A hydroxyalkyl starch complex, its preparation method and application

Abstract

The application discloses a hydroxyalkyl starch complex and a preparation method and application thereof. The hydroxyalkyl starch complex has a structure as shown in formula I: wherein, HAS' is a molecule of hydroxyalkyl starch without a terminal carbohydrate part; R1', R2' and R3' are independently any one of a group of formula II, a group of formula III, a hydrogen atom or a C1-C8 straight chain / branched chain hydroxyalkyl group, and the proportion of the group of formula II in R1', R2' and R3' is 10%-40%. The complex can exist in the form of nanoparticles, and through modification of a carbonic anhydrase inhibitor, expression of carbonic anhydrase of tumor cells can be inhibited, pH of tumor cells, tumor stem cells and tumor-related fibroblasts can be regulated, intracellular pH can be reduced, catalytic efficiency of chemical kinetics induced by metal ions is favorable, and the killing effect on tumor cells is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, more specifically, relates to a hydroxyalkyl starch complex and a preparation method and application thereof. BACKGROUND

[0002] Tumor is the main cause of human death and the biggest obstacle to improve life expectancy. The acid-base balance in the tumor microenvironment has an important influence on tumor growth and spread.

[0003] Intratumoral hypoxia leads to the activation of hypoxia-inducible factor 1 subunit alpha (HIF1A), which in turn triggers the re-adjustment of metabolism, producing acidic metabolites. The accumulation of these acidic metabolites not only affects the function and health of cells, but also has a negative impact on the tumor microenvironment (TME).

[0004] To counteract the effects of the increasingly severe hypoxic and acidified microenvironment, cancer cells will increase the concentration of a series of metabolites and up-regulate the enzyme and transporter network related to pH regulation. Among them, extracellular carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) are important molecules and are widely considered as representative markers of tumor hypoxia.

[0005] CA9 is not only highly expressed in tumor cells, but is also widely considered as an adverse prognostic indicator for a variety of solid tumors. It is not widely expressed in normal human tissues, thus becoming an attractive therapeutic target. A number of studies have confirmed that CA9 plays a key role in tumor growth and metastasis. Carbonic anhydrase inhibitors can be used as a component of multi-modal therapy in combination with traditional radiotherapy, chemotherapy, chemo-dynamic therapy or immunotherapy, etc. This multi-modal therapy can improve the therapeutic effect and reduce the risk of tumor resistance to treatment.

[0006] The activity of chemo-dynamic CDT depends largely on the concentration of H + and H2O2 in the tumor microenvironment. In fact, as a common microenvironmental stressor, the feature of tumor acidosis is that the extracellular pH is acidic (pHe≈6.5) and the intracellular pH is weakly alkaline (pHi≈7.2), which undoubtedly brings a huge chemical obstacle to the effective implementation of CDT in cells, thus leading to insufficient hydroxyl radicals required for tumor oxidative stress damage. Therefore, it is very important to develop a strategy to regulate the pH microenvironment of the tumor to improve chemo-dynamic therapy combined with chemotherapy for the treatment of tumors. SUMMARY

[0007] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a metal ion-doped hydroxyalkyl starch macromolecular conjugate for enhancing chemokinetics and combining with chemotherapy by regulating the tumor pH microenvironment, as well as its preparation and application. The metal ion-doped hydroxyalkyl starch macromolecular conjugate prepared by the above method functions to alter the tumor pH microenvironment, improve chemokinetic catalytic efficiency, and enhance the therapeutic effect of the chemotherapy drug doxorubicin.

[0008] To achieve the above objectives, the present invention provides a hydroxyalkyl starch complex having the structure shown in Formula I:

[0009]

[0010] HAS' is a molecule of hydroxyalkyl starch without terminal carbohydrate moiety; R1', R2' and R3' are independently a group of formula II, a group of formula III, a hydrogen atom, or any one of straight-chain / branched hydroxyalkyl groups of C1 to C8, and the sum of the proportions of the group of formula II in R1', R2' and R3' is 10% to 40%;

[0011]

[0012] Wherein, R4 and R5 are independently C1 to C8 alkyl groups, and A is a carbonic anhydrase inhibitor; at least one phenolic hydroxyl group and / or carbonyl group on all or part of the anthracene ring of Formula III are connected to a divalent metal ion by a coordinate bond.

[0013] Preferably, the hydroxyalkyl starch is hydroxyethyl starch.

[0014] Preferably, R1', R2' and R3' are each independently a group of formula II, a group of formula III, a hydrogen atom, or any one of straight-chain / branched hydroxyalkyl groups of C1 to C4, and R4 and R5 are each independently an alkyl group of C1 to C4.

[0015] Preferably, the sum of the proportions of the Formula III group in R1', R2' and R3' is 5% to 20%.

[0016] Preferably, the carbonic anhydrase inhibitor has a sulfonamide group, a sulfonamide group, or an amino group.

[0017] Preferably, the molar ratio of the divalent metal ion to the group of formula III is 1:(1 to 10).

[0018] Preferably, the divalent metal ion is Cu. 2+ Fe 2+ or Mn 2+ One or more of them.

[0019] Preferably, the average molecular weight of the hydroxyalkyl starch complex is 40-480 kDa.

[0020] Preferably, the hydroxyalkyl starch complex exists in the form of nanoparticles.

[0021] According to another aspect, the present application also discloses a preparation method of the above-mentioned hydroxyalkyl starch complex, and the initial structure of the hydroxyalkyl starch complex is shown as formula IV:

[0022]

[0023] wherein, HAS is the original molecule of the hydroxyalkyl starch without terminal carbohydrate moiety, R1, R2 and R3 are independently any one of hydrogen atom or C1-C8 linear / branched hydroxyalkyl group;

[0024] The preparation method is that, when R1, R2 and R3 are C1-C8 linear / branched hydroxyalkyl group, a carbonic anhydrase inhibitor is modified on the hydroxyl group of part of the hydroxyalkyl groups and doxorubicin is modified on the hydroxyl group of part of the hydroxyalkyl groups, to obtain the complex with the structure of formula I;

[0025] wherein, the modification of the carbonic anhydrase inhibitor on the hydroxyl group is specifically:

[0026]

[0027] A represents the carbonic anhydrase inhibitor;

[0028] The modification of the doxorubicin on the hydroxyl group is specifically:

[0029]

[0030] wherein, DMSO represents dimethyl sulfoxide, Adriamycin represents doxorubicin hydrochloride, DMF represents N,N-dimethylformamide, and Triethylamine represents triethylamine.

[0031] Preferably, the method further comprises complexing the solution containing the complex with the structure of formula I with metal ions in the solution of divalent metal ions by using a self-assembly method, to obtain the hydroxyalkyl starch complex.

[0032] According to another aspect, the present application also discloses the use of the above-mentioned hydroxyalkyl starch complex in the preparation of a medicine for treating and / or preventing tumors, and in the regulation of the pH microenvironment of tumors.

[0033] Overall, compared with the prior art, the above technical solutions conceived by the present application have the following beneficial effects:

[0034] 1. The present application provides a metal-hydroxyalkyl starch complex for adjusting the pH microenvironment of tumor to enhance the chemical kinetics combined with chemotherapy, which can exist in the form of nanoparticles, and the complex can inhibit the expression of carbonic anhydrase of tumor cells by modification of carbonic anhydrase inhibitors, adjust the pH of tumor cells, tumor stem cells and tumor-related fibroblasts, reduce the intracellular pH of tumor cells, and improve the catalytic efficiency of metal ion-induced chemical kinetics and the killing effect on tumor cells.

[0035] 2. The present application couples the chemotherapeutic drug doxorubicin to hydroxyalkyl starch through acid-labile hydrazone bonds, and modifies carbonic anhydrase inhibitors to obtain a corresponding macromolecular conjugate, which can release doxorubicin under acidic conditions, significantly improving the biological safety.

[0036] 3. The present application inhibits the high expression of carbonic anhydrase in tumor tissue, reduces the intracellular pH of tumor cells, and enhances the inhibition of tumor growth by chemical kinetics combined with chemotherapy.

[0037] 4. In the preferred embodiment of the present application, metal ions are doped into the CAi-HES-hyd-DOX macromolecular conjugate to obtain CHHD-metal nanoparticles, which release the chemotherapeutic drug doxorubicin under acidic conditions, and as the pH decreases, the catalytic efficiency of divalent metal ion-induced chemical kinetics is higher, the production of hydroxyl radicals is higher, and the killing effect on tumor cells, tumor stem cells and tumor-related fibroblasts is better.

[0038] It has been verified that the metal-hydroxyalkyl starch complex of the present application can inhibit the growth of tumor cells, tumor-related fibroblasts and tumor stem cells; the present application fills the gap in the use of tumor pH microenvironment-based chemical kinetics combined with chemotherapy for tumor cells, tumor stem cells and tumor-related fibroblasts, and provides a new effective strategy and technology for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The particle size of CHHD-Cu in Example 1;

[0040] Figure 2 The charge of HHD-Cu and CHHD-Cu in Example 1;

[0041] Figure 3 The electron microscope morphology of CHHD-Cu nanoparticles in Example 1;

[0042] Figure 4 The ability of CHHD-Cu to produce hydroxyl radicals under different pH conditions in Example 1;

[0043] Figure 5Effect of Example 1 CHHD-Cu vs. HHD-Cu on H22 cell viability;

[0044] Figure 6 Protein expression content map of Example 1 CHHD-Cu vs. HHD-Cu;

[0045] Figure 7 Effect of Example 1 CHHD-Cu vs. HHD-Cu on intracellular pH change of cells;

[0046] Figure 8 Effect of Example 1 CHHD-Cu vs. HHD-Cu on ROS detection fluorescence intensity;

[0047] Figure 9 Effect of Example 1 CHHD-Cu vs. HHD-Cu on stem cell marker expression;

[0048] Figure 10 Tumor inhibition effect of Example 1 CHHD-Cu vs. HHD-Cu. DETAILED DESCRIPTION

[0049] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and should not be used to limit the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0050] The present application provides a hydroxyalkyl starch complex and a metal-hydroxyalkyl starch complex prepared by the hydroxyalkyl starch complex, which can be stably present in the form of a nanoparticle dispersion liquid, and is applied to a drug for treating and / or preventing tumors and regulating tumor pH microenvironment; the average molecular weight of the hydroxyalkyl starch complex and the metal-hydroxyalkyl starch complex is preferably 40-480 kDa.

[0051] The metal-hydroxyalkyl starch complex provided by the present application has a structure as shown in Formula I:

[0052]

[0053] wherein HAS' is a molecule of hydroxyalkyl starch without a terminal carbohydrate moiety (since there is more than one structure of formula I for a hydroxyalkyl starch HAS, the other moieties than a certain structure of formula I are represented by HAS' instead of HAS), and preferably a molecule of hydroxyethyl starch, i.e. HES'; R1', R2' and R3' are each independently any one of a group of formula II, a group of formula III, a hydrogen atom, or a C1-C8 (preferably C1-C4) straight chain or branched hydroxyalkyl group, and the total proportion of groups of formula II in R1', R2' and R3' is 10-40%;

[0054]

[0055] wherein R4 and R5 are each independently a C1-C8 (preferably C1-C4) alkyl group, and A is a carbonic anhydrase inhibitor; at least one phenolic hydroxyl group and / or carbonyl group of the total or part of the anthracene ring of the group of formula III is / are linked to a divalent metal ion (such as Cu 2+ , Fe 2+ or Mn 2+ , etc.) in a coordination bond; the molar ratio of the divalent metal ion to the group of formula III is preferably 1 : (1-10).

[0056] The initial structure of said hydroxyalkyl starch complex is shown in formula IV:

[0057]

[0058] wherein HAS is a raw molecule of hydroxyalkyl starch without a terminal carbohydrate moiety, and preferably a raw molecule of hydroxyethyl starch, i.e. HES, and R1, R2 and R3 are each independently any one of a hydrogen atom or a C1-C8 straight chain or branched hydroxyalkyl group;

[0059] When R1, R2 and R3 are C1-C8 straight chain or branched hydroxyalkyl groups, a carbonic anhydrase inhibitor is modified on the hydroxyl group of part of the hydroxyalkyl groups and doxorubicin is modified on the hydroxyl group of part of the hydroxyalkyl groups to obtain a complex of formula I;

[0060] wherein the modification of the carbonic anhydrase inhibitor on the hydroxyl group is specifically:

[0061]

[0062] The neutral organic solvent is an organic solvent such as dimethyl sulfoxide (DMSO), and A represents a carbonic anhydrase inhibitor, which preferably has a sulfonamide group, a sulfonamide group or an amino group so as to be connected to the hydroxyalkyl starch, such as (4-(2-aminoethyl)benzenesulfonamide;

[0063] The modification of doxorubicin on the hydroxyl group is specifically:

[0064]

[0065] Adriamycin represents doxorubicin hydrochloride, DMF represents N,N-dimethylformamide, and Triethylamine represents triethylamine.

[0066] In some embodiments, since both modification of carbonic anhydrase inhibitor and doxorubicin require the use of p-nitrophenyl chloroformate (NPC) in the first step, this step can be performed simultaneously; the proportion of hydroxyl groups modified with carbonic anhydrase inhibitor can be controlled by adjusting the concentration of carbonic anhydrase inhibitor, and the proportion of hydroxyl groups modified with doxorubicin can be controlled by adjusting the concentration of hydrazine hydrate in the next step, thereby adjusting the sum of the proportions of groups of formula II and groups of formula III in R1', R2' and R3'.

[0067] After modification with groups of formula III, a solution containing the complex of formula I is combined with metal ions in a solution of divalent metal ions by self-assembly to obtain the hydroxyalkyl starch complex; the complex can exist in the form of nanoparticles, and specifically, by using the coordination of doxorubicin with metal ions, metal ion solution is added dropwise into the hydroxyalkyl starch complex to form metal ion-hydroxyalkyl starch complex nanoparticles by self-assembly, including the following steps:

[0068] (1) Dissolve the hydroxyalkyl starch conjugate in ultrapure water, add an organic solvent, and perform ultrasonic treatment under ice bath conditions, and then remove most of the organic solvent by rotary evaporation;

[0069] (2) Dissolve the divalent metal ions in ultrapure water, and slowly add them to the stirring hydroxyalkyl starch macromolecular conjugate aqueous solution using a micro-injection pump, and then purify the reaction product by dialysis using ultrapure water to obtain metal ion-hydroxyalkyl starch complex nanoparticles; each metal ion has one to three coordination bonds, and each coordination bond is connected to the phenolic hydroxyl group and the carbonyl group, respectively; each pair of coordination bonds on each divalent metal ion can be connected to the same group of formula III or different groups of formula III; taking the case where metal ion X has two pairs of coordination bonds connected to two groups of formula III as an example, the structure is shown in formula VIII:

[0070]

[0071] In some embodiments, the ratio of ultrapure water to organic solvent is 1:(1-100); in other embodiments, the reaction time of the metal ions with the hydroxyalkyl starch macromolecular conjugate is 12-96 h.

[0072] Example 1

[0073] The embodiment provides a hydroxyalkyl starch macromolecule conjugate nanoparticle for adjusting tumor pH microenvironment sensitive chemical kinetics combined with chemotherapy, which comprises a carbonic anhydrase inhibitor, a metal ion, doxorubicin and a main chain structure of hydroxyalkyl starch.

[0074] The carbonic anhydrase inhibitor is a benzene sulfonamide small molecule inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAi), which has a structure as shown in formula I.

[0075]

[0076] The preparation method of the carbonic anhydrase small molecule inhibitor-hydroxyalkyl starch-doxorubicin macromolecule conjugate comprises the following steps:

[0077] (1) 1g of hydroxyethyl (HES) starch is weighed and dissolved in ultrapure water, 1mL of 80mg / mL NaOH solution is added under the condition of ice bath, a needle is pushed into the reaction bottle, and stirring is performed; 0.4g (2mmol) of p-nitrophenyl chloroformate (NPC) is dissolved in 10mL of dichloromethane, and then slowly added into the reaction bottle; after the addition is completed, the system is moved to room temperature, and the reaction is continued for 1h; then the reaction system is slowly added into 200mL of isopropanol for precipitation, washed with isopropanol three times, centrifuged at 5000rpm for 5min, and the precipitate is collected and vacuum dried to obtain the hydroxyethyl starch (HES-NPC) modified by the NPC, and the specific structure of the part modified by the NPC is shown in formula II:

[0078]

[0079] (2) 500mg of HES-NPC obtained in the above step is weighed and placed in a reaction bottle, 5mL of dimethyl sulfoxide is dissolved, 80mg of 4-(2-aminoethyl) benzenesulfonamide is weighed and dissolved in dimethyl sulfoxide, 30μL of triethylamine is added, the reaction is carried out for 3h, then 500μL of hydrazine hydrate is added, stirring is carried out at room temperature for 3h, then the reaction system is slowly added into a system of 100mL of isopropanol+100mL of petroleum ether, precipitated and separated by centrifugation, the supernatant is discarded, the precipitate is washed with petroleum ether three times, collected and vacuum dried to obtain the target product, i.e., the carbonic anhydrase inhibitor-hydroxyalkyl starch macromolecule conjugate CAi-HES-NH-NH2 modified by hydrazine hydrate, and the specific structure of the part modified by hydrazine hydrate is shown in formula III:

[0080]

[0081]

[0082] (3) Take 200 mg of CAi-HES-NH-NH2 obtained in the previous step and place it in a reaction bottle, dissolve it in 3 mL of N,N-dimethylformamide (DMF), add 30 μL of triethylamine, add 2 drops of acetic acid, take 40 mg of a chemotherapeutic drug, doxorubicin hydrochloride, dissolve it in 1 mL of DMF solution, add 10 μL of triethylamine to the doxorubicin solution, and then slowly add it dropwise to the CAi-HES-NH-NH2 solution after ultrasonic dissolution. The system is protected by N2, and is stirred at room temperature for 36 h. After the reaction is completed, the reaction system is collected and dialyzed in ultrapure water for three days using a 3500 Da dialysis bag. The target product is obtained by freeze-drying, and the partial structure of the product is a carbonic anhydrase inhibitor-hydroxyalkyl starch-chemotherapeutic drug doxorubicin conjugate CAi-HES-hyd-DOX (CHHD), wherein the specific structural formula of the part modified by doxorubicin is shown in Formula Four. It should be noted that the carbonic anhydrase inhibitor and the doxorubicin branched chain do not necessarily appear in the same structural unit; in this embodiment, they are drawn in the same chemical formula only to illustrate the reaction principle:

[0083]

[0084] (4) Preparation and characterization of metal ion-doped carbonic anhydrase inhibitor-hydroxyalkyl starch-chemotherapeutic drug conjugate complex, including the following steps:

[0085] Dissolve CAi-HES-Hyd-DOX (100 mg) in 10 mL of ultrapure water, add 2 mL of dichloromethane, and perform ultrasonic treatment under ice bath conditions. Remove most of the organic solvent by rotary evaporation, and then inject CuCl2 aqueous solution (50 mM, 2 mL) into the reaction system at a flow rate of 1 mL / h under magnetic stirring. When the CuCl2 solution is added, the color of the solution gradually changes from orange red to purple. After stirring in the dark for 12 h, the copper-doped carbonic anhydrase inhibitor-hydroxyalkyl starch-chemotherapeutic drug conjugate complex nanoparticles (CHHD-Cu) are purified by dialysis using a 300 KDa dialysis bag, and then freeze-dried for subsequent use. The structure is characterized in that the phenolic hydroxyl and / or carbonyl group on the anthracycline of doxorubicin in Formula Four forms a coordination compound with copper ions, and the coordination form is shown in Formula Five:

[0086]

[0087] Comparative Example 1

[0088] Weigh 500 mg of HES-NPC obtained in step (I) of Example 1 and place it in a reaction flask. Dissolve it in 5 mL of dimethyl sulfoxide. Add 500 μL of hydrazine hydrate and stir at room temperature for 3 h. Slowly add the reaction system to a 100 mL isopropanol + 100 mL petroleum ether system. Precipitate will precipitate. Centrifuge, discard the supernatant, wash three times with petroleum ether, collect the precipitate and vacuum dry to obtain the target product, hydrazine hydrate modified hydroxyalkyl starch macromolecular conjugate HES-NH-NH2. Weigh 200 mg of HES-NH-NH2 obtained in the previous step and place it in a reaction flask. Dissolve it in 3 mL of N,N-dimethylformamide (DMF). Add 30 μL of triethylamine and 2 drops of acetic acid. Weigh 40 mg of the chemotherapy drug doxorubicin hydrochloride and dissolve it in 1 mL of N,N-dimethylformamide solution. Add 10 μL of triethylamine to the doxorubicin solution, sonicate to dissolve, and then slowly add it to the CAi-HES-NH-NH2 solution. The system is protected with N2 and stirred at room temperature for 36 h. After the reaction was completed, the reaction system was collected and dialyzed in ultrapure water for three days using a 3500 Da dialysis bag. The product, hydroxyalkyl starch-doxorubicin conjugate HES-hyd-DOX (HHD), was obtained by lyophilization.

[0089] HES-Hyd-DOX (100 mg) was dissolved in 10 mL of ultrapure water, and 2 mL of dichloromethane was added. The mixture was sonicated under ice bath conditions, and most of the organic solvent was removed by rotary evaporation. Subsequently, CuCl2 aqueous solution (50 mM, 2 mL) was injected into the reaction system at a flow rate of 1 mL / h under magnetic stirring. Upon addition of CuCl2 solution, the solution color gradually changed from orange-red to purple. After stirring for 12 h in the dark, the copper-doped carbonic anhydrase inhibitor-hydroxyalkyl starch-chemotherapeutic drug conjugate nanoparticles (HHD-Cu) were purified by dialysis using a 300 kDa dialysis bag, and then lyophilized for further use. Validation Example 1: Detection of HHD-Cu nanoparticle size and potential.

[0090] The particle size and potential of the prepared CHHD-Cu nanoparticles were detected using dynamic light scattering (DLS). The particle size of CHHD-Cu was approximately 170 nm, and the zeta potential was approximately 10 mV, which is about 5 mV lower than that of CHHD. Figure 1 and Figure 2 As shown, CHHD-Cu particles observed under a transmission electron microscope (TEM) all exhibit a compact spherical morphology, with a size of approximately 170 nm, consistent with the DLS detection results. Figure 3 As shown. Measurements of the copper content in CHHD-Cu using ICP-OES showed a molar ratio of approximately 1:1 with the chemotherapy drug doxorubicin.

[0091] Verification Example 2 Detection of the chemical kinetic performance of CHHD-Cu nanoparticles

[0092] Detection of the chemical kinetic performance of CHHD-Cu nanoparticles under different pH buffer conditions: methylene blue (MB) colorimetric method was used to detect hydroxyl radicals (·OH). Specifically, CHHD-Cu (Cu concentration of 1 mM) was mixed with GSH (1 mM) in 890 μL of buffer solution (pH 7.4, pH 6.5, pH 5). The mixture was shaken at 37°C for 2 h. Then, MB (10 μL, 1 mg / mL) was added to the mixed solution, and the absorption spectrum of the mixed solution was tested after incubation at 37°C for 5 h. The decrease in absorbance at 660 nm reflects the amount of ·OH produced.

[0093] Figure 4 The content is the ability of CHHD-Cu to produce hydroxyl radicals under different pH conditions. From the figure, it can be seen that the ability of CHHD-Cu to produce hydroxyl radicals is stronger as the pH decreases.

[0094] Detection of the cytotoxicity of CHHD-Cu nanoparticles: H22 cells were seeded in a 96-well plate at 5 × 10 3 cells per well and incubated at 37°C, 5% CO2 for 24 h. Then, the H22 cells were co-incubated with different concentrations of CHHD-Cu, HHD-Cu at 37°C for 48 h. The relative cell viability of different groups was determined by the standard CCK8 method.

[0095] Figure 5 The content is the effect of CHHD-Cu and HHD-Cu on the viability of H22 cells, which shows that CHHD-Cu can better inhibit the viability of H22 cells than HHD-Cu, with IC50 values of 1.12 μg / mL (CHHD-Cu) and 2.04 μg / mL (HHD-Cu), respectively.

[0096] Verification Example 4 Detection of the ability of CHHD-Cu nanoparticles to inhibit tumor-related fibroblast protein carbonic anhydrase 9

[0097] HHD-Cu and CHHD-Cu were used to treat hypoxic tumor-related fibroblasts for a period of time. After 24 hours, the cells were collected and lysed, and then the lysate was collected for protein analysis. Equal amounts of protein were determined by SDS polyacrylamide gel electrophoresis. Actin was detected with an anti-actin antibody as a control.

[0098] Figure 6The content is the protein expression content atlas of each group. From the figure, it can be seen that the expression of carbonic anhydrase in the HHD-Cu and CHHD-Cu groups is decreased compared with the blank control, and the expression of carbonic anhydrase 9 in the CHHD-Cu group is the lowest, indicating that the coupling of Cai can inhibit the expression of carbonic anhydrase 9 by tumor-related fibroblasts.

[0099] Example 5: Detection of the ability of CHHD-Cu nanoparticles to reduce intracellular pH of cells

[0100] BCECF-AM is a fluorescent dye that can penetrate the cell membrane. BCECF AM has no fluorescence, and after entering the cell, it can be cut by intracellular esterase to form BCECF, thereby being retained in the cell. The pH-sensitive probe BCECF-AM, its fluorescence emission intensity will decrease with the decrease of pH value, thereby showing the change of intracellular pH value. Tumor-related fibroblasts (4 million cells per well) were inoculated into CLSM special culture dishes and allowed to adhere overnight. Then the culture dishes were moved into a hypoxic incubator for 24 hours. Cells were co-incubated with CHHD-Cu and the HHD-Cu of the comparative example at 37°C for 6h. Phosphate buffer was used to wash away the drugs that did not enter the cells, followed by the addition of phosphate buffer containing 1 μΜ BCECF-AM for 30 min, phosphate buffer was washed for 3 times, and the cells were collected. The fluorescence intensity of BCECF in each group was monitored using a confocal microscope to detect the change of intracellular pH of cells.

[0101] Figure 7 The content is the intracellular pH change diagram of each group of cells. From the figure, it can be seen that the control group of cells shows the strongest green fluorescence compared with HHD-Cu and CHHD-Cu, because the intracellular of tumor-related fibroblasts is usually weakly alkaline. The green fluorescence intensity of CHHD-Cu and HHD-Cu groups is significantly reduced, which indicates that the addition of CAi can reduce the intracellular pH of cells, and the coupling of CAi can successfully inhibit the expression of CA9, reduce the intratumoral pH, and help to improve the Cu-induced chemical kinetics of killing tumor cells.

[0102] Example 6: Detection of the ability of CHHD-Cu nanoparticles and the comparative example 1 (HHD-Cu nanoparticles) to produce ROS in cells

[0103] 2',7'-dichlorodihydrofluorescein diacetate (DCFHDA) is used, which has cell membrane permeability and no fluorescence itself. Once it enters the cell, it is hydrolyzed by cell esterase to generate 2',7'-dichlorodihydrofluorescein and further oxidized to generate strong fluorescent 2',7'-dichlorofluorescein (DCF), which is a general ROS indicator.

[0104] Experimental procedure: H22 cells were seeded in 6-well plates at 400,000 cells per well and incubated at 37°C, 5% CO2 for 24 h. Then, H22 cells were incubated with CHHD-Cu and HHD-Cu of the comparative example at 37°C for 6 h. The uncellular drug was removed by phosphate buffer washing, followed by the addition of 10 μM DCFHDA, incubation for 30 min, phosphate buffer washing for 3 times, collection of cells, and quantification of the fluorescence intensity of each group using flow cytometry.

[0105] Figure 8 Content is the ROS detection flow chart of each group, and content b is the quantification of fluorescence intensity. It can be seen from the figure that the intracellular ROS level of each group is increased after treatment, and the CHHD-Cu group has the largest increase, and the ROS production ability of the CHHD-Cu group is stronger than that of the HHD-Cu group at the same concentration, which indicates that the inhibition of carbonic anhydrase 9 helps to accumulate ROS produced by CDT.

[0106] Verification example 7: Detection of the inhibition ability of CHHD-Cu nanoparticles on H22 cell stemness markers

[0107] H22 cells (400,000 cells per well) were seeded in 6-well plates, and then the culture dishes were moved into a hypoxic incubator for 24 hours. The cells were incubated with CHHD-Cu and HHD-Cu of the comparative example at 37°C for 6 h. The uncellular drug was removed by phosphate buffer washing, the cells were collected, and the CD13 and CD133 stem cell marker antibodies were incubated with the cells for 15 min, phosphate buffer was washed for 3 times, the cells were collected, and the expression amount of stem cell markers of each group was quantified using flow cytometry.

[0108] Figure 9 Content is the expression graph of stem cell markers of each group. It can be seen from the figure that the stem cell markers of each group are decreased after treatment, and the expression amount of stem cell markers of CHHD-Cu is significantly lower than that of HHD-Cu, which indicates that the inhibition of carbonic anhydrase 9 expression helps to reduce the proportion of stem cells in tumor cells.

[0109] Verification example 8: Anti-tumor efficacy of CHHD-Cu in a mouse subcutaneous H22 tumor model

[0110] Male BALB / c mice were inoculated subcutaneously with mouse H22 cell suspension on the right back of the back, 1×10 6 3 ​Mice were randomly divided into four groups of eight each, and injected intratumorally with saline, HHD-Cu, or CHHD-Cu, respectively. Doxorubicin was administered at a dose of 6 mg / kg. The first day of administration was designated as Day 1. A second intratumoral injection at the same dose was administered on Day 10. Starting from Day 1, the long side (a) and short side (b) of the subcutaneous tumor were measured every other day using calipers. The tumor volume was calculated using the formula: V = a × b × b / 2. A tumor volume-time curve was plotted. Figure 10 The results showed that CHHD-Cu had the best tumor-suppressive effect.

[0111] Following the same steps as in Example 1 of this invention, acetazolamide (Formula 6), SLC-0111 (Formula 7), and Indisulam (Formula 8) were used to replace 4-(2-aminoethyl)benzenesulfonamide in Example 1, and CuCl2 was partially replaced with other metal chlorides to obtain the carbonic anhydrase inhibitor-hydroxyalkyl starch-chemotherapeutic drug conjugate structures of Examples 2-4, and further obtained metal ion-doped carbonic anhydrase inhibitor-hydroxyalkyl starch-chemotherapeutic drug conjugate nanoparticles.

[0112] Examples 2-4

[0113] By replacing the 4-(2-aminoethyl)benzenesulfonamide in Example 1 with the compound of formula 6, the compound of formula 7 was obtained.

[0114]

[0115] Furthermore, the phenolic hydroxyl group and carbonyl group on the anthracene ring of doxorubicin in Formula 7 form a coordination compound with copper ions.

[0116]

[0117] Example 3

[0118] By replacing 4-(2-aminoethyl)benzenesulfonamide in Example 1 with compound of formula 8 and replacing copper chloride with ferrous chloride, compound of formula 9 was obtained.

[0119]

[0120] Furthermore, the phenolic hydroxyl group and carbonyl group on the anthracene ring of compound formula nine, and the Fe... 2+ Ions form coordination compounds.

[0121]

[0122] Example 4

[0123] By replacing 4-(2-aminoethyl)benzenesulfonamide in Example 1 with compound of formula 10 and replacing copper chloride with manganese chloride, compound of formula 11 was obtained.

[0124]

[0125] and the phenolic hydroxyl group and the carbonyl group of the doxorubicin anthracycline form a coordination compound with Fe 2+ ions.

[0126]

[0127] Examples 2-4 were tested and verified to have similar effects as Example 1.

[0128] It is readily understood by those skilled in the art that the foregoing description is only a preferred embodiment of the application and is not intended to limit the application thereto. Any modifications, equivalents and improvements made during the spirit and principles of the application shall be included within the scope of the application.

Claims

1. A hydroxyalkyl starch complex, characterized in that, having a structure as shown in Formula I: I wherein HAS' is a molecule of hydroxyalkyl starch without a terminal carbohydrate moiety; R1', R2' and R3' are each independently any one of a group of Formula II, a group of Formula III, a hydrogen atom, or a C1-C8 linear / branched hydroxyalkyl group, and the sum of the proportions of the group of Formula II in R1', R2' and R3' is 10% to 40%, and the sum of the proportions of the group of Formula III in R1', R2' and R3' is 5% to 20%; wherein R4 and R5 are each independently a C1-C8 alkyl group; at least one phenolic hydroxyl group and / or carbonyl group on the entire or part of the anthracene ring of the group of Formula III is connected to a divalent metal ion in a coordination bond form; The divalent metal ion is Cu 2+ .

2. The hydroxyalkyl starch complex of claim 1, wherein, the molar ratio of the divalent metal ion to the group of Formula III is 1: (1-10).

3. The hydroxyalkyl starch complex according to any one of claims 1 or 2, characterized in that, the average molecular weight of which is 40 kDa to 480 kDa.

4. The hydroxyalkyl starch complex according to any one of claims 1 or 2, wherein the hydroxyalkyl starch complex exists in the form of nanoparticles.

5. A method for preparing the hydroxyalkyl starch complex according to any one of claims 1-4, the initial structure of the hydroxyalkyl starch complex being shown in Formula IV: IV wherein HAS is an original molecule of hydroxyalkyl starch without a terminal carbohydrate moiety, and R1, R2 and R3 are each independently any one of a hydrogen atom or a C1-C8 linear / branched hydroxyalkyl group; characterized in that, when R1, R2 and R3 are C1-C8 linear / branched hydroxyalkyl groups, a carbonic anhydrase inhibitor is modified on the hydroxyl groups of part of the hydroxyalkyl groups, and doxorubicin is modified on the hydroxyl groups of part of the hydroxyalkyl groups, to obtain a complex having a structure of Formula I; wherein the modification of the carbonic anhydrase inhibitor on the hydroxyl groups is specifically: A is ; the modification of doxorubicin on the hydroxyl groups is specifically: 。 6. Use of the hydroxyalkyl starch complex according to any one of claims 1-4 in the preparation of a medicament for treating and / or preventing tumors.

Images