CN106267219A - The application of the application of targeting vector, targeted drug and targeted drug, targeted probes and targeted probes - Google Patents
The application of the application of targeting vector, targeted drug and targeted drug, targeted probes and targeted probes Download PDFInfo
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- CN106267219A CN106267219A CN201610643160.7A CN201610643160A CN106267219A CN 106267219 A CN106267219 A CN 106267219A CN 201610643160 A CN201610643160 A CN 201610643160A CN 106267219 A CN106267219 A CN 106267219A
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Classifications
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- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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Abstract
The invention discloses the application of the application of a kind of targeting vector, targeted drug and targeted drug, targeted probes and targeted probes, targeting vector includes double-strand poly ribonucleic acid and part；Part is connected on double-strand poly ribonucleic acid by adapter；Double-strand poly ribonucleic acid is formed by causing chain alternately to hybridize after causing by nucleic acid monomer H1 and nucleic acid monomer H2, and the sequence of nucleic acid monomer H1 is the sequence shown in SED ID NO.1, and the sequence of nucleic acid monomer H2 is the sequence shown in SED ID NO.2；Targeted drug includes above-mentioned targeting vector and drug molecule, can be applicable to prepare tumor-targeting drug；Targeted probes includes above-mentioned targeting vector and bio-imaging agent, can be applicable to prepare tumor imaging and the detection kit of cancerous cell targeting detection.It is high that the targeting vector of the present invention has drug loading, and the targeting ability being obviously enhanced and cell internalizing effect have good biocompatibility and biodegradability, good stability.
Description
Technical field
The present invention relates to field of biomedicine technology, particularly relate to a kind of targeting vector, targeted drug and targeted drug
Application, targeted probes and the application of targeted probes.
Background technology
Cancer due to its easily transfer, can convert and the characteristic such as infinite multiplication, become one of the most fearful disease of the mankind.Statistics
Show newly-increased cancer patient 14,000,000 people in 2012 and have 8,200,000 people dead, estimating that newly-increased cancer patient in 2025 is up to
19000000 people, the situation is tense for whole world pathogenesis of cancer, and M & M is the most in rising trend.Effectively improve diagnostic techniques
Become minimizing cancer morbidity with Therapeutic Method, reduce mortality rate in the urgent need to.Improve the diagnosis of cancer, it is possible to sending out of pole morning
Show the early stage patient of cancer and treat, thus alleviating patient suffering and spirit, financial burden, making cancer patient's health early
Multiple, developing a kind of the technical method of Sensitive Detection cancer can become the raising vital part of medical level.Cancer
In treatment, common method includes radiation therapy, surgical resection and chemotherapy.Chemotherapy is as a kind of systemic treatment method quilt
It is widely applied, especially when tumor development to middle and advanced stage or appearance transfer, radiation therapy and surgical resection treatment side
Method exists clearly disadvantageous, and chemotherapy even becomes the Therapeutic Method of unique feasible.But chemotherapeutics lacks targeting, is killing
Can cause damage healthy tissue and cell while tumor cell, secondly chemotherapeutics is due to poor stability, and dissolubility is low,
Easily cause the problems such as multidrug resistance.Therefore, select suitable carrier that image reagent or chemotherapeutics are transported target thin
Born of the same parents, are of great significance for the diagnosis and treatment tool improving tumor.
Along with the development of nanotechnology, different types of targeting vector (such as polymer, inorganic material etc.) is the most widely
It is applied to diagnosis and the treatment of tumor, but these carriers need high complicated design, the synthetic technology of specialty and need long
The preparation of time；Secondly, a lot of containing metal or the poor biocompatibility of material of silicon composition own, polymeric material is at organism
Interior being difficult to is degraded, and constantly assembles showing toxicity；Finally, these carriers are due to low targeting efficiency so that accumulate in tumor thin
The chemotherapeutics in intracellular portion and image reagent do not reach effective concentration, the most inadequate for diagnosis and the therapeutic effect of tumor
Preferable.Therefore, a kind of high struck capacity, high targeting ability, effective cell internalization and the carrier of good biocompatibility are developed
Problem above can be overcome, significantly improve in clinical using value.
Biomolecule is easily-synthesized the advantages such as easy modification because of its good good biocompatibility, biodegradability, easily design
Become wide variety of vector construction material.In targeting transports, active targeting can be by the part on carrier and target cell table
The receptor-specific in face combines, and bio-imaging agent and drug molecule is transported target cells, and is retained in target cell for a long time,
Thus on target cell, reach the enrichment of high concentration.Can significantly improve the sensitivity of diagnosis, improve drug effect, reduce poison
Property, thus can stand the extensive concern of researcher.That targeting transports it is crucial that improve the specific binding energy of carrier and target cell
Power, this depends on the high-affinity of part, the high combination selected, and obtaining high performance part is extremely difficult work.Cause
This, on present ligand base, improve the specific binding capacity of carrier, diagnosis highly sensitive for cancer clinically and treatment
Have great importance.
Conventional part includes antibody, folic acid and aptamer etc., and aptamer (Aptamer) is one section of oligomerization core
Thuja acid, have easily screen, be easily-synthesized, the easily advantage such as modification, high-affinity, high-biocompatibility and biodegradability, and energy
Efficiently, be specifically combined with target molecule.When aptamer is attached on medicine carrying skeleton, owing to both define high molecular
Complex, can significantly reduce the binding ability of aptamer, thus affect binding ability.Prior art is adaptive by nucleic acid
Body is used for targeted therapy, there is a series of defect, and such as drug loading is low, binding ability is weak, need the organic reaction of complexity, reaction to produce
Rate is low, poor stability and chemical modification cost high.These defects hinder they application clinically.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, utilizes multivalence to combine effect, by many times
Aptamer is connected on nanometer skeleton, and the multivalent carrier of formation significantly improves targeting binding ability and cell internalizing effect
Really, thus provide that a kind of drug loading is high, have good biocompatibility and the targeting of biodegradability, good stability carries
Body；Additionally provide a kind of targeted drug, to target cell lethal strong, to non-target cell lethal little, side effect is little, can conduct
The medicine of cancer.Present invention also offers a kind of targeted probes, can be used for the targeting detection of cancer, detection efficiency is high.
For solving above-mentioned technical problem, it is provided that a kind of targeting vector, described targeting vector includes double-strand poly ribose core
Acid and part；Described part is connected on described double-strand poly ribonucleic acid by adapter；Described double-strand poly ribonucleic acid
Being formed by causing chain alternately to hybridize after causing by nucleic acid monomer H1 and nucleic acid monomer H2, the sequence of described nucleic acid monomer H1 is
Sequence shown in SED ID NO.1, the sequence of described nucleic acid monomer H2 is the sequence shown in SED ID NO.2.
Above-mentioned targeting vector, it is preferred that described nucleic acid monomer H1 is the nucleic acid monomer H1 of biotin modification, described nucleic acid
Monomer H2 is the nucleic acid monomer H2 of biotin modification, and described part is the part of biotin modification, and described adapter is strepto-parent
And element；The sequence of described initiation chain is the sequence shown in SED ID NO.3.It is further preferred that described nucleic acid monomer H1 is
The nucleic acid monomer H1, described nucleic acid monomer H2 that FITC modifies is the nucleic acid monomer H2 that FITC modifies, and described part is that FITC modifies
Part.
Above-mentioned targeting vector, it is preferred that described part is Zy1, the sequence of described Zy1 is shown in SED ID NO.4
Sequence.
Above-mentioned targeting vector, it is preferred that described biotin is Biotin.
Above-mentioned targeting vector, it is preferred that the concentration of described initiation chain is 250 nM～5000 nM；Described nucleic acid monomer
The concentration of H1 is 25 μMs；The concentration of described nucleic acid monomer H2 is 25 μMs.
Above-mentioned targeting vector, it is preferred that the concentration ratio of described initiation chain, nucleic acid monomer H1 and nucleic acid monomer H2 be 1～
20∶100∶100.It is further preferred that the concentration ratio of described initiation chain, nucleic acid monomer H1 and nucleic acid monomer H2 is 1: 100: 100.
Above-mentioned targeting vector, it is preferred that the concentration of described part is 0.25 μM～50 μMs.It is further preferred that institute
The concentration stating part is 50 μMs.
As total technology design, present invention also offers a kind of targeted drug, including above-mentioned targeting vector
And drug molecule, described drug molecule is loaded on described targeting vector by covalent modification or Non-covalent binding.
Above-mentioned targeted drug, it is preferred that described drug molecule is amycin.2 μMs of amycin can be loaded in the load of 4 nM
On body, the maximum drug load of each carrier is 500 drug molecules.
As total technology design, present invention also offers above-mentioned targeted drug answering in preparing tumour medicine
With.
As the design of total technology, present invention also offers a kind of targeted probes, including above-mentioned targeting vector and
Bio-imaging agent, described bio-imaging agent is loaded on described targeting vector by covalent modification or Non-covalent binding.
Above-mentioned targeted probes, it is preferred that described bio-imaging agent is fluorescence molecule or SYBR Green I.Described fluorescence
Molecule is loaded on described targeting vector by covalent modification.Described SYBR Green I is loaded by Non-covalent binding and targeting
On carrier.The SYBR Green I of 1 times of concentration can be loaded on 5 nM carriers.
As total technology design, present invention also offers a kind of above-mentioned targeted probes and prepare tumor imaging and cancer
The detection kit of cell-targeting detection and preparation application in tumor.
Compared with prior art, it is an advantage of the current invention that:
(1) the invention provides a kind of targeting vector, there is low, the complicated synthesis step of drug loading in existing medicine carrying body, reaction is produced
Rate is low, chemical modification cost is high is combined the shortcomings such as establishment weak, poor selectivity, internalization DeGrain with target cell.In order to overcome
Above deficiency, the present invention utilizes the method for self assembly, by biotinylated nucleic acid, causes chain, biotinylated nucleic acid adaptive
Body and Streptavidin are assembled into the transport agent with targeting ability.Biotinylated nucleic acid is shape under the triggering causing chain
Becoming a series of length-adjustable double-strand poly ribonucleic acid, longer double-strand poly ribonucleic acid applies more loading in theory
Site, thus realize higher struck capacity, at the same time it can also be combine the biotinylated aptamer of many times, many times
Part can significantly enhance multivalence and combine effect, thus greatly improves the targets identification ability of carrier, it is achieved higher
Target cell binding ability, and greatly facilitate the internalization of cell, the imaging and treatment improving tumor had significantly
Effect.The adjustable targeting vector of this adjustable length, number of ligands, has a wide range of applications clinically.
(2) the invention provides a kind of targeting vector, built the structure of long-chain by the method for self assembly, it is only necessary to by pre-
First design puts into reactant and just can assemble by spontaneous carrying out, it is to avoid complicated synthesis step, professional synthesis knowledge and have
The use of machine solvent；Secondly, the material of structure is the biomaterials such as DNA, Streptavidin, due to the bio-compatible that they are good
Property and degradability make prepared nanometer Scolopendra also have high-biocompatibility and biodegradability.
(3) the invention provides a kind of targeting vector, due to the degradability of biomaterial, it is easy to by blood as carrier
Enzymatic degradation in liquid, thus lose the function of carrier.The present invention is through dexterous design, it is thus achieved that has good antienzyme and cuts energy
The targeting vector of power, and do not lose selectivity, good stability under the physiological environment of simulation.
(4) the invention provides a kind of targeting vector, can be prepared from by different types of nucleic acid, the special knowledge of utilizable energy
The aptamer of other various disease cell is prepared thus is realized personalized treatment, can be used for transporting diversified bio-imaging
Agent or drug molecule, have the highest universality.
(5) the invention provides a kind of targeted drug, including targeting vector and medicine, targeting vector has the longest trunk
Can be used to by the way of Non-covalent binding, it is achieved on targeting vector, load drug molecule.The present invention utilizes medicine with double
Specific bond between chain DNA, devises a lot of binding sites on the trunk of targeting vector, so that the longest nanometer
Can be in conjunction with a lot of drug molecules in Scolopendra structure.Combine the targeting vector of medicine, can be used for the targeted therapy of cancerous cell.This
The targeting vector of invention utilizes adjustable nanometer Scolopendra length and number of ligands, it is thus achieved that a series of tie the most by force with target cell
The carrier of conjunction ability, has the biggest drug loading.The targeted drug of the present invention has the specific toxicity to target cell simultaneously, and right
Non-target cell lethal the most less, thus improve the maximum tolerated concentration at non-target cell Chinese medicine；Reduce in health
The toxicity of cell or tissue medicine, thus decrease side effect and improve therapeutic effect.
(6) the invention provides a kind of targeted probes, including targeting vector and bio-imaging agent, can be used for the target of cancerous cell
To detection.
Accompanying drawing explanation
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is carried out clear, complete description.
Fig. 1 is the targeting vector synthesis schematic diagram of the embodiment of the present invention 1.
Fig. 2 is the targeting vector characterization result figure of the embodiment of the present invention 1；Wherein Fig. 2 A is initiation chain and the core of variable concentrations
The agarose gel electrophoresis analysis result of the double-strand poly ribonucleic acid that acid monomers H1, nucleic acid monomer H2 are formed；Fig. 2 B is for being formed
Agarose electrophoretic analysis result after targeting vector；Fig. 2 C is by the fluorescence polarization phenogram to produced targeting vector.
Fig. 3 is to use agarose gel electrophoresis to analyze the targeting vector of the embodiment of the present invention 1 stability result in enzyme
Figure.
Fig. 4 is that the binding ability that target cell is strong is tied by the targeting vector using flow cytometry to characterize the embodiment of the present invention 1
Fruit figure；Fig. 4 A is that the length of targeting vector affects result to binding ability；Fig. 4 B be on targeting vector Zy1 bar number to combining energy
Power affect result.
Fig. 5 is that flow cytometry characterizes the Zy1 binding ability result figure strong to target cell.
Fig. 6 is the targeted probes selectivity to target cell of flow cytometry embodiments of the invention 4；Fig. 6 A is 4
The targeted probes flow cytometry figure to target cell identification in buffer is combined at DEG C；Fig. 6 B is to combine buffer at 4 DEG C
The middle targeted probes flow cytometry figure to non-target cell identification；Fig. 6 C is the targeted probes combining in buffer at 37 DEG C
Flow cytometry figure to target cell identification；Fig. 6 D is that non-target cell is known by the targeted probes combining in buffer at 37 DEG C
Other flow cytometry figure；Fig. 6 E is the fluidic cell to target cell identification of the targeted probes in cell culture medium at 37 DEG C
Art analysis chart；Fig. 6 F is the flow cytometry figure to non-target cell identification of the targeted probes in cell culture medium at 37 DEG C.
Fig. 7 is the laser confocal imaging analysis chart of the targeted probes of the embodiment of the present invention 6；Fig. 7 A is that targeted probes is hatched
Cell fluorescence intensity；Fig. 7 B is the cell fluorescence intensity that Zy1 is hatched.
Fig. 8 is the laser confocal imaging analysis chart of the targeted probes of different price in the embodiment of the present invention 7.
Fig. 9 is the spectrofluorimetry figure of the targeted drug in the embodiment of the present invention 9.
Figure 10 is the laser scanning co-focusing microscope image of the targeted drug in the embodiment of the present invention 10；Thin including target
The drug treating figure that born of the same parents deliver through targeting vector；Target cell processes figure through free drug；Non-target cell delivers through targeting vector
Drug treating figure；Non-target cell processes figure through free drug.
Figure 11 is the targeted drug MTT experiment collection of illustrative plates of the embodiment of the present invention 11, and Figure 11 A is the targeted drug work to target cell
By result；Figure 11 B is the targeted drug exercising result to non-target cell；Figure 11 C is the MTT result of targeted drug itself.
Detailed description of the invention
Below in conjunction with Figure of description and concrete preferred embodiment, the invention will be further described, but the most therefore and
Limit the scope of the invention.
Embodiment
Material and instrument employed in following example are commercially available.
Embodiment 1
The targeting vector of a kind of present invention, sees Fig. 1, including double-strand poly ribonucleic acid and Zy1；Zy1 passes through Streptavidin
It is connected on double-strand poly ribonucleic acid form " nanometer Scolopendra "；Its double center chain poly ribonucleic acid is by biotin labeled nucleic acid
Monomer H1 and biotin labeled nucleic acid monomer H2 alternately hybridizes the trunk of formation " nanometer Scolopendra " by causing chain after causing；Body
Dry both sides are connected to the biotin labeled Zy1 of specific recognition targeted cells, form the foot of " nanometer Scolopendra ".Nucleic acid monomer H1
Sequence be the DNA sequence shown in SED ID NO.1 in table 1, the sequence of nucleic acid monomer H2 is in table 1 shown in SED ID NO.2
DNA sequence, cause chain sequence be the DNA sequence shown in SED ID NO.3 in table 1, the sequence of Zy1 is SED ID in table 1
DNA sequence shown in NO.4.
As can be seen from Figure 1: " nanometer Scolopendra " is made up of " trunk " and " foot " two parts, " trunk " of " nanometer Scolopendra "
Through design, can load chemotherapeutics and bio-imaging agent, it is can Selective recognition that its " foot " is positioned at the both sides of " trunk "
Targeting disease cells or the aptamer of tissue, foot can be connected on trunk by adapter easily.
Table 1 be respectively the H1 of nucleic acid monomer H1, FITC labelling, the H2 of nucleic acid monomer H2, FITC labelling, cause chain, Zy1,
The DNA sequence of the Zy-1 of FITC labelling.
Table one: DNA sequence
| Probe | Sequence |
| Nucleic acid monomer H1 | 5 '-Biotin-CGT CGT GCA GCA GCA GCA GCA GCA ACG GCT TGC TGC TGC TGC TGC TGC-3 ' (SED ID NO.1) |
| The H1 of FITC labelling | 5’-Biotin-CGT CGT GCA GCA GCA GCA GCA GCA ACG GCT TGC TGC TGC TGC TGC TGC-FITC-3’ |
| Nucleic acid monomer H2 | 5 '-Biotin-TGC TGC TGC TGC TGC TGC ACG ACG GCA GCA GCA GCA GCA GCA AGC CGT-3 ' (SED ID NO.2) |
| The H2 of FITC labelling | 5’-Biotin-TGC TGC TGC TGC TGC TGC ACG ACG GCA GCA GCA GCA GCA GCA AGC CGT-FITC-3’ |
| Cause chain | 5 '-TGC TGC TGC TGC TGC TGC ACG ACG-3 ' (SED ID NO.3) |
| Zy1 | 5’-ACG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACC GTG AGC GCG T(T)10-Biotin-3 ' (SED ID NO.4) |
| The Zy-1 of FITC labelling | 5’-FITC-ACG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACC GTG AGC GCG T(T)10-Biotin-3’ |
The preparation method of the targeting vector of a kind of embodiment 1, comprises the following steps:
(1) at phosphate buffer, (composition of phosphate buffer is: 10 mM phosphate buffer, 137 mM NaCl, 2.7
MM KCl, pH 7.4, in full text, the uniform component of phosphate buffer causes) in, by nucleic acid monomer H1 and the core of 25 μMs of 25 μMs
Acid monomers H2 mixture mixes, and respectively with 5000 nM, the initiation chain of 2500 nM, 1250 nM, 500 nM, 250 nM is placed in room
Temperature, reacts 24 hours, obtains double-strand poly ribonuclease A 1, A2, A3, A4, A5.
Analyzing double-strand poly ribonuclease A 1, A2, A3, A4, A5 with agarose gel electrophoresis, analysis result sees Fig. 2 A:
Along with the reduction of initiation chain concentration, the molecular weight of the double-strand poly ribonucleic acid of formation is the biggest, and namely length is the longest.Along with drawing
Sending out the decline of chain concentration, double-strand poly ribonucleic acid becomes increasingly to concentrate.It follows that the length of double-strand poly ribonucleic acid
By causing the chain concentration ratio with nucleic acid monomer to control, concentration ratio is the biggest, and the length of double-strand poly ribonucleic acid is the longest.
(2) take the double-strand poly ribonuclease A 5 in step (1), add Streptavidin and aptamer Zy1 is hatched
15 min obtain targeting vector.
Analyzing targeting vector with agarose gel electrophoresis, analysis result sees Fig. 2 B: add bar after Streptavidin and Zy1
Band concentrates on sample well, illustrates to define the product of larger molecular weight, illustrates that " nanometer Scolopendra " targeting vector is formed.
With fluorescence polarization assay nucleic acid monomer H1, nucleic acid monomer H2, double-strand poly ribonucleic acid and targeting vector, analyze knot
Fruit sees Fig. 2 C: by the fluorescence polarization sign to produced targeting vector；Nucleic acid list it is gradually introducing in nucleic acid monomer H1
Body H2, causes chain and Streptavidin and Zy1, it appeared that polarization signal is strengthened, illustrates that spontaneous assembling defines big
Complex, has ultimately formed targeting vector.
Embodiment 2
Investigate the antienzyme of targeting vector of embodiment 1 and cut stability:
Respectively by the double-strand poly ribonuclease A 5 in " nanometer Scolopendra " targeting vector of embodiment 1 and embodiment 1 at 37 DEG C,
React 0,1,2,4,8,12,24 hours at phosphate buffer with the exonuclease III of 0.05 unit/ μ L, with 20 mM EDTA
Stopped reaction.
Analyzing products therefrom with agarose gel electrophoresis, analysis result sees Fig. 3: targeting vector at exonuclease III
Still can keep after processing 2 hours complete (the seeing Fig. 3 A) of structure, and the most degradable (ginseng of double-strand poly ribonuclease A 5
See Fig. 3 B), illustrate that targeting vector has preferable antienzyme and cuts stability.
Embodiment 3
A kind of targeted probes, including targeting vector and bio-imaging agent, wherein targeting vector is the targeting vector of embodiment 1, raw
Thing preparation is that FITC, FITC are loaded on the part of targeting vector by covalent modification.
A kind of preparation method of the targeted probes of the present embodiment:
(1) in phosphate buffer, by the nucleic acid monomer H2 mixture mixing of the nucleic acid monomer H1 of 25 μMs and 25 μMs, respectively with
The initiation chain of 5000 nM, 2500 nM, 1250 nM, 500 nM, 250 nM is placed in room temperature, reacts 24 hours, obtains double-strand poly
Ribonucleic acid B1, B2, B3, B4, B5.
(2) in double-strand poly ribonucleic acid B1, B2, B3, B4, B5, all add the Streptavidin of 50 μMs and 50 μMs
The Zy1 that marked fluorescence molecule (fluorescence molecule is FITC) obtains targeted probes b1, b2, b3, b4, b5.
(3) taking targeted probes b1 in step (2), b2, b3, b4, b5 respectively, stepwise dilution becomes variable concentrations (final
Zy1 concentration is respectively 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0 nM).Targeting after dilution is visited
Pin and target cell SMMC-7721 cell are hatched half an hour on ice, and with lavation buffer solution, (composition of lavation buffer solution is: 10 mM
Phosphate buffer, 137 mM NaCl, 2.7 mM KCl, 250 mM glucoses, 5 mM MgCl2, pH 7.4；In full
The uniform component of lavation buffer solution causes) wash away free targeting vector, by the cell of gained through flow cytometry cell
Average fluorescent strength.Binding ability by the concentration (X) of average fluorescent strength (Y) and Zy1 according to formula Y=Bmax X/(Kd
+ X).
Analysis result sees the Zy1 number of Fig. 4 A: each targeting vector and becomes positive with double-strand poly ribonucleic acid length
Close, thus obtain the targeting vector of different length.At given nucleic acid monomer H1 and the nucleic acid monomer H2 of 25 μMs by 25 μMs
Under, the initiation chain (Trigger) of low concentration obtains longer " nanometer Scolopendra " (targeting vector), and result shows longer " nanometer
Scolopendra " induce higher binding ability, therefore, binding ability increases along with the length of targeting vector and strengthens.Drawing of its optimum
Sending out chain concentration is 250 nM.
Embodiment 4
A kind of targeted probes, including targeting vector and bio-imaging agent, wherein targeting vector is the targeting vector of embodiment 1, raw
Thing preparation is that FITC, FITC are loaded on the part of targeting vector by covalent modification.
A kind of preparation method of the targeted probes of the present embodiment:
(1) in phosphate buffer, by the nucleic acid monomer H1 of 25 μMs and the nucleic acid monomer H2 mixture mixing of 25 μMs, with 250
The initiation chain of nM is placed in room temperature, reacts 24 hours, obtains double-strand poly ribonucleic acid.
(2) double-strand poly ribonucleic acid is equally divided into 5 parts, adds the Streptavidin of concentration same concentrations and marked
The Zy1 of fluorescence molecule, their concentration is respectively 0.25 μM, 1.25 μMs, 2.5 μMs, 5 μMs, 12.5 μMs, 25 μMs, 50 μMs
Obtain targeted probes c1, c2, c3, c4, c5.
(3) taking targeted probes c1 in step (2), c2, c3, c4, c5 respectively, stepwise dilution becomes variable concentrations (final
Zy1 concentration is respectively 20,10,5,2,1,0.5,0.2,0 nM), by targeted probes with target cell SMMC-7721 cell on ice
Hatch half an hour, wash away free nanometer Scolopendra with lavation buffer solution, by the cell of gained through flow cytometry cell
Average fluorescent strength.Binding ability by the concentration (X) of average fluorescent strength (Y) and Zy1 according to formula Y=Bmax X/(Kd
+ X).
Analysis result sees Fig. 4 B: at the nucleic acid monomer H1 of given 25 μMs, the nucleic acid monomer H2 of 25 μMs and 250 nM
Causing under chain, add Streptavidin and the Zy1 of variable concentrations, it is thus achieved that the targeted probes of different number Zy1, each targeting is visited
In pin, Zy1 number becomes positive correlation with the concentration of Zy1, thus obtains the targeted probes of different number Zy1.Result shows greater number
The targeted probes of Zy1 induces higher binding ability；Binding ability along with in targeted probes the increase of Zy1 number and strengthen.Its
Optimum Streptavidin and Zy1 concentration are 50 μMs, by the optimum named Zy1-Nces of targeted probes.
Fig. 5 is that flow cytometry characterizes the Zy1 binding ability to target cell SMMC-7721 cell.Zy1 is diluted to difference
Concentration (100,50,20,10,5,2,1,0 nM) respectively with target cell SMMC-7721 Cell binding, flow cytometer measures average
Fluorescence intensity, calculating its affinity is 7.55 ± 1.10 nM.
Embodiment 5
A kind of targeted probes, including targeting vector and bio-imaging agent, wherein targeting vector is the targeting vector of embodiment 1, raw
Thing preparation is that FITC, FITC are loaded on the part of targeting vector by covalent modification.
A kind of preparation method of the targeted probes of the present embodiment:
(1) in phosphate buffer, by the nucleic acid monomer H1 of 25 μMs and the nucleic acid monomer H2 mixture mixing of 25 μMs, with 250
The initiation chain of nM is placed in room temperature, reacts 24 hours, obtains double-strand poly ribonucleic acid.
(2) add concentration at double-strand poly ribonucleic acid be the Streptavidin of 50 μMs and marked fluorescence molecule
Zy1 obtains targeted probes.
(3) by targeted probes respectively with target cell SMMC-7721 cell and non-target cell L02 cell incubation 30 min, adjust
Save incubation temperature and hatch environment；Then free nanometer Scolopendra is washed away, by the cell of gained through streaming with lavation buffer solution
The average fluorescent strength of cytometry cell.
Investigating result and seeing Fig. 6: Fig. 6 A is that to combine targeted probes in buffer solution at 4 DEG C thin to the streaming of target cell identification
Born of the same parents' art analysis chart；Fig. 6 B is to combine the targeted probes flow cytometry figure to non-target cell identification in buffer solution at 4 DEG C；
Fig. 6 C is to combine the targeted probes flow cytometry figure to target cell identification in buffer solution at 37 DEG C, and Fig. 6 D is at 37 DEG C
In conjunction with the flow cytometry figure to non-target cell identification of the targeted probes in buffer solution；Fig. 6 E is cell culture medium at 37 DEG C
The targeted probes flow cytometry figure to target cell identification in (Ox blood serum containing 10%), Fig. 6 F is that at 37 DEG C, cell is cultivated
The targeted probes flow cytometry figure to non-target cell identification in base (Ox blood serum containing 10%).Comparison diagram 6 is respectively schemed, table
Bright: flow cytometry shows under experimental conditions, targeted probes remains the aptamer choosing to targeting disease cells
Select identity.
Embodiment 6
A kind of targeted probes, including targeting vector and bio-imaging agent, wherein targeting vector is the targeting vector of embodiment 1, raw
Thing preparation is that FITC, FITC are loaded on the part of targeting vector by covalent modification.
A kind of preparation method of the targeted probes of the present embodiment:
(1) in phosphate buffer, by the nucleic acid monomer H1 of 25 μMs and the nucleic acid monomer H2 mixture mixing of 25 μMs, with 250
The initiation chain of nM is placed in room temperature, reacts 24 hours, obtains double-strand poly ribonucleic acid.
(2) in double-strand poly ribonucleic acid, add the Streptavidin that concentration is 50 μMs and the labelling that concentration is 50 μMs
The Zy1 of fluorescence molecule obtains targeted probes.
(3) targeted probes and Zy1 are hatched at cell culture incubator with target cell SMMC-7721 cell, respectively at 15 min,
30 min, 1 h, 2 h, take out after 3 h, wash away free targeting vector and Zy1 with lavation buffer solution, by the cell warp of gained
Co-focusing imaging analyzes cell surface or the fluorescence signal intensity of cell interior.
Testing result sees Fig. 7: under the same conditions, and the cell fluorescence intensity that targeted probes (such as Fig. 7 A) is hatched compares Zy1
(such as Fig. 7 B) is higher, and this analysis can verify that targeting vector has more preferable internalization effect than free Zy1, and targeted probes can promote target
The endocytosis of cell, this is using as the theoretical basis being improved drug delivery efficiency by targeting vector.
Embodiment 7
A kind of targeted probes, including targeting vector and bio-imaging agent, wherein targeting vector is the targeting vector of embodiment 1, raw
Thing preparation is that FITC, FITC are loaded on the part of targeting vector by covalent modification.
A kind of preparation method of the targeted probes of the present embodiment:
(1) in phosphate buffer, by the nucleic acid monomer H1 of 25 μMs and the nucleic acid monomer H2 mixture mixing of 25 μMs, with 250
The initiation chain of nM is placed in room temperature, reacts 24 hours, obtains double-strand poly ribonucleic acid.
(2) add concentration at double-strand poly ribonucleic acid be the Streptavidin of 250 nM and marked fluorescence molecule
Zy1 obtains targeted probes.
(3) targeted probes of fluorescence molecule and target cell SMMC-7721 cell and non-by Non-covalent binding in step (2)
Target cell L02 hatches 2 h at cell culture incubator respectively, with lavation buffer solution (lavation buffer solution in full is same) eluting
Fall free " nanometer Scolopendra ", by the cell of gained through analyzing cell surface or cell interior with laser scanning co-focusing microscope
Fluorescence signal intensity.Investigate the most after the same method simultaneously the Zy1-Nces(nucleic acid monomer H1 concentration of embodiment 4 be 25 μMs,
Nucleic acid monomer H2 concentration is 25 μMs, cause chain concentration is 250 nM, Streptavidin concentration is 50 μMs and marked fluorescence and divides
The concentration of the Zy1 of son is 50 μMs).
Analysis result sees the intracellular targeting than the present embodiment of Fig. 8: the Zy1-Nces target cell SMMC-7721 hatched and visits
Needle set has higher fluorescence, and its more preferable endocytosis effect is described, and the most glimmering for both compared with control cells L02 cells
Light, illustrates both can not enter compared with control cells.Show to depend on the Zy1 of more high price in the internalization of targeted probes.It is simultaneously targeting
Probe does not lose its selectivity after embedding fluorescent dye, has the specific recognition ability to target cell.
Embodiment 8
A kind of targeted probes, including targeting vector and bio-imaging agent, wherein targeting vector is the targeting vector of embodiment 1, raw
Thing preparation is that SYBR Green I, SYBR Green I is loaded on targeting vector by Non-covalent binding.
Embodiment 9
The medicine of a kind of present invention, including targeting vector and medicine, wherein targeting vector is the targeting vector of embodiment 1, medicine
Molecule is amycin.Amycin is loaded on the double-strand poly ribonucleic acid of targeting vector by Non-covalent binding mode.
The preparation method of the medicine of a kind of the present embodiment, comprises the following steps:
(1) in phosphate buffer, by the nucleic acid monomer H1 of 25 μMs and the nucleic acid monomer H2 mixture mixing of 25 μMs, with 250
The initiation chain of nM is placed in room temperature, reacts 24 hours, obtains double-strand poly ribonucleic acid.
(2) take the double-strand poly ribonucleic acid in step (1), add concentration and be Streptavidin and the nucleic acid of 50 μMs
Aptamers Zy1 obtains targeting vector.To cause chain for corresponding targeting vector concentration, step by step targeting vector dilution is obtained concentration and divide
It is not 0.2 nM, 0.4 nM, 1 nM, 2 nM, 4 nM, the targeting vector of 10 nM.
(3) amycin that concentration is 2 μMs and concentration are respectively 0 nM, 0.2 nM, 0.4 nM, 1 nM, 2 nM, 4 nM,
The targeting vector of 10 nM is hatched in phosphate buffer, by fluorophotometer, compares same concentrations drug level with the denseest
Degree targeting vector hatches the fluorescence intensity of rear gained mixture Chinese medicine.
Seeing Fig. 9, spectrofluorimetry shows that medicine (amycin, English abbreviation is Dox) fluorescence is being loaded into 4 nM
Targeting vector after be quenched, and be 500: 1 according to the molar concentration ratio of drug molecule and targeting vector, Scolopendra has nanometer
The highest drug load.
Embodiment 10
The medicine of a kind of present invention, including targeting vector and medicine, wherein targeting vector is the targeting vector of embodiment 1, medicine
Molecule is amycin.Amycin is loaded on the double-strand poly ribonucleic acid of targeting vector by Non-covalent binding mode.
The preparation method of the medicine of a kind of the present embodiment, comprises the following steps:
(1) in phosphate buffer, by the nucleic acid monomer H1 of 25 μMs and the nucleic acid monomer H2 mixture mixing of 25 μMs, with 250
The initiation chain of nM is placed in room temperature, reacts 24 hours, obtains double-strand poly ribonucleic acid.
(2) take the double-strand poly ribonucleic acid in step (1), add concentration and be Streptavidin and the nucleic acid of 50 μMs
Aptamers Zy1 obtains targeting vector.With initiation chain for corresponding targeting vector concentration, by the concentration ratio of amycin with targeting vector
The ratio of 500: 1, adds 125 μMs of amycin, obtains containing the targeting vector that doxorubicin concentration is 2 μMs.
(3) by thin with target cell SMMC-7721 cell and compared with control cells L02 containing the targeting vector that doxorubicin concentration is 2 μMs
Born of the same parents are hatched in cell culture fluid, after cultivating 2 hours in cell culture incubator, and laser scanning co-focusing microscope image.
Simultaneously according to identical condition by amycin and target cell SMMC-7721 cell and compared with control cells L02 cell at cell culture fluid
In hatch.
Laser scanning co-focusing microscope imaging sees Figure 10, and the first row is that target cell is at the medicine that targeting vector delivers
Reason figure, the second row be target cell through free drug process figure, the third line is the drug treating that non-target cell delivers through targeting vector
Figure, fourth line is that non-target cell is through free drug process figure.Contrast Figure 10 understand: by be mounted with fluorescence drug molecule (as
Amycin) on targeting vector, drug selectivity can be transported target cell SMMC-7721 cell by targeting vector, and can not
Transport is to compared with control cells L02 cell；Both drug release can be monitored in real time by the character that medicine fluorescence signal after release strengthens,
Targeting vector can be shown again to the Selective recognition of target cell and medicament transport；And amycin does not has selectivity freely.
Embodiment 11
The medicine of a kind of present invention, including targeting vector and medicine, wherein targeting vector is the targeting vector of embodiment 1, medicine
Molecule is amycin.Amycin is loaded on the double-strand poly ribonucleic acid of targeting vector by Non-covalent binding mode.
The preparation method of the medicine of a kind of the present embodiment, comprises the following steps:
(1) at phosphate buffer, (composition of phosphate buffer is: 10 mM phosphate buffer, 137 mM NaCl, 2.7
MM KCl, pH 7.4) in, by the nucleic acid monomer H1 of 25 μMs and the nucleic acid monomer H2 mixture mixing of 25 μMs, with 250 nM's
Cause chain to be placed in room temperature, react 24 hours, obtain double-strand poly ribonucleic acid.
(2) take the double-strand poly ribonucleic acid in step (1), add concentration and be respectively Streptavidin and the core of 50 μMs
Acid aptamers Zy1 obtains targeting vector.With initiation chain for corresponding targeting vector concentration, by the concentration ratio of amycin with targeting vector
The ratio of 500: 1, adds 125 μMs of amycin and obtains mixed solution.With the concentration of amycin for standard by dilute for above-mentioned mixed solution
Release；Obtaining containing doxorubicin concentration is 0.05 μM, 0.1 μM, 0.25 μM, 1 μM, 2.5 μMs, the targeting vector of 5 μMs.
(3) will contain doxorubicin concentration is to be not 0.05 μM, 0.1 μM, 0.25 μM, 1 μM, 2.5 μMs, the targeting of 5 μMs
Carrier is hatched in cell culture fluid with target cell SMMC-7721 cell and compared with control cells L02 cell, through in cell culture incubator
After cultivating 2 hours, remove the cell culture fluid in supernatant, replaced with fresh medium, and be then incubated for 48 h.Then try with MTT
Agent test cell multiplication rate, with the testing drug toxicity size to target cell and non-target cell.
Test result sees Figure 11, Figure 11 A and shows that the medicine transported through targeting vector (nanometer Scolopendra) has target cell
(SMMC-7721) specific cell growth inhibition function, Figure 11 B shows non-target cell (L02) then rejection ability less；Figure
11C shows the concrete well biocompatibility of targeting vector itself.Shown by MTT experiment, through the medicine of targeting vector transport
(including amycin) has target cell (SMMC-7721) specific cell growth inhibition function, and to non-target cell (L02)
Then rejection ability is less.
The above, be only presently preferred embodiments of the present invention, and the present invention not makees any pro forma restriction.Though
So the present invention discloses as above with preferred embodiment, but is not limited to the present invention.Any it is familiar with those skilled in the art
Member, in the case of without departing from the spirit of the present invention and technical scheme, may utilize in method and the technology of the disclosure above
Hold and technical solution of the present invention is made many possible variations and modification, or be revised as the Equivalent embodiments of equivalent variations.Therefore,
Every content without departing from technical solution of the present invention, according to the present invention technical spirit to made for any of the above embodiments any simply
Amendment, equivalent, equivalence change and modification, all still fall within the range of technical solution of the present invention protection.
Claims (10)
1. a targeting vector, it is characterised in that described targeting vector includes double-strand poly ribonucleic acid and part；Described part
It is connected on described double-strand poly ribonucleic acid by adapter；Described double-strand poly ribonucleic acid is by nucleic acid monomer H1 and nucleic acid
Monomer H2 is by causing chain alternately to hybridize formation after causing, and the sequence of described nucleic acid monomer H1 is the sequence shown in SED ID NO.1
Row, the sequence of described nucleic acid monomer H2 is the sequence shown in SED ID NO.2.
Targeting vector the most according to claim 1, it is characterised in that described nucleic acid monomer H1 is the nucleic acid of biotin modification
Monomer H1, described nucleic acid monomer H2 are the nucleic acid monomer H2 of biotin modification, and described part is the part of biotin modification, described
Adapter is Streptavidin；The sequence of described initiation chain is the sequence shown in SED ID NO.3.
Targeting vector the most according to claim 2, it is characterised in that described part is Zy1, the sequence of described Zy1 is SED
Sequence shown in ID NO.4.
Targeting vector the most according to any one of claim 1 to 3, it is characterised in that the concentration of described initiation chain is
250nM～5000nM；The concentration of described nucleic acid monomer H1 is 25 μMs；The concentration of described nucleic acid monomer H2 is 25 μMs；Described part
Concentration be 0.25 μM～50 μMs.
5. a targeted drug, it is characterised in that include that the targeting according to any one of drug molecule and Claims 1 to 4 carries
Body, described drug molecule is loaded on described targeting vector by covalent modification or Non-covalent binding.
Targeted drug the most according to claim 5, it is characterised in that described drug molecule is amycin.
7. the application in preparing tumor-targeting drug of the targeted drug described in a claim 5 or 6.
8. a targeted probes, it is characterised in that include the targeting according to any one of bio-imaging agent and Claims 1 to 4
Carrier, described bio-imaging agent is loaded on described targeting vector by covalent modification or Non-covalent binding.
Targeted probes the most according to claim 8, it is characterised in that described bio-imaging agent is fluorescence molecule or SYBR
Green I。
10. the targeted probes described in a claim 8 or 9 is preparing tumor imaging and the detectable of cancerous cell targeting detection
Application in box.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107764804A (en) * | 2017-10-09 | 2018-03-06 | 郑州大学 | A kind of self assembly poly- HRP probes, preparation method, kit and its application |
| CN111330022A (en) * | 2018-12-18 | 2020-06-26 | 深圳先进技术研究院 | Tumor-targeted DNA fluorescent probe and preparation method and application thereof |
| CN114681619A (en) * | 2020-12-30 | 2022-07-01 | 上海交通大学医学院附属仁济医院 | Aptamer compound based on Biotin-SA and application thereof |
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| CN104894243A (en) * | 2015-05-12 | 2015-09-09 | 湖南大学 | Nucleic acid aptamer probe and device for cancer cell detection and application of nucleic acid aptamer probe and device for cancer cell detection |
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| CN103301481A (en) * | 2013-06-24 | 2013-09-18 | 湖南大学 | Target nucleic acid drug delivery system and application thereof |
| CN104894243A (en) * | 2015-05-12 | 2015-09-09 | 湖南大学 | Nucleic acid aptamer probe and device for cancer cell detection and application of nucleic acid aptamer probe and device for cancer cell detection |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107764804A (en) * | 2017-10-09 | 2018-03-06 | 郑州大学 | A kind of self assembly poly- HRP probes, preparation method, kit and its application |
| CN107764804B (en) * | 2017-10-09 | 2020-02-07 | 郑州大学 | Self-assembled poly HRP probe, preparation method, kit and application thereof |
| CN111330022A (en) * | 2018-12-18 | 2020-06-26 | 深圳先进技术研究院 | Tumor-targeted DNA fluorescent probe and preparation method and application thereof |
| CN114681619A (en) * | 2020-12-30 | 2022-07-01 | 上海交通大学医学院附属仁济医院 | Aptamer compound based on Biotin-SA and application thereof |
| CN114681619B (en) * | 2020-12-30 | 2023-08-29 | 上海交通大学医学院附属仁济医院 | Aptamer complex based on Biotin-SA and application thereof |
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| CN106267219B (en) | 2019-03-29 |
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