CN118320117B - Molecular-based drug delivery system for multicellular organ targeting and in situ release - Google Patents
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Abstract
本发明提供了一种多细胞器靶向、原位释放的分子基药物递送系统，属于抗癌药物技术领域。该药物递送系统包括药物单元和与其共价键连接的靶向单元及阴离子单元。药物单元的母体药物结构中至少含有一个带活泼氢的杂原子基团，靶向单元为派罗宁及其类似物的结构。
The present invention provides a multi-organelle targeting, in-situ release molecular-based drug delivery system, belonging to the technical field of anticancer drugs. The drug delivery system comprises a drug unit and a targeting unit and an anion unit covalently bonded thereto. The parent drug structure of the drug unit contains at least one heteroatom group with active hydrogen, and the targeting unit is the structure of pyronin and its analogs.
Description
Technical Field
The invention belongs to the technical field of antitumor drugs, and particularly relates to a molecular-based drug delivery system for multi-organelle targeting and in-situ release.
Background
The research and development investment of the brand new medicine is large, and the output is small. The in-vivo metabolism and safety data of the medicines on the market are more detailed, so that the research and development cost can be saved, and the research and development period can be shortened. Accordingly, "old medicine new use" is receiving more and more attention. The new use of the old medicine refers to the development of new indications or new uses of medicines on the market, and becomes one of the most commonly used means in the research and development of new medicines. Mitochondria are specific organelles that produce energy in eukaryotic cells, and are also the primary sites of Reactive Oxygen Species (ROS), playing an important role in the processes of cell growth, metabolism, and death. Abnormal mitochondria in tumor cells represent glycolytic pathways in tumor cells that differ from normal cells, and are the origin of high ROS levels in tumor cells. Oxidative stress in tumor cells further exacerbates the differentiation of organelles such as endoplasmic reticulum, lysosomes, etc. in cells. In order to maintain self redox homeostasis, the level of bioreductive agent in tumor cells is also much higher than in normal cells, most notably abnormally high Glutathione (GSH) levels in tumor cells （R. Franco, J. A. Cidlowski, Apoptosis and glutathione beyond an antioxidant. Cell Death Differ. 2009, 16, 1303-1314）.
The molecular-based drug delivery system with multicellular organ targeting and in-situ release can effectively reduce the drug dosage and reduce the toxicity to normal cells. On the basis of multi-organelle targeted delivery, the in-situ release of drug molecules is realized under the action of high ROS and high GSH level in tumor cells, so that the drug action efficiency can be further improved, and the selective and efficient killing of tumor cells is realized. In tumor cells, the differentiation of various organelles such as mitochondria, endoplasmic reticulum, lysosomes, lipid droplets, golgi apparatus and the like plays an important role in the occurrence and development of tumors （A. Saminathan, M. Zajac, P. Anees, Y. Krishman, Organelle-level precision with next generation targeting technologies. Nat. Rev. Mater. 2022, 7, 355-371）.
Currently, the drug encapsulation and loading rates of organelle-targeted nano-based drug delivery systems are not precise, limiting the clinical prospects （W. Zhen, S. An, S. Wang, W. Hu, Y. Li, X. Jiang, J. Li, Precise subcellular organelle targeting for boosting endogenous-stimuli-mediated tumor therapy. Adv. Mater. 2021, 33, 210572）. of drug delivery systems in the relevant scientific fields of medicine, pharmacy, chemical biology, etc., and there is a need for a methodology that can precisely manipulate multicellular organelle-targeted, in situ released molecular-based drug delivery systems and for cancer treatment.
Disclosure of Invention
The invention provides a molecular-based drug delivery system for multi-organelle targeting and in-situ release, which can solve the technical problems in the background art.
The technical scheme provided by the invention is as follows:
In one aspect, a multicellular organ targeted, in situ released, molecular-based drug delivery system is presented that includes a drug unit and a covalently linked targeting unit. The medicine unit is parent medicine with active hydrogen removed, and the targeting unit is the structure of pyronine and analogues thereof. The structural general formula of the molecular-based drug delivery system is shown as follows:
Wherein Drug represents a Drug unit, and the structure of the Drug unit is a parent Drug structure with one active hydrogen removed; pyronine and analogues thereof are targeting units, wherein R 1-R4 is methyl or ethyl, X represents an exchangeable linking group in the aromatic ring backbone of pyronine and analogues thereof; a - represents an anionic unit.
In a preferred embodiment, the parent drug structure of the drug unit in the structure of the multicellular organ targeted, in situ released drug delivery system contains N, O and/or S nucleophilic groups and contains at least one active H.
In a preferred embodiment, the alternative linking group X in the aromatic ring backbone of pyronine and analogs thereof comprises O, C (CH 3)2 or Si (Me) 2.
In a preferred embodiment, R 1、R2、R3、R4 are the same or different hydrocarbyl groups.
In a preferred embodiment, the anion is a halogen anion, an inorganic or organic oxyacid radical anion.
In a preferred embodiment, the multicellular organelle targeted, in situ released drug delivery system targets organelles including at least two or more of mitochondrial, endoplasmic reticulum, lysosome, lipid droplet, or golgi organelle.
In another aspect, a method of preparing a multicellular organ targeted, in situ released drug delivery system is provided, the method comprising:
Sequentially adding 9-thiocarbonyl pyronine derivatives, a catalyst and a cocatalyst into a solvent according to the molar ratio of 1:1.2:10;
Stirring at normal temperature until the reaction is complete, and filtering the reaction mixture by using diatomite to obtain filtrate;
adding parent medicine and triethylamine in a molar ratio of 1:1.2 into the filtrate, reacting for 8 to 12 hours at room temperature, removing the solvent by screwing, and adding saturated saline;
Extracting and drying to obtain a filter material, and purifying the filter material to obtain the molecular-based drug delivery system with the multi-organelle targeting and in-situ release.
In a preferred embodiment, the method further comprises adjusting the structure of the anion units of the purified multicellular organ targeted, in situ released molecular-based drug delivery system by an anion replacement method to obtain the multicellular organ targeted, in situ released molecular-based drug delivery system containing different anions.
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The foregoing and other objects, features and advantages of the disclosure will become more apparent from the following more particular description of exemplary embodiments of the disclosure, as illustrated in the accompanying drawings.
FIG. 1 is a schematic diagram of 1 H NMR spectra of a multicellular organ targeted, in situ released, molecular-based drug delivery system (DDY).
FIG. 2 is a schematic diagram of 13 C NMR spectra of a multicellular organ targeted, in situ released, molecular-based drug delivery system (DDY).
FIG. 3 is a schematic diagram of the fluorescence response of a multicellular organ targeted, in situ released molecular-based drug delivery system (DDY) to various cellular endogenous substances such as reactive oxygen species, reactive Nitrogen Species (RNS), reactive Sulfur Species (RSS), and the like.
FIG. 4 is a schematic representation of fluorescence time course curves of a multicellular organelle targeted, in situ released molecular-based drug delivery system (DDY) at different concentrations of GSH.
FIG. 5 is a schematic diagram of liquid chromatography-mass spectrometry results analysis of a multicellular organ targeted, in situ released molecular-based drug delivery system (DDY) and GSH equilibrated mixture.
FIG. 6 is a schematic representation of the effect of multicellular organelle targeted, in situ released molecular-based drug delivery system (DDY) and parent drug Desloartadine on cell viability of human normal hepatocellular strain THLE-2 and human hepatoma cell strain Hep 3B.
FIG. 7 is a schematic diagram of an organelle localization experiment of a multicellular targeted, in situ released molecular-based drug delivery system (DDY).
FIG. 8 is a graphical representation of a graph of graft tumor growth after administration of a multicellular organ targeted, in situ released molecular-based drug delivery system (DDY) and a parent drug Desloartadine to tumor-bearing BALB/c nude mice.
FIG. 9 is a graphical representation of tumor volume and weight change of tumor-bearing BALB/c nude mice after 10 days of treatment with a multicellular organ targeted, in situ released, molecular-based drug delivery system (DDY).
Detailed Description
Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While embodiments of the present disclosure are illustrated in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
The term "comprising" and variations thereof as used herein means open ended, i.e., "including but not limited to. The term "or" means "and/or" unless specifically stated otherwise. The term "based on" means "based at least in part on". The terms "one example embodiment" and "one embodiment" mean "at least one example embodiment. The term "another embodiment" means "at least one additional embodiment". The terms "first," "second," and the like, may refer to different or the same object. Other explicit and implicit definitions are also possible below.
The molecular-based drug delivery system with multi-organelle targeting and in-situ release provided by the invention can target at least two organelles in organelles such as mitochondria, endoplasmic reticulum, lysosome, lipid droplets or golgi apparatus and release parent drug molecules in the organelles in situ. The invention changes the action part of the medicine by a modification method, thereby changing the activity of the medicine and endowing the parent medicine with new anti-tumor activity. The molecular-based drug delivery system with the multi-organelle targeting and in-situ release provided by the invention has the structure of pyronine and analogues thereof, the raw materials are cheap and easy to obtain, the method for modifying the drug units is simple and convenient to operate, the reaction is efficient, the reaction condition is mild, and the molecular-based drug delivery system has good inhibition effect on the growth of various tumor cells. The multi-organelle targeted, in situ released molecular-based drug delivery system provided by the present invention will be further explained and described by alternative embodiments.
In one aspect, the invention provides a multicellular organ targeted, in situ released, molecular-based drug delivery system, the multicellular organ targeted, in situ released, molecular-based drug delivery system comprising a drug unit, an organelle targeted unit and an anion unit, the structural general formula of which is shown as follows:
Wherein Drug represents a Drug unit, and the structure of the Drug unit is a parent Drug structure with one active hydrogen removed; pyronine and analogues thereof are targeting units, wherein R 1-R4 is methyl or ethyl, X represents an exchangeable linking group in the aromatic ring backbone of pyronine and analogues thereof; a - represents an anionic unit.
The molecular-based drug delivery system for multi-organelle targeting and in-situ release provided by the embodiment of the invention can inhibit the growth of tumor cells or inhibit the activity of tumor cells in vitro. The molecular-based drug delivery system for multi-organelle targeting and in-situ release provided by the embodiment of the invention has the effect of inhibiting tumor growth in a nude mouse subcutaneous transplantation tumor model. The molecular-based drug delivery system for targeted and in-situ release of multicellular organelles provided by the embodiment of the invention has at least the following advantages: the preparation method of the molecular-based drug delivery system for multi-organelle targeting and in-situ release provided by the embodiment of the invention has the characteristics of low-cost and easily obtained raw materials, simplicity and convenience in operation, mild reaction conditions, accurate and stable drug loading proportion and the like; the molecular-based drug delivery system for multi-organelle targeting and in-situ release provided by the embodiment of the invention can simultaneously target at least two organelles in mitochondria, endoplasmic reticulum, lysosomes, lipid droplets, golgi apparatus and the like, release parent drugs in situ, reduce the survival rate of tumor cells, and has definite clinical prospects of drugs; the molecular-based drug delivery system for multi-organelle targeting and in-situ release provided by the embodiment of the invention induces death of tumor cells and has almost no toxicity to normal cells.
In an alternative embodiment, the structure of the multicellular organ targeted, in situ released drug delivery system comprises N, O and/or S nucleophilic groups and at least one active H. Illustratively, the parent drug is desloratadine (Desloartadine) having an-NH group within its structure.
In an alternative embodiment, the pyronine and analogs thereof include an alternative linking group X in the aromatic ring backbone, wherein the alternative linking group X in the aromatic ring backbone includes O, C (CH 3)2 or Si (Me) 2. Illustratively, X is an O atom.
In an alternative embodiment, R 1-R4 is methyl or ethyl. Illustratively, R 1、R2、R3、R4 are each methyl.
In an alternative embodiment, the anionic units comprise halogen anions, inorganic or organic oxyacid radical anions. Illustratively, the anion is a chloride anion.
In an alternative embodiment, the multicellular organelle-targeted, in situ delivery system-targeted organelle comprises at least two organelles in the mitochondria, endoplasmic reticulum, lysosomes, lipid droplets, golgi apparatus, and the like. Illustratively, a drug delivery system that releases molecule groups in situ can target both the mitochondria and the endoplasmic reticulum.
In another aspect, embodiments of the present invention provide a method of preparing a multicellular organ targeted, in situ released drug delivery system, the method comprising:
And adding the 9-thiocarbonyl pyronine derivative, the catalyst and the cocatalyst into the solvent in a molar ratio of 1:1.2:10. Illustratively, the molar ratio of 9-thiocarbonylpyronine derivative to catalyst and cocatalyst may be 1:1.2:10.
Stirring is continued at room temperature, and after the reaction is completed, diatomite is used for filtering, so as to obtain filtrate.
Adding parent medicine and triethylamine in the molar ratio of 1:1.2 into the filtrate, reacting for 8-12 hours at room temperature, removing the solvent by screwing, and adding saturated saline.
Extracting and drying to obtain a filter material, and purifying the filter material to obtain the molecular-based drug delivery system with the multi-organelle targeting and in-situ release.
In an alternative embodiment, the solvent does not include a protic solvent, which may be acetonitrile, for example. Preferably, the catalyst is a transition metal salt. Illustratively, the catalyst may be CuCl 2 and the promoter may be KCl.
In an alternative embodiment, the molar ratio of parent drug to triethylamine may be 1:1.2.
In an alternative embodiment, it may comprise adjusting the structure of the anion units of the multicellular organ targeted, in situ released molecular-based drug delivery system by an anion replacement method, resulting in a multicellular organ targeted, in situ released molecular-based drug delivery system comprising different anions.
In an alternative embodiment, the steps of extracting and drying to obtain a filtrate include: extraction was performed with methylene chloride and drying was performed with anhydrous sodium sulfate.
On the other hand, the embodiment of the invention also provides application of the multi-organelle targeted and in-situ released molecular-based drug delivery system, which is prepared by adopting the preparation method of any multi-organelle targeted and in-situ released molecular-based drug delivery system, and the multi-organelle targeted and in-situ released molecular-based drug delivery system is applied to tablets, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar coating agents, granules, dry powder, oral solutions, small water injection, freeze-dried injection, large transfusion or small transfusion.
Example 1:
One embodiment of the invention discloses a method of preparing a multicellular organ targeted, in situ released, molecular-based drug delivery system (DDY).
The method comprises the following specific steps:
To 10mL deoxygenated, dried acetonitrile was added 1.0 mmol of 9-thiocarbonylpyronine, 1.2mmol of CuCl 2, followed by 10.0 mmol of KCl. Stirring was continued at room temperature until the completion of the thioketone reaction, and the reaction mixture was filtered through an appropriate amount of celite. To the filtrate, 1.0 mmol of Desloartadine (parent drug) and 1.2 of mmol of triethylamine were added. The reaction was carried out at room temperature for 12 hours. After the reaction, acetonitrile is removed by screwing, and 10mL saturated saline water is added; dichloromethane extraction and drying with anhydrous sodium sulfate. The organic solvent is dried by spin, and the residue is separated and purified by silica gel column chromatography to obtain the molecular-based drug delivery system (DDY) with the target multicellular organ and in-situ release. In this example, the yield of the multicellular organ targeted, in situ released, molecular-based drug delivery system (DDY) was 75%.
Wherein DDY nuclear magnetic resonance hydrogen spectrum (1 H NMR) is shown in fig. 1, the specific data are as follows:
1H NMR (300 MHz, CDCl3) δ: 8.40 (dd, J = 1.6, 4.8 Hz, 1H), 7.96 (d, J= 9.6 Hz, 2H), 7.49 (dd, J = 1.6, 7.8 Hz, 1H), 7.21 (s, 1H), 7.14 (d, J = 1.4 Hz, 3H), 6.87 (dd, J = 2.6, 9.6 Hz, 2H), 6.55 (d, J = 2.6 Hz, H), 4.23-4.15 (m, 2H), 4.05-3.97 (m, 2H), 3.57-3.44 (m, 2H), 3.19 (s, 12 H), 2.91-2.84 (m, 3H), 2.77-2.74 (m, 1H), 2.73-2.70 (m, 2H).
wherein DDY nuclear magnetic resonance carbon spectrum (13 C NMR) is shown in fig. 2, the specific data are as follows:
13C NMR (75 MHz, CDCl3) δ: 161.4, 157.9, 155.5, 146.2, 140.2, 139.3, 138.5, 137.2, 135.9, 134.5, 133.4, 130.1, 129.6, 129.2, 126.2, 122.8, 114.1, 111.8, 107.2, 96.8, 56.5, 56.4, 40.3, 31.5, 29.7.
as can be seen from fig. 1 and 2, embodiments of the present invention produce a multicellular organ targeted, in situ released, molecular-based drug delivery system (DDY).
Example 2: characterization of multicellular organ targeted, in situ released molecular-based drug delivery System (DDY) in vitro drug release
Test system: phosphate Buffer (PBS) with pH 7.4 and 50mM containing 1% by volume of dimethyl sulfoxide (DMSO). DDY concentration: 10. mu M; ROS concentration: 50. mu M; active nitrogen (RNS) concentration: 50. mu M; active sulfur species (RSS) (excluding glutathione GSH) concentration: 100. mu M; GSH concentration: 1.0 mM. Excitation wavelength: 492 nm. Test temperature: 22. + -0.5 deg.C. The excitation and emission slit widths were 5 nm a.
FIG. 3 is a graph showing fluorescence response spectra of DDY to ROS, RNS, RSS (containing GSH) and other cellular endogenous substances.
DDY presents weak green fluorescence in PBS buffer solution containing 1% DMSO (volume ratio), and only GSH can obviously enhance the fluorescence emission intensity of the system at 535nm in all detected endogenous species of cells such as ROS, RNS, RSS and the like.
FIG. 4 is a graph showing fluorescence time course of DDY at different concentrations of GSH.
Test system: PBS buffer containing 1% DMSO (volume ratio); DDY concentration: 10. mu M. Excitation wavelength: 492 nm. Test temperature: 22+/-0.5 ℃ inclusive; the excitation and emission slit widths were 5 nm a.
DDY has a concentration-and time-dependence on the fluorescence response of GSH; after 15 minutes of action with GSH, the GSH-induced fluorescence emission intensity at 535nm of 2.0 mM is 2.4 times the GSH value of 0.2 mM.
FIG. 5 is a liquid chromatography-mass spectrometry analysis of a reaction equilibrium mixture of DDY and GSH.
Test system: PBS buffer containing 1% DMSO (volume ratio).
To 1.0 mL of the above PBS buffer containing DMSO, 10.0. Mu. Mol of DDY followed by 1.0 mmol of GSH were added. Mix well for 30 minutes at room temperature. And carrying out liquid chromatography-mass spectrometry analysis on the balance mixture. Molecular ion peaks for GSH, the "addition-elimination" products of parent drugs Desloratadine, GSH and DDY, DDY were detected at mass-to-charge ratios 308.00, 310.04, 573.06, 575.08, respectively. The results indicate DDY is capable of releasing the parent drug molecule Desloratadine in situ under the action of GSH.
As can be seen from fig. 3, 4 and 5, the multicellular organ targeted in situ released molecular-based drug delivery system (DDY) provided by the embodiments of the present invention is capable of in situ releasing the parent drug molecule Desloratadine under the action of GSH.
Example 3: antitumor Activity assay of molecular-based drug delivery System (DDY) for Multi-organelle targeting, in situ Release (CCK-8 method)
Experimental cell lines: human normal liver cell strain THLE-2 and human liver cancer cell strain Hep3B.
DDY, desloratadine was dissolved separately in DMSO and the stock solution was stored at-80℃in the dark. In the experimental process, the culture solution of DMEM containing 1% of diabody and 10% of FBS is used for dilution, and the volume fraction of DMSO in the experiment is 0.1%.
The experimental steps are as follows:
The experimental cells were seeded in 96-well plates (density 7×10 3 cells/well) with 100 μl of cell suspension per well, 6 multiplex wells per group, while blank groups were added.
After 24 hours of cell attachment, the old culture broth was discarded and the complete culture broth (containing 0.1% DMSO) was added sequentially at different concentrations of DDY or Desloratadine. After further culturing for 24 hours, the supernatant was aspirated, 10. Mu.L of CCK-8 reagent and 100. Mu.L of complete culture solution were added to each well, and after thorough mixing, the wells were incubated in an incubator for 1 hour, the OD value of each well at 450nm was measured with an ELISA reader, and the cell viability was calculated.
Experimental results show that the molecular-based drug delivery system (DDY) for targeted and in-situ release of multicellular organelles provided by the embodiment of the invention has the effect of obviously inhibiting the growth of liver cancer cells, and has almost no toxicity to normal liver cells.
FIG. 6 is a schematic diagram showing the effect of DDY and parent drug Desloartadine on cell viability of human normal hepatocellular strain THLE-2 and human hepatoma cell strain Hep 3B.
Example 4: organelle targeting of multicellular targeted, in situ released molecular-based drug delivery system (DDY)
The Hep3B cells were inoculated into confocal imaging dishes, 10 μm DDY was added, incubated with commercial co-localized fluorescent probes such as mitochondrial red, endoplasmic reticulum red, lysosome red, etc. respectively for 30min, and fluorescent confocal imaging was performed on the cells. Wherein, DDY excitation wavelength 488 nm, emission band 500-570 nm (green fluorescence channel); commercial probe excitation wavelength 561 nm, emission band 600-1000 nm (red fluorescence channel).
Co-localization imaging analysis results show that Pearson coefficients between DDY green fluorescence and three commercial dyes of mitochondrial red, endoplasmic reticulum red and lysosome red are 0.80, 0.83 and 0.42 respectively, which indicates that the molecular-based drug delivery system (DDY) with multi-organelle targeting and in-situ release provided by the embodiment of the invention has the capability of simultaneously targeting mitochondria and endoplasmic reticulum.
FIG. 7 is a schematic diagram of an organelle localization experiment of DDY. Ruler: 20. μm.
Example 5: the molecular-based drug delivery system (DDY) for targeted and in-situ release of multicellular organ provided by the embodiment of the invention has the inhibition effect on the growth of subcutaneous transplanted tumor of nude mice
15 Female BALB/c nude mice were injected subcutaneously on the back of 4-6 weeks to inoculate Hep3B cells. When the tumor volume was 10×10mm 2, tumor-bearing nude mice were randomly divided into 3 groups of 5. Each group of nude mice was injected with physiological saline, DDY (10 mg/kg), desloratadine (10 mg/kg) via tail vein. After administration, tumor diameters were measured every two days, nude mice weights were measured, and transplanted tumor growth curves were drawn. After 10 days, nude mice were sacrificed, the major and minor diameters of the tumor were measured, the tumor volume was calculated, and the tumor weight was measured.
FIG. 8 is a graphical representation of the growth curve of a transplanted tumor following administration to a tumor-bearing BALB/c nude mouse. FIG. 9 is a schematic representation of tumor volume and weight of tumor-bearing BALB/c nude mice after 10 days of drug treatment.
Experimental results show that Desloratadine has little inhibition effect on the growth of tumors; after DDY provided by the embodiment of the invention is acted for 10 days, the tumor volume is obviously smaller than that of a physiological saline group and a Desloratadine group, and is equivalent to the volume at the initial time of administration, which shows that DDY can effectively inhibit the growth of tumors. On the other hand, DDY provided by the embodiment of the invention does not significantly affect the body weight of the nude mice, which shows that the toxic and side effects in vivo are equivalent to those of the parent drug Desloratadine.
The foregoing description of the embodiments of the present disclosure has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Any changes or substitutions that would be easily recognized by those skilled in the art within the technical scope of the present disclosure are intended to be covered by the present invention. The terminology used herein is chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of the prior art, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
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| CN108578708A (en) * | 2018-04-25 | 2018-09-28 | 中国科学院化学研究所 | A kind of double target polypeptide-drug conjugates and preparation method thereof and antitumor application thereof |
| CN113945552A (en) * | 2021-10-20 | 2022-01-18 | 延边大学 | Method for measuring concentration of reduced glutathione in mitochondria of living cells |
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| WO2025255943A1 (en) | 2025-12-18 |
| CN118320117A (en) | 2024-07-12 |
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