CN116813515A - Curcumin derivative prodrug, preparation method and application thereof - Google Patents
Curcumin derivative prodrug, preparation method and application thereof Download PDFInfo
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- CN116813515A CN116813515A CN202310561686.0A CN202310561686A CN116813515A CN 116813515 A CN116813515 A CN 116813515A CN 202310561686 A CN202310561686 A CN 202310561686A CN 116813515 A CN116813515 A CN 116813515A
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Abstract
The application discloses a curcumin derivative prodrug, a preparation method and application thereof, wherein the curcumin derivative prodrug is a compound with a monosulfur bond or a disulfide bond positioned at alpha and beta positions of adjacent carbonyl groups, which is obtained by connecting curcumin derivatives with oleyl alcohol through 2,2 '-thiodiacetic acid, 2' -thiodipropionic acid, 3 '-dithiodiacetic acid or 2,2' -dithiodipropionic acid, and can be obtained by adopting a nanoparticle precipitation method. The curcumin derivative prodrug can be used for treating breast cancer or liver cancer, and the obtained self-assembled nanoparticle has the advantages of high drug loading capacity, good stability, low toxic and side effect, quick drug release at the tumor site specificity, prolonged body circulation time, increased bioavailability and improved treatment effect.
Description
Technical Field
The application relates to the field of preparation of antitumor drugs, in particular to synthesis of a monosulfur bond/disulfide bond connected oleyl alcohol-curcumin derivative C210 prodrug, preparation of self-assembled nanoparticles thereof and application of the self-assembled nanoparticles in preparation of antitumor drugs.
Background
Curcumin is a polyphenol compound with a diketone structure extracted from turmeric tubers, has remarkable in-vitro anti-tumor activity on leukemia cells and the like, also has remarkable anti-proliferation activity on solid tumors such as colon cancer, lung cancer, liver cancer, breast cancer and other tumor cells, and has a wide anti-tumor spectrum. Curcumin has also been subjected to a number of clinical studies to show some effect in the treatment of many cancers. Besides stronger anti-tumor activity, the curcumin has higher safety, and clinical experiments show that the curcumin has no obvious toxic or side effect even at the dosage of 8 g/day. However, due to the rapid elimination of curcumin in vivo, poor oral bioavailability limits the use of curcumin. Based on the fact that the main metabolic pathway of curcumin in vivo is that phenolic hydroxyl on benzene ring is combined with glucuronic acid, so that curcumin metabolism is delayed and activity is not reduced, methoxy group is introduced into phenolic hydroxyl position to enhance activity of a compound, and accordingly, the inventor designs and synthesizes a novel curcumin derivative C210 (a chemical structure is shown as a compound No. 4 disclosed in patent CN 104030904A), researches show that C210 has stronger molecular chaperone function for inhibiting heat shock 90 protein and stronger in-vitro anti-tumor activity than curcumin, but pharmacokinetics characteristics of the compound are not obviously improved compared with curcumin, and the compound becomes a bottleneck for development and application.
Prodrugs are drugs which have low or no activity in vitro but are capable of metabolizing or releasing active ingredients in vivo, and release drugs in specific tissues can be achieved by modification of chemical modification, so that the selective action of the drugs is improved. The small molecular prodrug has the advantages of small molecular weight, high drug loading and capability of self-assembling to form nano-particles, so that the small molecular prodrug nano-particles are favored by more and more researchers. Some prodrugs based on fatty acids and fatty alcohol modifications can extend the systemic residence time of the drug, for example paliperidone palmitate is one of the lipid modified prodrugs currently marketed, and one injection can significantly extend the duration of effective concentration in vivo, suggesting that lipid modified drugs may be an effective strategy for improving pharmacokinetic behavior. Tumor tissue has a higher redox level than normal tissue, and the intracellular glutathione content of tumor cells is more than 10 times that of normal cells, and the concentration of Reactive Oxygen Species (ROS) in tumor cells is about 100 times higher than that of normal cells, so that release based on the redox response of tumor cells has been widely used.
Disclosure of Invention
Aiming at the development bottleneck of poor pharmacokinetic properties of the traditional curcumin compounds, the application provides a curcumin derivative C210 prodrug, self-assembled nanoparticles thereof, and a preparation method and application thereof, so as to improve the pharmacokinetic properties of C210 and the selectivity anti-tumor effect thereof. The C210 prodrug uses monosulfur bond or disulfide bond with different chain lengths to connect curcumin derivative C210 and oleic acid, and because the prodrug contains the monosulfur bond or disulfide bond with redox response, the prodrug can specifically release C210 in tumor cells with high redox level, plays a role in improving the specific distribution and release of drugs in tumor tissues, and enhances the selective anti-tumor effect. The oleic acid modification can enable the prodrug to have the capacity of self-assembling to form nano particles, so that the average residence time of the drug in the systemic circulation is prolonged, and the pharmacokinetic property of the drug is improved. The obtained nanoparticle has the characteristics of high stability, good pharmacokinetic properties, specific release of C210 in tumor cells, high tumor tissue distribution, strong in vivo anti-tumor activity and the like.
In particular, the application uses different redox intermediates to connect unsaturated fatty acid oleic acid and curcumin derivative C210 to prepare prodrug nano-drug delivery system, so as to improve the drug formation of curcumin derivative C210 and further improve the in-vivo curative effect. A total of 4 prodrugs were synthesized. These prodrugs are named according to the bond bridge containing a central element bond (monosulfur S, disulfide SS) and an ester bond (α is ethyl ester, β is propyl ester), and are accordingly abbreviated as α -C210-S-OA, α -C210-SS-OA, β -C210-S-OA, β -C210-SS-OA. The self-assembled nano-particles of the prodrug can remarkably improve the body circulation time and bioavailability of C210, and based on the background of high redox level in tumor cells and tumor microenvironment, the designed sulfur-containing bridge can enhance the targeted release capability of the drug, so that the prodrug nano-particles are specifically distributed in tumor cells or tumor tissues, and based on the intelligent release of C210 triggered by high redox of tumors, the distribution and release of the drug in the tumor tissues are improved, the selective antitumor activity is enhanced, and the in-vivo antitumor effect of the drug is improved.
To achieve the above object, the present application provides a prodrug of a monosulfur/disulfide-linked oleyl alcohol-curcumin derivative C210 having the following structural formula, wherein "/" stands for "or" herein and hereinafter, reference is made to "monosulfur/disulfide linkage".
Wherein n is an integer of 1 to 2, and R is a monosulfide bond or a disulfide bond.
Wherein the curcumin derivative C210 is described in CN104030904A, and the shorthand of the curcumin derivative C210 or the C210 prodrug is shown in the specification, and the structural formula is as follows:
the curcumin derivative C210 prodrug structure with a monosulfur bond and a diethyl ester structure comprises the following components:
the curcumin derivative C210 prodrug structure with disulfide bond and diethyl ester structure is as follows:
the curcumin derivative C210 prodrug structure with a mono-sulfur bond and a dipropyl ester structure is as follows:
the curcumin derivative C210 prodrug structure with disulfide bond and dipropyl ester structure is as follows:
the application provides a preparation method of a C210 prodrug containing a mono-sulfide/disulfide-bond connected oleyl alcohol-curcumin derivative, which comprises the following steps:
(1) 2,2 '-thiodiacetic acid, 2' -dithiodiacetic acid, 3 '-thiodipropionic acid or 3,3' -dithiodipropionic acid, oleyl alcohol, DMAP and EDCI are respectively weighed into a 100ml round bottom flask, 30ml of a reaction solvent methylene dichloride is added for reaction for 24 hours at room temperature under the protection of nitrogen, and the reaction progress is checked by thin layer chromatography. The solvent was removed by rotary evaporation under reduced pressure, and the concentrate was purified by silica gel column chromatography (petroleum ether: ethyl acetate=20:1) and dried under vacuum to give the intermediates α -S-OA, α -SS-OA, β -S-OA, β -SS-OA as white oils.
(2) And (2) weighing the alpha-S-OA, the alpha-SS-OA, the beta-S-OA, the beta-SS-OA and the curcumin derivative C210, DMAP and EDCI obtained in the step (1) respectively, putting the mixture into a 100ml round bottom flask, adding 10ml of methylene dichloride serving as a reaction solvent, reacting for 24 hours at room temperature under the protection of nitrogen, and checking the reaction progress by using a thin layer chromatography. The reaction mixture was concentrated, then, saturated brine and ethyl acetate were added thereto, followed by extraction three times, drying over anhydrous magnesium sulfate, filtration and concentration. The concentrate was purified by silica gel column chromatography (petroleum ether: ethyl acetate=5:1-3:1) and dried under vacuum. The target products of alpha-C210-S-OA, alpha-C210-SS-OA, beta-C210-S-OA and beta-C210-SS-OA are obtained.
The application also provides self-assembled nanoparticles of the monosulfur/disulfide bond connected oleyl alcohol-curcumin derivative C210 prodrug, which comprise PEG modified prodrug self-assembled nanoparticles, and the preparation method is a nano precipitation method.
The preparation method of the self-assembled nano-particle of the monosulfur bond/disulfide bond connected oleyl alcohol-curcumin derivative C210 prodrug is as follows: each prodrug and DSPE-PEG2000 were completely dissolved in acetone. Then adding the mixed solution into a proper amount of physiological saline drop by drop under magnetic stirring, and removing acetone by a reduced pressure evaporation method to obtain a nanoparticle solution without an organic solvent. Wherein, DSPE-PEG2000 is not added in the preparation method of the non-PEGylated C210 prodrug nanoparticle.
The application also provides application of the self-assembled nano-particles of the monosulfur/disulfide-linked oleyl alcohol-curcumin derivative C210 prodrug in preparation of a drug delivery system.
The application also provides application of the self-assembled nano-particles of the monosulfur/disulfide bond connected oleyl alcohol-curcumin derivative C210 prodrug in preparation of antitumor drugs.
The application also provides application of the self-assembled nano-particles of the monosulfur/disulfide-linked oleyl alcohol-curcumin derivative C210 prodrug in preparing injection, oral administration or local administration systems.
The technical problem solved by the application is that monosulfur bonds/disulfide bonds with different chain lengths are introduced into oleyl alcohol-curcumin derivative C210 prodrugs, different monosulfur bond/disulfide bond bridged redox double-sensitive C210 prodrugs are designed, and the C210 prodrugs are used for constructing self-assembled nanoparticles, so that the high drug loading, good stability, low toxic and side effects and tumor site specificity quick drug release are realized, the body circulation time is prolonged, the bioavailability is increased, and the treatment effect is improved. Meanwhile, the difference of the monosulfur bond/disulfide bond connecting chains with different lengths in the aspect of self-assembly of the C210 prodrug is examined, and the influence on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of the prodrug self-assembly nanoparticle is examined.
The application has the advantages that:
(1) The C210 prodrug containing the monosulfur bond/disulfide bond connecting chains with different lengths is designed and synthesized, and the synthesis method is simple and easy to implement.
(2) The preparation method is simple and feasible, realizes high-efficiency entrapment of the medicine and can reach about 50 percent of ultrahigh medicine carrying capacity.
(3) The difference of the monosulfur bond/disulfide bond connecting chains with different lengths in terms of self-assembly and the influence on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of the prodrug self-assembly nanoparticles are examined. Comprehensive experiment results show that the prodrug containing the monosulfide bond and the diethyl ester structure has proper particle size and chemical stability, and simultaneously has the best capacity of releasing C2120 in vitro in redox response, thereby remarkably improving the bioavailability of C210 and the in vivo anti-tumor effect. The application provides a new strategy and more choices for developing an intelligent response type drug delivery system for a tumor microenvironment, and meets the urgent need of high-efficiency selective anti-tumor drugs in clinic.
Drawings
Purity confirmation of the prodrug of fig. 1C 210;
FIG. 2C210 electron microscope characterization of prodrug self-assembled nanoparticles;
FIG. 3C210 stability results of prodrug self-assembled nanoparticles;
a: c210 prodrug nanoparticles were stable in PBS solution containing 10% fbs for 48 h;
b: stability of the C210 prodrug nanoparticles in a dark environment at 4 ℃ for 3 months;
FIG. 4C210 in vitro release assay of prodrug self-assembled nanoparticles under simulated tumor high redox conditions;
a: c210 prodrug nanoparticle containing 0mM H 2 O 2 Release in PBS (ph=7.4) medium;
b: c210 prodrug nanoparticle containing 1mM H 2 O 2 Release in PBS (ph=7.4) medium;
c: c210 prodrug nanoparticle containing 10mM H 2 O 2 Release in PBS (ph=7.4) medium;
d: c210 prodrug nanoparticles were released in PBS (ph=7.4) medium containing 0mM DTT;
e: c210 prodrug nanoparticles were released in PBS (ph=7.4) medium containing 1mM DTT;
f: c210 prodrug nanoparticles were released in PBS (ph=7.4) medium containing 10mM DTT;
FIG. 5C210 plasma C-T curve of prodrug self-assembled nanoparticles intravenously administered to rats;
FIG. 6C210 distribution of prodrug self-assembled nanoparticles in tumor tissue;
a: distribution of C210 prodrug in tumor tissue for 1h, 2h, 4h, 12h and C210 released therefrom;
b: tumor accumulation of C210 released by the C210 prodrug nanoparticle within 0-12 h;
FIG. 7C210 results of prodrug self-assembled nanoparticle in vivo anti-tumor experiments;
a: tumor volume growth curve of Balb/C tumor-bearing mice following C210 prodrug nanoparticle treatment;
b: tumor photographs of Balb/C tumor-bearing mice after C210 prodrug nanoparticle treatment;
c: tumor weights of Balb/C tumor-bearing mice following C210 prodrug nanoparticle treatment;
d: body weight change in Balb/C tumor-bearing mice following C210 prodrug nanoparticle treatment.
Detailed Description
The application is further illustrated by way of examples which follow, but are not thereby limited to the scope of the examples described.
Example 1: synthesis of C210 prodrugs (alpha-C210-S-OA) containing monosulfur and diethyl ester structures
Oleyl alcohol (268 mg,1 mmol), 2' -thiodiacetic acid (150 mg,1 mmol), EDCI (191 mg,1 mmol) and DMAP (24 mg,0.2 mmol) were added to a 10ml dichloromethane solution. The reaction mixture was stirred at room temperature under nitrogen. Detecting formation of a target product by adopting a thin layer chromatography; after the completion of the reaction, methylene chloride was evaporated, and saturated brine was added. The aqueous layer product was extracted with dichloromethane, washed with saturated NaCl solution and dried over anhydrous sodium sulfate. The concentrate was separated by silica gel column chromatography to give intermediate (E) -2- [ (2- (octadeca-9-en-1-yloxy) -2-oxyethyl) thio) acetic acid ] (α -S-OA) in 23% yield.
C210 (552 mg,1 mmol), α -S-OA (400 mg,1 mmol), EDCI (191 mg,1 mmol) and DMAP (24 mg,0.2 mmol) were dissolved in 10ml dichloromethane aqueous solution. The reaction mixture was then stirred at room temperature under nitrogen overnight. Detecting the formation of the target product by adopting a thin layer chromatography.
After the completion of the reaction, the reaction solution was extracted with methylene chloride, washed with a saturated solution of NaCl and dried over anhydrous sodium sulfate. The concentrate was separated by column chromatography on silica gel to give the desired product [ (2- { [ (10E) -octadeca-9-enyl ] oxy } -2-oxyethyleneethyl) thio ] acetic acid-2-methoxy-4- [ (4E) -3-oxyen-2- [ (2E) -1-oxyen-3- (3, 4, 5-trimethoxyphenyl) prop-2-enyl ] -5- (3, 4, 5-trimethoxyphenyl) pent-4-enyl ] phenyl ester (. Alpha. -C210-S-OA) in 78% yield.
The high-resolution mass spectrum, the nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum are adopted for carrying out structural confirmation, and the analysis result of the spectrogram is as follows: [ M-H ] is detected] - Peak 973.4774, demonstrating successful synthesis of alpha-C210-S-OA of formula C 55 H 74 O 13 S。
1 H NMR(500MHz,CDCl 3 )δ7.69(d,J＝15.3Hz,2H),6.93–6.86(m,2H),6.83(d,J＝15.3Hz,2H),6.67(s,3H),5.35(dqd,J＝7.9,4.6,4.0,2.1Hz,2H),4.13(t,J＝6.9Hz,2H),3.98(s,2H),3.93–3.82(m,19H),3.77(s,3H),3.62(d,J＝5.6Hz,2H),3.48(s,2H),2.06–1.97(m,4H),1.64(t,J＝7.3Hz,2H),1.58(s,2H),1.35–1.22(m,24H),0.88(t,J＝6.8Hz,3H)。
13 C NMR(126MHz,CDCl 3 )δ183.56,169.82,168.05,153.44,151.27,140.40,140.20,138.17,130.72,130.01,129.76,122.79,119.92,111.82,108.68,105.92,105.40,,65.75,61.00,56.24,56.19,55.90,33.39,33.23,31.91,31.78,29.77,29.72,29.53,29.40,29.32,29.20,28.53,27.22,27.18,25.81,22.69,14.13。
Example 2: synthesis of C210 prodrugs (alpha-C210-SS-OA) containing disulfide and diethyl ester structures
2-methoxy-4- [ (4E) -3-oxo-2- [ (2E) -1-oxo-3- (3, 4, 5-trimethoxyphenyl) prop-2-enyl ] -5- (3, 4, 5-trimethoxyphenyl) pent-4-enyl ] phenyl ester (. Alpha. -C210-SS-OA) was prepared by converting 2,2 '-thiodiacetic acid to 2,2' -dithiodiacetic acid using the procedure of example 1.
By usingThe high resolution mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum are used for carrying out structural confirmation, and the analysis result of the spectrogram is as follows: [ M-H ] is detected] - Peak 1005.4497, demonstrating successful synthesis of alpha-C210-SS-OA of formula C 55 H 74 O 13 S 2 。
1 H NMR(500MHz,CDCl 3 )δ7.69(d,J＝15.3Hz,2H),6.93–6.80(m,4H),6.67(s,3H),5.39–5.30(m,2H),4.13(dd,J＝7.3,6.2Hz,2H),3.98(s,2H),3.91–3.76(m,23H),3.62(s,2H),2.06–1.92(m,4H),1.68–1.60(m,3H),1.36–1.23(m,24H),0.88(t,J＝6.9Hz,3H)。
13 C NMR(126MHz,CDCl 3 )δ183.56,169.38,167.68,153.44,151.33,142.26,140.45,140.19,138.18,130.72,130.00,129.77,122.82,119.93,119.89,111.85,108.69,105.40,65.90,61.02,61.00,56.19,55.90,55.86,41.44,41.03,31.91,29.77,29.73,29.53,29.41,29.33,29.21,29.18,28.51,27.22,27.19,25.81,22.69,14.13。
Example 3: synthesis of C210 prodrugs (beta-C210-S-OA) containing monosulfur and dipropyl ester structures
2-methoxy-4- [ (4E) -3-oxo-2- [ (2E) -1-oxo-3- (3, 4, 5-trimethoxyphenyl) prop-2-enyl ] -5- (3, 4, 5-trimethoxyphenyl) pent-4-enyl ] phenyl ester (. Beta. -C210-S-OA) of 3- [ (3- { [ (10E) -nineteen-10-enyl ] oxy } -3-oxoidenpropyl) thio ] propanoic acid was prepared by the procedure of example 1 by converting 2,2 '-thiodiacetic acid into 3,3' -thiodipropionic acid.
The high-resolution mass spectrum, the nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum are adopted for carrying out structural confirmation, and the analysis result of the spectrogram is as follows: [ M-H ] is detected] - Peak 1001.5082, demonstrated successful synthesis of beta-C210-S-OA, molecular formula C 57 H 78 O 13 S。
1 H NMR(500MHz,CDCl 3 )δ7.62(d,J＝15.3Hz,2H),6.85–6.73(m,3H),6.60(s,3H),5.33–5.21(m,3H),4.01(t,J＝6.8Hz,2H),3.90(s,2H),3.85–3.77(m,18H),3.70(d,J＝7.1Hz,3H),2.87–2.74(m,6H),2.59–2.52(m,2H),1.94(t,J＝6.3Hz,3H),1.59–1.46(m,4H),1.22–1.18(m,24H),0.81(d,J＝1.8Hz,3H)。 13 C NMR(126MHz,CDCl 3 )δ182.53,170.86,169.05,168.95,152.44,152.41,150.34,141.19,139.15,139.13,137.20,129.71,128.96,128.75,121.92,118.94,118.85,110.75,107.71,104.89,104.37,63.94,59.99,59.97,55.21,55.16,54.87,33.78,33.60,30.90,30.88,30.75,30.41,29.16,28.74,28.71,28.68,28.63,28.50,28.39,28.34,28.30,28.25,28.19,27.56,26.19,26.16,26.06,25.96,24.86,21.66,13.10。
Example 4: synthesis of C210 prodrugs (beta-C210-SS-OA) containing disulfide and dipropyl ester structures
2-methoxy-4- [ (4E) -3-oxo-2- [ (2E) -1-oxo-3- (3, 4, 5-trimethoxyphenyl) prop-2-enyl ] -5- (3, 4, 5-trimethoxyphenyl) pent-4-enyl ] phenyl ester (. Beta. -C210-SS-OA) of 3- [ (3- { [ (10E) -octadec-9-enyl ] oxy } -3-oxoidenepropyl) disulfide ] propionic acid was prepared by the procedure of example 1, substituting 2,2 '-thiodiacetic acid into 3,3' -dithiodipropionic acid.
The high-resolution mass spectrum, the nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum are adopted for carrying out structural confirmation, and the analysis result of the spectrogram is as follows: [ M-H ] is detected] - Peak 1035.4805, demonstrated successful synthesis of beta-C210-SS-OA, molecular formula C 57 H 78 O 13 S 2 。
1 H NMR(500MHz,CDCl 3 )δ7.69(d,J＝15.3Hz,2H),6.92–6.78(m,4H),6.67(s,3H),5.40–5.30(m,2H),4.08(t,J＝6.8Hz,2H),3.97(s,1H),3.93–3.84(m,18H),3.76(s,3H),3.06–2.99(m,4H),2.95(t,J＝7.2Hz,2H),2.74(t,J＝7.2Hz,2H),2.17(d,J＝3.1Hz,2H),2.01(q,J＝6.7Hz,3H),1.63(h,J＝6.9Hz,4H),1.30–1.23(m,24H),0.88(d,J＝1.9Hz,3H)。
13 C NMR(126MHz,CDCl 3 )δ183.56,171.73,169.93,153.47,153.43,151.36,142.23,140.18,138.21,130.89,130.73,129.99,129.78,122.91,119.96,119.87,111.78,108.73,105.92,105.39,68.15,65.03,64.62,61.02,61.00,56.23,56.19,55.90,38.72,34.14,33.89,33.23,33.02,31.91,31.44,30.94,30.19,29.77,29.73,29.70,29.66,29.52,29.41,29.36,29.32,29.27,29.21,28.92,28.57,27.22,27.19,25.89,23.74,22.99,22.69,14.13。
EXAMPLE 5 purity confirmation of the C210 prodrug
The purity of the C210 prodrug is measured by utilizing high performance liquid chromatography, and the result is shown in figure 1, wherein the purity of the 4 prodrugs is above 98%, and the requirement of the subsequent experiment is met.
Example 6: preparation and stability investigation of C210 prodrug self-assembled nanoparticles
4mg of the prodrug (α -C210-S-OA, α -C210-SS-OA, β -C210-S-OA or β -C210-SS-OA, synthesized in examples 1-4, respectively) and 0.8mg DSPE-PEG2000 were weighed out and dissolved in 1ml acetone. The mixed solution was then added dropwise to 4ml of physiological saline under magnetic stirring and the acetone was removed by evaporation under reduced pressure. Mixing the nanoparticles with PBS containing 10% fetal bovine serum, placing the mixture in a shaking incubator at 37 ℃, sampling at 0, 2, 4, 6, 8, 12, 24 and 48 hours respectively, measuring the particle size of the nanoparticles after dilution, and taking the particle size and PDI as indexes for evaluating the stability to examine the short-term stability of the nanoparticles.
The nanoparticles were placed in a light-shielding environment at 4 ℃, sampled at 1, 3, 5, 7, 14, 28, 60, and 90 days, and after dilution, the particle size of the nanoparticles was measured, and the long-term stability was examined using the particle size and PDI as an index for evaluating stability. The results are shown in table 1 and fig. 2, the DSPE PEG2K modified prodrug nanoparticles are spherical, the particle sizes are all about 120nm, the particle size distribution is uniform, the surface charges are all about 36mV, the state of the nanoparticles is stable and are not easy to aggregate, the drug loading rate of each prodrug nanoparticle is about 50%, and the use of auxiliary materials and the potential risks brought by the auxiliary materials are effectively reduced. The stability of the prodrug nanoparticles was then examined, as shown in fig. 3, and the prodrug nanoparticles showed substantially no change in particle size in PBS (ph=7.4) containing 10% fetal bovine serum at 37 ℃ for 48 hours, with good stability. The prepared nanoparticle is kept in a dark environment at 4 ℃ for 3 months, and the particle size is not changed, so that the nanoparticle has very good stability and long-term storage potential.
TABLE 1 particle size, particle size distribution, surface charge, encapsulation efficiency and drug loading of prodrug self-assembled nanoparticles of C210
Example 7: in vitro simulated tumor high redox responsive release experiment of C210 prodrug self-assembled nanoparticle
PBS (pH 7.4) containing 5% SDS was used as a release medium to study the release profile of C210 from prodrug nanoparticles. 1ml of C210 prodrug nanoparticle was added to 30ml of each containing 0, 1, 10mM H 2 O 2 Or in a different release medium of DTT at 37 ℃. At predetermined time points 0, 2, 4, 6, 8, 12, 24h, 100 μl of solution was removed and an equivalent amount of release medium was replenished. The amount of C210 released was determined by high performance liquid chromatography. The oxidative release results are shown in FIGS. 4A-C, where each prodrug nanoparticle was incubated in PBS (pH 7.4) at 37deg.C for 24H with only a small amount of C210 (less than 10% w/w) released, but with 1mM H 2 O 2 Or 10mM H 2 O 2 The prodrug nanoparticles were able to release C210 rapidly when incubated in PBS at a rate of α -C210-S-OA > α -C210-SS-OA > β -C210-S-OA > β -C210-SS-OA in sequence. The results of the reduction release are shown in FIGS. 4D-F, where the disulfide bond containing prodrugs exhibited more sensitive reduction reactions with a response rates of a-C210-SS-OA > β -C210-S-OA > α -C210-S-OA, respectively, incubated in PBS containing 1mM/10mM DTT, where the α -C210-SS-OA was substantially completely reduced in about 2 hours. In contrast, nanoparticles slowly convert within 24 hours under non-reducing conditions. The results demonstrate that both the monosulfide and disulfide bonds have redox dual responsiveness, where the monosulfide bond is more sensitive to oxidizing environments and the disulfide bond is more sensitive to reducing environments, and long chain prodrugs release at a slower rate than short chain prodrugs.
Example 8: selective cytotoxicity of C210 prodrug self-assembled nanoparticles on tumor cells
The MTT assay was used to determine the cell viability of MCF-7 cells, MDA-MB-231 cells, 4T1 cells, MCF-10A cells (normal mammary epithelial cells) and HepG2 cells treated with C210 and prodrug nanoparticles. Cancer cells were seeded using 96-well plates, 3000 cells per well, and cultured for 12h until the cells were completely adherent. Then, the medium is replaced with a medium having C210 or prodrug nanoparticles having a concentration of C210 ranging from 0 to 200. Mu. Mol/l. The cells were then cultured for 48h or 72h. Then 15. Mu.l MTT solution was added to each well and the cells were further cultured at 37℃for 4 hours. After removal of the medium, the formazan crystals were dissolved with 200 μl DMSO. The absorbance of the plate was then measured with a microplate reader at 570nm wavelength.
As shown in Table 2, the 4 kinds of prodrug nanoparticles have more remarkable difference in cytotoxicity to breast cancer cells and liver cancer, and the alpha-C210-S-OANPs and the alpha-C210-SS-OANPs have stronger antitumor activity, while the beta-C210-S-OANPs and the beta-C210-SS-OANPs have weaker antitumor activity. From the cytotoxicity results of C210 and each prodrug nanoparticle against breast cancer and liver cancer, it was found that although C210 prodrug nanoparticle showed lower cytotoxicity than free C210, each prodrug nanoparticle was significantly selective against normal breast cell MCF-10A and breast cancer cell MCF-7, with a higher relative therapeutic index than the prodrug, as seen from table 3.
TABLE 2C210 and IC of each prodrug nanoparticle to tumor cells 50 Value of
TABLE 3C210 and IC of each prodrug nanoparticle to tumor cells and normal cells 50 Value (relative therapeutic index = normal cell IC) 50 Tumor cell IC 50 )
Example 9: pharmacokinetic and tumor tissue specific distribution research of C210 prodrug self-assembled nanoparticles
Pharmacokinetic studies were performed with SD rats. Rats fasted for 12 hours prior to the experiment and were free to drink water. The 18 rats were divided into 6 groups and the free C210 or C210 prodrug nanoparticles were intravenously injected in an equal amount of 20 μmol/kg C210, respectively. Blood samples were collected at predetermined time points (0.08, 0.15, 0.5, 1, 1.5, 2, 4, 6, 8, 12 h) and centrifuged to obtain plasma. LC-MS method determines the concentration of C210 and the corresponding prodrug. Pharmacokinetic parameters were analyzed using drug and statistics 2.0 (DAS) software. The biodistribution of C210 and prodrug nanoparticles was studied by determining the concentration of C210 and prodrug nanoparticles in the major organs of 4T1 tumor-bearing BALB/C mice. 60 mice were randomly divided into 5 groups and were given intravenous doses of free C210 or C210 prodrug nanoparticles, with an equivalent dose of 20. Mu. Mol/kg of C210. Mice were sacrificed 1, 2, 4, and 12 hours after intravenous administration, tumor tissues were taken, and LC-MS was used to determine the accumulation of C210 in tumors.
As a result, as shown in FIG. 5 and Table 4, free C210 was rapidly eliminated from blood. The Mean Residence Time (MRT) of the prodrug nanoparticle-released C210 in blood circulation was significantly prolonged compared to free C210, with MRTs for the α -C210-S-OA NPs, α -C210-SS-OA NPs, β -C210-S-OA NPs and β -C210-SS-OA NPs being 7.3, 6.8, 8.7 and 6.1 times that of the free C210 group, respectively. The C210 prodrug nanoparticles significantly increased AUC of C210 compared to the free C210 group, with AUC of α -C210-S-OA NPs, α -C210-SS-OA NPs, β -C210-S-OA NPs, and β -C210-SS-OA NPs being 9.9, 9.6, 3.4, and 2.2 times that of the free C210 group, respectively. Indicating that the prodrug nanoparticles can effectively improve the pharmacokinetic behavior of C210. The tumor tissue specific distribution is shown in FIG. 6, in which the accumulation of α -C210-S-OA NPs is much higher than in the free C210 group. The accumulation of C210 released by alpha-C210-S-OA NPs, alpha-C210-SS-OA NPs, beta-C210-S-OA NPs and beta-C210-S-OA NPs in tumor tissues is 3.3 times, 1.5 times, 1.2 times and 0.8 times that of free C210 groups, which indicates that the nano-drug delivery system can effectively increase the drug accumulation in tumor tissues.
Table 4C210 pharmacokinetic parameters of prodrug nanoparticles
Example 10: in-vivo anti-tumor study of C210 prodrug self-assembled nanoparticles
Construction of a mouse breast cancer 4T1 subcutaneous tumor model: will be 1X 10 6 4T1 cells were injected into the right abdomen of female BALB/C mice and C210 and prodrug nanoparticles were observedAnti-tumor therapeutic effect. When the tumor volume reached 100mm3, tumor-bearing mice were randomly divided into 7 groups (saline, CTX, C210 and α -C210-S-OA-NPs, α -C210-SS-OA-NPs, β -C210-S-OA-NPs, β -C210-SS-OA-NPs), 8 each. CTX (dose: 30 mg/kg) was injected intraperitoneally 1 time every 3 days. The remaining groups were intravenously injected 1 time per day (corresponding to C210. Mu. Mol/kg). Treatment for 14 days or tumor size up to 2000mm 3 Mice were sacrificed at that time. Tumor tissues were collected, weighed and photographed, and tumor inhibition rates were calculated.
As shown in FIG. 7, the anti-tumor effect of alpha-C210-S-OANPs was most pronounced in several prodrug nanoparticles, which were stronger than the effect of the equivalent dose of free C210, slightly stronger than the effect of the positive drug cyclophosphamide 30mg/kg, and the anti-tumor effect of alpha-C210-S-OANPs was stronger than that of beta-C210-S-OANPs, i.e., the short-chain prodrug activity was significantly better than the long-chain prodrug.
In summary, α -C210-S-OANPs have many advantages such as good colloidal stability, rapid prodrug release, efficient cellular uptake, longer blood circulation time, and higher tumor-specific accumulation.
Claims (10)
1. The prodrug is an oleyl alcohol-curcumin derivative connected by adopting monosulfur bonds or disulfide bonds with different chain lengths, and the structural formula is shown as a general formula (1):
wherein n is an integer of 1 to 2, R is a monosulfide bond or a disulfide bond;
the curcumin derivative has the following structural formula:
2. the curcumin derivative prodrug according to claim 1, wherein the curcumin derivative is a compound in which a monosulfur bond or a disulfide bond obtained by connecting 2,2 '-thiodiacetic acid, 2' -thiodipropionic acid, 3 '-dithiodiacetic acid or 2,2' -dithiodipropionic acid with oleyl alcohol is positioned at an alpha position and a beta position of adjacent carbonyl groups, and the structural formula is as follows:
3. a process for the preparation of a curcumin derivative prodrug as claimed in claim 2, comprising the steps of:
(1) Respectively adding 2,2 '-thiodiacetic acid, 2' -thiodipropionic acid, 3 '-dithiodiacetic acid, 2' -dithiodipropionic acid, oleyl alcohol, EDCI and DMAP into dichloromethane solution, stirring the reaction mixture at room temperature under the protection of nitrogen, and separating by silica gel column chromatography to obtain 4 intermediate products of alpha-S-OA, alpha-SS-OA, beta-S-OA and beta-SS-OA;
(2) Dissolving the alpha-S-OA, the alpha-SS-OA, the beta-S-OA and the beta-SS-OA obtained in the step (1) and curcumin derivatives, EDCI and DMAP respectively in dichloromethane, stirring the reaction mixture at room temperature under the protection of nitrogen, and separating the reaction mixture by silica gel column chromatography to obtain target products alpha-C210-S-OA, alpha-C210-SS-OA, beta-C210-S-OA and beta-C210-SS-OA.
4. Self-assembled nanoparticles carrying the curcumin derivative prodrug of claim 1, characterized in that the self-assembled nanoparticles are non-pegylated curcumin derivative prodrug self-assembled nanoparticles or PEG-modified curcumin derivative prodrug self-assembled nanoparticles; the particle size is about 120nm, the particle size distribution is uniform, and the surface charge is about 36 mV.
5. The self-assembled nanoparticle carrying a curcumin derivative prodrug according to claim 4, wherein the self-assembled nanoparticle is obtained by a nanoparticle precipitation method, the curcumin derivative prodrug and DSPE-PEG2000 are dissolved in solvent acetone, the obtained solution is slowly dropped into water under stirring, the curcumin derivative prodrug spontaneously forms uniform nanoparticles, and the solvent is removed by reduced pressure evaporation to obtain a PEG modified curcumin derivative prodrug self-assembled nanoparticle solution without an organic solvent;
wherein, DSPE-PEG2000 is not added in the preparation method of the non-PEGylated C210 prodrug nanoparticle.
6. A pharmaceutical composition comprising a curcumin derivative prodrug of claims 1-2 or a self-assembled nanoparticle carrying a curcumin derivative prodrug of claim 4, and a pharmaceutically acceptable excipient.
7. The use of a curcumin derivative prodrug according to claims 1-2 for preparing a medicament for treating tumors, wherein the curcumin derivative prodrug contains a redox-responsive monosulfur bond or disulfide bond, so that the prodrug can specifically release the curcumin derivative in tumor cells with high redox level, plays a role in improving the specific distribution and release of the medicament in tumor tissues, and enhances the selective anti-tumor effect.
8. The use according to claim 7, wherein the tumor is breast cancer or liver cancer.
9. Use of self-assembled nanoparticles carrying a prodrug of a curcumin derivative as defined in claim 4 for the preparation of a medicament for treating tumors, wherein the prodrug of a curcumin derivative is modified with oleic acid, which enables the prodrug to have the ability to self-assemble into nanoparticles, thereby prolonging the average residence time of the drug in the systemic circulation and improving the pharmacokinetic properties thereof.
10. The use according to claim 9, characterized in that the tumor is breast cancer or liver cancer.
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