CN113372904B - Glutathione response nanoprobe for tumor imaging and targeted cooperative therapy and construction method thereof - Google Patents
Glutathione response nanoprobe for tumor imaging and targeted cooperative therapy and construction method thereof Download PDFInfo
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Abstract
The invention discloses a glutathione response nanoprobe for tumor imaging and targeted cooperative therapy and a construction method thereof, wherein the glutathione response nanoprobe comprises gold nanoparticles and DNA Y-motif, the DNA Y-motif comprises DNA S1, DNA S2 and MUC1 aptamer DNA S3, a DNA sequence S1 is respectively complementary, matched and hybridized with bases of a DNA sequence S2 and a MUC1 aptamer DNA S3 to form a Y-shaped structure, a sulfydryl at the 5' end of the S1 sequence is connected to the surface of the gold nanoparticles to form an AuNP Y-motif, a photosensitizer is modified at the 5' end of an S2 sequence, a double-mode imaging group is modified at the 3' end of the S2 sequence and is close to the surface of the gold nanoparticles, and the imaging of fluorescence-surface enhanced Raman scattering double-mode tumor cells is realized by adjusting the distance between the double-mode imaging group and the gold nanoparticles. The aptamer is used as a target, the endogenous substance DNA is utilized to construct a nano probe carrying chemotherapeutic drugs and photosensitizers, and the cooperative treatment of CT-PDT is realized.
Description
The technical field is as follows:
the invention belongs to the field of tumor cell cooperative treatment and spectral imaging, and particularly relates to a glutathione response nanoprobe for tumor imaging and targeted cooperative treatment and a construction method thereof.
Background art:
glutathione (GSH) is a sulfhydryl compound, and abnormal change of GSH content in cells is directly related to the generation of some diseases, such as tumors, neurological diseases, AIDS and the like. GSH has become a recognized tumor cell marker, significantly higher than normal cells in many malignant tumors. Therefore, monitoring and imaging studies of GSH are an urgent need for early diagnosis of cancer. The content of GSH in cancer cells is 2-10mM, the content of GSH in healthy cells is 2-10 μ M, the content of GSH in tumor microenvironment is about 100-fold times of that in normal tissues, and the obvious difference of GSH level makes the nano-carrier of GSH response the first choice for targeting intracellular drug delivery. Wherein, the disulfide bond is widely applied to the design of the tumor specific stimulation response nano drug-loaded probe as a typical reduction sensitive bond.
As a non-invasive medical technique, PDT has become a reliable treatment for cancer. More and more research is aimed at ablating malignant tumors in PDT using harmless photosensitizers and visible light. PDT can also cause acute inflammation and leukocyte infiltration, thereby promoting an immune response. PDT relies on Reactive Oxygen Species (ROS) generated by Photosensitizers (PSs), and PDT may provide additional tissue selectivity compared to conventional chemotherapy because of the pre-determined accumulation of PSs and the placement of the illumination, the size of the treatment area being dependent on the size of the illuminated area, ensuring less side effects on adjacent healthy tissue. Chemotherapeutic drugs have strong toxic and side effects, limiting their use. In order to overcome the limitations, people develop a series of targeting nano platforms with good targeting capability, high treatment efficiency and small toxic and side effects, realize the specific treatment of tumors and improve the curative effect. Aptamers, short peptides and small molecules, have recently become promising targeting ligands for the design of novel drug delivery systems. Because they can specifically recognize different cell surface targets over-expressed in tumor cells, the construction of a targeted drug delivery controlled release system is crucial for the safety of targeted drug delivery in cancer therapy. PDT helps to overcome the multidrug resistance of chemotherapeutic drugs. Therefore, a drug delivery system that can achieve both effective synergistic delivery of photosensitizers and chemotherapeutic drugs, and reduce off-target toxicity of chemotherapy, has great potential in cancer treatment. Chlorin E6(Chlorin E6, Ce6) is commonly used as a conventional photosensitizer for PDT treatment of tumors.
The current optical sensor mainly relies on single signal dependence analysis and is easily interfered by some substances. In contrast, optical sensors based on dual signals, such as rate sensors, have a higher accuracy. More importantly, however, dual-mode optical sensors using different types of signals are more conducive to improved sensitivity and accuracy, and have considerable potential applications. In recent years, dual-signal optical sensors based on Surface Enhanced Raman Scattering (SERS) and fluorescence have been widely developed in the field of detection. For example, the task force group has recently developed fluorescence-SERS switch nanoprobes based on Cy3 and gold nanoparticles to monitor the dynamic behavior of cells and detect various miRNAs. The same colleagues prepared a cytochrome C activated dual-mode receptor sensor based on SERS and fluorescence signals, and the sensor is formed by self-assembly of a DNA modified Au nano triangle and a Cy5 modified complementary chain. Therefore, a large number of nano platform strategies are constructed by utilizing the SESR technology and the fluorescence technology to realize the diagnosis and treatment integration of the tumor, the tumor detection accuracy is improved, and the tumor treatment effect is enhanced.
The invention content is as follows:
based on the situation, the invention aims to design a DNA-based nano probe to carry chemotherapeutic drugs and photosensitizers to carry out drug release in response of a tumor microenvironment, so that the photosensitizers and the chemotherapeutic drugs are effectively delivered in a synergistic manner, the CT-PDT (computed tomography-PDT) synergistic treatment of tumors is realized, and the tumor treatment efficiency and the imaging analysis of tumor cells are improved.
In order to achieve the above purpose, the glutathione response nanoprobe for tumor imaging and targeted synergistic therapy, which is provided by the invention, comprises gold nanoparticles and DNA Y-motif, wherein the DNA Y-motif comprises DNA S1, DNA S2 and MUC1 aptamer DNA S3, the DNA sequence S1 is respectively hybridized with the DNA sequence S2 and the MUC1 aptamer DNA S3 in a base complementary pairing manner to form a Y-shaped structure, wherein the sequence S1 is 5'-SH-GCT ACG ATA ACG ACG AAA GGA TCA ACT G-3', the sequence S2 is 5'-CCA GGG AAT CCA TCG TCG SSA TCG TAG C-3', the sequence S3 is 5'-ACA CGG CAG TTG ATC CTT TGG ATA CCC TGG CGT GT-3', the sulfydryl at the 5 'end of the sequence S1 is connected to the surface of the gold nanoparticles to form AuNP Y-motif, and the 5' end of the sequence S2 is modified photosensitizer, the 3' end of the S2 sequence is modified with a dual-mode imaging group and is close to the surface of the gold nanoparticles, and the fluorescence-surface enhanced Raman scattering dual-mode tumor cell imaging is realized by adjusting the distance between the dual-mode imaging group and the gold nanoparticles.
When the glutathione response nanoprobe for tumor imaging and targeted cooperative therapy reaches the cell surface, the MUC1 aptamer is combined with MUC1 protein, so that the endocytosis of tumor cells to the nanoprobe is promoted, after the nanoprobe enters the cells, the disulfide bond is cut off by GSH, the DNA-Y-motif is completely melted to release the photosensitizer, and the accurate targeted therapy and photodynamic therapy of tumor microenvironment response are realized. A dual-mode imaging group in the AuNPs-Y-motif probe is close to AuNPs to generate a strong Raman signal, fluorescence quenching is performed, when a disulfide bond is cut off by GSH, the dual-mode imaging group is far away from the AuNPs to recover the fluorescence signal, namely the conformational change of Y-motif is caused by the action of GSH in tumor cells and the AuNP-Y-motif probe, the dual-mode imaging group generates the Raman signal to weaken the change of fluorescence signal enhancement, and the surface enhanced Raman scattering spectrum imaging analysis of the tumor cells is realized.
Specifically, the bimodal imaging groups include, but are not limited to, one of Cy3, Cy5, and ROX.
Specifically, the photosensitizer includes, but is not limited to, photosensitizer chlorin E6(Ce6), methylene blue, or hemin.
Further, the glutathione response nanoprobe for tumor imaging and targeted synergistic therapy further comprises a chemotherapeutic drug, wherein the chemotherapeutic drug is inserted into the DNA Y-motif base pair. Specifically, chemotherapeutic drugs include, but are not limited to, Doxorubicin (DOX). At the moment, the probe carries the photosensitizer and the chemotherapeutic drug, and endogenous substances are controlled to release the drugs by jointly delivering the photosensitizer and the chemotherapeutic drug, so that CT-PDT combined treatment of tumor cells is realized.
The invention relates to a construction method of a glutathione response nanoprobe for tumor imaging and targeted cooperative therapy, which comprises the following steps:
(1) modifying a photosensitizer at the 5 'end of a DNA S2 sequence, modifying a double-mode imaging group at the 3' end, annealing DNA S1, DNA S2 and MUC1 aptamer DNA S3, and then cooling in an ice bath for later use;
(2) vibrating and mixing the cooled DNA S1 and AuNPs to fix the DNA S1 on the surface of the AuNPs through a covalent gold-thiol bond to form AuNPs-S1;
(3) re-dispersing AuNPs-S1 in PBS, adding the DNA S2 and the mucin-1 aptamer DNA S3 processed in the step (1), shaking at room temperature, and after full reaction, respectively carrying out base complementary pairing and hybridization on the mucin-1 aptamer DNA S3 and the DNA S2 and the DNA S1 to form a Y-shaped structure, so as to obtain a probe AuNP-Y-motif;
(4) the chemotherapeutic drug is mixed with AuNP-Y-motif to insert the chemotherapeutic drug into the GC base pair.
Compared with the prior art, the GSH response nanoprobe provided by the invention has the following advantages when being used for tumor imaging and targeted cooperative therapy:
(1) the nano probe carrying the chemotherapeutic drug and the photosensitizer is constructed by utilizing endogenous substance DNA through the targeting effect of the aptamer, so that the effective synergistic delivery of the photosensitizer and the chemotherapeutic drug is realized, the targeted treatment efficiency of the tumor is improved, and the nano probe is simple to prepare and low in cost.
(2) The tumor cells are imaged by using the fluorescence and Raman dual-mode optical signals, so that the sensitivity and the accuracy are improved, and the interference of a complex environment in a single-signal strategy is overcome.
Description of the drawings:
fig. 1 is a schematic diagram of the principle of the present invention.
FIG. 2 is a transmission electron micrograph (A) of AuNPs, an ultraviolet spectrum (B) of a probe, a dynamic light scattering particle size map (C) and a Raman characterization map (D) in example 1.
FIG. 3 is a graph of the fluorescence spectra of GSH (a-f: 0, 1, 2, 3, 4, 5mM) and AuNP-Y-motif probe at different concentrations described in example 1, in response to release of DOX (A) and Cy3 (B).
FIG. 4 is an in vitro sample of AuNP-Y-motif probes described in example 1 at different irradiation times 1 O 2 A detection map is generated.
FIG. 5 shows intracellular expression of AuNP-Y-motif probe described in example 1 1 O 2 A detection map is generated, wherein DCF represents the fluorescence map of DCF, Bright represents the white light map, and Merge represents the superposition of the fluorescence map and the white light map.
FIG. 6 is a photograph showing fluorescence images of cells according to example 1, wherein Cy3 represents a fluorescence image of Cy3, DOX represents a fluorescence image of DOX, Bright represents a white light image, and Merge represents a superimposed image of the fluorescence image and the white light image.
Fig. 7 is a raman mapping chart of the cell according to example 1, wherein each column from left to right is a raman mapping chart of HeLa cell, a superposition chart of the raman mapping chart of HeLa cell and a white light image of the cell, the white light image of the cell and a corresponding intracellular Cy3 raman signal chart.
FIG. 8 is a graph of the activity of tumor cells detected by the probe described in example 1.
The specific implementation mode is as follows:
the present invention will be described in detail with reference to the following examples and accompanying drawings.
Example 1
In the embodiment, the dual-mode imaging group adopts Cy3, and the photosensitizer adopts Ce 6.
1. Synthesis of gold nanoparticles
2L of ethanol is taken, 500g of potassium hydroxide (KOH) is weighed, KOH is dissolved in 2L of ethanol, and an alkali cylinder is prepared for subsequent experiments. Adding a certain amount of ethanol and KOH every 10 days. AuNPs are used as a carrier, the synthesis method adopts the traditional citric acid reduction method (also called one-step reduction method) to prepare, and the sodium citrate with reducibility can be used for dissolving Au in chloroauric acid solution 3+ Reducing the Au into a simple substance of Au, further polymerizing the Au simple substance to grow, and further polymerizing the Au into gold nanoparticles with different particle sizes under the regulation and control of sodium citrate (controlling the volume ratio of the sodium citrate to the chloroauric acid). In the preparation process, all used glassware, magnetons and the like are firstly soaked in an alkali cylinder for 12 hours, then cleaned and dried in an oven, and then the dried glassware is soaked in aqua regia solution for 24 hours, cleaned and dried. Therefore, colloid instability caused by external factors can be effectively avoided, the synthesis of AuNPs is ensured not to be polluted, and the colloid state is more stable. Firstly, 1 percent of HAuCl is prepared 4 Dissolving, taking 1% HAuCl 4 1mL of the solution, and the volume is adjusted to 100mL in a volumetric flask to obtain 0.01% HAuCl 4 And (3) dissolving the sodium citrate with secondary water to obtain a 1% sodium citrate solution. Set up the reflux unit and then pour in 0.01% HAuCl 4 Heating the solution while stirring, adding trisodium citrate solution when the solution is slightly boiling (with large bubbles), observing the color of the solution, and gradually changing to wine redAnd fully reacting to obtain a stable AuNPs solution, continuously heating, stirring and refluxing for 30min, then closing heating, continuously stirring to naturally recover the AuNPs solution to the room temperature, and finally storing the cooled AuNPs solution in a refrigerator at 4 ℃. And finally, all the experimental glassware is put back into the alkali jar to be soaked for next use, so that the cleanliness of the experimental glassware is ensured.
2. Preparation of AuNP-Y-motif Probe
2.1 DNA sequence of the beacon, detailed information is shown in Table 1.
TABLE 1 Experimental use of DNA sequences
The construction process of S2 specifically comprises the following steps: firstly, Ce6 is modified at the 5 'end of an S2 sequence, Cy3 is modified at the 3' end, a GSH-responsive disulfide bond is inserted at one end close to Cy3 by optimizing the chain melting temperature of base complementary pairing, and the DNA Y-motif can be melted to release a drug and Cy3 after GSH response.
2.2 preparation of AuNP-Y-motif Probe
To successfully prepare the AuNP-Y-motif probe, thiol-modified DNA S1 (1.0. mu.M, 50.0. mu.L) was first annealed in a water bath at 95 ℃ for 5min to avoid base mismatches, and then cooled in an ice bath for 30min before use. The cooled S1 was mixed with 1mL of AuNPs in a 10mL beaker and shaken gently at 37 ℃ for about 16h with a shaker. After 16h, 200.0. mu.L of 0.05M NaCl solution was slowly and uniformly added dropwise to the mixture during the subsequent salt aging, and shaking was continued for 6h, after 6h, 200.0. mu.L of 0.1M NaCl solution was slowly and uniformly added dropwise to the mixture and further shaking was continued for 6h to enhance the stability of the probe. After the secondary salting for 6h, centrifuging for 30min at 10000rpm, then washing for 2 times to remove the free redundant S1 sequence, so that the S1 chain is fixed on the AuNPs surface through a covalent gold-thiol bond. Subsequently, the 2-pass washed pellet was redispersed in PBS (0.1M, pH 7.4), then the DNA S2 (1. mu.M, 50. mu.L) and the mucin-1 aptamer sequence (S3, 1. mu.M, 50. mu.L) were annealed as described above, and the annealed and cooled S2, S3 were added to the redispersed AuNP-S1 and shaken at room temperature for 6 h. After 6h the probe AuNP-Y-motif was obtained and mixed with 2. mu.L of 10mM doxorubicin hydrochloride DOX and shaken overnight at room temperature so that the doxorubicin hydrochloride could be inserted completely into the GC base pairs. The mixture was washed by centrifugation and then resuspended in PBS as described above, thus obtaining AuNP-Y-motif loaded with DOX and photosensitizer Ce6 for subsequent experiments. The prepared probe is used for detecting Cy3 signals by Raman detection to verify the success of the preparation.
3. Characterization of AuNP-Y-motif probes
The AuNP-Y-motif probe was characterized by UV, TEM, DLS and Raman. The TEM image as shown in FIG. 2-A shows the size and morphology of AuNPs, which have an average diameter of about 25nm, are spherical and are uniformly dispersed. DLS measurements further showed that the hydrated particle size diameter of AuNPs increased when surface-modifying the DNA Y-motif (fig. 2-B). UV-vis further showed that functionalization resulted in a slight red shift in the UV absorption of AuNPs (FIG. 2-C). The characteristic absorption red at 526nm of AuNPs modified by the nucleic acid chain is shifted to 529nm because the dielectric constant of the environment around the AuNPs is changed by functionalization. The Cy3 molecule is a raman signal molecule, and since the Cy3 molecule is close to AuNPs, a strong Cy3SERS signal can be detected. SERS spectrum is 1586cm -1 (characteristic peak of Cy 3) showed a strong Raman signal (FIG. 2-D). All of these characterization results above confirm the successful preparation of AuNP-Y-motif probes.
4. Fluorescence analysis of AuNP-Y-motif probe and GSH response
GSH solutions (0, 1, 2, 3, 4, 5mM) were prepared accurately at different concentrations and reacted with AuNP-Y-motif probe for 6 h. After the reaction, GSH and AuNP-Y-motif probe mixed solution with different concentrations is centrifuged (10000rpm, 30min), and the fluorescence intensity of Cy3 and DOX in the supernatant is measured.
Then, we investigated the drug release behavior of the reaction of GSH with AuNP-Y-motif at different concentrations, and as shown in fig. 3-A, B, fluorescence signals of DOX and Cy3 were successfully detected in the supernatant, the GSH concentration was in the range of 0-5 mM, and the fluorescence intensity of DOX and Cy3 was significantly increased with the increase of GSH concentration, so that it could be concluded that the higher the GSH concentration, the greater the release amount of DOX.
5. Cell culture of HeLa
HeLa cells were cultured in a DMEM medium (10% bovine serum albumin, 1% penicillin streptomycin) in a 100% humidified atmosphere containing 5% carbon dioxide at 37 ℃.
6. In vitro AuNP-Y-motif Probe 1 O 2 Generating a test
Determination of AuNP-Y-motif in vivo with 1, 3-Diphenylisobenzofuran (DPBF) 1 O 2 Generation capacity, 50. mu.L of LAuNP-Y-motif was added to a solution of 50. mu.g/mL of DPBF. The mixture was irradiated for various periods of time with a 650nm lamp. After irradiation, the fluorescence intensity at 485nm was recorded at an excitation wavelength of 403 nm. DPBF solution without AuNP-Y-motif was used as a control.
As shown in FIGS. 4-A, B, the fluorescence of DPBF was not reduced by any of DPBF, DPBF + light irradiation, and DPBF + AuNP-Y-motif, whereas the fluorescence intensity of DPBF rapidly decreased as the laser irradiation time was increased from 0 to 20min under the irradiation with 650nm laser light, indicating that ROS was generated until DPBF was oxidized under light irradiation, and the fluorescence intensity was reduced accordingly. The result shows that the AuNP-Y-motif nano probe has good generation in vitro solution 1 O 2 The ability of the cell to perform.
DCFH-DA is an intracellular ROS-sensing probe that can be specifically produced intracellularly 1 O 2 Oxidized and converted into 2, 7-Dichlorofluorescein (DCF) with a strong fluorescent signal. Detection of intracellular conditions using 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) 1 O 2 Generating the capabilities. AuNP-Y-motif was incubated with HeLa cells. After culturing for 4h, the cells were washed 2 times, and then 10. mu.M DCFH-DA was incubated with HeLa cells for 30min, and the cells were washed 2 times. Then, the culture dish cells were irradiated at a wavelength of 650nm for 20 min. AuNP-Y-motif, unilluminated culture dish cells were not added as a control. After 20min, on a confocal laser scanning microscope, the excitation wavelength is 488nm, and the emission wavelength acquisition range is 510-540 nm.
As shown in FIG. 5, DCF fluorescence was not observed in only DCFH-DA HeLa cells (5a), while DCF fluorescence was negligible in laser-irradiated HeLa cells (5b) with DCFH-DA, and laser irradiation was not usedSlight DCF fluorescence was observed in irradiated DCFH-DA and AuNP-Y-motif treated HeLa cells (5 c). In contrast, HeLa cells incubated with DCFH-DA and AuNP-Y-motif showed strong green fluorescence after laser irradiation (5d), confirming efficient generation in cancer cells 1 O 2 。
7. Fluorescence imaging of HeLa cells
HeLa cells were plated for 24 hours, and then 20. mu.L of AuNP-Y-motif solution was mixed with 1mL of culture solution and added to a confocal dish for culture. After incubation at 37 ℃ for various times, wash 3 times with PBS to reduce background fluorescence. And then confocal imaging is carried out, wherein the exciting light is 514nm, the fluorescence emission of Cy3 is collected from 560-590 nm positions of the band-pass filter, and the fluorescence emission of DOX is collected from 488nm positions and 590-650 nm positions of the collecting wavelength range.
The capability of releasing AuNP-Y-motif targeted drugs in tumor cells is researched, and fluorescent confocal imaging analysis is carried out on the HeLa cells. As shown in FIG. 6, after 1h incubation of AuNP-Y-motif with cells, Cy3 and weak DOX fluorescence signals were successfully detected in cytoplasm under the action of GSH. It was confirmed that DOX was successfully released from AuNP-Y-motif, and as the culture time was prolonged, the content of AuNP-Y-motif probe entering tumor cells was gradually increased, and the fluorescence intensity of Cy3 and DOX was also gradually increased. After 6h incubation, a distinct red fluorescence was observed in the nucleus, indicating that DOX accumulated in the nucleus, binding to chromosomes and killing cancer cells. This indicates that DOX was successfully encapsulated in AuNP-Y-motif probes and that GSH-controlled drug release in tumor cells was achieved. In conclusion, the development of GSH stimulus responsive nano-platforms enables activation imaging and controlled release of drugs in tumor cells.
8. Raman imaging of HeLa cells
And inoculating the HeLa cells on the SERS substrate gold, culturing for 24h in an incubator, washing once with PBS, and incubating the AuNP-Y-motif probe and the HeLa cells for different times (1h, 2h and 3 h). Free AuNP-Y-motif was then removed with PBS, imaged on a renisha invita raman microscope, 50 x objective, 10% laser intensity, using a 633nm HeNe laser,the exposure time is 5s, and the detection range of Raman signals is 1700cm -1 To 600cm -1 And (5) collecting the range.
As can be seen in fig. 7, a strong SERS signal of Cy3 was observed in HeLa cells at 1h, with a gradual attenuation of the raman signal of Cy3 over time. The results show that the AuNP-Y-motif probe enters the cell by endocytosis due to the targeting effect of MUC1, the raman signal is strongest at the beginning, and as time increases, a large amount of probe reacts with GSH, and the raman signal gradually decreases until the Cy3 labeled oligonucleotide is released from the AuNP-Y-motif.
9. Therapeutic study of AuNP-Y-motif Probe
The cell activity of AuNP-Y-motif probe on HeLa cell and HEK293T cell was determined by CCK-8 method. 2 cells were seeded in 96-well plates and grown for 24 h. After incubation with AuNP-Y-motif (0.1-0.9nM) at different concentrations for 16h, cells were washed with PBS and irradiated at 650nM for 20 min. Cells not irradiated with laser served as control. Thereafter, 10. mu.L of CCK-8 solution was added to each well, incubated for 3.5h, and the Optical Density (OD) at 450nm was measured in each well using a microplate reader. To further investigate the effect of the different treatment methods, HeLa cells were incubated with free DOX, free Ce6, AuNP-Y-motif loaded with DOX only (AuNP-Y-motif/DOX), AuNP-Y-motif loaded with Ce6 only (AuNP-Y-motif/Ce6) and AuNP-Y-motif (loaded with both drugs), and then the cells were measured by the above treatment. Cell viability was calculated as described for the cytotoxicity test kits.
The CCK-8 cell activity experiment is used for researching the synergistic treatment effect of CT-PDT of AuNP-Y-motif on tumor cells. First, the specific cell targeting tumor therapeutic ability was explored, HeLa cells overexpressing MUC1 receptor were selected as target models, and the AuNP-Y-motif side effects and therapeutic effects were evaluated using HEK293T cells as control groups. As shown in fig. 8-A, B, AuNP-Y-motif (without drug) was negligibly toxic to HeLa cells and HEK293T cells, indicating that AuNP-Y-motif has good biocompatibility and no toxicity. However, with increasing AuNP-Y-motif (drug-loaded) concentration and laser irradiation for 20min, the viability of HeLa cells decreased significantly. Under the same conditions, compared with a control group, the survival rate of the non-target HEK293T cell is higher, which shows that the AuNP-Y-motif has a certain toxic effect on the HeLa cell and realizes the specific release of the drug. At the same time, this also explains that non-targeted HEK293T cells have a low uptake efficiency for AuNP-Y-motif, which has a good selectivity for targeted HeLa cells due to the low expression of MUC1 receptor in HEK293T cells.
Cytotoxicity of different drug treatments was further tested in HeLa cells, as shown in fig. 8-C relative cell activity, the inhibition of tumor cell growth by chemotherapy-PDT synergistic treatment of AuNP-Y-motif loaded DOX with Ce6 group (8C-e) was significantly higher than that of Control group (Control), free Ce6 group (8C-a), free DOX group (8C-b), AuNP-Y-motif loaded DOX only group (8C-C), and AuNP-Y-motif loaded Ce6 only group (8C-d). The results indicate that the two therapeutic effects of the nano-carrier with the targeting property are far superior to that of the single drug therapeutic strategy without the targeting carrier.
Sequence listing
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Claims (4)
1. A glutathione response nanoprobe for tumor imaging and targeted synergistic therapy is characterized by comprising gold nanoparticles, DNA Y-motif and adriamycin, wherein the DNA Y-motif comprises DNA S1, DNA S2 and MUC1 aptamer DNA S3, the DNA S1 sequence is respectively complementary, paired and hybridized with the DNA S2 sequence and the MUC1 aptamer DNA S3 sequence to form a Y-shaped structure, the DNA S1 sequence is 5' -SH-GCT ACG ATA ACG ACG AAA GGA TCA ACT G-3', the DNA S2 sequence is 5' -CCA GGG AAT CCA TCG TCG SSA TCG TAG C-3', the DNA S3 sequence is 5'-ACA CGG CAG TTG ATC CTT TGG ATA CCC TGG CGT GT-3', DNA Y-motif, and the DNA S1 sequence is connected with the surface of the gold nanoparticles through a sulfydryl at the 5' end of the DNA S1 sequence to form AuNP-Y-motif, the 5 'end of the DNA S2 sequence is modified with a photosensitizer, the 3' end of the DNA S2 sequence is modified with a dual-mode imaging group and is close to the surface of the gold nanoparticles, and the fluorescence-surface enhanced Raman scattering dual-mode tumor cell imaging is realized by adjusting the distance between the dual-mode imaging group and the gold nanoparticles; adriamycin was inserted into the GC base pair in AuNP-Y-motif.
2. The glutathione-responsive nanoprobe for tumor imaging and targeted co-therapy according to claim 1, wherein the bi-modal imaging group is one of Cy3, Cy5 and ROX.
3. The glutathione-responsive nanoprobe for tumor imaging and targeted co-therapy according to claim 2, wherein the photosensitizer is chlorin E6, methylene blue or hemin.
4. The construction method of the glutathione response nanoprobe for tumor imaging and targeted co-therapy according to claim 3 comprises the following steps:
(1) modifying a photosensitizer at the 5 'end of a DNA S2 sequence, modifying a dual-mode imaging group at the 3' end, annealing DNA S1, DNA S2 and MUC1 aptamer DNA S3, and cooling in an ice bath for later use;
(2) vibrating and mixing the cooled DNA S1 and the gold nanoparticles to fix the DNA S1 on the surfaces of the gold nanoparticles through covalent gold-thiol bonds to form AuNP-S1;
(3) redispersing AuNP-S1 in PBS, adding the DNA S2 and the MUC1 aptamer DNA S3 processed in the step (1), shaking at room temperature, fully reacting, and respectively carrying out base complementary pairing and hybridization on the MUC1 aptamer DNA S3 and the DNA S2 with the DNA S1 to form a Y-shaped structure so as to obtain AuNP-Y-motif;
(4) adriamycin was mixed with AuNP-Y-motif to insert the Adriamycin into the GC base pair in AuNP-Y-motif.
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