Drug delivery system, preparation method thereof and method for delivering drugs by using drug delivery system

Abstract

The invention discloses a drug delivery system, a preparation method of the drug delivery system and a method for delivering drugs by using the drug delivery system, the drug delivery system is composed of two parts, i.e., nanocrystals of indissolvable drugs and conjugates of polypeptides and the drugs, and the conjugates are coated on the outer layers of the nanocrystals. The drug delivery system provided by the invention can realize the effects of improving the drug stability, accurately delivering the drug, improving the drug curative effect and reducing the toxic and side effects of the drug. According to the preparation method of the drug delivery system provided by the invention, the introduction of polypeptide targeting molecules (pHA-VAP) is synchronously completed in the preparation process of the nanocrystals, and additional steps are not needed, so that a simpler prescription and a simpler process are provided, and clinical application is more convenient.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and specifically relates to a delivery system, a preparation method thereof, a method for delivering drugs using the same, and an application of a conjugate of a polypeptide and a drug as a stabilizer in nanocrystals. Background Art

[0002] Drug nanocrystals are drug crystalline materials with a particle size of 1-1000nm formed by the drug itself. Compared with other nano delivery methods, drug nanocrystals do not require carrier materials, only a small amount of surfactant as a stabilizer, thus avoiding the toxic and side effects caused by carrier materials, while providing extremely high drug loading, which can meet the effective concentration of the drug even at low doses. In addition, constructing the drug into nanocrystals also helps to improve the solubility, dissolution rate and bioavailability of the drug, which is conducive to the drug penetrating into the lesion site through the biological barrier in the human body, thereby improving the in vivo absorption, distribution, metabolism and excretion of the drug, and obtaining better therapeutic effects. Therefore, in recent years, drug nanocrystals have become one of the hot spots in the field of drug preparation research and development, and have received the attention of preparation researchers.

[0003] However, due to the small size of drug nanocrystal particles and their large specific surface area, they have high interfacial free energy, are thermodynamically unstable, and are easy to aggregate. In addition, Ostwald Ripening, a phenomenon in which smaller crystal particles gradually dissolve and redeposit on larger crystal particles when supersaturated during the crystal state change, leads to an increase in the size of crystal particles, which makes the physical stability of drug nanocrystals worse. There is a risk of vascular occlusion and embolism and shortened blood circulation after intravenous injection of drug nanocrystals. Therefore, how to improve the stability of drug nanocrystals has always been the key and difficulty of research in this field. At present, the method of stabilizing drug nanocrystals is mainly to add suitable stabilizers to the nanocrystal system to reduce the surface free energy and prevent the aggregation of drug nanocrystals. Commonly used stabilizers can be divided into several categories: ionic surfactants, non-ionic surfactants, polymer stabilizers and other stabilizers. Ionic surfactants mainly include sodium dodecyl sulfate (SDS); non-ionic surfactants include Tween, vitamin E polyethylene glycol succinate (TPGS) and decyl glucoside; polymer stabilizers include hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (CMC-Na), polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) and poloxamer, etc.

[0004] In order to achieve precise drug delivery, improve drug efficacy and reduce drug toxicity, active targeting strategies have also been widely used in the research of drug nanocrystals in recent years. It achieves active targeted delivery of drug nanocrystals by modifying specific targeting ligands, targeting molecules or coating biofilms on the surface of drug nanocrystals. Summary of the invention

[0005] In order to solve the problems of low precision, low drug efficacy and large drug toxicity and side effects in drug delivery in the prior art, the present invention provides a drug delivery system, a preparation method thereof and a method for delivering drugs using the same, the drug delivery system provided by the present invention, and the drug delivery system provided by the present invention can achieve the effects of improving drug stability, accurately delivering drugs, improving drug efficacy and reducing drug toxicity and side effects. The preparation method of the drug delivery system provided by the present invention simultaneously completes the introduction of polypeptide targeting molecules (pHA-VAP) during the preparation of nanocrystals, without the need for additional steps, thereby providing a simpler prescription and process, which is more convenient for clinical application.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides a drug delivery system, which includes a nanocrystal and a conjugate, wherein the nanocrystal is a nanocrystal of a poorly soluble drug, the conjugate is formed by the conjugation of a polypeptide and the poorly soluble drug, and the conjugate is coated on the outer layer of the nanocrystal.

[0007] The delivery system of the present invention is coated on the surface of nanocrystals constructed by covalently linking the poorly soluble drug and the hydrophilic targeting polypeptide to form an amphiphilic conjugate, thereby reducing the surface energy of the nanocrystals and improving the stability of the drug nanocrystals; at the same time, the polypeptide on the surface of the nanocrystal can mediate the drug delivery system to target the lesion site, so that the drug is enriched at the lesion site and the effect of drug treatment is enhanced. In vivo and in vitro research data show that the polypeptide-drug conjugate can be successfully coated on the surface of the drug nanocrystal to improve the stability of the drug nanocrystal. Through the targeting effect of the polypeptide on the surface of the nanocrystal, the drug can be effectively delivered to the target tissue (such as increasing the transport capacity across the BBB by targeting brain capillary endothelial cells, increasing the transport capacity across the BBTB by targeting tumor neovascularization, etc.), and significantly enhancing the efficacy of the drug (such as showing better anti-subcutaneous tumor and brain in situ tumor effects in vivo).

[0008] The polypeptide-drug conjugate-coated drug nanocrystal drug delivery system of the present invention can select corresponding hydrophilic polypeptide targeting molecules and poorly soluble therapeutic drugs according to the specific disease conditions, prepare polypeptide-drug conjugates through appropriate covalent connection methods, and further construct polypeptide-drug conjugate-coated drug nanocrystal drug delivery systems for targeted treatment of diseases.

[0009] In some embodiments of the present invention, the poorly soluble drug is an anti-tumor drug.

[0010] In some preferred embodiments of the present invention, the anti-tumor drug is selected from one or more of the following: anthracyclines, taxanes, camptothecins and vinca alkaloids.

[0011] In some more preferred embodiments of the present invention, the anthracycline drugs are selected from one or more of the following: doxorubicin and epirubicin; the taxane drugs are selected from one or more of the following: paclitaxel, cabazitaxel and docetaxel; the camptothecin drugs are selected from one or more of the following: camptothecin, hydroxycamptothecin, 9-nitrocamptothecin and irinotecan; the vinca alkaloid drugs are selected from one or more of the following: vinblastine and vincristine.

[0012] In some further more preferred embodiments of the present invention, the anti-tumor drug is cabazitaxel or docetaxel.

[0013] In some embodiments of the present invention, the nanocrystal is a nanocrystal prepared from the poorly soluble drug and excipients.

[0014] In some preferred embodiments of the present invention, the excipient is selected from one or more of an ionic surfactant, a non-ionic surfactant and a polymer stabilizer; for example, the ionic surfactant includes sodium dodecyl sulfate; the non-ionic surfactant is selected from one or more of Tween, vitamin E, polyethylene glycol succinate and decyl glucoside; the polymer stabilizer is selected from one or more of hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and poloxamer.

[0015] In some more preferred embodiments of the present invention, the excipient is poloxamer 407.

[0016] In some embodiments of the present invention, the conjugate is formed by connecting the hydrophilic targeting polypeptide and the poorly soluble drug via a covalent bond, wherein the covalent bond includes a dissociable and an indissociable covalent bond, and is preferably a hydrazone bond.

[0017] In some preferred embodiments, the mass ratio of the poorly soluble drug, the conjugate and the excipient is 1:(0-0.17):(1.6-5.0), and the mass of the conjugate is not 0; the mass ratio is, for example, 6:1:20.

[0018] In some embodiments of the present invention, the hydrophilic targeting polypeptide is selected from one or more of a tumor targeting polypeptide and a brain targeting polypeptide.

[0019] In some preferred embodiments of the present invention, the tumor targeting polypeptide includes VAP polypeptide, cVAP polypeptide, S VAP peptide, DVAP polypeptide, A7R polypeptide, cA7R polypeptide, D A7R polypeptide, RGD polypeptide, Stapled-RGD polypeptide, RW polypeptide, mn polypeptide, RAP12 polypeptide and D RAP12 polypeptide and its derivatives; the brain targeting polypeptides include p-hydroxybenzoic acid (pHA) derivatives, D8 polypeptide, TGN polypeptide, D TGN peptide, CDX peptide, D CDX peptide, T7 peptide and D T7 polypeptide and its derivatives.

[0020] In some more preferred embodiments of the present invention, the hydrophilic targeting polypeptide is VAP and / or pHA-VAP.

[0021] In order to solve the above technical problems, the second aspect of the present invention provides a method for preparing a drug delivery system as described in the first aspect of the present invention, the method comprising the following steps: dissolving the poorly soluble drug, conjugate and excipients, concentrating them into a thin film and hydrating them, and then ultrasonicating them.

[0022] In some embodiments of the present invention, the mass ratio of the poorly soluble drug, the conjugate and the excipient is 6:1:20; the dissolution is performed by dissolving in alcohol or dichloromethane, and the alcohol is preferably methanol or ethanol; the concentration is performed by rotary concentration under reduced pressure, preferably at 37°C; the hydration is performed by adding PBS buffer or polyvinyl alcohol; and the ultrasound is performed by using an ultrasonic cell crusher.

[0023] In some preferred embodiments of the present invention, the power of the ultrasonic cell disruptor is 80-200 W, such as 160 W, and the ultrasonic time is 20-60 min, such as 20 min.

[0024] The drug delivery system prepared by the above method has a particle size of 100-200nm, good stability, and good targeting ability. The dosage of poloxamer 407 (F-127) needs to reach a certain dosage to form a relatively stable nanocrystal solution. Below this dosage, nanocrystals cannot be formed, or the formed nanocrystals are unstable. Within the dosage that can form nanocrystals, as the dosage increases, the particle size of the nanocrystals will increase and the drug loading will decrease. If the peptide-drug conjugate is not used, nanocrystals can be formed, but the particle size will be significantly larger than the nanocrystals using peptide-conjugated drugs. If used in excess, some peptide-drug conjugates will self-assemble to form a nanoparticle system with a dual particle size distribution and instability. Within the appropriate range of use, the larger the dosage, the larger the particle size of the resulting nanocrystals. If the probe power is insufficient or the time is insufficient, the nanocrystals will have a larger particle size and insufficient stability, and will settle if left for a long time. If the probe power is too high or the time is too long, the degradation of the peptide-conjugated drug will occur.

[0025] In order to solve the above technical problem, the third aspect of the present invention provides a method for delivering a drug, the method comprising the following steps: delivering the drug by using the drug delivery system as described in the first aspect of the present invention.

[0026] In order to solve the above technical problems, the fourth aspect of the present invention provides the use of the drug delivery system as described in the first aspect of the present invention in the preparation of drugs for treating diseases, wherein the diseases include brain tumors and peripheral tumors.

[0027] In order to solve the above technical problems, the fifth aspect of the present invention provides a use of a conjugate of a polypeptide and a poorly soluble drug as a stabilizer in the preparation of a nanocrystalline drug of the poorly soluble drug.

[0028] In some preferred embodiments of the present invention, the conjugate is formed by connecting the hydrophilic targeting polypeptide and the poorly soluble drug via a covalent bond, wherein the covalent bond includes a dissociable and an indissociable covalent bond, and is preferably a hydrazone bond.

[0029] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0030] The reagents and raw materials used in the present invention are commercially available.

[0031] The positive and progressive effects of the present invention are: the drug delivery system of the present invention can give the drug delivery system the ability to actively target tumors and / or cross barriers, while retaining the high drug loading characteristics of the nanocrystal system, which can significantly improve the therapeutic effect. The method of preparing the drug delivery system of the present invention is simpler to implement, and the introduction of the polypeptide targeting molecule (pHA-VAP) is simultaneously completed during the preparation of the nanocrystals, without the need for additional steps. This provides a simpler prescription and process, and fewer obstacles will be encountered when developing clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The synthetic route of peptide-drug conjugates VAP-HA-DTX and pHA-VAP-HA-DTX.

[0033] Figure 2 HPLC and ESI-MS spectra of peptide-drug conjugates VAP-HA-DTX and pHA-VAP-HA-DTX; wherein, Figure A is the HPLC and ESI-MS spectra of VAP-HA-DTX; and Figure B is the HPLC and ESI-MS spectra of pHA-VAP-HA-DTX.

[0034] Figure 3Figure 1 is the particle size distribution and electron microscope image of the drug nanocrystal delivery system; Figure A is the particle size distribution of the drug nanocrystal delivery system detected by a laser particle size scattering instrument; Figure B is the electron microscope image of NC@VD.

[0035] Figure 4 Figure 1 is an X-ray powder diffraction and differential scanning calorimetry analysis diagram; Figure A is an X-ray powder diffraction diagram; Figure B is a differential scanning calorimetry analysis diagram.

[0036] Figure 5 Figure 3 is the stability of NC@VD under different pH conditions; Figure A is the particle size and distribution of NC@VD at different times in pH 5.0; Figure B is the change of NC@VD particle size over time under different pH conditions.

[0037] Figure 6 The hydrolysis of VAP-HA-DTX under different pH conditions.

[0038] Figure 7 Figure 2 is the in vitro release curve of the nanocrystal drug delivery system; Figure A is the release curve of each nanocrystal drug delivery system in pH 7.4 medium; Figures BD are the release curves of NC@pVD, NC@VD and NC in pH 7.4 and 6.0 media, respectively.

[0039] Figure 8 The quantitative results of U87 cells' uptake of the nanocrystal drug delivery system.

[0040] Fig. 9 These are the quantitative results of the in vitro trans-BBB and BBTB transport of nanocrystal drug delivery systems, which are grouped into DTX, NC, NC@VD and NC@pVD in the horizontal axis direction; Figure A is the quantitative results of the in vitro trans-BBB transport of DTX drug solution and each nanocrystal drug delivery system; Figure B is the quantitative results of the in vitro trans-BBTB transport of DTX drug solution and each nanocrystal drug delivery system.

[0041] Fig.10 This is the pharmacokinetic curve of the nanocrystal drug delivery system.

[0042] Fig.11 Figure 2 is the efficacy of the nanocrystal drug delivery system on subcutaneous transplanted tumors; Figure A is the curve of the change of U87 subcutaneous tumor volume over time; Figure B is the statistical result of in vitro tumor weighing; Figure C is the photographic result of the in vitro tumor.

[0043] Fig.12 This is the survival curve of U87 orthotopic brain glioma model mice. DETAILED DESCRIPTION

[0044] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0045] Example 1 Preparation and characterization of peptide-drug conjugates

[0046] The synthetic route of peptide-drug conjugates is as follows Figure 1 , the specific steps are as follows:

[0047] 1.1 Esterification of docetaxel (LEV-DTX)

[0048] 61 μL levulinic acid (1.5 eq) and 120 mg dicyclohexylcarbodiimide (DCC, 1.5 eq) were dissolved in 15 ml acetonitrile and placed in an ice-water bath and stirred for 0.5 h. Then 310 mg docetaxel (DTX, 1.0 eq) and 11.7 mg dimethylaminopyridine (DMAP, 0.25 eq) were added, and after stirring for 4 h in an ice-water bath, 12 mL of pure water was added to quench the reaction. Filter to remove the by-product dicyclohexylurea (DCU). The filtrate was purified by preparative liquid phase and freeze-dried to obtain the product LEV-DTX.

[0049] 1.2 Docetaxel hydrazone bond (HA-DTX)

[0050] 69.1 mg LEV-DTX (1.0 eq) and 70 mg maleimidohexanoic acid hydrazine trifluoroacetate (2.5 eq) were dissolved in 5 mL methanol by stirring at room temperature. Then 20 μL glacial acetic acid was added and stirred at room temperature for 5 h. The reaction solution was then added dropwise to an alkaline aqueous solution (4 mL 0.2 M ammonium acetate aqueous solution, 5 mL acetonitrile and 150 μL ammonia water). The reaction solution was purified by preparative liquid phase and freeze-dried to obtain the product HA-DTX.

[0051] 1.3 Preparation of peptide-drug conjugates VAP-HA-DTX or pHA-VAP-HA-DTX

[0052] 29.6 mg HA-DTX (1.0 eq) was dissolved in 4 mL acetonitrile / PBS (1:1, v / v) mixed solution, and 27 mg VAP-Cys (all-D-configuration polypeptide, amino acid sequence: D P D A D V D R D T D N D S D C, 1.2 eq) or pHA-VAP (peptide derivative, sequence is pHA-Acp- D C D PD A D V D R D T D N D S) 40 mg (1.2 eq), stirred at room temperature for 15 min. The reaction solution was purified by preparative liquid phase to obtain VAP-HA-DTX or pHA-VAP-HA-DTX. The purity of the product was detected by HPLC, and the molecular weight was characterized by MS. The results are as follows Figure 2 , indicating that the obtained VAP-HA-DTX and pHA-VAP-HA-DTX have high purity and correct molecular weight.

[0053] Example 2 Construction of a drug delivery system of peptide-drug conjugate coated drug nanocrystals

[0054] 2.1 Construction of VAP-HA-DTX coated DTX nanocrystal drug delivery system (NC@VD)

[0055] NC@VD was prepared by film-forming hydration plus probe ultrasound. The specific process is as follows:

[0056] Accurately weigh 0.5 mg of VAP-HA-DTX, 3.0 mg of DTX, and 10 mg of poloxamer 407 (F-127), add appropriate amount of methanol to dissolve, place in an eggplant-shaped bottle, and concentrate under reduced pressure and rotation at 37°C to form a thin film. Add 3 mL of PBS buffer for hydration, and place under the ultrasonic probe of an ultrasonic cell disruptor and ultrasonicate at a power of 160 w for 20 min.

[0057] After laboratory formulation process exploration, a uniform and stable DTX nanocrystal drug delivery system coated with the peptide-conjugated drug VAP-HA-DTX can be formed within the feasible parameter range shown in Table 1.

[0058] Table 1 Nanocrystal drug delivery system process exploration

[0059]

[0060] Experiments have shown that a certain amount of poloxamer 407 relative to the drug DTX is required to form a relatively stable nanocrystalline solution. When the amount is too high, the drug loading capacity will be reduced, which is not conducive to drug delivery.

[0061] Experiments have shown that if peptide-coupled drugs are not used, nanocrystals can be formed, but the particle size will be significantly larger than that of nanocrystals using peptide-coupled drugs. If used in excess, some peptide-coupled drugs will self-assemble to form a nanoparticle system with a dual particle size distribution and instability.

[0062] Experiments have shown that insufficient probe ultrasonic power or insufficient time will result in a stable nanocrystal solution; too high probe ultrasonic power or too long a time will lead to degradation of the peptide-conjugated drug.

[0063] Experiments have confirmed that if the peptide-conjugated drug VAP-HA-DTX remains unchanged and only the drug core is changed from docetaxel to cabazitaxel, a uniform and stable nanocrystal drug delivery system cannot be formed under the above-mentioned formulation process.

[0064] 2.2 Construction of pHA-VAP-HA-DTX coated DTX nanocrystal drug delivery system (NC@pVD)

[0065] Replace VAP-HA-DTX with pHA-VAP-HA-DTX, and follow the same procedure as NC@VD to construct NC@pVD.

[0066] 2.3DTX Nanocrystal (NC) Construction

[0067] Except for not adding peptide-drug conjugate, the other processes were the same as NC@VD to construct NC.

[0068] Example 3 Characterization of the Peptide-Drug Conjugate Coated Drug Nanocrystal Delivery System

[0069] 3.1 Determination of particle size of nanocrystal drug delivery system

[0070] The particle size and particle size distribution of each nanocrystal drug delivery system were determined by dynamic light scattering (instrument model: ZetaSizerNano, Malvern), and the morphology was observed by electron microscopy (instrument model: Tecnai-D1270, FEI). The results showed that NC@VD and NC@pVD had smaller particle sizes, indicating that the coating of peptide-drug conjugates was beneficial to the stability of drug nanocrystals; electron microscopy showed that the morphology of NC@VD was round ( Figure 3 ).

[0071] 3.2 X-ray powder diffraction and differential scanning calorimetry analysis

[0072] DTX, F-127, NC, NC@VD and NC@pVD solid powders (the three nanocrystalline solid powders are freeze-dried products of the prepared nanocrystalline solutions after ultrafiltration and desalting) were characterized by X-ray diffractometer (instrument model: Bruker Corp Billerica; measurement range: 0-50°; scanning parameters: 0.02° / min Cu (40 kV, 40 mA) and differential scanning calorimetry (instrument model: Perkin–Elmer Pyris-1; measurement range: 30-300°C; reference material: air; heating rate: 10°C / min). The results are shown in Figure 2. Figure 4It shows that the X-ray diffraction patterns of NC, NC@VD and NC@pVD mainly show the characteristic diffraction peak of the auxiliary material F-127, and the characteristic diffraction peak of DTX is very weak, suggesting that DTX in NC, NC@VD and NC@pVD mainly exists in an amorphous state; the differential scanning calorimetry patterns of NC, NC@VD and NC@pVD mainly show the characteristic endothermic peak of the auxiliary material F-127, and the characteristic peak of DTX is very weak, suggesting the same result.

[0073] 3.3 Encapsulation efficiency and drug loading detection

[0074] Accurately weigh NC, NC@VD and NC@pVD solid powders, add appropriate amount of acetonitrile to dissolve them, take samples for HPLC detection, and calculate the DTX concentration using the standard curve. Calculate the encapsulation efficiency and drug loading according to the following formulas:

[0075] Encapsulation rate (%) = (DTX mass in nanocrystal drug delivery system / DTX mass in prescription) × 100%

[0076] Drug loading (%) = (DTX mass in nanocrystal drug delivery system / mass of nanocrystal drug delivery system) × 100%

[0077] The results are shown in Table 2. The encapsulation efficiency of the three is about 90%, and the drug loading is about 20%.

[0078] Table 2 Drug loading and encapsulation efficiency of drug nanocrystal delivery system

[0079]

[0080] 3.4 Detection of free peptide-drug conjugates

[0081] 1 mL of NC@VD solution was taken and added to an ultrafiltration tube with a molecular weight cutoff of 30 KDa. It was centrifuged at 12000 rpm for 15 min, and 1 mL of PBS was added to the retentate to resuspend it. The ultrafiltrate, resuspended solution and NC@VD samples were taken respectively, and the concentration of VAP-HA-DTX therein was detected by HPLC. The results showed that about 95% of VAP-HA-DTX in NC@DTX was retained by the ultrafiltration tube, and only about 5% could pass through the ultrafiltration tube, indicating that in NC@VD, the peptide-drug conjugate mainly exists in the nanocrystal drug delivery system, and only a small amount of VAP-HA-DTX exists in a free form.

[0082] Example 4 Stability Study of NC@VD

[0083] An appropriate amount of NC@VD solution was added to buffer media of pH 5.0, 6.0 and 7.4, respectively, and placed in an air bath shaker at 37°C and shaken at 100 rpm. Samples were taken at each time point to measure the particle size and its distribution.

[0084] VAP-HA-DTX was added to buffered salt media of pH 7.4, 6.0, and 5.0 containing 0.5% Tween-80, respectively, and placed in an air bath shaker at 37°C and shaken at 100 rpm. Samples were taken at each time point, and the degree of hydrolysis of VAP-HA-DTX was detected by HPLC.

[0085] The results showed that the particle size and distribution of NC@VD in different pH buffer media did not change significantly over time ( Figure 5 ). As for VAP-HA-DTX, since it covalently links the peptide to the drug through a pH-sensitive hydrazone bond, the hydrolysis rate is significantly different under different pH conditions. The lower the pH, the faster the hydrolysis ( Figure 6 This phenomenon may be because the acid-sensitive hydrazone bond in VAP-HA-DTX belongs to the non-polar fragment of the molecular structure, which is hidden inside the nanoparticles and is not easily + Therefore, NC@VD is not greatly affected by pH.

[0086] Example 5 In vitro release study of nanocrystal drug delivery system

[0087] Accurately pipette 1 mL of the nanocrystal drug delivery system (NC@pVD or NC@VD or NC) solution into a dialysis bag with a molecular weight cutoff of 14 KDa, then place it in 10 mL of a buffer medium containing 1% Tween-80 at pH 7.4 or 6.0, and shake it at 100 rpm in a 37°C water bath shaker. Samples of the liquid outside the dialysis bag were taken at 1, 2, 4, 8, 12, 24, 36, 48, and 60 h, respectively. After each sampling, the liquid was replenished with an equal volume of the corresponding buffer medium. The drug concentration of the sampled solution was determined by HPLC, and the release curve was drawn. The results are shown in Figure 7 There was no significant difference in the release of NC@pVD and NC@VD in pH 7.4 buffer medium, and both were significantly faster than NC without peptide-drug conjugate coating. The release of NC@pVD and NC@VD in pH 6.0 medium was slightly faster than that in pH 7.4 medium, while the release of NC at two different pH values ​​was almost the same.

[0088] Example 6 U87 cells uptake of nanocrystal drug delivery system

[0089] U87 cells in the logarithmic growth phase were digested and dispersed, with 2×10 cells per well. 5The cells were inoculated at a density of 100 cells / well in a 6-well plate. After adding 2 mL of cell culture medium to each well, the plates were transferred to a cell culture incubator and cultured for 24 hours. After the cells were completely attached to the wall, the culture medium was discarded, and a drug solution containing 30 μM DTX, NC, NC@VD, and NC@pVD prepared with cell culture medium was added, 2 mL per well, and the drugs were administered in triplicate. The plates were incubated at 37°C in a cell culture incubator for 4 hours, the drug solution was discarded, the plates were washed twice with PBS, 400 μL of trypsin was added for digestion, and then the cells were resuspended with 600 μL of PBS. The cell suspension was counted using an automatic cell counter, and the drug concentration was determined by HPLC to calculate the drug uptake per unit cell. The results are shown in Figure 8 , the uptake of U87 cells by the nanocrystal drug delivery system is significantly higher than that by free DTX. This shows that the construction of a DTX nanocrystal drug delivery system can improve the ability of drugs to enter cells.

[0090] Example 7 Investigation of in vitro transport capacity across BBB (blood-brain barrier) and BBTB (blood-brain tumor barrier)

[0091] 7.1 In vitro trans-BBB transport capacity

[0092] The brains of 4-week-old SD rats were removed after decapitation, and the cerebral cortex was quickly separated in ice-cold D-Hanks solution. The meninges and large blood vessels of the brain were removed and cut into pieces, and collagenase and DNase were added. After digestion at 37°C for 90 min, centrifuge at 1000 r / min for 8 min, discard the supernatant, transfer to 20% BSA, centrifuge at 1000 g / min at 4°C for 20 min, discard the upper solution, transfer the bottom microvessel to the culture solution, centrifuge at 1000 r / min for 5 min, prepare the bottom microvessel segment with DMEM culture solution containing 20% ​​fetal bovine serum to form a microvessel segment suspension, inoculate in a 24-well transwell pre-coated with rat tail collagen, move the transwell into a carbon dioxide incubator, culture at 37°C, 5% CO2 and saturated humidity for 24 h to allow the microvessel segment to adhere to the wall, then change to endothelial-specific culture solution containing puromycin for further culture for 72 h, and then change to endothelial-specific culture solution without puromycin for culture for 72 h, and measure the transmembrane electrical resistance (TEER) exceeding 250 Ω·cm 2 The results showed that the in vitro BBB modeling was successful. 100 μL of drug solution containing 30 μmol / L DTX, NC, NC@VD and NC@pVD solution prepared with cell culture medium were administered to the upper chamber of Transwell, and 600 μL of blank culture medium was added to the lower chamber. The cells were incubated at 37°C, and 20 μL of culture medium from the lower chamber was taken out at 0.5, 1, 2 and 4 h after administration for quantification by LC-MS / MS. The results are shown in Fig. 9A, NC@pVD has a significantly stronger ability to transport across the BBB than NC@VD and NC. After 4 hours of administration, the transport amounts of NC@pVD, NC@VD and NC across the BBB were 17.84±1.90%, 13.05±2.15% and 6.36±0.36% of the total drug amount, respectively. The results show that the surface coating of drug nanocrystals with peptide-drug conjugate pHA-VAP-HA-DTX can enhance the trans-BBB transport ability of nanocrystals through the affinity of pHA for dopamine receptors.

[0093] 7.2 In vitro trans-BBTB transport capacity

[0094] HUVEC cells and U87 cells in the logarithmic growth phase were taken, and the monolayer culture cells were digested with 0.25% trypsin, and a single cell suspension was prepared with DMEM culture medium containing 10% fetal bovine serum. HUVEC cells were inoculated in the upper chamber of the 24-well transwell, with a volume of 0.5 mL per well; U87 cells were inoculated in the lower chamber of the transwell, with a volume of 1.5 mL per well, and the number ratio of HUVEC to U87 cells in each well was 1:5. The transwell culture plate was moved into a carbon dioxide incubator, and after three days of culture at 37°C, 5% CO2 and saturated humidity, a BBTB model was formed. 100 μL of a drug solution containing 30 μmol / LDTX, NC, NC@VD and NC@pVD solutions prepared with cell culture medium were given to the upper chamber of the Transwell, and 600 μL of blank culture medium was added to the lower chamber, and incubated at 37°C. 20 μL of the culture medium in the lower chamber was taken at 0.5, 1, 2 and 4 h after administration, respectively, and quantified by LC-MS / MS. Results are shown in Fig. 9 B, NC@pVD and NC@VD have significantly stronger trans-BBTB transport capabilities than NC. After 4 hours of administration, the trans-BBTB transport amounts of NC@pVD, NC@VD, and NC were 18.78±2.03%, 17.38±3.31%, and 7.26±0.79% of the total drug dose, respectively. The results indicate that pHA-VAP-HA-DTX or VAP-HA-DTX coated drug nanocrystals can mediate the affinity of VAP for heat shock protein GRP78 to enhance the trans-BBTB transport capacity.

[0095] Example 8 Pharmacokinetics Study

[0096] ICR mice were used to investigate the in vivo pharmacokinetic of each nanocrystal drug delivery system. Four groups, DTX solution, NC, NC@VD and NC@pVD, were set up, with 3 mice in each group. The tail vein was injected, and the dose was 5 mg / kg of DTX. Blood was collected from the eye socket at each time point, and the blood drug concentration was detected by LC-MS / MS, and the pharmacokinetic curve was drawn. The blood drug concentration at each time point was analyzed by DAS2.0 software, and the area under the curve (AUC), mean retention time (MRT) and half-life (t1 / 2 ). The pharmacokinetic curve is as follows Fig.10 The pharmacokinetic parameters are shown in Table 3. The results showed that the in vivo circulation time (MRT) of the nanocrystals coated with the peptide-drug conjugate was (0-∞) and t 1 / 2 ) and AUC (0-∞) Significantly higher than uncoated nanocrystals and drug solutions.

[0097] Table 3 Statistical moment parameter results of main pharmacokinetic parameters (n=3)

[0098]

[0099] Example 9 Pharmacodynamic Evaluation

[0100] 9.1 Evaluation of drug efficacy in subcutaneous transplanted tumors

[0101] U87 cells in the logarithmic growth phase were trypsinized and the cell concentration was adjusted to 3×10 7 / mL, 100 μL was inoculated into the subcutaneous part of the right dorsal side of nude mice near the armpit. After inoculation, the mice were kept in SPF grade and the tumor size was observed regularly. 3 The tumor-bearing mice were randomly divided into 7 groups, 5 mice in each group, and the tail veins were injected with normal saline, DTX solution, NC, NC@VD (high, medium and low dose groups) and NC@pVD. Among them, the total dose of DTX in the high dose group of NC@VD was 16 mg / kg, the low dose group was 6 mg / kg, and the dose of DTX in the other groups was 10 mg / kg. The drugs were administered on days 0, 3, 6 and 9, and the long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper every other day. The formula V 瘤体积 =0.5(a×b 2 ) Calculate the tumor volume of each group and draw a curve of tumor volume change over time. When the average tumor volume of the saline group reached 1000mm 3 At the above time, all nude mice were killed by cervical dislocation, subcutaneous tumors were removed and weighed, and the statistical differences among the groups were calculated.

[0102] Results Fig.11 , the effect of NC@VD in inhibiting tumor growth increases with increasing dose, showing a dose-dependent manner. The anti-tumor effects of NC@VD and NC@pVD are significantly stronger than those of NC and DTX solutions at the same dose. This is because both drug delivery systems have tumor-targeting peptides VAP on their surfaces, which can promote the targeted distribution of drugs to tumors, thus having a stronger anti-tumor effect.

[0103] 9.2 Evaluation of drug efficacy in situ brain glioma

[0104] U87 cells in the logarithmic growth phase were taken and 5×10 5cells (dispersed in 5 μL PBS buffer). After the nude mice were anesthetized, they were fixed with a brain stereotaxic instrument, and the cells were inoculated in the right part of the striatum (0.6 mm in front of the anterior fontanelle, 1.8 mm on the side, and 3 mm deep). On the 7th day after the nude mice were inoculated with tumor cells, they were randomly divided into 5 groups, with 10 mice in each group. On the 7th, 10th, 13th and 16th days, normal saline, DTX solution, NC, NC@VD and NC@pVD were injected into the tail vein respectively. The total dose of each group contained 16 mg / kg of DTX. The survival time of the nude mice in each group was recorded, and the survival curve was drawn. The results are shown in Fig.12 . The results showed that compared with normal saline, the drug-treated groups all showed varying degrees of inhibition of brain tumor growth. Among them, NC@pVD had a median survival of 51 days, and had the best anti-brain tumor effect, significantly better than NC@VD (median survival of 46.5 days), NC (median survival of 46 days) and DTX solution (median survival of 44.5 days). This is because the pVD polypeptide on the surface of NC@pVD can improve the ability of the nanocrystal drug delivery system to transport across the BBB, while targeting tumor cells, thereby showing the best anti-brain tumor effect.

Claims

1. A drug delivery system, characterized in that: The drug delivery system comprises nanocrystals and conjugates. The nanocrystals are nanocrystals of poorly soluble drugs. The conjugates are formed by coupling a polypeptide with the poorly soluble drugs. The conjugates are coated on the outer layer of the nanocrystals.

2. The drug delivery system according to claim 1, wherein: The poorly soluble drug is an anti-tumor drug; Preferably, the anti-tumor drug is selected from one or more of the following: anthracyclines, taxanes, camptothecins and vinca alkaloids; More preferably, the anthracycline drugs are selected from one or more of the following: doxorubicin and epirubicin; the taxane drugs are selected from one or more of the following: paclitaxel, cabazitaxel and docetaxel; the camptothecin drugs are selected from one or more of the following: camptothecin, hydroxycamptothecin, 9-nitrocamptothecin and irinotecan; the vinca alkaloid drugs are selected from one or more of the following: vinblastine and vincristine; Even more preferably, the anti-tumor drug is cabazitaxel or docetaxel.

3. The drug delivery system according to claim 1 or 2, characterized in that: The nanocrystals are nanocrystals prepared from the poorly soluble drug and excipients; Preferably, the excipient is selected from one or more of an ionic surfactant, a nonionic surfactant and a polymer stabilizer; for example, the ionic surfactant includes sodium dodecyl sulfate; the nonionic surfactant is selected from one or more of Tween, vitamin E, polyethylene glycol succinate and decyl glucoside; the polymer stabilizer is selected from one or more of hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and poloxamer; More preferably, the excipient is poloxamer 407.

4. The drug delivery system according to any one of claims 1 to 3, characterized in that: The conjugate is formed by connecting the hydrophilic targeting polypeptide and the poorly soluble drug via a covalent bond, wherein the covalent bond includes a dissociable and an indissociable covalent bond, and is preferably a hydrazone bond; Preferably, the mass ratio of the poorly soluble drug, the conjugate and the excipient is 1:(0-0.17):(1.6-5.0), and the mass of the conjugate is not 0; the mass ratio is, for example, 6:1:

20.

5. The drug delivery system according to any one of claims 1 to 4, characterized in that: The hydrophilic targeting polypeptide is selected from one or more of a tumor targeting polypeptide and a brain targeting polypeptide; Preferably, the tumor targeting polypeptide includes VAP polypeptide, cVAP polypeptide, S VAP peptide, D VAP polypeptide, A7R polypeptide, cA7R polypeptide, D A7R polypeptide, RGD polypeptide, Stapled-RGD polypeptide, RW polypeptide, mn polypeptide, RAP12 polypeptide and D RAP12 polypeptide and its derivatives; the brain targeting polypeptides include p-hydroxybenzoic acid (pHA) derivatives, D8 polypeptide, TGN polypeptide, D TGN peptide, CDX peptide, D CDX peptide, T7 peptide and D T7 polypeptide and its derivatives; More preferably, the hydrophilic targeting polypeptide is VAP and / or pHA-VAP.

6. A method for preparing the drug delivery system according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: dissolving the poorly soluble drug, the conjugate and the auxiliary materials, concentrating them into a film, hydrating them and then ultrasonicating them.

7. The method according to claim 6, characterized in that The mass ratio of the poorly soluble drug, the conjugate and the excipient is 6:1:20; the dissolution is performed by using alcohol or dichloromethane, and the alcohol is preferably methanol or ethanol; the concentration is performed by vacuum rotary concentration, preferably at 37°C; the hydration is performed by adding PBS buffer or polyvinyl alcohol; and the ultrasound is performed by using an ultrasonic cell pulverizer; Preferably, the power of the ultrasonic cell disruptor is 80-200W, and the ultrasonic time is 20-60min.

8. A method for delivering a drug, characterized in that: The method comprises the following steps: delivering the drug by using the drug delivery system according to any one of claims 1-5.

9. Use of the drug delivery system according to any one of claims 1 to 5 in the preparation of drugs for treating diseases, wherein the diseases include brain tumors and peripheral tumors.

10. Use of a conjugate of a polypeptide and a poorly soluble drug as a stabilizer in the preparation of a nanocrystalline drug of the poorly soluble drug; Preferably, the conjugate is formed by connecting the hydrophilic targeting polypeptide and the poorly soluble drug via a covalent bond, wherein the covalent bond includes a dissociable and an indissociable covalent bond, and is preferably a hydrazone bond.

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