CN114870031A - CD44 targeted taxane nanocrystal and preparation method and application thereof - Google Patents
CD44 targeted taxane nanocrystal and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a CD44 targeted taxane nanocrystal, which comprises taxane drugs and glycosaminoglycan derivative carriers. The invention also discloses a preparation method of the CD44 targeted taxane nano crystal, wherein the nano crystal obtains a highly dispersed nano crystal by utilizing one or more of hydrogen bond action, hydrophobic action and pi-pi accumulation action of Fmoc group of glycosaminoglycan derivative carrier and taxane drugs. The invention also discloses application of the CD44 targeted taxane nanocrystals in preparation of a medicament for treating CD44 receptor high-expression diseases, and the nanocrystals can be prepared into a freeze-dried preparation. The taxane nano crystal actively targets the medicine to specific tumor cells by virtue of the high affinity of the polysaccharide skeleton and the CD44 receptor highly expressed on the surface of the tumor cells, acts on a treatment target, and can remarkably improve the medicine concentration of the tumor part, thereby enhancing the anti-tumor curative effect.
Description
Technical Field
The invention relates to the field of medicinal preparations, in particular to a CD44 targeted taxane nanocrystal and a preparation method and application thereof.
Background
The taxane medicines are broad-spectrum antitumor medicines with cytotoxicity, and inhibit the depolymerization of microtubules by promoting the formation of microtubules, thereby inhibiting the mitosis and proliferation of tumor cells. The taxanes approved by FDA to be marketed include Paclitaxel (PTX) and Docetaxel (DTX), both of which have good therapeutic effects on tumors such as ovarian cancer and breast cancer. Wherein, paclitaxel is a fat-soluble drug, the solubility in water is extremely low, and the commercial paclitaxel injection takes polyoxyethylene castor oil and absolute ethyl alcohol as solvents for solubilization, which may cause severe hypotension, anaphylactic reaction and neurotoxicity; DTX is also poor in water solubility, and is mainly solubilized by Tween 80 and ethanol, and a large amount of Tween 80 is easy to cause adverse reactions such as hemolysis and allergy. Therefore, it is important to improve the solubility of the chemotherapeutic drug, reduce the toxic and side effects and improve the targeting property of the drug.
Tumor cells often have many different types of receptor overexpression on their surface compared to normal cells, such as the CD44 receptor. CD44 is a multifunctional transmembrane glycoprotein associated with tumor cell invasion, metastasis and resistance to chemotherapeutic drugs. Glycosaminoglycans such as chondroitin sulfate, hyaluronic acid and heparin are natural targeting ligands of CD44 and are commonly used for the targeted treatment of tumors. The glycosaminoglycan such as chondroitin sulfate, hyaluronic acid and heparin is natural mucopolysaccharide, has the characteristics of no toxicity, no immunogenicity, good biocompatibility, degradability and the like, can be specifically combined with a CD44 receptor on a tumor cell membrane, and a constructed drug delivery system shows excellent tumor targeting characteristics.
The nano-crystal drug contains no or a small amount of carrier materials, can improve drug-loading capacity, reduce toxic and side effects of the carrier, and can well solve the problem of solubilization of insoluble drugs.
Based on the above background, a CD44 targeted nanocrystal delivery system was designed. The natural polysaccharide is used as a framework, lipid-soluble micromolecules with Fmoc groups are coupled on the polysaccharide through covalent bonds to form amphiphilic glycosaminoglycan derivative carriers with CD44 targeting property, and taxane drugs are loaded by a bottom-up precipitation method to prepare the nanocrystal preparation, and the nanocrystal preparation has the following characteristics: the Fmoc group wraps the insoluble drug through pi-pi accumulation with the taxane drugs, so that the solubility, the stability and the in-vivo circulation time of the drug are remarkably improved. Secondly, the medicine is actively targeted to the tissues and specific cells with high expression of CD44 by virtue of high affinity of glycosaminoglycan and CD44 receptor, and the medicine concentration of the focal tissues is improved. And the glycosaminoglycan derivative carrier can improve the drug-loading rate of the taxane drugs and reduce the toxic and side effects caused by auxiliary materials.
Disclosure of Invention
The invention aims to provide a CD44 targeted taxane nanocrystal and a preparation method and application thereof, which are used for improving the solubility of chemotherapeutic drugs in the prior art, reducing toxic and side effects and improving the targeting property of drugs.
To achieve the above objects, in one embodiment, the present invention provides a CD44 targeted taxane nanocrystal, the nanocrystal comprising a taxane and a glycosaminoglycan derivative carrier.
In a preferable scheme of the invention, the glycosaminoglycan derivative carrier is prepared by coupling a polysaccharide skeleton and a fat-soluble small molecule with an Fmoc group through an amido bond; the taxane medicine is selected from one or two of paclitaxel and docetaxel.
In a preferred embodiment of the present invention, the glycosaminoglycan material of the glycosaminoglycan derivative carrier is selected from any one or two of hyaluronic acid, chondroitin sulfate and low molecular weight heparin, and the molecular weight of the glycosaminoglycan material is 1kDa to 500 kDa; the fat-soluble micromolecules with Fmoc groups are selected from one or more of Fmoc-Val-Gly-OH, Fmoc-Gly, Fmoc-AEEA and Fmoc-GABA containing Fmoc groups.
In a preferred embodiment of the present invention, a method for preparing a glycosaminoglycan derivative support, comprises the steps of:
step (1): dissolving glycosaminoglycan containing carboxyl in a reaction solvent, adopting a connecting arm with amino at two ends, carrying out condensation reaction by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine or 1-ethyl- (3-dimethylaminopropyl) carbodiimide and hydroxysuccinimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole as activating agents, and reacting polysaccharide with the amino at one end of the connecting arm to obtain an intermediate;
step (2): dissolving the intermediate and the fat-soluble micromolecule containing the Fmoc group in a reaction solvent, and generating an amido bond by the amino group of the intermediate and the carboxyl group of the fat-soluble micromolecule containing the Fmoc group to obtain the amphiphilic glycosaminoglycan carrier targeting the CD44 receptor;
in the step (1), the connecting arm with amino at two ends is any one of ethylenediamine, adipic dihydrazide, diaminohexane and cystamine.
Based on the CD44 targeted taxane nanocrystal disclosed by the invention, the invention also discloses a preparation method of the CD44 targeted taxane nanocrystal, and the nanocrystal utilizes one or more of hydrogen bonding action, hydrophobic action and pi-pi stacking action of Fmoc group of glycosaminoglycan derivative carrier and taxane drugs to obtain highly dispersed nanocrystals.
In a preferred embodiment of the present invention, the method for preparing nanocrystals specifically comprises the following steps:
step (1): dispersing taxane medicine in good solvent, stirring in dark place until the solution is clear
Step (2): dispersing the solution of step (1) in a poor solvent, and adding a glycosaminoglycan derivative carrier to the poor solvent;
and (3): and (3) removing the good solvent in the step (1) to obtain a suspension, wherein the mass ratio of the taxane drugs to the glycosaminoglycan derivative carrier is 1: 2-1: 10;
and (4): freeze drying to obtain nanometer crystal preparation of taxane medicine.
In a preferable scheme of the invention, the good solvent is selected from any one or a mixture of more of ethanol, acetone, dimethyl sulfoxide, tetrahydrofuran and dimethylformamide; the poor solvent is water, and the mixing ratio of the good solvent to the poor solvent is 2: 1-1: 200.
in a preferred embodiment of the present invention, before the taxane nanocrystal preparation prepared in step (4) is used, an injection solvent is added to redissolve and disperse uniformly, so that the taxane nanocrystal preparation can be used.
The invention also discloses application of the CD44 targeted taxane nanocrystals in preparation of a medicament for treating CD44 receptor high-expression diseases, and the nanocrystals can be prepared into a freeze-dried preparation.
In one preferable scheme of the invention, a freeze-drying protective agent is added in the process of preparing the nano crystal into a freeze-drying preparation, wherein the freeze-drying protective agent is one or more of mannitol, trehalose, lactose, sucrose and glucose, and the mass ratio of the freeze-drying protective agent to the medicine is 0: 1-1: 2.
in conclusion, the beneficial effects of the invention are as follows:
1. the nano crystal preparation actively targets the medicine to specific tumor cells by virtue of the high affinity of the polysaccharide skeleton and the CD44 receptor highly expressed on the surface of the tumor cells, acts on a treatment target, and can remarkably improve the medicine concentration of tumor parts, thereby enhancing the anti-tumor curative effect.
2. The glycosaminoglycan derivative carrier can effectively improve the drug loading rate of the taxane drugs, the drug loading rate can reach 30-50%, and the carrier is natural polysaccharide, so that the toxic and side effects can be reduced, and the biocompatibility can be improved.
3. The taxane nano crystal prepared by the invention has CD44 targeting property due to the fact that the surface of the taxane nano crystal is covered by the glycosaminoglycan derivative carrier, and the effective concentration of the medicine in a target tissue is improved.
4. The taxane nano crystal can be suspended in water, glucose injection or physiological saline in the form of freeze-dried powder injection to form suspension for intravenous injection. Therefore, when the medicinal solution is prepared, additional solubilizer such as polyoxyethylene castor oil and Tween 80 is not needed, so that the toxic and side effects caused by the solubilizer are removed, and the safety of the preparation is improved.
5. The invention has the advantages of good biocompatibility, high drug loading, good safety, strong stability and simple preparation process.
6. Cell experiments show that cells with high expression of CD44 receptors, such as pancreatic cancer cells, melanoma cells and breast cancer cells, have higher uptake efficiency on the taxane nanocrystal preparation prepared by the invention.
Drawings
FIG. 1 is a structural formula of paclitaxel and docetaxel in accordance with one embodiment of the present invention;
FIG. 2 shows CS-NH in an embodiment of the present invention 2 Structure and nuclear magnetic hydrogen spectrum of (a);
FIG. 3 shows an embodiment of the present invention in which CS-NH is present 2 (ii) an infrared spectrum of;
FIG. 4 is a graph of the structure and nuclear magnetic hydrogen spectrum of CS-Fmoc in accordance with an embodiment of the present invention;
FIG. 5 shows HA-NH according to an embodiment of the present invention 2 Structure and nuclear magnetic hydrogen spectrum of (a);
FIG. 6 is a graph of the structure and nuclear magnetic hydrogen spectra of HA-Fmoc in accordance with an embodiment of the present invention;
FIG. 7 is a graph of particle size of PTX NC @ CS-Fmoc in accordance with an embodiment of the present invention;
FIG. 8 is a Zeta potential diagram of PTX NC @ CS-Fmoc in accordance with an embodiment of the present invention;
FIG. 9 is a graph of the particle size of DTX NC @ CS-Fmoc in accordance with an embodiment of the present invention;
FIG. 10 is a Zeta potential diagram of DTX NC @ CS-Fmoc in accordance with an embodiment of the present invention;
FIG. 11 is a graph of reconstituted particle sizes of a lyophilized sample without lyoprotectant in accordance with an embodiment of the present invention;
FIG. 12 is a graph of reconstituted particle size of a lyophilized sample with sucrose added in accordance with one embodiment of the present invention;
FIG. 13 is a graph of reconstituted particle size of a lyophilized sample to which mannitol has been added according to one embodiment of the present invention;
FIG. 14 is a graph of the change in particle size of a lyophilized sample after reconstitution in accordance with one embodiment of the present invention;
FIG. 15 is an appearance of reconstitution of a lyophilized sample according to one embodiment of the present invention;
FIG. 16 is an X-ray diffraction pattern of PTX NC @ CS-Fmoc nanocrystals in accordance with an embodiment of the present invention;
FIG. 17 is an electron micrograph of nanocrystals according to one embodiment of the present invention. (A) A transmission electron microscope picture of PTX NC @ CS-Fmoc, (B) a scanning electron microscope picture of PTX NC @ CS-Fmoc, (C) a transmission electron microscope picture of DTX NC @ CS-Fmoc, (D) a transmission electron microscope picture of PTX NC @ HA-Fmoc;
FIG. 18 is a graph showing the uptake of PTX NC @ CS-Fmoc by B16F10 cells in accordance with an embodiment of the present invention;
FIG. 19 is a graph showing the uptake of PTX NC @ CS-Fmoc by Panc02 cells in accordance with one embodiment of the present invention;
FIG. 20 is a graph showing the uptake of PTX NC @ CS-Fmoc by 4T1 cells in accordance with one embodiment of the present invention;
FIG. 21 is a graph of the energy of bioluminescence following administration of various formulations in one embodiment of the present invention;
FIG. 22 is a graph of the change in body weight of C57bl/6 after administration of different formulations in one embodiment of the invention;
FIG. 23 is a graph of the quality of the pancreas following administration of various formulations in one embodiment of the present invention;
FIG. 24 is a graph of tumors after completion of dosing in one embodiment of the invention;
FIG. 25 is a graph of immunohistochemistry following administration of various formulations in one embodiment of the present invention.
Detailed Description
The invention provides a CD44 targeted taxane nanocrystal, which comprises taxane drugs and glycosaminoglycan derivative carriers. Wherein the taxane is selected from one or two of paclitaxel and docetaxel, and the structural formula of paclitaxel and docetaxel is shown in figure 1.
The glycosaminoglycan derivative carrier is prepared by coupling polysaccharide skeleton and liposoluble micromolecules with Fmoc group through amido bond. The glycosaminoglycan material of the glycosaminoglycan derivative carrier is selected from one or two of hyaluronic acid, chondroitin sulfate and low molecular weight heparin, the molecular weight of the glycosaminoglycan material is 1 kDa-500 kDa, and preferably the molecular weight of the glycosaminoglycan material is 1 kDa-50 kDa.
The fat-soluble micromolecule with Fmoc group is selected from one or more of Fmoc-Val-Gly-OH, Fmoc-Gly, Fmoc-AEEA and Fmoc-GABA containing Fmoc group, preferably, the fat-soluble micromolecule with Fmoc group is Fmoc-Gly or Fmoc-AEEA.
A method for preparing a glycosaminoglycan derivative support, comprising the steps of:
step (1): dissolving glycosaminoglycan containing carboxyl in a reaction solvent, adopting a connecting arm with amino at two ends, carrying out condensation reaction by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine or 1-ethyl- (3-dimethylaminopropyl) carbodiimide and hydroxysuccinimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole as activating agents, and reacting polysaccharide with the amino at one end of the connecting arm to obtain an intermediate;
step (2): and dissolving the intermediate and the fat-soluble micromolecule containing the Fmoc group in a reaction solvent, and generating an amido bond by the amino group of the intermediate and the carboxyl group of the fat-soluble micromolecule containing the Fmoc group to obtain the amphiphilic glycosaminoglycan carrier targeting the CD44 receptor.
Wherein in the step (1), the connecting arm containing amino groups at two ends is any one of ethylenediamine, adipic acid dihydrazide, diaminohexane and cystamine, and the reaction solvent in the step (2) is any one of water, methanol, N-dimethylformamide, formamide, dimethyl sulfoxide, a mixed solvent of water and methanol, a mixed solvent of water and N, N-dimethylformamide, a mixed solvent of water and formamide and a mixed solvent of N, N-dimethylformamide and formamide.
The particle size of the nanometer crystal is 100 nm-500 nm, wherein the short diameter is 20 nm-50 nm, the long diameter is 100 nm-500 nm, and the shape of the nanometer crystal preparation is rod-shaped or square.
The taxane nanocrystal can also be prepared into a freeze-drying preparation, the freeze-drying stability is good, and a freeze-drying protective agent can be added or not added in the process of preparing the freeze-drying preparation. The added freeze-drying protective agent is one or more selected from mannitol, trehalose, lactose, sucrose and glucose, and preferably, the freeze-drying protective agent is mannitol.
The mass ratio of the freeze-drying protective agent to the medicine is 0: 1-1: 2, preferably, the mass ratio of the freeze-drying protective agent to the medicine is 0: 1-0.2: 1.
the invention also discloses a preparation method of the CD44 targeted taxane nano crystal, wherein the nano crystal obtains a highly dispersed nano crystal by utilizing one or more of hydrogen bond action, hydrophobic action and pi-pi accumulation action of Fmoc group of glycosaminoglycan derivative carrier and taxane drugs.
The invention prepares taxane nanocrystals by a top-down precipitation method, and the preparation method of the nanocrystals specifically comprises the following steps:
step (1): dispersing the taxane drugs in a good solvent, and stirring in the dark until the solution is clear;
step (2): dispersing the solution of step (1) in a poor solvent, and adding a glycosaminoglycan derivative carrier to the poor solvent;
and (3): and (3) removing the good solvent in the step (1) to obtain a suspension, wherein the mass ratio of the taxane drugs to the glycosaminoglycan derivative carrier is 1: 2-1: 10, freeze-drying to obtain taxane medicine nanocrystal freeze-dried powder;
and (4): before use, adding an injection solvent to redissolve the taxane nanocrystal freeze-dried powder obtained in the step (4), and uniformly dispersing to obtain the taxane nanocrystal freeze-dried powder for use.
Wherein, the good solvent in the step (1) includes but is not limited to one or a mixture of ethanol, acetone, dimethyl sulfoxide, tetrahydrofuran or dimethylformamide, and ethanol or dimethyl sulfoxide is preferred; the poor solvent in the step (2) is water, the good solvent in the step (3) is removed in any one of freeze drying, vacuum drying, reduced pressure rotary evaporation and dialysis, preferably reduced pressure rotary evaporation or dialysis, and the dispersion mode includes but is not limited to one or more of rapid stirring, water bath ultrasound, probe ultrasound or high pressure homogenization, preferably rapid stirring or probe ultrasound; a freeze-drying protective agent can also be added in the step (3), the specific step is to disperse the solution obtained in the step (3) in the freeze-drying protective agent, wherein the freeze-drying protective agent is selected from one or more of mannitol, trehalose, lactose, sucrose and glucose, preferably mannitol, and the mass ratio of the freeze-drying protective agent to the medicine is 0: 1-1: 2, preferably 0: 1-0.2: 1.
the invention also discloses application of the CD44 targeted taxane nanocrystal in preparation of a medicine for treating CD44 receptor high-expression diseases.
Example 1: synthesis and characterization of CS vector (CS-Fmoc)
(1) First, CS intermediate (CS-NH) is synthesized 2 ) 1545mg of CS was weighed out and dissolved in 30mL of ultrapure water, 4.023g of EDC was weighed out and dissolved in 15mL of ultrapure water, 2.415g of NHS was weighed out and dissolved in 12mL of ultrapure water, and added to the CS solution, and the carboxyl group was activated by stirring at room temperature for 30 min. Adding 5.58mL of ethylenediamine into a 250mL flask, adding 45mL of ultrapure water, adjusting the pH to about 7 with 12mL of concentrated hydrochloric acid, slowly adding activated CS into the ethylenediamine solution with a constant-pressure dropping funnel under an ice bath condition, dropwise adding for 60min under the ice bath condition, heating the reaction system to room temperature to react for 3.5h, dialyzing for 4d in water with an MWCO5000 dialysis bag, centrifuging, taking the supernatant, and freeze-drying to obtain a product CS-NH 2 And confirming CS-NH by nuclear magnetic hydrogen spectrum and infrared spectrum 2 The structure of (1). FIG. 2 shows CS-NH 2 The characteristic peak of CS was N-acetyl (a,1.90 ppm). The signals of primary (b, 2.73ppm) and secondary (c, 3.39ppm) amine groups of ethylenediamine indicate that ethylenediamine successfully introduced CS; FIG. 3 shows CS-NH 2 The characteristic peak of C-H vibration of the infrared spectrum of (1) is 2925cm -1 The characteristic peak of the amide I wave band is 1640cm -1 The characteristic peak of the amide II wave band is 1558cm -1 . CS-NH in contrast to CS 2 At 1560cm -1 The peak at (a) is significantly increased. Indicating successful attachment of ethylenediamine to CS.
(2) Then CS-Fmoc was synthesized, 823mg of Fmoc-AEEA was weighed and dissolved in 10ml of DMF, 1242mg of EDC was weighed and dissolved in 40ml of DMF, 757.6mg of NHS was weighed and dissolved in 8ml of DMF, and activation was carried out at room temperature for 1 hour. Weighing 400mg of CS-NH 2 Dissolving in 24mL formamide at 50 deg.C, adding the above activated solution to CS-NH 2 And the room temperature is 44 h. Precipitating with 5 times volume of cold acetone, centrifuging, dissolving precipitate with UP water, dialyzing with ultrapure water (ethanol, v/v ═ 1: 1) for 1d, dialyzing with ultrapure water for 5d, centrifuging (4700r, 10min), collecting supernatant, and lyophilizing to obtain CS-Fmoc product. FIG. 4 shows the nuclear magnetic hydrogen spectrum, CS-NH, of CS-Fmoc 2 The characteristic peaks of (A) appear at 1.90 and 3.27-4.25ppm respectively. CS-NH was confirmed by Fmoc motif peaks at 7.25-8.1ppm 2 Introduction of Fmoc-AEEA.
Example 2: synthesis and characterization of HA Carrier (HA-Fmoc)
(1) Synthesis of HA intermediate (HA-NH) 2 ): 403.3mg HA (1mmol,1eq,8kD) was weighed and dissolved in 10mL of ultrapure water; 1.3419g EDC (7mmol,7eq) was weighed out and dissolved in 5mL pure water, 0.8046g NHS (7mmol,7eq) was dissolved in 5mL ultrapure water; EDC and NHS solution were added to CSA solution and the carboxyl groups were activated for 30min with stirring at room temperature. 1.86mL (28mmol,28eq) of ethylenediamine was placed in a 100mL flask, 15mL of ultrapure water was added, about 4mL of concentrated hydrochloric acid was added, and the pH was adjusted to about 7 using a pH paper. Slowly adding activated CS into the ethylenediamine solution with a constant pressure dropping funnel under ice bath condition, dropwise adding for 60min under ice bath condition, heating the reaction system to room temperature for reaction for 3.5h, dialyzing in water with a dialysis bag of MWCO5000 for 4 days, centrifuging at 4000rpm and 4 ℃ for 10min, and taking supernatant for freeze-drying.
(1) Synthesis of HA-Fmoc: 385.41mg of Fmoc-AEEA (1.0mmoL,3eq) were weighed out and dissolved in 8mL of DMF, 575.1mg (3.0mmoL,9eq) of EDC was weighed out and dissolved in 24mL of DMF, 345.26mg (3.0mmoL,9eq) of NHS was weighed out and dissolved in 8mL of DMF, and activation was carried out at room temperature for 1 hour. 141mg of HA-NH were weighed 2 Dissolving in 40mL of ultrapure water at 50 ℃ to obtain HA-NH 2 The mixture was added to the above activated solution and reacted at room temperature for 44 hours. Precipitating with 5 times volume of cold acetone, centrifuging at 4000rpm and 4 deg.C for 15min, re-dissolving the precipitate with ultrapure water, dialyzing with 50% ethanol for 2d, dialyzing with ultrapure water for 5d, centrifuging at 4000rpm and 4 deg.C for 15min, and lyophilizing the supernatant. FIG. 5 shows HA-NH 2 FIG. 6 shows the nuclear magnetic hydrogen spectrum of HA-Fmoc.
Example 3: preparation and characterization of paclitaxel nanocrystals
(1) Preparation of paclitaxel nanocrystals (PTX NC @ CS-Fmoc): PTX NC @ CS-Fmoc is prepared by a bottom-up precipitation method and an ultrasonic method. 48mg of CS-Fmoc is weighed, dissolved in 6mL of ultrapure water, and dissolved by ultrasonic for 15min, 2mL of the solution is added into a penicillin bottle, 12mg of PTX is added dropwise into the solution, the solution is stirred for 30min at room temperature and is transferred into a 10mLep tube, 1mL of ultrapure water is added into the solution, and ultrasonic treatment is carried out on an ice bath probe (180w, 5s, 5s and 13 min). And (5) freeze-drying to obtain a PTX NC @ CS-Fmoc freeze-dried sample.
(2) Particle size and potential: the particle size and potential diagram of the sample measured by Zetasizer dynamic light scattering method are shown in FIG. 7 and FIG. 8, respectively, and the particle size is shown in Table 1.
(3) Appearance and re-dissolubility of the freeze-dried powder injection: the PTX NC @ CS-Fmoc is subjected to freeze drying on the basis of adding or not adding a freeze-drying protective agent, and a freeze-dried sample is good in redissolution property, and basically has no change in particle size and potential, and is shown in figures 11, 12, 13, 14 and 15.
(4) Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), TEM of PTX NC @ CS-Fmoc is shown in FIG. 17A; SEM of PTXNC @ CS-Fmoc is shown in FIG. 17B.
(5) X-ray diffraction confirms the crystal structure of PTX NC @ CS-Fmoc: the X-ray diffraction pattern of PTX NC @ CS-Fmoc is shown in FIG. 16.
Example 4: preparation and characterization of docetaxel nanocrystal
(1) Preparation of docetaxel nanocrystals (DTX NC @ CS-Fmoc): DTX NC @ CS-Fmoc is prepared by a bottom-up precipitation method and an ultrasonic method. 48mg of CS-Fmoc is weighed and dissolved in 6mL of ultrapure water, after ultrasonic dissolution is carried out for 15min, 2mL of the solution is added into a penicillin bottle, 8mg of DTX is dropwise added into the solution, the solution is stirred for 30min at room temperature and is transferred into a 10mLep tube, 1mL of ultrapure water is added into the solution, and ice bath probe ultrasonic treatment is carried out (180w, 5s, 5s and 13 min). And (5) freeze-drying to obtain a DTX NC @ CS-Fmoc freeze-dried sample.
(2) Particle size and potential: the particle size and potential diagram of the sample measured by Zetasizer dynamic light scattering method are shown in FIGS. 9 and 10, respectively, and the particle size is shown in Table 1.
(3) Appearance and re-dissolubility of the freeze-dried powder injection: the DTX NC @ CS-Fmoc is freeze-dried on the basis of adding or not adding a freeze-drying protective agent, and the freeze-dried sample has good re-solubility and basically unchanged particle size and potential.
(4) A TEM of DTX NC @ CS-Fmoc is shown in FIG. 17C.
Example 5: preparation and characterization of paclitaxel nanocrystals
Preparation of paclitaxel nanocrystals (PTX NC @ HA-Fmoc): PTX NC @ HA-Fmoc is prepared by a bottom-up precipitation method and an ultrasonic method. 48mg of CS-Fmoc is weighed, dissolved in 6mL of ultrapure water, and dissolved by ultrasonic for 15min, 2mL of the solution is added into a penicillin bottle, 12mg of PTX is added dropwise into the solution, the solution is stirred for 30min at room temperature and is transferred into a 10mLep tube, 1mL of ultrapure water is added into the solution, and ultrasonic treatment is carried out on an ice bath probe (180w, 5s, 5s and 13 min). And (5) freeze-drying to obtain a PTXNC @ HA-Fmoc freeze-dried sample.
(1) Particle size and potential: zetasizer dynamic light scattering method measures the particle size of the sample, and the particle size is shown in Table 1.
(2) TEM of PTX NC @ HA-Fmoc is shown in FIG. 17D.
Example 6: preparation and characterization of docetaxel nanocrystal
Preparation of docetaxel nanocrystals (DTX NC @ HA-Fmoc): DTX NC @ HA-Fmoc is prepared by a bottom-up precipitation method and an ultrasonic method. 48mg of CS-Fmoc is weighed and dissolved in 6mL of ultrapure water, after ultrasonic dissolution is carried out for 15min, 2mL of the solution is added into a penicillin bottle, 8mg of DTX (anhydrous ethanol) is dropwise added into the solution, stirring is carried out for 30min at room temperature, the solution is transferred into a 10mLep tube, 1mL of ultrapure water is added into the solution, and ice bath probe ultrasonic treatment is carried out (180w, 5s, 5s and 13 min). And (5) freeze-drying to obtain a DTXNC @ HA-Fmoc freeze-dried sample.
Particle size and potential: zetasizer dynamic light scattering method measures the particle size of the sample, and the particle size is shown in Table 1.
Table 1: particle size potential of different formulations
Example 7: in vitro cell study of PTX NC @ CS-Fmoc
Flow assay of PTX NC @ CS-Fmoc uptake by B16F10 cells, Panc02 cells, 4T1 cells: taking cells in logarithmic growth phase, and treating with trypsin solutionDigesting, making into single cell suspension, and counting cells with cell counter at 2 × 10 5 Cell concentration per well was seeded in 12-well plates and at 37 ℃ with 5% CO 2 And incubating for 24 h. The medium was then aspirated, washed with PBS and added Did @ CS-Fmoc and Did/PTX NC @ CS-Fmoc solutions, the final concentration of DiD in the well plates being 0.4. mu.g/mL each. Incubating at 37 ℃ for 30min and 2h, removing the culture medium, washing with PBS for 3 times, digesting with pancreatin, terminating digestion with a serum-containing culture medium and dispersing cells, centrifuging at 3000rpm for 3min, removing the supernatant, washing with PBS once, centrifuging, removing the supernatant, reselecting with PBS, collecting the cells with a flow cytometer, and measuring the fluorescence intensity in the cells. The cellular uptake patterns are shown in FIGS. 18, 19 and 20. Wherein the uptake rate of Did/PTX NC @ CS-Fmoc by the three cells is more advantageous than that of Did @ CS-Fmoc. Did @ CS-Fmoc is a spherical micelle, Did/PTX NC @ CS-Fmoc is a rod-like nanocrystal. It has been reported in the literature that rod-like structures may be more favorable for entry into cells. The possible reason is that the rod-like structure having a large aspect ratio has an increased surface area due to the edge effect, thereby increasing the contact area with the cell; also, the elongated structure is more able to escape phagocytosis by phagocytic cells than the spherical structure. Did/PTX NC @ CS-Fmoc showed that PTX NC @ CS-Fmoc was more accessible for entry into the cell.
Example 8: experiment on antitumor Activity
PTX NC @ CS-Fmoc nanocrystal prepared in example 1 is taken to carry out in-situ pancreatic cancer in-vivo antitumor effect experimental investigation. Well-conditioned C57bl/6 female mice were selected and inoculated with Panc02-luc cells at logarithmic growth phase. Anaesthetizing a mouse by using 2% 2,2, 2-tribromoethanol, cutting the left side of the mouse downwards, cutting a small opening, gently taking out a spleen together with a pancreas by using a forceps, injecting 200 ten thousand Panc02-luc cells at the tail part of the pancreas, putting the spleen and the pancreas back into the mouse body, and sewing the skin. After two weeks, the PTX NC @ CS-Fmoc (PTX NC) solution group, the albumin paclitaxel (Nab-PTX) solution group, and the Gemcitabine (GEM) solution group were administered to the tail vein. The administration was performed every 3 days for 5 times. The body weight was measured every 3 times, during which the bioluminescence intensity was measured by in vivo imaging and the growth of the tumor was observed. After dosing, mice were sacrificed, tumors were isolated and tumor mass was recorded.
As is clear from FIGS. 21, 22, 23, 24 and 25, the PTX NC @ CS-Fmoc, Nab-PTX and GEM were smaller in tumor size than normal saline at the concentrations set, and the PTX NC @ CS-Fmoc showed better antitumor effect than GEM and Nab-PTX. And there was substantially no significant change in the body weight of the mice during the administration. The PTX NC @ CS-Fmoc provided by the invention has a certain anti-tumor effect and higher safety.
While the present invention has been described in detail with reference to the illustrated embodiments, it should not be construed as limited to the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (10)
1. A CD 44-targeted taxane nanocrystal, comprising: the nanocrystals include a taxane and a glycosaminoglycan derivative carrier.
2. The CD 44-targeted taxane nanocrystal of claim 1, wherein: the glycosaminoglycan derivative carrier is prepared by coupling a polysaccharide skeleton and fat-soluble micromolecules with Fmoc groups through amido bonds; the taxane medicine is selected from one or two of paclitaxel and docetaxel.
3. The CD 44-targeted taxane nanocrystal of claim 2, wherein: the glycosaminoglycan material of the glycosaminoglycan derivative carrier is selected from one or two of hyaluronic acid, chondroitin sulfate and low molecular weight heparin, and the molecular weight of the glycosaminoglycan material is 1-500 kDa; the fat-soluble micromolecules with Fmoc groups are selected from one or more of Fmoc-Val-Gly-OH, Fmoc-Gly, Fmoc-AEEA and Fmoc-GABA containing Fmoc groups.
4. The CD 44-targeted taxane nanocrystal of claim 1, wherein: the preparation method of the glycosaminoglycan derivative carrier comprises the following steps:
step (1): dissolving glycosaminoglycan containing carboxyl into a reaction solvent, adopting a connecting arm with two ends containing amino, carrying out condensation reaction by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine or 1-ethyl- (3-dimethylaminopropyl) carbodiimide and hydroxysuccinimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole as activating agents, and reacting polysaccharide with the amino at one end of the connecting arm to obtain an intermediate;
step (2): dissolving the intermediate and the fat-soluble micromolecule containing the Fmoc group in a reaction solvent, and generating an amido bond by the amino group of the intermediate and the carboxyl group of the fat-soluble micromolecule containing the Fmoc group to obtain the amphiphilic glycosaminoglycan carrier targeting the CD44 receptor;
the connecting arm with amino groups at two ends in the step (1) is any one of ethylenediamine, adipic dihydrazide, diaminohexane and cystamine.
5. A preparation method of CD44 targeted taxane nanocrystals is characterized by comprising the following steps: the nano-crystal utilizes one or more of hydrogen bond function, hydrophobic function and pi-pi accumulation function of Fmoc group of glycosaminoglycan derivative carrier and taxane drugs to obtain highly dispersed nano-crystal.
6. The method of claim 5, wherein the method for preparing the CD 44-targeted taxane nanocrystal comprises the steps of:
step (1): dispersing taxane medicine in good solvent, stirring in dark place until the solution is clear
Step (2): dispersing the solution of step (1) in a poor solvent, and adding a glycosaminoglycan derivative carrier to the poor solvent;
and (3): and (3) removing the good solvent in the step (1) to obtain a suspension, wherein the mass ratio of the taxane drugs to the glycosaminoglycan derivative carrier is 1: 2-1: 10;
and (4): freeze drying to obtain nanometer crystal preparation of taxane medicine.
7. The method of claim 6, wherein the CD 44-targeted taxane nanocrystal is prepared by: the good solvent is selected from any one or a mixture of ethanol, acetone, dimethyl sulfoxide, tetrahydrofuran and dimethylformamide; the poor solvent is water, and the mixing ratio of the good solvent to the poor solvent is 2: 1-1: 200.
8. the method of claim 6, wherein the CD 44-targeted taxane nanocrystal is prepared by: and (4) adding an injection solvent to redissolve the taxane medicine nanocrystal preparation prepared in the step (4) before use, and dispersing the taxane medicine nanocrystal preparation uniformly to obtain the taxane medicine nanocrystal preparation for use.
9. Use of the CD 44-targeted taxane nanocrystals according to any one of claims 1-4, wherein: the nano-crystal can be applied to the preparation of a medicine for treating CD44 receptor high-expression diseases, and the nano-crystal can be prepared into a freeze-dried preparation.
10. The use of a CD 44-targeted taxane nanocrystal according to claim 9, wherein: the nano crystal is prepared into a freeze-drying preparation, a freeze-drying protective agent is required to be added in the process of preparing the freeze-drying preparation, the freeze-drying protective agent is one or more of mannitol, trehalose, lactose, sucrose and glucose, and the mass ratio of the freeze-drying protective agent to the medicine is 0: 1-1: 2.
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