they were able to penetrate the nasal mucosa in vivo and in vitro.
(4) At the apex of tFNA, two groups Cy3 and BHQ2 are attached, tFNA is non-fluorescent when tFNA is structurally intact, and tFNA emits Cy3 fluorescence when tFNA is broken. Using this specially designed tFNA for nasal administration, after collection of brain tissue sections, the results indicated (fig. 13) that only a small portion of tFNA structure was disrupted and a large portion was intact after tFNA nasal entry into the brain. tFNA are capable of maintaining structural stability in brain tissue for at least 9 hours after nasal brain access.
Test example 4 biosafety of nasal administration of tetrahedral framework nucleic acids of the invention
1. Experimental method
(1) Cell CCK8 assay
BV2 cells were plated uniformly into 96-well plates (2500 cells per well), tFNA prepared in example 1 at different concentrations (100, 200, 300, 400, 500 nM) were added after cell attachment, after 24 hours of incubation, the cells were washed with PBS, cell counting reagent (CCK-8: PBS=1:9 (volume ratio), cell Counting Kit-8 from Medchemexpress, product number: HY-K0301) was added, and the plates were co-cultured for 0.5 hours. Finally, the absorbance of the solution at 450 nm in each well was measured, and the cell activity was calculated.
(2) HE staining of nasal mucosa and vital organs
TFNA was prepared as described in example 1. Balb/c mice are adopted for experiments, the prepared tFNA is diluted to 1 mu M by using TM buffer solution, then the diluted solution is dripped into one side of the mice through nasal cavities, the dripped dosage is 25 mu L, the mice are killed after 2 hours of medicine dripping, nasal tissues and important organ tissues of the mice are collected, and the tissues are fixed in formalin for more than 24 hours. Subsequently, the tissue was dehydrated, embedded and cut into 4 micron thick sections. These sections were stained with hematoxylin and eosin and photographed with an FSX100 microscope (olympus).
2. Experimental results
(1) From fig. 14, tFNA did not affect cell activity at the treatment concentration.
(2) HE staining observed nasal tissues on the dosing side and the non-dosing side (fig. 15), indicating tFNA nasal dosing was safe. As can be seen from the results of HE staining of important organs in FIG. 16, tFNA was also safe for nasal administration.
In summary, the invention uses the framework nucleic acid as a drug carrier to realize the drug delivery from nose to brain for the first time, and no research is currently performed on using the framework nucleic acid or the framework nucleic acid carrier for the drug delivery from nose to brain, and the drug delivery mode can bypass the obstruction of the blood brain barrier to realize the brain tissue delivery of the drug. The nasal-to-brain framed nucleic acid drug delivery route allows for noninvasive delivery of drugs to brain tissue, reducing the incidence of invasive drug delivery complications. The research result of the invention shows that the tetrahedral framework nucleic acid can still maintain the complete tetrahedral structure after entering the brain through nose, and the tetrahedral framework nucleic acid administration system can also protect the integrity of the medicine after carrying the medicine (such as polypeptide) and reduce the loss of the medicine before entering brain tissue. Because of the way of transnasal administration, non-invasive administration can be realized, the compliance of patients is high, and the method has obvious advantages in treating chronic diseases in nerve and mental systems due to non-invasive operation.
Claims (10)
1. Use of a tetrahedral framework nucleic acid for the preparation of a carrier for nasal administration to the brain, characterized in that: the tetrahedral framework nucleic acid is formed by base complementary pairing of nucleotide sequences shown in SEQ ID NO. 1-4.
2. Use according to claim 1, characterized in that: the preparation method of the tetrahedral framework nucleic acid comprises the following steps: after dissolving four DNA single strands in TM buffer solution in equimolar manner, the mixture is maintained at 85-105 ℃ for 5-15min, and then maintained at 2-8 ℃ for 10-30min.
3. Use according to claim 2, characterized in that: four DNA single strands were equimolar dissolved in TM buffer, and maintained at 95℃for 10min and then at 4℃for 20min.
4. Use according to claim 3, characterized in that: the concentration of Tris-HCl in the TM buffer solution is 10mM, the concentration of MgCl 2 is 50mM, and the pH value is 8.0; the concentration of the four DNA single strands was 1. Mu.M.
5. Use according to any one of claims 1 to 4, characterized in that: the carrier for transnasal administration is a carrier for transnasal administration through olfactory bulb and trigeminal nerve.
6. Use of tetrahedral framework nucleic acid for the preparation of a medicament for nasal administration into the brain, characterized in that: the medicine is obtained by taking tetrahedral framework nucleic acid formed by base complementation pairing of nucleotide sequences shown in SEQ ID NO. 1-4 as a carrier and carrying the medicine.
7. Use according to claim 6, characterized in that: the preparation method of the tetrahedral framework nucleic acid comprises the following steps: after four DNA single strands are equimolar dissolved in TM buffer, the mixture is maintained for 5 to 15 minutes at the temperature of between 85 and 105 ℃ and then maintained for 10 to 30 minutes at the temperature of between 2 and 8 ℃;
preferably, after equimolar dissolution of the four DNA strands in TM buffer, they are maintained at 95℃for 10min and then at 4℃for 20min.
8. Use according to claim 7, characterized in that: the concentration of Tris-HCl in the TM buffer solution is 10mM, the concentration of MgCl 2 is 50mM, and the pH value is 8.0; the concentration of the four DNA single strands was 1. Mu.M.
9. Use according to any one of claims 6 to 8, characterized in that: the medicine is used for treating brain diseases.
10. Use according to claim 9, characterized in that: the medicine is used for treating sepsis encephalitis, parkinson's disease, alzheimer's disease, cerebral ischemia and epilepsy.
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