A nano-targeted drug-loaded complex, a preparation method and application thereof

Abstract

The application belongs to the technical field of nanocomposites, and particularly relates to a nano-targeting drug-loaded composite, a preparation method and application thereof. In order to solve the problems that the prior art is deficient in tumor active targeting specificity, has fewer targets, and has application limitations, the nano-targeting drug-loaded composite takes Ti3C2 as a carrier, loads anticancer drugs on the surface thereof, and is coupled with a nucleic acid aptamer capable of recognizing specific proteins on the surface of cells. The planar size of Ti3C2 is 80-200 nm, the anticancer drug is doxorubicin, and the loading amount is 15wt.%-50wt.% of Ti3C2. The provided doxorubicin / Ti3C2 / nucleic acid aptamer nano drug-loaded system has excellent active targeting effect, has stronger killing capacity on targeted tumor cells, and can also reduce the toxic and side effects of drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanocomposites, and particularly relates to a nano-targeting drug-loaded composite and a preparation method and application thereof. BACKGROUND

[0002] Cancer is one of the main diseases threatening human life at present. With the development of nanotechnology, nano materials with special properties are continuously developed to overcome the limitations of traditional cancer treatment. Ti3C2 belongs to a two-dimensional layered MXene nanomaterial, which has strong absorption capacity and high photothermal conversion performance in the near-infrared light region, and has the characteristics of high specific surface area and low toxicity, and therefore can be used for tumor photothermal therapy, and can also load and deliver tumor therapeutic agents. At present, most Ti3C2 nanomaterials are accumulated in the tumor site in vivo through passive targeting, i.e. high permeability and long retention. However, most of the Ti3C2 nanomaterials are rapidly phagocytosed by the reticuloendothelial system after entering the body, which reduces the number of nanoparticles reaching tumor cells and reduces drug efficacy, which greatly limits the research and application of Ti3C2 nanomaterials in the medical field. Therefore, active targeting can more effectively transport Ti3C2 nanomaterials or composite nanomaterials to the tumor site.

[0003] So far, there have been reports on the preparation of MXenes nanomaterials with active targeting. Chinese patent document CN108273058A provides a tumor-targeted sustained-release preparation and a preparation method thereof, which comprises co-reacting MXene nanosheets with a mixed aqueous solution of cetyltrimethylammonium chloride (CTAC) and triethanolamine (TEA); then adding tetraethyl orthosilicate (TEOS) and reacting at 80°C, centrifuging, washing, and obtaining mesoporous silica-coated MXene nanosheets. The mesoporous silica-coated MXene nanosheets are subjected to polyethylene glycol surface modification, and then covalently combined with arginine-glycine-aspartic acid polypeptide (RGD) to load drugs. However, the chemical nature of RGD is protein, which is affected by temperature, pH and enzyme inhibitors, thereby inhibiting the exertion of its targeting effect. Chinese patent document CN113144206A provides a folate-pluronic-MXenes tumor-targeted drug delivery system, and the preparation method comprises the preparation of MXenes, the activation of folate, the preparation and purification of folate-MXenes system and activated folate-pluronic-MXenes, the esterification reaction of the hydroxyl group contained in the pluronic and the carboxyl group on the surface of the folate in the presence of a condensing agent to form a folate-pluronic-MXenes tumor-targeted drug delivery system with tumor targeting effect. However, the traditional folate-targeted preparation modification method has disadvantages, especially the modification method makes most of the folate groups wrapped in the hydrophobic core of the preparation, which cannot be fully exposed to affect the interaction with the target cell surface receptors. Moreover, the above-mentioned method of MXenes achieving active targeting through surface modification of RGD or folate has some deficiencies in targeting specificity, and the target is less, which has application limitations. SUMMARY

[0004] In view of the problems that the prior art has some deficiencies in tumor active targeting specificity, and the target is less, which has application limitations, the present application provides a nano-targeted drug delivery complex, a preparation method and application thereof.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] A nano-targeted drug delivery complex, which takes Ti3C2 nanomaterial as a carrier, loads an anticancer drug on the surface thereof, and couples a nucleic acid aptamer (Aptamer, Apt) capable of recognizing specific proteins on the surface of cells on the surface thereof.

[0007] Further, the particle size of the Ti3C2 nanomaterial is 80-200 nm.

[0008] Further, the anticancer drug is doxorubicin (DOX), and the loading amount is 15wt.%-50wt.% of MXenes.

[0009] Further, the nucleic acid aptamer is coupled to the surface of the Ti3C2 nanomaterial by a covalent binding method.

[0010] A preparation method of a nano-targeting drug-loaded complex, comprising the following steps:

[0011] Step 1, preparation of Ti3C2-based MXenes nanomaterial: taking Ti3AlC2-based MAX as raw material, preparing multilayer Ti3C2 (M-Ti3C2) by HF acid etching method, and then preparing few-layer Ti3C2 nanomaterial (F-Ti3C2) by using tetrapropylammonium hydroxide (TPAOH) for intercalation dispersion.

[0012] Step 2, loading of anticancer drugs: loading DOX onto F-Ti3C2 by using electrostatic interaction to prepare DOX / Ti3C2;

[0013] Step 3, PEG surface modification: mixing and stirring DOX / Ti3C2 prepared in step 2 with double-carboxyl PEG to prepare DOX / Ti3C2-PEG;

[0014] Step 4, coupling of nucleic acid aptamer: using EDC / NHS reagent to couple Apt (Apt-M) capable of targeting tumor cell surface MUC1 mucin to the surface of DOX / Ti3C2-PEG by a covalent binding method, so as to obtain the nano-targeting drug-loaded complex DOX / Ti3C2 / Apt-M.

[0015] The application of the nano-targeting drug-loaded complex is applied to the combined treatment of photothermal therapy and chemotherapy for tumors.

[0016] Compared with the prior art, the present application has the following advantages:

[0017] (1) The present application uses Ti3C2-based MXenes nanomaterial as an antitumor drug carrier, and is modified by double-carboxyl PEG, so as to effectively load DOX and improve the dispersion and biocompatibility in physiological environment;

[0018] (2) The present application uses Apt as a targeting group to modify the surface of DOX / Ti3C2-PEG, so as to effectively target the loaded drug to tumor cells in the human body and make them die.

[0019] (3) The Apt used in the present application can be changed according to different target cells and specific antigens on the surface of different cells, so it can be applied to the treatment of various cancers.

[0020] (4) One aspect of the present application is to use Ti3C2-based MXenes nanomaterial as a carrier to realize the delivery of anti-tumor drug DOX to tumor cells, and at the same time to achieve the purpose of chemotherapy / photothermal therapy; another aspect is to use Apt as a targeting group to covalently couple and modify DOX / Ti3C2-PEG, so as to have the ability to target target cells, thereby achieving the purpose of targeted therapy. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 TEM image of F-Ti3C2 nanosheet in Example 1 in the present application;

[0022] Figure 2 EDS analysis result of F-Ti3C2 nanosheet in Example 1 in the present application;

[0023] Figure 3 XRD analysis result of Ti3AlC2 material, and M-Ti3C2 and F-Ti3C2 nanosheet in Example 1 in the present application;

[0024] Figure 4 Dynamic light scattering analysis result of F-Ti3C2, DOX / Ti3C2, DOX / Ti3C2-PEG and DOX / Ti3C2 / Apt-M nanosheet in the present application;

[0025] Figure 5 Zeta potential analysis result of F-Ti3C2, DOX / Ti3C2, DOX / Ti3C2-PEG and DOX / Ti3C2 / Apt-M nanosheet in the present application;

[0026] Figure 6 FT-IR analysis result of F-Ti3C2, DOX / Ti3C2, DOX / Ti3C2-PEG and DOX / Ti3C2 / Apt-M nanosheet in the present application;

[0027] Figure 7 Temperature change result of aqueous solution of DOX / Ti3C2 / Apt-M nanosheet with different concentrations under 808nm laser irradiation in the present application;

[0028] Figure 8 DOX release curve result of DOX / Ti3C2 / Apt-M nanosheet under laser irradiation in PBS in the present application;

[0029] Figure 9 Comparison chart of uptake of DOX / Ti3C2 / Apt-M nanosheet in MCF-7 and HepG2 cells at different times (2, 4 and 6 hours) in the present application;

[0030] Figure 10Figure for comparison of uptake of MCF-7 and HepG2 cells after incubation with different nanomaterials for 4 hours in the present application;

[0031] Figure 11 Figure for MCF-7 cell viability results after treatment with each drug and nanomaterial in the present application;

[0032] Figure 12 Figure for MCF-7 cell apoptosis results after treatment with each drug and nanomaterial in the present application;

[0033] Figure 13 Figure for the effect of each drug and nanomaterial on mouse tumor proliferation in the present application.

[0034] Figure 14 Figure for mouse tumor dissection results after treatment with each drug and nanomaterial in the present application;

[0035] Figure 15 Figure for mouse tumor weight results after treatment with each drug and nanomaterial in the present application; DETAILED DESCRIPTION

[0036] Example 1

[0037] A nano-targeted drug-loaded complex, with Ti3C2-based MXenes as a carrier, loading an anticancer drug doxorubicin on the surface, with a loading capacity of 38.3wt.%, and coupling Apt on the surface which can recognize specific proteins on the cell surface, so as to have targeting ability.

[0038] A preparation method of a nano-targeted drug-loaded complex, comprising the following steps:

[0039] Step 1, preparation of few-layer Ti3C2nanomaterial: 1.0g of Ti3AlC2was immersed in 20mL of 40% hydrogen fluoride aqueous solution at room temperature and stirred for 3d. The multi-layer Ti3C2(M-Ti3C2) precipitate was collected by centrifugation and washed with water several times until the HF was removed. Then, the M-Ti3C2precipitate was dispersed in 20mL of TPAOH aqueous solution (25wt.%) and stirred at room temperature for 3d. After ultrasonic treatment for 15min, few-layer Ti3C2(F-Ti3C2) nanosheets were obtained, and then the F-Ti3C2precipitate was collected by centrifugation at 3500rpm for 60min. The prepared F-Ti3C2nanosheets were dispersed in deionized water and stored at 4°C. As shown in Figure 1 , the particle size of the obtained F-Ti3C2nanosheets was 80-200nm, and the average particle size was 100nm. Figure 2 and 3 As shown in, the Al element in the obtained F-Ti3C2nanosheets has been removed.

[0040] Step 2, loading of anticancer drug: F-Ti3C2nanosheets (2.5 mL, 1.0 mg / mL) were slowly added to a DOX aqueous solution (2.5 mL, 0.4 mg / mL) and stirred overnight in the dark. DOX / Ti3C2was centrifuged and washed with deionized water three times to remove excess DOX. From Figure 4 and Figure 5 It can be seen that the hydrated particle size and zeta potential of DOX / Ti3C2were higher than those of F-Ti3C2due to the surface loading of DOX. In addition, from Figure 6 FT-IR of DOX / Ti3C2, it can be seen that DOX / Ti3C2had a characteristic absorption peak at ~1469 cm -1 . The above results show that DOX was successfully loaded onto the surface of F-Ti3C2.

[0041] Step 3, PEG surface modification: The surface of DOX / Ti3C2nanosheets was modified with COOH-PEG-COOH, and DOX / Ti3C2aqueous solution (2.5 mL, 1.0 mg / mL) was slowly added to COOH-PEG-COOH solution (2.5 mL, 10.0 mg / mL) and stirred for 6 hours in the dark. PEGylated DOX / Ti3C2nanosheets (DOX / Ti3C2-PEG) were collected by centrifugation (12000 rpm, 10 min) and washed with water twice to remove excess PEG. From Figure 4 and Figure 5 It can be seen that the hydrated particle size of DOX / Ti3C2-PEG was higher than that of DOX / Ti3C2, and the zeta potential was lower than that of DOX / Ti3C2, due to the loading of COOH-PEG-COOH on the surface of DOX / Ti3C2. In addition, from Figure 6 it can be seen that DOX / Ti3C2had a characteristic absorption peak at ~1727 cm -1 . The above results show that COOH-PEG-COOH was successfully modified on the surface of DOX / Ti3C2.

[0042] Step 4, coupling of Apt: DOX / Ti3C2-PEG (1.0 mL, 2.0 mg / mL) nanosheets were activated with EDC-HCl (500 μL, 500 mM) and NHS (500 μL, 100 mM) under magnetic stirring for 30 min, and the activated DOX / Ti3C2-PEG nanosheets were obtained by centrifugation followed by 3-4 washes. Then, Apt-M (10 OD) was denatured at 85 °C for 10 min and refolded in an ice bath for 10 min. The activated DOX / Ti3C2-PEG nanosheets were reacted with Apt-M under shaking for 5 h, and then centrifuged and washed with water 3 times to obtain the Apt-M modified DOX / Ti3C2 / Apt-M nanomaterial; at the same time, to perform a control experiment later, a random sequence (Apt-C) without targeting performance was reacted under the same conditions to prepare a DOX / Ti3C2 / Apt-C nanomaterial. The Apt-M modified DOX / Ti3C2 / Apt-M nanomaterial was characterized by TEM, zeta potential, FTIR and Raman spectroscopy. Figure 4 and Figure 5 It can be seen that, due to the successful connection of Apt-M to the surface of DOX / Ti3C2-PEG, the hydrated particle size of DOX / Ti3C2 / Apt-M is higher than that of DOX / Ti3C2-PEG, and the zeta potential is lower than that of DOX / Ti3C2-PEG. In addition, it can be seen that, due to the successful connection of Apt-M to the surface of DOX / Ti3C2-PEG, the FTIR spectrum of DOX / Ti3C2 / Apt-M has a characteristic peak of amide bond at ~ 1650 cm Figure 6 It can be seen that, due to the successful connection of Apt-M to the surface of DOX / Ti3C2-PEG, the hydrated particle size of DOX / Ti3C2 / Apt-M is higher than that of DOX / Ti3C2-PEG, and the zeta potential is lower than that of DOX / Ti3C2-PEG. In addition, it can be seen that, due to the successful connection of Apt-M to the surface of DOX / Ti3C2-PEG, the FTIR spectrum of DOX / Ti3C2 / Apt-M has a characteristic peak of amide bond at ~ 1650 cm -1 , ~ 1573 cm -1 and ~ 1644 cm -1 There are obvious characteristic peaks of amide bond. It shows that the DOX / Ti3C2 / Apt-M nanomaterial is successfully prepared, and Apt-M is connected to the nanomaterial by covalent binding.

[0043] Example 2

[0044] The aqueous solution (1.0 mL) of DOX / Ti3C2 / Apt-M with different concentrations (0-100 μg / mL) was irradiated with an 808 nm laser at a power density of 1.5 w / cm 2 for 10 min, and the temperature change was recorded. It can be seen from Figure 7 that the solution temperature of DOX / Ti3C2 / Apt-M with different concentrations can be raised under 808 nm laser irradiation, and the solution temperature rise is related to the concentration of DOX / Ti3C2 / Apt-M, which shows that DOX / Ti3C2 / Apt-M can be used for tumor photothermal therapy.

[0045] DOX / Ti3C2 / Apt-M (0.5 mL, 1.0 mg / mL) was encapsulated in a dialysis bag with a cut-off molecular weight of 3000 daltons, and the dialysis bag was immersed in 30 mL of PBS with different pH values (pH 7.4, 6.0, and 4.5) and then incubated at 37°C and 150 rpm. The dialysis bag immersed in PBS was irradiated with an 808 nm laser (1.5 W / cm 2 , 10 min) at time points of 5 h and 10 h, respectively, and the absorbance of the dialysate was measured with a UV-Vis spectrophotometer to calculate the DOX release rate. It can be seen from Figure 8 that the release of DOX from DOX / Ti3C2 / Apt-M is related to the pH in the solution, and an acidic environment of pH facilitates the release of DOX, and laser irradiation also promotes the release of DOX.

[0046] Example 3

[0047] Cancer cells were seeded in 6-well plates (1 x 10 5 cells / well) and cultured for 24 h. The original culture medium was replaced with 2.0 mL of culture medium containing DOX / Ti3C2 / Apt-M nanosheets (100 μg / mL), and then cultured for 2, 4, and 6 h. The cells were gently washed with PBS for 7-8 times, and the DOX fluorescence intensity of the cells in each group was analyzed by flow cytometry. It can be seen from Figure 9 that the uptake of DOX / Ti3C2 / Apt-M by MCF-7 and HepG2 cells gradually increased with time, but due to the targeting effect of the presence of Apt-M, MCF-7 completed the uptake of DOX / Ti3C2 / Apt-M within 4 h.

[0048] To verify the targeting performance, cancer cells were seeded in 6-well plates (1 x 10 5 cells / well) and cultured for 24 h. The culture medium in the 6-well plates was replaced with culture medium containing DOX / Ti3C2-PEG (100 μg / mL), DOX / Ti3C2 / Apt-M (100 μg / mL), DOX / Ti3C2 / Apt-M (100 μg / mL) + 2O D Apt-M, and DOX / Ti3C2 / Apt-M (100 μg / mL) + 2O D Apt-C, respectively, and then cultured for 4 h. Then, the DOX fluorescence intensity of the cells in each group was analyzed by flow cytometry. It can be seen from Figure 10 that the uptake of DOX / Ti3C2 / Apt-M by MCF-7 is improved due to the targeting effect of the presence of Apt-M, and if the cell surface of MCF-7 is occupied by free Apt-M, DOX / Ti3C2 / Apt-M cannot effectively exert its targeting effect.

[0049] Example 4

[0050] MCF-7 cells were seeded in 96-well plates (5 x 10 3 cells / well) and incubated for 24 h. The original culture medium was discarded and fresh culture medium containing each group of drugs and nanomaterials was added, respectively, and incubated for another 4 h. Subsequently, the cells were washed slowly with PBS and all laser treatment groups were irradiated with 808 nm laser at a power intensity of 1.5 W / cm 2 for 10 min. After another 12 h incubation, cell viability was detected by MTT method. It can be seen that, due to the targeting effect of Apt-M, the DOX / Ti3C2 / Apt-M+Laser treatment group has a significantly better inhibitory effect on the growth of MCF-7 cells under the combined action of chemotherapy and photothermal therapy than other groups. Figure 11

[0051] Example 5

[0052] A nude mouse tumor model was established with MCF-7 cells, and 100 μL of MCF-7 cells (5 x 10 7 cells / mL) suspended in PBS solution were injected subcutaneously into the mice. When the tumor volume reached about 100 mm 3 , the mice were randomly divided into six groups (n = 5 for each group), and PBS or nanomaterials were injected into the tail vein: (1) control group (PBS), (2) DOX group (DOX dose = 3 mg / kg), (3) Ti3C2-PEG+Laser group (Ti3C2-PEG dose = 12 mg / kg), (4) DOX / Ti3C2-PEG+Laser group (DOX / Ti3C2-PEG dose = 12 mg / kg), (5) DOX / Ti3C2 / Apt-C+Laser group (DOX / Ti3C2 / Apt-C dose = 12 mg / kg), (6) DOX / Ti3C2 / Apt-M+Laser group (DOX / Ti3C2 / Apt-M dose = 12 mg / kg). After 4 h, the mice in groups (3), (4), (5) and (6) were irradiated with 808 nm laser (1.5 W / cm 2 ) for 10 min. The tumor size and the body weight of the mice were measured and recorded every two days, and the tumor volume was determined by the following equation: tumor volume = (tumor length) x (tumor width) 2 / 2. It can be seen that, the tumor volume of the mice in the DOX / Ti3C2 / Apt-M+Laser treatment group was effectively inhibited, indicating that under the active targeting effect, combined chemotherapy and photothermal therapy can achieve better therapeutic effect. Figure 13 , Figure 14 and Figure 15 It can be seen that, the tumor volume of the mice in the DOX / Ti3C2 / Apt-M+Laser treatment group was effectively inhibited, indicating that under the active targeting effect, combined chemotherapy and photothermal therapy can achieve better therapeutic effect.

[0053] Example 6

[0054] ​A nano-targeting drug-loaded complex, taking Ti3C2-based MXenes as a carrier, loading anticancer drug doxorubicin on the surface, the loading amount being 14.46wt.%, and coupling Apt capable of recognizing specific proteins on the cell surface on the surface, so as to have a targeting capacity.

[0055] A preparation method of a nano-targeting drug-loaded complex, comprising the following steps:

[0056] Step 1, preparation of few-layer Ti3C2 nanomaterials: 1.0g Ti3AlC2 is immersed in 20mL 40% hydrogen fluoride aqueous solution at room temperature and stirred for 3d. The multi-layer Ti3C2 (M-Ti3C2) precipitate is collected by centrifugation and washed with water for several times until the HF is removed. Then, the M-Ti3C2 precipitate is dispersed in 20mL TPAOH aqueous solution (25wt.%) and stirred at room temperature for 3d. After ultrasonic treatment for 15min, few-layer Ti3C2 (F-Ti3C2) nanosheets are obtained, and then centrifuged at 3500rpm for 60min. The prepared F-Ti3C2 nanosheets are dispersed in deionized water and stored at 4℃.

[0057] Step 2, loading of anticancer drugs: F-Ti3C2 nanosheets (2.5mL, 1.0mg / mL) are slowly added to a DOX aqueous solution (2.5mL, 0.15mg / mL) and stirred overnight in the dark. DOX / Ti3C2 is centrifuged and washed with deionized water three times to remove excess DOX.

[0058] Step 3, PEG surface modification: the surface of DOX / Ti3C2 nanosheets is modified with COOH-PEG-COOH, and DOX / Ti3C2 aqueous solution (2.5mL, 1.0mg / mL) is slowly added to COOH-PEG-COOH solution (2.5mL, 10.0mg / mL) and stirred in the dark for 6 hours. Polyglycolated DOX / Ti3C2 nanosheets (DOX / Ti3C2-PEG) are collected by centrifugation (12000rpm, 10min) and washed with water twice to remove excess PEG.

[0059] Step 4, coupling of Apt: The DOX / Ti3C2-PEG (1.0 mL, 2.0 mg / mL) nanosheets were activated with EDC-HCl (500 μL, 500 mM) and NHS (500 μL, 100 mM) under magnetic stirring for 30 min of reaction, the activated DOX / Ti3C2-PEG nanosheets were obtained by centrifugation, followed by 3-4 washings. Afterwards, the Apt (4 OD) was denatured at 85 °C for 10 min and then refolded in an ice bath for 10 min. The activated DOX / Ti3C2-PEG nanosheets were reacted with Apt under shaking conditions for 5 h, then centrifuged and washed with water 3 times to obtain the Apt-modified DOX / Ti3C2 / Apt-M nanomaterials.

[0060] Example 7

[0061] A nano-targeting drug-loaded complex, taking Ti3C2-based MXenes as a carrier, loading an anti-cancer drug doxorubicin on the surface, the loading amount being 47.15 wt.%, and coupling Apt capable of recognizing specific proteins on the cell surface on the surface, so as to have a targeting ability.

[0062] A preparation method of a nano-targeting drug-loaded complex, comprising the following steps:

[0063] Step 1, preparation of few-layer Ti3C2 nanomaterials: 1.0 g of Ti3AlC2 was immersed in 20 mL of 40% hydrogen fluoride aqueous solution at room temperature and stirred for 3 d. The multi-layer Ti3C2 (M-Ti3C2) precipitate was collected by centrifugation and washed with water several times until the HF was removed. Then, the M-Ti3C2 precipitate was dispersed in 20 mL of TPAOH aqueous solution (25 wt.%) and stirred at room temperature for 3 d. After ultrasonic treatment for 15 min, few-layer Ti3C2 (F-Ti3C2) nanosheets were obtained, and then centrifuged at 3500 rpm for 60 min. The prepared F-Ti3C2 nanosheets were dispersed in deionized water and stored at 4 °C.

[0064] Step 2, loading of anti-cancer drugs: the F-Ti3C2 nanosheets (2.5 mL, 1.0 mg / mL) were slowly added to the DOX aqueous solution (2.5 mL, 0.5 mg / mL) and stirred overnight in the dark. The DOX / Ti3C2 was centrifuged and washed with deionized water three times to remove excess DOX.

[0065] Step 3, PEG surface modification: The surface of DOX / Ti3C2 nanosheets was modified with COOH-PEG-COOH, and the aqueous solution of DOX / Ti3C2 (2.5 mL, 1.0 mg / mL) was slowly added to the COOH-PEG-COOH solution (2.5 mL, 10.0 mg / mL) and stirred in the dark for 6 hours. The PEGylated DOX / Ti3C2 nanosheets (DOX / Ti3C2-PEG) were collected by centrifugation (12000 rpm, 10 min) and washed twice with water to remove excess PEG.

[0066] Step 4, coupling of Apt: The DOX / Ti3C2-PEG (1.0 mL, 2.0 mg / mL) nanosheets were activated with EDC-HCl (500 μL, 500 mM) and NHS (500 μL, 100 mM) and the reaction was carried out under magnetic stirring for 30 min. The activated DOX / Ti3C2-PEG nanosheets were obtained by centrifugation and subsequently washed 3-4 times. Afterwards, the Apt (20 OD) was denatured at 85°C for 10 min and then refolded in an ice bath for 10 min. The activated DOX / Ti3C2-PEG nanosheets were reacted with Apt under shaking conditions for 5 h, then centrifuged and washed 3 times with water to obtain the Apt-modified DOX / Ti3C2 / Apt-M nanomaterial.

[0067] In the above examples, the sequences of Apt-M and Apt-C are as follows:

[0068] The sequence of Apt-M is:

[0069] NH2-(CH2)6-GCAGTTGATCCTTTGGATACCCTGGTTTTTTTTTT;

[0070] The sequence of Apt-C is:

[0071] NH2-(CH2)6-ATTGCACTTACTATATTGCACTTACTATATTGCAC.

[0072] The content not described in detail in the specification of the present application belongs to the prior art known to those skilled in the art. The above description is only the preferred embodiment of the present application, and the present application is not limited to the scope of the specific embodiments. For those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

Claims

1. A nano-targeted drug-loaded complex, characterized in that, Ti3C2 nanomaterial is used as a carrier to load anticancer drugs on its surface and couple nucleic acid aptamer which can recognize specific proteins on cell surface; the particle size of the Ti3C2 nanomaterial is 80-200 nm; the anticancer drug is doxorubicin, and the loading amount is 15 wt.%-50 wt.% of the Ti3C2 nanomaterial; the nucleic acid aptamer is Apt-M which can specifically recognize MUC1 protein on the surface of tumor cells, and its sequence is NH2-(CH2)6-GCAGTTGATCCTTTGGATACCCTGGTTTTTTTTTT, and it is coupled to the surface of the Ti3C2 nanomaterial by covalent bonding; the preparation method of the nano-targeted drug-loaded complex comprises the following steps: Step 1, preparation of Ti3C2 nanomaterial: taking Ti3AlC2 as raw material, etching Ti3AlC2 material by HF acid, and then intercalating and dispersing by tetrapropylammonium hydroxide, after ultrasonic treatment for 15 min, preparing few-layer Ti3C2 nanomaterial; Step 2, loading of anticancer drugs: loading doxorubicin onto Ti3C2 nanomaterial by electrostatic interaction to prepare doxorubicin / Ti3C2; Step 3, PEG surface modification: mixing and stirring doxorubicin / Ti3C2 prepared in step 2 with double-carboxyl PEG to prepare doxorubicin / Ti3C2-PEG; Step 4, coupling of nucleic acid aptamer: using EDC / NHS reagent to couple nucleic acid aptamer to the surface of doxorubicin / Ti3C2-PEG by covalent bonding, i.e. to prepare nano-targeted drug-loaded complex; wherein the nucleic acid aptamer Apt-M is denatured at 85℃ for 10 minutes, and then refolded in ice bath for 10 minutes.

Images