CN116712557A - EpCAM aptamer targeting Dox hyaluronic acid nanogel and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of nanoparticle medicines, and discloses an EpCAM aptamer-targeted Dox hyaluronic acid nanogel which is formed by coupling an EpCAM aptamer and a Dox-entrapped HA nanogel, wherein the HA nanogel is modified with tetrazole and methacrylic acid. The EpCAM aptamer-targeted Dox hyaluronic acid nanogel can realize a double-active targeting effect, and the Dox is released directionally under the reduction condition, so that the drug effect is improved. Compared with internalization of Dox hyaluronic acid loaded nanogel before and after modification of EpCAM aptamer and induction and killing of apoptosis, experimental results show that the modified nanogel has obviously improved effect and can be used for preparing tumor targeted drugs. The invention also discloses a preparation method and application of the EpCAM aptamer-targeted Dox hyaluronic acid nanogel.

Description

EpCAM aptamer targeting Dox hyaluronic acid nanogel and its preparation method and application

Technical field

The invention belongs to the field of nanoparticle drugs, and in particular relates to an EpCAM aptamer-targeted Dox hyaluronic acid nanogel and its preparation method and application.

Background technique

Chemotherapy is considered to be one of the most effective methods for treating human cancer, and drug therapy is also a very important means of tumor treatment. However, serious drug-related side effects, such as poor physical and chemical properties, low tumor targeting, insufficient cellular internalization, and acquired drug resistance, seriously limit the application of anticancer drugs. Severe toxic side effects and limited efficacy have always been A problem that is difficult to eradicate with chemotherapy drugs. To reduce the side effects of chemotherapy drugs, cancer-specific targeted therapy is the first choice. In this regard, people have developed a method of using drug carrier systems for tumor-targeted delivery. In addition to the enhanced penetration and retention effect (EPR effect) of nano-sized carriers at the tumor site, the surface of the drug carrier is modified with groups that can be specifically recognized by tumor tissue cells. , which can effectively improve the concentration of drugs in tumor sites. Among them, aptamers have attracted more and more attention from researchers due to their high affinity and easy modification. The use of environmentally sensitive stimulus-responsive drug delivery systems can further improve tumor visual effects and reduce toxic and side effects. Among them, reduction sensitivity is one of the most widely used sensitive stimulus methods.

HA is a natural polysaccharide molecule with good biodegradability and biocompatibility, and can target malignant tumor cells with overexpression of CD44, such as MCF-7 breast cancer cells, A549 lung cancer cells, and LP-1 Human multiple myeloma cells and ALM-2 acute leukemia cells.

HA nanogel is derived from two types of HA: hyaluronic acid-cystamine-methacrylate derivative (HA-Cys-MA) and hyaluronic acid-lysine-tetrazole derivative (HA-Lys-Tet) The material was prepared by nanoprecipitation method and "tetrazole-ene" photo-click chemical cross-linking method. This photoclick chemical reaction has the advantages of high efficiency, strong selectivity, and does not require any catalyst. At the same time, it was reported that this reaction can be used to selectively label proteins in mammalian cells, showing good cytocompatibility. The MA group is connected through cystamine and HA, which gives the nanogel bioresponsiveness, which makes the nanogel stable during blood circulation, but will degrade under strong reducing conditions (2-10mM glutathione) after entering the cells. The encapsulated protein drug is quickly released due to the cleavage of the disulfide bond. In addition, the formed nanogel has strong fluorescence because it contains pyrazoline cycloadduct, which facilitates our tracking and observation of the nanogel in vivo and in vitro.

In order to improve the targeting and efficacy of the poorly water-soluble drug doxorubicin (Dox), Lin Zhu et al. developed a method consisting of polyethylene glycol (PEG), matrix metalloproteinase 2 (MMP2)-sensitive peptide linker (Pp), and cell A novel drug release platform (PEG) composed of penetrating peptide (TAT) and model drug (doxorubicin).

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein with a molecular weight of 40,000. It plays a role in the process of epithelial carcinogenesis as a homophilic calcium-independent inter-epithelial cell adhesion molecule. . EpCAM was first screened from colon cancer. The epithelial cell adhesion molecule EpCAM is located on the cell surface and plays an important role in the process of epithelial carcinogenesis. EpCAM mainly has three domains, namely extracellular domain, transmembrane domain and intracellular structure. area. EpCAM is highly expressed on the surface of various tumor cells, while under physiological conditions EpCAM is weakly expressed on most normal epithelia except squamous epithelial cells and hepatocytes, and is mostly located at cell junctions. It is not expressed in normal tissues, such as brain, fat, cardiac muscle, skeletal muscle, lymph node, ovary, oral cavity, uterine basal layer and gastrointestinal smooth muscle. EpCAM is a highly practical and specific tumor marker. EpCAM has been recognized as a malignant tumor-related antigen and is highly expressed in malignant solid tumors such as lung cancer, liver cancer, and gastric cancer. EpCAM has become an immunotherapy target for a variety of solid tumors, and the development and transformation of EpCAM antibodies has become an inevitable trend. However, more and more targeted therapies suggest that some patients are insensitive to antibodies or develop drug resistance. Therefore, new EpCAM-targeted treatment options need to be studied.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings and defects mentioned in the above background technology and provide an EpCAM aptamer-targeted Dox hyaluronic acid nanogel and a preparation method thereof.

In order to solve the above technical problems, the technical solutions proposed by the present invention are:

An EpCAM aptamer targeting Dox hyaluronic acid nanogel, consisting of an EpCAM aptamer coupled with a hyaluronic acid nanogel (HA nanogel) containing doxorubicin (Dox), the transparent Hydronic acid nanogels are modified with autofluorescent tetrazole and methacrylic acid.

In the present invention, hyaluronic acid (HA) is modified to couple with methacrylic acid (MA), and at the same time, hyaluronic acid is modified to carry autofluorescent tetrazole (Tet), through tetrazole-ene click Autofluorescent hyaluronic acid nanogels (HA-NG) were chemically obtained and then coupled to EpCAM aptamers through amidation for cancer cell capture to achieve active tumor targeting. Hyaluronic acid targets CD44, and specific aptamers target EpCAM. Both CD44 and EpCAM are specific and highly expressed molecules expressed by cancer cells, thereby achieving dual targeting and synergistic effects on tumor cells and improving tumor targeting. tropism and enhanced cellular internalization. At the same time, Dox is wrapped with hyaluronic acid, and drugs (such as Dox) can be wrapped in nanogels that can be passively and actively targeted at the same time, thereby achieving targeted therapy of drugs, reducing non-specific adsorption in the circulatory system, increasing circulation time, and reducing systemic damage. Improve drug efficacy and reduce systemic toxicity.

The above-mentioned EpCAM aptamer targets Dox hyaluronic acid nanogel. Preferably, the DNA sequence of the EpCAM aptamer is: CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG (as shown in SEQ ID NO: 1).

EpCAM aptamer is a single-stranded DNA obtained through SELEX screening. It can selectively bind to EpCAM molecules with high specificity and strong affinity. It is a new type of EpCAM targeting molecule. Epidermal cell adhesion molecule (EpCAM) is overexpressed on the surface of most circulating tumor cells, and EpCAM aptamers can specifically recognize EpCAM to capture CTCs. Since MCF-7 and T47D cells highly express EpCAM, we selected these two cells as model cells for CTCs. The cells in the control group were selected from MB-MDA-231 and MCF-10A cells that expressed low EpCAM. The cells in the negative control group were selected from cells that did not express EpCAM. EpCAM human embryonic kidney (HEK) 293T cells.

Preferably, the mass ratio of hyaluronic acid, tetrazole, and methacrylic acid in the HA nanogel is 10:1:1.

Preferably, the mass ratio of the EpCAM aptamer and Dox-encapsulated HA nanogel is 10:1.

Preferably, the Dox-encapsulated HA nanogel is also coupled with Cy5.5-NH ₂ and/or Cy3-NH ₂ .

Based on a general inventive concept, the present invention also provides a preparation method for EpCAM aptamer-targeted Dox hyaluronic acid nanogel, which includes the following steps:

(1) Prepare single-end Boc protected cystamine NH ₂ -Cys-Boc, react the NH ₂ -Cys-Boc with methacryloyl chloride to prepare N-tert-butylcarbonyl-N'-methacryloyl- Cystamine small molecule, namely MA-Cys-Boc;

(2) Remove the Boc protecting group from the MA-Cys-Boc obtained in step (1) to prepare MA-Cys-NH ₂ , and use hyaluronic acid and MA-Cys-NH ₂ to undergo an amidation reaction to prepare hyaluronic acid Acid-cystamine-methacrylate derivative, namely HA-Cys-MA;

(3) Prepare HA-Lys-NH ₂ and use HA-Lys-NH ₂ and Tet to prepare HA-Lys-Tet through amidation reaction;

(4) Dissolve the HA-Cys-MA obtained in step (2) and the HA-Lys-Tet obtained in step (3) in PB buffer solution to form a polymer solution, add Dox and mix evenly, and pass through the coupling Dox-encapsulated HA nanogel was prepared using nanoprecipitation method and catalyst-free "tetrazole-ene" photoclick chemical reaction;

(5) Perform an amidation reaction between the Dox-encapsulated HA nanogel obtained in step (4) and the EpCAM aptamer to obtain the EpCAM aptamer-targeted Dox hyaluronic acid nanogel.

The above preparation method, preferably, in step (3), the preparation method of HA-Lys-NH ₂ is as follows: hyaluronic acid (HA) and N-tert-butoxycarbonyl-L-lysine methyl ester The hydrochloride Lys(Boc) reacts to obtain HA-Lys(Boc), and then the HA-Lys(Boc) is deprotected by the action of TFA and HCl, dialyzed and freeze-dried to obtain HA-Lys-NH ₂ ;

The preparation method of HA-Lys-Tet is prepared through amidation reaction of HA-Lys-NH ₂ and Tet as follows: Dissolve tetrazole Tet in DMSO and add DCC to activate the carboxyl group overnight, and then dissolve HA-Lys-NH ₂ in formazan. Amide is added to the activated Tet solution, and DMAP is added for amidation reaction, followed by dialysis and freeze-drying to obtain HA-Lys-Tet.

Preferably, in step (4), the preparation method of the Dox-encapsulated HA nanogel is as follows: dissolve HA-Lys-Tet and HA-Cys-MA in a PB buffer solution at a molar ratio of 1:1. Obtain the solution, add Dox nanoparticles and mix evenly; then inject the obtained polymer solution into acetone, and then irradiate it with ultraviolet light; remove the acetone by rotary evaporation, dialyze the aqueous phase with deionized water, and freeze-dry to obtain HA nanogel.

Preferably, in step (5), the amidation reaction specifically includes the following steps: dissolve the Dox-encapsulated HA nanogel in water, add EDC and NHS, adjust pH = 5.0, activate carboxyl groups for 15 minutes, Then add it dropwise to the DMSO solution containing NH ₂ -Apt-Cy5.5 or NH ₂ -Apt-Cy3, and react in the dark at 40°C for 24 hours; after the reaction is completed, add water/methanol v/v=1:1 respectively. The solution was dialyzed against pure water and finally freeze-dried to obtain aptamer-modified nanogels.

Based on a general inventive concept, the present invention also provides the application of an EpCAM aptamer-targeted Dox hyaluronic acid nanogel in the preparation of tumor-targeted drugs.

Preferably, the tumor-targeting drug is an A549 cell (lung cancer human alveolar basal epithelial cell)-targeting drug.

Compared with the prior art, the beneficial effects of the present invention are:

1. The present invention develops a nano-delivery platform, which encapsulates doxorubicin (Dox) in hyaluronic acid nanogel (HA-NG) that can target CD44 through light click, and further combines it with the EpCAM aptamer (Apt) combination to achieve a dual active targeting effect; the present invention introduces cysteine (Cys) containing a disulfide bond into HA-NG, thereby achieving directional release of Dox under reducing conditions and improving drug efficacy. The internalization, induction and killing of apoptosis of Dox-loaded hyaluronic acid nanogel (Dox@HA-NG) before and after EpCAM aptamer modification were compared. The experimental results showed that the modified nanogel (Dox@Apt-HA -NG) effect is significantly improved and can be used to make tumor-targeting drugs, especially A549 cell-targeting drugs. At the same time, the present invention realizes the tracing of nanoparticles by modifying Cy3 and Cy5.5 on the aptamer, showing good diagnostic and therapeutic effects.

2. The preparation method of the present invention is simple to operate, the nanogel remains stable 48 hours after light click, and the quality of the drug is guaranteed.

Description of the drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are: For some embodiments of the present invention, those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting creative efforts.

Figure 1 is a schematic diagram of the principle of obtaining EpCAM-specific targeting and traceable nanogels through amidation reaction;

Figure 2 is the Boc-Cys-NH ₂ NMR spectrum;

Figure 3 is the MA-Cys-Boc NMR spectrum;

Figure 4 is the hydrogen nuclear magnetic resonance spectrum of tetrazole;

Figure 5 is the carbon NMR spectrum of tetrazole;

Figure 6 is the NMR spectrum of HA-Lys-Tet;

Figure 7 is the UV absorption spectrum before and after cross-linking;

Figure 8 is a diagram of apoptosis detected by flow cytometry;

Figure 9 shows the stability of nanogels at different concentrations and after adding disulfide bond cross-linking;

Figure 10 shows the binding efficiency of EpCAM-Apt-Cy3 and A549 cells;

Figure 11 is the hydrogen nuclear magnetic resonance spectrum of HA-Cys-MA;

Figure 12 is the hydrogen nuclear magnetic resonance spectrum of HA-Cys-MA-Apt;

Figure 13 is the hydrogen nuclear magnetic resonance spectrum of HA-Cys-MA-Apt-Cy3;

Figure 14 shows the viability of DOX cells treated with different concentrations;

Figure 15 shows the viability of HA-NG cells treated with different concentrations;

Figure 16 shows the binding efficiency of Cy3-modified EpCAM aptamer to EpCAM-positive cells A549 and EpCAM-negative cells 293T at different aptamer concentrations;

Figure 17 shows the affinity of A549 cells for Cy3-Apt before and after washing;

Figure 18 shows the internalization of Dox and Dox@Apt-NG by A549 cells;

Figure 19 shows the changes in nanoparticles when the reductive disulfide bond of GSH is destroyed;

Figure 20 is the fluorescence spectrum of Dox released by Dox@HA-NG in 10mM PB (Px in the figure represents the emission spectrum after treatment in PB solution for x hours);

Figure 21 is the fluorescence spectrum of Dox released by Dox@HA-NG in 10mM GSH (Gx in the figure represents the emission spectrum after treatment in GSH solution for x hours);

Figure 22 is the Dox release fluorescence spectrum of Dox@HA-NG in PBS and GSH (Px in the figure represents the emission spectrum after treatment in PB solution for x hours, Gx represents the emission spectrum after treatment in GSH solution for x hours);

Figure 23 shows the fluorescence intensity of different concentrations of Dox under 480nm excitation (dox1 in the figure represents the fluorescence intensity of 0.4nm Dox, dox2 represents the fluorescence intensity of 0.2nm Dox, dox3 represents the fluorescence intensity of 0.1nm Dox, and dox4 represents the fluorescence intensity of 0.05nm Dox , dox5 represents the fluorescence intensity of 0.025nm Dox);

Figure 24 is the relationship between Dox concentration and maximum fluorescence intensity;

Figure 25 is the laser confocal of HA-NG and Cy3-Apt-HA-NG entering A549;

Figure 26 shows the changes in UV absorption before and after light click;

Figure 27 shows the efficiency of DOX packaging with HA-NG and Apt-HA-NG;

Figure 28 shows the change in particle size after GSH treatment of HA-NG;

Figure 29 shows the change in potential after GSH treats HA-NG;

Figure 30 shows the cytotoxicity of different concentrations of DOX encapsulated in nanogels on A549 and 293T.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more comprehensively and in detail below with reference to the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

Unless otherwise defined, all technical terms used below have the same meanings as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased in the market or prepared by existing methods.

Example 1:

An EpCAM aptamer targeting Dox hyaluronic acid nanogel, the preparation method includes the following steps:

1. Synthesis of N-tert-butylcarbonyl-N’-methacryloyl-cystamine (MA-Cys-Boc) small molecule

(1) Preparation of single-end Boc protected cystamine (NH ₂ -Cys-Boc)

First, cystamine dihydrochloride (10.00g, 44.40mmol) was added to 200mL of anhydrous methanol at room temperature. After sufficient stirring, triethylamine (13.48g, 133.25mmol) was added and reacted for 15 minutes to obtain desalted cystamine. ; Subsequently, di-tert-butyl dicarbonate (9.71g, 44.45mmol) was added to the desalted cystamine solution and reacted for 2.5 hours to obtain single-ended or double-ended Boc-protected cystamine; then, after the above reaction solution was spin-dried, 300 mL was added KH ₂ PO ₄ (1M, pH 4.2) solution and stirred thoroughly, then washed with diethyl ether (2 × 50 mL) to remove the Boc-protected cystamine at both ends. Adjust the pH of the remaining aqueous phase to about 9 with 1M NaOH solution, and extract with ethyl acetate (6 × 30 mL); finally, add anhydrous magnesium sulfate to the organic phase and dry overnight, filter with suction, and spin dry to obtain a yellow oil. Single-end Boc protected cystamine (NH ₂ -Cys-Boc), Boc-Cys-NH ₂ NMR spectrum is shown in Figure 2.

(2) Prepare MA-Cys-Boc by reacting NH ₂ -Cys-Boc with methacryloyl chloride

Under nitrogen atmosphere, methacryloyl chloride (1.80 mL, 22.15 mmol) was dissolved in 50 mL of dry chloroform under ice bath conditions, and then 50 mL of triethylamine (3.21 mL, 023.12 mmol) and NH ₂ -Cys -The chloroform solution of Boc (2.77g, 10.97mmol) was added dropwise to methacryloyl chloride (it takes about 1.5 hours); after reacting at room temperature for 24 hours, spin the reaction solution to dryness, then wash it with 120mL of water, and then Extract with chloroform (3×50mL); the organic phase is concentrated and passed through the column (developing solvent is ethyl acetate: petroleum ether = 5:1). After spin drying, the white powder MA-Cys-Boc product is obtained, with a yield of 20%. . The MA-Cys-Boc NMR spectrum is shown in Figure 3.

2. Synthesis of small molecule monomer tetrazole (Tet)

(1) Under nitrogen protection, dissolve p-formylbenzoic acid (0.75g, 5mmol) in 50mL ethanol, stir for about 10 minutes, then add benzenesulfonyl hydrazide (0.86g, 5mmol) and react for 30 minutes; the reaction is completed Then use 15-20 times of secondary water to precipitate, and then vacuum dry to obtain a light yellow phenylhydrazone solid.

(2) Add 0.23mL aniline to the mixed solution of 2mL water, 2mL ethanol, and 0.65mL concentrated HCl under ice-water bath conditions and stir to mix evenly. Then add 1mL of aqueous solution containing 0.175g NaNO ₂ dropwise to the above aniline mixed solution. Continue stirring for about 15 minutes to obtain a light yellow diazobenzene solution.

(3) Dissolve phenylhydrazone (0.6g) in 15mL of pyridine under ice bath conditions, then add the diazobenzene solution prepared in step (2) drop by drop into the phenylhydrazone solution and continue the reaction for 6 hours. After the reaction is completed, use acetic acid to The organic phase was separated by extraction with ethyl ester (10ml×4), and then HCl (3M, 120ml) was added to extract the upper floc, filtered, and vacuum dried to obtain pink tetrazole small molecule solid powder with a yield of about 26%. As can be seen from Figures 4 and 5, tetrazole was prepared.

3. Synthesis of hyaluronic acid-cystamine-methacrylate derivative (HA-Cys-MA)

(1) Use MA-Cys-Boc to remove the Boc protecting group to prepare MA-Cys-NH ₂

In a nitrogen atmosphere, MA-Cys-Boc (21.1 mg, 66 nmol) was dissolved in 0.5 mL trifluoroacetic acid/methanol (v/v=l:l) under ice bath conditions, stirred for 6 hours, and the solvent was removed by rotary evaporation. .

(2) Use HA and MA-Cys-NH ₂ to prepare HA-Cys-MA through amidation reaction

Dissolve HA (50 mg, 0.132 mmol carboxyl group) in 5 mL water, add EDC (75.9 mg, 0.396 mmol) and NHS (22.8 mg, 0.198 mmol), adjust pH = 5.0, activate the carboxyl group for 15 minutes, and then add dropwise to the above MA-Cys-NH ₂ (14.5 mg, 66 pmol) in DMSO (1 mL) solution, react in the dark at 40°C for 24 hours; after the reaction is completed, dialyze in water/methanol (v/v=l:1) and pure water. (Spectra/Pore, MWCOof3500), and finally freeze-dried to obtain the HA-Cys-MA product.

4. Synthesis of hyaluronic acid-lysine-tetrazole derivative (HA-Lys-Tet)

(1) Preparation of HA-Lys-NH ₂

First, triethylamine (85 mg, 0.84 mmol) was added to a fully stirred solution of anhydrous methanol (2.0 ml) containing H-Lys(Boc)-OMe·HCl (240 mg, 0.80 mmol) at room temperature; the mixture continued to stir for 1 hours; containing HA (300 mg, 0.79 mmol carboxyl), EDC (460 mg, 2.40 mmol) and NHS (140 mg, 1.22 mmol); water was added to adjust pH=8.5; the mixture was stirred at room temperature for 24 hours. HA-Lys(Boc) was isolated by dialysis against deionized water (Spectra/Pore, MWCO of 3500) and freeze-dried. Then, use TFA/1M HCl (v/v 1/1) to remove BOC from HA-Lys(BOC), and the reaction proceeds for 6 hours; then use 4M NaOH to adjust the pH value of the solution to about 7.0, and isolate HA-Lys- NH ₂ ; dialyzed with deionized water (Spectra/Pore, MWCO of 3500) and freeze-dried to obtain HA-Lys-NH ₂ with a yield of 91%.

(2) Use HA-Lys-NH ₂ and Tet to prepare HA-Lys-Tet through amidation reaction

First, Tet (519 mg, 1.95 mmol) was fully dissolved in 10 mL dimethyl sulfoxide under a nitrogen atmosphere, and then DCC (232 mg, 1.12 mmol) was added to react overnight to activate the carboxyl group; at the same time, HA-Lys-NH ₂ (400mg, 195nmol amino group) was added to 30mL formamide to fully dissolve, then added dropwise to the above carboxyl-activated Tet solution, and then DMAP (80mg, 655unol) was added and reacted at 40°C for 48 hours; after the reaction was completed , followed by dialysis in water/dimethyl sulfoxide (v/v=l:l) and pure water (Spectra/Pore, MWCO of 3500), and finally freeze-drying to obtain HA-Lys-Tet with a yield of 69%. It can be seen from Figure 6 that there is a specific Tet peak, and the present invention synthesized HA-Lys-Tet.

5. Preparation of reduction-sensitive autofluorescent nanogels

HA nanogels were prepared by combining nanoprecipitation method and catalyst-free "tetrazole-ene" photoclick chemical reaction. details as follows:

First, HA-Lys-Tet and HA-Cys-MA were dissolved in PB buffer solution (pH=7.4, 10mM) at a molar ratio of 1:1 to prepare a polymer solution with a concentration of 1.25 mg/mL, and then 1 mL of the The polymer solution was injected into 100 mL acetone, and then illuminated in a UV curing box (320-390 nm, 50 mW/cm ² ) for 3 minutes; after the reaction, the acetone was removed by rotary evaporation, and the aqueous phase was dialyzed against deionized water (Spectra/Pore , MWCO=3500), and finally freeze-dried to obtain HA nanogel (HA-Nanogel, abbreviated as HA-NG) with a yield of 89%. The preparation process is shown in Figure 1.

The preparation method of Dox-encapsulated nanogels (Dox@HA-NG) is similar to this. Dox only needs to be added to the HA-Lys-Tet and HA-Cys-MA polymer solutions in advance, and then mixed thoroughly according to the above HA nanogel preparation method. Just follow the steps for making the gel.

6. Nanoparticle-aptamer combination

As shown in Figure 1, EpCAM-specific targeting and traceable nanogels were obtained through the amidation reaction of NH ₂ -Apt-Cy5.5 or NH ₂ -Apt-Cy3 with HA-NG/Dox@HA-NG ( Apt-HA-NG/Dox@Apt-HA-NG), the details are as follows:

The DNA sequence of the EpCAM aptamer (AptEpCAM, abbreviated as Apt) is: CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG (as shown in SEQ ID NO: 1).

Dissolve HA-NG/Dox@HA-NG (100 mg, 0.264 mmol carboxyl group) in 5 mL water, and add EDC (75.9 mg, 0.396 mmol) and NHS (22.8 mg, 0.198 mmol), adjust pH = 5.0, and activate carboxyl group 15 minutes, then add dropwise to the DMSO (1mL) solution of the above NH ₂ -Apt-Cy5.5 or NH ₂ -Apt-Cy3 (30 mg, 60 mol), and react in the dark at 40°C for 24 hours; after the reaction is completed, place it in water /methanol (v/v=l:1) and dialyzed in pure water (Spectra/Pore, MWCOof3500), and finally freeze-dried to obtain EpCAM aptamer-modified traceable HA nanogel.

In the obtained HA nanogel, the mass ratio of hyaluronic acid, tetrazole, and methacrylic acid was 10:1:1, and the mass ratio of EpCAM aptamer and Dox-encapsulated HA nanogel was 10:1.

7. Experimental data

(1)Synthesis and characterization of HA-NG

Figure 7 is the UV absorption spectrum before and after cross-linking; Figure 8 is the flow cytometry detection of apoptosis. As can be seen from Figures 7-8, the present invention synthesizes HA nanogel and encapsulates the drug Dox, which can induce tumor cell death at the cellular level.

As shown in Figure 9, the nanogel has good stability after adding cysteine (Cys) to introduce disulfide bonds.

It can be seen from Table 1 that the particle size increased after coating Dox, but the potential did not change much.

Table 1: Particle size potential measured by dynamic light scattering

particle size PDI Potential Not cross-linked 1368.9±20.1 0.307 -35.11±2.83 HA-NG 265.3±24.5 0.236 -40.70±0.53 Dox@HA-NG 597.7±45.1 0.437 -40.74±1.25

It can be seen from Table 2 that the nanogel remains stable 48h after light click.

Table 2: DLS particle size and NTA particle size of nanogels 48h after light click

(2) Binding efficiency of EpCAM-Apt-Cy3 at different concentrations and A549 cells

As shown in Figure 10, as the concentration increases, the binding ratio increases, and when it reaches 4umol/ml, more than 90% of the cells are labeled, indicating that the binding efficiency of EpCAM-Apt-Cy3 to A549 cells is high.

(3) Aptamer-modified HA-Cys-MA obtained through amidation reaction between -COOH on HA-Cys-MA and NH ₂ -Apt or NH ₂ -Apt-Cy3

Figures 11-13 are hydrogen nuclear magnetic resonance spectra of HA-Cys-MA, HA-Cys-MA-Apt and HA-Cys-MA-Apt-Cy3. As shown in Figure 11-13, the comparison found that a new peak was obtained, indicating that Apt is connected to HA-Cys-MA through amidation.

(4) CCK8 detects the cytotoxicity of HA-NG and free Dox

Figures 14-15 show the viability of DOX and HA-NG cells treated with different concentrations. As shown in Figure 14-15, free DOX becomes more cytotoxic as the concentration increases, while HA-NG has no cytotoxicity.

(5) Binding efficiency of Cy3-modified EpCAM aptamer to EpCAM-positive cells A549 and EpCAM-negative cells 293T under different aptamer concentrations

As shown in Figure 16, the Cy3-modified EpCAM aptamer can specifically bind to A549 in a concentration-dependent manner, but hardly binds to 293T.

(6) Photo-click synthesis and purification to obtain Apt-HA-NG (Apt), Cy3-Apt-HA-NG (Cy3), Dox@Apt-HA-NG (Dox@Apt), Dox@Cy3-Apt-HA- NG(Dox@Cy3)

Figure 17 shows the affinity of A549 cells for Cy3-Apt before and after washing; the nanogel potential before and after wrapping was negative and the particle size did not change much. After filtration, the particle size of DOX-wrapped nanoparticles did not exceed 300 nm.

(7) Internalization of Dox and Dox@Apt-NG by A549 cells

Figure 18 is the internalization of Dox and Dox@Apt-NG by A549 cells; Figure 19 is the change of nanoparticles when the reductive disulfide bond of GSH is destroyed; Figure 20 is Dox@HA-NG in 10mM PB Dox release fluorescence spectrum; Figure 21 is Dox release fluorescence spectrum of Dox@HA-NG in 10mM GSH; Figure 22 is Dox release fluorescence spectrum of Dox@HA-NG in PBS and GSH; Figure 23 is different concentrations of Dox excited at 480nm Fluorescence intensity under; Figure 24 is the relationship between Dox concentration and maximum fluorescence intensity. As shown in Figure 18-24, under reducing conditions, the disulfide bonds in the nanogel open to release Dox, and after aptamer modification, more nanogel enters the A549 cells and is released.

(8) Confocal observation of Cy3-Apt internalization in A549 cells

Figure 25 is the laser confocal of HA-NG and Cy3-Apt-HA-NG entering A549; Figure 26 is the change of UV absorption before and after light click; Figure 27 is the efficiency of DOX wrapping by HA-NG and Apt-HA-NG; Figure 28-29 are the changes in particle size and potential after GSH treatment of HA-NG. As shown in Figure 25-29, the nanogel is released triggered by reducing conditions. After being modified by aptamers, it can promote the directional internalization of the nanogel into tumor cells and release. Figure 30 shows the cytotoxicity of different concentrations of DOX encapsulated in nanogels on A549 and 293T. As shown in Figure 30, it was found that the cytotoxicity to A549 at the same concentration was significantly higher than that of 293T, indicating that the aptamerized nanogel can specifically recognize A549 and can be used to prepare targeted drugs for A549 cells.

Claims (10)

1. An EpCAM aptamer-targeted Dox hyaluronic acid nanogel, characterized in that it consists of an EpCAM aptamer coupled with a Dox-encapsulated HA nanogel, and the HA nanogel is modified with tetrazole and methacrylic acid. 2. The EpCAM aptamer targeting Dox hyaluronic acid nanogel according to claim 1, wherein the DNA sequence of the EpCAM aptamer is: CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGG GGGTTGGCCTG. 3. EpCAM aptamer targeting Dox hyaluronic acid nanogel according to claim 1, characterized in that the mass ratio of hyaluronic acid, tetrazole and methacrylic acid in the HA nanogel is 10: 1:1. 4. The EpCAM aptamer-targeted Dox hyaluronic acid nanogel according to claim 1, characterized in that the mass ratio of the EpCAM aptamer and Dox-encapsulated HA nanogel is 10:1. 5. EpCAM aptamer-targeted Dox hyaluronic acid nanogel according to any one of claims 1-4, characterized in that the Dox-encapsulated HA nanogel is also coupled with Cy5.5 -NH ₂ and/or Cy3-NH ₂ . 6. A method for preparing EpCAM aptamer-targeted Dox hyaluronic acid nanogel, which is characterized by comprising the following steps: (1) Prepare single-end Boc protected cystamine NH ₂ -Cys-Boc, react the NH ₂ -Cys-Boc with methacryloyl chloride to prepare N-tert-butylcarbonyl-N'-methacryloyl- Cystamine small molecule, namely MA-Cys-Boc; (2) Remove the Boc protecting group from the MA-Cys-Boc obtained in step (1) to prepare MA-Cys-NH ₂ , and use hyaluronic acid and MA-Cys-NH ₂ to undergo an amidation reaction to prepare hyaluronic acid Acid-cystamine-methacrylate derivative, namely HA-Cys-MA; (3) Prepare HA-Lys-NH ₂ and use HA-Lys-NH ₂ and Tet to prepare HA-Lys-Tet through amidation reaction; (4) Dissolve the HA-Cys-MA obtained in step (2) and the HA-Lys-Tet obtained in step (3) in PB buffer solution to form a polymer solution, add Dox and mix evenly, and pass through the coupling Dox-encapsulated HA nanogel was prepared using nanoprecipitation method and catalyst-free "tetrazole-ene" photoclick chemical reaction; (5) Perform an amidation reaction between the Dox-encapsulated HA nanogel obtained in step (4) and the EpCAM aptamer to obtain the EpCAM aptamer-targeted Dox hyaluronic acid nanogel. 7. The preparation method according to claim 6, characterized in that, in step (3), the preparation method of the HA-Lys- _NH is as follows: hyaluronic acid HA and N-tert-butoxycarbonyl-L - Lysine methyl ester hydrochloride Lys(Boc) reacts to obtain HA-Lys(Boc), and then HA-Lys(Boc) is deprotected by the action of TFA and HCl, dialyzed and freeze-dried to obtain HA-Lys-NH ₂ ; The preparation method of HA-Lys-Tet is prepared through amidation reaction of HA-Lys-NH ₂ and Tet as follows: Dissolve tetrazole Tet in DMSO and add DCC to activate the carboxyl group overnight, and then dissolve HA-Lys-NH ₂ in formazan. Amide is added to the activated Tet solution, and DMAP is added for amidation reaction, followed by dialysis and freeze-drying to obtain HA-Lys-Tet. 8. The preparation method according to claim 6, characterized in that, in step (4), the preparation method of the Dox-loaded HA nanogel is as follows: combine HA-Lys-Tet and HA-Cys-MA Dissolve the solution in PB buffer solution at a molar ratio of 1:1, add Dox nanoparticles and mix evenly; then inject the resulting polymer solution into acetone, and then irradiate it with UV light; remove the acetone by rotary evaporation, and use the water phase to Dialyzed in deionized water and freeze-dried to obtain HA nanogel. 9. The preparation method according to claim 6, characterized in that in step (5), the DNA sequence of the EpCAM aptamer is: CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG, and the amidation reaction specifically includes the following steps: loading the inclusion Dox's HA nanogel is dissolved in water, and EDC and NHS are added, the pH=5.0, the carboxyl group is activated for 15 minutes, and then added dropwise to the DMSO solution containing NH ₂ -Apt-Cy5.5 or NH ₂ -Apt-Cy3 in the dark at 40°C for 24 hours; after the reaction, it was dialyzed in a solution of water/methanol v/v = 1:1 and pure water, and finally freeze-dried to obtain aptamer-modified nanogels. 10. An EpCAM aptamer targeting Dox hyaluronic acid nanogel as described in any one of claims 1-5 or an EpCAM aptamer prepared by the preparation method as described in any one of claims 6-9. Application of ligand-targeted Dox hyaluronic acid nanogel in the preparation of tumor-targeting drugs.