CN118239999A - Preparation method and application of disulfiram prodrug based on cholesterol and diethyl Dithiocarbamate (DTC) - Google Patents
Preparation method and application of disulfiram prodrug based on cholesterol and diethyl Dithiocarbamate (DTC) Download PDFInfo
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- CN118239999A CN118239999A CN202311557010.0A CN202311557010A CN118239999A CN 118239999 A CN118239999 A CN 118239999A CN 202311557010 A CN202311557010 A CN 202311557010A CN 118239999 A CN118239999 A CN 118239999A
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Abstract
The invention discloses a diethyl dithiocarbamic acid-cholesterol conjugate which is used as a prodrug compound of a disulfiram degradation product diethyl dithiocarbamic acid (DTC), has anti-tumor activity and can also play a synergistic anti-tumor role together with conventional chemotherapeutics such as doxorubicin hydrochloride and the like. The invention also discloses a preparation method of the diethyl dithiocarbamic acid-cholesterol conjugate, and discloses a method for preparing various nano preparations by using the diethyl dithiocarbamic acid-cholesterol conjugate and application of the diethyl dithiocarbamic acid-cholesterol conjugate in anti-tumor aspect. The conjugate has drug-assisted dual-purpose property, and can be used for constructing various nanoparticle, liposome and other microparticle drug delivery systems. Taking the constructed liposome formulation as an example, the constructed doxorubicin liposome has high encapsulation efficiency and good tumor targeting property, and can obtain better effects in cell and animal experiments.
Description
Technical Field
The invention belongs to the field of new drug synthesis/pharmaceutical preparations, and discloses a preparation method of diethyl Dithiocarbamate (DTC) and cholesterol conjugate, a microparticle preparation prepared based on the compound and application of the microparticle preparation in resisting tumors.
Background
Disulfiram (Disulfiram, DSF) has been used clinically for over 70 years as an alcohol-stopping drug, etc., and has the advantages of perfect pharmacokinetics, safety, tolerance, low cost, etc. DSF, as a new typical drug for the old, has great potential in tumor treatment and prevention. After oral ingestion, more than 99% of DSF is rapidly converted to diethyldithiocarbamate (Diethyldithiocarbamate, DTC) in the acidic environment of the stomach. Both DSF and DTC are rapidly absorbed into the portal circulation through the gastrointestinal mucosa and are enriched in the liver and rapidly metabolized and degraded. DTCs are powerful metal chelates that can chelate with a variety of heavy metal ions through sulfhydryl groups. DTC chelates with copper ions to form Cu (DTC) 2 with anticancer activity, so that DTC is the main active ingredient of disulfiram for exerting antitumor effect. Compared with normal tissue cells, many tumor cells have higher copper ion concentration, and the disulfiram can form higher concentration of Cu (DTC) 2 in tumor tissues after administration, thereby specifically killing the tumor cells. However, no clinical experiments related to disulfiram have been reported to achieve a significant therapeutic effect. The drug is probably characterized by poor pharmaceutical property of disulfiram, cu (DTC) 2 and other compounds, quicker degradation and elimination after administration, insufficient targeting property or drug release for tumor tissues and tumor cells and the like. Therefore, there is a need to develop more effective compounds and dosage forms thereof around DSF or DTC, to improve effectiveness, drug formation, targeting, stability, etc., to fully exert therapeutic effects.
Disclosure of Invention
In view of the above, it is an object of the present invention to overcome the drawbacks of the prior art by synthesizing a cholesteryl-DTC conjugate, a prodrug compound of the disulfiram active metabolite DTC, which is more potent and/or pharmaceutically acceptable and/or less toxic, and to provide a viable method of preparation.
It is a further object of the present invention to provide the use of the compounds and dosage forms thereof, including as carriers for the preparation of nano-formulations and as pharmaceutical ingredients for the treatment of tumors in combination with chemotherapeutic agents.
The third objective of the present invention is to investigate the possible mechanism of anti-tumor of the compounds and to point out the feasibility of co-anti-tumor of common chemotherapeutics such as DOX.
One of the purposes of the invention is realized by adopting the following technical scheme:
Cholesterol groups are introduced into the molecular structure, and DTCs are connected with the cholesterol groups through chemical bonds which can be degraded in vivo. Thus, the novel compounds will have some advantages of cholesterol, such as increased affinity to cells, enhanced pharmaceutical properties of the nano-formulations, etc., thus achieving our design objectives.
From the standpoint of synthesis and degradation in vivo, the compound may have a basic structure of the following formula.
Wherein X is a group that is chemically inert during synthesis, including but not limited to alkyl or aryl. A is a stimulus-responsive, sensitive bond, including but not limited to, one that is responsive to pH, glutathione, reactive oxygen species, or highly expressed enzymes in the tumor microenvironment or tumor cells; the GSH response linkages include, but are not limited to, -S-S-structures; the ROS-responsive sensitive bonds include, but are not limited to, phenylboronic acid bonds, thioketal bonds, or oxalate bonds; the enzymes highly expressed by the tumor tissue include, but are not limited to, matrix metalloproteinase, furin, legumain, FAP-alpha, or cathepsin, etc.
Still further, compounds include, but are not limited to, compounds having the following structure (C-DTC):
the preparation method of the C-DTC comprises the following basic reaction processes:
the basic preparation process is that (1) diethylamine, mercaptoethanol, carbon disulfide, triethylamine and carbon tetrabromide are mixed and reacted in methylene dichloride to obtain DTC-OH.
(2) In dichloromethane, mixing DTC-OH, cholesterol ester formyl chloride and pyridine, and carrying out esterification reaction to obtain the C-DTC with the structure shown in the formula I.
In the step (1), the mol ratio of diethylamine to mercaptoethanol to carbon disulfide to triethylamine to carbon tetrabromide is 1:1:1 (1-2); the temperature of the reaction is 10-35 ℃; the reaction time is 1 to 4 hours, preferably 2 to 4 hours.
In the step (2), the molar ratio of DTC-OH, cholesterol ester formyl chloride and pyridine is 1 (1-2): 1-2; the reaction temperature is 10-35 ℃; the reaction time is 1 to 8 hours, preferably 4 to 8 hours.
The second object of the present invention is to provide a method for preparing a cholesteryl-DTC conjugate as a drug and a carrier for nano-preparation, taking C-DTC as an example. The C-DTC can be used for preparing liposome drug delivery systems by using the entrapped drug together with phospholipid materials, or preparing other types of microparticle drug delivery systems by using the entrapped drug together with other carrier auxiliary materials. Depending on the nature of the drug, each dosage form may be prepared by a method that meets the purpose of the formulation. For example, liposome may be prepared by film dispersion method, freeze drying method, reverse phase evaporation method, and ultrasonic method.
For example, the preparation of liposome drug delivery system by using C-DTC and phospholipid material together by using a film dispersion method is also in accordance with the basic preparation process, and the basic process is as follows:
When the medicine to be encapsulated is a fat-soluble medicine, the preparation process comprises the steps of placing C-DTC, phospholipid, fat-soluble medicine and the like in an eggplant-shaped bottle, adding an organic solvent for dissolution, and rotationally evaporating the organic mixed solution under reduced pressure to form a continuous and uniform film; adding the aqueous solution and uniformly dispersing to obtain the liposome drug delivery system prepared by the C-DTC and phospholipid material together and encapsulating the liposoluble drug.
When the medicine to be coated is a compound such as doxorubicin hydrochloride, an active medicine-carrying method can be utilized. For example, the ammonium sulfate gradient method is adopted to prepare the doxorubicin hydrochloride (DOX) liposome: the basic preparation process comprises placing C-DTC, phospholipid, etc. in eggplant-shaped bottle, adding organic solvent for dissolving, and rotary evaporating the organic mixed solution under reduced pressure to form continuous and uniform film; adding an ammonium sulfate aqueous solution into the obtained film, dispersing uniformly to form ammonium sulfate liposome, removing unencapsulated ammonium sulfate by means of dialysis and the like, adding DOX aqueous solution into the dialyzed liposome, and encapsulating for a period of time to obtain the DOX-entrapped C-DTC liposome drug delivery system.
For another example, the C-DTC self-assembled entrapped drug can be prepared as microparticles by emulsion solvent evaporation, which is basically prepared as follows: C-DTC is dissolved in an organic solvent to form an organic phase, and water-soluble auxiliary materials are dissolved in water to form a water phase. Mixing the water phase with an oil tank to obtain a suspension; and dispersing the suspension by adopting methods such as shearing, ultrasonic and the like, and removing the organic solvent by adopting modes such as decompression rotary evaporation and the like to obtain the C-DTC self-assembled nanoparticle preparation.
When the preparation is combined with other carrier auxiliary materials, the corresponding preparation method can be flexibly adopted according to the properties of the carrier and the medicine. For example, when the drug to be entrapped is a fat-soluble drug and the carrier auxiliary material is solid lipid, the lipid can be heated and melted, and then the drug and the C-DTC are added and mixed uniformly; when the co-adjuvant is a liquid lipid, such as for example in the preparation of an emulsion, the drug and C-DTC may be dissolved in the liquid lipid; when the contracted auxiliary material is PLGA, the medicine, C-DTC and PLAG can be jointly dissolved in an organic solution. Then mixing the mixture with water phase, dispersing uniformly, removing organic solvent,
Wherein the phospholipid material comprises a mixture of one or more of various types of natural phospholipids, semisynthetic phospholipids and fully synthetic phospholipids.
The other carrier auxiliary materials are common particle preparation carrier auxiliary materials such as PLGA, mPEG-PLGA, fatty acid monoglyceride, fatty acid diglyceride, fatty acid triglyceride, fatty acid and the like, and also comprise one or a mixture of auxiliary materials modified by PEG and/or various targeting groups with long circulation effect, and auxiliary materials such as ionizable lipid, cationic lipid and the like. Besides the usual support materials mentioned, inorganic supports or metal supports can also be prepared by corresponding preparation methods.
The water-soluble auxiliary materials comprise auxiliary materials such as PVA, PEG, poloxamer 188, poloxamer 407, tween 80, vc or SDS which meet the requirements of the administration route or auxiliary materials which increase the stability, regulate the osmotic pressure and the like. The formulation may be freeze-dried to prolong stability.
The prepared nano preparation can be added with PEG and other modified long-circulation modification materials, hyaluronic acid and other medicinal targeting materials, and can ionize lipid so as to achieve the purposes of reducing toxic and side effects and enhancing curative effect.
The microparticle preparation, also called microparticle drug delivery system (microparticle drug DELIVERY SYSTEM, MDDS), refers to a drug or a solid, liquid, semisolid or gaseous drug preparation composed of microparticles with a certain particle size (micro-scale or nano-scale) prepared by a certain dispersion embedding technology with a proper carrier (generally biodegradable material), and has the effects of covering bad smell and taste of the drug, solidifying the liquid drug, reducing compatibility change of the compound drug, improving solubility of the insoluble drug, improving bioavailability of the drug, improving stability of the drug, reducing adverse reaction of the drug, delaying release of the drug, improving targeting of the drug and the like.
The preferred microparticle delivery system of the present invention comprises primarily one or a mixture of several of nanoparticles, microparticles, liposomes, emulsions or polymeric micelles.
The third objective of the present invention is to investigate the possible mechanism of C-DTC anti-tumor and to point out the feasibility of co-ordinating anti-tumor with common chemotherapeutics such as DOX. Because of the excellent auxiliary material function of the nano preparation of the C-DTC, the C-DTC is used as an active ingredient for treating tumors in combination with chemotherapeutics and the like, and the applicable dosage forms are very wide. In addition, the anti-tumor agent can also cooperate with different types of anti-tumor drugs from the aspect of anti-tumor mechanism to play a more effective anti-tumor role, and is used for treating tumors of the types of breast cancer, gastric cancer, glioblastoma, liver cancer, pancreatic cancer, prostate cancer and the like.
In theory, the compounds of the present invention can exert not only tumor therapeutic effects but also other pharmacological effects related to DTC. For example, disulfiram has been shown to have anti-radiation, anti-fibrosis, anti-inflammatory, aortic protection, etc., and is inevitably first degraded to DTC in vivo, and thus the compounds of the present patent invention may also have similar therapeutic effects.
In addition to the conjugate itself, the pharmaceutically acceptable metal complex, chelate, deuterate or prodrug thereof should also have an antitumor effect. Especially the chelate with copper ions has stronger anti-tumor effect.
In summary, to overcome the limitations of disulfiram and DTC, the present invention provides a novel DTC prodrug, exemplified by C-DTC, having the following advantages:
Advantage 1: compared with disulfiram, the C-DTC antitumor activity is obviously improved.
Advantage 2: the C-DTC can improve the anti-tumor effect of the anti-tumor medicament from various aspects, including but not limited to inhibiting the clone formation of tumor cells, inhibiting the migration capacity of the tumor cells, promoting the apoptosis of the tumor cells, inhibiting the stem cells of the tumor and the like.
Advantage 3: the addition of the C-DTC can obviously improve the uptake rate of the tumor cells to the nano preparation.
Advantage 4: after intravenous administration, the aggregation and retention of the C-DTC-containing nano-preparation in tumor tissues can be obviously improved.
The advantages are 5:C-DTC has better drug property than disulfiram and DTC related preparations. The C-DTC has the characteristic of dual purposes of medicine assistance, can be self-assembled into nano particles, can carry medicine, and can be prepared into nano preparations such as liposome together with other auxiliary materials.
Advantage 6: C-DTC can completely or partially replace cholesterol, and can form vesicle structure with phospholipid for preparing liposome. Thus, C-DTC also has some of the advantages of cholesterol.
Drawings
FIG. 1 is a 1 H-NMR spectrum of a C-DTC according to the invention.
FIG. 2 is a 13 C-NMR spectrum of the C-DTC of the invention.
FIG. 3 is a transmission electron microscope image of example 3 according to the present invention.
FIG. 4 is a graph showing cumulative release of doxorubicin hydrochloride at various GSH concentrations for example 3 according to the invention.
FIG. 5 is a graph of experimental results of the clonogenic capacity of 4T1 cells by different formulations.
FIG. 6 is a statistical plot of cell mobility after scratch experiments on 4T1 cells for different formulations.
FIG. 7 is a graph of 4T1 cell uptake for different DOX formulations.
FIG. 8 is a bar graph of total apoptosis rate of 4T1 cells for different formulations.
Figure 9 is a graph showing the results of WB experiments for different formulations.
Fig. 10 is a graph showing tumor appearance of different formulations to H22 tumor-bearing mice after intravenous administration.
Fig. 11 is a graph showing the change of body weight of H22 tumor-bearing mice over time for different formulations after intravenous administration.
FIG. 12 is a photograph showing in vivo imaging experiments of tumor-bearing mice after intravenous administration of different formulations for 2-48 hours.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1C Synthesis of DTC
1.095G of diethylamine (15.0 mmol) and 1.17g of mercaptoethanol (15.0 mmol) were weighed into a one-necked flask, 20.0mL of anhydrous methylene chloride was added, the mixture was cooled in an ice bath, 1.14g of carbon disulfide (15.0 mmol) was added dropwise to the one-necked flask, and then 1.515g of triethylamine (16.5 mmol) was slowly added. After 5min, a solution of carbon tetrabromide in 9.94g (15.0 mmol) of anhydrous methylene chloride was added thereto, and the reaction was stirred at room temperature for 2 hours. After the reaction, the mixture was washed 3 times with water, added with an appropriate amount of anhydrous sodium sulfate, and dried overnight. And (3) carrying out suction filtration on the dried reaction liquid, placing the eggplant-shaped bottle filled with the filtrate on a rotary evaporator, and evaporating the organic solvent under reduced pressure. Petroleum ether/ethyl acetate is used as an eluent, silica gel column chromatography is used for purification, and the solvent is distilled off under reduced pressure to obtain a brown yellow oily product which is named as DTC-OH.
4.49G of cholesterol ester formyl chloride (10 mmol) was weighed into a one-necked flask, 10.0mL of anhydrous methylene chloride was added, the solution was cooled with an ice bath, and then 3.58g of DTC-OH (15 mmol) and 2mL of pyridine were added. The mixture was stirred at room temperature for 4h. After the reaction, the mixture was washed 3 times with a saturated sodium chloride solution, and then dried overnight by adding an appropriate amount of anhydrous sodium sulfate. And (3) carrying out suction filtration on the dried reaction liquid, placing the eggplant-shaped bottle filled with the filtrate on a rotary evaporator, and evaporating the organic solvent under reduced pressure. Petroleum ether/ethyl acetate is used as an eluent, silica gel column chromatography is carried out, the solvent is distilled off under reduced pressure, and a white solid is obtained, the yield is about 53%, and the product is named as C-DTC. 1 H-NMR and 13 C-NMR are shown in FIGS. 1 and 2, respectively.
1 The assignment of the H-NMR peaks is ：1H NMR(400MHz,Acetone-d6)δ5.42(dt,J＝5.0,2.0Hz,1H),4.46 -4.37(m,1H),4.34(t,J＝6.4Hz,2H),4.07(m,2H),3.95(m,2H),3.12(t,J＝6.3Hz,2H),2.47 -2.29(m,2H),2.06-1.96(m,5H),1.96-1.82(m,3H),1.68-1.20(m,13H),1.20-1.07(m,4H),1.06(s,3H),0.96(d,J＝6.5Hz,3H),0.88(dd,J＝6.6,2.1Hz,6H),0.73(s,3H).
According to the carbon spectrum, analysis is carried out on the chemical shift of carbon atoms in the synthetic structure, delta 194.03 is C37, delta 154.12 is C19, delta 139.57 is C4, delta 122.56 is C7, delta 64.92 is C41, and the chemical shift of the rest carbon can be found in the carbon spectrum of the product.
EXAMPLE 2C preparation of DTC liposomes
Precisely weighing 40mg of egg yolk lecithin and 10mg of C-DTC, placing into a eggplant-shaped bottle, adding 2mL of dichloromethane and 1mL of methanol to dissolve the egg yolk lecithin and the C-DTC completely, and removing the organic solvent (50 ℃) by rotary evaporation under reduced pressure until a layer of uniform and continuous film is formed on the bottle wall. 4mL of PBS solution is added into the mixture, the mixture is hydrated by water bath ultrasonic at 40 ℃ to form uniform milky crude liposome, an ultrasonic cell grinder (5 min, work for 3s, intermittent 4s and power 300W) is used for further dispersion, and then 100nm of membrane is passed, so that the C-DTC liposome, abbreviated as C-DTC@Lips, is obtained, and the average particle size of the C-DTC liposome is 73.3nm.
EXAMPLE 3 preparation of doxorubicin hydrochloride C-DTC liposomes
40Mg of egg yolk lecithin and 10mg of C-DTC were precisely weighed into a eggplant-shaped bottle, and then 2mL of methylene chloride and 1mL of methanol were added to dissolve them completely. The eggplant-shaped bottle is placed in a rotary evaporator, and the organic solvent is removed by decompression rotary evaporation (50 ℃) until a layer of uniform and continuous film is formed on the bottle wall. Then the eggplant-shaped bottle is taken down, 4mL of ammonium sulfate solution (250 mmoL) is added, and the mixture is subjected to water bath ultrasonic treatment at the temperature of 40 ℃ to form uniform milky crude liposome. Then, the mixture was dispersed by using an ultrasonic cell mill for 5 minutes (3 s for operation, 4s for intermittent operation, and 300W for power), and then was passed through a 100nm membrane. To remove the ammonium sulphate solution outside the liposomes, the prepared liposomes were transferred to dialysis bags (8000-14000 Da) and placed in a beaker containing 1L of ultra pure water, stirred at constant speed, changed water every 1h, dialyzed for 4h. Finally, 2mg DOX solution (dissolved in 200. Mu.L of ultra pure water) was added to the dialysis bag and the solution was loaded for 2 hours. In order to remove unencapsulated DOX, the liposome after drug loading is transferred to another dialysis bag (3000 Da) and placed in a beaker filled with 1L of ultrapure water, the mixture is stirred at constant speed, water is changed every 1h, and dialysis is carried out for 4h, so that a delivery system of the C-DTC liposome loaded hydrophilic doxorubicin hydrochloride, abbreviated as DOX@C-DTC Lips (figure 3), and the average particle size is 70.18nm.
EXAMPLE 4 preparation of paclitaxel-loaded C-DTC liposomes
40Mg of egg yolk lecithin, 8mg of C-DTC and 1mg of taxol were placed in a eggplant-shaped bottle, and then 3mL of methylene chloride and 1mL of methanol were added to dissolve them completely. The eggplant-shaped bottle is placed in a rotary evaporator, the rotary evaporator is used for removing the organic solvent by decompression rotary evaporation in a water bath at 50 ℃ to form a layer of uniform lipid film. Adding 4ml PBS solution into eggplant-shaped bottle, performing ultrasonic treatment in water bath at 40deg.C to hydrate to obtain uniform cream yellow coarse liposome, performing ultrasonic dispersion for 5min (working 3s, intermittent and power 360W), and passing through 100nm membrane to obtain paclitaxel-loaded C-DTC liposome preparation with average particle diameter of 166.3nm.
Example 5 preparation of C-DTC nanoparticles
10Mg of C-DTC is placed in an eggplant-shaped bottle, 1.5mL of dichloromethane and 0.5mL of ethanol are added, then 4mL of F68 aqueous solution (2%) solution is added, ultra-dispersion is detected for 5min (working for 3s, intermittent for 5s and power is 360W), and the organic solvent is removed by decompression rotary evaporation, so that the C-DTC nanoparticle preparation with the average particle size of 50.23nm is obtained.
EXAMPLE 6 preparation of paclitaxel-loaded C-DTC nanoparticles
5Mg of taxol is dissolved in 1ml of dichloromethane to prepare 5mg/ml taxol mother liquor, 4mg of C-DTC is taken, 100 μl taxol mother liquor is removed, and 650 μl dichloromethane and 250 μl ethanol are added. Then adding 2ml of PVA aqueous solution (3%), ultra-dispersing for 5min (working for 3s, intermittent for 5s and power for 300W), and evaporating the organic solvent in a water bath at 50 ℃ under reduced pressure by using a rotary evaporator to obtain taxol-carrying C-DTC nanoparticles with average particle size of 168.2nm.
EXAMPLE 7 preparation of docetaxel-loaded C-CDT nanoparticles
Docetaxel was taken out in a solution of 5mg in 1ml of methylene chloride to prepare a docetaxel mother liquor of 5mg/ml, 4mg of C-DTC was taken out, 100. Mu.l of docetaxel mother liquor was removed, and 00. Mu.l of methylene chloride and 200. Mu.l of ethanol were added. Then adding 2ml of PVA aqueous solution (3%), detecting for more than 5min (working for 3s, intermittent for 5s and power for 300W), and evaporating the organic solvent in a water bath at 50 ℃ under reduced pressure by using a rotary evaporator to obtain the docetaxel-entrapped C-DTC nanoparticles, wherein the average particle size is 127.1nm.
Example 8 preparation of C-DTC micelle
8Mg of mPEG2K-PLGA50/50 18k,6mg of C-DTC were weighed into an EP tube and 800. Mu.l of methylene chloride and 200. Mu.l of absolute ethanol were added. Then 3ml of PVA aqueous solution (1.5%) was added and the mixture was subjected to a supercritical fluid for 10 minutes (working for 3s, intermittent for 5s, power 210W), the organic solvent was distilled off under reduced pressure in a water bath at 50℃by a rotary evaporator, and 100nm of the film was passed, to obtain C-DTC micelles having an average particle diameter of 121.5nm.
EXAMPLE 9 preparation of C-DTC Co-carried paclitaxel micelle
5Mg of paclitaxel is weighed and dissolved in 1ml of dichloromethane to prepare 5mg/ml of paclitaxel mother liquor. 8mg of MPEG2K-PLGA50/50 18k,6mg of C-DTC were weighed into an EP tube, 150. Mu.l of paclitaxel mother liquor was removed, and 650. Mu.l of dichloromethane and 200. Mu.l of absolute ethanol were added. Then adding 3mlPVA aqueous solution (1.5%), detecting for more than 10min (working for 3s, intermittent for 5s, power 210W), and evaporating the organic solvent in water bath at 50deg.C under reduced pressure by rotary evaporator to obtain C-DTC loaded paclitaxel micelle with average particle diameter of 128nm.
EXAMPLE 10 preparation of Hyaluronic Acid (HA) modified Lipid Nanoparticles (LNP)
4Ml of C-DTC liposome was prepared as in example 2, and then 0.4ml of aqueous solution containing 4% hyaluronic acid was added and mixed uniformly to obtain a final preparation having an average particle size of 80.5nm.
EXAMPLE 11C preparation of solid lipid nanoparticles of DTC
100Mg of glyceryl monostearate, 5mg of C-DTC and 100mg of F68 are weighed, heated and melted, and uniformly mixed. To the mixture was added 5ml of ultrapure water. Dispersing in 80deg.C constant temperature water bath for more than 10min (working for 3s and intermittent for 5s at power of 210W), and standing at room temperature to obtain C-DTC solid lipid nanoparticle preparation with average particle diameter of 71.06nm.
EXAMPLE 12C preparation of DTC-Co-carried paclitaxel solid lipid nanoparticle
100Mg of glyceryl monostearate, 5mg of C-DTC, 100mg of F68 and 1mg of taxol are weighed, heated and melted, uniformly mixed and 5ml of ultrapure water are added to the mixture. The solid-liquid mixture is subjected to ultra-dispersion in a water bath at 80 ℃ for 10min (working for 3s, intermittent for 5s and power for 210W), and is placed at room temperature to obtain the taxol-entrapped C-DTC solid lipid nanoparticle, wherein the average particle size of the taxol-entrapped C-DTC solid lipid nanoparticle is 56.04nm.
The above representative examples are selected below and related experiments are performed to illustrate the characteristics and advantages of C-DTC and its formulations. Grouping, formulation, conclusion making, etc. are all arranged and analyzed according to the relevant basic principles of scientific experimentation, and therefore reference is made to basic principles if specific details in the experiments describe inattention.
Experimental example 1 Transmission Electron microscopy the appearance of example 3
The appearance of example 3 was observed using a JEOL 100CX type II electron microscope. Briefly, DOX@C-DTC Lips of example 3 was diluted ten times with ultrapure water and then dropped onto a copper mesh placed on a filter paper. After 10 minutes, 10 μl of 3% uranium acetate stain was added. After 1.5 minutes, the excess staining solution on the copper mesh was removed using filter paper. The sample was allowed to stand at room temperature for 10 minutes and then photographed under an electron microscope. The preparation form is shown in figure 3. The preparation accords with the electron microscope characteristics of the liposome.
Experimental example 2 reduction-responsive drug release behavior experiment of example 3
The release of DOX in example 3 was determined by fluorescence spectrophotometry in solutions released at different Glutathione (GSH) concentrations. The basic experimental method comprises the following steps: example 3 was placed in dialysis bags (mw=3000 Da), placed in PBS solutions with GSH concentrations of 0,2 and 10mM, ph=7.4, and dialyzed continuously for 48 hours, samples were taken at different times, the DOX content in the samples was measured using a fluorescence spectrophotometer, and finally, the DOX standard curve was substituted, and the release amounts of each group were calculated.
The in vitro release test results of DOX in example 3 are shown in FIG. 4, in which the release amount of example 3 and GSH concentration show a remarkable dependence, and the higher the GSH content, the faster the DOX release and the higher the total release amount. This experiment demonstrates that example 3, which has a composition sensitive to GSH, should be such that after the liposome encounters GSH, disulfide bonds of C-DTCs in the liposome membrane gradually break, resulting in DTCs and cholesterol derivatives, which disrupt the bilayer structure of the liposome, and the greater the GSH content in the release solution, the faster the DOX release. Thus, this experiment demonstrates that example 3 has a reductive responsive drug release profile, while also laterally speaking the liposomes can release DTCs rapidly in the presence of GSH.
Experimental example 3 cytotoxicity experiment (MTT experiment)
In order to examine the effect of various drugs and formulations on tumor cell activity, MTT experiments were performed. The basic process is as follows: 4T1 cells in the logarithmic growth phase were digested in a petri dish, centrifuged, counted and diluted to an appropriate concentration. Taking 96-well plates, inoculating 4X 10 3 4T1 cells in each well, adding a complete culture medium, culturing for 12 hours, sucking out the culture medium in the holes when the cell adherence state is normal, diluting each group of medicines to a proper concentration by using the complete culture medium, adding 200 mu L of each well into the 96-well plates, and culturing for 24 hours or 48 hours in a culture box under proper conditions again. After incubation, adding 5mg/mL MTT solution under the dark condition, continuously culturing for 4 hours, taking out the 96-well plate, gently sucking the liquid in the well by using a syringe, avoiding sucking the formazan crystal at the bottom, finally adding 200 mu L of DMSO, placing the 96-well plate on an oscillator, vibrating for 15 minutes in dark condition to enable the formazan crystal to be fully dissolved, and setting the absorbance value (OD value) of each well when the detection wavelength of an enzyme-labeled instrument is 490 nm. From the experimental results, cell viability and IC50 of each group of 4T1 breast cancer cells were calculated.
The subjects examined included A) disulfiram solution, B) C-DTC solution, C) example 5 (C-DTC nanoparticles), D) example 2 (C-DTC liposomes), E) example 3 (DOX-entrapped C-DTC liposomes), F) example 5+copper gluconate solution (where the molar ratio of C-DTC to copper ions was 2: 1) G) copper gluconate solution.
The results of 24h IC50 showed that the IC50 values for the amount concentration of total active species in A, B, C, E, F, G were 0.186. Mu. Mol.L -1, respectively (1 molecule disulfiram can be decomposed into 2 molecules of DTC, so that the concentrations of DTC are 0.37μmol·L-1)、0.155μmol·L-1、36.1nmol·L-1、3.50nmol·L-1、1.72nmol·L-1、0.64nmol·L-1 and 122.5 nmol.L -1, and the average values are significantly different from each other.
The above results demonstrate that the cytotoxicity of the synthesized product C-DTC is significantly higher than disulfiram. The C-DTC is prepared into nano particles, liposome or combined with copper ions, so that the activity of the compound on tumor cells can be obviously improved.
In addition, in order to determine the optimal combination ratio of C-DTC and DOX, the content of DOX was changed in the same manner as in example 3, except that the content was not changed, and C-DTC was prepared in the same manner: the DOX ratio is 5:1 and 8:1, DOX-loaded C-DTC liposomes. Examine whether DOX and C-DTC have anti-tumor synergistic effect, wherein 5:1 is example 3. We examined that the mass ratio of C-DTC to DOX in C-DTC@DOX Lip is respectively as follows: 1. 8:1 were combined on 4T1 cells for 48h and cell viability and CI values were calculated at each ratio. When C-DTC: dox=5: 1 or 8: at 1, all CI values were less than 1, exhibiting significant synergy. Thus, C-DTC: DOX has remarkable anti-tumor synergistic effect under the proper proportion.
Experimental example 4 cloning experiments
4T1 cells in the logarithmic growth phase were digested in a petri dish, centrifuged, counted and diluted to an appropriate concentration. Taking a 6-hole plate, inoculating 1X 10 3 4T1 cells in each hole, adding a complete culture medium, shaking the cells in the 6-hole plate uniformly, culturing for 48 hours, observing the cell adhesion and dispersing uniformly, sucking out the culture medium in the holes, diluting each group of medicines to a proper concentration by using the complete culture medium, adding 2mL of each hole, replacing the medicine-containing culture medium every 2 days, and culturing until obvious cell aggregation is formed in a control hole, thereby stopping the experiment. Then, sucking out the culture medium in the holes, adding 1mL of 4% cell tissue fixing solution into each hole, fixing for 15min, and washing off residual cell tissue fixing solution by using PBS; next, 1mL of 0.1% crystal violet solution was added to each well, and after 10min of staining, the residual crystal violet was washed off with PBS, and the colony formation per well was recorded and photographed.
Experimental grouping: a) DOX liposome group, B) DSF liposome group, C) example 2 (C-DTC liposome group), D) DSF liposome+dox liposome, E) example 2+dox liposome group, F) example 3, and saline Control group, each group was provided with 3 duplicate wells. Wherein DOX liposome and DSF liposome were prepared in the same manner as in example 4, C-DTC was replaced with cholesterol, and the drug was loaded. The preparation method similar to that of the experimental example is adopted in the rest experimental example when the preparation is related to the preparation. DOX concentrations were 0.5ng/mL for each group, and DSF and C-DTC concentrations were 2.5ng/mL.
The results of the cloning experiments are shown in FIG. 5. Compared with a blank control group, the C-DTC group has a certain inhibition effect on clone formation, while the C-DTC Lip and DOX Lip combined group and the C-DTC@DOX Lip group have almost no clone formation, and the result also shows that the inhibition capability of the C-DTC and DOX combined group on cell proliferation is superior to that of the DSF and DOX combined group.
Experimental example 5 cell scratch experiment
4T1 cells in the logarithmic growth phase were taken, digested from a petri dish, centrifuged, counted and diluted to a proper concentration. Taking a 6-hole plate, reversely placing the hole plate, transversely scribing the hole plate by using a marker pen, enabling each hole to pass through 5 straight lines, inoculating 5X 10 5 4T1 cells in each hole, adding a complete culture medium, shaking the cells in the 6-hole plate uniformly, culturing for 12 hours, sucking out the culture medium in the hole when the cell density reaches 80%, vertically scribing by using a 200 mu L gun head, scraping the cells in the hole, and forming cell scratches vertically crossing the bottom scribing. Next, the cells were gently washed 3 times with PBS, the scraped cells were washed away, and each group of drugs was diluted to an appropriate concentration with serum-free medium, and then added to a 6-well plate, and 2mL of each well was used for further culture. Scratch healing was recorded by microscopic observation at 0, 24, 48h, respectively. Experimental grouping: a) DOX liposome group, B) DSF liposome group, C) example 2 (C-DTC liposome group), D) DSF liposome+dox liposome, E) example 2+dox liposome group, F) example 3, and saline Control group, each group was provided with 3 duplicate wells. DOX concentrations were 0.05. Mu.g/mL for each group, and DSF and C-DTC concentrations were 0.25. Mu.g/mL.
A statistical graph of the scratch results is shown in fig. 6. As can be seen from the figure, the cell mobilities for 24h were, in order from high to low, control, A, B, C, D, E and F, with very significant differences (p less than 0.0001) between any of the dosing groups compared to the saline group, no significant differences between the A, B, D groups, no significant differences between the C, E, F groups, any of A, B, D groups compared to any of C, E, F groups (p at least less than 0.01). The overall trend for 48h was substantially consistent with 24 h. Thus, C-DTCs have a significant ability to inhibit migration, in combination with DOX, the inhibition of which is synergistic.
Experimental example 6 cell uptake experiment
Taking 4T1 cells in logarithmic growth phase, digesting and centrifuging the 4T1 cells from a culture dish, diluting the culture dish to a proper concentration after counting, taking a 6-pore plate, dripping one drop of PBS at the center of each pore, then putting a cell climbing sheet to enable the cell climbing sheet to cling to the bottom of the 6-pore plate, inoculating 1X 10 5 4T1 cells in each pore, adding a complete culture medium, shaking the cells in the 6-pore plate uniformly, culturing for 12 hours, and sucking out the culture medium in the pore when observing cell adhesion and uniform dispersion. Then, diluting each group of medicines to a proper concentration by using a complete culture medium, and adding the medicines into a 6-hole plate, wherein each hole is 2mL; after incubation for 1,2 and 4 hours respectively, sucking out the culture medium containing the medicine, and rinsing with PBS for 3 times; 1mL of 4% cell tissue fixing solution is added, after fixing for 15min, the residual cell tissue fixing solution is washed off by PBS; then, 1mL of DAPI solution is added, and after dyeing is carried out for 10min, the residual dyeing liquid is washed by PBS; and finally, dripping a drop of anti-fluorescence quenching attenuator on the glass slide, taking out the cell climbing sheet in the hole, enabling the surface with cells to cling to the glass slide, and observing the cell uptake condition of each group by using a confocal microscope.
Experimental grouping: a) DOX solution group, B) DOX liposome group, C) example 3 (C-DTC@DOX Lip). DOX concentration was 0.5. Mu.g/mL for each group, incubation time: 4h. The results are shown in FIG. 7. The fluorescence ranges of DAPI-stained nuclei and DOX overlap, indicating that DOX entering tumor cells can enter nuclei faster. From the red fluorescence intensity results of DOX, it can be seen that C-DTC has a remarkable effect of promoting tumor cells to ingest liposome, and the specific mechanism of the promotion of the uptake is yet to be studied.
Experimental example 7 Reactive Oxygen Species (ROS) detection assay
Referring to Reactive Oxygen Species (ROS) detection kit instruction, taking 4T1 cells in logarithmic growth phase, digesting and centrifuging from a culture dish, diluting to a proper concentration after counting, taking a 6-well plate, inoculating 1×10 5 4T1 cells in each well, adding a complete culture medium, shaking the cells in the 6-well plate uniformly, culturing for 12 hours, and sucking out the culture medium in the holes when observing cell attachment and uniform dispersion. Then, each group of drugs was diluted to an appropriate concentration with complete medium, added to a 6-well plate with 2mL per well, and cultured for 12 hours. After the cultivation is finished, sucking out the medicine-containing culture medium, rinsing 3 times by PBS, diluting the DCFH-DA probe by serum-free culture solution according to the proportion of 1:1000, adding 1mL of diluted DCFH-DA probe into each hole, then placing the mixture into a cell culture box for incubation for 20min, washing cells by serum-free cell culture solution for three times, fully removing DCFH-DA which does not enter the cells, and finally observing the generation condition of the ROS of the cells by a confocal microscope under the parameters of FITC and photographing. Experimental grouping: a) DOX liposome group, B) DSF liposome group, C) example 2 (C-DTC liposome group), D) DSF liposome+dox liposome, E) example 2+dox liposome group, F) example 3, and saline Control group, each group was provided with 3 duplicate wells. DOX concentrations were 0.1. Mu.g/mL for each group, and DSF and C-DTC concentrations were 0.5. Mu.g/mL.
The results show that the C, E, F groups containing C-DTCs were all significantly more fluorescent than the other groups, indicating that C-DTCs were able to significantly up-regulate ROS levels. This will be advantageous in that it exerts an anti-tumour effect.
Experimental example 8 apoptosis experiments
Referring to the instruction book of an Annexin V-FITC/PI apoptosis detection kit, taking 4T1 cells in a logarithmic growth phase, digesting and centrifuging the 4T1 cells from a culture dish, diluting the 4T1 cells to a proper concentration after counting, taking a 6-hole plate, inoculating 5X 10 4 4T1 cells in each hole, adding a complete culture medium, shaking the cells in the 6-hole plate uniformly, culturing for 12 hours, sucking out the culture medium in the holes when the cell adherence state is good, diluting each group of medicines to the proper concentration by using the complete culture medium, adding 2mL of each hole, and continuously culturing for 48 hours. After the culture is finished, collecting the culture solution, adding 1mL of pancreatin digested cells without EDTA, incubating at room temperature until the adherent cells can be detached by gentle blowing, and collecting the cells after pancreatin digestion, wherein excessive digestion of pancreatin needs to be avoided. The collected cells were washed 3 times with pre-chilled PBS, centrifuged at 800r/min for 10min, resuspended in 195. Mu.L of Annexin V-FITC conjugate solution, then gently mixed with 5. Mu.L of Annexin V-FITC, stained with 10. Mu.L of PI dye for 10min, and stained at room temperature (20-25 ℃) in the dark for 15min. Finally, the sample is placed in an ice box in a dark place and analyzed by a flow cytometer as soon as possible. Experimental grouping: a) a DOX liposome group, B) a DSF liposome group, C) example 2 (C-DTC liposome group), D) DSF liposome+dox liposome, E) example 2+dox liposome group, F) example 3, and saline Control group, each group being provided with 3 duplicate wells; DOX concentrations were 0.1. Mu.g/mL for each group, and DSF and C-DTC concentrations were 0.5. Mu.g/mL. The statistical graph of the results is shown in FIG. 8.
The apoptosis rate of the single administration group (A, B, C group) is not more than 20%, but the apoptosis degree of the two drug combination group D, E and the apoptosis degree of the F group are sequentially enhanced, and are respectively 27.5%, 34.98% and 53.0%. Therefore, the ability of the C-DTC to promote apoptosis of tumor cells is significantly enhanced after the combination of the C-DTC and DOX.
Experimental example 9 Western immunoblotting experiment (Western blot, WB)
In the experiment, 4T1 cells in logarithmic growth phase are taken, digested and centrifuged from a culture dish, counted, diluted to proper concentration, a10 cm diameter cell culture dish is taken, 2X 10 6 4T1 cells are inoculated in each dish, a complete culture medium is added, the cell culture dish cells are uniformly shaken and then put into a culture box under proper conditions for culture for 12 hours, when the cell adherence state is good, the culture medium in the hole is sucked out, each group of medicines is diluted to proper concentration by the complete culture medium and then added into the cell culture dish, and 8mL of each dish is continuously cultured for 48 hours; after incubation, cells were collected into centrifuge tubes, washed 2-3 times with PBS, 100 μl of cell lysate (RIPA cell lysate: PMSF: protease inhibitor=100:1:1, ready-to-use) was added, the tubes were gently flicked to allow the lysate and cells to come into full contact, ice-bath for 30min, after ice-bath lysis was completed, the tubes were placed in a refrigerated centrifuge, centrifuged at 10000r/min at 4 ℃ for 5min, and the supernatant was taken for use. And carrying out subsequent protein sample preparation, gel preparation, electrophoresis, membrane transfer, sealing, antibody incubation and imaging according to a standard procedure of a western blot experiment.
Experimental grouping: a) example 2 (C-DTC liposomes), B) DOX liposomes, C) example 2+dox liposomes, D) example 3 and Control incubated with complete medium. Wherein, DOX concentration is 0.1 mug/mL and C-DTC concentration is 0.5 mug/mL. In addition to the enhancement of DOX's endogenous apoptosis in 4T1 cells by C-DTC, it was simply verified at the protein level whether NF- κB pathways were inhibited by detecting whether phosphorylated P65 (P-P65) proteins were produced. The results showed that DOX liposome group had little effect on P-P65 protein, and that expression of P-P65 protein in A, C and D groups containing C-DTC was significantly increased, thereby supposing that C-DTC could also inhibit NF- κB expression and inhibit generation of P65 protein, resulting in an increase in P-P65 expression level. DOX enhances the effect of C-DTC on the protein.
From the results, it can be seen that three groups of liposomes containing the entrapped C-DTC can also inhibit the expression of the dry related factor ALDHA1, so that the C-DTC has the capacity of inhibiting the dryness of the breast cancer CSCs, and is helpful for inhibiting the growth and metastasis of tumor cells. DOX enhances the effect of C-DTC on the protein. The results are shown in FIG. 9.
Experimental example 10C-DTC Liposome hemolysis experiment
Example 2 was diluted to the appropriate concentration and mixed with an equal volume of 2% erythrocyte suspension to give final C-DTC concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.15625mg/mL and incubated at 37℃for 4h. Meanwhile, a negative control (physiological saline group) free from hemolysis and a positive control (ultrapure water group) completely hemolyzed were set. After the incubation, the tubes were placed in a centrifuge and centrifuged at 3000rpm for 10min. Finally, a 96-well plate was taken, 200. Mu.l of the supernatant was pipetted into the well, absorbance values at a wavelength of 540nm were measured with a microplate reader, and the hemolysis rate was calculated. As a result, the hemolysis rate of example 2 was lower than 5%. Thus, the lipids of C-DTC are able to meet the requirements of intravenous administration.
Experimental example 11 experiment for inhibiting tumor in H22 tumor mice in example 2 and example 5
An armpit H22 tumor model of Kunming mice (female mice, about 20 g) was established according to a conventional method, and the animals were divided into 5 groups when tumors were grown to 100mm 3 and the animals were administered in groups. The saline group was Control group, the administration group was C-DTC nanoparticle group (example 5) at an administration dose of 25mg/kg of group A, and the B, C, D group was C-DTC liposome group (example 2) at an administration dose of 12.5 mg/kg, 25mg/kg, 50mg/kg, respectively. Animals in each group were dosed 4 times every two days, 6 times at the tail vein, and animals were sacrificed one day after the last dose. The tumor appearance and the body weight change of the mice are shown in fig. 10 and 11, respectively. Wherein, the tumor weight inhibition rate of the A group nano particle group is 25%, and the tumor weight inhibition rates of the C-DTC liposome group at the administration doses of 12.5, 25 and 50mg/kg are 47%,72% and 79%, respectively. The body weight of animals in each dosing group was not significantly different from that in the normal saline group, indicating that neither the C-DTC nanoparticle group nor the liposome group had significant systemic toxicity to animals after intravenous dosing. For H22 transplanted tumors, both the C-DTC nanoparticles and the liposome group can obviously inhibit the growth of the tumors, and the liposome group with the same dosage is superior to the nanoparticle group. An inhibition of more than 70% was achieved in example 2 at a dose of 25 mg/kg.
Experimental example 12 Living body imaging experiment
First, preparing C-DTC@IR780 Lip: precisely weighing 40mg of egg yolk lecithin and 10mg of C-DTC powder in a eggplant-shaped bottle, 200 mu L of IR780 ethanol solution (the concentration is 1 mg/mL), then adding 2mL of dichloromethane and 1mL of methanol to completely dissolve the egg yolk lecithin and the C-DTC, placing the eggplant-shaped bottle on a rotary evaporator, heating to 50 ℃ in a water bath kettle, evaporating the organic solvent under reduced pressure until a layer of uniform continuous film is formed on the bottle wall, then taking down the eggplant-shaped bottle, adding 4mL of PBS solution, carrying out ultrasonic hydration at 40 ℃ to form uniform milky crude liposome, then using an ultrasonic cell grinder (5 min, working for 3s, intermittent for 4s and power 300W) to ensure that the particle size of the liposome is uniform, and finally extruding the film through 100nm high pressure to obtain the C-DTC@IR780 Lip. The C-DTC is changed into cholesterol, and the rest is the same as above, so that the IR780 Lip is prepared.
The experimental groupings were as follows: a) IR780 group, B) IR780Lip group, C) C-DTC@IR780 Lip group, 4 mice in each group, and the administration concentration of IR780 was 2mg/kg, and the administration was by tail vein injection. After the anesthetic treatment of the mice, the mice were subjected to live photographing for 2-48 hours using a live animal imager. As shown in fig. 12, the liposomes were more likely to accumulate at the tumor site and eliminated slowly without a decrease in 48h concentration compared to the IR780 solution group. The C-DTC@IR780 Lip group has faster tumor tissue targeting speed and more accumulation compared with IR780Lip, and the drug content of the C-DTC@IR780 Lip group at the lung and kidney parts is obviously lower than that of the IR780 Lip. It is demonstrated that C-DTC does not increase aggregation at non-tumor sites while improving liposomal tumor targeting, and even reduces targeting in normal lung and kidney.
Experimental example 13 encapsulation efficiency measurement of example 3
And measuring the DOX content by adopting a fluorescence spectrophotometry. Wherein DOX has an excitation wavelength of 475nm and an emission wavelength of 596nm. The method for measuring the encapsulation efficiency comprises the following steps: taking 250 mu L of example 3, fixing the volume to 5mL by using methanol, swirling for 5min and carrying out ultrasonic treatment for 20min, taking 200 mu L of demulsified filtrate, fixing the volume to 10mL by using methanol again, measuring the fluorescence intensity of the filtrate, substituting the fluorescence intensity into a standard curve, calculating the concentration and calculating the total dosage; 200 μl of example 3 was placed in a 3kDa ultrafiltration centrifuge tube; centrifuging at 12000r/min for 30min, quantitatively measuring the solution at the bottom of the centrifuge tube, fixing the volume to 10mL by using methanol, and calculating the free drug amount. The encapsulation efficiency of the liposome was 99.8% calculated according to the encapsulation efficiency formula. Therefore, the liposome prepared by the C-DTC has good encapsulation effect on DOX.
In this patent, the doses of disulfiram and C-DTC are the same in many animal and cell experiments, and are administered in the same mass dose. The molar ratio of the isobaric disulfiram to the C-DTC, converted to the amount of DTC, is greater than 8.84:1, the effect of C-DTC in each of the above-mentioned aspects of investigation is therefore significantly better than DSF.
In summary, the present invention provides a novel DTC prodrug with the following advantages: (1) Compared with disulfiram, the C-DTC antitumor activity is obviously improved. (2) The C-DTC can improve the anti-tumor effect of the anti-tumor medicament from various aspects, including but not limited to inhibiting the clone formation of tumor cells, inhibiting the migration capacity of the tumor cells, promoting the apoptosis of the tumor cells, inhibiting the stem cells of the tumor and the like. (3) The addition of the C-DTC can obviously improve the uptake rate of the tumor cells to the nano preparation. (4) After intravenous administration, the aggregation and retention of the C-DTC-containing nano-preparation in tumor tissues can be obviously improved. (5) C-DTC has better pharmaceutical property than disulfiram and DTC related preparation. The C-DTC has the characteristic of dual purposes of medicine assistance, can be self-assembled into nano particles, can carry medicine, and can be prepared into nano preparations such as liposome together with other auxiliary materials. (6) C-DTC can completely or partially replace cholesterol, and can form vesicle structure with phospholipid for preparing liposome. Thus, C-DTC also has some of the advantages of cholesterol. In conclusion, the C-DTC brings new possibility for the research and application of tumor treatment, and is a great innovation of anti-tumor drugs.
It is noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and the present invention is not limited thereto, and that any person skilled in the art can make modifications or variations within the scope of the present invention without departing from the scope of the present invention.
Claims (10)
1. A diethyldithiocarbamate-cholesterol conjugate and a pharmaceutically acceptable metal complex, chelate, deuterate or prodrug thereof. Characterized in that the structure of the compound contains both a cholesterol group and a diethyl dithiocarbamate group (DTC).
2. A compound according to claim 1, which may have the basic structure of formula i:
Wherein X is a group that is chemically inert during synthesis, including but not limited to alkyl or aryl. A is an in vivo degradable chemical bond, preferably a stimulus responsive sensitive bond. Such stimulus-responsive sensitive bonds include, but are not limited to, those that are responsive to pH, glutathione, reactive oxygen species, or highly expressed enzymes in the tumor microenvironment or tumor cells; the GSH response linkages include, but are not limited to, -S-S-structures; the ROS-responsive sensitive bonds include, but are not limited to, phenylboronic acid bonds, thioketal bonds, or oxalate bonds; the enzymes highly expressed by the tumor tissue include, but are not limited to, matrix metalloproteinase, furin, legumain, FAP-alpha, or cathepsin, etc.
3. The compound of claim 2, including, but not limited to, compounds of the structure:
4. the use of a compound according to claims 1-3, characterized by having a tumor therapeutic effect.
5. The use of a compound according to claim 4, wherein the compound has a synergistic effect in combination with a tumour therapeutic agent, including but not limited to doxorubicin hydrochloride.
6. The use of a compound according to claims 1-5, wherein the compound is used for the preparation of nanoparticulate, liposome or like particulate drug delivery systems, preferably having a particle size in the range of 10-500nm.
7. The use of a compound according to claim 6 for the preparation of a microparticle delivery system, including but not limited to: self-assembled nanoparticles of the compound; or the compound is singly used as a carrier to encapsulate other medicines to form nanoparticles; or the compound and other carrier materials are prepared into a liposome, emulsion, nanoparticle, lipid nano carrier (LNP), micelle and other particle drug delivery systems; or the compound and various phospholipid materials are used together to prepare liposome drug delivery systems; or the compound and other carrier auxiliary materials are used together to prepare microparticle drug delivery systems such as emulsion, nanoparticle, LNP, micelle and the like.
8. The use of a compound as claimed in claims 6 and 7, wherein the nano-preparation containing the compound has the effect of promoting tumor cell uptake and improving tumor targeting after administration.
9. Use of a compound according to claims 1-3, characterized in that it has the potential for therapeutic capacity in relation to diethyl Dithiocarbamate (DTC) groups.
10. A compound according to claim 3, which is prepared by the following method:
(1) In methylene dichloride, diethylamine, mercaptoethanol, carbon disulfide, triethylamine and carbon tetrabromide are mixed and reacted, wherein the mol ratio of the diethylamine to the mercaptoethanol to the carbon disulfide to the triethylamine to the carbon tetrabromide is 1:1:1 (1-2); the temperature of the reaction is 20-32 ℃; the reaction time is 1-4h; the intermediate DTC-OH is obtained.
(2) In dichloromethane, mixing DTC-OH, cholesterol ester formyl chloride and pyridine, and carrying out esterification reaction, wherein the molar ratio of DTC-OH to cholesterol ester formyl chloride to pyridine is 1 (1-2); the reaction temperature is 10-35 ℃; the reaction time is 1-8h. Obtaining the diethyldithiocarbamate-cholesterol conjugate of claim 3.
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