CN115558039B

CN115558039B - Glycyrrhetinic acid-carboxymethyl chitosan-ketal-rhetinic acid conjugate, preparation method and application thereof

Info

Publication number: CN115558039B
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: 2023-05-12
Anticipated expiration: 2041-07-02
Also published as: CN115558039A

Abstract

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

the micelle formed by the conjugate can be used for loading the medicine tripterine, and the obtained micelle loaded with the medicine shows targeting to liver tissues and tumor tissues and has the function of slowly releasing the medicine in vivo. The characteristics enable the conjugate and the micelle of the invention to have the following properties in the preparation of liver cancer drugsHigh application potential.

Description

Glycyrrhetinic acid-carboxymethyl chitosan-ketal-rhetinic acid conjugate, 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-ketal-rhein conjugate, a preparation method and application thereof.

Background

Tripterine (Cela) is one of the effective components extracted from root of Tripterygium wilfordii (Trepterygium) belonging to Celastraceae, and has red needle crystal shape, and is easily dissolved in organic solvent. The Cela has a plurality of pharmacological actions, is used as a natural proteasome inhibitor, is found to have obvious anticancer action by a plurality of pharmacological experiments, has proliferation inhibiting action on a plurality of cancer cells, can play a role in inhibiting tumor growth by regulating a plurality of mechanisms such as signal paths, oncogenic molecular targets and the like, and is expected to be a very promising drug for treating cancers.

In the process of seeking new safe and effective liver cancer chemotherapeutics, cela becomes one of the important points of research. As further studies on Cela have found that Cela can regulate apoptosis-related factors at a cytological level to induce apoptosis, and block growth of HepG 2. In addition, research shows that Cela can obviously increase the expression of transmembrane glycoprotein Fas and FasL in liver cancer cells, obviously increase the content of cytochrome C (Cyt C) in cytoplasm, and simultaneously obviously increase the expression of activated caspase3, caspase8, caspase9 and other proteins, thus proving that the Cela can activate endogenous and exogenous apoptosis pathways of cells at the same time and induce apoptosis of liver cancer Bel-7402 cells. Animal experimental study shows that Cela can obviously reduce the activity of glutamic-oxaloacetic transaminase (Astate, AST), glutamic-pyruvic transaminase (Alanine aminotransferase, ALT) and alkaline phosphatase (Alkaline phosphatase, ALP) in hepatocellular carcinoma (hepatocellular carcinoma, HCC), reduce the level of Alpha-fetal protein (AFP) in serum, reduce the number of liver cancer cells and tumor volume of HCC rats, improve the expression of liver tissue tumor suppressor gene P53 and Bax protein of rats, and show obvious effect of resisting HCC of rats. The result shows that Cela has a certain curative effect on hepatocellular carcinoma, and can be used for further anti-liver cancer research.

However, the defects of low water solubility, poor bioavailability, large 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 novel tripterine-dendrimer conjugate and a preparation method and application thereof, wherein a novel tripterine dosage form is prepared from PAMAM nano-carrier, polyethylene glycol and surface targeting ligand, and then the tripterine is loaded by the polymer. The tripterine dosage form provided by the patent application has a targeting effect in treating cancers with abundant EpCAM surface membrane proteins such as colon cancer, liver cancer, breast cancer and the like, can improve the selectivity and specificity of the medicine, and reduces the toxicity of the tripterine. PAMAM, however, belongs to the class of polyamide-amine dendrimers, which have the disadvantage of being easily associated with negatively charged non-characteristic cells and proteins. And the formulations in this patent application are targeted against EpCAM surface membrane proteins, which are selectively and specifically directed against a variety of cancer cells. In addition, the targeting effect of the preparation on liver cancer cells is not ideal. In summary, the formulations in the above patent applications are still deficient in targeting selectivity and specificity for liver cancer. Therefore, the preparation of the tripterine is necessary to be further developed aiming at liver cancer, and more choices are provided for 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-ketal-rhein conjugate, a preparation method and application thereof, and aims to provide a carrier for loading tripterine, which has good targeting to liver tissues and tumor tissues after the tripterine is loaded, 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-ketal-rhein conjugate, GCTR conjugate), which is carboxymethyl chitosan substituted with two substituents:

preferably, the substituents

The molar substitution of (2) is 5.43-9.05% and 1.79-2.93%, respectively.

Preferably, the modified carboxymethyl chitosan is obtained by connecting rhein at one end of ketal, and grafting glycyrrhetinic acid and the ketal onto carboxymethyl chitosan respectively.

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

rhein and ketal are 1:1-1:2;

and/or carboxymethyl chitosan and glycyrrhetinic acid in the ratio of 1:1-1:2;

and/or carboxymethyl chitosan and ketal in the ratio of 1:1.2-1:2.4;

And/or rhein, ketal, carboxymethyl chitosan and glycyrrhetinic acid 1.2:1.8:1:1.5.

Preferably, the rhein is linked to the carboxyl group at one end of the ketal via a polyamino compound; the polyamino compound is preferably one of ethylenediamine, 1, 3-propylenediamine or 1, 4-butylenediamine.

Preferably, the carboxymethyl chitosan is selected from O-carboxymethyl chitosan; and/or the molecular weight of the carboxymethyl chitosan is 1 ten thousand 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 make one amino group of ethylenediamine react with carboxyl of rhein;

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

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

(4) And (3) reacting glycyrrhetinic acid with the ketal-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 cross-linking agent and a catalyst ₃ 、Na ₂ CO ₃ At least one of sodium acetate, sodium phosphate or sodium hydrogen phosphate, and/or 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 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), the ketal is dissolved by using a solvent dimethylformamide and then added 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 ketal-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 hours;

And/or, in the step (4), the glycyrrhetinic acid is dissolved by using a solvent dimethylformamide and then reacts with the carboxymethyl chitosan-ketal-rhein conjugate; and/or the reaction is carried out under the action of a catalyst, wherein the catalyst is at least one selected from 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 medicines for preventing and/or treating liver cancer.

Preferably, the modified carboxymethyl chitosan is used for loading drug molecules as micelles.

The micelle of the GCTR conjugate provided by the invention is sensitive to active oxygen, and the prepared drug Cela/GCTR micelle shows targeting property on liver tissues and tumor tissues and has the function 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. This provides a new choice for clinical treatment of liver cancer.

Compared with the existing targeting preparation, the preparation provided by the invention has active targeting of glycyrrhetinic acid receptor on the surface of liver cancer cells, and has a passive targeting effect due to small particle size, so that the targeting effect of the preparation provided by the invention is better.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a schematic diagram of the synthetic procedure of example 1;

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

FIG. 3 is the FT-IR pattern (left) and sum of glycyrrhetinic acid (a), rhein (b), ketal (c), carboxymethyl chitosan (d) and GCTR conjugate (e) of example 1 ¹ H-NMR spectrum (right);

FIG. 4 is a particle size distribution plot of Cela/GCTR micelle of example 2;

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

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

fig. 7 is an in vitro release profile of Cela/GCTR micelles in experimental example 5 (n=3);

FIG. 8 shows cytotoxicity (n=6) of GCTR conjugate and Cela/GCTR micelle on HepG2, BEL-7402, L-02 cells in experimental example 6;

FIG. 9 is a CLSM graph of HepG2, L-02 cell uptake in Experimental example 6;

FIG. 10 is a flow cytometry and fluorescence intensity profile (n=3) of uptake by HepG2 cells of experimental example 6, wherein a: control group; group P4; c, a (P4+Cela)/GCTR micelle group after GA pretreatment for 1 h; d (P4+Cela)/CR micelle group; e (P4+Cela)/GCTR micelle group;

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

FIG. 12 is a graph showing the blood concentration versus time after intravenous injection of Cela formulations into the tail of a mouse in Experimental example 7;

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

FIG. 14 is a fluorescence imaging result of a mouse in experimental example 7;

FIG. 15 shows the results of in vivo fluorescence intensity of mice imaged at various time points in Experimental example 7;

FIG. 16 shows the results of tumor fluorescence intensity at various time points in vivo imaging of mice in Experimental example 7;

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

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

Detailed Description

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

glycyrrhetinic Acid (GA), pharmaceutical grade (98%), shanghai-derived leaf biotechnology limited;

o-carboxymethyl chitosan (CMCS) with a molecular weight of 1 ten thousand is prepared by Qingdao sea biotechnology Co., ltd;

ketal (TK), pharmaceutical grade (98%), sierra xi biotechnology limited;

rhein (R), pharmaceutical grade (98%), australia source biotechnology limited of shanxi;

ethylenediamine hydrochloride, analytical grade (99%), microphone Biochemical technologies Co., ltd;

n-hydroxysuccinimide (NHS), analytical grade (98%), shanghai Ala Biochemical technologies Co., ltd;

1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), analytical grade (98%), shanghai Ala Biochemical technologies Co., ltd;

anhydrous ethylenediamine, analytical grade, fuzhou macro new glassware limited;

pyrene, analytical grade (99%), shanghai Ala Biochemical technologies Co., ltd;

dialysis bags, MWCO3500, biosharp;

celastrol (Cela), pharmaceutical grade (98%), nanjing cassis pharmaceutical technologies limited;

DMSO, ethanol, methanol, analytically pure, national pharmaceutical chemicals limited;

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

glucose, analytically pure, national drug group chemical company, inc;

mannitol, analytically pure, national drug group chemical reagent company, inc;

0.9% sodium chloride injection, 190407A44, fuzhou sea Wang Fuyao pharmaceutical Co., ltd;

5% glucose injection, analytically pure, fuzhou sea Wang Fuyao pharmaceutical Co., ltd;

dialysis bags, MWCO3500, biosharp;

p2 and P4 fluorescent probes were synthesized according to the method of 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; ketal-rhein conjugate: TER; carboxymethyl chitosan-ketal-rhein conjugate: CTR; glycyrrhetinic acid-carboxymethyl chitosan-ketal-rhein conjugate: GCTR conjugate; tripterine/glycyrrhetinic acid-carboxymethyl chitosan-ketal-rhein conjugate micelle: cela/GCTR micelles; carboxymethyl chitosan-rhein conjugate: a CR conjugate; tripterine/carboxymethyl chitosan-rhein conjugate: cela/CR micelle; active oxygen: ROS.

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

The synthesis of GCTR conjugates is shown in figure 1, and four-step synthesis is contemplated:

the first step: 0.1mmol R with 1% NaCO ₃ Heating for dissolving, adding 0.12mmol EDC and 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. 0.15mmol of EDA is added to dissolve with water, R-NHS is added dropwise to the EDA aqueous solution, and the reaction is carried out for 6 hours to obtain R-EDA.

And a second step of: dissolving 0.15mmol of TK in DMF, adding 0.18mmol of EDC and HCl, reacting at room temperature for 20min, adding 0.18mmol of NHS, reacting at room temperature for 10min, adding into R-EDA solution, and reacting at room temperature to obtain TER.

And a third step of: 0.083mmol CMCS was dissolved in water. Taking the TER solution, centrifuging to obtain supernatant, adding 0.12mmol EDC and HCl to react for 20min at room temperature, adding 0.12mmol NHS to react for 10min at room temperature, adding the mixture into CMCS aqueous solution, and reacting for 24h to obtain CTR.

Fourth step: dissolving 0.12mmol of GA in DMF, reacting 0.14mmol of EDC and HCl at room temperature for 20min,0.14mmol NHS min, adding into CTR solution, reacting at room temperature for 24h, dialyzing the reaction solution in dialysis bag (MWCO 3500) with distilled water for 72h, ultrasonically treating with probe for 20min, and freeze-drying to obtain the final product 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 particle size of micelles formed by self-assembly in water is smaller, the micelles are (220.17 +/-5.50) nm (figure 2), the distribution is uniform, and the potential is (-12.03+/-0.82) mV.

FT-IR characterization and characterization of each starting material and resulting GCTR conjugate ¹ The results of the H-NMR characterization are shown in FIG. 3, where the GCTR conjugate was successfully synthesized as indicated by the changes in the infrared spectrum peak and proton peak.

EXAMPLE 2 preparation of Cela/GCTR micelles

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

Cela/GCTR micelles were prepared by the following method: weighing 12mg of GCTR conjugate, adding 2.4mL of distilled water, stirring and dissolving the conjugate for a strong time, dropwise adding 20mg/mL of Cela DMSO solution under vigorous stirring after ultrasonic treatment for 10min, continuing stirring at a high speed for 20min, performing ultrasonic treatment for 20min by using the ice water bath probe, dialyzing the conjugate by using distilled water for 12h to remove organic solvent, performing ultrasonic treatment for 20min by using the ice water bath probe after dialyzing, centrifuging for 10min at 2500 r/min, and filtering the supernatant by using a 0.8 mu m filter membrane to obtain the Cela/GCTR micelle solution.

Three batches of the preparation were repeated according to the optimal drug-loading process, and the results are shown in Table 1 and FIG. 4, and the Cela/GCTR micelle prepared in the embodiment has the advantages of small particle size, small particle size distribution (PDI) range, large drug-loading rate (DL), high Encapsulation Efficiency (EE), and high repeatability, thus indicating that the drug-loading process is reliable and stable.

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

To further illustrate the beneficial effects of the present invention, the following experiments were performed, with the GCTR conjugate of example 1 being used without illustration, and the Cela/GCTR micelle of example 2 being used.

Experimental example 1 Synthesis condition screening of GCTR conjugate

Based on the method of example 1, the particle size was calculated asAnd Distribution (PDI), zeta potential, degree of substitution of R and GA (DS _R 、DS _GA ) As an index for the investigation of the GCTR conjugate synthesis process, the detection results of GCTR conjugates synthesized at different feed ratios are shown in table 2.

When CMCS and GA are fixed, the molar substitution degree of R and GA is reduced along with the increase of TK feeding when the quantity of R and TK feeding is changed (1:1-1:2), and when R is TK=1:1, the substitution degree of GTCR conjugate micelle R and GA is maximum, the particle size is smaller, the particle size is (237.67 +/-21.51) nm, and the distribution is more uniform.

When the R and TK charge were fixed and the CMCS and GA charges varied (1:1-1:2), the molar substitution of R, GA of the GCTR conjugate varied greatly, and the potential increased with increasing GA charge. When CMCS: ga=1:1.5, R, GA molar substitution was the largest and the particle size and distribution effects were not apparent.

The reaction time investigation results show that as the reaction time is prolonged (6-24 h), the molar substitution degree of R and GA on the GCTR conjugate is increased, the particle size is smaller, the particle size distribution is more uniform, and the increase of the reaction time is presumed to be beneficial to the synthesis of the product, but if the reaction time is too long, the synthesis efficiency is reduced. Therefore, 24h was chosen as the reaction time.

And comprehensively considering the factors such as the substitution degree, the particle size, the PDI, the potential and the like of R, GA, and finally determining that R is the optimal synthesis feeding ratio of TK to CMCS to GA (1.2:1.8:1:1.5), and reacting R with TK for 24 hours.

TABLE 2 investigation index results for different synthetic process products

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Experimental example 2 ROS-sensitive Properties of GCTR conjugate

The GCTR conjugate obtained in example 1 was prepared into GCTR conjugate micelle by direct dispersion dissolution method, and its morphology was subjected to TEM characterization, as shown in fig. 5 a. As can be seen, the GCTR conjugate micelles have a spherical structure, and the particle size distribution is relatively uniform and is about 220 nm. GC was takenThe TR conjugate micelle solution contains 10mmol/L H ₂ O ₂ Diluting the solution to 1mg/mL, placing in a shaking table at 37deg.C, treating for 6 hr, observing TEM image, and passing through H as shown in FIG. 5b ₂ O ₂ The particle size of the GCTR conjugate micelle becomes large after treatment, which indicates that TK bonds in the GCTR conjugate may be broken, so that the micelle structure is changed, the volume becomes large, and the GCTR conjugate micelle has the characteristic of ROS sensitivity.

The GCTR conjugate micelle was taken out at various time points to determine the particle size, and the results are shown in Table 3, in which the particle size of the GCTR conjugate micelle is as follows with H ₂ O ₂ The contact time is prolonged, the particle size is rapidly increased, and the dispersity is also increased. It was further demonstrated that GCTR-conjugate micelles could be found at low concentrations of H ₂ O ₂ Depolymerization in solution, ROS sensitivity.

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

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Experimental example 3 stability of GCTR conjugate micelle

The stability investigation result of the GCTR conjugate micelle of the embodiment 1 is shown in fig. 6, and the particle size of the GCTR polymer micelle is basically unchanged under the indoor natural illumination and light-shielding conditions within one week, flocculation, precipitation and other phenomena are avoided, so that the stability is good. The GCTR conjugate micelle has good stability when being placed in water for one week at room temperature.

Experimental example 4 preparation condition screening of Cela/GCTR micelle

And (3) taking the drug loading capacity, the encapsulation efficiency, the particle size and the distribution index as indexes to examine the influence of factors such as the Cela solvent, the drug loading ratio and the carrier concentration on the GCTR conjugate micelle entrapped Cela.

1. Cela solvent screening

The effect of the solvents on the drug carrying capacity of GCTR conjugates was examined using methanol, ethanol, DMF, DMSO as solvents for Cela, and the experimental results are shown in table 4. As can be seen from the table, DMSO is more favorable for the loading of Cela than methanol, ethanol, DMF. When DMSO is used as a Cela solvent, the prepared Cela/GCTR micelle has the particle size of (182.83 +/-4.37) nm, the PDI of 0.017+/-0.010, the drug loading rate of (38.73 +/-4.64) percent and the encapsulation rate of (77.72+/-9.15). Taken together, the Cela/GCTR micelle has the advantages of small particle size, uniform distribution, large drug loading and high encapsulation efficiency, so DMSO is selected as a solvent for Cela dissolution in the study.

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

2. Drug to carrier feed ratio

The effect of drug to carrier dosing ratio (drug loading ratio) on GCTR conjugate-entrapped Cela, results are shown in table 5. The drug loading rate increases with the increase of the drug loading ratio, and the encapsulation efficiency also increases with the increase of the drug loading ratio. The encapsulation efficiency decreased significantly when the drug loading ratio increased to 1:1.6, indicating that the GCTR conjugate-entrapped Cela had reached saturation. And when the drug load ratio is 1:1.4, the particle size is smaller than other ratios, so that 1:1.4 is selected as the optimal feeding ratio of the Cela and GCTR conjugate.

TABLE 5 influence of different drug amounts and carrier dose ratios on the drug carrying capacity of GCTR conjugates

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3. Carrier concentration

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

TABLE 6 Effect of different carrier concentrations on the drug carrying capacity of GCTR conjugates

4. Screening of lyoprotectants

In order to prevent adverse conditions such as loose structure, poor appearance, scattering along with water and the like of the drug in the freeze-drying process, a freeze-drying protective agent can be generally added to improve the product form and the solubility of the freeze-dried preparation. The experiment takes the appearance, redispersibility, particle size, potential, drug loading and encapsulation efficiency of the freeze-dried preparation as evaluation indexes, and examines the influence of two freeze-drying protective agents, namely mannitol and glucose with the concentration of 0.2 percent on Cela/GCTR micelle freeze-drying.

The evaluation index is as follows:

TABLE 7 evaluation index of appearance and resolubility

The results show that the Cela/GCTR micelle freeze-dried preparation without the freeze-drying protective agent is orange floccule, has a rough surface and loose texture, and can observe that part of the medicine is scattered along with water in the freeze-drying process. After the freeze-drying protective agent is added, the appearance, the re-solubility, the particle size, the drug loading rate and the encapsulation efficiency of the Cela/GCTR micelle freeze-drying preparation are all improved. In the two freeze-drying protective agents, the glucose has better effect, the appearance is flat and cake-shaped, the re-solubility is stable, the particle size is (135.40 +/-35.4) nm, the drug loading rate and the encapsulation efficiency are (36.71+/-6.71)%, and (79.77+/-9.77)%, respectively, and no obvious difference exists compared with the preparation without freeze-drying. Compared with the freeze-dried preparation without the freeze-dried protective agent, the particle size is reduced, and the encapsulation efficiency and the drug loading rate are both increased. In summary, 0.2% glucose was selected as a protectant for the Cela/GCTR micelle lyophilized formulation.

TABLE 8 Cela/GCTR micelle lyophilized formulation prescription screening

Experimental example 5 Cela/GCTR micelle in vitro Release

The behavior of Cela released from GCTR conjugate in vitro was studied by dialysis to simulate in vivo environment, using phosphate buffer solution at pH 5.0 and pH 5.0+10mmol/L H, respectively ₂ O ₂ The phosphate buffer solution of (2) simulates the environment of tumor tissues and the phosphate buffer solution of pH 7.4 simulates the neutral environment of blood, and the in vitro release kinetics behavior of Cela/GCTR micelle is examined.

1. Experimental method

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

Taking particle size as an inspection index, inspecting the stability of Cela/GCTR micelle in phosphate buffers with different pH values. Weighing a proper amount of Cela/GCTR micelle, dissolving with phosphate buffers with different pH values, detecting for 10min until the mixture is uniformly dispersed, placing the mixture in a water bath shaking table at 37 ℃, measuring the particle size of the Cela/GCTR micelle after 100r/min for 0, 6, 12, 24, 48 and 72h, and examining the stability of the Cela/GCTR micelle.

2. Cela/GCTR micelle in vitro release experiment

Precisely weighing Cela/GCTR micelle, adding 0.9% sodium chloride injection for dissolution, adding release medium for dilution until the concentration of Cela is 250 mug/mL, performing ultrasonic treatment for 10min, taking 1mL of Cela/GCTR micelle solution, placing into 3500Da dialysis bag, placing into a beaker containing 100mL of dissolution medium, paralleling 3 parts, and performing Cela/GCTR micelle in vitro at a vibration speed of 100r/min and a medium temperature of 37 DEG CRelease experiments. Taking 4mL of release liquid outside a dialysis bag according to set time intervals of 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60 and 72 hours, adding the release liquid into a 10mL centrifuge tube volatilized by adding 10 mu L of internal standard Emodin (300 mu g/mL), and simultaneously adding a blank release medium with the same volume. Extracting the sample with 3mL of dichloromethane, vortexing for 5min, collecting the lower layer liquid into a 5mL centrifuge tube, volatilizing under reduced pressure, redissolving with 200 μL of methanol, vortexing for 3min, centrifuging for 20min at 14000r/min, collecting appropriate amount of supernatant, measuring Cela content by high performance liquid chromatography, calculating accumulated release amount, drawing release curve, and collecting accumulated release rate (Q _t ) The calculation formula of (2) is as follows:

wherein C is _n For the concentration of Cela in the release solution at the nth sampling, V is the total release medium volume, V ₀ For the sampling volume, M is the total Cela for release.

2. Experimental results

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

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

TABLE 9 variation of particle size of Cela/GCTR micelles in vitro Release Medium

2. In vitro Release study

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

As can be seen from the release results in fig. 7, the cumulative release rate of the Cela solution for 12h at pH 7.4 reached more than 70%, with almost complete release for 24 h. Whereas the cumulative release rate of Cela/GCTR micelle 12h was about 29% and that of 24h was about 37%, a steady release was maintained for 72 h. No abrupt release phenomenon exists in the release process, which indicates that the Cela is completely entrapped in the hydrophobic inner core of the conjugate micelle, and the Cela/GCTR micelle has a slow release effect in a neutral environment of blood. Under the condition of pH 5.0, the cumulative release rate of the Cela/GCTR micelle 12h is about 37%, the cumulative release rate of the Cela/GCTR micelle 12h is about 50%, the release rate of the Cela/GCTR micelle is faster in the first 2 hours, the Cela/GCTR micelle slowly releases after 2 hours, and compared with the release amount of the Cela/GCTR micelle under the condition of pH 7.4, the release amount of the drug increases along with the decrease of pH, and the Cela/GCTR micelle has certain pH sensitivity. Having a particle size of 10mmol/L H ₂ O ₂ When present, cela/GCTR micelles had a cumulative release rate of about 58% at 12h and about 81% at 24 h. As can be seen, 10mmol/L H is added ₂ O ₂ After that, the release rate was faster and the release amount was more, indicating that Cela/GCTR micelle had 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 acidic environment and a high ROS environment with the characteristics of tumor microenvironment, and can improve the drug release rate.

Experimental example 6 cytobiological Effect of Cela/GCTR micelle

1. Experimental method

1. Synthesis of CR conjugates and GCTR conjugates

CR conjugate synthesis:

(1) Weighing carboxymethyl chitosanAdding sugar into a reaction bottle, adding 10mL of distilled water, and swelling for 30min; rhein powder was taken in a jar and 10mL of 1% NaHCO was added ₃ Heating the solution to dissolve rhein completely, cooling to room temperature, adding EDC and HCl, activating for 20min, and adding NHS; then adding the mixed solution into carboxymethyl chitosan solution while stirring, and stirring and reacting for 24 hours in a dark place; wherein the molar ratio of the substances is carboxymethyl chitosan, rhein, EDC, HCl, NHS=1:1:3:1; (2) Precipitating the reaction solution with 95% ethanol, standing, suction filtering, and washing the precipitate with 95% ethanol until the washing solution is colorless; dissolving the pumped precipitate in water, performing ultrasonic treatment with a probe under ice water bath condition for 20min, centrifuging at 3000rpm for 10min, collecting supernatant, filtering with 0.8 μm filter membrane, and dialyzing the filtrate in dialysis bag for 72 hr; (3) After dialysis is finished, placing the filtrate in an ice water bath, performing probe ultrasound for 20min, centrifuging at 3000rpm for 10min, taking supernatant, passing through a 0.8 mu m filter membrane, and freeze-drying the filtrate to obtain the carboxymethyl chitosan-rhein conjugate.

GCTR conjugate synthesis:

(1) 0.1mmol of R is dissolved by heating with 1% NaCO3, 0.12mmol of EDC and HCl are added under stirring, the reaction is carried out for 20min at room temperature, and then 0.12mmol of NHS is added for reaction for 10min at room temperature, so that R-NHS is obtained. 0.15mmol of EDA is added to dissolve with water, R-NHS is added dropwise to the EDA aqueous solution, and the reaction is carried out for 6 hours to obtain R-EDA.

(2) Dissolving 0.15mmol of TK in DMF, adding 0.18mmol of EDC and HCl, reacting at room temperature for 20min, adding 0.18mmol of 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. Taking the TER solution, centrifuging to obtain supernatant, adding 0.12mmol EDC and HCl to react for 20min at room temperature, adding 0.12mmol NHS to react for 10min at room temperature, adding the mixture into CMCS aqueous solution, and reacting for 24h to obtain CTR.

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

2. Cell culture

Preparation of cell complete medium: the culture medium of HepG2 cells is DMEM culture solution, 10% FBS and 1%Penicilin Streptomycin Solution are added and mixed uniformly; the BEL-7402 cells are prepared by adding 10% FBS and 1%Penicilin Streptomycin Solution into RPIM1640 culture solution, and mixing; the culture medium of L-02 cells is RPIM1640 culture solution, 20% FBS and 1%Penicilin Streptomycin Solution are added, and the mixture is uniformly mixed.

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

Cell passage: when the cell adhesion reaches about 80%, subculturing is carried out, the old culture medium is discarded, PBS is washed for 2 times, 1mL of trypsin solution is added for digestion, and the culture medium is placed in an incubator for 2min; observing under a microscope, if the cell gap becomes larger, rounding the cells, immediately adding 3mL of a culture medium containing serum, lightly blowing the wall of the bottle by using a gun, and enabling the cells to fall into the culture solution in the bottle; transferring the suspension into a 15mL centrifuge tube, centrifuging at 1000r/min for 4 mm, and removing the supernatant; and adding a proper amount of complete culture medium to the cell sediment in the centrifuge tube to resuspend the cells, and carrying out passage according to the ratio of 1:1.2-1:4 to continue culturing.

3. Cytotoxicity investigation

Cytotoxicity of drug-loaded micelles was determined by thiazole blue colorimetric method (MTT assay), and toxicity of CR conjugate, GCTR conjugate, cela/CR micelle, cela/GCTR micelle, free Cela and its solvent Cremophor EL: etOH (50%: 50%, v/v) to HepG2, BEL-7402, L-02 cells was examined.

Inoculating HepG2, BEL-7402, L-02 cells in logarithmic growth phase into 96-well plate with each well being 5×10 ⁴ The cells, the border wells were filled with blank PBS, incubated in an incubator for 24h to allow adherence, and medium was aspirated off. Different concentrations of CR conjugate, GCTR conjugate, cela/CR micelle, cela/GCTR micelle, free Cela, crEmpor EL: etOH (50%: 50%, v/v) 150. Mu.L of test solution was added to 96-well plates at a concentration of 6 wells in parallel. Incubating in an incubator, and setting blank control hole without cell and normal hole without cell to give medicine. The plates were removed 24, 48, 72 hours after dosing, 100. Mu.L of MTT solution at a concentration of 1. Mu.g/mL was added to each well and incubated for 4 hours in the incubator. The plates were removed and the supernatant carefully aspirated. 150 μl DMSO was added to each well to dissolve the blue-violet crystals. Absorbance (OD) was measured at 570nm using a microplate reader. Cell viability was calculated according to the following formula and IC was calculated using Graphpad software ₅₀ Values.

4. Cellular uptake

4.1 preparation of P4-loaded micelles

Since Cela itself does not have fluorescence, in order to compare uptake capacity of tumor cells to the Cela/CR micelle and the Cela/GCTR micelle, the (p4+cela)/CR micelle and the (p4+cela)/GCTR micelle, which co-load P4 and Cela, were prepared with a hydrophobic organic dye P4 (environmental response type fluorescent probe) as a fluorescent indicator, in a loading method similar to the Cela-loading method. The method comprises the following steps: 12mg of CR conjugate or GCTR conjugate is weighed, 2.4mL of distilled water is added, and probe ultrasound is used to fully dissolve the conjugate and form CR micelle or GCTR micelle solution. Weighing a small amount of Cela, dissolving in DMSO solution containing 90 mu g P4, uniformly mixing, dropwise adding into CR micelle or GCTR micelle solution under high-speed stirring, continuously stirring at high speed for 20min, performing ultrasonic treatment with an ice water bath probe for 20min, dialyzing for 24h, performing ultrasonic treatment with the probe for 20min, centrifuging at 2500 r/min for 10min, and taking supernatant to obtain (P4+Cela)/CR micelle and (P4+Cela)/GCTR micelle.

4.2 qualitative investigation by laser confocal microscope

Uptake of both free P4 and P4-loaded micelles (P4+Cela)/CR and (P4+Cela)/GCTR micelles (P4 concentration 0.5. Mu.g/mL) by HepG2 cells was examined by confocal microscopy. Taking HepG-2, L-02 cells in logarithmic growth phase at 2×10 ⁵ Density of individual/mL inoculated in confocal laser culture dishIn the above, after the adherence was cultured, the culture solution was discarded, PBS preheated at 37℃was added to gently wash for 2 times, 1mL of each group of test solutions was added (when the influence of GA was examined, GA 5. Mu.g/mL of solution was added 1 hour in advance, GA receptors on HepG-2 cells were saturated, and then drug-loaded micelles were added), after incubation for 4 hours, the drug solution was discarded, and the uptake was terminated by adding ice-cold HBSS at 4℃and the cells were washed 3 times. The HBSS was discarded, fixed with 4% paraformaldehyde for 20min, and the HBSS was washed 2-3 times. 1mL of PBS solution containing Hoechst33258 (10. Mu.g/mL) was added to each well, the nuclei were stained for 15min, and after 3 washes of cold HBSS, 200. Mu.L of PBS was added, and observed under a laser confocal microscope and photographed.

4.3 quantitative investigation by flow cytometer

HepG2 cells in exponential growth phase were taken at 4X 10 ⁵ Inoculating the culture medium into six-hole plates at the density of each mL, culturing for 24h, sucking and discarding the culture medium, adding 1.5mL of each of the prepared (P4+Cela)/CR micelle and (P4+Cela)/GCTR micelle solution containing the same concentration of P4, continuously culturing for 4h, sucking and discarding the liquid medicine, adding ice-cold HBSS at 4 ℃ to stop ingestion, washing for 2 times, adding digestive juice into the six-hole plates, collecting cells, centrifuging for 3min at 2000r/min, discarding the culture solution, and adding PBS again for repeating for 2 times. The objective is to remove fluorescent substances that may not be phagocytized by cells and to remove digestive juice. After washing, 1mL of PBS was added, and the mixture was blown and dispersed uniformly to prepare a suspension. Filtering with 400 mesh sieve, exciting at 480nm, emitting at 575nm, sampling, detecting by flow cytometry, and analyzing the result by software.

4.4 cell cycle experiments

HepG2 cells in logarithmic growth phase were taken at 2X 10 ⁵ The density of individual cells was seeded in 6-well plates, the incubator was incubated overnight, the medium was aspirated and washed twice with PBS. And (3) respectively setting a Control group, a free Cela group and a Cela/GCTR micelle group, setting 3 compound holes in each group, culturing in an incubator for 24 hours, performing sample treatment by using a cell cycle detection kit, and detecting by using a flow cytometer.

2. Experimental results

1. Cytotoxicity test results

HepG2, BEL-7402 and L-02 cells are selected as cell models, and the effects of CR conjugate, GCTR conjugate, cela/CR micelle, cela/GCTR micelle, free Cela and solvent Cremophor EL thereof on proliferation of HepG-2, BEL-7402 and L-02 cells are examined by adopting an MTT method, so that cytotoxicity of carrier materials and drug-loaded micelles is evaluated. A series of test reagent solutions with different concentrations are incubated with cells for 24 hours, 48 hours and 72 hours, and the cell survival rate is detected.

As can be seen from the MTT test results of L-02 in FIG. 8, the CR conjugate and the GCTR conjugate with different concentrations have no significant effect 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 cause damage to the cells and has good biocompatibility. Under the concentration of 0.01-1 mug/mL, cela/CR micelle, solvent group EtOH+EL and Cela/GCTR micelle have no toxicity to L-02 cells, and free Cela shows a certain inhibition effect after 48 hours under the concentration range. Cela, cela/CR micelle, cela/GCTR micelle all showed L-02 cell inhibition at two concentrations of 2.5. Mu.g/mL and 5. Mu.g/mL, and inhibition was gradually enhanced over time. In conclusion, the results show that the GCTR conjugate carrier has no cytotoxicity to L-02 cells in the experimental concentration range, and Cela/GCTR micelle has no obvious proliferation inhibition effect to L-02 cells in the concentration range of 0.01-1 mug/mL, and the GCTR conjugate and the Cela/GCTR micelle have good safety.

From MTT experimental results of HepG2 cells, it can be seen that within the experimental concentration range, etOH+EL, CR conjugate and GCTR conjugate with different concentrations have no obvious influence on the survival rate of HepG2 cells. The free Cela, the Cela/CR micelle and the Cela/GCTR micelle have certain proliferation inhibition effect on HepG2 cells, and are shown as time and dose dependence, and the inhibition effect is stronger with the increase of time and concentration. When the concentration of Cela is low, the free Cela, the Cela/CR micelle and the Cela/GCTR micelle have no obvious inhibition effect on HepG2 cells, and when the concentration of Cela reaches 0.5 mug/mL, the growth inhibition effect of the free Cela, the Cela/CR micelle and the Cela/GCTR micelle on the HepG2 cells is different. The Cela/GCTR micelle is obviously larger than the free Cela and has better proliferation inhibition effect compared with the Cela/CR micelle. At 72h, the concentration was 0.5. Mu.g/mL, the Cela/GCTR micelle cell viability was (41.58.+ -. 6.14)%, the Cela/CR micelle cell viability was (71.73.+ -. 2.83)%, and the free Cela cell viability was (87.06.+ -. 5.78)%. Compared with free Cela and Cela/CR micelle, the survival rate of the Cela/GCTR micelle cells is reduced by about 2.1 times and about 1.7 times respectively. The Cela/GCTR micelle has more obvious effect of inhibiting HepG2 cell proliferation along with the prolongation of time and the increase of concentration, and shows stronger cytotoxicity.

As can be seen from the MTT results of BEL-7402 cells, the EtOH+EL, CR conjugate and GCTR conjugate at different concentrations had no significant effect on BEL-7402 cell viability. When the Cela concentration reaches 0.25 mug/mL, the free Cela, the Cela/CR micelle and the Cela/GCTR micelle show the effect of inhibiting BEL-7402 cell proliferation, and the inhibition effect is stronger than that of HepG2 cells when the Cela/GCTR micelle is taken at the same concentration, wherein the inhibition effect is strongest. At a Cela concentration of 0.5 μg/mL, 24 hours, the survival rate of free Cela cells is about 1.5 times that of Cela/CR micelle, about 1.6 times that of Cela/GCTR micelle, indicating that the ability of Cela to inhibit cell proliferation is enhanced after entrapped into micelle.

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

TABLE 10 Cela and different micelles with different cytotoxicity effects IC ₅₀ Value (μg/mL) (n=6)

2. Results of cell uptake experiments

The experiment uses P4 as a fluorescent probe for cell uptake experiments. P4 is an environmentally-responsive aza BODIPY dye that exhibits red fluorescence in a hydrophobic environment, but is fluorescent quenched in water by molecular aggregation. The results of the confocal laser qualitative study are shown in FIG. 10, wherein red is the fluorescence of P4 and blue is the fluorescence of Hoechst 33342. In HepG2 cells, there was no apparent red fluorescence in the cells after 4h incubation of the free P4 solution, whereas red fluorescence appeared in HepG2 cells after incubation of (p4+cela)/CR micelles, (p4+cela)/GCTR micelles, indicating that the micelles encapsulated Cela and P4 into the cells in a complete micelle morphology. And no obvious red fluorescence appears in all groups in the L-02 cells, which indicates that the normal liver cells have less uptake to the micelle, so that the drug-loaded micelle has less toxic and side effects on the liver.

In order to study the uptake of GA modified micelles, GA solution is added 1h in advance, after GA receptors on HepG2 cells are saturated, the (P4+Cela)/GCTR micelles are added, and the (P4+Cela)/CR micelles without GA modification are arranged, so that the uptake condition of the HepG2 cells on the micelles is examined. As can be seen from FIG. 11, GA-pretreated (P4+Cela)/GCTR micelle has the weakest red fluorescence and the average fluorescence intensity of 225.62 + -13.45, indicating that the uptake rate of (P4+Cela)/GCTR micelle by tumor cells after GA pretreatment is low. In the same time, GA modified (P4+Cela)/GCTR micelle has more obvious cellular uptake than GA-modified (P4+Cela)/CR micelle, and the average fluorescence intensities of the GA modified (P4+Cela)/GCTR micelle and the GA-modified (P4+Cela)/CR micelle are 606.79 +/-49.94 and 293.37 +/-13.94 respectively.

In combination with the above results, it was shown that GA-mediated active targeting facilitates uptake of the nanomicelle by liver tumor cells.

3. Cell cycle test results

After incubation of HepG2 cells with Cela, cela/GCTR micelle solution for 24h, respectively, cell populations at different stages of the cell cycle were detected using a flow cytometer, and cell cycle distribution was determined by staining DNA with PI. The results in FIG. 11 and Table 11 show that the Control group cells were mostly in the G0/G1 phase, and that the ratio of the G0/G1 phase cells was increased and the ratio of the S phase cells was decreased when both groups of the pharmaceutical preparation were administered. The Cela/GCTR micelle treatment group also blocked some cells in the G2/M phase compared to the Cela alone treatment group. Thus, cela/GCTR micelle can block the cell cycle, inhibit the mitosis of tumor cells and promote apoptosis.

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

The results of this experimental example show that the GCTR conjugate is a safe drug carrier. Cela/GCTR micelle has no obvious toxicity to normal liver cells L-02 in a proper concentration range, and has obvious proliferation inhibition effect to liver cancer cells HepG2 and BEL-7402. The GA modified Cela/GCTR micelle has a recognition 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 targeting tumor inhibition effect.

Experimental example 7 in vivo pharmacokinetic and tissue distribution study of Cela/GCTR micelle

1. Experimental method

1. Preparation of plasma samples

Blood is taken from eyeballs of mice, placed in a 1.5mL centrifuge tube containing heparin sodium, centrifuged at 4000r/min for 5min, and plasma is taken for later use. Precisely sucking 200 mu L of blank plasma of a mouse, adding 10 mu L of an internal standard substance (tansineII A5 mu g/mL), adding 3mL of ethyl acetate for extraction after vortex mixing uniformly, vortex oscillating for 3min, centrifuging for 10min at 3500r/min in a high-speed centrifuge, taking supernatant into A5 mL centrifuge tube, volatilizing, vortex oscillating for 5min for redissolving with 100 mu L of methanol, centrifuging for 10min at 14000r/min, and taking 70 mu L of supernatant for UPLC-MS analysis.

2. Living body imaging experiment

2.1 preparation of Co-carried P2 and Cela micelles

12mg of GCTR conjugate was weighed into a penicillin bottle and dissolved in 2.4mL of water. 2mg of P2 is taken in a 250mL volumetric flask, dissolved with methanol to a fixed volume, and prepared into mother liquor with the volume of 8 mug/mL for standby. Precisely measuring 11.25mL of P2 mother liquor, volatilizing methanol in a centrifuge tube, adding 450 mu L of DMSO solution containing a small amount of Cela for re-dissolution, dropwise adding the solution into a GCTR conjugate water solution stirred at high speed at room temperature, continuously stirring for 20min, ultrasonically treating for 20min by an ice water bath probe, transferring the solution into a dialysis bag, dialyzing for 12h, and freeze-drying to obtain the (P2+Cela)/GCTR micelle carrying the P2 and the Cela together. The preparation method of the (P2+Cela)/CR micelle is the same as that of the (P2+Cela)/CR micelle.

2.2 Experimental methods

To examine the in vivo distribution of GCTR micelles, we examined the in vivo distribution using GCTR micelles co-loaded with P2 and Cela. Tumor volume is larger than 100mm ³ The mice of (3) were randomly divided into 4 groups, each group was injected with P2 physiological saline solution, (P2+Cela)/CR micelle and (P2+Cela)/GCTR micelle via tail vein, and after injection, the in vivo fluorescent material distribution of the mice was observed under a living body imager at time points of 0h, 0.5h, l h,2h,4h,6h,12h and 24h, and recorded by photographing.

2. Experimental results

1. Blood concentration time curve

FIG. 12 is a graph showing blood concentration versus time, taken as abscissa, of blood concentration of Cela in mice at various time points, after intravenous injection of Cela, cela/CR micelle and Cela/GCTR micelle into the tail of the mice. The results show that the blood concentration of Cela in plasma gradually decreases after administration, and the blood concentration of Cela/CR micelle and Cela/GCTR micelle changes slowly, and gradually keeps stable release after 2 hours. The free Cela has a rapid removal rate in blood, shows a rapid decline trend in 2 hours, is almost undetectable in blood plasma at 24 hours, has a rapid release rate in front of a Cela/CR micelle and a Cela/GCTR micelle group for 1 hour, starts to release slowly after 1 hour, and still maintains stable blood concentration for 24 hours, so that the Cela is entrapped into the micelle, the existing form of the drug is changed, the Cela can exist in the body for a long time and is kept released slowly, and the retention time of the Cela in the blood is obviously prolonged.

2. Living body imaging research of Cela/GCTR micelle

The results of fluorescence imaging of tissues at 24h after intravenous injection of P2, (P2+Cela)/CR micelle and (P2+Cela)/GCTR micelle in physiological saline solution into the tail of mice, in vivo, are shown in FIGS. 14 to 18. As can be seen from fig. 15, after intravenous injection of (p2+cela)/GCTR micelles in the tail of the mice, the fluorescence signal gradually accumulated in the liver from 0.5h and reached the maximum and then gradually decreased at 1h, and the fluorescence signal still remained for 24 h. The (P2+Cela)/CR micelle fluorescence signal also accumulated in the liver from 0.5h, with 2h reaching a maximum. As can be seen from fig. 16, when the fluorescence intensity of the whole body of the mice is 1h and 2h, (p2+cela)/GCTR micelle group is larger than (p2+cela)/CR micelle group, which indicates that both p2+cela/CR micelle and (p2+cela)/GCTR micelle show liver targeting, and (p2+cela)/GCTR micelle liver targeting is stronger, probably due to GA binding with hepatocyte surface receptor in GCTR conjugate, so that affinity of (p2+cela)/GCTR micelle to liver tissue is increased, and more concentration is carried out in liver. As can be seen from fig. 17, with the lapse of time, the fluorescence signal gradually accumulated at the tumor site, the (p2+cela)/CR micelle group reached the maximum fluorescence intensity at 12h, the (p2+cela)/GCTR micelle was the strongest at 8h, and the fluorescence intensity was significantly higher than that of the (p2+cela)/CR micelle. The result shows that the (P2+Cela)/GCTR micelle has a targeting effect on tumor cells, and the targeting effect is better than that of the (P2+Cela)/CR micelle.

The mice were sacrificed after 24h of dosing, and the organs and tumor tissues were dissected and removed for in vivo fluorescence analysis. As a result of tissue fluorescence imaging, at 24h, a small amount of fluorescence signal is also present in liver tissue, and the fluorescence signal in tumor tissue is relatively strong. The same results can be seen from the fluorescence intensity plot, which shows that the residual fluorescence intensity in liver tissue is slightly higher than that of the (P2+Cela)/CR micelle group, and the analysis is probably due to the fact that GA in the carrier GCTR conjugate can be combined with GA receptor in liver, so that fluorescence is stronger. The higher residual fluorescence intensity of the (P2+Cela)/GCTR micelle in the tumor tissue is probably due to the smaller size of the Cela/GCTR micelle, better water solubility and more contact with the tumor tissue, so that the residual fluorescence is stronger.

Experiments of the experimental example show that the Cela/GCTR micelle has long circulation in vivo, can obviously improve the residence time of the Cela in blood and improve the bioavailability of the Cela. In addition, cela/GCTR micelle can reach liver and tumor sites in vivo, can stay for more than 24 hours, and shows targeting to liver tissues and tumor tissues.

The above examples 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 to liver tissues and tumor tissues and has the function 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 by the following two substituents through amidation reaction:

and->

。

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

The molar substitution degree of (2) is 5.43-9.05%, and the substituent is

The molar substitution of (2) is 1.79 to 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 ketal, and grafting glycyrrhetinic acid and the ketal onto carboxymethyl chitosan respectively.

4. A modified carboxymethyl chitosan according to claim 3, characterized in that the molar feed ratio of the raw materials for preparing the modified carboxymethyl chitosan is:

rhein and ketal are 1:1-1:2;

and/or carboxymethyl chitosan and glycyrrhetinic acid in a ratio of 1:1-1:2;

and/or carboxymethyl chitosan and ketal in the ratio of 1:1.2-1:2.4;

5. A modified carboxymethyl chitosan according to claim 3, characterized in that: the rhein is connected to the carboxyl at one end of the ketal through ethylenediamine.

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 1-10 ten thousand.

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

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

8. The method of preparing as claimed in claim 7, wherein: in step (1), the reaction is carried out under the action of a base, a cross-linking agent and a catalyst, wherein the base is selected from NaHCO ₃ 、Na ₂ CO ₃ At least one of sodium acetate, sodium phosphate or sodium hydrogen phosphate, and/or the cross-linking agent is selected from 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochlorideOr at least one of 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 hours;

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

9. Use of the modified carboxymethyl chitosan according to any one of claims 1 to 6 for 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 for loading drug molecules as micelles.