CN108753770A - A kind of gene nano probe and its preparation method and application for lung cancer-targeted treatment - Google Patents
A kind of gene nano probe and its preparation method and application for lung cancer-targeted treatment Download PDFInfo
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- CN108753770A CN108753770A CN201810559188.1A CN201810559188A CN108753770A CN 108753770 A CN108753770 A CN 108753770A CN 201810559188 A CN201810559188 A CN 201810559188A CN 108753770 A CN108753770 A CN 108753770A
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Abstract
The gene nano probe and its preparation method and application that the present invention relates to a kind of for lung cancer-targeted treatment, belongs to biochemical nano field of material technology.Gene nano probe provided by the present invention for lung cancer-targeted treatment includes RNA nano-hydrogels and the DOX and/or TMPyP4 that are supported on the RNA nano-hydrogels；The RNA nano-hydrogels include single-stranded DNA self assemblies, cyclic DNA profiling and targeting aptamers.Gene nano probe specificity recognition capability of the present invention is strong, and targeting is accurate, and toxic side effect is small, big to the killing intensity of lung carcinoma cell, and therapeutic effect is good, has great potentiality in the application of lung cancer therapy.
Description
Technical field
The present invention relates to biochemical nano field of material technology, and in particular to a kind of gene for lung cancer-targeted treatment
Nano-probe and its preparation method and application.
Background technology
Lung cancer is one of most common malignant tumour, and the death rate, which occupy all cancers, leads to dead forefront.In recent years,
Lung cancer is even more that can not be ignored to the threat of human health, more seriously.Therefore effective therapeutic strategy is studied and defined to improve
It is very important to the treatment dynamics of lung cancer.
Treatment for lung cancer, current existing treatment technology mostly use operative treatment and radiotherapy.And operative treatment
It is big that there are wounds, and risk is high, the defects of being also easy to produce complication, and damage of the radiotherapy for body normal structure is then larger,
And treatment cycle is relatively long.Simple leans on a kind of therapeutic modality, function and effect bad.And it is easy in the same for the treatment of cancer
When, other injuries are caused to body, cause complication, exists in specific experimental study and clinical application and can not be ignored
The drawbacks of.
Invention content
The purpose of the present invention is to provide a kind of gene nano probe for lung cancer-targeted treatment and preparation method thereof and
Using.Gene nano probe specificity recognition capability of the present invention is strong, and targeting is accurate, and toxic side effect is small, to lung carcinoma cell
Killing intensity is big, and therapeutic effect is good, has great potentiality in the application of lung cancer therapy.
The present invention provides a kind of gene nano probe for lung cancer-targeted treatment, the gene nano probe includes
RNA nano-hydrogels and the DOX and/or TMPyP4 being supported on the RNA nano-hydrogels；
The RNA nano-hydrogels include single-stranded DNA self assemblies, cyclic DNA profiling and targeting aptamers；
The single-stranded DNA self assemblies include nucleotide sequence ASM-DNA-1, nucleotides sequence as shown in SEQ ID NO.1
Arrange ASM-DNA-2 and the nucleotide sequence ASM-DNA-3 as shown in SEQ ID NO.3 as shown in SEQ ID NO.2, three
DNA is single-stranded respectively with, for node, both ends complementary pairing two-by-two constitutes Trident Type self-assembled structures among sequence；
The cyclization DNA profiling includes the phosphorylated nucleotide sequence Three- as shown in SEQ ID NO.4 in 5 ' ends
The phosphorylated ends nucleotide sequence Three-miR 34a as shown in SEQ ID NO.5 and 5 ' in let7a, 5 ' ends are phosphorylated
Nucleotide sequence such as SEQ ID NO.6 shown in it is one or more in Three-miR 145, the cyclization DNA profiling is logical
The T7 promoter complementary series at sequence both ends is crossed, it is mutual with the T7 promoter sequences of the branches end of the Trident Type self-assembled structures
It mends and combines；
The targeting aptamers are 5 ' of sequence described in SEQ ID NO.7 terminal modified to have fluorophor, 3 ' terminal modified courages solid
The substance of alcohol；The targeting aptamers are combined by T7 promoters complementary series with cyclic DNA profiling.
Preferably, the grain size of the RNA nano-hydrogels is 80~200nm.
Preferably, the fluorophor includes FAM, FITC or Cy3.
Preferably, the carrying volume ratio of RNA nano-hydrogels and DOX are (0~100) in the gene nano probe：
100；The carrying volume ratio of the RNA nano-hydrogels and TMPyP4 are (0~25)：5.
The present invention also provides the preparation methods of gene nano probe described in above-mentioned technical proposal, include the following steps：
1) single-stranded mixed in Tris-HCl buffer solutions with cyclic DNA profiling of DNA self assemblies is made annealing treatment, it is described
The condition of annealing is：95 DEG C, 5min, then 16~28 DEG C are down to the speed of 0.5 DEG C/min, obtain Trident Type self assembly knot
Structure carrier；
2) the Trident Type self-assembled structures carrier that step 1) obtains is mixed with T4 ligases and T4 ligase buffer solutions, 16
DEG C carry out annulation, obtain the Trident Type self-assembled structures carrier containing circular template；
3) the Trident Type self-assembled structures carrier containing circular template that step 2) obtains is mixed with nucleotide triphosphoric acid
Liquid, RNase inhibitor and T7 polymerases and the mixing of T7 polymerase buffers, 37 DEG C of progress rolling ring 0.5~3h of responsive transcription, obtain
In conjunction with the carrier of cyclic DNA；
4) carrier for the combination cyclic DNA that step 3) obtains is mixed with targeting aptamers, is made annealing treatment, it is described to move back
Fire processing condition be：65 DEG C, 5min, then 16~28 DEG C are down to the speed of 0.5 DEG C/min, obtain RNA nano-hydrogels；
5) the RNA nano-hydrogels that step 4) obtains are mixed with DOX and/or TMPyP4,37 DEG C of reaction 2h obtain gene
Nano-probe.
Preferably, the single-stranded mixed volume ratio with cyclic DNA profiling of the DNA self assemblies is 1:(0.8~1.2).
Preferably, it is 1 in conjunction with the mixed volume ratio of the carrier of cyclic DNA and targeting aptamers described in step 4):(90
~110).
Preferably, in step 5) the gene nano probe carrying volume ratio of RNA nano-hydrogels and DOX be (0~
100)：100；The carrying volume ratio of the RNA nano-hydrogels and TMPyP4 are (0~25)：5.
The present invention also provides preparation methods described in gene nano probe described in above-mentioned technical proposal or above-mentioned technical proposal
Application of the obtained gene nano probe in preparing targeted inhibition lung carcinoma cell drug.
Preferably, the lung carcinoma cell includes A549 cell lines.
Preferably, the effective dose of gene nano probe is 30~150 μ L in the drug.
The present invention provides a kind of gene nano probes for lung cancer-targeted treatment.Gene nano probe of the present invention
By combining RNA nano-hydrogels and the DOX being supported on the RNA nano-hydrogels and/or TMPyP4, collection base is formd
Because of the multiple action system for the treatment of, chemotherapy and/or optical dynamic therapy, therapeutic effect is more preferable.RNA nano-hydrogel energy
Enough so that there is the gene nano probe targets identification effect and gene therapy effect, DOX can play Chemo-Therapy to lung cancer
Treatment acts on, and TMPyP4 can play photodynamic therapy to lung cancer.Gene nano probe specificity recognition capability of the present invention
By force, targeting is accurate, and toxic side effect is small, big to the killing intensity of lung carcinoma cell, and therapeutic effect is good.Test result shows the present invention
Gene nano probe can to targeting cell can precisely identify, and have good synergistic therapeutic action.
Description of the drawings
Fig. 1 is that gene nano probe provided by the invention synthesizes schematic diagram；
Fig. 2 is the TEM phenograms of RNA nano-hydrogels provided by the invention；
Fig. 3 is optimization figure of the RNA nano-hydrogels provided by the invention to DOX and TMPyP4 bearing capacities；
Fig. 4 is that RNANHs-T provided by the invention and RNANHs acts on the flow cytomery figure after cell；
Fig. 5 is that RNANHs-D-T provided by the invention detects the CLSM of A549 cell therapy mechanism under illumination effect
Figure；
Fig. 6 be RNANHs-D-T provided by the invention and RNA nano-hydrogels, DOX, TMPyP4, RNANHs-D and
After RNANHs-T are respectively acting on A549 cells, the detection figure of cytotoxicity；
Fig. 7 is lung cancer provided by the invention (A549) tumor-bearing mice after PBS (left side) and RNA NHs-D-T (right side) administrations
Living body fluorescent detection figure；
Fig. 8 be the tumour picture provided by the invention being finally stripped out out of Mice Body and by different injections it
The variation diagram of opposite gross tumor volume afterwards.
Specific implementation mode
The present invention provides a kind of gene nano probe for lung cancer-targeted treatment, the gene nano probe includes
RNA nano-hydrogels and the DOX and/or TMPyP4 being supported on the RNA nano-hydrogels；The RNA nano-hydrogels packet
Include single-stranded DNA self assemblies, cyclic DNA profiling and targeting aptamers；
The single-stranded DNA self assemblies include nucleotide sequence ASM-DNA-1, nucleotides sequence as shown in SEQ ID NO.1
Arrange ASM-DNA-2 and the nucleotide sequence ASM-DNA-3 as shown in SEQ ID NO.3 as shown in SEQ ID NO.2, three
DNA is single-stranded respectively with, for node, both ends complementary pairing two-by-two constitutes Trident Type self-assembled structures among sequence；
The cyclization DNA profiling includes the phosphorylated nucleotide sequence Three- as shown in SEQ ID NO.4 in 5 ' ends
The phosphorylated ends nucleotide sequence Three-miR 34a as shown in SEQ ID NO.5 and 5 ' in let7a, 5 ' ends are phosphorylated
Nucleotide sequence such as SEQ ID NO.6 shown in it is one or more in Three-miR 145, the cyclization DNA profiling is logical
The T7 promoter complementary series at sequence both ends is crossed, it is mutual with the T7 promoter sequences of the branches end of the Trident Type self-assembled structures
It mends and combines；
The targeting aptamers are 5 ' of sequence described in SEQ ID NO.7 terminal modified to have fluorophor, 3 ' terminal modified courages solid
The substance of alcohol；The targeting aptamers are combined by T7 promoters complementary series with cyclic DNA profiling.
In the present invention, the DOX is adriamycin, and the present invention does not have the source of adriamycin special restriction, using this
Adriamycin conventional commercial product known to field technology personnel.TMPyP4 of the present invention is photosensitizer, similarly, the present invention
There is no special restriction to the source of photosensitizer TMPyP4, using photosensitizer TMPyP4 routines city well known to those skilled in the art
Sell product.In the present invention, in the gene nano probe carrying volume ratio of RNA nano-hydrogels and DOX be (0~
100)：100, more preferably 80:100；The concentration of DOX of the present invention preferably uses 20 μM.In the present invention, the RNA receives
The carrying volume ratio of rice hydrogel and TMPyP4 are (0~25)：5, more preferably 20:5；The concentration of TMPyP4 of the present invention is excellent
Choosing uses 25 μM.
In the present invention, the nucleotide sequence that above-mentioned DNA self assemblies are single-stranded, cyclic DNA profiling and targeting aptamers are related to
As shown in table 1：
1 gene nano probe correlated series of table
In the present invention, one end of three single-stranded ASM-DNA sequences of DNA self assemblies respectively carries T7 promoter sequences,
The both ends of the cyclization DNA profiling carry the complementary series of T7, and cyclic DNA profiling can be by this complementary series and DNA from group
Fill single-stranded combination.It is each in three-let 7a, three-miR34a and the cyclic DNA template sequences of three-miR 145 3
The self-contained complementary series for having corresponding microRNA, therefore can subsequently generate corresponding hairpin RNA knot respectively by transcription
Phosphate group is contained at 5 ' ends of structure, three kinds of cyclic DNA profilings, and the phosphate group can be such that DNA chain both ends are attached, be conducive to
Cyclization.Targeting aptamers of the present invention introduce the tract with lung carcinoma cell specificity S6 aptamers by base pairing
Section, the sequence one end are modified with fluorophor, and one end is modified with cholesterol, targets the T7 promoter complementary series energy in aptamers
The targeting aptamers are enough made to be incorporated on the hairpin structure after above-mentioned transcription.
The cyclic DNA profiling of RNA nano-hydrogels of the present invention includes that can play what gene regulation acted on
MicroRNA (let-7a and/or miR 34a and/or miR 145), wherein the presence of let-7a and miR 34a can make
Two kinds of proto-oncogenes of bcl-2 and c-my (proto-oncogene can inhibit apoptosis of tumor cells) are significantly lowered, and miR 145 can make Oct-
4 expression are lowered, and the high expression of Oct-4 can also inhibit apoptosis of tumor cells, so Oct-4 is made to lower, then can promote tumour cell
Apoptosis；When a variety of microRNA are existed simultaneously in RNA nano-hydrogels, the effect of enhancing gene therapy can be cooperateed with, and is worked as
When three kinds of microRNA are existed simultaneously in the RNA nano-hydrogels, the regulating and controlling effect of the RNA nano-hydrogels is most strong,
Therapeutic effect is preferably also.Targeting aptamers of the present invention, can to include the aptamers of the cytotropic sequence of target in sequence
Make the hydrogel that there is targets identification effect.In the present invention, the addition for targeting fluorophor in aptamers is conducive to glimmering
Light imaging observation；In the present invention, the fluorophor preferably includes FAM, FITC or Cy3, specifically, in the embodiment of the present invention
It is specifically chosen FAM fluorophors.In the present invention, the modification that the targeting aptamers carry out cholesterol can make it solid using courage
The hydrophobicity of alcohol makes RNA nano-hydrogel structure both shrinks, grain size become smaller.In the present invention, the grain size of the RNA nano-hydrogels
For 80~200nm, most preferably 150nm.
RNA nano-hydrogels itself of the present invention have the ability to form G-quadruplex (G4), Neng Gouwei
TMPyP4 provides carrying site, so that TMPyP4 is passed through physisorption and is directly embedded into RNA nano-hydrogels, in laser
Under (650nm) irradiation condition, TMPyP4 can cause active oxygen in living cells (ROS) molecule to generate, subsequent cell death.DOX energy
It is enough to be combined with RNA nano-hydrogel double center chain GC or CG sequences, RNA nano-hydrogels are directly embedded by physisorption
In double-strand GC or CG base-pair in, to lung carcinoma cell carry out chemotherapy.In the present invention, when the RNA nano-hydrogels
When loading DOX and TMPyP4 simultaneously, the combination of multiple action system enables to gene nano probe to have stronger drug effect, real
Now spend the combination therapy effect of lung cancer.
The present invention also provides the preparation methods of gene nano probe described in above-mentioned technical proposal, include the following steps：
1) single-stranded mixed in Tris-HCl buffer solutions with cyclic DNA profiling of DNA self assemblies is made annealing treatment, it is described
The condition of annealing is：95 DEG C, 5min, then 16~28 DEG C are down to the speed of 0.5 DEG C/min, obtain Trident Type self assembly knot
Structure carrier；
2) the Trident Type self-assembled structures carrier that step 1) obtains is mixed with T4 ligases and T4 ligase buffer solutions, 16
DEG C carry out annulation, obtain the Trident Type self-assembled structures carrier containing circular template；
3) the Trident Type self-assembled structures carrier containing circular template that step 2) obtains is mixed with nucleotide triphosphoric acid
Liquid, RNase inhibitor and T7 polymerases and the mixing of T7 polymerase buffers, 37 DEG C of progress rolling ring 0.5~3h of responsive transcription, obtain
In conjunction with the carrier of cyclic DNA；
4) carrier for the combination cyclic DNA that step 3) obtains is mixed with targeting aptamers, is made annealing treatment, it is described to move back
Fire processing condition be：65 DEG C, 5min, then 16~28 DEG C are down to the speed of 0.5 DEG C/min, obtain RNA nano-hydrogels；
5) the RNA nano-hydrogels that step 4) obtains are mixed with DOX and/or TMPyP4,37 DEG C of reaction 2h obtain gene
Nano-probe.
The synthesis of RNA nano-hydrogels is transcribed based on DNA self assemblies and rolling ring in gene nano probe of the present invention
React (Rollig Circle Transcription, RCT) realization, by physisorption, the RNA nanometers of water-setting
Glue can load DOX and/or TMPyP4.Fig. 1 is that gene nano probe provided by the invention synthesizes schematic diagram.In Fig. 1 of the present invention
Genetic fragment and sequence between be not present any proportionate relationship, picture be intended merely to image signal needs.
The present invention makes annealing treatment single-stranded mixed in Tris-HCl buffer solutions with cyclic DNA profiling of DNA self assemblies,
The condition of the annealing is：95 DEG C, 5min, then 16~28 DEG C, more preferably 25 DEG C are down to the speed of 0.5 DEG C/min,
Obtain Trident Type self-assembled structures carrier.In the present invention, the annealing operation enables to three single-stranded generations of DNA self assemblies
Self assembly forms Trident Type self-assembled structures carrier, specifically, tri- DNA points of ASM-DNA-1, ASM-DNA-2, ASM-DNA-3
It is not node both ends complementary pairing two-by-two with centre, forms the self-assembled structures of " Trident Type " type.In the present invention, described
The pH value of Tris-HCl buffer solutions is preferably 7.8, and the concentration of the buffer solution is preferably 30mM, in the present invention, the Tris-
HCl buffer solutions preferably include the MgCl of 10mM2.In the present invention, the single-stranded mixing with cyclic DNA profiling of the DNA self assemblies
Volume ratio is preferably 1：(0.8~1.2), more preferably 1:1, in the present invention, the DNA self assemblies are single-stranded and at circular DNA mould
Concentration in each comfortable buffer solution of plate is preferably independently 1.0 × 10-4Mol/L~1.0 × 10-7Mol/L, more preferably 1.0 ×
10-6mol/L。
After obtaining Trident Type self-assembled structures carrier, the present invention is by Trident Type self-assembled structures carrier and T4 ligases and T4
Ligase buffer solution mixes, and 16 DEG C of progress annulations obtain the Trident Type self-assembled structures carrier containing circular template.This hair
The bright source to the T4 ligases and its buffer solution does not have special restriction, using conventional commercial T4 ligases and its buffer solution
?.The present invention is to the application method of the T4 ligases also not special restriction, using conventional T4 ligases application method
?.In the present invention, phosphate group is contained at 5 ' ends of three kinds of cyclic DNA profilings, and phosphate group can be such that DNA chain both ends carry out
Connection is conducive to cyclization；And contains T7 in the end of the Trident Type self-assembled structures carrier branch of the single-stranded formation of DNA self assemblies and start
Subsequence into circular DNA template both ends there is the sequence with T7 complementary pairings, the two to interact by base pairing, be formed
Cyclic annular masterplate (circular template is made of respective microRNA complementary series and T7 promoter complementary series respectively), is contained
There is the Trident Type self-assembled structures carrier of circular template.
After obtaining the Trident Type self-assembled structures carrier containing circular template, the present invention is by the Trident Type containing circular template
Self-assembled structures carrier is mixed with nucleotide triphosphoric acid mixed liquor, RNase inhibitor and T7 polymerases and T7 polymerase buffers,
37 DEG C of progress rolling ring responsive transcription 1.5h, obtain the carrier in conjunction with cyclic DNA.The present invention is to nucleotide triphosphoric acid mixed liquor, RNA
The enzyme inhibitor and source of T7 polymerases and its buffer solution, dosage and application method do not have special restriction, using this field skill
Conventional commercial product known to art personnel and conventional amount used, application method.In the present invention, the rolling ring responsive transcription
(RCT) using the Trident Type self-assembled structures carrier containing circular template as template, by 37 DEG C, cyclization is realized in the reaction of 1.5h
The amplification of DNA profiling.This cyclic DNA profiling is augmented with conducive to the combination of targeting aptamers.
After obtaining the carrier in conjunction with cyclic DNA, the present invention mixes the carrier for combining cyclic DNA with targeting aptamers, into
Row annealing, the condition of the annealing are：65 DEG C, 5min, then it is down to 16~28 with the speed of 0.5 DEG C/min, it is more excellent
Choosing is down to 25 DEG C, obtains RNA nano-hydrogels.In the present invention, the carrier of the combination cyclic DNA is mixed with targeting aptamers
Volume ratio be preferably 1:(90~110), more preferably 1：100.In the present invention, the carrier and target of the combination cyclic DNA
It is preferably independently 1.0 × 10 to the respective concentration of aptamers-4Mol/L~1.0 × 10-7Mol/L, more preferably 1.0 × 10- 6mol/Lmol/L.In the present invention, in the annealing process, the T7 targeted in the circular DNA template of aptamers and amplification starts
Subsequence complementation combines, and obtains RNA nano-hydrogels.
After obtaining RNA nano-hydrogels, the present invention mixes RNA nano-hydrogels with DOX and/or TMPyP4, and 37 DEG C anti-
2h is answered, gene nano probe is obtained.In the present invention, the carrying volume ratio of the RNA nano-hydrogels and DOX be (0~
100)：100, more preferably 80：100.In the present invention, the concentration of the DOX is preferably 20 μM.In the present invention, the RNA
The carrying volume ratio of nano-hydrogel and TMPyP4 are preferably (0~25)：5, more preferably 20:5, it is in the present invention, described
The concentration of TMPyP4 is preferably 25 μM.After the present invention reacts 2h at 37 DEG C, preferably product is cleaned, the cleaning is preferably adopted
It is cleaned with DPBS, the number of the cleaning is preferably 2~3 times.
The present invention also provides preparation methods described in gene nano probe described in above-mentioned technical proposal or above-mentioned technical proposal
Application of the obtained gene nano probe in preparing targeted inhibition lung carcinoma cell drug.
In the present invention, the corresponding cell line of the lung cancer preferably includes A549 cell lines.The present invention is verifying the RNA
When the effect of hydrogel, it is preferred to use following methods：The RNA nano-hydrogels are acted on into A549 cell lines：By RNANHs
It is placed in incubator and is incubated with cultured A549 cells, culture environment condition is 37 DEG C, 5%CO2Concentration is incubated certain
After time, coherent detection is carried out.
In the present invention, the effective dose of gene nano probe is 30~150 μ L in the drug.
With reference to specific embodiment to a kind of gene nano probe for lung cancer-targeted treatment of the present invention and
Preparation method and application are further described in detail, and technical scheme of the present invention includes but not limited to following embodiment.
Embodiment 1
The preparation of RNA nano-hydrogels：
Respectively by three DNA for synthesizing Trident Type self-assembled structures carrier it is single-stranded (ASM-DNA-1, ASM-DNA-2,
ASM-DNA-3) and for cyclic Three-let7a, 145 3 kinds of Three-miR 34a, Three-miR is at circular DNA mould
Version mixes, and adds Tris-HCl buffer solutions to be settled to 20 μ L, it is 1 × 10 to make the concentration of above-mentioned substance-6mol/L.It will be upper
State mixed liquor by annealing (95 DEG C, 5min, then it is per minute drop 0.5 DEG C be down to 25 DEG C) after, Trident Type self assembly knot
Structure carrier；
Addition T4 ligases and its buffer solution carry out cyclization under the conditions of being placed in 16 DEG C, obtain the Trident Type containing circular template
Self-assembled structures carrier.
Then nucleotide triphosphoric acid mixed liquor (rNTP), RNase inhibitor, T7 polymerases and its buffer solution is added in 37 DEG C
After lower progress RCT reactions 1.5h, the carrier in conjunction with cyclic DNA is obtained；
With 1：100 volume ratio mixes the carrier for combining cyclic DNA with targeting aptamers, is made annealing treatment, described
The condition of annealing is：65 DEG C, 5min, then 25 DEG C are down to the speed of 0.5 DEG C/min, obtain RNA nano-hydrogels
(RNANHs).Obtained RNA nano-hydrogels are characterized with transmission electron microscope (TEM), characterization result such as Fig. 2 institutes
Show, as seen from the figure, RNA nano-hydrogels are in the hydrogel of nano flower-like, and for average grain diameter in 150nm or so, grain size is more uniform, real
Show it successfully to synthesize.
The preparation of gene nano probe：
Use water dissolution spare respectively DOX and TMPyP4, by RNA nano-hydrogels obtained by the above method and DOX and/or
TMPyP4 is mixed, and the DOX of the TMPyP4 and/or 100 μ L20 μM of every 80 μ LRNA nano-hydrogels load, 25 μM of 20 μ L, 37 DEG C anti-
2h is answered, is acted on by the physical carrier in tetra- serobila results of G- and so that DOX and/or TMPyP4 is combined with RNA nano-hydrogels,
It is cleaned with DPBS, respectively obtains the gene nano probe (RNANHs-D) of load DOX, loads the gene nano probe of TMPyP4
(RNANHs-T) and simultaneously the gene nano probe (RNANHs-D-T) of DOX and TMPyP4 is loaded.
Embodiment 2
Carry out the carrying and optimization of DOX and TMPyP4：
25 μM are taken, the TMPyP4 solution of 5 μ L detects its UV absorption being located at 421nm, is then respectively adding as required
The different volume (0~20 μ L) of RNA nano-hydrogels synthesized by ratio, reacts 2h under the conditions of 37 DEG C after mixing.So
High speed centrifugation takes supernatant to carry out uv absorption spectra detection afterwards.
Equally, aforesaid operations are similar to, the RNANHs of different volumes (0~80 μ L) is added in 20 μM, 100 μ L DOX,
Then with increasing for RNANHs amounts is added, the DOX being loaded into above can increase, the remaining DOX meetings being not loaded on hydrogel
It is free in supernatant, using the fluorescent characteristic of DOX itself, the supernatant after high speed centrifugation is taken to carry out fluoroscopic examination.
The amount of the optimization of the DOX that can be carried on RNANHs and TMPyP4 can be detected by the above method.RNA
Nano-hydrogel is as shown in Figure 3 to the optimum results of DOX and TMPyP4 bearing capacities, wherein Fig. 3 A are that RNANHs holds TMPyP4
The ultraviolet spectra optimization figure of carrying capacity, the big enables of RNA NHs of 5 25 μM of μ L TMPyP4 and 20 μ L reach complete carrying, i.e.,
TMPyP4 and RNANHs carryings are than being 5:20.Fig. 3 B are that RNANHs optimizes figure to the fluorescence spectrum of DOX bearing capacities.When RNANHs's
When volume reaches 80 μ L, 20 μM, the fluorescence intensity of the DOX of 100 μ L has reached relatively low value, and (fluorescence signal value is 200 left
It is right), show the state for reaching relative saturation between the two, i.e. DOX and RNANHs carryings are than being 100:80.
Embodiment 3
The detection of intracellular ROS level, specific operation process are as follows：
A549 cells are placed in the glass button culture dish of 35mm and are incubated 12h under the conditions of 37 DEG C, waits for that cell density reaches
It is separately added into when to 80% or so and cultivates 2h containing RNANHs, RNANHs-T, it is then clear with the DPBS buffer solutions containing 1%BSA
It washes.Then it is added and is used as molecular probe with 50 μM of DCFH-DA (2', 7'- dichlorofluorescein diacetate) that pure culture liquid dissolves
After cultivating 40min, it is placed under 650nm laser and irradiates 50min.Finally, cell is resuspended in 1mL DPBS, uses fluidic cell
Instrument is detected.
RNANHs-T and RNANHs act on the flow cytomery after cell the results are shown in Figure 4, testing result table
Bright, treated that A549 cell fluorescence signal intensities are apparent by RNANHs or RNANHs-T, and the cell of RNANHs-T effects is glimmering
Optical signal is more stronger.This is because being unable to permeabilized cells film since DCFH-DA can hydrolyze to generate under esterase acts in the cell
DCFH, inducing cell produces ROS after the TMPyP4 that RNANHs-T is carried enters cell, and ROS then aoxidizes DCFH and generates
The DCF for having fluorescence, makes fluorescence signal enhance.RNANHs-T is also demonstrated compared with RNANHs simultaneously, it is thin after RNANHs-T effects
The presence of intracellular ROS.Fluorescence intensity is stronger, then the ROS generated into the cell by TMPyP4 inductions is more, then is easier to lead to cell
It is dead.
Embodiment 4
For the synergy mechanism of cell after being loaded on RNANHs for DOX and TMPyP4, specific experiment is as follows：
Spare A549 lung carcinoma cells and the culture medium containing RNANHs-D-T are cultivated into 2h in 37 DEG C of incubators, then
50 μM of the DCFH-DA that the pure culture base dissolving of serum-free is added cultivates 20min in incubator, then under 650nm laser
50min is irradiated, meanwhile, as a control group, the cell handled without laser irradiation is set.Two groups of cells are all with DAPI to nucleus
It is marked.It finally carries out laser scanning co-focusing microscope and carries out image checking.
RNANHs-D-T is as shown in Figure 5 to the CLSM testing results of A549 cell therapy mechanism under illumination effect, wherein
Fig. 5-1,5-3,5-5 do not carry out the DAPI nuclei dyeing chromatic graphs of laser irradiation, DOX action diagrams and the two stacking chart respectively；Figure
5-2,5-4,5-6 are respectively the DAPI nuclei dyeing chromatic graphs carried out after laser irradiation, DOX action diagrams and the two stacking chart.DAPI
Nuclear targeting it is apparent, do not carry out the cell of laser treatment, DOX is mostly in cytoplasm, and pass through laser irradiation handle
Cell later, DOX are then applied to the position of nucleus.This illustrates under illumination effect that DOX is discharged from RNANHs-D-T, with
TMPyP4 has played effect simultaneously.
Embodiment 5
Cytotoxicity experiment is carried out using CCK-8 kits：
100 μ L cell suspensions are configured in 96 orifice plates first, culture plate are placed on 37 DEG C, 5%CO2Preculture in incubator
For 24 hours, be added into culture plate the different types of drugs to be measured of 10 μ L (DOX, TMPyP4, RNANHs, RNANHs-D, RNANHs-T,
RNANHs-D-T), culture plate is put and is incubated in the incubator after a certain period of time, discarding original culture solution, to change 100 μ L new
Culture solution, 10 μ L CCK-8 solution then are added to each hole, and (attention cannot generate bubble in hole, otherwise can influence OD values
Reading), continue after being incubated one section of reasonable time in the incubator, the absorbance in 450nm is measured with microplate reader.Select cell
Suspension is only added CCK-8 and is added without the hole of test substance hole as a contrast, and culture solution of the selection without containing cell is added CCK-8 and makees
For blank group, tested.Final cell viability %=[A (dosing)-A (blank)]/[A (0 dosing)-A (blank)] ×
100%.
RNANHs-D-T and RNA nano-hydrogels, DOX, TMPyP4, RNANHs-D and RNANHs-T are respectively acting on
After A549 cells, the testing result of cytotoxicity is as shown in fig. 6, under same experiment condition, the cell after RNANHs-D-T effects
Survival rate is significantly lower than the cell survival rate after RNANHs, RNANHs-D, RNANHs-T effects.That is RNANHs-D-T is for targeting
The therapeutic effect effect of lung carcinoma cell is best.For RNANHs-D and RNANHs-T for RNA NHs, therapeutic effect is slightly bright
It is aobvious, but the optimum therapeuticing effect still not as good as RNANHs-D-T.
Embodiment 6
Vivo detection is tested
Bearing mouse model under A549 cell skins is initially set up, one group is set as experimental group, intratumor injection RNANHs-D-T
Drug, another group is as a control group, and the same PBS that injects carries out contrast experiment, and two groups of injection dosage is 30 μ L of per injection,
It is consistent to inject the frequency.After carrying out injection operation, continuous tracing study tracks the gross tumor volume and record of decimal.Experiment terminates knot
Shu Hou puts to death mouse using carbon dioxide smother play, removes tumour.
Living body fluorescent testing result of lung cancer (A549) tumor-bearing mice after PBS (left side) and RNANHs-D-T (right side) administrations is such as
Shown in Fig. 7, tumor region shows the strong fluorescence of tumour institute band, and the tumour after RNA NHs-D-T administrations is apparent
Less than the tumor size after PBS comparison administrations.The high contrast of its tumor imaging is attributed to the targets identification of RNANHs-D-T
And synergistic therapeutic action.It is opposite after the tumour picture being finally stripped out out of Mice Body and the different injection of process
The result of variations of gross tumor volume is as shown in Figure 8, wherein Fig. 8 A are mouse In vivo detection fluorescence imaging figure, and Fig. 8 B are in Mice Body
Tumor Volume Changes figure.Wherein, Fig. 8 A illustrate, for mice with tumor under same experiment condition, the tumour of experimental group dissection gained is bright
It is aobvious to be less than control group, the two significant difference.It monitors known to the variation of relative tumour volume (Fig. 8 B), is handled with RNANHs-D-T swollen
After tumor mouse, tumour growth obviously slows down, and effectively has evaluated cylinder therapeutic effects of the RNANHs-D-T to tumour.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
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Claims (11)
1. a kind of gene nano probe for lung cancer-targeted treatment, which is characterized in that the gene nano probe includes that RNA receives
Rice hydrogel and the DOX and/or TMPyP4 being supported on the RNA nano-hydrogels；
The RNA nano-hydrogels include single-stranded DNA self assemblies, cyclic DNA profiling and targeting aptamers；
The DNA self assemblies it is single-stranded include nucleotide sequence ASM-DNA-1, nucleotide sequence as shown in SEQ ID NO.1 such as
ASM-DNA-2 and the nucleotide sequence ASM-DNA-3 as shown in SEQ ID NO.3, three DNA shown in SEQ ID NO.2 are mono-
Chain is respectively with, for node, both ends complementary pairing two-by-two constitutes Trident Type self-assembled structures among sequence；
The cyclization DNA profiling includes the phosphorylated nucleotide sequence Three- as shown in SEQ ID NO.4 in 5 ' ends
The phosphorylated ends nucleotide sequence Three-miR 34a as shown in SEQ ID NO.5 and 5 ' in let7a, 5 ' ends are phosphorylated
Nucleotide sequence such as SEQ ID NO.6 shown in it is one or more in Three-miR 145, the cyclization DNA profiling is logical
The T7 promoter complementary series at sequence both ends is crossed, it is mutual with the T7 promoter sequences of the branches end of the Trident Type self-assembled structures
It mends and combines；
The targeting aptamers are 5 ' of sequence described in SEQ ID NO.7 terminal modified to have fluorophor, 3 ' terminal modified cholesterol
Substance；The targeting aptamers are combined by T7 promoters complementary series with cyclic DNA profiling.
2. gene nano probe according to claim 1, which is characterized in that the grain size of the RNA nano-hydrogels be 80~
200nm。
3. gene nano probe according to claim 1, which is characterized in that the fluorophor include FAM, FITC or
Cy3。
4. gene nano probe according to claim 1, which is characterized in that RNA nanometer waters in the gene nano probe
The carrying volume ratio of gel and DOX are (0~100)：100；The carrying volume ratio of the RNA nano-hydrogels and TMPyP4 are (0
~25)：5.
5. the preparation method of gene nano probe, includes the following steps described in Claims 1 to 4 any one：
1) single-stranded mixed in Tris-HCl buffer solutions with cyclic DNA profiling of DNA self assemblies is made annealing treatment, the annealing
The condition of processing is：95 DEG C, 5min, then 16~28 DEG C are down to the speed of 0.5 DEG C/min, obtain Trident Type self-assembled structures load
Body；
2) the Trident Type self-assembled structures carrier that step 1) obtains is mixed with T4 ligases and T4 ligase buffer solutions, 16 DEG C into
Row annulation obtains the Trident Type self-assembled structures carrier containing circular template；
3) the Trident Type self-assembled structures carrier containing circular template for obtaining step 2) and nucleotide triphosphoric acid mixed liquor,
RNase inhibitor and the mixing of T7 polymerases and T7 polymerase buffers, 37 DEG C of progress rolling ring 0.5~3h of responsive transcription, are tied
Close the carrier of cyclic DNA；
4) carrier for the combination cyclic DNA that step 3) obtains is mixed with targeting aptamers, is made annealing treatment, at the annealing
The condition of reason is：65 DEG C, 5min, then 16~28 DEG C are down to the speed of 0.5 DEG C/min, obtain RNA nano-hydrogels；
5) the RNA nano-hydrogels that step 4) obtains are mixed with DOX and/or TMPyP4,37 DEG C of reaction 2h obtain gene nano
Probe.
6. preparation method according to claim 5, which is characterized in that the DNA self assemblies are single-stranded with cyclic DNA profiling
Mixed volume ratio is 1:(0.8~1.2).
7. preparation method according to claim 5, which is characterized in that described in step 4) in conjunction with cyclic DNA carrier with
The mixed volume ratio for targeting aptamers is 1:(90~110).
8. preparation method according to claim 5, which is characterized in that RNA nanometers in step 5) the gene nano probe
The carrying volume ratio of hydrogel and DOX are (0~100)：100；The carrying volume ratio of the RNA nano-hydrogels and TMPyP4 is
(0~25)：5.
9. preparation method described in gene nano probe or claim 5~8 any one described in Claims 1 to 4 any one
Application of the obtained gene nano probe in preparing targeted inhibition lung carcinoma cell drug.
10. application according to claim 9, which is characterized in that the lung carcinoma cell includes A549 cell lines.
11. application according to claim 9, which is characterized in that the effective dose of gene nano probe is in the drug
30~150 μ L.
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