CN114904013A - Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate - Google Patents

Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate Download PDF

Info

Publication number
CN114904013A
CN114904013A CN202110171207.5A CN202110171207A CN114904013A CN 114904013 A CN114904013 A CN 114904013A CN 202110171207 A CN202110171207 A CN 202110171207A CN 114904013 A CN114904013 A CN 114904013A
Authority
CN
China
Prior art keywords
heparan sulfate
calcitriol
conjugate
heparanase
ctl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110171207.5A
Other languages
Chinese (zh)
Inventor
李亚平
黄鑫
郎天群
郑众
尹琦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institute of Materia Medica of CAS
Original Assignee
Shanghai Institute of Materia Medica of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Institute of Materia Medica of CAS filed Critical Shanghai Institute of Materia Medica of CAS
Priority to CN202110171207.5A priority Critical patent/CN114904013A/en
Publication of CN114904013A publication Critical patent/CN114904013A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • Polymers & Plastics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention provides a heparan sulfate-calcitriol conjugate, heparanase responsive nanoparticles containing the conjugate, a preparation method and application of the conjugate. The heparanase responsive nanoparticles containing the heparan sulfate-calcitriol conjugate are spherical nanoparticles with regular particle size and round and uniform shape. The heparanase responsive nanoparticles can obviously inhibit the proliferation, migration, invasion and scratch healing of tumor cells and the tumor growth of a breast cancer mouse model, so the heparanase responsive nanoparticles have a good application prospect in the field of tumor treatment.

Description

Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a heparan sulfate-calcitriol conjugate, heparanase responsive nanoparticles containing the heparanase conjugate, a preparation method and application of the heparanase responsive nanoparticles.
Background
Breast cancer is the first incidence of cancer in women, and chemotherapy remains the classic clinical choice for breast cancer treatment. However, cytotoxic chemotherapy drugs in traditional dosage forms have non-selective toxicity to all rapidly dividing cells, resulting in low systemic therapeutic efficacy and severe toxic side effects, greatly limiting the clinical use of chemotherapy.
Calcitriol (CTL) is the most bioactive vitamin D metabolite, and has various biological activities, such as anti-tumor cell proliferation, apoptosis promotion, differentiation promotion, and tumor metastasis inhibition by binding to Vitamin D Receptor (VDR). The combined use of CTL and chemotherapeutic drugs can enhance the anti-tumor effect and can resist the metastasis caused by taxane chemotherapeutic drugs. Clinically, however, free vitamin D in effective doses can cause hypercalcemia.
CTLs therefore require a suitable vector to promote their accumulation in tumor cells and to reduce their distribution in normal tissues.
Disclosure of Invention
The present invention covalently couples CTL with Heparan Sulfate (HS) to obtain a heparan sulfate-calcitriol conjugate (HS-CTL), which can be used as a prodrug of CTL to promote accumulation of CTL in tumor cells and reduce distribution in normal tissues.
Research shows that the heparan sulfate-docetaxel conjugate (HS-DTX) obtained by covalently coupling HS and Docetaxel (DTX) has strong toxicity to breast cancer cells, has low toxicity to non-cancer breast epithelial cells, and obviously reduces the toxic and side effects of DTX in tumor-bearing mice.
The research of the invention discovers that HS-DTX and HS-CTL can be self-assembled into nanoparticles in aqueous medium. Preliminary studies show that the obtained nanoparticles can obviously inhibit the proliferation, migration, invasion and scratch healing of tumor cells, and obviously inhibit the tumor growth of a breast cancer mouse model.
Accordingly, it is an object of the present invention to provide a heparan sulfate-calcitriol conjugate.
Another object of the present invention is to provide a method for preparing the heparan sulfate-calcitriol conjugate.
It is another object of the present invention to provide the use of the heparan sulfate-calcitriol conjugate described above for the preparation of a medicament.
It is another object of the present invention to provide a pharmaceutical composition comprising the heparan sulfate-calcitriol conjugate according to the invention.
It is another object of the present invention to provide a pharmaceutical composition comprising a heparan sulfate-calcitriol conjugate according to the invention and a heparan sulfate-docetaxel conjugate.
It is still another object of the present invention to provide heparanase-responsive nanoparticles comprising the above-described heparan sulfate-calcitriol conjugate and heparan sulfate-docetaxel conjugate.
Still another object of the present invention is to provide a method for preparing the heparanase-responsive nanoparticle.
Still another object of the present invention is to provide the use of the heparanase-responsive nanoparticles described above for the preparation of a medicament.
It is still another object of the present invention to provide a pharmaceutical composition comprising the heparanase-responsive nanoparticle described above.
In one aspect, the invention provides a heparan sulfate-calcitriol conjugate (HS-CTL), wherein calcitriol is covalently bound to heparan sulfate.
In particular, the heparan sulfate-calcitriol conjugate of the present invention may have a weight average molecular weight of 5000 to 20000, preferably 10000 to 20000, and a molecular weight distribution of 2 to 4, preferably 2 to 3, wherein the molecular weight distribution is a value obtained by dividing the weight average molecular weight by the number average molecular weight.
In particular, in the heparan sulfate-calcitriol conjugate according to the present invention, the content of calcitriol is 5 wt% to 15 wt%, preferably 8 wt% to 15 wt%, based on the total weight of the heparan sulfate-calcitriol conjugate.
Heparan Sulfate (HS) is an extracellular matrix component that can be produced by all cell types. It is a glycosaminoglycan that can be covalently linked to core protein to form proteoglycans. Its structure is very diverse because it undergoes extensive sulfation and epimerization during the synthesis, and therefore does not have a single HS structure.
In the present invention, the heparan sulfate can be commercially available heparan sulfate or can be obtained from commercially available heparan sulfate salts. For example, commercially available sodium heparan sulfate may be acidified (e.g., by treatment with a cationic resin column) to provide heparan sulfate.
In general, heparan sulfate may be represented by formula I below, but is not limited thereto;
Figure BDA0002938973930000031
where n is a positive integer representing the number of repeat monomer units, relative to the molecular weight of the polymer.
Calcitriol may be represented by formula II below:
Figure BDA0002938973930000032
in embodiments, the HS-CTL may have the structure:
Figure BDA0002938973930000033
wherein HS represents heparan sulfate, CTL represents calcitriol, and m is a positive integer representing the number of calcitriol linked to the heparan sulfate chain, wherein m is preferably 2 to 5. In embodiments, the HS-CTL may be obtained as follows: the method comprises the following steps of carrying out esterification reaction on heparan sulfate and succinic anhydride to obtain succinic anhydride heparan sulfate with side chains having carboxyl, and then carrying out esterification reaction on calcitriol and the carboxyl of the succinic anhydride heparan sulfate through any one of hydroxyl on the calcitriol.
On the basis of the heparan sulfate-calcitriol conjugate provided by the invention, one skilled in the art can select an appropriate organic reaction in the field to covalently couple heparan sulfate and calcitriol to obtain the heparan sulfate-calcitriol conjugate provided by the invention. Thus, the method of making the heparan sulfate-calcitriol conjugates of the invention is not limited to the synthetic routes disclosed herein.
In particular, the invention provides a preparation method of a heparan sulfate-calcitriol conjugate, which comprises the following steps:
(1) the method comprises the following steps of carrying out esterification reaction on heparan sulfate and succinic anhydride to obtain succinic anhydride heparan sulfate, wherein the synthesis reaction formula is as follows:
Figure BDA0002938973930000041
(2) the succinic anhydride heparan sulfate and calcitriol are subjected to esterification reaction to obtain a heparan sulfate-calcitriol conjugate, and the synthetic reaction formula is as follows:
Figure BDA0002938973930000042
more particularly, the method for preparing heparan sulfate-calcitriol conjugate of the present invention comprises the following steps:
step A: synthesis of tributylated heparan sulfate
Regulating the pH value of the heparan sulfate aqueous solution to 6-7 by using tributylamine, and drying to obtain tributylated heparan sulfate;
and B: synthesis of succinic anhydride heparan sulfate
B, carrying out esterification reaction on the tributylated heparan sulfate prepared in the step A and succinic anhydride to obtain succinic anhydride-modified heparan sulfate;
and C: synthesis of heparan sulfate-calcitriol conjugate
And D, performing esterification reaction on the succinic acid anhydrization heparan sulfate and calcitriol prepared in the step B to obtain a heparan sulfate-calcitriol conjugate.
Preferably, step B may be carried out in an organic reagent, which may be anhydrous dimethylformamide. Preferably, step B may be carried out in the presence of 4-dimethylaminopyridine. Preferably, the reaction time of step B may be 18 to 24 hours. Preferably, step B may also comprise a purification step. The purification can be carried out by dialysis, for example, the reaction solution can be added into a dialysis bag with molecular weight cutoff of 3500Da, and dialyzed in water for 24-36 hours, and the water can be replaced every 3-4 hours.
Preferably, step C may be carried out in an organic reagent, which may be anhydrous dimethylformamide. Preferably, step C may be carried out in the presence of 4-dimethylaminopyridine and dicyclohexylcarbodiimide. Preferably, the reaction time of step C may be 18 to 24 hours. Preferably, step C may comprise a purification step. Preferably, the purification can be carried out by a dialysis method, for example, the reaction solution can be added into a dialysis bag with the molecular weight cutoff of 3500Da, and dialyzed in water for 36-48 hours, and the water is replaced every 3-4 hours.
In another aspect, the present invention provides the use of a heparan sulfate-calcitriol conjugate according to the invention or of a heparan sulfate-calcitriol conjugate prepared according to the method for the preparation of a heparan sulfate-calcitriol conjugate according to the invention for the preparation of a medicament. In one embodiment, the medicament may be for the treatment of cancer, such as breast cancer.
In another aspect, the present invention provides a pharmaceutical composition comprising the heparan sulfate-calcitriol conjugate according to the present invention or the heparan sulfate-calcitriol conjugate prepared according to the method for preparing the heparan sulfate-calcitriol conjugate according to the present invention.
Research shows that the heparan sulfate-docetaxel conjugate (HS-DTX) of HS and Docetaxel (DTX) has strong toxicity to breast cancer cells, has low toxicity to non-cancer mammary gland epithelial cells, and obviously reduces the toxic and side effects of DTX in tumor-bearing mice. The invention combines HS-DTX and HS-CTL to improve the combination effect of DTX and CTL.
Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising a heparan sulfate-calcitriol conjugate according to the present invention or a heparan sulfate-calcitriol conjugate prepared according to the method for preparing a heparan sulfate-calcitriol conjugate according to the present invention, and a heparan sulfate-docetaxel conjugate.
In the invention, the heparan sulfate-docetaxel conjugate refers to that a part of hydroxyl groups of HS are converted into carboxyl groups by reacting with succinic anhydride, and then react with 2-hydroxyl groups of DTX to form ester bond connection.
Heparan sulfate-docetaxel conjugates can be prepared by methods described in the prior art references (e.g., Lang T, Ran W, Dong X, Zheng Z, Liu Y, Yin Q, Li Y. tumor cells-selective biochemical extraction heparin complexes Mater substrates metallic research cancer. adv Funct Mater 2018; 28: 1707289.).
Alternatively, the heparan sulfate-docetaxel conjugate may be prepared with reference to the preparation method of the heparan sulfate-calcitriol conjugate as described above.
In particular, heparan sulfate-docetaxel conjugates can be prepared as follows:
step A: synthesis of tributylated heparan sulfate
Regulating the pH value of the heparan sulfate aqueous solution to 6-7 by using tributylamine, and drying to obtain tributylated heparan sulfate;
and B: synthesis of succinic anhydride heparan sulfate
B, carrying out esterification reaction on the tributylated heparan sulfate prepared in the step A and succinic anhydride to obtain succinic anhydride-modified heparan sulfate;
step C': and D, performing esterification reaction on the succinic anhydride heparan sulfate prepared in the step B and docetaxel to obtain a heparan sulfate-docetaxel conjugate.
Preferably, step B may be carried out in an organic reagent, which may be anhydrous dimethylformamide. Preferably, step B may be carried out in the presence of 4-dimethylaminopyridine. Preferably, the reaction time of step B may be 18 to 24 hours. Preferably, step B may also include a purification step. The purification can be carried out by dialysis, for example, the reaction solution can be added into a dialysis bag with molecular weight cutoff of 3500Da, and dialyzed in water for 24-36 hours, and the water can be replaced every 3-4 hours.
Preferably, step C' may be carried out in an organic reagent, which may be anhydrous dimethylformamide. Preferably, step C' may be carried out in the presence of 4-dimethylaminopyridine and dicyclohexylcarbodiimide. Preferably, the reaction time of step C' may be 18 to 24 hours. Preferably, step C' may comprise a purification step. The purification can be carried out by dialysis, for example, the reaction solution can be added into a dialysis bag with molecular weight cutoff of 3500Da, and dialyzed in water for 36-48 hours, and the water can be replaced every 3-4 hours.
Further research shows that the heparan sulfate-calcitriol conjugate and the heparan sulfate-docetaxel conjugate can be self-assembled into nanoparticles in an aqueous medium. After administration, HS is degraded by Hpa and free DTX and CTL are released in the tumor, synergistically killing tumor cells and inhibiting metastasis, improving the therapeutic effect of breast cancer.
Therefore, in another aspect, the present invention provides a heparanase-responsive nanoparticle, wherein the nanoparticle is formed by self-assembly of a heparan sulfate-calcitriol conjugate (HS-CTL) according to the present invention or a heparan sulfate-calcitriol conjugate prepared according to the preparation method of the present invention and a heparan sulfate-docetaxel conjugate (HS-DTX) into a mixed micelle in a solvent (e.g., water).
In particular, the heparanase-responsive nanoparticles may have an average particle size of 200nm to 400nm, preferably 200nm to 300nm, and a particle size distribution of 0.1 to 0.5, preferably 0.1 to 0.3.
In another aspect, the present invention provides a method for preparing the heparanase-responsive nanoparticle, comprising the following steps:
dissolving the heparan sulfate-calcitriol conjugate according to the present invention or the heparan sulfate-calcitriol conjugate and the heparan sulfate-docetaxel conjugate prepared according to the method for preparing a heparan sulfate-calcitriol conjugate according to the present invention in a solvent (e.g., deionized water), and filtering the solution to obtain the heparanase-responsive nanoparticle HDC.
Preferably, in the above preparation method, the filtering method is to filter the solution 2 times through a 0.22 micron water-based syringe filter.
In a further aspect, the invention provides the application of the heparanase-responsive nanoparticles in preparing a tumor treatment drug, in particular a drug for treating breast cancer.
In yet another aspect, the present invention provides a pharmaceutical composition comprising the heparanase-responsive nanoparticle described above. The pharmaceutical composition can be used for treating breast cancer.
Drawings
FIG. 1 is a NMR chart of calcitriol in example 2.
FIG. 2 is the NMR spectrum of succinic anhydride-modified heparan sulfate synthesized in example 2.
FIG. 3 is a gel exclusion chromatogram of succinic anhydride-sulfated heparan sulfate synthesized in example 2.
FIG. 4 is the NMR spectrum of HS-CTL synthesized in example 3.
FIG. 5 is a gel exclusion chromatogram of HS-CTL synthesized in example 3.
FIG. 6 is a NMR spectrum of HS-DTX synthesized in example 4.
FIG. 7 is a gel exclusion chromatogram of HS-DTX synthesized in example 4.
FIG. 8 is a graph showing the particle size distribution of HDC nanoparticles of example 6.
FIG. 9 is a transmission electron micrograph of HDC nanoparticles of example 6.
Fig. 10 is a graph showing the effect of the HDC nanoparticles on the proliferation inhibition of tumor cells in example 7.
Fig. 11 is a graph of the migration inhibitory effect of the HDC nanoparticles on tumor cells in example 8 (× p < 0.001).
Fig. 12 is a graph of the invasion inhibitory effect of the HDC nanoparticles on tumor cells in example 9 (× p < 0.001).
Fig. 13 is a graph of the inhibition of scratch healing of tumor cells by HDC nanoparticles in example 10 (. about.. p < 0.001).
FIG. 14 is a photograph of the 4T1 tumor tissue obtained in example 11.
FIG. 15 is a graph showing the change in tumor volume of 4T1 in example 11.
Fig. 16 shows the 4T1 tumor suppression rate (× p <0.01) in example 11.
FIG. 17 is a graph showing the change in body weight of mice in example 11.
Detailed Description
The present invention will be described with reference to the following specific examples, but the present invention is not limited to these specific examples.
Reagents and instrumentation: heparan Sulfate (HS) was purchased from rohm biotechnology limited, tokyo; docetaxel (Docetaxel, DTX), regenerated cellulose dialysis bag (MWCO 3.5kDa), PBS buffer (pH 7.4), Tris-HCl solution (pH 7.4), ampicillin, streptomycin sulfate double antibody solutions were purchased from gangrenes biotechnology limited; succinic anhydride, 4-dimethylaminopyridine, anhydrous dimethylformamide, dicyclohexylcarbodiimide were obtained from Shanghai Bailingwei science and technology Co., Ltd; heparanase (Hpa) was purchased from Thermo Fisher, USA; 0.2% uranium acetate dye solution and carbon supporting film for electron microscope were purchased from Beijing Zhongjing Objective Instrument Co., Ltd; RMPI 1640 medium, Fetal Bovine Serum (FBS), 0.25% Trypsin-EDTA, Trypan blue dye solution purchased from Life Technology, USA; calcitriol was purchased from south beijing luomai biotechnology limited; other reagents are purchased from national pharmaceutical group chemical reagent limited unless otherwise specified, and all solvents are analytically pure and are directly used for experiments without treatment.
Magnetic heating stirrer (MS-H-PRO, national medicine group, Shanghai, China); rotary evaporator (R-300, Buchi, Switzerland); electronic balance (Quintix 224-1, Sartorius, Germany); nuclear magnetic resonance spectrometer (500MHz, Bruker, USA); high performance liquid chromatography (e2695, Waters, USA); ultraviolet spectrophotometer (multistkan GO, Thermo Fisher, USA); freeze drier (laboconco, USA); water purifiers (MILL I-Q, Millipore, USA); transmission electron microscopy (TEM, Tecnai F20, FEI, USA); desktop centrifuges (Centrifuge 5702, Eppendorf, Germany); water jacket type CO 2 Incubators (Forma Series II, Thermo Fisher, USA); biological safety cabinets (laboconco, USA); a cell counter (Countess, Thermo Fisher, USA).
Mouse breast cancer cell line 4T1 was purchased from shanghai cell bank of chinese academy of sciences.
SPF grade Balb/c mice (18-22g, female) were purchased from Shanghai laboratory animals center, Chinese academy of sciences. Feeding at 25 deg.C under 12h light-dark alternating environment, and feeding animals with free diet. All animal experimental procedures strictly follow the requirements of the Committee for the management and use of laboratory animals (IACUC) of the Shanghai pharmaceutical research institute of Chinese academy of sciences.
Example 1 Synthesis of Tributylated heparan sulfate
732 injecting cation exchange resin into the chromatographic column and flattening. 450mL of 1M HCl solution, 450mL of 1M NaOH solution, 200mL of water, 300mL of 1M HCl solution, and 170mL of water were added in this order, and the effluent pH was measured with pH paper to be 6. Weighing 1g of heparan sulfate (in the form of sodium salt), adding 100mL of water for dissolving, adding into a chromatographic column, collecting an effluent, putting into a 250mL flask, adding 2mL of tributylamine, measuring the pH value to be 6-7, and performing rotary evaporation to obtain the tributylated heparan sulfate.
Example 2 Synthesis of succinic anhydrified heparan sulfate
1g of the tributylated heparan sulfate prepared in example 1 was added to 12mL of anhydrous dimethylformamide, dissolved by sonication, and transferred to a 50mL flask. Weighing 1.38mg of 4-dimethylaminopyridine and 11.16mg of succinic anhydride, adding 1mL of anhydrous dimethylformamide into each solution to dissolve, adding the 4-dimethylaminopyridine solution into a flask, dropwise adding the succinic anhydride solution into the flask, and stirring at room temperature for 24 hours. After the reaction, the reaction solution was transferred to a 3500Da dialysis bag and dialyzed in pure water for 24 hours. And after the dialysis is finished, freeze-drying to obtain the succinic anhydride heparan sulfate. The calcitriol and the obtained product were characterized by NMR hydrogen spectra, and the results are shown in FIGS. 1 and 2. The molecular weights and distributions of the obtained products were determined by gel exclusion chromatography, and as shown in FIG. 3 and the following table, the weight average molecular weight of succinic acid-anhydrified heparan sulfate was 11796, and the molecular weight distribution (PDI) was 1.77.
Figure BDA0002938973930000091
Example 3 Synthesis of HS-CTL
1g of succinic anhydridized heparan sulfate obtained in example 2 was dissolved in 15mL of anhydrous dimethylformamide and transferred to a 50mL flask. 71.69mg of dicyclohexylcarbodiimide, 8.38mg of 4-dimethylaminopyridine and 100mg of CTL were weighed and dissolved in 700. mu.L of anhydrous dimethylformamide, respectively. Under the stirring of 400rpm, the 4-dimethylaminopyridine and dicyclohexylcarbodiimide solution is added firstly, stirred for 1min, then CTL is dropwise added into the flask, and stirred for 24h at room temperature. After the reaction, the reaction solution was transferred to a 3500Da dialysis bag and dialyzed in pure water for 48 hours. And after dialysis, freeze-drying for 48 hours to obtain the product HS-CTL. The obtained product is characterized by a nuclear magnetic resonance hydrogen spectrum, the result is shown in figure 4, and the nuclear magnetic resonance spectrogram shows that the HS-CTL comprises characteristic peaks of two parts of HS and CTL, wherein the characteristic peaks belonging to CTL are as follows: ppm 6.00 and 6.20; characteristic peaks belonging to HS are: ppm 3.60-4.0, 4.10-4.80 and 4.80-5.40, indicating successful CTL ligation with HS. The obtained product was measured for molecular weight and distribution thereof by gel exclusion chromatography, and as a result, as shown in fig. 5 and the following table, the HS-CTL weight average molecular weight was 12993, and the molecular weight distribution (PDI) was 2.35, and further, the CTL content in the HS-CTL was calculated to be 9.21%.
Figure BDA0002938973930000092
Example 4 Synthesis of HS-DTX
1g of succinic anhydridized heparan sulfate obtained in example 2 was dissolved in 15mL of anhydrous dimethylformamide and transferred to a 50mL flask. 71.69mg of dicyclohexylcarbodiimide, 8.38mg of 4-dimethylaminopyridine and 163.9mg of DTX were weighed and dissolved in 700. mu.L of anhydrous dimethylformamide, respectively. Under the stirring of 400rpm, firstly adding a 4-dimethylaminopyridine and dicyclohexylcarbodiimide solution, stirring for 1min, then dropwise adding DTX into the flask, and stirring for 24h at room temperature. After the reaction, the reaction solution was transferred to a 3500Da dialysis bag and dialyzed in pure water for 48 hours. After dialysis, the mixture is freeze-dried for 48 hours to obtain the product HS-DTX. The obtained product was characterized by nmr hydrogen spectrum, and the results are shown in fig. 6. The obtained product was measured for molecular weight and distribution by gel exclusion chromatography, and as a result, as shown in fig. 7 and the following table, the weight average molecular weight of HS-DTX was 13426, and the molecular weight distribution (PDI) was 1.94, and further, DTX content in HS-DTX was calculated to be 12.14%.
Figure BDA0002938973930000101
Example 5 preparation of nanoparticles
Weighing 20mg of HS-DTX prepared in the embodiment 4, adding 5mL of water, dissolving by ultrasonic, sucking by a syringe, and filtering by a 0.22 mu m polyethersulfone filter membrane for 2 times to obtain the HS-DTX nanoparticles.
Weighing 2.5mg of HS-CTL prepared in the embodiment 3 and 20mg of HS-DTX prepared in the embodiment 4, adding 5mL of water, dissolving by ultrasonic, sucking by using a syringe, and filtering by using a 0.22 mu m polyether sulfone filter membrane for 2 times to obtain the HDC nanoparticles.
Example 6 particle size and morphology of HDC nanoparticles
The average particle size and surface potential of the HDC nanoparticles prepared in example 5 were measured using a light scattering particle sizer, and the results are shown in fig. 8, where the average particle size of the HDC nanoparticles was 254.57 ± 8.18nm, the particle size distribution (PDI) was 0.271, and the surface potential was-18.53 ± 1.5 mV.
And (3) dropwise adding 7 mu L of the HDC nanoparticles prepared in the embodiment 5 onto a carbon support film special for a transmission electron microscope, and sucking the mixture for 90 seconds by using filter paper. 7 mu L of uranium acetate dye solution is dripped on the carbon supporting membrane, and the dye solution is sucked dry by filter paper after 90 s. After the carbon support film is dried for 30min, the film is shot by a transmission electron microscope, and the result is shown in fig. 9, the HDC nanoparticles are spherical nanoparticles with regular particle size and round and uniform shape.
Example 7 cytotoxicity of HDC nanoparticles
After digestion and counting of 4T1 cells, the cell density was adjusted to 5X 10 4 Each cell/mL, 100 μ L of cell suspension was added to each well in a 96-well plate while setting a blank, i.e., 100 μ L of medium was added to the 96-well plate, and the 96-well plate was placed in an incubator for overnight incubation.
HDC nanoparticles prepared in example 5 were diluted and added to 96-well plates to give DTX final concentrations of 18.62. mu.g/mL, 3.72. mu.g/mL, 1.862. mu.g/mL, 0.372. mu.g/mL, 0.1862. mu.g/mL and CTL final concentrations of 1.35. mu.g/mL, 0.27. mu.g/mL, 0.135. mu.g/mL, 0.027. mu.g/mL, 0.0135. mu.g/mL, and a saline group was set, and after the addition of the drug, the 96-well plates were returned to the incubator to continue the incubation for 24 hours.
And after 24h, taking out the 96-well plate, discarding the culture medium in the 96-well plate, adding 100 mu L of CCK8 working solution into each well, putting the well into an incubator, incubating for 1-4h, and measuring the absorbance at 450nm by using an enzyme-labeling instrument.
Cell Viability (Cell Viability) was calculated according to the following formula:
Figure BDA0002938973930000111
as: absorbance Ac of the test group of wells: absorbance Ab of wells of control without dosing: absorbance of blank solvent well
Cell survival as shown in fig. 10, HDC nanoparticles significantly inhibited 4T1 cell proliferation, and the inhibitory potency increased with increasing HDC nanoparticle concentration.
Example 8 inhibition of cell migration by HDC nanoparticles
After digestion and counting of 4T1 cells, the cell density was adjusted to 7.5X 10 with incomplete medium 5 Individual cells/mL. 0.5mL of complete medium containing 10% Fetal Bovine Serum (FBS) was added to each well of a 24-well plate, a 24-well plate-adapted Transwell chamber was placed in the 24-well plate, 0.2mL of cell suspension was added to each well of the chamber, and a 24-well plate was placed in the culture mediumCulturing in a culture box for 4 h.
After the cells are cultured for 4 hours, the preparations of normal saline, free DTX + CTL, HS-DTX and HDC are added into each group of cells respectively, the final concentration of DTX is 0.28 mug/mL, the final concentration of CTL is 0.02 mug/mL, and after the medicines are added, the 24-hole plate is put back into the incubator to be continuously cultured for 24 hours.
After 24h, the 24-well plate was removed, the medium was discarded, the chamber was washed 1 time with Phosphate Buffered Saline (PBS), the cells on the upper layer of the chamber were wiped off with a cotton swab, and the chamber was fixed with 95% ethanol for 20 min. After ethanol fixation, the chamber was washed 1 time with PBS, and crystal violet dye was added to the chamber and stained for 50 min. After crystal violet staining, the chamber was washed several times with PBS, excess water was blotted with filter paper, and the chamber was photographed under a microscope.
The result is shown in fig. 11, compared with HS-DTX nanoparticles, HDC nanoparticles can significantly inhibit cell migration, with a cell migration rate of 15.99%, and the effect is comparable to that of the free drug group.
Example 9 inhibition of cell invasion by HDC nanoparticles
The 24-well plate-adapted Transwell chamber is placed into a 24-well plate, 120 mu L of Matrigel is diluted by 20 times, 100 mu L of diluted Matrigel is added into each chamber, and the 24-well plate is placed into an incubator and fixed for 24 hours to promote the solidification of the Matrigel. After 24h of fixation the 24-well plate was removed and the liquid above the matrigel was carefully discarded. After digestion and counting of 4T1 cells, the cell density was adjusted to 10 using incomplete medium 6 Individual cells/mL. 0.5mL of complete medium containing 10% FBS was added to each well of a 24-well plate, 0.2mL of cell suspension was added to each well of the chamber, and the 24-well plate was placed in an incubator for 4 h.
After the cells are cultured for 4 hours, the preparations of Saline, free DTX + CTL, HS-DTX and HDC are added into each group of cells, so that the final concentration of DTX is 0.28 mu g/mL and the final concentration of CTL is 0.02 mu g/mL, and after the medicines are added, the 24-hole plate is put back into the incubator to be continuously cultured for 24 hours.
After 24h, the 24-well plate was removed, the medium was discarded, the chamber was washed 1 time with PBS, the cells on the upper layer of the chamber were wiped off with a cotton swab, and the chamber was fixed with 95% ethanol for 20 min. After ethanol fixation, the chamber was washed 1 time with PBS, and crystal violet dye was added to the chamber and stained for 50 min. After crystal violet staining, the chamber was washed several times with PBS, excess water was blotted with filter paper, and the chamber was photographed under a microscope.
The result is shown in fig. 12, compared with the HS-DTX nanoparticles, the HDC nanoparticles can significantly inhibit cell invasion, the cell invasion rate is 10.02%, and the effect is equivalent to that of the free drug group.
Example 10 inhibition of cell scratch healing by HDC nanoparticles
After digestion and counting of 4T1 cells, the cell density was adjusted to 2X 10 5 Each cell/mL, 1mL of cell suspension was added to each well of a 24-well plate, and the 24-well plate was placed in an incubator for 24 hours. After 24h of cell culture, scratching each well of cells by using a medium-sized pipette tip, washing the cells once by using PBS, and photographing the scratches under a microscope. Adding the preparation Saline, free DTX, free CTL, free DTX + CTL, HS-DTX and HDC into each group of cells respectively to ensure that the final concentration of DTX is 0.28 mu g/mL and the final concentration of CTL is 0.02 mu g/mL, and after adding the medicines, putting the 24-hole plate back into the incubator to continue culturing for 24 hours. After 24h, the 24-well plate was removed, the medium was discarded, and the scratch was photographed under a microscope.
The result is shown in fig. 13, compared with HS-DTX nanoparticles, HDC nanoparticles can significantly inhibit healing of cell scratches, the healing rate of cell scratches is 8.84%, and the effect is equivalent to that of a free drug group.
Example 11 HDC nanoparticles inhibit Breast cancer growth
After digestion and counting of 4T1 cells, the cell density was adjusted to 10 7 One cell/mL, the second pair of mammary glands of each female Balb/c mouse was inoculated with 100. mu.L of cells to a tumor volume of about 100mm 3 The administration was started in Saline group, free DTX group, free CTL group, free DTX + CTL group, HS-DTX group and HDC group, DTX administration dose was 2.39mg/kg, CTL administration dose was 0.17mg/kg, each group was administered by tail vein injection, 3 times per week in weeks 1 and 2, and 2 times in week 3. Before each administration and before the mice are dissected at the test end point, the maximum diameter (Major axis) and the minimum diameter (Minor axis) of the Tumor of the mice are measured by using a vernier caliper, the Tumor Volume (Tumor Volume) is calculated and recorded, and the Tumor growth inhibition capacity of each group of the preparation is examined.
Tumor volume was calculated according to the following formula:
Figure BDA0002938973930000121
on day 21 after the 1 st dose, CO 2 The mice of each group were sacrificed by asphyxiation, tumor tissues of the mice were dissected and harvested, superficial blood was washed off with cold PBS, after blotting with filter paper, the tumor tissues of each group were photographed, weighed (W) using an electronic balance and recorded. Tumor Inhibition Rate (TIR) was calculated according to the following formula:
Figure BDA0002938973930000131
the tumor growth inhibition of the preparations in each group is shown in figures 14, 15 and 16, and the tumor growth inhibition effect of free DTX and free CTL is poor, and the TIRs are 11.19 percent and 15.85 percent respectively; free DTX + CTL can inhibit the tumor growth to a certain extent, and the TIR of the free DTX + CTL is 30.89%; compared with the free medicine group, the HS-DTX nano-particle has slightly improved anti-tumor growth effect, and the TIR is 41.64%; the HDC nanoparticles showed good anti-tumor proliferative capacity with a TIR of 67.8%.
Body weight reflects the health of the mice, and the change in body weight of the mice after administration is shown in fig. 17, and the body weight of the mice in free CTL, free DTX + CTL and free DTX groups is significantly reduced. The weight of the mice is not obviously reduced after the HS-DTX and HDC preparations are administrated for a plurality of times, the state of the mice is good, and the HS-DTX and HDC preparations are preliminarily proved not to cause obvious toxicity to the mice.

Claims (10)

1. A heparan sulfate-calcitriol conjugate (HS-CTL), wherein calcitriol is covalently bound to heparan sulfate.
2. Heparan sulphate-calcitriol conjugate according to claim 1, wherein the HS-CTL has a weight average molecular weight of 5000 to 20000, preferably 10000 to 20000, a molecular weight distribution of 2 to 4, preferably 2 to 3,
in particular, the calcitriol is present in an amount ranging from 5% to 15% by weight, preferably from 8% to 15% by weight, based on the total weight of HS-CTL.
3. The heparan sulfate-calcitriol conjugate of claim 1, wherein,
the HS-CTL has the following structure:
Figure FDA0002938973920000011
wherein HS represents heparan sulfate, CTL represents calcitriol, and m is a positive integer representing the number of calcitriol linked to the heparan sulfate chain, wherein m is preferably 2 to 5.
4. A preparation method of a heparan sulfate-calcitriol conjugate comprises the following steps:
(1) the method comprises the following steps of carrying out esterification reaction on heparan sulfate and succinic anhydride to obtain succinic anhydride heparan sulfate, wherein the synthesis reaction formula is as follows:
Figure FDA0002938973920000012
(2) the succinic anhydride heparan sulfate and calcitriol are subjected to esterification reaction to obtain a heparan sulfate-calcitriol conjugate, and the synthetic reaction formula is as follows:
Figure FDA0002938973920000021
5. use of a heparan sulfate-calcitriol conjugate according to any of claims 1 to 3 or of a heparan sulfate-calcitriol conjugate prepared according to the method of preparation of a heparan sulfate-calcitriol conjugate according to claim 4 for the preparation of a medicament.
6. A pharmaceutical composition comprising the heparan sulfate-calcitriol conjugate of any one of claims 1 to 3 or the heparan sulfate-calcitriol conjugate prepared by the method of preparation of the heparan sulfate-calcitriol conjugate of claim 4; preferably, the conjugate comprises the heparan sulfate-calcitriol conjugate as defined in any one of claims 1 to 3 or the heparan sulfate-calcitriol conjugate as defined in claim 4 prepared by the method for preparing the conjugate, and the heparan sulfate-docetaxel conjugate.
7. A heparanase-responsive nanoparticle formed by self-assembly of a heparan sulfate-calcitriol conjugate prepared from a heparan sulfate-calcitriol conjugate according to any one of claims 1 to 3 or a method of preparing a heparan sulfate-calcitriol conjugate according to claim 4 in a solvent with a heparan sulfate-docetaxel conjugate into a mixed micelle.
8. The method of claim 7, comprising the steps of:
dissolving the heparan sulfate-calcitriol conjugate as defined in any one of claims 1 to 3 or the heparan sulfate-calcitriol conjugate as defined in claim 4 and the heparan sulfate-docetaxel conjugate in a solvent, and filtering the solution to obtain the heparanase-responsive nanoparticle HDC.
9. Use of heparanase-responsive nanoparticles according to claim 7 for the preparation of a medicament for the treatment of tumors.
10. A pharmaceutical composition comprising the heparanase-responsive nanoparticle of claim 7.
CN202110171207.5A 2021-02-08 2021-02-08 Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate Pending CN114904013A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110171207.5A CN114904013A (en) 2021-02-08 2021-02-08 Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110171207.5A CN114904013A (en) 2021-02-08 2021-02-08 Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate

Publications (1)

Publication Number Publication Date
CN114904013A true CN114904013A (en) 2022-08-16

Family

ID=82761710

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110171207.5A Pending CN114904013A (en) 2021-02-08 2021-02-08 Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate

Country Status (1)

Country Link
CN (1) CN114904013A (en)

Similar Documents

Publication Publication Date Title
JP6550008B2 (en) Derivatized hyperbranched polyglycerols
CN101254308B (en) Biogastrone acid-polyethyleneglycol /chitosan liver target composite drug administration system and preparation thereof
Chen et al. Saporin-loaded CD44 and EGFR dual-targeted nanogels for potent inhibition of metastatic breast cancer in vivo
KR102237234B1 (en) Integrated nanosystem for co-transporting genes/drugs with liver targeting and method for manufacturing the same
CN107669632B (en) Drug carrier, micelle, drug preparation, preparation method and application thereof
CN113264906B (en) Docetaxel dimer micromolecular prodrug and construction of self-assembled nanoparticle thereof
CN103705534A (en) Preparation of natural active substance constructed polymer composite medicine and application thereof in inhibiting angiogenesis
CN108670954B (en) Chemotherapeutic drug co-loaded glycyrrhetinic acid prodrug micelle and preparation method thereof
CN115252560B (en) Self-assembled nanoparticle based on natural product and preparation method and application thereof
CN112089845B (en) Taxane drug-adriamycin prodrug self-assembly nanoparticles and application thereof
CN105963706A (en) Branched polyHPMA copolymer-DOX conjugate, and preparation method and application thereof
CN113616803A (en) GSH response type gemcitabine nano-particle and preparation method and application thereof
KR101429668B1 (en) Nanoparticles comprising amphiphilic low molecular weight hyaluronic acid complex and a process for the preparation thereof
CN111249473B (en) Delivery system and preparation method of polymerized chloroquine fluorene methyl carbonyl nanogel
Xu et al. The design and synthesis of redox-responsive oridonin polymeric prodrug micelle formulation for effective gastric cancer therapy
CN109953974B (en) Preparation method of enzyme-reduction dual-responsiveness hyaluronic acid-polypropylene sulfide copolymer nanocapsule
CN114904013A (en) Heparan sulfate-calcitriol conjugate, heparanase-responsive nanoparticle containing heparanase-responsive nanoparticle, and preparation method and application of heparanase-calcitriol conjugate
CN115969992A (en) Phenylboronic acid copolymer for remodeling tumor immune microenvironment and application thereof
KR101332001B1 (en) Nanoparticles comprising amphiphilic low molecular weight hyaluronic acid complex and a process for the preparation thereof
CN113041355B (en) Co-delivery nano-drug capable of accurately regulating and controlling ratio of combined drug and application
CN110279856B (en) PEG-Peptide photodynamic-chemotherapy combined drug delivery system and preparation method and application thereof
CN115154422B (en) CD44 targeting and ROS response nano micelle pharmaceutical composition, and preparation method and application thereof
CN109568592B (en) Nano gel NG1 and application thereof
CN115192523B (en) Tripterine prodrug self-assembled nanoparticle taking phenylboronate as connecting unit, and preparation method and application thereof
KR20120120712A (en) Water souble Heparin-DTX-TCA conjugates improved targeting and preparing method for the same

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination