CN115558039A

CN115558039A - Glycyrrhetinic acid-carboxymethyl chitosan-keto thiol-rhein conjugate, and preparation method and application thereof

Info

Publication number: CN115558039A
Application number: CN202110753678.7A
Authority: CN
Inventors: 王晓颖; 张雪; 徐伟
Original assignee: Fujian University of Traditional Chinese Medicine
Current assignee: Fujian University of Traditional Chinese Medicine
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2023-01-03
Anticipated expiration: 2041-07-02
Also published as: CN115558039B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a glycyrrhetinic acid-carboxymethyl chitosan-thioketal-rhein conjugate, and a preparation method and application thereof. The conjugate of the invention is a modificationCarboxymethyl chitosan, which is carboxymethyl chitosan substituted with two substituents:

the micelle formed by the conjugate can be used for loading a medicament tripterine, shows targeting on liver tissues and tumor tissues and has the function of slowly releasing the medicament in vivo. The characteristics enable the conjugate and the micelle of the invention to have high application potential in the preparation of liver cancer drugs.

Description

Glycyrrhetinic acid-carboxymethyl chitosan-ketothiol-rhein conjugate, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a glycyrrhetinic acid-carboxymethyl chitosan-thioketal-rhein conjugate, and a preparation method and application thereof.

Background

Celastrol (Cela) is one of effective components extracted from root of Tripterygium wilfordii (tripterygium wilfordii) of Celastraceae, is red needle crystal, and is easily soluble in organic solvent. The results show that the Cela is expected to be a very promising drug for treating cancers.

In the process of seeking new safe and effective liver cancer chemotherapeutic drugs, cela becomes one of the important research points. With further research on Cela, cela can regulate apoptosis-related factors at a cytological level to induce apoptosis and block the growth of HepG 2. In addition, researches show that Cela can obviously increase the expression of Fas and FasL in transmembrane glycoproteins in liver cancer cells, obviously increase the content of cytochrome C (Cyt C) in cytoplasm, and obviously increase the expression of activated proteins such as caspase3, caspase8, caspase9 and the like, thereby proving that endogenous and exogenous apoptosis pathways of the cells can be simultaneously activated to induce apoptosis of the Bel-7402 cells of the liver cancer. Animal experiment research shows that Cela can obviously reduce the activity of Aspartate (AST), alanine Aminotransferase (ALT) and Alkaline phosphatase (ALP) in hepatocellular carcinoma (HCC), reduce the level of Alpha-fetoprotein (AFP) in serum, reduce the number of liver cancer cells and the tumor volume of HCC rats, improve the expression of liver tissue tumor suppressor genes P53 and Bax protein of the rats, and show obvious effect of resisting the HCC of the rats. The results show that Cela has a certain treatment effect on hepatocellular carcinoma, and can be used for further anti-liver cancer research on hepatocellular carcinoma.

However, the defects of low water solubility, poor bioavailability, larger toxic and side effects and the like of the tripterine limit the research, development and clinical application of the tripterine.

The Chinese patent application CN108888774B discloses a tripterine-tree macromolecule conjugate, a preparation method and application thereof, which discloses a novel tripterine dosage form, wherein a dendritic organic macromolecule is prepared from PAMAM nano-carrier, polyethylene glycol and surface-targeted ligand, and the macromolecule is used for loading tripterine. The tripterine preparation provided by the patent application has a targeting effect in treating cancers such as colon cancer, liver cancer, breast cancer and the like with abundant EpCAM surface membrane proteins, can improve the selectivity and specificity of the medicine, and reduces the toxicity of the tripterine. However, PAMAM belongs to the polyamidoamine dendrimer, and has a disadvantage of being easily bound to negatively charged non-specific cells and proteins. And the preparation in the patent application targets EpCAM surface membrane protein, and the preparation is selectively and specifically directed to various cancer cells. In addition, the preparation has an unsatisfactory targeting effect on hepatoma cells. In summary, the targeting selectivity and specificity of the formulations in the above patent applications for liver cancer is still insufficient. Therefore, the preparation of tripterine is needed to be further developed aiming at the liver cancer, and more choices are provided for the clinical treatment of the liver cancer.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a glycyrrhetinic acid-carboxymethyl chitosan-thioketal-rhein conjugate, a preparation method and application thereof, and aims to provide a carrier for loading tripterine, wherein the carrier has good targeting property on liver tissues and tumor tissues after loading the tripterine, is particularly suitable for being used as a medicament for preventing or treating liver cancer, and provides a new choice for clinic.

A modified carboxymethyl chitosan (i.e. glycyrrhetinic acid-carboxymethyl chitosan-ketothiol-rhein conjugate, GCTR conjugate) is a carboxymethyl chitosan substituted with two substituents:

preferably, the substituents

The molar substitution degrees of (A) are 5.43-9.05% and 1.79-2.93%, respectively.

Preferably, the modified carboxymethyl chitosan is obtained by connecting rhein to one end of a thioketal and grafting glycyrrhetinic acid and the thioketal to the carboxymethyl chitosan respectively.

Preferably, the molar charge ratio of the preparation raw materials of the modified carboxymethyl chitosan is as follows:

rhein and ketodithiol 1;

and/or, the ratio of carboxymethyl chitosan to glycyrrhetinic acid 1;

and/or, the ratio of carboxymethyl chitosan to ketathiol 1.2-1;

and/or rhein, ketamine, carboxymethyl chitosan and glycyrrhetinic acid 1.2.

Preferably, the rhein is linked to the carboxyl group at one end of the ketal thiol through a polyamino compound; the polyamino compound is preferably selected from one of ethylenediamine, 1, 3-propanediamine or 1, 4-butanediamine.

Preferably, the carboxymethyl chitosan is selected from O-carboxymethyl chitosan; and/or the molecular weight of the carboxymethyl chitosan is 1 to 10 ten thousand.

The invention also provides a preparation method of the modified carboxymethyl chitosan, which comprises the following steps:

(1) Reacting rhein with ethylenediamine or its salt to react one of the amino groups of ethylenediamine with the carboxyl group of rhein;

(2) Adding ketomercaptan into the reaction system obtained in the step (1) to enable the ketomercaptan to react with the other amino group of the ethylenediamine to obtain a ketomercaptan-rhein conjugate;

(3) Reacting the ketothiol-rhein conjugate obtained in the step (2) with carboxymethyl chitosan to obtain a carboxymethyl chitosan-ketothiol-rhein conjugate;

(4) And (3) reacting glycyrrhetinic acid with the ketamine-rhein-carboxymethyl chitosan conjugate obtained in the step (3) to obtain the modified carboxymethyl chitosan.

Preferably, in step (1), the reaction is carried out under the action of a base selected from NaHCO, a crosslinking agent and a catalyst ₃ 、Na ₂ CO ₃ At least one of sodium acetate, sodium phosphate or sodium hydrogen phosphate, and/or the crosslinking agent is selected from at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride or N, N-diisopropylcarbodiimide hydrochloride; and/or, the catalyst is selected from at least one of N-hydroxysuccinimide or 4-dimethylaminopyridine; and/or, the reaction is carried out in solvent water; and/or the temperature of the reaction is room temperature; and/or the reaction time is 6-12 h;

and/or, in the step (2), dissolving the ketal thiol by using a solvent dimethylformamide, and adding the dissolved ketal thiol into the reaction system obtained in the step (1); and/or the reaction is carried out under the action of a cross-linking agent, wherein the cross-linking agent is selected from at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride or N, N-diisopropylcarbodiimide hydrochloride; and/or the temperature of the reaction is room temperature;

and/or, in the step (3), the carboxymethyl chitosan is dissolved by solvent water and then reacts with the ketothiol-rhein conjugate; and/or the reaction is carried out under the action of a cross-linking agent, wherein the cross-linking agent is selected from at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride or N, N-diisopropylcarbodiimide hydrochloride; and/or the temperature of the reaction is room temperature; and/or the reaction time is 12-24 h;

and/or, in the step (4), the glycyrrhetinic acid is dissolved by a solvent dimethylformamide and then reacts with the carboxymethyl chitosan-ketothiolane-rhein conjugate; and/or, the reaction is carried out under the action of a catalyst, and the catalyst is selected from at least one of N-hydroxysuccinimide or 4-dimethylaminopyridine; and/or the temperature of the reaction is room temperature; and/or the reaction time is 12-24 h.

The invention also provides application of the modified carboxymethyl chitosan in preparing a medicament for preventing and/or treating liver cancer.

Preferably, the modified carboxymethyl chitosan is used as micelle to load drug molecules.

The micelle of the GCTR conjugate provided by the invention is sensitive to active oxygen, and the prepared drug Cela/GCTR micelle shows targeting on liver tissues and tumor tissues and has the effect of slowly releasing the Cela drug in vivo. The characteristics enable the GCTR conjugate and the Cela/GCTR micelle to have high application potential in the preparation of liver cancer drugs. It provides a new choice for the clinical treatment of liver cancer.

Compared with the existing targeting preparation, the preparation has active targeting for the glycyrrhetinic acid receptor on the surface of the liver cancer cell, and has passive targeting effect due to small particle size, so that the preparation has better targeting effect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a schematic of the synthesis of example 1;

FIG. 2 is a graph of the size distribution of micelles formed by the GCTR conjugate of example 1;

FIG. 3 shows FT-IR spectra (left) and (right) of glycyrrhetinic acid (a), rhein (b), thioketal (c), carboxymethyl chitosan (d) and GCTR conjugate (e) of example 1 ¹ H-NMR spectrum (right);

FIG. 4 is a graph of the size distribution of Cela/GCTR micelles of example 2;

FIG. 5 shows the micelles and H of GCTR conjugate (a) in Experimental example 2 ₂ O ₂ TEM images of GCTR conjugate (b) micelles of 6h were processed;

fig. 6 is a graph of the change in particle size of GCTR conjugate micelles in aqueous solution in experimental example 3 (n = 3);

FIG. 7 is the in vitro Cela/GCTR micelle release profile for Experimental example 5 (n = 3);

FIG. 8 shows the cytotoxicity of the GCTR conjugate with Cela/GCTR micelles on HepG2, BEL-7402, L-02 cells in Experimental example 6 (n = 6);

FIG. 9 is a CLSM map of the uptake by HepG2, L-02 cells in Experimental example 6;

FIG. 10 is a flow cytometry and fluorescence intensity distribution graph (n = 3) of HepG2 cell uptake in Experimental example 6, wherein a is the Control group; b is P4 group; c, performing GA pretreatment on the (P4 + Cela)/GCTR micelle group after 1 h; d (P4 + Cela)/CR micelle group; e, (P4 + Cela)/GCTR micelle group;

FIG. 11 is a graph showing the flow cytometry detection of the effect of Cela/GCTR micelles on the HepG2 cell cycle in Experimental example 6;

fig. 12 is a graph of plasma concentration versus time for the mouse tail of experimental example 7 after intravenous injection of each of the formulations of Cela;

FIG. 13 is a graph showing the distribution of different Cela preparations in each tissue of tumor-bearing mice at different time points in Experimental example 7;

FIG. 14 shows the results of fluorescence imaging of a mouse in vivo in Experimental example 7;

FIG. 15 shows the results of fluorescence intensity of whole body at different time points in the live body imaging of the mouse in Experimental example 7;

FIG. 16 shows the fluorescence intensity results of the tumors at different time points in the live body imaging of the mouse in Experimental example 7;

FIG. 17 shows fluorescence imaging of 24h tissue of the mouse in Experimental example 7 (tissue fluorescence images are, from top to bottom, heart, liver, spleen, lung, kidney, and tumor);

FIG. 18 shows the results of fluorescence intensity of 24h tissue of the mouse in Experimental example 7.

Detailed Description

In the following examples and experimental examples, the reagents and materials used are commercially available, specifically as follows:

glycyrrhetinic Acid (GA), pharmaceutical grade (98%), shanghai-derived phyllotonics ltd;

o-carboxymethyl chitosan (CMCS) with the molecular weight of 1 ten thousand, qingdao Honghai biotechnology limited;

ketodithiols (TK), pharmaceutical grade (98%), sienna ruixi biotechnology limited;

rhein (R), pharmaceutical grade (98%), aoyuan biotechnology, shanxi;

ethylenediamine hydrochloride, analytical grade (99%), mclin biochemistry science and technology ltd;

n-hydroxysuccinimide (NHS), analytical grade (98%), shanghai Allantin Biotechnology Ltd;

1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), analytical grade (98%), shanghai Allantin Biotechnology Ltd;

anhydrous ethylenediamine, analytical grade, fuzhou macro new glass instruments ltd;

pyrene, analytical grade (99%), shanghai alading biochem technologies, ltd;

dialysis bags, MWCO3500, biosharp;

tripterine (Cela), pharmaceutical grade (98%), tokasi pharmaceutical technology ltd;

DMSO, ethanol, methanol, analytical grade, national drug group chemical reagents, inc.;

n, N-dimethylformamide, analytically pure, national chemical group chemical reagent limited;

glucose, analytical pure, national drug group chemical reagents limited;

mannitol, analytical pure, national pharmaceutical group chemical reagents ltd;

0.9% sodium chloride injection, 190407a44, hawangfu, fuzhou pharmaceutical limited;

5% glucose injection, analytically pure, fuzhou Haiwang medicine pharmacy Co., ltd;

dialysis bag, MWCO3500, biosharp;

p2 and P4 fluorescent probes, synthesized according to the method of the literature Nanomedicine: nanotechnology, biology, and Medicine,2015 (11): 1939-1948.

In the following examples and experimental examples, the following abbreviations are also used:

rhein-N-hydroxysuccinimide adduct: R-NHS; rhein-ethylenediamine adduct: R-EDA; ketothiol-rhein conjugates: TER; carboxymethyl chitosan-ketothiol-rhein conjugate: CTR; glycyrrhetinic acid-carboxymethyl chitosan-keto thiol-rhein conjugate: a GCTR conjugate; tripterine/glycyrrhetinic acid-carboxymethyl chitosan-thioketal-rhein conjugate micelle: cela/GCTR micelles; carboxymethyl chitosan-rhein conjugate: a CR conjugate; tripterine/carboxymethyl chitosan-rhein conjugate: cela/CR micelles; active oxygen: and (4) ROS.

Example 1 Synthesis of GCTR conjugate (i.e., modified carboxymethyl chitosan)

Synthesis of GCTR conjugate as shown in figure 1, a four-step synthesis is envisaged:

the first step is as follows: 0.1mmol R1% NaCO ₃ Heating to dissolve, adding 0.12mmol EDC & HCl under stirring, reacting at room temperature for 20min, and then adding 0.12mmol NHS to react at room temperature for 10min to obtain R-NHS. Adding 0.15mmol of EDA to dissolve with water, dripping R-NHS into EDA water solution, and reacting for 6h to obtain R-EDA.

The second step is that: dissolving 0.15mmol TK with DMF, adding 0.18mmol EDC & HCl, reacting at room temperature for 20min, adding 0.18mmol NHS, reacting at room temperature for 10min, adding into R-EDA solution, and reacting at room temperature to obtain TER.

The third step: 0.083mmol CMCS is dissolved in water. And (3) centrifuging the TER solution, taking the supernatant, adding 0.12mmol of EDC & HCl, reacting at room temperature for 20min, then adding 0.12mmol of NHS, reacting at room temperature for 10min, then adding into the CMCS aqueous solution, and reacting for 24h to obtain the CTR.

The fourth step: dissolving 0.12mmol of GA with DMF, reacting 0.14mmol of EDC & HCl at room temperature for 20min, reacting 0.14mmol of NHS at room temperature for 10min, adding into CTR solution, reacting at room temperature for 24h, dialyzing the reaction solution in a dialysis bag (MWCO 3500) with distilled water for 72h, performing ultrasonic treatment on a probe for 20min, and performing freeze drying to obtain the GCTR conjugate.

In the GCTR conjugate structure synthesized by the synthesis process, the molar substitution degree of R is (7.24 +/-1.81)%, the molar substitution degree of GA is (2.36 +/-0.57)%, the micelle particle size formed by self-assembly in water is smaller and is (220.17 +/-5.50) nm (figure 2), the distribution is uniform, and the potential is (-12.03 +/-0.82) mV.

FT-IR characterization of each starting material and the resulting GCTR conjugate and ¹ and H-NMR characterization is carried out, the result is shown in figure 3, and the GCTR conjugate is successfully synthesized according to changes of infrared spectrum peaks and proton peaks in the figure.

Example 2 preparation of Cela/GCTR micelles

The GCTR conjugate in this example was prepared using the method of example 1.

Cela/GCTR micelles were prepared by the following method: weighing 12mg of GCTR conjugate, adding 2.4mL of distilled water, stirring for dissolving, performing ultrasonic treatment on an ice-water bath probe for 10min to uniformly disperse, dropwise adding 20mg/mL of Cela DMSO solution under vigorous stirring, continuing stirring at high speed for 20min, performing ultrasonic treatment on the ice-water bath probe for 20min, dialyzing the distilled water for 12h to remove the organic solvent, performing ultrasonic treatment on the ice-water bath probe for 20min, performing centrifugal treatment at 3500r/min for 10min after dialysis is finished, and filtering a supernatant through a 0.8-micrometer filter membrane to obtain the Cela/GCTR micellar solution.

The results of repeating three batches according to the optimal drug loading process are shown in table 1 and fig. 4, and the Cela/GCTR micelle prepared in the embodiment has small particle size, small particle size distribution (PDI) range, large drug loading amount (DL), high Encapsulation Efficiency (EE) and high repeatability, which indicates that the drug loading process is reliable and stable.

Table 1 best drug loading process verification results (n = 3)

To further illustrate the beneficial effects of the present invention, experiments were conducted in which the GCTR conjugate used in the unexplained case was the GCTR conjugate of example 1 and the Cela/GCTR micelles used were the Cela/GCTR micelles of example 2.

Experimental example 1 Synthesis Condition screening of GCTR conjugates

Based on the method of example 1, the particle size and distribution (PDI), zeta potential, and degree of substitution of R with GA (DS) were used _R 、DS _GA ) As an index for the synthesis process investigation of the GCTR conjugate, the detection results of the GCTR conjugate synthesized by different feed ratios are shown in Table 2.

When CMCS and GA are fed fixedly and the feeding amount of R: TK is changed (1-1.

When the feeding amounts of R and TK are fixed, and the feeding amounts of CMCS and GA are changed (1. When CMCS is GA = 1.5, the molar substitution degree of R and GA is maximum, and the influence of particle size and distribution is not obvious.

The reaction time examination result shows that the molar substitution degree of R and GA on the GCTR conjugate is increased along with the extension of the reaction time (6-24 h), the particle size is reduced, the particle size distribution is more uniform, and the increase of the reaction time is supposed to be beneficial to the synthesis of the product, but the synthesis efficiency is reduced if the reaction time is too long. Therefore, 24h was chosen as the reaction time.

And finally, considering factors such as the substitution degree, the particle size, the PDI, the potential and the like of R and GA comprehensively, determining the optimal synthesis charge ratio of R to TK to CMCS to GA (1.2.

TABLE 2 examination of the results of the different synthesis processes

Experimental example 2 ROS-sensitive Properties of GCTR conjugates

Example 1The obtained GCTR conjugate is made into GCTR conjugate micelle by direct dispersion and dissolution method, and its morphology is TEM characterized as shown in fig. 5 a. As can be seen, the micelles of the GCTR conjugate are all spherical structures, and the particle size distribution is relatively uniform and is about 220 nm. Taking the micelle solution of the GCTR conjugate, adding H with the concentration of 10mmol/L ₂ O ₂ The solution was diluted to 1mg/mL, placed in a shaker at 37 ℃ and after 6H of treatment, TEM images were observed, as shown in FIG. 5b, over H ₂ O ₂ The treated GCTR conjugate micelle has larger particle size, which indicates that TK bonds in the GCTR conjugate can be broken, so that the micelle structure is changed and the size is larger, and indicates that the GCTR conjugate micelle has ROS-sensitive characteristics.

The GCTR conjugate micelles are taken out at different time points to measure the particle size, and the results are shown in Table 3, wherein the particle size of the GCTR conjugate micelles is changed along with the particle size of H ₂ O ₂ The contact time is prolonged, the particle size is sharply increased, and the degree of dispersion is also increased. It was further demonstrated that micelles of GCTR conjugate can be loaded with low concentrations of H ₂ O ₂ Depolymerisation in solution, ROS sensitive.

TABLE 3 10mmol/LH ₂ O ₂ Particle size distribution characteristics of GCTR conjugate micelles in solution

Experimental example 3 stability of GCTR conjugate micelles

The observation result of the stability of the GCTR conjugate micelle in example 1 is shown in fig. 6, and within one week, it can be observed that the GCTR polymer micelle has substantially no change in particle size under indoor natural illumination and dark conditions, and does not generate flocculation, precipitation and other phenomena, and has good stability. Indicating that the GCTR conjugate micelles are stable well in water at room temperature for one week.

Experimental example 4 screening of preparation conditions for Cela/GCTR micelles

The influence of various factors of Cela solvent, drug loading ratio and carrier concentration on the GCTR conjugate micelle loaded Cela is examined by taking the drug loading rate, the encapsulation efficiency, the particle size and the distribution index as indexes.

1. Cela solvent screening

Methanol, ethanol, DMF and DMSO are used as solvents of Cela, the influence of the solvents on the drug-loading capacity of the GCTR conjugate is examined, and the experimental results are shown in Table 4. As can be seen, DMSO is more favorable for Cela loading than methanol, ethanol, DMF. When DMSO is used as the Cela solvent, the prepared Cela/GCTR micelle has the particle size of (182.83 +/-4.37) nm, PDI of 0.017 +/-0.010, drug-loading rate of (38.73 +/-4.64)%, and encapsulation rate of (77.72 +/-9.15)%. In conclusion, the Cela/GCTR micelle has small particle size, uniform distribution, large drug-loading rate and high encapsulation rate, so DMSO is selected as a solvent for Cela dissolution in the research.

TABLE 4 Effect of different Cela solvents on the drug loading Capacity of GCTR conjugates

2. The feeding ratio of the medicine and the carrier

The effect of drug-to-carrier charge ratio (drug loading ratio) on Cela entrapment in GCTR conjugate is shown in table 5. The drug loading rate increases with the increase of the drug loading ratio, and the encapsulation efficiency also increases. The encapsulation efficiency dropped significantly when the drug loading ratio was increased to 1.6, indicating that the GCTR conjugate-encapsulated Cela had reached saturation. And when the drug loading ratio is 1.4, the particle size is smaller than other ratios, so that 1.

Table 5 influence of different drug amounts to vehicle dosing ratio on the drug loading capacity of GCTR conjugates

3. Concentration of Carrier

The effect of carrier concentration on Cela loading of GCTR conjugate is shown in table 6. The results show that when the carrier concentration is between 4 and 8mg/mL, the contact chance of Cela and the conjugate micelle is increased along with the increase of the carrier concentration, so the drug loading is increased. When the carrier is 5mg/mL, the drug loading rate and the encapsulation rate are larger than those of 6-8 mg/mL, and the particle size is smaller. Therefore, the optimal carrier concentration was determined to be 5mg/mL.

TABLE 6 Effect of different Carrier concentrations on the drug Loading Capacity of GCTR conjugates

4. Screening of lyoprotectants

In order to prevent the occurrence of adverse conditions such as loose structure, poor appearance, and water dispersion during the lyophilization process, a lyoprotectant can be added to improve the product form and solubility of the lyophilized preparation. The experiment takes the appearance, the redispersibility, the particle size, the potential, the drug loading rate and the encapsulation rate of the freeze-dried preparation as evaluation indexes, and inspects the influence of two freeze-drying protective agents, namely 0.2 percent mannitol and glucose, on the Cela/GCTR micelle freeze-drying.

The evaluation indexes are as follows:

TABLE 7 evaluation indexes of appearance and resolubility

The results show that the Cela/GCTR micelle lyophilized preparation without the lyoprotectant is orange floccule, rough in surface, loose in texture, and some of the drug is observed to scatter with water during the lyophilization process. After the freeze-drying protective agent is added, the appearance, re-solubility, particle size, drug loading rate and encapsulation rate of the Cela/GCTR micelle freeze-drying preparation are improved. In the two freeze-drying protective agents, the glucose effect is better, the appearance is smooth and cake-shaped, the redissolution is stable, the particle size is (135.40 +/-35.4) nm, the drug loading rate and the encapsulation rate are respectively (36.71 +/-6.71)%, (79.77 +/-9.77)%, and no obvious difference exists compared with a non-freeze-dried preparation. Compared with the freeze-dried preparation without the freeze-drying protective agent, the particle size is reduced, and the encapsulation efficiency and the drug-loading rate are both increased. In conclusion, 0.2% glucose was selected as a protectant for Cela/GCTR micelle lyophilized formulations.

TABLE 8 Cela/GCTR micelle lyophilized formulation screening

Experimental example 5 Cela/GCTR micelle in vitro Release

The dialysis method is adopted to study the in vitro release behavior of Cela from GCTR conjugate, simulate the in vivo environment, and respectively use phosphate buffer solution with pH 5.0, pH 5.0+10mmol/L H ₂ O ₂ The phosphate buffer solution simulates the tumor tissue environment and the phosphate buffer solution with the pH value of 7.4 simulates the blood neutral environment, and the in vitro release kinetic behavior of the Cela/GCTR micelle is investigated.

1. Experimental methods

1. Stability of Cela/GCTR micelles in a Release Medium

The stability of Cela/GCTR micelles in phosphate buffer solutions with different pH values is examined by taking the particle size as an examination index. Weighing a proper amount of Cela/GCTR micelles, dissolving the Cela/GCTR micelles by using phosphate buffer solutions with different pH values, detecting the pH value for more than 10min until the dispersion is uniform, placing the mixture in a water bath shaker at 37 ℃, measuring the particle sizes of the Cela/GCTR micelles after 0, 6, 12, 24, 48 and 72h at 100r/min, and inspecting the stability of the Cela/GCTR micelles.

2. Cela/GCTR micelle in vitro release experiment

Precisely weighing Cela/GCTR micelle, adding 0.9% sodium chloride injection for dissolving, adding release medium for diluting until the content of C isela concentration is 250 mu g/mL, probe ultrasonic treatment is carried out for 10min, 1mL of Cela/GCTR micelle solution is placed in a 3500Da dialysis bag and placed in a beaker filled with 100mL of dissolution media, 3 parts are paralleled, and Cela/GCTR micelle in-vitro release degree experiments are carried out at the vibration speed of 100r/min and the medium temperature of 37 ℃. At set time intervals of 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72h, 4mL of the release fluid from the dialysis bag was placed in 10mL centrifuge tubes containing 10. Mu.L of internal standard Emodin (300. Mu.g/mL) in a volatile state, and the same volume of blank release medium was added. Adding 3mL of dichloromethane into a sample for extraction, vortexing for 5min, collecting the subnatant to a 5mL centrifuge tube, evaporating under reduced pressure, redissolving with 200 mu L of methanol, vortexing for 3min, and centrifuging at 14000r/min for 20min, taking a proper amount of supernatant, measuring the Cela content by high performance liquid chromatography, calculating the cumulative release amount, drawing a release curve, and determining the cumulative release rate (Q) _t ) The calculation formula of (a) is as follows:

wherein, C _n The concentration of Cela in the release solution at the time of the n-th sampling, V is the total release medium volume, V ₀ For the sample volume, M is the total amount of Cela used for release.

2. Results of the experiment

1. Stability of Cela/GCTR micelles in an in vitro release medium

The stability of Cela/GCTR micelles in different release media is shown in Table 9, and the particle size of Cela/GCTR micelles in NaCl and pH 7.4 buffer media is not obviously changed for 72 h. In a buffer medium with pH 5.0, the particle size begins to increase after 6h, exceeds 200nm after 12h and reaches 259nm after 72h; pH 5.0+10mmol/L H ₂ O ₂ The particle size of 3H in the buffer medium reaches more than 200nm, the particle size becomes larger and larger with the time, almost no micelle exists at 12H, which is similar to the ROS sensitivity determination result of the GCTR conjugate, and shows that Cela/GCTR micelle has pH 5.0+10mmol/L H ₂ O ₂ The release of the drug from the buffered media will be faster. No visible precipitation or flocculation appears in the medium, and the stability is good.

TABLE 9 particle size variation of Cela/GCTR micelles in an in vitro release medium

2. In vitro Release Studies

The in vitro release behavior of Cela from Cela/GCTR micelles was studied by dialysis. Three different conditions of phosphate buffer salt (pH 7.4, pH 5.0+10mmol/L H are used in this experiment ₂ O ₂ ) As release media, the neutral blood environment, the tumor tissue acid environment and the tumor tissue high ROS internal environment are simulated respectively, and the release degree of Cela from Cela/GCTR micelles at different time points is examined.

As can be seen from the release results in FIG. 7, the cumulative release rate of Cela solution at pH 7.4 for 12h was more than 70%, and the release was almost complete for 24h. While the cumulative release rate of Cela/GCTR micelle 12h is about 29 percent, the cumulative release rate of Cela/GCTR micelle 24h is about 37 percent, and the release is still maintained stably for 72 h. There was no burst release during the release process, indicating that Cela was completely entrapped in the hydrophobic core of the conjugate micelle, and that Cela/GCTR micelle has a sustained release effect in blood neutral environment. Under the condition of pH 5.0, the cumulative release rate of Cela/GCTR micelle 12h is about 37%, the cumulative release rate of Cela/GCTR micelle 12h is about 50%, the release speed in the first 2h is high, the release is slow after 2h, and compared with the release rate of Cela/GCTR micelle under the condition of pH 7.4, the release of the drug is increased along with the reduction of the pH value, and a certain pH sensitivity is presented. When the concentration is 10mmol/L H ₂ O ₂ When existing, the Cela/GCTR micelle has a cumulative release rate of about 58% in 12h and about 81% in 24h. Thus, it can be seen that 10mmol/L H was added ₂ O ₂ Later, the release rate is faster, and the release amount is more, indicating that Cela/GCTR micelle has ROS sensitivity.

Experimental results show that the Cela/GCTR micelle has a slow-release effect in a blood pH environment, is sensitive to a weak acid environment with the characteristics of a tumor microenvironment and a high ROS environment, and can improve the drug release rate.

Experimental example 6 cellular biological Effect of Cela/GCTR micelles

1. Experimental methods

1. Synthesis of CR conjugates, GCTR conjugates

CR conjugate synthesis:

(1) Weighing carboxymethyl chitosan into a reaction bottle, and adding 10mL of distilled water to swell for 30min; adding rhein powder into jar, adding 1mL of NaHCO ₃ Heating the solution to completely dissolve rhein, cooling to room temperature, adding EDC & HCl, activating for 20min, and adding NHS; then adding the mixed solution into the carboxymethyl chitosan solution while stirring, and stirring and reacting for 24 hours in a dark place; wherein the molar ratio of carboxymethyl chitosan to rhein to EDC HCl to NHS = 1; (2) Precipitating the reaction solution with 95% ethanol, standing, filtering, and washing the precipitate with 95% ethanol until the washing solution is colorless; dissolving the dried precipitate in water, performing ultrasonic treatment with probe in ice water bath for 20min, centrifuging at 3000rpm for 10min, filtering the supernatant with 0.8 μm filter membrane, and dialyzing the filtrate in dialysis bag for 72 hr; (3) And after dialysis, putting the filtrate in ice water bath, performing ultrasonic treatment for 20min by using a probe, centrifuging at 3000rpm for 10min, taking supernate, filtering by using a 0.8-micron filter membrane, and freeze-drying the filtrate to obtain the carboxymethyl chitosan-rhein conjugate.

GCTR conjugate synthesis:

(1) Dissolving 0.1mmol of R in 1% of NaCO3 under heating, adding 0.12mmol of EDC & HCl under stirring, reacting at room temperature for 20min, and reacting with 0.12mmol of NHS at room temperature for 10min to obtain R-NHS. Adding 0.15mmol of EDA to dissolve with water, dripping R-NHS into EDA water solution, and reacting for 6h to obtain R-EDA.

(2) Dissolving 0.15mmol TK with DMF, adding 0.18mmol EDC & HCl, reacting at room temperature for 20min, adding 0.18mmol NHS, reacting at room temperature for 10min, adding into R-EDA solution, and reacting at room temperature to obtain TER.

(3) 0.083mmol CMCS was dissolved in water. And centrifuging the TER solution, taking the supernatant, adding 0.12mmol of EDC & HCl, reacting at room temperature for 20min, adding 0.12mmol of NHS, reacting at room temperature for 10min, adding into CMCS aqueous solution, and reacting for 24h to obtain CTR.

(4) Dissolving 0.12mmol GA in DMF, reacting 0.14mmol EDC & HCl at room temperature for 20min, reacting 0.14mmol NHS at room temperature for 10min, adding into CTR solution, reacting at room temperature for 24h, dialyzing the reaction solution in dialysis bag (MWCO 3500) with distilled water for 72h, performing ultrasonic treatment with probe for 20min, and freeze-drying to obtain GCTR conjugate.

2. Cell culture

Preparation of a complete cell culture medium: the medium of HepG2 cells is DMEM medium supplemented with 10% FBS, 1% Penicilin Streptomycin Solution and mixed well; the culture medium of BEL-7402 cells is RPIM1640 culture medium, 10% of FBS, 1% of Penicilin Streptomycin Solution, and mixing; the culture medium of L-02 cells is RPIM1640 culture medium, to which 20% FBS, 1% Penicilin Streptomycin Solution was added and mixed.

Cell recovery: taking out cells from liquid nitrogen, immediately placing into 37 deg.C constant temperature water bath, rapidly shaking until frozen stock solution is completely dissolved, transferring into a centrifuge tube, adding about 5 times volume of culture solution, repeatedly and lightly beating, centrifuging for 4min (1000 r/min), discarding supernatant, adding appropriate amount of culture solution into the centrifuge tube, repeatedly and lightly beating, transferring into a culture flask, placing at 37 deg.C, and 5% CO ₂ And (5) changing the liquid every other day in an incubator.

Cell passage: subculturing when the cell adherence reaches about 80%, discarding the old culture medium, washing with PBS for 2 times, adding 1mL of trypsin solution for digestion, and placing in an incubator for 2min; observing under a microscope, if the cell gap becomes larger, the cells become round, immediately adding 3mL of culture medium containing serum, and slightly blowing the bottle wall by using a gun to make the cells fall into the culture solution in the bottle; transferring the suspension into a 15mL centrifuge tube, centrifuging at 1000r/min for 4mim, and removing the supernatant; the cells were suspended by adding an appropriate amount of complete medium to the cell pellet in the centrifuge tube, passaged at a ratio of 1.2 to 1.

3. Investigation of cytotoxicity

The cytotoxicity of the drug-loaded micelles is determined by adopting a thiazole blue colorimetric method (MTT assay), and the toxicity of CR conjugates, GCTR conjugates, cela/CR micelles, cela/GCTR micelles, free Cela and a solvent thereof Cremophor EL: etOH (50%: 50%, v/v) on HepG2, BEL-7402 and L-02 cells is examined.

HepG2, BEL-7402 and L-02 cells in logarithmic growth phase are inoculated in a 96-well plate5X 10 per hole ⁴ The cells, marginal wells filled with blank PBS, were incubated in the incubator for 24h to allow adherence, and the medium was discarded. mu.L of different concentrations of CR conjugate, GCTR conjugate, cela/CR micelles, cela/GCTR micelles, free Cela, cremophor EL: etOH (50%: 50%, v/v) test solution was added to a 96-well plate in 6 wells per concentration. Incubate in incubator, experimental set with blank control wells containing no cells and normal wells containing cells without drug. At 24, 48, and 72 hours after the addition of the drug, the plates were removed, 100. Mu.L of MTT solution at a concentration of 1. Mu.g/mL was added to each well, and the plates were returned to the incubator for incubation for 4 hours. The plate was removed and the supernatant carefully aspirated. The bluish violet crystals were dissolved by adding 150. Mu.L of DMSO to each well. The absorbance value (OD value) was measured at 570nm with a microplate reader. Cell viability was calculated according to the following equation and IC was calculated using Graphpad software ₅₀ The value is obtained.

4. Cellular uptake

4.1 preparation of P4-loaded micelles

Since Cela does not have fluorescence, in order to compare the uptake capacity of tumor cells to Cela/CR micelles and Cela/GCTR micelles, (P4 + Cela)/CR micelles and (P4 + Cela)/GCTR micelles carrying P4 and Cela together are prepared by taking a hydrophobic organic dye P4 (an environmental response type fluorescent probe) as a fluorescent indicator, and the carrying method is similar to the Cela carrying method. The method comprises the following specific steps: weighing 12mg of CR conjugate or GCTR conjugate, adding 2.4mL of distilled water, and performing ultrasonic treatment by using a probe to fully dissolve the CR conjugate or the GCTR conjugate to form a CR micelle or GCTR micelle solution. Weighing a small amount of Cela, dissolving the Cela in a DMSO solution containing 90 microgram of P4, uniformly mixing, dropwise adding the solution into a CR micelle or GCTR micelle solution under high-speed stirring, continuously stirring at high speed for 20min, performing ultrasonic treatment by an ice-water bath probe for 20min, dialyzing for 24h, performing ultrasonic treatment by the probe for 20min, and centrifuging at 3500r/min for 10min, and taking supernate to obtain (P4 + Cela)/CR micelle and (P4 + Cela)/GCTR micelle.

4.2 qualitative examination by confocal laser microscopy

Examination of HepG2 cells for dissociation by means of confocal microscopyUptake of P4 and two micelles carrying P4 (P4 + Cela)/CR micelles and (P4 + Cela)/GCTR micelles (P4 concentration 0.5. Mu.g/mL). Taking HepG-2, L-02 cells in logarithmic growth phase at 2X 10 ⁵ Inoculating the cells/mL of the mixture into a laser confocal culture dish, culturing the cells to adhere to the wall, discarding the culture solution, adding PBS preheated at 37 ℃ for gently washing for 2 times, adding 1mL of each test solution (when the influence of GA is considered, adding 2mL of GA 5 mu g/mL solution 1h in advance to saturate GA receptors on HepG-2 cells, then adding drug-loaded micelles), incubating for 4h, discarding the drug solution, adding ice-cold HBSS at 4 ℃ to stop uptake, and washing the cells for 3 times. HBSS is discarded, fixed with 4% paraformaldehyde for 20min, and cleaned for 2-3 times by HBSS. 1mL of a solution containing Hoechst33258 PBS (10. Mu.g/mL) was added to each well, nuclei were stained for 15min, and 200. Mu.L of PBS was added after 3 washes with cold HBSS, observed under a confocal laser microscope, and photographed.

4.3 flow cytometry quantitative investigation

Taking HepG2 cells in exponential growth phase at 4X 10 ⁵ Inoculating the cells at a density of one/mL into a six-well plate, culturing for 24h, removing the culture medium by aspiration, adding 1.5mL of solutions containing the same concentration of P4 and prepared (P4 + Cela)/CR micelles and (P4 + Cela)/GCTR micelles, continuously culturing for 4h, removing the liquid medicine by aspiration, adding ice-cold HBSS at 4 ℃ to stop the uptake, washing for 2 times, adding the digestive juice into the six-well plate, collecting the cells, centrifuging at 2000r/min for 3min, removing the culture solution, and adding PBS for 2 times. The aim is to remove fluorescent substances that may not be phagocytosed by the cells, and to remove digestive juices. After washing, 1mL of PBS was added, and the mixture was dispersed by pipetting to obtain a suspension. Filtering with 400-mesh screen, exciting wavelength 480nm, emission wavelength 575nm, sampling, detecting by flow cytometry, and analyzing the result by software.

4.4 cell cycle experiments

HepG2 cells in logarithmic growth phase were selected at 2X 10 ⁵ The density of individual cells was plated in 6-well plates, the incubator was incubated overnight, the medium was aspirated and washed twice with PBS. Respectively setting a Control group, a free Cela group and a Cela/GCTR micelle group, wherein each group is provided with 3 multiple holes, culturing for 24h in an incubator, then carrying out sample treatment by adopting a cell cycle detection kit, and detecting by using a flow cytometer.

2. Results of the experiment

1. Results of cytotoxicity experiments

HepG2, BEL-7402 and L-02 cells are selected as cell models, and the MTT method is adopted to investigate the influence of CR conjugate, GCTR conjugate, cela/CR micelle, cela/GCTR micelle, free Cela and solvent Cremophor EL thereof, namely EtOH (50%: 50%, v/v), on the proliferation of the HepG-2, BEL-7402 and L-02 cells respectively so as to evaluate the cytotoxicity of carrier materials and drug-loaded micelles. And (3) incubating a series of test drug solutions with different concentrations with the cells for 24h, 48h and 72h, and detecting the cell survival rate.

As can be seen in the result chart 8 of the MTT experiment on L-02, the CR conjugates and GCTR conjugates with different concentrations have no significant influence on the survival rate of L-02 cells, and after 72 hours of action, no obvious toxic effect appears, which indicates that the carrier material does not damage the cells and has good biocompatibility. At the concentration of 0.01-1 mu g/mL, cela/CR micelles, solvent group EtOH + EL and Cela/GCTR micelles have no toxicity to L-02 cells, and free Cela starts to show a certain inhibition effect within 48 hours at the concentration range. At both concentrations of 2.5. Mu.g/mL and 5. Mu.g/mL, the isolated Cela, cela/CR micelles, cela/GCTR micelles all showed L-02 cell inhibitory effect, and the inhibitory effect gradually increased with time. The results show that the GCTR conjugate carrier has no cytotoxicity to L-02 cells in the experimental concentration range, the Cela/GCTR micelles have no obvious proliferation inhibiting effect on the L-02 cells in the concentration range of 0.01-1 mu g/mL, and the GCTR conjugate and the Cela/GCTR micelles have good safety.

As can be seen from the MTT experimental results of HepG2 cells, the survival rates of the HepG2 cells are not significantly influenced by different concentrations of EtOH + EL, CR conjugates and GCTR conjugates within the experimental concentration range. The free Cela, cela/CR micelles and Cela/GCTR micelles have certain proliferation inhibition effects on HepG2 cells, are shown as time-dependent and dose-dependent, and have stronger inhibition effects along with the increase of time and concentration. When the Cela concentration is lower, the free Cela, the Cela/CR micelles and the Cela/GCTR micelles have no obvious inhibition effect on the HepG2 cells, and when the Cela concentration reaches 0.5 mu g/mL, the growth inhibition effects of the free Cela, the Cela/CR micelles and the Cela/GCTR micelles on the HepG2 cells show difference. The proliferation inhibition effect of the gel is that Cela/GCTR micelle is obviously larger than free Cela, and the proliferation inhibition effect is better than that of Cela/CR micelle. At 72h, the survival rate of Cela/GCTR micelle cells is (41.58 +/-6.14)%, the survival rate of Cela/CR micelle cells is (71.73 +/-2.83)%, and the survival rate of free Cela cells is (87.06 +/-5.78)%, when the concentration is 0.5 mu g/mL. Compared with the free Cela and Cela/CR micelles, the cell survival rate of the Cela/GCTR micelles is reduced by about 2.1 times and 1.7 times respectively. With the prolonging of time and the increase of concentration, the Cela/GCTR micelle has more obvious effect of inhibiting the proliferation of HepG2 cells and shows stronger cytotoxicity.

As can be seen from the results of BEL-7402 cell MTT, different concentrations of EtOH + EL, CR conjugate and GCTR conjugate have no significant effect on BEL-7402 cell survival rate. When the Cela concentration reaches 0.25 mu g/mL, the free Cela, cela/CR micelles and Cela/GCTR micelles show the effect of inhibiting BEL-7402 cell proliferation, and the inhibition effect is stronger than the inhibition effect on HepG2 cells at the same administration concentration, wherein the inhibition effect on the Cela/GCTR micelles is strongest. The survival rate of the free Cela cells is about 1.5 times of that of Cela/CR micelles and about 1.6 times of that of Cela/GCTR micelles in 24 hours when the Cela concentration is 0.5 mu g/mL, which shows that the ability of Cela to inhibit cell proliferation is enhanced after the micelles are coated.

As can be seen from Table 10, free Cela and Cela/CR micelles, cela/GCTR micelle group IC ₅₀ The result of the comparison shows that each group of ICs ₅₀ Has larger difference, and proves that Cela/CR micelle and Cela/GCTR micelle group have good killing effect on liver cancer cells. Under the same Cela concentration, the proliferation inhibition effect of the Cela/GCTR micelle on the liver cancer cells is stronger than that of the normal liver cells, which indicates that the Cela/GCTR micelle has certain liver cancer cell selectivity. Compared with Cela/CR micelles, cela/GCTR micelles have stronger inhibition and proliferation effects on HepG2 cells and BEL-7402 cells, and have stronger killing effects over time.

TABLE 10 Cela and its different micelles with different cytotoxic Effect IC ₅₀ Value (μ g/mL) (n = 6)

2. Results of cell uptake experiments

In the experiment, P4 is used as a fluorescent probe to carry out a cell uptake experiment. P4 is an environment-responsive aza BODIPY dye, exhibits red fluorescence in hydrophobic environment, but is quenched in water due to molecular aggregation. The results of the confocal laser qualitative study are shown in FIG. 10, where red is the fluorescence of P4 and blue is the fluorescence of Hoechst 33342. In HepG2 cells, after 4h incubation of free P4 solution, no red fluorescence is obvious in the cells, while red fluorescence appears in HepG2 cells after (P4 + Cela)/CR micelle incubation and (P4 + Cela)/GCTR micelle incubation, which indicates that the micelles wrap Cela and P4 into the cells in an intact micelle form. And no obvious red fluorescence appears in all groups in the L-02 cells, which indicates that normal liver cells take less micelles, so the drug-loaded micelles can generate less toxic and side effects on livers.

To study the uptake of GA-modified micelles, the GA solution was added 1h in advance to saturate the GA receptors on HepG2 cells, then (P4 + Cela)/GCTR micelles were added, and (P4 + Cela)/CR micelles without GA modification were set up to examine the uptake of micelles by HepG2 cells. As can be seen in FIG. 11, the GA-pretreated (P4 + Cela)/GCTR micelles have the weakest red fluorescence and the mean fluorescence intensity of 225.62. + -. 13.45, indicating that the uptake rate of (P4 + Cela)/GCTR micelles by tumor cells is lower after GA pretreatment. In the same time, the GA-modified (P4 + Cela)/GCTR micelles showed more significant cellular uptake than the non-GA-modified (P4 + Cela)/CR micelles, and the mean fluorescence intensities of the two were 606.79. + -. 49.94 and 293.37. + -. 13.94, respectively.

The results show that the GA-mediated active targeting is beneficial to the nano-micelle to be taken up by liver tumor cells.

3. Results of cell cycle experiments

After incubating HepG2 cells with Cela and Cela/GCTR micellar solutions for 24h, cell populations at different stages of the cell cycle were examined by flow cytometry, and the cell cycle distribution was determined by staining DNA with PI. FIG. 11 and the results in Table 11 show that the cells in the Control group are mostly in the G0/G1 phase, and when the two groups of pharmaceutical preparations are administered, the proportion of the cells in the G0/G1 phase is increased, and the proportion of the cells in the S phase is decreased. The Cela/GCTR micelle-treated group also blocked some cells in the G2/M phase compared to the Cela alone treated group. Therefore, cela/GCTR micelle can block the cell cycle, thereby inhibiting the mitosis of tumor cells and promoting the apoptosis of the cells.

TABLE 11 Effect of Cela and Cela/GCTR micelles on the HepG2 cell cycle (n = 3)

The results of this experimental example show that the GCTR conjugate is a safe drug carrier. The Cela/GCTR micelle has no obvious toxicity to normal liver cells L-02 in a proper concentration range, and has obvious proliferation inhibiting effect on liver cancer cells HepG2 and BEL-7402. The GA-modified Cela/GCTR micelle has an identification effect on liver tumor cells, increases the uptake rate of the liver tumor cells, further enhances the killing efficiency on the liver tumor cells, and proves that the Cela/GCTR micelle has an in-vitro targeted tumor inhibition effect.

Experimental example 7 in vivo pharmacokinetics and tissue distribution study of Cela/GCTR micelles

1. Experimental method

1. Preparation of plasma samples

Blood is taken from mouse eyeballs, the mouse eyeballs are placed in a 1.5mL centrifuge tube containing heparin sodium, centrifugation is carried out for 5min at 4000r/min, and plasma is taken for standby. Precisely absorbing 200 mu L of mouse blank plasma, adding 10 mu L of internal standard substance (Tanshinone IIA 5 mu g/mL), vortex mixing uniformly, adding 3mL of ethyl acetate for extraction, vortex oscillating for 3min, placing in a high-speed centrifuge for 3500r/min and centrifuging for 10min, taking supernatant into A5 mL centrifuge tube, volatilizing, vortex oscillating for 5min for redissolution with 100 mu L of methanol, centrifuging for 10min at 14000r/min, taking 70 mu L of supernatant for UPLC-MS analysis.

2. In vivo imaging experiments

2.1 preparation of Co-loaded P2 and Cela micelles

Weighing 12mg of GCTR conjugate in a penicillin bottle, and adding 2.4mL of water for dissolving. 2mg P2 is taken out of a 250mL volumetric flask, dissolved by methanol to a constant volume and prepared into mother liquor of 8 mug/mL for standby. Precisely measuring 11.25mL of P2 mother solution, putting the mother solution into a centrifuge tube, volatilizing methanol, adding 450 mu L of DMSO solution containing a small amount of Cela for redissolving, dropwise adding the solution into GCTR coupler aqueous solution stirred at a high speed at room temperature, continuously stirring for 20min, carrying out ultrasonic treatment on an ice-water bath probe for 20min, transferring the solution into a dialysis bag, dialyzing for 12h, and freeze-drying to obtain the (P2 + Cela)/GCTR micelle carrying P2 and Cela together. (P2 + Cela)/CR micelles were prepared as described above.

2.2 Experimental methods

To examine the distribution of GCTR micelles in vivo, we used GCTR micelles co-loaded with P2 and Cela to examine their distribution in vivo. Tumor volume is larger than 100mm ³ The mice were randomly divided into 4 groups of 3 mice, and the mice were injected with P2 physiological saline solution, (P2 + Cela)/CR micelles and (P2 + Cela)/GCTR micelles via the tail vein, and the distribution of the fluorescent substance in the mice was observed under a live imager at 0h, 0.5h, lh, 2h,4h,6h,12h and 24h after the injection, and recorded by photographing.

2. Results of the experiment

1. Time curve of blood concentration

Fig. 12 is a graph of plasma concentration-time curves plotted by taking the sampling time point as the abscissa and the plasma concentration as the ordinate after Cela, cela/CR micelle and Cela/GCTR micelle were injected into the tail vein of mice and measuring the concentration of Cela in the blood of the mice at different time points. The results show that the plasma Cela blood drug concentration gradually decreases after the administration, and the Cela blood drug concentration of Cela/CR micelles and Cela/GCTR micelles slowly changes and gradually keeps a stable trend to release after 2 hours. Free Cela is cleared in blood quickly, shows a rapid descending trend within 2h and can not be detected in plasma at 24h, while the Cela/CR micelle and Cela/GCTR micelle group release slowly at a high rate for 1h and release slowly after 1h, and the stable plasma concentration is still maintained for 24h, which shows that the form of the drug after the Cela is coated and loaded in the micelle is changed, so that the Cela can exist in the body for a long time and keep slow release, and the retention time of the Cela in the blood is obviously prolonged.

2. In vivo imaging study of Cela/GCTR micelles

The fluorescence imaging results of the mouse living body and the 24h tissue after the mouse tail vein injection of the physiological saline solution of P2, (P2 + Cela)/CR micelle and (P2 + Cela)/GCTR micelle are shown in FIGS. 14-18. As can be seen from FIG. 15, after the mice are injected with (P2 + Cela)/GCTR gel via tail vein, the fluorescence gradually accumulates in the liver from 0.5h, and the fluorescence signal reaches the maximum at 1h and then gradually weakens, and the fluorescence signal still exists at 24h. The (P2 + Cela)/CR micelle fluorescence signal also accumulated in the liver starting at 0.5h and reached a maximum at 2 h. As can be seen from fig. 16, the (P2 + Cela)/GCTR micelle group is greater than the (P2 + Cela)/CR micelle group at 1h and 2h of the mouse total body fluorescence intensity, indicating that both the P2+ Cela/CR micelle and the (P2 + Cela)/GCTR micelle exhibit liver targeting, and the (P2 + Cela)/GCTR micelle has stronger liver targeting, probably due to the fact that the affinity of the (P2 + Cela)/GCTR micelle with liver tissue is increased and more concentrated at the liver part because GA in the GCTR conjugate binds to the liver cell surface receptor. As can be seen from FIG. 17, the fluorescence signal gradually accumulated at the tumor site with time, and the fluorescence intensity of the (P2 + Cela)/CR micelle group reached the maximum at 12h, and the fluorescence intensity of the (P2 + Cela)/GCTR micelle was the strongest at 8h, and was significantly higher than that of the (P2 + Cela)/CR micelle. The result shows that the (P2 + Cela)/GCTR micelle has targeting effect on tumor cells, and the targeting effect is better than that of the (P2 + Cela)/CR micelle.

After 24h of administration, the mice were sacrificed by cervical dislocation, and the visceral organs and tumor tissues were dissected and removed for in vivo fluorescence analysis. Tissue fluorescence imaging results show that at 24h, a small amount of fluorescence signals still exist in liver tissues, and the fluorescence signals in tumor tissues are relatively strong. The same result can be seen from the fluorescence intensity plot, and the residual fluorescence intensity in liver tissue is slightly higher than that of the (P2 + Cela)/CR micelle group, which is probably because GA in the carrier GCTR conjugate can bind to GA receptors in liver, so that the fluorescence is stronger. The higher residual fluorescence intensity of (P2 + Cela)/GCTR micelles in tumor tissues is probably due to the fact that the Cela/GCTR micelles have smaller particle sizes, better water solubility and more contact with the tumor tissues, and therefore the residual fluorescence is stronger.

Experiments in the experimental example show that the Cela/GCTR micelle has a long circulation effect in vivo, can obviously improve the retention time of the Cela in blood, and improves the bioavailability of the Cela. In addition, the Cela/GCTR micelle can reach the liver and a tumor part in vivo, can stay for more than 24 hours, and shows targeting on liver tissues and tumor tissues.

The embodiments and experimental examples show that the micelle of the GCTR conjugate provided by the invention is sensitive to active oxygen, and the prepared drug Cela/GCTR micelle shows targeting on liver tissues and tumor tissues and has the effect of slowly releasing the Cela drug in vivo. The characteristics enable the GCTR conjugate and the Cela/GCTR micelle to have high application potential in the preparation of liver cancer drugs.

Claims

1. A modified carboxymethyl chitosan is characterized in that: it is carboxymethyl chitosan substituted with two substituents:

2. the modified carboxymethyl chitosan according to claim 1, wherein: in the structure of the modified carboxymethyl chitosan, substituent groups

The molar degree of substitution of (a) is 5.43-9.05%, the substituents

The molar substitution of (a) is 1.79-2.93%.

3. The modified carboxymethyl chitosan according to claim 1, wherein: the modified carboxymethyl chitosan is obtained by connecting rhein at one end of thioketal and grafting glycyrrhetinic acid and the thioketal to the carboxymethyl chitosan respectively.

4. The modified carboxymethyl chitosan according to claim 3, wherein the molar charge ratio of the raw materials for preparing the modified carboxymethyl chitosan is as follows:

rhein and ketodithiol 1;

and/or, the ratio of carboxymethyl chitosan to glycyrrhetinic acid 1;

and/or, the ratio of carboxymethyl chitosan to ketathiol 1 is (1.2-1);

and/or rhein, ketothiolane, carboxymethyl chitosan, and glycyrrhetinic acid 1.2.

5. The modified carboxymethyl chitosan according to claim 3, wherein: the rhein is connected to a carboxyl at one end of the ketal thiol through a polyamino compound; the polyamino compound is preferably selected from one of ethylenediamine, 1, 3-propanediamine or 1, 4-butanediamine.

6. The modified carboxymethyl chitosan according to claim 1, wherein: the carboxymethyl chitosan is selected from O-carboxymethyl chitosan; and/or the molecular weight of the carboxymethyl chitosan is 1 to 10 ten thousand.

7. The method for preparing modified carboxymethyl chitosan according to any one of claims 1 to 6, comprising the steps of:

8. The method for preparing a conjugate according to claim 7, wherein: in the step (1), the reaction is carried out under the action of alkali, a cross-linking agent and a catalyst, wherein the alkali isSelected from NaHCO ₃ 、Na ₂ CO ₃ At least one of sodium acetate, sodium phosphate or sodium hydrogen phosphate, and/or the crosslinking agent is selected from at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride or N, N-diisopropylcarbodiimide hydrochloride; and/or, the catalyst is selected from at least one of N-hydroxysuccinimide or 4-dimethylaminopyridine; and/or, the reaction is carried out in solvent water; and/or the temperature of the reaction is room temperature; and/or the reaction time is 6-12 h;

and/or, in the step (3), the carboxymethyl chitosan is dissolved by solvent water and then reacts with the ketal thiol-rhein conjugate; and/or the reaction is carried out under the action of a cross-linking agent, wherein the cross-linking agent is selected from at least one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride or N, N-diisopropylcarbodiimide hydrochloride; and/or the temperature of the reaction is room temperature; and/or the reaction time is 12-24 h;

9. Use of the modified carboxymethyl chitosan of any one of claims 1 to 6 in the preparation of a medicament for preventing and/or treating liver cancer.

10. Use according to claim 9, characterized in that: the modified carboxymethyl chitosan is used as micelle loaded drug molecules.