CN103301481A - Target nucleic acid drug delivery system and application thereof - Google Patents
Target nucleic acid drug delivery system and application thereof Download PDFInfo
- Publication number
- CN103301481A CN103301481A CN2013102527522A CN201310252752A CN103301481A CN 103301481 A CN103301481 A CN 103301481A CN 2013102527522 A CN2013102527522 A CN 2013102527522A CN 201310252752 A CN201310252752 A CN 201310252752A CN 103301481 A CN103301481 A CN 103301481A
- Authority
- CN
- China
- Prior art keywords
- drug
- nucleic acid
- nanometer
- train
- loading system
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a target nucleic acid drug delivery system. The target nucleic acid drug delivery system (nanometer train) disclosed by the invention maintains the recognition specificity on a target cell by an original aptamer and can deliver a loaded drug to a disease target cell in a targeting manner; a biological imaging agent delivered to the disease cell can be applied to biological diagnosis; and the drug delivered to the target disease cell can be gradually released to specifically kill the target cell. The specific killing effect reduces side effect and improves curative effect. The preparation process of the system disclosed by the invention is simple, economical and efficient, and the system is especially suitable for highly sensitive diagnosis and target administration of a disease.
Description
Technical field
The invention belongs to the bio-pharmaceutical technical field, be specifically related to a kind of targeting nucleic acid drug-loading system and application thereof.This drug-loading system be a kind of general, based on " nanometer train " nucleic acid and aptamer, that can be used for diagnosis of disease.
Background technology
The quick diagnosis of disease and effective treatment have become the key point of medical treatment.Yet existing medical diagnosis on disease technology still can not sensitively must detect a small amount of disease cell or tissue.But then, scientific research shows, the cure rate of a lot of diseases is positively related with diagnosis promptness to this disease: disease is diagnosed more early, and cure rate is then higher.The technical method of researching and developing a kind of energy Sensitive Detection disease has become and has improved the vital part of medical level.Meanwhile, traditional disease treatment also has many defectives.Wherein one of most important defective is the traditional treatment Chinese medicine, especially the high toxic and side effects of chemotherapeutics.Take cancer as example, chemotherapy is present clinical most popular cancer therapy.Yet, the non-specific of tradition chemotherapeutics causes them not only to the cancerous cell of rapid growth, and can induce toxicity and suppress cell amplification or tissue growth at healthy cell or the tissue of rapid growth, these normal cells comprise myeloid element, hair follicle, the cell of oral cavity, digestive tract, reproductive system etc.This toxicity to healthy cell causes chemotherapy to have obvious side effect, and and then so that can only tolerate dosage with very limited maximum clinically.Another large defective of tradition disease treatment is to lack the therapy that is applicable to the given patient individuality, yet, studies show that a lot of diseases (such as cancer) have very high individual variation.This so that development for patient individual's methods for the treatment of diseases, and personalized treatment (Personalized Medicine) has very high application prospect.In addition, the bioavailability of traditional micromolecule chemotherapeutics is very low.These factors have all reduced traditional chemotherapeutics to the therapeutic effect of a lot of diseases.Therefore, the delivery system that development has target function, high drug load and stable medicine-carrier complexes will help to overcome this series of problems, and can improve the effectiveness of clinically disease treatment.
Because fluorescence is to the safety of human body and the ease for operation that fluorescence molecule is used for fluoroscopic examination, fluoroscopic examination plays an important role in medical diagnosis on disease.Yet the muting sensitivity of fluoroscopic examination means is so that most important to the amplification of fluorescence signal at present.
Aspect targeted therapy, the chemotherapeutics carrier that is used at present targeted therapy mainly comprises nano material (as: liposome) or has the macromolecule of better biocompatibility.Chemotherapeutics by coated to nano material or be loaded in the pharmaceutical carrier with the mode of macromolecule formation complex and be transported to target cell.Yet these medicament carrier systems have a series of defectives: traditional targeting transportation can only be carried out by passive target the targeting transportation of medicine, and the poor efficiency of this targeting is also so that the effect of targeted therapy is very limited; Complicated synthetic, purification and further functionalization have lowered the clinical practice feasibility of product; The own biocompatibility of nano material of a lot of containing metals or silicon composition is poor; At last, a large amount of drug-loading systems can only with medicament transport to cell surface, lack the further active transportation of medicine to intracellular function.
The development of poly DNA (deoxyribonucleic acid) (DNA) nanotechnology is overcome above-mentioned many defectives.The DNA nano material that this technology produces has good biocompatibility, high stability, biodegradability, easily the advantages such as easily modification are easily synthesized in design.In addition, in targeted therapy, initiatively targeting can also be retained in the sick point of targeting with the pharmaceutical carrier transportation for a long time by part and the special interaction between target cell that is used for targeting.Molecule with target function commonly used has two large classes: antibody and aptamer.Aptamer (Aptamer) is one section oligonucleotide, and the phyletic evolution technology screening by the part index concentration receives.Compare with antibody, aptamer have easy screening, easily synthetic, easily store, the advantages such as easily modification, high-affinity, high-biocompatibility and biodegradability, and can be efficiently, be combined with target molecule specifically.Specifically for aptamer, U.S. FDA obtained extensive approval to examining of first aptamer class medicine to its biological safety along with 2004.In addition, have can be by the effect of target cell endocytosis for a large amount of aptamers.These advantages so that aptamer be with a wide range of applications at aspects such as medical diagnosis treatment, SARS drug design.
Prior art is used for targeted therapy with aptamer itself as carrier.Yet these technology all have a series of defectives, for example (,) drug loading is low, the DNA consumption reach greatly cost high, need that complicated organic reaction, reaction yield are low, the chemical modification cost is high.These defectives have hindered their application clinically.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, and a kind of targeting nucleic acid drug-loading system and application are provided.
In order to achieve the above object, technical scheme provided by the invention is:
Described targeting nucleic acid drug-loading system is mutually alternately to hybridize the double-stranded poly ribonucleic acid of length that forms by several nucleic acid monomers, and double-stranded poly ribonucleic acid one termination of described length has the aptamer of specific recognition targeted cells.Formed thus " the nanometer train " that comprise " headstock " (aptamer) and " compartment " (long double-stranded poly ribonucleic acid).The aptamer of specific recognition targeted cells is by after designing with molecular engineering, but the formation of excitation nano train.
Bio-imaging agent, drug molecule or nano material are loaded on the double-stranded poly ribonucleic acid of above-mentioned length by the mode of covalent modification or non-covalent combination.Described bio-imaging agent comprises fluorescence molecule or fluorescent nano material.Described drug molecule includes but not limited to amycin (Doxorubicin), daunorubicin (Daunorubicin), epirubicin (Epirubicin).Described nano material includes but not limited to nanogold particle.Described aptamer includes but not limited to sgc8c, AS1411 or TD05.
Described nucleic acid monomer comprises nucleic acid monomer M1 and nucleic acid monomer M2, and nucleic acid monomer M1 and nucleic acid monomer M2 be alternately hybridization mutually; The sequence of described nucleic acid monomer M1 is shown in SEQ ID NO.1, and the sequence of described nucleic acid monomer M2 is shown in SEQID NO.2.
Above-mentioned drug-loading system can be used for preparing the medicine of targeting diagnosis and treatment cancer and infected by microbes disease.By medicine or bio-imaging agent being loaded on the carrier and targeting is transported to the disease cell or tissue, and then realize medical diagnosis on disease or targeting disease treatment; The nanometer train that has loaded goods has higher stability, and institute's lade targeting can be transported to the specified disease cell or tissue, and then realizes medical diagnosis on disease or targeted therapy.Medical diagnosis on disease can realize by the detection to signals such as fluorescence, magnetic, and wherein, the detection of change in fluorescence both can by being marked at the fluorescence molecule on the nanometer train, also can be undertaken by drug molecule or nano material with photoluminescent property.
The invention will be further described below in conjunction with design principle and advantage:
Drug-loading system drug loading in the prior art is low in order to overcome, the DNA consumption reach greatly cost high, need that complicated organic reaction, reaction yield are low, chemical modification high in cost of production shortcoming, the present invention proposes based on nucleic acid, have strong selectivity by the aptamer guiding, have the ability, high stability of high load large number of biological preparation or drug molecule and delivery system cheaply.
At first, the present invention made up a kind of simple economy, high-biocompatibility and biodegradability be used for nano-carrier in medical diagnosis on disease and treatment transmission medicine and bio-imaging agent.The present invention prepares nano-carrier with Polynucleotide as material, specifically, prepares the nanometer train with DNA as biomaterial.The biocompatibility of DNA and degradability are so that prepared nanometer train also has high-biocompatibility and biodegradability.Other Polynucleotides, RNA for example, LNA etc. also has similarity.By structural design cleverly, the nanometer train has very long duplex structure, and these long structures only have short chain DNA to assemble, thus, realizing when the nanometer train loads large number of biological preparation or drug molecule, saving time and the cost (seeing Table) of DNA preparation.
Table one nanometer train and traditional simple aptamer structure are used for the cost compare of targeted therapy
Secondly, the invention provides a kind of energy selectivity identification targeting disease cell, can transport medicine to the disease cell by targeting, and can further medicament transport be arrived intracellular drug-loading system.The present invention utilize can specific recognition the aptamer of disease cell, with aptamer modified to each nanometer train, thereby medicine or the bio-imaging agent that will be loaded on the nanometer train carrier are transported to targeted cells.And then, by a lot of aptamers can by the characteristic of targeted cells endocytosis with medicament transport in cell, and the further efficient disease Growth of Cells that suppresses.
Described aptamer comprises sgc8c, AS1411, TD05.Its sequence is shown in subordinate list two.It is fit that the preparation of nanometer train is not limited to deoxyribonucleotide (DNA).
The sequence that table two DNA is fit
The 3rd, the invention provides a kind of good stability and have the targeting transport agent of very large drug loading.The nanometer train has very long duplex structure can be used for mode by covalent modification or non-covalent combination, realizes loading large number of biological preparation or drug molecule at the nanometer train.The present invention utilizes the specific bond between medicine and the particular sequence double-stranded DNA, at a lot of drug binding sites of nanometer train design, thus so that can be in conjunction with a lot of drug molecules on the very long nanometer train structure.The very strong adhesion that all has between nanometer train itself and medicine and the specific site determined prepared loading the nanostructured of medicine have very high stability.Described medicine includes but not limited to amycin, daunorubicin, epirubicin etc.
The 4th, the invention provides the practical application of this class pharmaceutical carrier.The present invention is used for target cancer cell detection and target on cancer treatment with this drug-loading system.The inventor has confirmed formation, successful modification and long nanometer train double-stranded DNA " compartment " structure of aptamer on each nanometer train of nanometer train by gel electrophoresis and atomic force microscope.The inventor has also proved that by flow cytometry this nanometer train can the specific recognition target cell, and can in connection with the fluorescence signal of target cell of nanometer train amplify.The inventor has proved nanometer train energy selective binding target cell, has discharged gradually by the target cell endocytosis and with medicine by laser scanning co-focusing microscope.The inventor has confirmed that by spectrofluorimetry the drug molecule with fluorescent characteristic can successfully be loaded on the nanometer train, and has very high stability, has proved that by projection electron microscope gold nano grain can be loaded onto on the nanometer train.Further: the inventor has also proved this class adduction physical ability by cell endocytic, and can be gradually with drug release and recovered the fluorescence of medicine.The inventor has proved that by In vitro cell experiment prepared nanometer train has the special toxicity to target cell, lethal then less to non-target cell, thus improved maximum tolerated concentration at the non-target cell Chinese medicine.
In sum, the invention provides the nanometer transport agent system that a class can be used for diagnosis of disease.
Compared with prior art, the beneficial effect of targeting nucleic acid drug-loading system of the present invention is:
1, preparation technology is simple, and cost is low.The preparation of the synthetic and nanometer train of Polynucleotide all has simply, economy, advantage fast;
2, high drug load: but a large amount of medicine carryings site in the nanometer train is given the site of their very high medicine useful loads, a large amount of chemical modifications the site of more modified biological preparations or other goods is provided;
3, stability is high;
4, can be used for sensitive diagnosis to disease, nanometer train can load the characteristics of a plurality of bio-imaging agent so that combine the disease cell signal of these nanometer trains and amplified, thereby realizes the Sensitive Detection of disease;
5, the nanometer train can be entered cell by endocytosis.Thus, the medicine that is loaded on the nanometer train is further delivered into cell, then discharges gradually, has strengthened like this medicine in intracellular valid density, and the fluorescence recovery of release medicine then helps the drug detection after the medication;
6, reduce drug side effect.By aptamer can specific bond the characteristic of target cell, reduced at healthy cell or organize the toxicity of medicine and improved the maximum tolerated concentration of organizing Chinese medicine at these, thereby reduced side effect and improved therapeutic effect;
7, the nanometer train carrier based on nucleic acid has very high biocompatibility and biodegradability;
8, universality: the nanometer train can be prepared from by different types of nucleic acid, thereby the preparation of the aptamer of utilizable energy specific recognition various disease cell realizes personalized treatment (Personalized Medicine), can be used for transporting diversified bio-imaging agent or drug molecule.
These advantages will make this class New Type of Diseases diagnosis and treatment instrument provided by the invention have great application prospect in clinical.
Description of drawings:
Fig. 1: " nanometer train " (Apt-NTrs) prepares schematic diagram; One end of " nanometer train " is the aptamer of energy selectivity identification targeting disease cell or tissue; The other end is by the mutual duplex structure of the length that forms of hybridization alternately of chain rupture Polynucleotide, and through designing, these structures have with the bio-imaging agent and drug molecule is interactional much can modify a little.
Fig. 2: by the sign of agarose gel electrophoresis to " the nanometer train " that produce;
Fig. 3: flow cytometry figure, Fig. 3 A are the flow cytometry figure that " nanometer train " identified target cell, and Fig. 3 B is that " nanometer train " is to the flow cytometry figure of non-target cell identification; Flow cytometry show produce kept the selective recognition of aptamer to targeting disease cell at " compartment " inner " nanometer train " of having modified a lot of bio-imaging agent (fluorescence molecule);
Fig. 4: Fig. 4 A is laser scanning co-focusing imaging analysis figure, and Fig. 4 B is the enlarged drawing of Fig. 4 A; The laser scanning co-focusing imaging analysis shows prepared having and can be arrived intracellular ability by endocytosis at " compartment " inner " nanometer train " of having modified a lot of bio-imaging agent (fluorescence molecule);
Fig. 5: spectrofluorimetry figure; Spectrofluorimetry show medicine (as the fluorescence of amycin/Dox) after being loaded into " nanometer train " by cancellation, and according to the molar ratio of drug molecule and " nanometer train " as can be known, the nanometer train has very high medicine useful load;
Fig. 6: tem study figure; Tem study shows that gold nano grain successfully has been loaded onto on " nanometer train " as a pattern goods;
Fig. 7: drug release experimental patterns; The drug release experiment shows, the quick release with respect to free drug, and the drug release rate that is loaded in " nanometer train " is very slow, shows that medicine and " nanometer train " complex have very high stability;
Fig. 8: laser scanning co-focusing microscope image; The laser scanning co-focusing microscope imaging shows: 1) arrive on " nanometer train " by the drug molecule (such as amycin) that is mounted with fluorescence, through " nanometer train " drug selectivity is transported to target cell, both can by the character Real Time Monitoring drug release of medicine fluorescence signal enhancing after discharging, can show again " nanometer train " to selectivity identification and the medicament transport of target cell; Fig. 8 A is target cell, free drug processing figure, and Fig. 8 B is target cell, through the drug treating figure of nanometer train delivery, and Fig. 8 C is non-target cell, target cell, free drug processing figure, and Fig. 8 D is non-target cell, through the drug treating figure of nanometer train delivery;
Fig. 9: the MTS experimental patterns, wherein, Fig. 9 A, 9C, 9E are respectively amycin, daunorubicin, epirubicin to the MTS experimental result picture of target cell, and Fig. 9 B, D, F are respectively amycin, daunorubicin, epirubicin to the MTS experimental result picture of non-target cell; Show by the MTS experiment, the medicine (comprising amycin, daunorubicin, epirubicin) that transports through the nanometer train has the specific cell growth inhibition function of target cell (CEM), non-target cell (Ramos) is then suppressed ability less;
Figure 10: mouse experiment is figure as a result; " nanometer train " has medicament transport to the target tumor of intravital mouse, suppresses tumor growth and reduces the ability of toxic and side effects; Meta-tumor size figure when Figure 10 A is drug treating, meta-survival rate figure when Figure 10 B is drug treating, Figure 10 C is body weight variation diagram in ten days.
The specific embodiment:
Described targeting nucleic acid drug-loading system is mutually alternately to hybridize the double-stranded poly ribonucleic acid of length that forms by several nucleic acid monomers, and double-stranded poly ribonucleic acid one termination of described length has the aptamer of specific recognition targeted cells.Formed thus " nanometer train " (in following examples 4 to 10, targeting nucleic acid drug-loading system being called " nanometer train ") that comprises " headstock " (aptamer) and " compartment " (long double-stranded poly ribonucleic acid).
The bio-imaging agent is loaded on the double-stranded poly ribonucleic acid of above-mentioned length by the mode of covalent modification or non-covalent combination.Described bio-imaging agent is fluorescence molecule.Described aptamer is sgc8c.
Described nucleic acid monomer comprises nucleic acid monomer M1 and nucleic acid monomer M2, and nucleic acid monomer M1 and nucleic acid monomer M2 be alternately hybridization mutually; The sequence of described nucleic acid monomer M1 is shown in SEQ ID NO.1, and the sequence of described nucleic acid monomer M2 is shown in SEQID NO.2.
Described targeting nucleic acid drug-loading system is mutually alternately to hybridize the double-stranded poly ribonucleic acid of length that forms by several nucleic acid monomers, and double-stranded poly ribonucleic acid one termination of described length has the aptamer of specific recognition targeted cells.
Drug molecule or nano material are loaded on the double-stranded poly ribonucleic acid of above-mentioned length by the mode of covalent modification or non-covalent combination.Described drug molecule comprises amycin, daunorubicin, epirubicin.Described aptamer is AS1411.
Described nucleic acid monomer comprises nucleic acid monomer M1 and nucleic acid monomer M2, and nucleic acid monomer M1 and nucleic acid monomer M2 be alternately hybridization mutually; The sequence of described nucleic acid monomer M1 is shown in SEQ ID NO.1, and the sequence of described nucleic acid monomer M2 is shown in SEQ ID NO.2.
Embodiment 3
Described targeting nucleic acid drug-loading system is mutually alternately to hybridize the double-stranded poly ribonucleic acid of length that forms by several nucleic acid monomers, and double-stranded poly ribonucleic acid one termination of described length has the aptamer of specific recognition targeted cells.
Nano material is loaded on the double-stranded poly ribonucleic acid of above-mentioned length by the mode of covalent modification or non-covalent combination.Described nano material is nanogold particle.Described aptamer is TD05.
Described nucleic acid monomer comprises nucleic acid monomer M1 and nucleic acid monomer M2, and nucleic acid monomer M1 and nucleic acid monomer M2 be alternately hybridization mutually; The sequence of described nucleic acid monomer M1 is shown in SEQ ID NO.1, and the sequence of described nucleic acid monomer M2 is shown in SEQ ID NO.2.
Use automatic dna synthesizer, comprise aptamer or the nanometer train monomer of modification by the synthetic required short chain DNA of 1 μ mole of solid phase synthesis process.Thereby the product of gained gone to protect splits away off DNA used bead from synthetic, and with ethanol (70%, 2.5 times of dna solution volume) and NaCl(3M, 0.1 times of dna solution volume) DNA is precipitated.The DNA of precipitation is dissolved in TEAA(0.1M) in.
Above-mentioned product is passed through the HPLC purification, and dry.Remove DMT blocking group on the DNA with acetic acid, again with above-mentioned ethanol precipitation DNA is precipitated drying.Gained DNA is dissolved in (" DNA water " refers to not contain the water of degradation of dna ribozyme) in the DNA water, and it is for subsequent use to be stored in low temperature (20 ℃).
Embodiment 5
In phosphate buffer, be made into following reaction system: aptamer excites body 1 μ M, each 10 μ M of monomer M 1 and M2.Above-mentioned reaction system is placed room temperature, reacted 12 hours.
Analyze products therefrom (Fig. 2) with agarose gel electrophoresis.Excite body or monomer relatively with aptamer, " nanometer train " have a series of that does not wait, greater than the molecular weight of any monomer, this is to be arrived by the monomer number difference that comprises in " nanometer train ".If excite the different fluorescence molecule of labelling on body or the monomer at aptamer, also can analyze fluorescence distribution in the nanometer train by fluorescence imaging.
Products therefrom can also characterize by the atomic force imaging." nanometer train " will show as the nanostructured of wire under mirror.
Referring to Fig. 1, an end of " nanometer train " is the aptamer of energy selectivity identification targeting disease cell or tissue; The other end is by the mutual duplex structure of the length that forms of hybridization alternately of chain rupture Polynucleotide, and through designing, these structures have with the bio-imaging agent and drug molecule is interactional much can modify a little.
With " the nanometer train " of the flow cytometry gained selectivity identification ability to target cell.With labelling the nanometer train of fluorescence molecule and target cell and non-target cell hatch half an hour on ice respectively, wash away free nanometer train with lavation buffer solution, with the cell of the gained fluorescence signal intensity through the flow cytometry cell.This analysis both can have been verified the nanometer train to the specific recognition ability of target cell, can prove that again the nanometer train amplifies the fluorescence signal of target cell, improves the sensitivity that detects the disease target cell thus.
Referring to Fig. 3, flow cytometry show produce kept the selective recognition of aptamer to targeting disease cell at " compartment " inner " nanometer train " of having modified a lot of bio-imaging agent (fluorescence molecule);
Embodiment 7
With laser scanning co-focusing microscope analysis " nanometer train " to the binding ability of target cell and by the ability (see figure 4) of endocytosis.With labelling the nanometer train of fluorescence molecule and target cell and non-target cell hatch not equal time (0.5h at cell culture incubator respectively, 1h, 2h), wash away free " nanometer train " with lavation buffer solution, with the cell of the gained fluorescence signal intensity through co-focusing imaging analysis of cells surface or cell interior.This analysis both can have been verified " nanometer train " to the specific recognition ability of target cell, can prove that again " nanometer train " can be by target cell selectivity endocytosis, and this will be as the theoretical basis that improves drug conveying efficient by " nanometer train ".
Medicine and " nanometer train " are hatched and are realized the medicine loading in phosphate buffer.By fluorophotometer, relatively same concentrations drug level (2 μ M) and variable concentrations " nanometer train " are hatched the fluorescence intensity of rear gained mixture Chinese medicine.The medicine and the free drug that are loaded on " nanometer train " of gained are placed in the Dialysis tubing, through different time sections (0,15min, 30min, 1h, 2h, 4h, 8h) after, come medicine quantitative by the fluorescence signal of analyzing medicine, and calculate the drug ratios that has discharged, the data point of gained is fitted to next stage drug release model:
F
released=α[1-exp(-ln(2)t/t
1/2)]
(wherein t is the time, t
1/2Be a half required time of maximum burst size for discharging medication amount, α for maximum the maximum fluorescence intensity of release medicine).(seeing Fig. 6 to 8).
Referring to Fig. 5, spectrofluorimetry show medicine (as the fluorescence of amycin/Dox) after being loaded into " nanometer train " by cancellation, and according to the molar ratio of drug molecule and " nanometer train " as can be known, the nanometer train has very high medicine useful load.
Referring to Fig. 6, tem study shows that gold nano grain successfully has been loaded onto on " nanometer train " as a pattern goods.
Referring to Fig. 7, the drug release experiment shows, the quick release with respect to free drug, and the drug release rate that is loaded in " nanometer train " is very slow, shows that medicine and " nanometer train " complex have very high stability.
Referring to Fig. 8, the laser scanning co-focusing microscope imaging shows: 1) arrive on " nanometer train " by the drug molecule (such as amycin) that is mounted with fluorescence, through " nanometer train " drug selectivity is transported to target cell, both can by the character Real Time Monitoring drug release of medicine fluorescence signal enhancing after discharging, can show again " nanometer train " to selectivity identification and the medicament transport of target cell.
With the cell of In vitro culture, test " nanometer train " is used for the ability of targeted therapy.Take with amycin to the targeting of cancerous cell as example, cultured target cell and non-target cell are layered on (100 μ L in the 96 porocyte culture plates, 50,000 cells/well), in the hole, add respectively finite concentration (0,0.1 μ M, 0.2 μ M, 0.4 μ M, 0.8 μ M, the amycin of pure amycin 1.6 μ M, 3.2 μ M) or the delivery of nanometer train.Centrifugal after in cell culture incubator, cultivating 2 hours, remove the cell culture fluid in the supernatant, change fresh culture, and then cultivate 48h.Then use MTS reagent test cell proliferation speed, with the toxicity size of testing drug to target cell and non-target cell.
Referring to Fig. 9, show by the MTS experiment, the medicine (comprising amycin, daunorubicin, epirubicin) that transports through the nanometer train has the specific cell growth inhibition function of target cell (CEM), non-target cell (Ramos) is then suppressed ability less.
With the targeted therapy ability of animal model test " nanometer train ".Be built with the mouse model of the target cell tumor that is formed by the target cell development with target cell and immunodefiiciency mice.After the tumor on the mice occurs, begin respectively three groups of drug treating: 1) free drug, 2) free nanometer train and 3) loaded " the nanometer train " of medicine.The concentration of its Chinese medicine or " nanometer train " is consistent between group and group.In the periodicity drug treating, measure size and the Mouse Weight of mouse tumor, the latter is in order to characterize medicine to the toxic and side effects of mice.
Referring to Figure 10, the mouse experiment result shows that " nanometer train " has medicament transport to the target tumor of intravital mouse, suppresses tumor growth and reduces the ability of toxic and side effects.
Claims (8)
1. targeting nucleic acid drug-loading system, it is characterized in that, described drug-loading system is mutually alternately to hybridize the double-stranded poly ribonucleic acid of length that forms by several nucleic acid monomers, and double-stranded poly ribonucleic acid one termination of described length has the aptamer of specific recognition targeted cells.
2. drug-loading system as claimed in claim 1 is characterized in that, bio-imaging agent, drug molecule or nano material are loaded on the double-stranded poly ribonucleic acid of length claimed in claim 1 by the mode of covalent modification or non-covalent combination.
3. drug-loading system as claimed in claim 2 is characterized in that, described bio-imaging agent comprises fluorescence molecule or fluorescent nano material.
4. drug-loading system as claimed in claim 2 is characterized in that, described drug molecule comprises amycin, daunorubicin, epirubicin.
5. drug-loading system as claimed in claim 2 is characterized in that, described nano material comprises nanogold particle.
6. drug-loading system as claimed in claim 1 is characterized in that, described aptamer comprises sgc8c, AS1411 or TD05.
7. drug-loading system as claimed in claim 1 is characterized in that, described nucleic acid monomer comprises nucleic acid monomer M1 and nucleic acid monomer M2, and nucleic acid monomer M1 and nucleic acid monomer M2 be alternately hybridization mutually; The sequence of described nucleic acid monomer M1 is shown in SEQ ID NO.1, and the sequence of described nucleic acid monomer M2 is shown in SEQ ID NO.2.
8. such as claim 1 to 7 drug-loading system application in the medicine of preparation targeting diagnosis and treatment cancer and infected by microbes disease as described in each.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102527522A CN103301481A (en) | 2013-06-24 | 2013-06-24 | Target nucleic acid drug delivery system and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102527522A CN103301481A (en) | 2013-06-24 | 2013-06-24 | Target nucleic acid drug delivery system and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103301481A true CN103301481A (en) | 2013-09-18 |
Family
ID=49127485
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013102527522A Pending CN103301481A (en) | 2013-06-24 | 2013-06-24 | Target nucleic acid drug delivery system and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103301481A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106267219A (en) * | 2016-08-08 | 2017-01-04 | 湖南大学 | The application of the application of targeting vector, targeted drug and targeted drug, targeted probes and targeted probes |
| WO2018082143A1 (en) * | 2016-11-02 | 2018-05-11 | 四川大学 | Dna tetrahedron modified by aptamer as1411 and preparation method therefor |
| CN108187062A (en) * | 2017-12-11 | 2018-06-22 | 青岛科技大学 | A kind of construction method of the nano-medicament carrier based on nucleic acid |
| CN108837286A (en) * | 2018-05-04 | 2018-11-20 | 清华大学 | Degradable diagnosis and treatment robot |
| CN108866161A (en) * | 2018-06-27 | 2018-11-23 | 南京师范大学 | A kind of spherical shape nucleic acid probe and its preparation method and application |
| CN111363822A (en) * | 2019-11-20 | 2020-07-03 | 深圳市鲲鹏未来科技有限公司 | Solution containing blood-stable nanoparticles, preparation method thereof and detection method of miRNA marker |
-
2013
- 2013-06-24 CN CN2013102527522A patent/CN103301481A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| GUIZHI ZHU ET AL.: "Self-assembled, aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics", 《PNAS》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106267219A (en) * | 2016-08-08 | 2017-01-04 | 湖南大学 | The application of the application of targeting vector, targeted drug and targeted drug, targeted probes and targeted probes |
| CN106267219B (en) * | 2016-08-08 | 2019-03-29 | 湖南大学 | Targeting vector, the application of targeted drug and targeted drug, the application of targeted probes and targeted probes |
| WO2018082143A1 (en) * | 2016-11-02 | 2018-05-11 | 四川大学 | Dna tetrahedron modified by aptamer as1411 and preparation method therefor |
| US10563205B2 (en) | 2016-11-02 | 2020-02-18 | Sichuan University | Nucleic acid aptamer AS1411 modified DNA tetrahedron and preparation method thereof |
| CN108187062A (en) * | 2017-12-11 | 2018-06-22 | 青岛科技大学 | A kind of construction method of the nano-medicament carrier based on nucleic acid |
| CN108187062B (en) * | 2017-12-11 | 2020-10-02 | 青岛科技大学 | Construction method of nucleic acid-based nano-drug carrier |
| CN108837286A (en) * | 2018-05-04 | 2018-11-20 | 清华大学 | Degradable diagnosis and treatment robot |
| CN108837286B (en) * | 2018-05-04 | 2020-06-02 | 清华大学 | Degradable diagnosis and treatment robot |
| CN108866161A (en) * | 2018-06-27 | 2018-11-23 | 南京师范大学 | A kind of spherical shape nucleic acid probe and its preparation method and application |
| CN111363822A (en) * | 2019-11-20 | 2020-07-03 | 深圳市鲲鹏未来科技有限公司 | Solution containing blood-stable nanoparticles, preparation method thereof and detection method of miRNA marker |
| CN111363822B (en) * | 2019-11-20 | 2024-03-19 | 深圳市鲲鹏未来科技有限公司 | Solution containing blood stability nano particles, preparation method thereof and detection method of miRNA markers |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Advances in biological applications of self-assembled DNA tetrahedral nanostructures | |
| CN107469088B (en) | Construction method for accurately identifying targeted nano-carrier based on DNA origami and application thereof | |
| Mohammadinejad et al. | Shedding light on gene therapy: Carbon dots for the minimally invasive image-guided delivery of plasmids and noncoding RNAs-A review | |
| Ding et al. | A self-assembled RNA-triple helix hydrogel drug delivery system targeting triple-negative breast cancer | |
| Duangrat et al. | Tetrahedral DNA nanostructures as drug delivery and bioimaging platforms in cancer therapy | |
| CN103301481A (en) | Target nucleic acid drug delivery system and application thereof | |
| Xiao et al. | DNA self-assembly of targeted near-infrared-responsive gold nanoparticles for cancer thermo-chemotherapy | |
| Daniels et al. | Sterically stabilized siRNA: gold nanocomplexes enhance c-MYC silencing in a breast cancer cell model | |
| Kanwar et al. | Chimeric aptamers in cancer cell-targeted drug delivery | |
| JP5866119B2 (en) | Template nanocomposite | |
| Feng et al. | Folate-conjugated boron nitride nanospheres for targeted delivery of anticancer drugs | |
| CN101180400B (en) | Nanoparticles comprising RNA ligands | |
| Liang et al. | Specific activation of cGAS-STING pathway by nanotherapeutics-mediated ferroptosis evoked endogenous signaling for boosting systemic tumor immunotherapy | |
| US20130058984A1 (en) | Single-walled carbon nanotube/bioactive substance complexes and methods related thereto | |
| Zhang et al. | Aptamer-coded DNA nanoparticles for targeted doxorubicin delivery using pH-sensitive spacer | |
| Bi et al. | Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7 | |
| Jaleel et al. | Carbon dot festooned and surface passivated graphene-reinforced chitosan construct for tumor-targeted delivery of TNF-α gene | |
| Li et al. | Nuclease-resistant signaling nanostructures made entirely of DNA oligonucleotides | |
| Karimi et al. | Advances in Nanomaterials for Drug Delivery: Polymeric, nanocarbon and bio-inspired | |
| Chen et al. | Nucleic acid based nanocomposites and their applications in biomedicine | |
| CN102716494B (en) | Nucleic acid medicine loading system for targeted therapy and preparation method of nucleic acid medicine loading system | |
| Lin et al. | Repression of multiple myeloma cell growth in vivo by single-wall carbon nanotube (SWCNT)-delivered MALAT1 antisense oligos | |
| Kantak et al. | Integration of DNA barcoding and nanotechnology in drug delivery | |
| US20240209424A1 (en) | Heteromultivalent Spherical Nucleic Acids and Uses in Therapeutic and Diagnostic Applications | |
| Navarro et al. | Defined covalent attachment of three cancer drugs to DNA origami increases cytotoxicity at nanomolar concentration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130918 |