WO2023280128A1 - Docetaxel micelle nano-drug, and preparation method therefor and use thereof - Google Patents

Abstract

A docetaxel (DTX) micelle nano-drug. A method for preparing the nano-drug is: a polymer is dissolved in oligopolyethylene glycol to obtain a polymer mother liquor; DTX is dissolved in oligopolyethylene glycol to obtain a DTX mother liquor; the polymer mother liquor is mixed with the DTX mother liquor to obtain a mixed solution of DTX, the polymer and oligopolyethylene glycol; the mixed solution of DTX, the polymer and oligopolyethylene glycol is mixed with a buffer solution to obtain the DTX micelle nano-drug. Compared with a free drug, the nano-drug has increased solubility and prolonged circulation time in vivo, the biological distribution of the drug is changed, enrichment of normal tissue is reduced, and targeted treatment and inhibition effects on prostate cancer are achieved.

Description

A kind of docetaxel micellar nano-medicine and its preparation method and application technical field

The invention belongs to nano-medicines, in particular to docetaxel (DTX) micellar nano-medicines and its preparation method and application.

Background technique

Currently, chemotherapy is the main treatment for castration-resistant prostate cancer (CRPC). However, chemotherapy drugs generally have low bioavailability, high toxicity and side effects, and poor curative effect. The vigorous development of nanomedicine provides a way of thinking for the treatment of CRPC. However, drug delivery for tumor therapy faces many obstacles, including insufficient blood circulation time, low accumulation in tumor tissue, difficulty in penetrating deep into tumor tissue, and difficulty in being specifically endocytosed by tumor cells; researchers have developed multiple A multifunctional drug delivery system to overcome these challenges. However, the design of complex multifunctional delivery systems not only makes the synthesis of polymers time-consuming and difficult to quality control, but also may lead to problems such as poor biocompatibility, making it difficult to achieve clinical translation. So far, biodegradable polymer micelles based on polyethylene glycol-poly-D,L-lactide (PEG-PLA) block copolymers are one of the most important nano-delivery systems for anticancer drugs. PEG-PLA-based nano-medicines Genexol-PM and Nanoxel-PM have entered the clinic, but it should be pointed out that although these nano-medicines have reduced toxicity in clinical treatment, the prolongation of patient survival has not reached expectations. The possible reasons are premature drug release, insufficient uptake by tumor cells, etc.

technical problem

Aiming at the problems and challenges of the prior art, the present invention designs surface-coupled targeting molecules, disulfide cross-linking based on amphiphilic polymers, small-sized DTX micellar nanomedicines, and is used for the treatment of castration-resistant prostate cancer CRPC. highly effective targeted therapy. For example, the copolymers PEG-P(LA-DTC), PEG-P(CL-DTC) or PEG-P(TMC-DTC) are prepared by controlled ring-opening polymerization, and the molecular weight of the polymer is similar to that of Nanoxel-PM, which can be obtained The micelles with small size but disulfide cross-linking use dithiolane trimethylene carbonate (DTC) to spontaneously form disulfide cross-linking, stably load docetaxel (DTX), and can be absorbed by glutathione in the cell The peptide was quickly reduced and released, and it showed surprising curative effect in the treatment of mice bearing human CRPC tumors and PDX models of prostate cancer patients.

technical solution

The present invention adopts the following technical scheme: docetaxel (DTX) micellar nano-medicine, mixing the mixed solution of DTX, polymer, oligoethylene glycol and buffer solution to obtain DTX micelle nano-medicine; the oligoethylene glycol The molecular weight of the diol is 200-600 Da.

In the present invention, the molecular weight ( M _n ) of the oligoethylene glycol is 200-600 Da, preferably 300-500 Da; the molecular weight of the polymer is 2000-15000 Da, preferably 3000-8000 Da. The molecular weight of the polymer of the present invention is the number average molecular weight ( M _n ) determined by NMR, and the unit is Dalton (Da).

In the present invention, the polymer is a non-targeting polymer, or the polymer is a non-targeting polymer and a targeting polymer; wherein, the non-targeting polymer is PEG-P (CL-DTC), PEG-P (TMC -DTC), PEG-P(LA-DTC), etc.; targeting polymers are B-PEG-P(CL-DTC), B-PEG-P(TMC-DTC), B-PEG-P(LA-DTC ), B-PEG-PCL, B-PEG-PTMC or B-PEG-PLA, etc., B is a targeting molecule, preferably, the targeting molecule is a polypeptide, such as a cRGD polypeptide, a polypeptide targeting gonadotropin-releasing hormone ( LHRH polypeptide). In the polymer, the molecular weight ( M _n ) of PEG is 1000-6000 Da.

In the present invention, the polymer is dissolved in oligoethylene glycol to obtain a polymer mother liquor; DTX is dissolved in oligoethylene glycol to obtain a DTX mother liquor; then the polymer mother liquor and DTX mother liquor are mixed to obtain DTX, polymer, low A mixed solution of polyethylene glycol. Preferably, at 25-70° C., the mixed solution of DTX, polymer, and oligoethylene glycol is mixed with buffer to obtain DTX micellar nanomedicine. Further preferably, the mixed solution of DTX, polymer and oligoethylene glycol is added to the buffer, the volume concentration of oligoethylene glycol is not more than 20 vol.%, the concentration of DTX is 1-100 mg/mL, and the polymerization The concentration of the substance is 1-500 mg/mL; the buffer is a conventional substance, which can be PB, Hepes, PBS and other buffers, which will not affect the realization of the technical effect.

In the present invention, when the polymer is a non-targeting polymer and a targeting polymer, the molar ratio of the non-targeting polymer to the targeting polymer is (5-50):1.

The invention discloses the application of the above-mentioned DTX micellar nano-medicine in the preparation of antitumor drugs. The DTX micellar nano-medicine is a chemotherapeutic drug, and the tumor is prostate cancer.

Beneficial effect

The prostate is a gland in the male reproductive system. It is located below the bladder and in front of the rectum. It is as big as a walnut. Prostate cancer is a disease in which malignant (cancer) cells form in the prostate tissue. It is not easy to detect in the early stage. And dense tumors lead to poor accumulation of general nanomedicine in tumors and less uptake by tumor cells. Compared with the existing nano-drugs, the nano-drugs prepared by loading chemotherapeutic drugs in the present invention can increase the solubility of hydrophobic drugs, increase the circulation time of drugs, change the biodistribution of drugs, reduce the enrichment of normal tissues, etc., and the designed active tumor targeting Ligand-modified nano-drugs deliver drugs to tumor cells through receptor-mediated endocytosis, which solves the problems of poor drug accumulation in tumors and less uptake by tumor cells, and designs responsive to tumors and the internal environment of tumor cells. Chemical cross-linking can obtain nano-drugs with stable circulation, and release the cross-linking drug under the stimulation of tumor and tumor cells, prolong the circulation time and improve the drug release efficiency in tumor cells.

Description of drawings

Figure 1 shows the preparation process and structure of cRGD-MDTX and its targeting of cancer cells.

Figure 2 is PEG-P(LA-DTC) ((CDCl ₃ , 400 MHz) A), Mal-PEG-PLA (CDCl ₃ , 400 MHz) (B) and cRGD-PEG-PLA (DMSO- d ₆ , 400 MHz MHZ) (C) ¹ H NMR spectrum.

Figure 3. Determination of micellar morphology and structure by TEM (A) and SLS (B), (C) CMC of cRGD-Ms, Ms and ncMs.

Figure 4 shows the physicochemical properties of cRGD-MDTX and MDTX. (A) Particle size distribution. (B) Ultraviolet spectrum. The DMF solution (1.0 mg/mL) of PEG-P(LA-DTC) was used as the control group. Particle size change at 37 ^o C (C) or in the presence of 10% FBS for 24 h (D). Release profile (E) and change in particle size (F) of DTX with and without 10 mM GSH.

Figure 5 shows the cellular uptake and toxicity studies of cRGD-MDTX. (A) FACS analysis of PC3 cells incubated with Cy5-MDTX or cRGD-/Cy5-MDTX with different cRGD densities for 4 h. (B) CLSM plots of PC3 cells incubated with cRGD/Cy5-MDTX or Cy5-MDTX for 4 h. cRGD/Cy5-MDTX was incubated with cRGD-pretreated PC3 cells as a control. Scale bar: 75 μm. The survival rate of PC3 (C) and MCF-7 cells (D) treated with free DTX, MDTX or cRGD-MDTX was determined by MTT assay. (E) Toxicity of empty micelles to PC3 cells. (F) FACS measurement of apoptosis in PC3 cells free from DTX, MDTX and cRGD-MDTX treatment. Panels E-F: After 4 hours of culture, culture was continued for 44 hours in fresh medium.

Figure 6 is the study of cRGD-MDTX on the inhibition of tubulin in PC3 cells (incubation for 4 hours). (A) The expression of tubulin in the cells of the group with PBS, DTX, MDTX or cRGD-MDTX. (B) The effect of MDTX or cRGD-MDTX on the microtubule organization of cells. Scale bar: 25 μm.

Figure 7 shows the pharmacokinetics, in vivo imaging and biodistribution of cRGD-MDTX and MDTX in mice (7.5 mg/kg). (A) Pharmacokinetics in healthy mice. (B) In vivo fluorescence images of nude mice bearing PC3 tumors after tail vein injection of cRGD/Cy5-MDTX and Cy5-MDTX (7.5 mg DTX/kg, 0.02 mg Cy5/kg). Ex vivo fluorescence images (C) and semiquantification of fluorescence intensity (D) of major organs and tumors of mice 24 hours after injection. DTX biodistribution (E) and tumor-to-normal tissue (T/N) ratio (F) measured by HPLC in major organs and tumors 24 hours after injection. (G) Tumor accumulation of DTX 3 and 24 hours after injection. ns, no significant difference; ** p <0.01; *** p < 0.001.

Fig. 8 is a micellar tumor penetration study of PC3-bearing mice injected with Cy5-labeled cRGD-MDTX and MDTX 24 hours after tail vein injection. CLSM images of the peripheral region (A) or central region (B) of the tumor. Nuclei were stained with DAPI (blue), blood vessels were stained with Alexa Flour 488-labeled anti-CD31 antibody (green), and micelles were shown in red. Scale bar: 75 μm.

Figure 9 is the Masson staining pictures of tumor sections of mice bearing PC3 (A) and U87MG (B) tumors, and the immunofluorescence characterization of α-SMA in PC3 tumor tissues. Scale bar: 100 μm.

Figure 10 is a study of the anti-tumor efficacy of MDTX or cRGD-MDTX containing different cRGD densities in mice bearing PC3 tumors (n = 5). They were administered on days 0, 3, 6 and 9 (7.5 mg DTX/kg, 0.2 mL PBS). ncMDTX, DTX and PBS were used as controls. (A) Tumor volume and (B) relative body weight of mice over time. (C) Mean tumor inhibition rate (TIR) of each group at day 14. (D) TUNEL staining of tumor sections. Scale bar: 50 μm. Statistical analysis: One-way ANOVA and Tukey's multiple comparison test, no significant difference in ns, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 11 is the H&E staining diagram of the major organ sections of the mice on the 14th day of treatment. (Ⅰ) PBS, (Ⅱ) Free DTX, (Ⅲ) ncMDTX, (Ⅳ) MDTX, (Ⅴ) 2.5% cRGD-MDTX, (Ⅵ) 5% cRGD-MDTX or (VII) 10% cRGD-MDTX. Scale bar: 100 μm.

Figure 12 shows the concentrations of calcium (A) and phosphorus (B) in mouse serum on the 14th day of treatment. Statistical analysis: One-way ANOVA and Tukey's multiple comparison test, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 13 shows the antitumor activity of cRGD-MDTX on mice bearing PDX subcutaneous tumors. Administer on days 0, 3, 6, and 9 (7.5 mg DTX/kg, 0.2 mL PBS). Changes in tumor volume (A) and body weight (B). Statistical analysis: One-way ANOVA and Tukey's multiple comparison test, no significant difference in ns, ** p < 0.01, *** p < 0.001.

Figure 14 shows the anti-tumor study of LHRH-MDTX on mice bearing PC3 subcutaneous tumors. Administer on days 0, 3, 6, and 9 (7.5 mg DTX/kg, 0.2 mL PBS). Changes in tumor volume (A) and body weight (B). Statistical analysis: One-way ANOVA and Tukey's multiple comparison test, * p < 0.05.

Figure 15 is the treatment scheme of micellar cRGD-MS-DTX based on PEG-P(CL-DTC) (A) in mouse prostate cancer PDX model, (B) tumor volume monitoring, (C) body weight monitoring; except the last The dose of other DTX was 7.5 mg/kg except for the group 10 mg/kg. Statistical analysis was performed using one-way analysis of variance and Tukey's multiple comparison test.

Fig. 16 is an in vitro photograph of (A) PDX model treated mouse tumors on the 17th day of treatment in Fig. 15, (B) tumor inhibition rate (TIR). Statistical analysis was performed using one-way analysis of variance and Tukey's multiple comparison test.

Embodiments of the present invention

Monomethoxypolyethylene glycol (PEG, M _n = 2 kg/mol, TCI) was used after drying toluene to remove water; d,l-lactide (LA, > 99%, TCI) was dried in toluene Used after recrystallization; ε-caprolactone (ε-CL, 99%, Alfa Aesar) was dried with calcium hydride and distilled under reduced pressure; trimethylene carbonate (TMC, Jinan Daigang) was reconstituted twice from dry toluene Crystallized and stored in a nitrogen glove box until use; maleimide polyethylene glycol (Mal-PEG, M _n = 2 kg/mol, Beijing Jiankai Technology Co., Ltd.) was purchased and used directly. 1,2-Dithiolantrimethylene carbonate (DTC) is a compound disclosed by the applicant. Docetaxel (DTX, > 99%) was purchased from Shanghai Jinhe Biopharmaceutical Co., Ltd. 1,8-Diazabicycloundec-7-ene (DBU, >97%) was purchased from Sigma Aldrich (USA). cRGD cyclic peptide (cRGDfC, > 95%, Qiangyao Bio), PEG350 (oligoethylene glycol, M _n = 350 Da), etc. are used directly after purchase. Blood calcium and phosphorus detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. β-tubulin antibody (CST, USA), GAPDH antibody (Service bio, Wuhan), goat anti-rabbit secondary antibody (Service bio, Wuhan), primary antibody α-tubulin antibody (Santa Cruz, USA), Alexa-680 labeled anti -rat IgG (Molecular Probes, USA) were purchased and used directly. Human prostate cancer PC3, non-small cell lung cancer A549 and ovarian cancer SKOV-3 were purchased from the Cell Bank of Shanghai Chinese Academy of Sciences. The medium was RPMI 1640 (HyClone) containing 1% penicillin, streptomycin (Geno Bio) and 10% fetal bovine serum (Gibco), and the cells were placed in a monolayer in a 3111 incubator at 37ºC and 5% _CO2 to cultivate. All cells were digested with 0.25% (w/v) trypsin containing 0.03% (w/v) ethylenediaminetetraacetic acid (EDTA), and centrifuged at 1000 rpm for 3 minutes with an L-420 low-speed centrifuge.

Proton nuclear magnetic resonance spectrum ( ¹ H NMR ) was measured with a nuclear magnetic resonance spectrometer model of Agilent DD2 or Unity Inova 400, and the deuterated solvent was chloroform (CDCl ₃ ) or dimethyl sulfoxide (DMSO- d 6 ), and the chemical shifts were represented by solvent Signal is standard. Polymer molecular weight and molecular weight distribution were measured by Waters 1515 gel permeation chromatography (GPC), the mobile phase was DMF, the flow rate was 0.8 mL/min, the temperature was 40°C, and the standard sample was polymethyl methacrylate (PMMA) . Micelle particle size, particle size distribution, and surface zeta potential were measured at 25 ^° C using a ZetaSizer Nano-ZS nanoparticle size analyzer (Malvern Instruments) equipped with a 633 nm wavelength He-Ne laser source and a 173 ^° backscattering detector. The structure of the micelles was characterized by transmission electron microscopy (TEM, Tecnai G220, 200 kv, USA), and 10 μL of 0.5 mg/mL nanoparticle solution was dropped on the copper grid, and then stained with 1.0 wt.% phosphotungstic acid to prepare samples. The radius of gyration (Rg) was measured on a Wyatt Dualtec AF4 instrument connected to a Shimadzu LC-2030 Prominence-i using the asymmetric flow field-flow fractionation-UV-QELS (AF4-UV-QELS) technique. The endocytosis of cells and the distribution of micelles in the tumor were observed by confocal laser scanning microscope (CLSM, Leica, Germany), and the cell uptake and apoptosis were measured by BD FACSVerse flow cytometer (Becton Dickinson, FACSVerse, USA). The concentration of DTX was measured by high performance liquid chromatography, the mobile phase was acetonitrile/water (60/40), the flow rate was 1 mL/min, the detection temperature was 30 ^o C, and the detection wavelength was 227 nm. In vivo imaging was tested using the IVIS® Lumina III small animal in vivo imaging system.

Referring to Figure 1, as a specific technical solution of the present invention, a surface-coupled cRGD, disulfide cross-linked based on PEG-P (LA-DTC), and small-sized DTX micellar nanomedicine (cRGD-MDTX) are designed for Efficient targeted therapy in human-bearing CRPC mice.

Synthesis example Synthesis of polymers is a conventional method: PEG-P (LA-DTC) is synthesized by ring-opening polymerization of DTC and LA in anhydrous DCM with PEG as a macroinitiator and DBU as a catalyst. For example, stir MeO-PEG-OH (0.4 g, 200 μmol), DTC (0.14 g, 7.1 mmol), and LA (0.2 g, 13.9 mmol) in DCM (10 mL) in a glove box under nitrogen atmosphere until completely dissolved. DBU (30 mg, 2 mmol) was added under stirring, then the reactor was sealed and transferred out of the glove box, and placed in an oil bath at 37°C for 3 hours to react. Then the reaction solution was added dropwise to a 20-fold excess of cold anhydrous ether to precipitate, after centrifugation, it was dissolved with acetonitrile, and the precipitation was repeated twice. The obtained PEG-P (LA-DTC) was vacuum-dried for 24 hours to obtain a light yellow Lumpy solid. Yield: 76%. The relative molecular weight and molecular weight distribution of the polymers were measured by ¹ H NMR and GPC. NMR characteristic peaks are as follows: PEG: δ 3.6 (-CH ₂ CH ₂ O-) and δ 3.3 (CH ₃ O-), LA: δ 1.5 (CH ₃ -) and δ 5.1 (-COCHO-), DTC: δ 3.0 (-SCH ₂ C-) and δ 4.2 (-OCH ₂ C-).

Add MeO-PEG or Mal-PEG (0.1 g, 50 μmol), LA (0.1 g, 6.9 mmol), and anhydrous toluene into a 25 mL closed reactor, followed by Sn(Oct) ₂ (50 μmol, 16 μL ) was sealed, stirred and reacted overnight in an oil bath at 100°C, and the purification method of precipitation and filtration was the same as above to obtain PEG-PLA and Mal-PEG-PLA respectively.

The targeting polymer cRGD-PEG-PLA is obtained by coupling cRGD to Mal-PEG-PLA through Michael addition reaction. Dry DMF (2 mL) was bubbled with nitrogen for 10 min, Mal-PEG-PLA (0.1 g, 25 μmol) was added and stirred under nitrogen until completely dissolved, then cRGD (23.1 mg, 37.5 μmol) was added to the polymer The solution was sealed and reacted at 37°C for 24 hours. The reaction mixture was dialyzed against DMF (MWCO 1000) for 4 hours and DCM for 24 hours, then precipitated in cold ether, filtered, and dried in vacuo to obtain cRGD-PEG-PLA.

Replace cRGD with a polypeptide that can target the gonadotropin-releasing hormone (LHRH) receptor (sequence: Pyr-HWSYk(c)LRP-NH ₂ , >95%; lower case k, c are D-type amino acids, where The primary amine of k is connected to the side chain through amidation reaction with the carboxyl group of c, and the carboxyethylamine of the polypeptide is capped. Beijing Zhongke Yaguang Biotechnology Co., Ltd.)) to obtain LHRH-PEG-PLA.

The synthesis of PEG-P(CL-DTC) block copolymers is based on MeO-PEG-OH ( M _n = 2.0 kg/mol) as a macroinitiator and diphenyl phosphate (DPP) as a catalyst to initiate DTC and CL. It is obtained by ring-opening polymerization, and PEG-PCL is obtained only by triggering CL ring-opening; the synthesis of cRGD-PEG-P(TMC-DTC) block copolymer is initiated by Mal-PEG-OH as a macroinitiator and DPP as a catalyst The ring-opening polymerization of DTC and CL is obtained by reacting with cRGD. The synthesis of PEG-P(TMC-DTC) block copolymer was obtained by ring-opening polymerization of DTC and TMC initiated by MeO-PEG-OH ( M _n = 2.0 kg/mol) as the initiator and DPP as the catalyst; cRGD- The synthesis of PEG-P(TMC-DTC) block copolymer is obtained by reacting with cRGD after ring-opening polymerization of DTC and TMC initiated by Mal-PEG-OH as initiator and DPP as catalyst. For specific prior art, please refer to CN2021106266013 or other existing documents.

Fig. 2 is the NMR figure of above-mentioned typical product, the target molecular weight of PEG-P (LA-DTC) is 2.0-1.0-0.7 kg/mol, and the polymer molecular weight of Nanoxel-PM (PEG-PLA, 2.0-1.7 kg/mol ), the characterization results of some polymers are shown in Table 1, where the functionalization degree of cRGD in the polymer is 100%.

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^a Calculated based on ¹ H NMR peak area, ^b Determined by GPC.

Cy5-labeled PEG-P(LA-DTC) was synthesized in two steps. First, PEG-P(LA-DTC) was reacted with NPC in the presence of pyridine. Finally, PEG-P(LA-DTC)-NPC was reacted with Cy5-NH ₂ overnight to obtain Cy5-labeled PEG-P(LA-DTC), and then the target targeting polymer was purified by dialysis (MWCO 1000) in DMF and DCM .

Example 1 Preparation and characterization of DTX micellar nanomedicine: PEG _2k -P(LA _1k -DTC _0.7k ), cRGD-PEG _{2k -PLA 1.7k} and DTX were prepared at 200 mg/mL, 50 mg/mL and 100 mg/mL, respectively. The concentration of mg/mL was dissolved in PEG350 to configure three mother solutions. After mixing evenly according to the designed ratio, take 0.05 mL of the mixed solution and heat it to 60°C and inject it into 0.95 mL of phosphate buffer (PB, 10 mM, pH 7.4) preheated to 60°C to obtain a uniform and clear gel. The beam solution cRGD-MDTX is a surface-coupled cRGD, PEG-P (LA-DTC)-based disulfide cross-linked, small-sized DTX micellar nanomedicine.

By changing the ratio of the three mother solutions, a series of micelles with different cRGD density and DTX drug loading can be obtained. Among them, cRGD-PEG-PLA and PEG-P (LA-DTC) are mixed according to the molar ratio of 0/100, 2.5/97.5, 5/95, and 10/90, and the cRGD surface density can be obtained as 0, 2.5%, 5%, respectively. % and 10% micelles. DTX drug loading (DLC) is the mass of DTX/(mass of DTX+mass of polymer)×100.

Untargeted micellar MDTX (without cRGD-PEG-PLA), empty micellar cRGD-Ms (without DTX), untargeted empty micellar Ms (without cRGD-PEG-PLA, plus DTX), non-cross-linked empty micellar ncMs (with PEG-PLA instead of PEG-P(LA-DTC) and without cRGD-PEG-PLA, without DTX) and non-cross-linked micellar ncMDTX (with PEG-PLA instead of PEG-P (LA-DTC) and without cRGD-PEG-PLA).

Cy5-labeled micelles Cy5-MDTX, 2.5% cRGD/Cy5-MDTX, 5% cRGD/Cy5-MDTX and 10% cRGD/Cy5-MDTX.

DLS was used to determine the size of micelles, nanoparticles in serum-containing medium or diluted to different concentrations, and the size distribution over time. CMC is determined by measuring the fluorescence intensity of pyrene in a series of polymer micellar solutions with pyrene as a fluorescent probe. The concentration of the polymer is from 1.0×10 ^-4 to 1 mg/mL. Both were 1.0 µM. Fluorescent samples with an excitation wavelength of 330 nm were measured by a spectrophotometer. The ratio of the fluorescence intensity at 373 nm to 383 nm is the ordinate, and the micelle concentration is the abscissa. The concentration at the inflection point of the low concentration area in the figure is the CMC value of the polymer micelle. To determine whether the micelles were cross-linked, the DMF solution (1 mg/mL) of the polymer PEG-P(LA-DTC) and the PB solution of 1 mg/mL micelles were detected by UV to track the characteristics of disulfide rings in them. Referring to Fig. 3, the empty micelles all have small and uniform hydrodynamic sizes (PDI 0.05-0.11) of 22-27 nm.

cRGD-MDTX exhibited excellent DTX loading capacity and small and uniform size with a small increase in particle size and corresponding empty micelles (Table 2). Figure 4 shows the physical and chemical properties of cRGD-MDTX and MDTX. Its UV spectrum shows that the dithiolane of DTC in PEG-P (LA-DTC) solution has a significant absorption peak at 330 nm, while in the cRGD-MDTX and MDTX In micelles, this absorption peak decreased significantly (Fig. 4B), also showing that cross-linking occurred in micelles. In addition, MDTX and cRGD-MDTX were stable in particle size at 37°C in PB for at least 4 days, and neither showed a large increase in particle size after 24 hours in 10% FBS (Fig. 4C,D). In contrast, ncMDTX without DTC showed a marked increase in size during placement. According to the instructions for Nanoxel-PM, Nanoxel-PM must be used within 4 hours after reconstitution, indicating that the stability of this formulation after 4 hours cannot be guaranteed. It is also recorded in the literature: Nanoxel-PM only guarantees that DTX will not precipitate within 6 hours. The cRGD-MDTX preparation developed by the present invention has high stability, can prevent micelle dissociation and DTX leakage, and is conducive to maintaining the small size of nano-medicines under physiological conditions, which is the key to its ability to penetrate deep into tumors and treat tumors efficiently.

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DTX release experiments from drug-loaded micelles were performed in two media: PB buffer (10 mM, pH 7.4) containing 0.5% Tween 80, and the same solution plus 10 mM GSH. will be 0.5 mL of cRGD-MDTX (1.0 mg/mL, the concentration of DTX is 110 μg/mL) into a release bag with a MWCO of 3500 Da, and placed in 25 mL of the corresponding release medium, placed in a constant temperature shaker (100 rpm, 37°C). At pre-set time points, 5.0 mL of medium was taken each, and a corresponding volume of fresh medium was added. The removed medium was freeze-dried with 0.1 After redissolving in mL of acetonitrile, the concentration of DTX was measured by HPLC, and the cumulative release of the drug was calculated. Each group has three parallel samples.

The results of drug release experiments found that at pH 7.4 and 37°C, only about 15% of DTX leaked from cRGD-MDTX and MDTX within 24 hours, while 85% of DTX leaked from ncMDTX. When 10 mM GSH was added, cRGD-MDTX and MDTX released DTX rapidly, releasing about 80% of DTX in 24 hours (Fig. 4E), and ncMDTX did not release more DTX due to the addition of GSH. Importantly, this controlled decrosslinking and timely release triggered by the intracellular concentration of GSH is more beneficial in tumor therapy than non-responsive drug release by diffusion.

According to the above preparation method, PEG _2k -P(LA _1k -DTC _0.7k ) and cRGD-PEG _{2k -PLA 1.7k were} replaced with other polymers in Table 1, and the obtained targeted DTX micelles (cRGD surface density were 2.5 %, 5% and 10%) or non-targeting DTX micelles with a particle size of 20-32nm, belonging to small-sized nano-drugs, with a drug loading of 5-15%.

Extended example: PEG _2k -P(LA _1k -DTC _0.7k ) and DTX were dissolved in PEG350 at concentrations of 100 mg/mL and 100 mg/mL respectively to prepare two mother solutions. Then mix evenly according to the ratio of 15% DLC, take 0.05 mL of the mixed solution and heat it to 60°C, and inject it into 0.95 mL of phosphate buffer solution (PB, 10 mM, pH 7.4) preheated to 60°C to obtain Uniform and clear micellar solution, non-targeting PEG-P (LA-DTC)-based disulfide cross-linked, small-sized DTX micellar nanomedicine, with a test particle size of 29nm and a PDI of 0.2.

Dissolve PEG _2k -P(LA _1k -DTC _0.7k ) and DTX in PEG350 at concentrations of 200 mg/mL and 100 mg/mL, respectively, to prepare two stock solutions. Then, after mixing evenly according to the proportion of 10% DLC, take 0.05 mL of the mixed solution and heat it to 50°C, and inject it into 0.95 mL of phosphate buffer (PB, 10 mM, pH 7.4) preheated to 50°C to obtain Uniform and clear micellar solution, non-targeting PEG-P (LA-DTC)-based disulfide cross-linked, small-sized DTX micellar nanomedicine, with a test particle size of 26nm and a PDI of 0.23.

Dissolve PEG _2k -P(LA _1k -DTC _0.7k ) and DTX in PEG350 at concentrations of 200 mg/mL and 100 mg/mL, respectively, to prepare two stock solutions. Then mix evenly according to the ratio of 10% DLC to obtain a mixed solution, inject 0.95 mL of phosphate buffer (PB, 10 mM, pH 7.4) preheated to 60°C into 0.05 mL of the mixed solution heated to 60°C, A uniform and clear micellar solution was obtained, which is a non-targeting PEG-P (LA-DTC)-based disulfide cross-linked, small-sized DTX micellar nanomedicine with a test particle size of 23nm and a PDI of 0.10.

Example 2 Cell uptake experiment of DTX micellar nanomedicine: PC3 cells were selected. In the flow cytometry (FACS) test, 2 mL of PC3 cells were first spread in a 6-well plate (3×10 ⁵ cells/well) and cultured for 24 hours. After adding 200 µL of Cy5-MDTX, 2.5% cRGD/Cy5-MDTX, 5% cRGD/Cy5-MDTX or 10% cRGD/Cy5-MDTX and incubating for 4 hours, the medium and micelles were removed. After washing with PBS, add 0.25% (w/v) trypsin and 0.03% (w/v) EDTA to digest to a single cell suspension, centrifuge the cell suspension at 1000 rpm for 3 minutes, wash with PBS twice, and finally suspend the cells In 500 µL PBS, the fluorescence intensity of cell-associated Cy5 was detected by FACS. MCF-7 cells were used as negative control. In confocal laser microscopy (CLSM) assays, PC3 cells were plated in 24-well plates (3.0×10 ⁵ cells/well) with small round coverslips and cultured for 24 hours. Then add 200 µL of 5% cRGD/Cy5-MDTX or Cy5-MDTX and continue to culture for 4 hours. After removing the culture medium, fix with 4% formaldehyde for 15 minutes, and then stain the nuclei with DAPI for 10 minutes. Wash 3 times with PBS between each step. Finally, observe and photograph with CLSM (TCS SP5). In the receptor blocking experiment, PC3 cells were pre-incubated with 1 mg/mL cRGD for 4 hours before adding cRGD/Cy5-MDTX, and the subsequent treatment was the same.

FACS analysis showed that the fluorescence intensity of cells in the cRGD group was higher than that in the no-target group, while the cells containing 5% cRGD/Cy5-MDTX of cRGD has the strongest cell fluorescence intensity (Fig. 5 A), which is 2.5 times that of Cy5-MDTX, showing the best ability to enhance endocytosis. In addition, CLSM images showed that the fluorescence intensity of Cy5 on the cell membrane and cytoplasm of PC3 cells in the cRGD/Cy5-MDTX group was significantly higher than that in the MDTX group. Pretreatment of PC3 cells with cRGD will lead to a significant decrease in the fluorescence of cell-bound cRGD-MDTX, which is basically reduced to a level similar to that of MDTX (Figure 5 B), indicating that cRGD-MDTX can enter more PC3 cells through cRGD and has active targeting ability.

The present invention uses 5% cRGD cRGD-MDTX to carry out subsequent cell and animal experiments.

Example 3 In vitro cytotoxicity and apoptosis study: PC3 cells were seeded in a 96-well plate at a density of 2×10 ³ cells per well and cultured for 24 hours. After adding 20 μL of a series of concentrations of cRGD-MDTX, MDTX or free DTX and incubating for 4 hours, the drug-containing medium was removed and replaced with fresh medium to continue culturing for 44 hours. Then add 10 μL of MTT in PBS solution (5 mg/mL) and incubate for 4 hours. Remove the medium, add 150 μL DMSO and incubate for 15 minutes to dissolve formazan produced by living cells. Absorbance at 570 nm was measured using a microplate reader. Absorbance of control wells containing DMSO alone was used as background signal, and relative cell viability was determined by comparing the absorbance of the samples to the absorbance of the PBS group. The toxicity of empty micellar cRGD-Ms and Ms was assessed in the same way.

To evaluate the ability of cRGD-MDTX to induce apoptosis in PC3 cells, PC3 cells were seeded in 24-well plates at a density of ⁵ × 104 cells/well and cultured overnight. Add cRGD-MDTX, MDTX or free DTX (30 ng/mL) and culture for 4 hours, then discard the medium and add fresh medium for 44 hours. Cells treated with PBS served as negative control group. Trypsinize without EDTA, collect cells and resuspend in 200 μL binding buffer. Afterwards, add 5 μL of Annexin V-FITC and 10 μL of PI to stain in the dark for 15 min. Single positive control: In the single positive early apoptosis group, 1 mL of cell fluid was divided into two equal portions, one was left untouched, and the other was boiled in a 50°C water bath for 5-10 minutes; the single late apoptosis positive group was divided into two 1 mL of cell fluid was equally divided into two parts, one part was left untouched, and one part was treated with 300 μL of 4% formaldehyde for 5 minutes, and the last two parts were mixed, and finally detected by flow cytometry.

The toxicity of cRGD-MDTX and MDTX to PC3 cells was further investigated by MTT and FACS tests. MTT results showed that the cRGD-MDTX group exhibited the highest cytotoxicity among the three groups, with a semi-lethal concentration (IC ₅₀ ) of 29 ng DTX/mL on PC3 cells, which was 1.5 and 2.0 times lower than free DTX and MDTX, respectively (Fig. 5C). But in MCF-7 cells, cRGD-MDTX and MDTX had similar toxicity, both lower than the free DTX group (Fig. 5D). The cells in the empty cRGD-Ms and Ms groups had nearly 100% cell viability (Figure 5E), indicating that the micelles themselves were nontoxic and had good biocompatibility.

The effects of cRGD-MDTX and MDTX on cell apoptosis were analyzed by flow cytometry. The results showed that under the same conditions, cRGD-MDTX induced a large number of PC3 cell apoptosis, and both early and late apoptosis were significantly more than MDTX. And the free DTX group (Fig. 5 F), consistent with the results of the MTT experiment.

The above results confirmed that cRGD-MDTX enters PC3 cells to deliver DTX, and induces apoptosis more efficiently.

For prostate cancer cells LNCaP and 22RV1, MDTX also has certain toxicity, and the IC ₅₀ tested by the same method is 2.5 and 0.15 μg DTX/mL, respectively.

Example 4 Study on inhibition of cellular microtubules: The inhibitory effect of DTX on cellular microtubules was studied by western blot and immunofluorescence. In the western blot experiment, after 1.8 mL PC3 cells were plated in a 6-well plate (5×10 ⁵ cells/well) and cultured for 24 hours, 200 μL of cRGD-MDTX, MDTX or free DTX was added (the concentration of DTX was 2 μg/well). mL) after 4 hours of incubation, replace the medium with 2 mL of fresh medium and incubate for another 44 hours. Cells were then lysed with RIPA lysate, incubated on ice for 15 minutes, centrifuged at 12,000 rpm for 15 minutes, the supernatant was collected, and the protein concentration was determined with a BCA protein kit for uniform sample loading. Then, add bromophenol blue to the protein sample and boil for 5 minutes to denature the protein, then add it to the SDS-PAGE gel for electrophoresis. After the gel was transferred to a PVDF membrane, the membrane was incubated in blocking solution containing 5% nonfat dry milk for 1 hour at room temperature. Finally, β-tubulin antibody and GAPDH antibody were added and incubated overnight at 4ºC. Then add goat anti-rabbit secondary antibody and incubate for 90 minutes. The membrane was washed with Tris-buffered saline/0.1% Tween-20 and finally developed. The results of WB experiments showed that the tubulin band in PC3 cells in the free DTX group was very shallow, and the MDTX group did not show a stronger protein inhibitory effect than free DTX, while the β-tubulin in PC3 cells treated with cRGD-MDTX The content is significantly lower and basically negligible (Figure 6A), because after incubation with cRGD-MDTX or free DTX, almost all tubulin in PC3 cells is in the form of aggregates, resulting in the molecular weight of free α-tubulin No stripes anywhere. Immunofluorescence images showed that, compared with the scattered microtubules (red) in PC3 cells in the MDTX group, the clustered very bright microtubules around the nucleus of PC3 cells in the cRGD-MDTX group were significantly increased (Fig. 6 B), showing that DTX around the nucleus Increased expression of effects on microtubule depolymerization. Fluorescence semi-quantitative analysis found that there was a significant difference in the intracellular fluorescence intensity between the cRGD-MDTX and MDTX groups (Figure 6C). In addition, the proportion of cells with microtubule aggregation in the cRGD-MDTX group was about 74%, much higher than the 36% in the MDTX group (Fig. 6D). Thus, cRGD-MDTX can promote free tubulin polymerization and prevent microtubule depolymerization, thereby inhibiting mitosis and leading to apoptosis.

Animal models: All animal operations were approved by the Ethics Committee of Soochow University. PC3 subcutaneous tumor-bearing mice were obtained by subcutaneously inoculating PC3 cells (1.2×10 ⁷ /mouse) on the right side near the ribs of male nude mice (17-21 g, 6 weeks, from Vertonilever). In the patient-derived tumor xenograft model (Patient-derived tumor xenograft, PDX), the patient was a first-diagnosed and followed-up prostate cancer patient (57 years old, PSMA negative, non-malignant and metastatic) from the First Affiliated Hospital of Soochow University. The tumor mass Obtained with the informed consent of the patient. PDX tumor modeling method for prostate cancer patients: by implanting surgically resected patient prostate cancer tissue fragments subcutaneously in the right shoulder and back of NOD SCID mice, and cultivating them as breeding mice, when the tumor grows to about 1000 mm ³ , the tumor is removed The block was cut into small pieces of about 50-100 mm ³ and planted subcutaneously in NOD SCID mice to obtain the PDX model. After repeated inoculation for 3 times, the patient's tumor block could be stably passaged in the mouse. The stably passaged prostate cancer PDX model was amplified and passaged to a sufficient amount for drug evaluation.

Example 5 Pharmacokinetics and biodistribution: In the pharmacokinetic study of cRGD-MDTX, 5-week-old male Balb/c white mice were randomly divided into groups (n = 3), and 200 μL of cRGD-MDTX, MDTX and Free DTX (7.5 mg/kg) was injected into the mouse body through the tail vein, and about 50 μL of blood was collected from the orbit at the predetermined time point to the EP tube treated with sodium heparin, after centrifugation, 20 μL of serum was collected and added to 1 mL of acetonitrile for extraction DTX, placed in 4 ℃ refrigerator for 24 hours. After high-speed centrifugation (13,000 rpm, 20 minutes), take the supernatant, evaporate the acetonitrile to dryness, and then add 0.1 mL of acetonitrile to dissolve. Finally, the concentration of DTX was detected by HPLC, and the content of DTX in the sample was calculated by comparing with the standard curve. The standard curve was obtained by mixing 20 μL of healthy mouse plasma with known concentrations of DTX in acetonitrile and then determined in the same way. The blood circulation curve obtained by plotting DTX content against time is fitted by the quadratic exponential decay of Origin 8 software, and the formula is: y = A1×exp(-x/t1) + A2×exp(-x/t2), t _{1 /2, α} = 0.693×t1; t _{1/2, β} = 0.693×t2, the distribution half-life and elimination half-life t _{1/2, α} and t _{1/2, β} can be calculated. The pharmacokinetics of cRGD-MDTX and MDTX in healthy Balb/c mice were studied. As shown in Figure 7 A, the pharmacokinetics of both followed a two-compartment model, and their elimination half-lives (t _{1/2, β} ) were 4.5 and 5.0 hours, respectively. In sharp contrast, free DTX was rapidly eliminated (t _{1/2, β} , 0.4 h), and DTX was undetectable after 2 h. The t _{1/2, β} of the clinical DTX formulations Nanoxel-PM and Taxotere were reported to be 2.10 and 1.98 hours in mice, respectively. It is shown that cRGD-MDTX and MDTX of the present invention exhibit longer circulation times.

The in vivo targeting and tumor penetration effects of cRGD/Cy5-MDTX were evaluated using subcutaneous PC3 Balb/c nude mice as a model. The details are as follows: when the tumor volume reached about 300 mm ³ (n = 3), cRGD/Cy5-MDTX and Cy5-MDTX were injected into tumor-bearing nude mice (7.5 mg DTX/kg, 10 μg Cy5/kg). After the mice were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg) at 1, 2, 4, 8 and 24 hours after the injection, the mice were scanned with the IVIS imaging system to observe the distribution of Cy5 in vivo. After 24 hours, mice were sacrificed by neck dislocation, and tumors and major organs were collected, ex vivo fluorescence imaging and semi-quantitative fluorescence intensity. Afterwards, the tumor was sectioned, and the distribution of micelles in the tumor was observed by immunofluorescence. The specific steps are as follows: the tumor slices were dewaxed in xylene for 3 times, 10 minutes each time, and then rehydrated twice by soaking in absolute ethanol, 95% ethanol and secondary water respectively, 10 minutes each time. At room temperature, soak the slices in antigen retrieval solution (Tris/EDTA buffer, 10 mM Tris, 1 mM EDTA solution, 0.05% Tween-20, pH 9.0), add it in a microwave oven to make it boil, and keep it at 95-99 ^o C 10 minutes. Take out the slices and let them cool naturally at room temperature. Cover the tissue with 10% goat serum solution, keep the tissue in a moist state, and keep it in the dark overnight at 4 ^o C. Rinse with 0.1% PBST and soak in PBS twice, 15 minutes each time. Then add CD31 antibody (1000-fold dilution) solution to cover the tissue, incubate at 37 ^o C for 1 hour and wash with PBS three times. Then, they were incubated with Alexa-680-labeled anti-mouse IgG (diluted 1000 times) at 37 ^o C for 1 hour and then washed 3 times with PBS. Finally, nuclei were stained with DAPI, washed and sealed with glycerol, and observed with CLSM.

In order to study the distribution of nanomedicine in PC3 tumor-bearing mice, when the tumor volume reached about 300 mm ³ , cRGD-MDTX and MDTX were injected into PC3 tumor-bearing nude mice (7.5 mg/kg DTX, n = 3) through the tail vein. . Sacrifice mice at 3 and 24 h, take tumors and major organs, homogenize in 2 mL of methanol, and incubate at 4 ^° C for 24 h to extract DTX. After the supernatant was obtained by centrifugation and evaporated to dryness, 0.1 mL of acetonitrile was added to the residue to fully dissolve DTX. The concentration of DTX was measured by HPLC, and the percentage of the amount of DTX in the injected dose per gram of tissue (%ID/g) was calculated according to the standard curve. The standard curve is obtained by homogenizing a certain mass of each organ with acetonitrile solution of known concentration of DTX and measuring according to the above steps.

The real-time biodistribution and tumor accumulation of Cy5-labeled cRGD-MDTX and MDTX in PC3 subcutaneous tumor-bearing nude mice were monitored using noninvasive in vivo fluorescence imaging. The images showed that both rapidly accumulated in the tumor at 1 hour and reached higher levels at 2-4 hours. The intensity of MDTX decreased rapidly, while cRGD-MDTX still maintained a high level in the tumor at 8-24 hours (Fig. 7 B), indicating that it can not only be rapidly enriched in the tumor but also stay for a long time. The mice were sacrificed 24 hours after injection, and the in vitro images and semi-quantitative analysis of the main organs and tumors showed that the fluorescence intensity of the tumors in the cRGD-MDTX group was significantly higher than that in the MDTX group (** p ＜ 0.01, Figure 7 C, D), it is not enriched in major organs, especially the liver, which is different from the phenomenon observed in other systems.

After 24 hours of cRGD-MDTX injection, the main organs and tumors of the mice were collected, ground and extracted for DTX, and the content of DTX was determined by HPLC. The results showed that the accumulation of DTX in the tumor in the cRGD-MDTX group reached 8.3 %ID/g, about 3.1 times that of the MDTX group (*** p ＜ 0.001, Figure 7E). In PC3 tumor-bearing mice, the ratio of tumor to normal tissue (T/N) in the cRGD-MDTX group was 1.8-3.8 times that of the MDTX group (Fig. 7F). Because the T/N ratio is an important indicator of drug targeting in vivo, the high T/N ratio of cRGD-MDTX also confirmed its excellent tumor selectivity in vivo. DTX in tumors of cRGD-MDTX group significantly increased from 3 hours to 24 hours (*** p < 0.001), while MDTX did not change much (Fig. 7G), confirming the role of cRGD-MDTX in mouse PC3 tumors. Higher enrichment and longer retention.

24 hours after intravenous injection, tumor slices were taken, and the distribution of Cy5-labeled cRGD-MDTX and MDTX in the tumor was observed with a CLSM fluorescence microscope. In the tumor slice, the two micelles were mainly observed in the outer edge area and central area of the tumor. The distribution and relative position of blood vessels (vessels were stained green with anti-CD31 antibody). It was observed that the distribution of two types of micelles (red) could be observed no matter in the outer edge or central area of the tumor (Figure 8). In the outer edge of the tumor (Fig. 8 A), MDTX was slightly less and stayed in the blood vessels in large quantities, while cRGD-MDTX had significantly higher fluorescence intensity, was farther away from the blood vessels, and the fluorescence signal diffused to the entire field of view. The major difference was in the central area of the tumor (Fig. 8 B), cRGD-MDTX still had an extremely abundant and uniform distribution, and the fluorescence intensity was almost the same as that in the edge area; but the fluorescence intensity of MDTX was greatly reduced, and it was only distributed in the blood vessels. This phenomenon is exciting, especially considering that prostate cancer is reported to be a less vascularized tumor, resulting in a poor EPR response. PC3 tumor sections were also stained with Masson and α-SMA, and it was found that PC3 tumors were indeed very dense tumors, which contained a large number of collagen fibers stained blue-black and filled with tumor-associated fibroblast marker α-SMA (Fig. 9 A, C). It is generally believed in the prior art that the extracellular barrier of tumor tissue, such as high interstitial pressure and low pressure gradient between capillaries, will prevent nanoparticles from penetrating deep into tumor tissue. However, the small-sized DTX micellar nanomedicine of the present invention has strong penetrating ability and can penetrate into the central area of the tumor. Result of joint action.

Example 6 Anti-tumor activity and histological analysis of cRGD-MDTX in PC3 tumor-bearing nude mice: When the tumor volume reached about 100 mm ³ , the tumor-bearing mice were randomly divided into 7 groups (n = 5). Inject 200 μL of 2.5% cRGD-MDTX, 5% cRGD-MDTX, 10% cRGD-MDTX, MDTX, ncMDTX, or free DTX at 7.5 mg DTX/kg or PBS tail vein on days 0, 3, 6, and 9 . Tumor volume (V = W ² × L/2, where W and L are width and length, respectively) was measured and calculated every two days. Body weights were measured every two days and relative tumor volumes relative to initial were calculated. Sacrifice mice on day 14, collect blood, major organs, and tumors, as well as ribs, spine, limb bones, and fix with 4% formaldehyde. Major organs were stained with hematoxylin and eosin (H&E), and tumors were stained with TUNEL. Bone tissue was x-rayed in mice to assess bone damage and osteolysis. The contents of calcium and phosphorus in the blood were tested by corresponding ELSIA kits according to the instructions, and healthy mice were used as controls.

The results showed that, compared with the PBS group, ncMDTX and free DTX had similarly weaker antitumor abilities, while MDTX was significantly better than ncMDTX (* p < 0.05, Fig. 10A). Considering the similar size and surface properties of the two, the higher antitumor efficacy of MDTX is attributed to the strong cross-linked core constructed by DTC, which prevents the premature release of DTX as well as the slow release inside the cancer cells; 3 The inhibitory effect of cRGD-MDTX on tumor growth was significantly stronger than that of MDTX, ncMs or free DTX (*** p < 0.001). The tumor suppressive effect of 5% cRGD-MDTX was significantly better than that of 2.5% cRGD-MDTX (** p < 0.01) and 10% cRGD-MDTX (* p < 0.05) (Fig. 10A). Furthermore, all mice had a constant body weight during treatment (Fig. 10B), confirming the low toxicity of this micellar nanomedicine. The results confirmed that the cRGD density of nanomedicines plays a crucial role in targeting and antitumor efficacy in vivo. The prior art ( Biomaterials , 2014, 35, 3005) reported that DTX-loaded hyaluronic acid nanoparticles (targeted to CD44) at a dose of 10 mg DTX/kg, in the same PC3-loaded nude mouse model, only The tumor growth was slightly inhibited, but there was no significant difference from the PBS group. Even the tumors in the dual targeting group are slowly increasing, and the curative effect is much lower than that of the present invention.

Tumors were resected on day 14 and weighed to calculate the tumor inhibition rate (TIR) compared with the tumor mass in the PBS group. It was found that the TIR of the 5% cRGD-MDTX group was as high as 94.6% (Figure 10 C), which was much higher than that of MDTX, ncMDTX, free DTX (*** p <0.001), 2.5% cRGD-MDTX (** p <0.01) and 10% cRGD-MDTX (* p <0.05). Tumor sections were stained with TUNEL, and histological analysis showed that both cRGD-MDTX with 5% and 10% cRGD induced extensive and massive tumor cell apoptosis, which was significantly stronger than other groups (Fig. 10D). As a result of H&E staining, it was found that the preparations of each group had no obvious damage to the heart, liver, spleen, lung and kidney (Fig. 11).

Due to the bone metastases of PC3 tumors, when the mice were dissected, it was found that the ribs and spine near the tumor in the PBS group suffered severe damage, and even the ribs were broken, while the other treatment groups improved. The concentrations of calcium and phosphorus in the blood of the treated mice were then measured to quantitatively assess bone damage. It was found that the serum concentrations of calcium and phosphorus in the PBS group were indeed significantly higher than those in healthy mice. The treatment of free DTX, ncMDTX, MDTX, and 2.5% cRGD-MDTX significantly reduced the serum calcium and phosphorus in mice, while 5% cRGD-MDTX The concentration of the group is very low, and there is no significant difference with the healthy group (Fig. 12 A, B), indicating that it can protect the bones of mice from damage.

Example 7 Anti-tumor activity of cRGD-MDTX in PDX model mice: When the tumor volume of PDX mice reached about 100 mm ³ , the mice were randomly divided into 5 groups (n = 5), and the mice were randomly divided into 5 groups (n = 5). On day 9, 200 μL of cRGD-MDTX, MDTX, ncMDTX, free DTX was injected into mice at 7.5 mg DTX/kg or PBS. Tumor volume and body weight were measured every two days. Observe and record the status and survival period of the mice. Mice were considered dead when they died or their body weight decreased by more than 15%. The patient PDX model is the closest relevant tumor model to clinical research so far. This model that mimics human tumor specificity is of great significance to the preclinical assessment, treatment and prognosis of tumors. The experimental results showed that on the 16th day, the tumor volume in the PBS group grew to 800 mm ³ , which was far smaller than that in each administration group. It is worth noting that the tumor volumes of the free DTX and ncMDTX groups were not much different (ns), in contrast, the antitumor activity of MDTX was significantly improved (** p < 0.01), and the volume remained basically unchanged (Fig. 13 A). In this PDX model, the antitumor activity of the cRGD-MDTX group was also significantly better than that of the other groups (*** p < 0.001), and the tumor volume not only did not increase, but the tumor volume shrunk by 43% compared with the initial volume on day 16. While the tumor volume was significantly reduced, the body weight of the mice was not significantly reduced (Fig. 13 B). This result is consistent with the results of the CDX model of PC3 subcutaneous tumor.

Example 10 Anti-tumor activity of LHRH-MDTX in PC3 tumor-bearing mice: The preparation of LHRH-MDTX is similar to that of cRGD-MDTX, that is, cRGD-PEG-PLA is replaced by LHRH-PEG-PLA, and the rest are the same, prepared in the water phase, The particle sizes of LHRH-MDTX and cRGD-MDTX are similar, so it can be considered that there is no difference. The mouse experiment is the same, similar to cRGD-MDTX (Figure 14), the anti-tumor activity of the optimal micelle is the best when the surface density of LHRH is 5%, showing significantly stronger anti-tumor activity than other groups. At a surface density of LHRH of 10%, the therapeutic effect was similar to that of 5%.

Example 11 Antitumor activity of cRGD-MS-DTX in PDX model mice: DTX and PEG _2k -P(CL _{1.0k -DTC 1.1k} ) were mixed according to different mass ratios (5/100, 10/100, 20 /100, 30/100) were dissolved in PEG 350 with a polymer concentration of 50 mg/mL. At room temperature, 100 μL of this mixed solution was injected into the bottom of 900 μL of PB solution (pH 7.4, 10 mM) to obtain DTX micellar nanomedicine with a theoretical DTX drug loading of 4.8 wt. %-23.1 wt. %. It is a non-targeting small micellar nanomedicine, called MS-DTX, and its particle size is 25-30nm as determined by DLS.

Replace part of PEG _2k -P (CL _1.0k -DTC _1.1k ) with cRGD-PEG _2k -P (CL _0.9k -DTC _1.0k ) to obtain DTX gels with cRGD surface densities of 2.5%, 5% and 10%, respectively Beam nano-medicine is called cRGD-MS-DTX, and its particle size measured by DLS is 26-30nm.

Using PEG _2k -PCL 2.0k as a polymer, _ncMS -DTX was obtained.

The patient PDX model is the closest relevant tumor model to clinical research so far. This model that mimics human tumor specificity is of great significance to the preclinical assessment, treatment and prognosis of tumors. PBS, free DTX, ncMS-DTX, MS-DTX and cRGD-MS-DTX (7.5 mg/kg or 10 mg/kg ) were administered via tail vein when the tumor volume of PDX mice reached about 50-100 mm ³ (n= 5), give one injection every three days, a total of five injections. The experimental results showed that, on the 16th day, the tumor volume in the PBS group grew to 500 mm ³ , which was much smaller than that in each administration group. It is worth noting that the tumor volumes of free DTX, ncMS-DTX and MS-DTX were not significantly different (ns), and in this PDX model, the antitumor activity of the cRGD-MS-DTX group was significantly better than that of PBS, free DTX and ncMS-DTX and MS-DTX groups (* p < 0.05), the tumor volume did not increase significantly; the antitumor activity of cRGD-MS-DTX (10 mg /kg) was significantly better than that of PBS, free DTX, ncMS-DTX and MS-DTX groups ( ** p ＜ 0.01) and cRGD-MS-DTX(7.5 mg /kg) (*** p ＜ 0.001), the tumors of 4 mice were too small to be measured when dissected on the 16th day, the results are shown in Figure 15 . cRGD-MS-DTX has a stable micellar core, which enhances stability and DTX cycle time, and active targeting enhances the enrichment of micelles in tumors, penetration into tumor depths, and tumor cell-specific endocytosis, which can Shrink tumors in PDX model mice.

See Figure 16, after the 17th day of treatment, it can be seen from the tumor photos of the mice that the tumor volume of the mice in the cRGD-MS-DTX (7.5 mg/kg) treatment group was smaller than that of the tumors in the free DTX, ncMs-DTX and MS-DTX groups Among them, the cRGD-MS-DTX (10 mg/kg) group had the smallest tumor volume, and no tumor was found in the other 4 mice, which was consistent with the tumor volume measurement results. In addition, tumors were resected on day 17 and weighed to calculate the tumor inhibition rate (TIR) compared with the tumor mass in the PBS group. It was found that the TIR of cRGD-MS-DTX (10 mg/kg) group was as high as 99.4%, which was much higher than that of free DTX, ncMs-DTX and MS-DTX groups (** p < 0.01) and cRGD-MS-DTX (7.5 mg /kg) (* p ＜ 0.05); the TIR of cRGD-MS-DTX(7.5 mg /kg) group was as high as 91.2% higher than that of free DTX, ncMs-DTX and MS-DTX groups (* p ＜ 0.05); MS- The TIR of the DTX group was 79.8% higher than that of the ncMs-DTX group (* p < 0.05).

On the 17th day of administration, whole blood was collected from the sacrificed mice for blood routine testing, serum was separated for blood biochemical testing, and major organs were dissected for observation and sectioned, and H&E staining was used for histological analysis. Blood biochemical tests showed that each function of the main liver (alkaline phosphatase ALP, glutamyl transferase GGT, aspartate aminotransferase AST, alanine aminotransferase ALT), kidney (creatinine CRE, urea URE) functions There was no significant difference in the index among the groups, and by searching the literature, compared with healthy mice, it was found that the values of alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase ALP were all the same. It was lower than that of healthy mice; blood routine results showed that red blood cell count (RBC), platelet count (PLT) and white blood cell count (WBC) had no significant difference among the groups, and there was no significant difference with healthy mice. The H&E stained sections of the main organs of the tumor-bearing mice showed that there was no obvious tissue damage in the hearts, livers and kidneys of the mice in each group.

All data in the present invention are expressed as mean ± standard deviation (Mean ± SD). Differences between groups were calculated by ANOVA one-way analysis of variance. Among them, * p <0.05 means that there is a statistically significant difference, ** p <0.01 and *** p <0.001 mean that there is a significant difference.

The present invention designed and prepared DTX micelles cRGD-MDTX based on PEG-P (LA-DTC) and coupled with cRGD, which has high and stable DTX drug loading, small particle size, and can actively target tumor cells and tumor neovascularization. Intracellular DTX can be released quickly, has long blood circulation time, high tumor accumulation and deep tumor penetration, and has realized the targeted therapy and efficient inhibition of human castration-resistant prostate cancer PC3, and has been shown to be effective in prostate cancer patients with PDX Surprising efficacy was shown in the treatment of the model. This cRGD-MDTX is biodegradable, easy to prepare, and scalable, providing a platform for its efficient treatment.

Claims (10)

1. A kind of docetaxel micellar nano-medicine, it is characterized in that, the mixed solution of docetaxel, polymer, oligoethylene glycol is mixed with damping fluid, obtains docetaxel micellar nano-medicine; The low The molecular weight of polyethylene glycol is 200-600Da.
2. The docetaxel micelle nanomedicine according to claim 1, wherein the molecular weight of the oligoethylene glycol is 300-500 Da; the molecular weight of the polymer is 2000-15000 Da.
3. The docetaxel micelle nanomedicine according to claim 1, wherein the polymer is a non-targeting polymer, or the polymer is a mixture of a non-targeting polymer and a targeting polymer.
4. According to the described docetaxel micellar nano-medicine of claim 3, it is characterized in that, non-targeting polymer comprises PEG-P (LA-DTC), PEG-P (CL-DTC) or PEG-P (TMC-DTC ).
5. According to the described docetaxel micellar nanomedicine of claim 3, it is characterized in that, targeting polymer comprises B-PEG-P (LA-DTC), B-PEG-P (CL-DTC), B-PEG- P(TMC-DTC), B-PEG-PLA, B-PEG-PCL or B-PEG-PTMC, B is the targeting molecule.
6. The preparation method of docetaxel micellar nano-medicine according to claim 1, wherein the polymer is dissolved in oligoethylene glycol to obtain a polymer mother liquor; docetaxel is dissolved in oligoethylene glycol, Obtain the docetaxel mother liquor; Mix the polymer mother liquor and the docetaxel mother liquor again, obtain the mixed solution of docetaxel, polymer, oligoethylene glycol; Mix docetaxel, polymer, oligoethylene glycol The mixed solution of alcohol is mixed with the buffer to obtain the docetaxel micellar nanomedicine.
7. The preparation method of docetaxel micellar nanomedicine according to claim 6, wherein the polymer is a non-targeting polymer, or the polymer is a mixture of a non-targeting polymer and a targeting polymer.
8. According to the preparation method of the described docetaxel micellar nanomedicine of claim 6, it is characterized in that, when the polymer is a non-targeting polymer and a targeting polymer, the molar ratio of the non-targeting polymer and the targeting polymer For (5 ~ 50): 1.
9. The application of the docetaxel micellar nano-medicine in claim 1 in the preparation of anti-tumor drugs.
10. The application according to claim 9, wherein the docetaxel micellar nanomedicine is a chemotherapeutic drug.