CN114870031B - 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 a taxane drug and a glycosaminoglycan derivative carrier. The invention also discloses a preparation method of the CD44 targeted taxane nanocrystals, and the nanocrystals are highly dispersed by utilizing one or more of hydrogen bonding, hydrophobic effect and pi-pi stacking effect of Fmoc groups of the glycosaminoglycan derivative carrier and taxane drugs. The invention also discloses an application of the CD44 targeted taxane nanocrystals in preparing medicaments for treating CD44 receptor high-expression diseases, wherein the nanocrystals can be prepared into freeze-dried preparations. The taxane nanocrystals actively target the drug to specific tumor cells by means of the high affinity of the polysaccharide skeleton and the CD44 receptor highly expressed on the surface of the tumor cells, act on a treatment target, and can remarkably improve the drug concentration of tumor parts, thereby enhancing the anti-tumor curative effect.
Description
Technical Field
The invention relates to the field of pharmaceutical preparations, in particular to a CD44 targeted taxane nanocrystal and a preparation method and application thereof.
Background
Taxane drugs are broad-spectrum antitumor drugs with cytotoxicity, which inhibit the depolymerization of microtubules by promoting the formation of microtubules, thereby inhibiting the mitosis and proliferation of tumor cells. The taxane drugs approved by the FDA to be marketed comprise Paclitaxel (PTX) and Docetaxel (DTX), and the taxane drugs have good curative effects on tumors such as ovarian cancer, breast cancer and the like. Wherein, the taxol is a fat-soluble medicine, the solubility in water is extremely low, and the taxol injection sold in the market is solubilized by using polyoxyethylated castor oil and absolute ethyl alcohol as solvents, which may cause serious hypotension, anaphylactic reaction and neurotoxicity; DTX has poor 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, allergy and the like. Therefore, it is important to improve the solubility of chemotherapeutic drugs, reduce toxic and side effects, and improve the targeting of drugs.
There are often many different types of receptor over-expression on the surface of tumor cells, such as the CD44 receptor, compared to normal cells. CD44 is a multifunctional transmembrane glycoprotein that is associated with tumor cell invasion, metastasis and chemotherapy drug resistance. Glycosaminoglycans such as chondroitin sulfate, hyaluronic acid and heparin are natural targeting ligands for CD44 and are commonly used for targeted therapy of tumors. The glycosaminoglycans such as chondroitin sulfate, hyaluronic acid and heparin are natural mucopolysaccharide, have the characteristics of no toxicity, no immunogenicity, good biocompatibility, degradability and the like, can be specifically combined with CD44 receptors on tumor cell membranes, and the constructed drug delivery system shows excellent tumor targeting characteristics.
The nano crystal medicine contains no or a small amount of carrier material, can improve the medicine carrying amount, reduce the toxic and side effects of the carrier and can well solve the problem of solubilization of insoluble medicine.
Based on the above background, a nanocrystal delivery system with CD44 targeting was designed. The preparation method takes natural polysaccharide as a framework, and lipid-soluble small molecules with Fmoc groups are coupled on the polysaccharide through covalent bonds so as to form an amphipathic carrier with CD44 targeting glycosaminoglycan derivatives, and taxane medicines are loaded by a 'bottom-up' precipitation method to prepare a nanocrystal preparation, wherein the nanocrystal preparation has the following characteristics: (1) the Fmoc group wraps the insoluble medicine through pi-pi stacking action with the taxane medicine, so that the solubility, stability and in-vivo circulation time of the medicine are obviously improved. (2) By means of the high affinity of glycosaminoglycan and CD44 receptor, the medicine is actively targeted to the tissue and specific cells with high expression of CD44, and the medicine concentration of focus tissue is increased. (3) The glycosaminoglycan derivative carrier can improve the drug-loading rate of taxane drugs and reduce the toxic and side effects generated by auxiliary materials.
Disclosure of Invention
The invention aims to provide a CD44 targeted taxane nanocrystal, a preparation method and application thereof, which are used for improving the solubility of chemotherapeutic drugs, reducing toxic and side effects and improving the targeting of the drugs in the prior art.
To achieve the above object, in one embodiment of the present invention, there is provided a CD 44-targeted taxane nanocrystal, the nanocrystal including a taxane and a glycosaminoglycan derivative carrier.
In one of the preferred schemes of the invention, the glycosaminoglycan derivative carrier is prepared by coupling a polysaccharide skeleton with a fat-soluble small molecule with Fmoc groups through an amide bond; the taxane is selected from one or two of paclitaxel and docetaxel.
In one of the preferred embodiments 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 1 kDa-500 kDa; the fat-soluble small molecule with Fmoc group is selected from any one or more of Fmoc-Val-Gly-OH, fmoc-Gly, fmoc-AEEA and Fmoc-GABA containing Fmoc group.
In one of the preferred embodiments of the present invention, a method for preparing a glycosaminoglycan derivative carrier comprises the steps of:
step (1): dissolving glycosaminoglycan containing carboxyl into a reaction solvent, adopting a connecting arm with amino groups at two ends, carrying out condensation reaction by taking 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 one end amino group of the connecting arm to obtain an intermediate;
step (2): dissolving an intermediate and a fat-soluble small molecule containing an Fmoc group in a reaction solvent, wherein an amino group of the intermediate and a carboxyl group of the fat-soluble small molecule containing the Fmoc group generate an amide bond, and thus the amphiphilic glycosaminoglycan carrier of the targeted CD44 receptor is obtained;
the connecting arms with amino groups at the two ends in the step (1) are 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 is a highly dispersed nanocrystal obtained by utilizing one or more of hydrogen bonding, hydrophobic effect and pi-pi stacking effect of Fmoc groups of a glycosaminoglycan derivative carrier and taxane drugs.
In one of the preferred embodiments of the present invention, the preparation method of nanocrystals specifically comprises the steps of:
step (1): dispersing taxane in good solvent, and stirring in 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;
step (3): removing the good solvent in the step (1) to obtain a suspension, wherein the mass ratio of the taxane medicine to the glycosaminoglycan derivative carrier is 1: 2-1: 10;
step (4): freeze drying to obtain nanometer taxane crystal preparation.
In one of the preferred schemes 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.
according to one of the preferred schemes of the invention, before the taxane medicine nanocrystal preparation prepared in the step (4) is used, the injection solvent is added for re-dissolution, and the preparation is dispersed uniformly, so that the taxane medicine nanocrystal preparation can be used.
The invention also discloses an application of the CD44 targeted taxane nanocrystals in preparing medicaments for treating CD44 receptor high-expression diseases, wherein the nanocrystals can be prepared into freeze-dried preparations.
According to one of the preferred schemes, a freeze-drying protective agent is added in the process of preparing the nano crystal into a freeze-drying preparation, wherein one or more of mannitol, trehalose, lactose, sucrose and glucose are added in the freeze-drying protective agent, and the mass ratio of the freeze-drying protective agent to the medicine is 0:1 to 1:2.
in summary, the beneficial effects of the invention are as follows:
1. the nanocrystal preparation actively targets the medicine to specific tumor cells by means of the high affinity of the polysaccharide skeleton and the CD44 receptor highly expressed on the surfaces 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 toxic and side effects can be reduced, and biocompatibility can be improved.
3. The taxane nanocrystal prepared by the invention has CD44 targeting property due to the coverage of the glycosaminoglycan derivative carrier on the surface, and improves the effective concentration of the drug in target tissues.
4. The taxane nanocrystals of the invention 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, no extra solubilizer such as polyoxyethylene castor oil and tween 80 is needed when the medicine solution is prepared, 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 capacity, good safety, strong stability and simple preparation process.
6. Cell experiments show that cells which highly express CD44 receptors, such as pancreatic cancer cells, melanoma cells, breast cancer cells and the like, have higher uptake efficiency on the taxane nanocrystal preparation prepared by the invention.
Drawings
FIG. 1 is a structural formula of paclitaxel and docetaxel according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of CS-NH according to one embodiment of the present invention 2 The structure and nuclear magnetic hydrogen spectrogram of (2);
FIG. 3 is a schematic diagram of CS-NH according to one embodiment of the present invention 2 Is an infrared spectrum of (2);
FIG. 4 is a diagram showing the structure and nuclear magnetic resonance hydrogen spectrum of CS-Fmoc in one embodiment of the present invention;
FIG. 5 is a schematic illustration of HA-NH according to one embodiment of the present invention 2 The structure and nuclear magnetic hydrogen spectrogram of (2);
FIG. 6 is a diagram of the structure and nuclear magnetic resonance hydrogen spectrum of HA-Fmoc in one embodiment of the present invention;
FIG. 7 is a graph of particle size of PTX NC@CS-Fmoc in one embodiment of the invention;
FIG. 8 is a Zeta potential diagram of PTX NC@CS-Fmoc in one embodiment of the invention;
FIG. 9 is a graph of particle size for DTX NC@CS-Fmoc in one embodiment of the invention;
FIG. 10 is a Zeta potential diagram of DTX NC@CS-Fmoc in one embodiment of the invention;
FIG. 11 is a graph of reconstituted particle size of a lyophilized sample without lyoprotectant according to one embodiment of the invention;
FIG. 12 is a graph of reconstituted particle size of a lyophilized sample with sucrose added in one embodiment of the invention;
FIG. 13 is a graph of reconstituted particle size of a lyophilized sample with mannitol added in accordance with one embodiment of the present invention;
FIG. 14 is a graph showing the particle size change after reconstitution of a lyophilized sample according to one embodiment of the present invention;
FIG. 15 is a reconstituted appearance of a lyophilized sample according to one embodiment of the invention;
FIG. 16 is an X-ray diffraction pattern of PTX NC@CS-Fmoc nanocrystals according to one embodiment of the present invention;
FIG. 17 is an electron microscope image of nanocrystals in one embodiment of the present invention. (A) a transmission electron microscope image of PTX NC@CS-Fmoc, (B) a scanning electron microscope image of PTX NC@CS-Fmoc, (C) a transmission electron microscope image of DTX NC@CS-Fmoc, (D) a transmission electron microscope image of PTX NC@HA-Fmoc;
FIG. 18 is a graph showing uptake of PTX NC@CS-Fmoc by B16F10 cells according to one embodiment of the invention;
FIG. 19 is a graph showing uptake of PTX NC@CS-Fmoc by Panc02 cells according to one embodiment of the present invention;
FIG. 20 is a graph showing uptake of PTX NC@CS-Fmoc by 4T1 cells according to one embodiment of the present invention;
FIG. 21 is an energy diagram of bioluminescence following administration of various formulations in one embodiment of the present invention;
FIG. 22 is a graph showing the change in body weight of C57bl/6 following administration of different formulations in one embodiment of the invention;
FIG. 23 is a mass graph of pancreas after administration of various formulations in accordance with an embodiment of the present invention;
FIG. 24 is a graph showing tumor formation after administration according to one embodiment of the present invention;
FIG. 25 is a diagram of immunohistochemistry after administration of different formulations in one embodiment of the present invention.
Detailed Description
The invention provides a CD44 targeted taxane nanocrystal, which comprises a taxane drug and a glycosaminoglycan derivative carrier. Wherein the taxane is selected from one or two of paclitaxel and docetaxel, and the structural formulas of paclitaxel and docetaxel are shown in figure 1.
The glycosaminoglycan derivative carrier is prepared by coupling a polysaccharide skeleton with a fat-soluble small molecule with Fmoc groups through an amide bond. 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 1 kDa-500 kDa, preferably the molecular weight of the glycosaminoglycan material is 1 kDa-50 kDa.
The small fat-soluble molecule with Fmoc group is selected from any one or more of Fmoc-Val-Gly-OH, fmoc-Gly, fmoc-AEEA and Fmoc-GABA containing Fmoc group, preferably, the small fat-soluble molecule with Fmoc group is Fmoc-Gly or Fmoc-AEEA.
A method for preparing a glycosaminoglycan derivative carrier, comprising the steps of:
step (1): dissolving glycosaminoglycan containing carboxyl into a reaction solvent, adopting a connecting arm with amino groups at two ends, carrying out condensation reaction by taking 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 one end amino group of the connecting arm to obtain an intermediate;
step (2): and (3) dissolving the intermediate and the fat-soluble micromolecule containing the Fmoc group in a reaction solvent, wherein an amide bond is formed by the amino group of the intermediate and the carboxyl group of the fat-soluble micromolecule containing the Fmoc group, so that the amphiphilic glycosaminoglycan carrier targeting the CD44 receptor is obtained.
In the step (1), the connecting arm with amino groups at two ends is any one of ethylenediamine, adipic 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 taxane nanocrystal of the invention has the grain diameter of 100 nm-500 nm, wherein the short diameter is 20 nm-50 nm, the long diameter is 100 nm-500 nm, and the nanocrystal preparation is bar-shaped or square.
The taxane nanocrystals can also be prepared into lyophilized preparation with good lyophilization stability, and can be added with or without lyoprotectant during the preparation process. The added lyoprotectant is selected from one or more of mannitol, trehalose, lactose, sucrose and glucose, and preferably, the lyoprotectant is mannitol.
The mass ratio of the freeze-drying protective agent to the medicine is 0:1 to 1:2, preferably, the mass ratio of the lyoprotectant to the drug is 0:1 to 0.2:1.
the invention also discloses a preparation method of the CD44 targeted taxane nanocrystals, and the nanocrystals are highly dispersed by utilizing one or more of hydrogen bonding, hydrophobic effect and pi-pi stacking effect of Fmoc groups of the 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 taxane medicine in good solvent, and stirring in 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;
step (3): removing the good solvent in the step (1) to obtain a suspension, wherein the mass ratio of the taxane medicine to the glycosaminoglycan derivative carrier is 1: 2-1: 10, freeze-drying to obtain taxane nanometer crystal freeze-dried powder;
step (4): before use, the taxane nanocrystal freeze-dried powder obtained in the step (4) is added with a solvent for injection for redissolution, and the mixture is uniformly dispersed, so that the taxane nanocrystal freeze-dried powder can be used.
Wherein, the good solvent in the step (1) comprises one or a mixture of more than one of ethanol, acetone, dimethyl sulfoxide, tetrahydrofuran or dimethylformamide, preferably ethanol or dimethyl sulfoxide; the poor solvent in the step (2) is water, and the method for removing the poor solvent in the step (3) is any one of freeze drying, vacuum drying, reduced pressure rotary evaporation and dialysis, preferably reduced pressure rotary evaporation or dialysis, and the dispersion method comprises one or more of rapid stirring, water bath ultrasonic wave, probe ultrasonic wave or high pressure homogenization, preferably rapid stirring or probe ultrasonic wave; the step (3) can be further added with a lyoprotectant, and the specific steps are that the solution obtained in the step (3) is dispersed in the lyoprotectant, wherein the lyoprotectant is one or more selected from the group consisting of mannitol, trehalose, lactose, sucrose and glucose, preferably mannitol, and the mass ratio of the lyoprotectant to the medicine is 0:1 to 1:2, preferably 0:1 to 0.2:1.
the invention also discloses application of the CD44 targeted taxane nanocrystals in preparing medicaments for treating CD44 receptor high-expression diseases.
Example 1: synthesis and characterization of CS Carrier (CS-Fmoc)
(1) First, the CS intermediate (CS-NH) 2 ) 1545mg of CS was dissolved in 30mL of ultrapure water, 4.023g of EDC was dissolved in 15mL of ultrapure water, 2.415g of NHS was dissolved in 12mL of ultrapure water, and the solution was added to CS, and the carboxyl group was activated by stirring at room temperature for 30 minutes. 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 ice bath condition, dropwise adding for 60min under ice bath condition, and reactingThe system is heated to room temperature for reaction for 3.5h, dialyzed for 4d in water by a dialysis bag of MWCO5000, centrifugated, and the supernatant is taken and freeze-dried to obtain the product CS-NH 2 CS-NH was confirmed by nuclear magnetic hydrogen spectroscopy and infrared spectroscopy 2 Is a structure of (a). FIG. 2 is CS-NH 2 Is characterized by the characteristic peak of CS being N-acetyl (a, 1.90 ppm). The signals of primary (b, 2.73 ppm) and secondary (c, 3.39 ppm) amine groups of ethylenediamine indicate that ethylenediamine was successfully incorporated into CS; FIG. 3 is CS-NH 2 Is characterized by 2925cm -1 The characteristic peak of the amide I band is 1640cm -1 The characteristic peak of the amide II wave band is 1558cm -1 . CS-NH compared to CS 2 At 1560cm -1 The peak at which increases significantly. Showing successful attachment of ethylenediamine to CS.
(2) Then synthesizing CS-Fmoc, weighing 823mgFmoc-AEEA and dissolving in 10mLDMF, weighing 1242mg EDC and dissolving in 40mLDMF, weighing 757.6mgNHS and dissolving in 8mLDMF, and activating for 1h at room temperature. 400mg of CS-NH was weighed out 2 Dissolving in 24mL formamide at 50deg.C, adding the activated solution to CS-NH 2 In the above, the room temperature was 44h. Precipitating with 5 times volume of cold acetone, centrifuging, redissolving the precipitate with UP water, dialyzing with (ultrapure water: ethanol, v/v=1:1) for 1d, dialyzing with ultrapure water for 5d, centrifuging (4700 r,10 min), collecting supernatant, and lyophilizing to obtain CS-Fmoc product. FIG. 4 shows a nuclear magnetic hydrogen spectrum of CS-Fmoc, CS-NH 2 Characteristic peaks of (2) appear at 1.90 and 3.27-4.25ppm, respectively. CS-NH was confirmed by Fmoc motif peaks at 7.25-8.1ppm 2 Fmoc-AEEA.
Example 2: synthesis and characterization of HA vector (HA-Fmoc)
(1) Synthesis of HA intermediate (HA-NH) 2 ): 403.3mgHA (1 mmol,1eq,8 kD) was dissolved in 10mL of ultra pure water; 1.3419g of EDC (7 mmol,7 eq) are weighed out in 5mL of pure water, 0.8046g of NHS (7 mmol,7 eq) are dissolved in 5mL of ultrapure water; EDC and NHS solutions were added to CSA solution and the carboxyl groups were activated by stirring at room temperature for 30min. 1.86mL (28 mmol,28 eq) of ethylenediamine was added to 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 pH paper. Slowly adding activated CS into the ethylenediamine solution with constant pressure dropping funnel under ice bath condition, dropwise adding for 60min under ice bath condition, and lifting reaction systemThe reaction was carried out at room temperature for 3.5h, dialyzed against MWCO5000 dialysis bag in water for 4 days, centrifuged at 4000rpm at 4℃for 10min, and the supernatant was lyophilized.
(1) Synthesis of HA-Fmoc: 385.41mgFmoc-AEEA (1.0 mmol,3 eq) was weighed into 8mM LDMF, 575.1mg (3.0 mmol,9 eq) EDC was weighed into 24 mM LDMF, 345.26mg (3.0 mmol,9 eq) NHS was weighed into 8mM LDMF and activated for 1h at room temperature. 141mg of HA-NH was weighed out 2 Dissolving HA-NH in 40mL of ultra-pure water at 50deg.C 2 Adding the mixture into the activated solution, and reacting for 44h at room temperature. Precipitating with 5 times volume of cold acetone, centrifuging at 4000rpm and 4 deg.C for 15min, redissolving the precipitate with ultrapure water, dialyzing with 50% ethanol for 2d, dialyzing with ultrapure water for 5d,4000rpm and 4 deg.C, centrifuging for 15min, collecting supernatant, and lyophilizing. FIG. 5 is 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 combining a 'bottom-up' precipitation method and an ultrasonic method. 48mg of CS-Fmoc was weighed and dissolved in 6mL of ultrapure water, after 15min of ultrasonic dissolution, 2mL of the solution was added to a penicillin bottle, 12mg of PTX was added dropwise to the solution, stirred at room temperature for 30min, transferred to a 10mLep tube, 1mL of ultrapure water was added thereto, and an ice bath probe was sonicated (180 w,5s, 13 min). And freeze-drying to obtain a PTX NC@CS-Fmoc freeze-dried sample.
(2) Particle size and potential: the Zetasizer dynamic light scattering method was used to determine the particle size of the sample, the particle size and the potential diagram are shown in FIG. 7 and FIG. 8, respectively, and the particle size is shown in Table 1.
(3) Appearance and re-solubility of freeze-dried powder injection: the freeze-drying is carried out on the PTX NC@CS-Fmoc on the basis of adding or not adding a freeze-drying protective agent, so that the freeze-dried sample has good re-solubility, 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; the 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 nanocrystals
(1) Preparation of docetaxel nanocrystals (DTX NC@CS-Fmoc): and adopting a 'bottom-up' precipitation method and an ultrasonic method to prepare DTX NC@CS-Fmoc. 48mg of CS-Fmoc was weighed and dissolved in 6mL of ultrapure water, after ultrasonic dissolution for 15min, 2mL of the solution was added to a penicillin bottle, 8mg of DTX was added dropwise to the solution, stirring was carried out at room temperature for 30min, transferred to a 10mLep tube, 1mL of ultrapure water was added thereto, and an ice bath probe was subjected to ultrasonic treatment (180 w,5s, 13 min). And freeze-drying to obtain a DTX NC@CS-Fmoc freeze-dried sample.
(2) Particle size and potential: the Zetasizer dynamic light scattering method was used to determine the particle size of the sample, the particle size and the potential diagram are shown in FIG. 9 and FIG. 10, respectively, and the particle size is shown in Table 1.
(3) Appearance and re-solubility of freeze-dried powder injection: and freeze-drying DTX NC@CS-Fmoc on the basis of adding or not adding a freeze-drying protective agent to obtain a freeze-dried sample, wherein the freeze-dried sample has good re-solubility and basically has no change in particle size and potential.
(4) 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): the PTX NC@HA-Fmoc is prepared by combining a 'bottom-up' precipitation method and an ultrasonic method. 48mg of CS-Fmoc was weighed and dissolved in 6mL of ultrapure water, after 15min of ultrasonic dissolution, 2mL of the solution was added to a penicillin bottle, 12mg of PTX was added dropwise to the solution, stirred at room temperature for 30min, transferred to a 10mLep tube, 1mL of ultrapure water was added thereto, and an ice bath probe was sonicated (180 w,5s, 13 min). And freeze-drying to obtain a PTXNC@HA-Fmoc freeze-dried sample.
(1) Particle size and potential: the Zetasizer dynamic light scattering method was used to determine 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 nanocrystals
Preparation of docetaxel nanocrystals (DTX NC@HA-Fmoc): and adopting a 'bottom-up' precipitation method and an ultrasonic method to prepare DTX NC@HA-Fmoc. 48mg of CS-Fmoc was weighed and dissolved in 6mL of ultrapure water, after ultrasonic dissolution for 15min, 2mL of the solution was taken and added to a penicillin bottle, 8mg of DTX (absolute ethanol) was added dropwise to the solution, stirring was carried out at room temperature for 30min, transferred to a 10mLep tube, 1mL of ultrapure water was added thereto, and an ice bath probe was used for ultrasonic treatment (180 w,5s, 13 min). And freeze-drying to obtain a DTXNC@HA-Fmoc freeze-dried sample.
Particle size and potential: the Zetasizer dynamic light scattering method was used to determine the particle size of the sample, and the particle size is shown in Table 1.
Table 1: particle size potential size of different formulations
Example 7: in vitro cell Studies of PTX NC@CS-Fmoc
Flow assay uptake of PTX NC@CS-Fmoc by B16F10 cells, panc02 cells, 4T1 cells: taking cells in logarithmic growth phase, digesting with trypsin solution to obtain single cell suspension, and counting with cell counter to obtain 2×10 5 Cell concentration of each well was inoculated into 12-well plates and incubated at 37℃with 5% CO 2 Is incubated for 24h in an incubator. The medium was then aspirated, washed with PBS and Did@CS-Fmoc and Did/PTX NC@CS-Fmoc solutions were added, with the final concentration of DiD in the well plate being 0.4. Mu.g/mL. After incubation at 37℃for 30min, 2h, the medium was discarded, PBS was washed 3 times, the digestion was stopped with serum-containing medium and the cells were dispersed, the supernatant was discarded after centrifugation at 3000rpm for 3min, PBS was washed once, the supernatant was discarded after centrifugation, the cells were collected with a flow cytometer after reselection with PBS, and the fluorescence intensity in the cells was measured. The cell 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 superior to that of Did@CS-Fmoc. The TEM morphology of Did@CS-Fmoc is a spherical micelle, and the TEM morphology of Did/PTX NC@CS-Fmoc is a rod-shaped nanocrystal. It has been reported that rod-like structures may be more favorable for cell entry. The possible reason is that the rod-like structure with a large aspect ratio increases the surface area due to the edge effect, thereby increasing the contact area with the cells; and, relative to a spherical structure, is elongatedThe structure can further evade phagocytosis of phagocytes. A high uptake of Did/PTX NC@CS-Fmoc indicates that PTX NC@CS-Fmoc is easier to enter the cell.
Example 8: antitumor Activity assay
The PTX NC@CS-Fmoc nanocrystalline prepared in the embodiment 1 is taken for in-situ pancreatic cancer in-vivo anti-tumor efficacy experimental investigation. C57bl/6 female mice in good physical condition were selected and inoculated with Panc02-luc cells in the logarithmic growth phase. After the mice were anesthetized with 2%2, 2-tribromoethanol, the left side was turned down, a small opening was cut, the spleen with pancreas was gently removed with forceps, 200 ten thousand Panc02-luc cells were injected into the tail of the pancreas, the spleen and pancreas were returned to the mice, and the skin was sewn. Two weeks later, the tail vein was given with PTX NC@CS-Fmoc (PTX NC) solution, albumin paclitaxel (Nab-PTX) solution, and Gemcitabine (GEM) solution. The administration was 5 times every 3 days. Body weight was measured every 3 times, and bioluminescence intensity was measured by in vivo imaging during this period to observe the growth of tumor. Mice were sacrificed after dosing, tumors were isolated, and tumor mass was recorded.
As can be seen from fig. 21, 22, 23, 24 and 25, the tumor sizes of PTX nc@cs-Fmoc, nab-PTX and GEM are smaller than those of physiological saline at the set administration concentrations, and the antitumor effect of PTX nc@cs-Fmoc is better than that of GEM and Nab-PTX. And there was essentially no significant change in the body weight of the mice during the dosing period. The PTX NC@CS-Fmoc provided by the invention has a certain anti-tumor effect and is higher in safety.
Although specific embodiments of the invention have been described in detail with reference to the accompanying drawings, it should not be construed as limiting the scope of protection of the present patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.
Claims (8)
1. A CD44 targeted taxane nanocrystal characterized by: the nanocrystals comprise taxane drugs and glycosaminoglycan derivative carriers;
the glycosaminoglycan derivative carrier is prepared by coupling a polysaccharide skeleton with a fat-soluble small molecule with Fmoc groups through an amide bond; the taxane medicine is selected from one or two of paclitaxel and docetaxel;
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 1 kDa-500 kDa; the fat-soluble small molecule with Fmoc group is selected from any one or more of Fmoc-Val-Gly-OH, fmoc-Gly, fmoc-AEEA and Fmoc-GABA containing Fmoc group.
2. A CD44 targeted taxane nanocrystal according to 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 amino groups at two ends, carrying out condensation reaction by taking 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 one end amino group of the connecting arm to obtain an intermediate;
step (2): dissolving an intermediate and a fat-soluble small molecule containing an Fmoc group in a reaction solvent, wherein an amino group of the intermediate and a carboxyl group of the fat-soluble small molecule containing the Fmoc group generate an amide bond, and thus the amphiphilic glycosaminoglycan carrier of the targeted CD44 receptor is obtained;
the connecting arms with amino groups at the two ends in the step (1) are any one of ethylenediamine, adipic dihydrazide, diaminohexane and cystamine.
3. A method for preparing the CD44 targeted taxane nanocrystals according to any one of claims 1 to 2, wherein: the nanocrystals are highly dispersed by utilizing one or more of hydrogen bonding, hydrophobic and pi-pi stacking of Fmoc groups of glycosaminoglycan derivative carriers and taxane drugs.
4. A method for preparing a CD44 targeted taxane nanocrystal according to claim 3, wherein the method for preparing the nanocrystal comprises the steps of:
step (1): dispersing taxane medicine in good solvent, and stirring in 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;
step (3): removing the good solvent in the step (1) to obtain a suspension, wherein the mass ratio of the taxane medicine to the glycosaminoglycan derivative carrier is 1: 2-1: 10;
step (4): freeze drying to obtain nanometer taxane crystal preparation.
5. The method for preparing the CD44 targeted taxane nanocrystal, as recited in claim 4, wherein: 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 poor solvent to the poor solvent is 2:1-1:200.
6. the method for preparing the CD44 targeted taxane nanocrystal, as recited in claim 4, wherein: before the taxane medicine nanocrystal preparation prepared in the step (4) is used, adding an injection solvent for re-dissolution and uniformly dispersing, so that the taxane medicine nanocrystal preparation can be used.
7. Use of a CD44 targeted taxane nanocrystal according to any one of claims 1-2, characterized in that: the nanometer crystal can be used for preparing medicines for treating CD44 receptor high-expression diseases, and the nanometer crystal can be prepared into freeze-dried preparations.
8. Use of a CD44 targeted taxane nanocrystal according to claim 7, wherein: the nano crystal is added with a lyoprotectant in the process of preparing the freeze-dried preparation, wherein the lyoprotectant is one or more of mannitol, trehalose, lactose, sucrose and glucose, and the mass ratio of the lyoprotectant to the medicine is 0:1 to 1:2.
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