Mixed micelle of pegylated biotin and rhein conjugate for oral delivery of paclitaxel as well as preparation method and application of mixed micelle

Abstract

The invention provides a pegylated biotin and rhein conjugate mixed micelle for oral delivery of paclitaxel as well as a preparation method and application of the pegylated biotin and rhein conjugate mixed micelle, and belongs to the field of biological medicines. According to the invention, PTX is selected as a model drug and is entrapped in TCR-BPB to prepare a PTX / TCR-BPB mixed micelle, and the PTX / TCR-BPB micelle has good entrapment capability on PTX, small particle size, uniform distribution, slow release effect, good killing effect on tumor cells, capability of targeting tumor cells, small toxic and side effects and capability of promoting absorption of PTX in intestinal tracts, and the absorption path is irrelevant to P-gp. The PTX / TCR-BPB micelle improves the oral bioavailability of PTX, has a good anti-tumor effect, reduces the toxic and side effects of PTX, and is a nano drug delivery system with a good development prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a mixed micelle of PEGylated biotin and rhein conjugate for oral delivery of paclitaxel and a preparation method thereof. Background Art

[0002] Cancer is a common disease that threatens human life and health. For a long time, people have tried many methods to treat cancer, such as chemotherapy, surgical treatment, radiotherapy, etc. Among them, chemotherapy is the mainstay of standard cancer treatment. Chemotherapy is often administered orally, but due to the presence of P-glycoprotein (P-gp) in the epithelial cell membrane of the human gastrointestinal tract, the drug absorption rate is very low, which seriously affects the enrichment of anticancer drugs in the tumor site, making it impossible for the drug to produce sufficient therapeutic effects. In order to overcome the above-mentioned defects in the use of anti-tumor drugs, the commonly used formulation strategy is to use nanoscale drug delivery systems, such as liposomes, nanoparticles and polymer micelles. Among them, polymer micelles (PMs) are an emerging technology that overcomes the current obstacles to oral drug delivery.

[0003] Paclitaxel (PTX) is a compound isolated from Pacific yew (Taxus brevifolia Nutt.). It is a very common chemotherapy drug and is widely used in the treatment of cancer, especially for breast cancer. In order to solve the problem of oral use of paclitaxel, the "Preparation and Characterization of TPGS-modified Carboxymethyl Chitosan-Rhein Polymer Micelles" published in the Journal of Shenyang Pharmaceutical University (Volume 26, Issue 4, 2019) discloses a TPGS-modified carboxymethyl chitosan-rhein (TPGS-CR) conjugate, which can self-assemble into polymer micelles in water. The micelles can effectively improve the absorption of paclitaxel in the gastrointestinal tract, but the effect is still unsatisfactory. At the same time, the micelles have weak targeting, resulting in strong side effects.

[0004] Therefore, there is an urgent need to develop PTX drug polymer micelles with low systemic toxicity and side effects, strong targeting function, good absorption effect and good tumor treatment effect. Summary of the invention

[0005] The object of the present invention is to provide a drug polymer micelle with small systemic toxicity and side effects, strong targeting function, good absorption effect and good tumor treatment effect.

[0006] The present invention provides a carrier for drug loading, characterized in that it is a carrier prepared with PEGylated biotin and TCR conjugate as raw materials, and the mass ratio of PEGylated biotin to TCR conjugate is 3:(1-9).

[0007] Preferably, the ratio of PEGylated biotin to TCR conjugate in the carrier is 1:3.

[0008] Preferably, the mixed micelle is obtained by dissolving PEGylated biotin and TCR conjugate in water and ultrasonicating in an ice-water bath until dissolved.

[0009] Preferably, the carrier is prepared by adding drugs.

[0010] Preferably, in the mixed micelles, the mass ratio of the carrier to the drug is 1:(0.8-1.6); preferably, in the mixed micelles, the mass ratio of the carrier to the drug is 1:1; preferably, the drug is paclitaxel.

[0011] Preferably, the micelle is prepared by the following method: dissolving the drug in a solvent, adding dropwise to the carrier solution according to any one of claims 1 to 3, stirring, ultrasonicating, dialyzing, filtering, and freeze-drying; the solvent is an organic solvent, preferably anhydrous ethanol.

[0012] Preferably, the TCR conjugate is a TPGS-modified carboxymethyl chitosan-rhein conjugate.

[0013] The structure of the conjugate is shown in Formula I:

[0014]

[0015] wherein A and B are independently selected from rhein or TPGS;

[0016] n = 436-512;

[0017] Preferably, in the conjugate, the molar substitution degree of rhein is 6.44±0.83%; and the molar substitution degree of TPGS is 0.28±0.06%.

[0018] Preferably, the critical micelle concentration of the conjugate is 54.33 μg / mL.

[0019] A lyoprotectant is added in the freeze-drying step; the lyoprotectant is any one or more of glucose, mannitol and sucrose; preferably, the lyoprotectant is sucrose; preferably, the lyoprotectant is 0.2% sucrose.

[0020] The present invention also provides a method for preparing the mixed micelle, which is characterized by: dissolving the drug in a solvent, adding the solution dropwise to the carrier solution according to any one of claims 1 to 3, stirring, ultrasonicating, dialysis, filtering, and freeze-drying.

[0021] The present invention also provides the use of the mixed micelles in preparing a drug for treating tumors; preferably, the drug for treating tumors is a drug for treating breast cancer.

[0022] The present invention uses TCR conjugates and pegylated biotin (BPB) as mixed carriers, self-assembles into polymer micelles in water, physically encapsulates PTX, and forms PTX / TCR-BPB micelles, which can improve the solubility of PTX, enhance its tissue permeability, promote the absorption of PTX in the intestine, prolong the circulation half-life in the body, and improve the oral bioavailability; it can also avoid the recognition of P-gp, reverse multidrug resistance, and improve the sensitivity of tumor cells; further, the passive targeting of the PTX / TCR-BPB micelles based on the EPR effect and the active targeting based on biotin can reduce the systemic toxic side effects of PTX and obtain better tumor treatment effects.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .High performance liquid chromatogram (HPLC) A. PTX reference; B. TCR-BPB blank vector; C. PTX / TCR-BPB micelles; where (TCR:BPB = 3:1), the concentration is 30 μg / mL (in terms of PTX) The concentration of the vector contained in the PTX / TCR-BPB micelles

[0026] Figure 2 .Particle size distribution of PTX / TCR-BPB micelles

[0027] Figure 3 .Potential diagram of PTX / TCR-BPB micelles

[0028] Figure 4 TEM image of PTX / TCR-BPB micelles

[0029] Figure 5 .DSC images A. PTX; B. TCR-BPB; C. PTX+TCR-BPB; D. PTX / TCR-BPB micelles

[0030] Figure 6 .HPLC images A. PTX standard; BK-R solution and phenol red; C. Sample solution

[0031] Figure 7.Phenol red UV full wavelength scan

[0032] Figure 8 .The adsorption of drugs by pipelines ( n=4)

[0033] Fig. 9 .(A) Absorption rate constant (Ka) and (B) apparent permeability coefficient (Peff) of PTX preparations in each intestinal segment ( n = 3, and Compared with the same group, *P<0.05, **P<0.01, ***P<0.001)

[0034] Fig.10 (A) Absorption rate constant (Ka) and (B) apparent permeability coefficient (Peff) of PTX preparations in each intestinal segment ( n=3, ***P<0.001 compared with duodenum, #P<0.05 compared with ileum)

[0035] Fig.11 .Schematic diagram of subcutaneous tumor in mice

[0036] Fig.12 .Tumor volume change curve of tumor-bearing mice (n=8) (*P<0.05; **P<0.01; ***P<0.001)

[0037] Fig.13 .Therapeutic effect of tumor-bearing mice (n=8)

[0038] Fig.14 .Tumor weight of tumor-bearing mice (n=8) Note: *P<0.05; **P<0.01; ***P<0.001

[0039] Fig.15 . Tumor inhibition rate of each drug-treated group (n=8) (*P<0.05; ***P<0.001)

[0040] Fig.16 Body weight changes of tumor-bearing mice during treatment (n=8)

[0041] Fig.17 Results of organ index effects on tumor-bearing mice (n=8)

[0042] Fig.18 Pathological sections of organs and tumors of Balb / c mice bearing 4T1 after oral administration of each preparation (400×) DETAILED DESCRIPTION

[0043] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0044] The preparation method of TCR of the present invention is as follows:

[0045] The TCR conjugate of the present invention is a Chinese patent with patent number ZL201811603795.X, and the patent name is: A TPGS-modified carboxymethyl chitosan-rhein conjugate and its synthesis process and use, the TPGS-CR conjugate.

[0046] Detailed steps:

[0047] 1. Synthesis of TPGS-CR conjugate (i.e., TCR conjugate)

[0048] (1) Synthesis of TPGS-SA intermediate

[0049] Weigh TPGS, succinic anhydride (SA) and 4-dimethylaminopyridine (DMAP), place in a vial, add triethylamine (TEA) dropwise, the feeding amount is calculated based on the molar ratio of TPGS:SA:DMAP:TEA = 1:2:1:1, add appropriate amount of pyridine to dissolve. After stirring at 1500rpm for 24h at room temperature, remove pyridine by rotary evaporation. Dissolve the rotary evaporation residue with appropriate amount of dichloromethane, let it stand, and rotate at 4000r·min -1 Centrifuge for 10 minutes, evaporate the upper liquid to obtain a light yellow jelly, dissolve the jelly in appropriate amount of water, stir overnight at 1500 rpm, dialyze for 72 hours, remove the precipitate by centrifugation, and freeze-dry the supernatant to obtain a light yellow loose TPGS-SA crude product.

[0050] (2) Synthesis of TPGS-CR conjugate

[0051] Weigh CMCS (carboxymethyl chitosan) and place it in a reaction bottle. Add 5 mL of distilled water to swell it and stir at 1500 rpm until it dissolves. Make the CMCS concentration be 0.1 mmol / mL solution. Weigh the crude TPGS-SA and add 5 mL of distilled water to dissolve it to a concentration of 0.05 mmol / mL. Add 143.8 mg of EDC·HCl and activate it for 20 min. Then add 28.8 mg of N-hydroxysuccinimide (NHS). Take another 142.1 mg of R powder and place it in a wide-mouth bottle. Add 5 mL of 1% NaHCO 3The solution was heated to dissolve, cooled to room temperature, 287.6 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) was added, and after activation for 20 minutes, 57.7 mg NHS was added. 5 mL of the activated TPGS-SA solution and 5 mL of the R solution were added to 5 mL of the CMCS solution under stirring at 1500 rpm, and stirred at 1500 rpm in the dark for 24 hours, and then 95% ethanol was added for precipitation, filtered, and washed until the filtrate was nearly colorless. The precipitate was dissolved in water, probed for 30 minutes in an ice water bath, and dialyzed for 3 days. After the dialysis was completed, the probe was ultrasonicated again, filtered with a 0.8 μm filter membrane, and the filtrate was freeze-dried to obtain the TPGS-CR conjugate (i.e., TCR conjugate).

[0052] 1. Abbreviations

[0053] PTX: paclitaxel; TPGS: α-tocopheryl polyethylene glycol 1000 succinate; CMCS: carboxymethyl chitosan; R: rhein; PEG: polyethylene glycol; BPB: pegylated biotin; PMs: polymer micelles; TCR conjugate: PGS-modified carboxymethyl chitosan-rhein conjugate; TCR-BPB conjugate: TPGS-modified carboxymethyl chitosan-rhein and pegylated biotin mixed conjugate; Verapamil: verapamil, molecular formula C 27 H 38 N 2 O 4 , a derivative of papaverine, is a calcium channel blocker; DUODENUM: duodenum; jejunum: jejunum; HUM: ileum; colon: colon; DTX, docetaxel; UPLC-MS / MS: ultra-performance liquid chromatography-tandem mass spectrometry; ALT: glutamate; AST: aspartate aminotransferase; BUN: blood urea nitrogen; CRE: creatinine; CA15-3: cancer antigen 15-3.

[0054] 2. Chromatographic conditions 1

[0055] Chromatographic column: Dikma 5HC-C18 (250×4.6 mm, 5 μm), mobile phase: methanol-water (80:20), flow rate: 1.0 mL / min, column temperature: 30°C, detection wavelength: 227 nm, injection volume 20 μL.

[0056] 3. Chromatographic conditions 2

[0057] Chromatographic column: Diamonsil C18 (150×4.6 mm, 5 μm), mobile phase: acetonitrile-water 55:45, flow rate: 1.0 mL / min, column temperature: 30°C, detection wavelength: 227 nm, injection volume 20 μL.

[0058] Example 1 Preparation of PTX / TCR-BPB micelles of the present invention

[0059] The TCR conjugate and BPB were dissolved and mixed in a mass ratio of 3:1 (13.5 mg of TCR conjugate was weighed and added to 3 mL of ultrapure water, and magnetic stirring was used to dissolve; 4.5 mg of BPB was weighed and added to 1 mL of ultrapure water to dissolve. After the two were mixed, the probe was ultrasonicated at 280 W for 10 min in an ice water bath. 0.6 mL of a 30 mg / mL PTX anhydrous ethanol solution was slowly added dropwise to the mixed carrier solution. After high-speed stirring at 1500 rpm for 20 min, the probe was ultrasonicated at 280 W for 30 min in an ice water bath, placed in a dialysis bag (MWCO14000), and dialyzed with distilled water for 24 h. After the dialysis was completed, the solution was ultrasonicated at 280 W for 20 min in an ice water bath, filtered with a 0.8 μm microporous filter membrane, and freeze-dried to obtain PTX / TCR-BPB micelles.

[0060] Example 2 Preparation of PTX / TCR-BPB micelles of the present invention

[0061] The TCR conjugate and BPB were dissolved and mixed according to a mass ratio of 3:1 (13.5 mg of TCR conjugate was weighed and added to 3 mL of ultrapure water, and magnetic stirring was used to dissolve. 4.5 mg of BPB was weighed and added to 1 mL of ultrapure water to dissolve. After the two were mixed, the probe was ultrasonicated at 280 W for 10 min in an ice water bath. 0.6 mL of a 30 mg / mL PTX anhydrous ethanol solution was slowly added dropwise to the mixed carrier solution. After high-speed stirring at 1500 rpm for 20 min, the probe was ultrasonicated at 280 W for 30 min in an ice water bath, placed in a dialysis bag (MWCO14000), and dialyzed with distilled water for 24 h. After the dialysis was completed, the solution was ultrasonicated at 280 W for 20 min in an ice water bath, filtered with a 0.8 μm microporous filter membrane, sucrose with a freeze-drying protective agent concentration of 0.2% was added, and freeze-dried to obtain PTX / TCR-BPB micelles.

[0062] Example 3 Preparation of PTX / TCR-BPB micellar solution of the present invention

[0063] The TCR conjugate and BPB were dissolved and mixed in a mass ratio of 3:1 (13.5 mg of TCR conjugate was weighed and added to 3 mL of ultrapure water, and magnetic stirring was used to dissolve; 4.5 mg of BPB was weighed and added to 1 mL of ultrapure water to dissolve. After the two were mixed, the probe was ultrasonicated at 280 W for 10 min in an ice water bath. 0.6 mL of a 30 mg / mL PTX anhydrous ethanol solution was slowly added dropwise to the mixed carrier solution. After high-speed stirring at 1500 rpm for 20 min, the probe was ultrasonicated at 280 W for 30 min in an ice water bath, placed in a dialysis bag (MWCO14000), and dialyzed with distilled water for 24 h. After the dialysis was completed, the solution was ultrasonicated at 280 W for 20 min in an ice water bath, and filtered with a 0.8 μm microporous filter membrane to obtain a PTX / TCR-BPB micelle solution.

[0064] Example 4 Preparation of TCR-BPB micellar solution

[0065] Weigh TCR conjugate and BPB in a mass ratio of 3:1 and mix them as a mixed carrier. Weigh 18 mg of the mixed carrier, add 4 mL of ultrapure water, and dissolve it in an ice-water bath at 280 W for 10 min to obtain a mixed carrier solution. Alternatively, weigh 13.5 mg of TCR conjugate, add 3 mL of ultrapure water, stir and dissolve until clear, weigh 4.5 mg of BPB, add 1 mL of ultrapure water to dissolve, mix the two, and then dissolve it in an ice-water bath at 280 W for 10 min to obtain a mixed carrier solution.

[0066] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0067] Experimental Example 1: Screening of PTX / TCR-BPB micelles

[0068] 1. Experimental Methods

[0069] (I) Study on the optimal ratio of TCR conjugate to BPB in PTX / TCR-BPB micelles

[0070] 1. PTX / TCR-BPB micelles prepared with different mass ratios of TCR conjugates and BPB

[0071] According to the mass ratio of TCR conjugate and BPB in Table 1, weigh 18 mg of TCR conjugate and BPB as mixed carrier materials, add 4 mL of ultrapure water, and dissolve them in an ice-water bath with ultrasonic wave at 280 W for 10 min (or according to the mass ratio of TCR conjugate and BPB in Table 1, weigh TCR conjugate and BPB as mixed carrier materials, the total weight of TCR conjugate and BPB is 18 mg, and dissolve TCR conjugate and BPB in equal proportions of ultrapure water, wherein the total weight of ultrapure water is 4 ml. After mixing the two solutions, ultrasonic wave at 280 W for 10 min in an ice-water bath. According to the mass ratio of drug to carrier in Table 1, slowly add 600 μL of 30 mg / mL PTX anhydrous ethanol solution to the mixed carrier solution, stir at 1500 rpm for 20 min, ultrasonic wave at 280 W for 0.5 h in an ice-water bath, and place in a dialysis bag (MWCO 14000), dialyzed with distilled water for 24 h. After the dialysis, ultrasonicated at 280W for 20 min in an ice-water bath, filtered with a 0.8 μm microporous membrane, and freeze-dried to obtain PTX / TCR-BPB micelles.

[0072] 2. Determination of drug loading and encapsulation efficiency

[0073] Accurately weigh about 3 mg of the above-mentioned PTX / TCR-BPB micelle freeze-dried powder of different ratios, add 2 mL of ultrapure water, dissolve with ice water bath probe ultrasound at 280W for 10 min, filter with 0.8 μm filter membrane to remove insoluble impurities, take 200 μL and dilute to 10 mL with methanol, centrifuge at 14000 rpm for 5 min, take the supernatant and measure the peak area according to chromatographic condition 1, calculate the drug loading (DL) and encapsulation efficiency (EE) of PTX by formula (1) (2), respectively. The particle size, polydispersity index (PDI) and Zeta potential of PTX / TCR-BPB drug-loaded micelles of different ratios were measured by laser particle size analyzer (DLS).

[0074]

[0075]

[0076] (II) Exploration of the optimal feed ratio of PTX and TCR-BPB micelles in PTX / TCR-BPB micelles

[0077] 1. PTX / TCR-BPB micelles prepared with different mass ratios of PTX and TCR-BPB

[0078] TCR conjugate and BPB were mixed at a mass ratio of 3:1 as a mixed carrier. Weigh 13.5 mg of TCR conjugate, add 3 mL of ultrapure water, stir and dissolve until clear, weigh 4.5 mg of BPB, add 1 mL of ultrapure water to dissolve, mix the two, and ultrasonically probe at 280W for 10 min in an ice water bath. According to the feed ratio of PTX and mixed carrier in Table 2, slowly add 30 mg / mL of PTX anhydrous ethanol solution to the mixed carrier solution, stir at 1500 rpm for 20 min, ultrasonically probe at 280W for 30 min in an ice water bath, place in a dialysis bag (MWCO 14000), and dialyze with distilled water for 24 h. After the dialysis is completed, ultrasonically probe at 280W for 20 min in an ice water bath, filter with a 0.8 μm microporous filter membrane, and freeze-dry to obtain PTX / TCR-BPB micelles.

[0079] 2. Determination of drug loading and encapsulation efficiency

[0080] Same as the determination of drug loading and encapsulation efficiency in Section 2 of Item (I).

[0081] 2. Experimental Results

[0082] (A) The effect of the mixing ratio of TCR conjugate and BPB on the encapsulation of PTX is shown in Table 1.

[0083] Table 1 Effects of different ratios of TCR conjugates to BPB on the drug loading capacity of PTX / TCR-BPB micelles ( n=3)

[0084]

[0085]

[0086] As can be seen from Table 1, when different ratios of TCR conjugates and BPB are mixed, the particle size and Zeta potential of each PTX / TCR-BPB micelle are slightly different, the particle size distribution is uniform, and all have a high drug loading. When the mixing ratio is 3:1, 1:2 and 1:3, the drug loading is higher than that of other ratios, especially when the ratio is 1:2, the drug loading reaches 48%, but too high a drug loading may affect the stability of the micelles. Within the range of the investigated ratio, as the BPB ratio increases, the encapsulation rate gradually decreases. When the mass ratio of the TCR conjugate to BPB is 3:1, the drug loading and encapsulation rate are both high. Therefore, 3:1 is selected as the mass feed ratio of the TCR conjugate to BPB.

[0087] (ii) The effect of the feed ratio of drug to mixed carrier material on the PTX encapsulation capacity of mixed micelles is shown in Table 2.

[0088] Table 2 Effects of different drug amounts and mixed carrier feed ratios on the drug loading capacity of mixed micelles ( n=3)

[0089]

[0090] As shown in Table 2, within the investigated ratio range, as the ratio of the mixed carrier increases, the drug loading gradually decreases; when the drug-carrier ratio is 1:0.8 and 1:1, the drug loading and encapsulation efficiency are greater than those of other ratios, but when the drug-carrier ratio is 1:0.8, the TCR-BPB micelle particle size is larger than that of other ratios, the absolute value of the Zeta potential is smaller than that of other ratios, and the stability is poor. Therefore, considering the particle size, potential, drug loading and encapsulation efficiency, 1:1 is selected as the drug-carrier feed ratio.

[0091] The experimental results show that, considering the particle size, potential, drug loading capacity and encapsulation efficiency comprehensively, when the mass ratio of TCR conjugate to BPB in PTX / TCR-BPB micelles is 3:1 and the mass ratio of PTX to TCR-BPB is 1:1, the drug loading capacity and encapsulation efficiency of PTX / TCR-BPB micelles are large at this ratio.

[0092] Experimental Example 2: Investigation on the stability of the preparation process of PTX / TCR-BPB micelles

[0093] 1. Experimental Methods

[0094] The optimal drug loading process of Experimental Example 1 (ie, the method of Example 1) was repeated three times to test the particle size, polydispersity index (PDI), Zeta potential, drug loading (DL) and encapsulation efficiency (EE) of each batch.

[0095] Experimental Results

[0096] 2. Experimental Results

[0097] The verification results are shown in Table 3, and the DLS graphs of particle size and Zeta potential are shown in Figure 2 and Figure 3 The results showed that the prepared PTX / TCR-BPB micelles had a smaller particle size, a larger absolute value of negative charge, a uniform distribution, and a higher drug loading and encapsulation efficiency. It was also determined that the PTX / TCR-BPB micelle process was stable and highly reproducible.

[0098] Table 3 Validation results of the optimal preparation process of PTX / TCR-BPB micelles (n=3)

[0099]

[0100] The experimental results show that when the mass ratio of TCR conjugate to BPB in PTX / TCR-BPB micelles is 3:1 and the mass ratio of PTX to TCR-BPB is 1:1, the drug loading capacity and encapsulation rate of PTX / TCR-BPB micelles are large and the stability is good at this ratio, which is the best process choice.

[0101] Experimental Example 3: Characterization of PTX / TCR-BPB micelles

[0102] 1. Experimental methods:

[0103] (I) Characterization of PTX / TCR-BPB micelle morphology

[0104] The morphology of PTX / TCR-BPB micelles was observed by TEM. An appropriate amount of micelle solution was taken and dropped on a copper mesh covered with a carbon support film. The solution was left there for 3 minutes. Excess micelle solution was absorbed with filter paper. A 2% phosphotungstic acid solution was added for staining. The solution was dried at room temperature after 3 minutes and observed and photographed under TEM.

[0105] (II) DSC characterization of PTX / TCR-BPB micelles

[0106] The thermodynamic characteristics of PTX / TCR-BPB micelles were determined by a synchronous thermal analyzer. Under an argon atmosphere, the sample was heated from 40°C to 500°C at a heating rate of 10 K / min, and the scanning curve was recorded.

[0107] 2. Experimental results (I) Characterization of PTX / TCR-BPB micelle morphology

[0108] TEM image of PTX / TCR-BPB micelles (see Figure 4 ) showed that the micelles were nearly spherical with a relatively uniform particle size distribution of about 150 nm, which was smaller than the particle size measured by the DLS method. This may be because the transmission electron microscope was measured in a dry state, while the particle size analyzer was measured under swelling conditions.

[0109] (II) DSC characterization of PTX / TCR-BP micelles

[0110] Differential Scanning Calorimetry (DSC) is a thermal analysis method, and the curve obtained by scanning is called a DSC curve. Figure 5 DSC curves of PTX, TCR-BPB blank mixed carrier, physical mixture of PTX and TCR-BPB, and PTX / TCR-BPB micelles.

[0111] In the figure, the DSC spectrum of PTX shows two characteristic peaks, a strong exothermic peak and a strong endothermic peak at around 222°C and 241°C respectively; TCR-BPB has a weak exothermic peak and a weak endothermic peak at around 219°C and 253°C respectively; PTX+TCR-BPB has the characteristic peaks of both PTX and TCR-BPB, appearing at around 220°C and 247°C, with a slight offset; the DSC curve of PTX / TCR-BPB micelles is basically consistent with that of TCR-BPB, with an exothermic peak and an endothermic peak at around 218°C and 253°C respectively, and no characteristic peaks of PTX are shown, indicating that PTX is encapsulated in the TCR-BPB mixed carrier in an amorphous form to form PTX / TCR-BPB mixed micelles.

[0112] The experimental results show that the PTX / TCR-BPB micelles are nearly spherical, and the micelle particle size is small and evenly distributed. In summary, the present invention provides a nearly spherical PTX / TCR-BPB micelle and a preparation method thereof, wherein the PTX / TCR-BPB micelle particle size is small and evenly distributed, and the PTX / TCR-BPB micelle particle size measured by TEM is about 150nm. The micelles were measured by DLS method to have a particle size of about (213.27 ± 8.20) nm, a PDI of 0.09 ± 0.01, a Zeta potential of (-25.43 ± 1.47) mV, a drug loading of (41.94 ± 2.47)%, and an encapsulation efficiency of (55.72 ± 6.62)%. The process of the present invention prepares PTX / TCR-BPB micelles, and the method is reliable and stable, and the ratio of each component in Example 1 is the optimal ratio.

[0113] Experimental Example 4: Screening of PTX / TCR-BPB micellar lyophilization protectants

[0114] 1. Experimental methods:

[0115] (I) Screening of PTX / TCR-BPB micellar lyoprotectants

[0116] The protective effects of glucose, mannitol and sucrose at different concentrations (0.1%, 0.2% and 0.4%) as freeze-dried protective agents were investigated respectively. The PTX content in the PTX / TCR-BPB micelles was determined according to chromatographic condition 1, and the drug loading and encapsulation efficiency were calculated. The evaluation indicators of the appearance and solubility of the PTX / TCR-BPB micelles are shown in Table 4. The above freeze-dried protective agents were screened using the appearance, solubility, drug loading, encapsulation efficiency, particle size and distribution of the PTX / TCR-BPB micelles as evaluation indicators.

[0117] (II) Study on the stability of the reconstituted solution

[0118] 6 mg of PTX / TCR-BPB micelle freeze-dried product with 0.2% sucrose as freeze-drying protectant was taken, reconstituted with water and stored at 4°C. The drug loading, encapsulation efficiency, Zeta potential, particle size and distribution were measured within three days to investigate its reconstitution stability.

[0119] (III) Long-term storage stability study

[0120] The PTX / TCR-BPB micelle lyophilized product with 0.2% sucrose as lyoprotectant was stored in a refrigerator at 4°C under sealed conditions. 6 mg of the micelle lyophilized product was taken out at 0, 3, and 6 months, respectively, and reconstituted with water to measure the drug loading, encapsulation efficiency, particle size and distribution to investigate its long-term storage stability.

[0121] 2. Experimental results (I) Screening results of PTX / TCR-BPB micellar lyoprotectants

[0122] The evaluation indicators for the appearance and solubility of drug-loaded micelles are as follows:

[0123] Table 4 Appearance and resolubility evaluation indexes

[0124]

[0125]

[0126] The indicators of the PTX / TCR-BPB micellar freeze-dried preparations using different concentrations (0.1%, 0.2% and 0.4%) of glucose, mannitol and sucrose as freeze-drying protectants are shown in Table 5.

[0127] Table 5 Lyophilization protective agent screening results ( n=3)

[0128]

[0129] Note: G: glucose; M: mannitol; S: sucrose

[0130] The looseness of the texture of the PTX / TCR-BPB micelle freeze-dried products without protective agent, with glucose, mannitol and sucrose as freeze-dried protective agents was as follows: no freeze-dried protective agent group < glucose group < sucrose group < mannitol group. The surface of the micelle freeze-dried products in the group without protective agent was rough, flocculent, easy to crack and fly; the surface of the micelle freeze-dried products with glucose as freeze-dried protective agent was delicate, slightly collapsed, loose in texture but easy to fly; the texture of the micelle freeze-dried products with sucrose as freeze-dried protective agent was moderate, but became more compact as the sucrose concentration increased; the texture of the PTX / TCR-BPB micelle freeze-dried products with mannitol as protective agent was the most dense, easy to shrink, and easy to clump after being taken out.

[0131] As can be seen from Table 5, there is little difference in the particle size and distribution of PTX / TCR-BPB micelles before and after freeze-drying. After freeze-drying, the Zeta potential and drug loading of PTX / TCR-BPB micelles decreased slightly, and the encapsulation rate changed significantly. Among them, the encapsulation rate of drug-loaded micelles with 0.2% and 0.4% sucrose as freeze-drying protectants was significantly improved. Moreover, the appearance and solubility of these two groups of PTX / TCR-BPB micelle freeze-dried products are better than those of other groups, and the absolute value of Zeta potential is larger and more stable. The difference between the two groups is that the surface of the freeze-dried product in the 0.2% sucrose group is more delicate, the texture is loose and not easy to fly, and the freeze-dried product in the 0.4% sucrose group is too dense, not soft enough, and difficult to weigh.

[0132] Combining the results of various indicators and considering cost savings, 0.2% sucrose was finally selected as the optimal lyoprotectant for PTX / TCR-BPB micelles.

[0133] (II) Results of stability study of reconstituted solution

[0134] The results of the stability test of the reconstituted solution are shown in Table 6.

[0135] Table 6 Reconstitution stability of PTX / TCR-BPB micelle freeze-dried formulations ( n=3)

[0136] Time (d) Particle size (nm) PDI Zeta potential (mV) DL(%) EE(%) 1 232.83±9.29 0.11±0.03 -27.07±1.62 42.36±1.37 80.91±2.09 2 232.00±9.04 0.09±0.01 -26.27±0.69 42.08±0.82 80.39±1.87 3 233.47±13.61 0.08±0.04 -28.31±0.68 40.96±0.50 78.24±0.89

[0137] As can be seen from the table, the particle size of the PTX / TCR-BPB micelle solution reconstituted within three days was stable, the PDI was less than 0.2, the particle size distribution was uniform, and there was no significant change in the Zeta potential, drug loading and encapsulation efficiency.

[0138] (III) Results of long-term storage stability study

[0139] The results of long-term storage stability test are shown in Table 7. As can be seen from the table, with the extension of storage time, the particle size of PTX / TCR-BPB micelles changes little, the PDI is less than 0.2, the particle size is stable, the Zeta potential, drug loading and encapsulation efficiency are basically unchanged, and the micelles can be stably stored.

[0140] Table 7 Long-term storage stability of PTX / TCR-BPB micelle lyophilized formulations ( n=3)

[0141] Time (month) Particle size (nm) PDI Zeta potential (mV) DL(%) EE(%) 0 232.83±9.29 0.11±0.03 -27.07±1.62 42.36±1.37 80.91±2.09 3 225.63±3.43 0.10±0.01 -27.29±1.13 42.50±1.01 80.77±1.81 6 226.23±4.04 0.08±0.02 -27.29±0.98 42.92±0.88 80.37±3.50

[0142] The experimental results show that 0.2% sucrose was selected as the lyoprotectant of PTX / TCR-BPB micelles. The particle size of the prepared lyophilized preparation was (232.83±9.29) nm, the PDI was 0.11±0.03, the potential was (-27.07±1.62) mV, the drug loading and encapsulation efficiency were (42.36±1.37)% and (80.91±2.09)%, respectively. The surface of the lyophilized preparation was smooth, flat and full. After reconstitution, it was a clear and transparent liquid with good stability and long-term storage stability.

[0143] Experimental Example 5: Study on the absorption of PTX / TCR-BPB micelles in rat intestine

[0144] 1. Experimental methods:

[0145] (I) Preparation of each drug solution

[0146] 1. Preparation of KR solution (blank control)

[0147] Accurately weigh 7.8 g NaCl, 0.35 g KCl, 0.22 g MgCl2, 1.37 g NaHCO3, 0.22 g NaH2PO4, and 1.47 g glucose and dissolve them in an appropriate amount of distilled water as liquid I; weigh 0.32 g CaCl2 and dissolve it in an appropriate amount of distilled water as liquid II; mix liquid II with liquid I under stirring at 1500 rpm, and add distilled water to make up to 1000 mL.

[0148] 2. Preparation of PTX-containing perfusion fluid

[0149] Group: Perfusate: Precision aspiration 1. Add KR solution to the phenol red stock solution to make the volume so that the concentration of PTX in the solution is 30 μg / mL and the concentration of phenol red is 60 μg / mL.

[0150] +Verapamil Group: +Verapamil perfusion solution: precise aspiration Add appropriate amount of Verapamil and phenol red stock solution, and add KR solution to make up the volume so that the concentration of PTX in the solution is 30μg / mL, the concentration of phenol red is 60μg / mL, and the concentration of Verapamil is 400μg / mL.

[0151] +TCR-BPB group: +TCR-BPB perfusion solution: Take TCR-BPB (the amount of TCR-BPB is the same as that in the PTX / TCR-BPB micelle + Verapamil perfusion solution below), add and phenol red stock solution, add KR solution to make up the volume so that the concentration of PTX in the solution is 30μg / mL and the concentration of phenol red is 60μg / mL.

[0152] PTX / TCR micelle group: PTX / TCR micelle perfusion solution: accurately aspirate PTX / TCR micelle solution, add phenol red mother solution, and add KR solution to make up the volume so that the concentration of PTX in the solution is 30 μg / mL and the concentration of phenol red is 60 μg / mL.

[0153] PTX / TCR-BPB micelle group: PTX / TCR-BPB micelle perfusion solution: accurately aspirate PTX / TCR-BPB micelle solution, add phenol red mother solution, and add KR solution to make up the volume so that the concentration of PTX in the solution is 30 μg / mL and the concentration of phenol red is 60 μg / mL.

[0154] PTX / TCR-BPB micelles + Verapamil group: PTX / TCR-BPB micelles + Verapamil perfusion solution: Accurately aspirate the PTX / TCR-BPB micelle solution prepared in Example 3, add Verapamil and phenol red mother solution, and add KR solution to make up the volume so that the solution contains PTX concentration of 30 μg / mL, phenol red concentration of 60 μg / mL, and verapamil concentration of 400 μg / mL.

[0155] (II) In vivo intestinal absorption experiment in rats

[0156] 1. Experimental steps of intestinal absorption in rats

[0157] To avoid drug adsorption by the pipeline during the experiment, the pipeline should be saturated with the test solution before the experiment. Rats were randomly divided into 6 groups, 3 in each group, and fasted for 12 hours, but not water. Urethane solution was injected intraperitoneally for anesthesia, and the rats were fixed on the operating table with the ventral surface facing up. The abdominal cavity was opened along the midline of the abdomen for about 3 cm, and the stomach was found first. The duodenum started about 2 cm below the pylorus; the jejunal segment started from 20 cm below the pylorus; 2 cm above the cecum was the end of the ileum, and 10 cm above this point was the ileum segment; the colon segment was adjacent to the cecum to the rectum, and the duodenum, jejunum, ileum, and colon segments were separated, each about 10 cm, and a "V"-shaped small incision was cut at the beginning and end of the intestinal segment, and the infusion tube was inserted. The intestinal segment and the inlet and outlet were marked on the infusion tube, and it was tied with cotton thread, and the abdominal wound of the rat was covered with gauze soaked in saline. Slowly push physiological saline to clean the intestinal contents, then connect a constant flow pump, pre-saturate the perfusion pipeline and intestinal segment with blank KR solution at a flow rate of 0.2mL / min, and connect the drug solution for perfusion at a flow rate of 0.2mL / min after 30 minutes. The moment of the first drop of drug solution at the outlet after filling the entire pipeline is taken as the zero time. The perfusion fluid is collected at the outlet with a microcentrifuge tube (EP tube) every 15 minutes. The experiment lasts for 120 minutes. After the experiment, the rats were killed, the intestinal segments were cut off, and their length and radius were measured. The concentrations of PTX and phenol red were determined respectively, and the drug absorption rate constant (Ka) and apparent permeability coefficient (Peff) were calculated using the phenol red method.

[0158] 2. Drug stability in intestinal fluid

[0159] Blank KR solution was used as perfusion fluid, and the in vivo rat intestinal absorption test method was used for perfusion for 2 hours, and the blank intestinal perfusion fluid was collected for use. The blank intestinal perfusion fluid was used to prepare the drug perfusion fluids of each group containing 30 μg / mL of PTX, and the PTX content at 0, 1, and 2 hours was measured in a 37°C constant temperature water bath.

[0160] 3. Drug adsorption by intestinal segments

[0161] After the rats were killed, the target intestinal segments were taken out, each about 10 cm, washed with physiological saline, turned over, placed in 20 mL of perfusion fluid, and placed in a constant temperature water bath shaker at 37°C and a shaking speed of 100 rpm. After 2 hours, the intestinal segments were taken out, the PTX content in the incubation fluid was determined, and the changes in the drug concentration in the incubation fluid were investigated.

[0162] 4. Adsorption of drugs by pipelines

[0163] Cut a tube of the same length as that used in the in vivo intestinal absorption experiment. The rest of the operation is the same as "Item 1, Item (ii) in Example 5", except that the rat intestinal segment is missing in the middle. The perfusion fluid is collected every 15 minutes to determine the content, and the adsorption percentage of the drug by the tube is calculated.

[0164] 5. Determination of “steady state” in in vivo intestinal absorption experiment

[0165] In the initial stage of the in vivo intestinal absorption experiment, due to the adsorption of the drug in the pipeline, the propulsion of the drug solution in the pipeline has not reached equilibrium, and the dilution of the drug solution by the remaining KR solution in the pipeline, the phenol red concentration in the outflowing perfusate, the fraction of drug adsorbed in the pipeline, and the apparent absorption fraction of the drug vary greatly, and the entire system is in a non-steady state. In order to ensure the reliability and reproducibility of the experimental data, it is necessary to determine the time to reach a relative steady state.

[0166] (III) Calculation of apparent absorption rate constant and apparent permeability coefficient

[0167] Calculate the apparent absorption kinetic parameters of different intestinal segments of rats, compare the differences in the absorption kinetic behavior of the same preparation in different intestinal segments and the differences between different preparations in the same intestinal segment (to ensure the accuracy and reproducibility of the experimental results, remove the pipeline and intestinal wall adsorption parts during calculation). Apparent absorption rate constant (K a ) and apparent permeability coefficient (P eff ) is calculated as follows:

[0168]

[0169]

[0170] In the above formula, C pin and C pout Respectively represent the drug concentration in the perfusion solution entering and exiting the intestine (μg / mL); C PRin and C PRout represent the concentration of phenol red in the perfusion fluid entering and leaving the intestine (μg / mL); v is the perfusion rate (0.2mL / min); r and l are the radius and length of the perfused intestinal segment (cm), respectively.

[0171] 2. Experimental results:

[0172] (I) Stability of drugs in intestinal fluid

[0173] The stability of the drug in intestinal fluid was investigated. The experimental results showed that The PTX content in the intestinal perfusate of the PTX / TCR-BPB group was (98.80±1.69)% (n=3) of that at 0h 1 hour after drug administration, and was (98.18±1.11)% (n=3) of that at 0h 2 hours after drug administration. The PTX content in the intestinal perfusate of the PTX / TCR-BPB group was (99.05±0.67)% (n=3) of that at 0h 1 hour after drug administration, and was (96.90±1.83)% (n=3) of that at 0h 2 hours after drug administration. There was no significant change in drug content, so PTX is stable in blank intestinal fluid and is less affected by enzyme degradation.

[0174] (II) Adsorption of drugs by the tube and rat intestinal wall

[0175] 1. Adsorption of drugs by pipelines

[0176] The results of the drug adsorption by the pipeline are shown in Figure 8 , the results show that, The PTX remaining amount in the group was (90.61%±7.48)% at 45 minutes, with a strong adsorption effect. The PTX remaining amount at 60 minutes was (98.87%±2.14)%, and the PTX content was stable after 60 minutes. The pipeline had almost no adsorption effect on PTX / TCR-BPB micelles, and the PTX remaining amount at 60 minutes was (97.46%±1.85)%.

[0177] 2. Drug adsorption by intestinal segments

[0178] 1h The percentage of PTX in the drug incubation solution of the PTX group relative to the test solution at 0h was (97.54±2.82)% (n=3), and at 2h it was (98.79±1.74)% (n=3). At 1h, the percentage of PTX in the PTX / TCR-BPB group relative to the test solution at 0h was (98.52±1.81)% (n=3), and at 2h the adsorption rate was (97.01±0.28)% (n=3). There was no obvious physical adsorption of PTX in the isolated intestinal segment.

[0179] 3. Determination of “steady state” in in vivo intestinal absorption experiment

[0180] Combined with the stability of the drug in intestinal fluid and the results of the investigation on the adsorption of the drug by the pipeline and the intestinal wall of rats, during the in vivo intestinal experiment, the PTX content in the intestinal perfusion fluid collected after 60 minutes was stable, so 60 minutes was used as the steady-state time point, and the data before 60 minutes were not processed.

[0181] (III) Ka and Peff of different PTX preparations in intestinal absorption

[0182] Depend on Fig. 9 It can be seen that compared to, The Ka of PTX in each intestinal segment of the +Verapamil group increased by 2.44, 1.75, 1.18 and 1.49 times, and Peff increased by 2.13, 1.54, 1.05 and 1.34 times, respectively, indicating that the addition of Verapamil can increase the oral absorption of P-gp substrate PTX by inhibiting the activity of intestinal mucosal P-gp. and There was no statistically significant difference in Ka and Peff between the two groups +TCR-BPB, indicating that TCR-BPB micelles without PTX encapsulation had no effect on promoting absorption.

[0183] and Compared with the control group, the Ka and Peff of PTX in the whole intestinal segment of the PTX / TCR micelle group and the PTX / TCR-BPB micelle group were significantly increased. a and P eff There were no significant differences.

[0184] Compared to The Ka of the PTX / TCR-BPB micelle group was significantly increased in the duodenum, jejunum and colon (P<0.001), and significantly increased in the ileum (P<0.05), respectively. The Peff in the duodenum and colon was significantly increased (P<0.001), and that in the jejunum was significantly increased (P<0.01), respectively. The results showed that PTX / TCR-BPB micelles had a good intestinal absorption promoting effect on PTX.

[0185] There was no significant difference in Ka and Peff between the PTX / TCR-BPB micelle group and the PTX / TCR-BPB micelle + Verapamil group. It is speculated that the PTX / TCR-BPB micelles were internalized into the cells in the form of complete micelles, without direct contact with the P-gp protein, thus avoiding P-gp-mediated efflux.

[0186] like Fig.10 SPSS statistical results showed that the absorption kinetic parameters Ka and Peff in the duodenum were better than those in the other three intestinal segments (P<0.001), and Ka and Peff in the ileum were better than those in the jejunum (P<0.05). Therefore, the duodenum was determined to be the superior intestinal segment for PTX / TCR-BPB micelle absorption; the ileum was slightly better than the jejunum and colon.

[0187] The experimental results show that the PTX / TCR-BPB micelles of the present invention can promote the absorption of PTX in the whole intestine, with the duodenum being the dominant absorption segment, by using phenol red as the volume marker and the Ka and Peff of the drug in the intestine as the evaluation indicators through in vivo intestinal absorption experiments. Moreover, the absorption pathway of PTX / TCR-BPB glue has nothing to do with P-gp.

[0188] Experimental Example 6: In vivo pharmacodynamic evaluation of PTX / TCR-BPB micelles

[0189] 1. Experimental Methods

[0190] (I) Tumor inoculation in mice

[0191] The Balb / c tumor-bearing mouse model was established with 4T1 mouse breast cancer cells. The mice were female, 5-8 weeks old, and weighed 18±2 g. 4T1 cells in the logarithmic growth phase were collected and washed twice with 0.9% sodium chloride injection and adjusted to 1×10 7 / mL. 0.2mL was subcutaneously inoculated under the right forelimb of mice. After 7 days, the tumor grew to 100-250cm 3 Start dosing.

[0192] (II) Dosage design and administration cycle

[0193] 1. Grouping and dosage design

[0194] 1) Control group: normal saline;

[0195] 2) Group: 20 mg / kg;

[0196] 3) +TCR-BPB group: 20 mg / kg;

[0197] 4) PTX / TCR micelle group: 20 mg / kg;

[0198] 5) PTX / TCR-BPB micelle group: 20 mg / kg. PTX / TCR-BPB micelles were prepared according to the method of Example 3.

[0199] 2. Dosage cycle

[0200] Mice with similar tumor volumes were randomly divided into 5 groups, 8 mice in each group. The first administration was marked as day 0. According to the above grouping and dosage, the drug was orally administered at a volume of 0.2 mL / 10 g on days 0, 2, 4, 6, and 8, for a total of 5 times. The mice were killed on day 10 and the tumor tissues were dissected.

[0201] (III) Evaluation of tumor inhibition effect

[0202] The first administration was recorded as day 0, and the long and short diameters of the tumor were measured every other day. The tumor volume was calculated according to formula 5, and the tumor growth volume-time curve was drawn.

[0203] V tumor =(a 2 ×b) / 2 (5)

[0204] Among them, a is the short diameter of the tumor, and b is the long diameter of the tumor.

[0205] On the 10th day, the tumor-bearing mice were killed, and the tumor tissues were removed and weighed. The tumor inhibition rate was calculated according to Formula 6, the therapeutic effect was evaluated, and mathematical statistics were performed.

[0206] IR(%)=(W saline -W drug ) / W saline ×100% (6)

[0207] Among them, IR is the tumor inhibition rate, W drug is the tumor weight of the drug-treated group, W saline is the tumor weight of the control group (normal saline group).

[0208] (IV) Mouse weight examination

[0209] Starting from day 0, the weight of mice was measured at the same time every day, and the weight change curve of mice was drawn to measure the toxic effects of the drug on mice.

[0210] (V) Organ Index

[0211] After administration, the weight of tumor-bearing mice was measured every other day, and the weight change curve of mice was drawn. After weighing the mice on the 10th day, the heart, liver, spleen, lung, kidney, and tumor were immediately removed after being killed, weighed, recorded, and the organ index was calculated according to Formula 7. The changes in the organ index were used to examine the toxic and side effects of each group of drugs on the organs.

[0212] Organ index = organ mass (g) / mouse body weight (g) × 100% (Formula 7)

[0213] (VI) Morphological observation of tissue sections by HE staining

[0214] The freshly obtained tissue was fixed with fixative for 24 hours, taken out, trimmed and flattened, dehydrated and dipped in wax, and the wax-soaked tissue was embedded in an embedding machine. After the wax solidified, it was taken out and the wax was trimmed. After freezing at -20℃ overnight, 4μm thin slices were cut. The cut tissue was transferred to a glass slide, dried in an oven, stained and sealed, and examined under a microscope to collect images.

[0215] (VII) Serum biochemical index detection

[0216] About 0.5 mL of blood was collected from the mouse eyeball and placed in an EP tube. The tube was centrifuged at 14,000 rpm for 5 min, and the upper serum was collected and stored in a -20°C refrigerator for later use.

[0217] 1. Tumor marker CA15-3 detection

[0218] Serum carbohydrate antigen 15-3 (CA15-3) is a breast cancer tumor marker with high sensitivity and specificity. Mouse breast cancer marker-CA153 ELISA kit was used according to the kit instructions. The absorbance (OD value) was measured at a wavelength of 450nm using an ELISA instrument, and the mouse breast cancer antigen (CA15-3) concentration in the sample was calculated using the standard curve.

[0219] 2. Liver and kidney function tests

[0220] To study whether oral administration of PTX / TCR-BPB micelles causes functional damage to the organs of mice, the liver detection indicators are alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the kidney detection indicators are urea nitrogen (BUN) and creatinine (CRE). The serum of each group was tested under an automatic biochemical analyzer to determine the concentration of ALT, AST, BUN and CRE in the serum of each group, reflecting the degree of damage to liver and kidney cells after drug treatment in each group.

[0221] 2. Experimental Results

[0222] (I) Tumor growth curve

[0223] Mouse subcutaneous tumors Fig.11 The tumor volume change curves of each group of tumor-bearing mice are shown in Fig.12 As shown in Figure 2, the tumor volume of tumor-bearing mice on day 10 was 5.38 times larger than that on day 0. Group and The tumor volume of the +TCR-BPB group increased by 3.99 times and 3.64 times, respectively; the PTX / TCR micelle group and the PTX / TCR-BPB micelle group increased by 3.41 times and 3.16 times, respectively. The results showed that compared with the Contol group, each treatment group could inhibit tumor growth to varying degrees, among which the PTX / TCR-BPB micelle group had the most obvious therapeutic effect. Comparing the tumor volume of tumor-bearing mice on the 10th day, the tumor volume of the Control group and other groups was significantly different (compared with group P < 0.01, and the rest of the groups P < 0.001); +TCR-BPB group tumor volume is slightly smaller than The difference between the two groups was not significant, but it was greater than that of the PTX / TCR-BPB micelle group (P<0.001), indicating that the TCR-BPB micelles without PTX encapsulation had no enhancing effect on the anti-tumor effect of PTX, indicating that the TCR-BPB micelles had no intestinal absorption promoting effect in the in vivo intestinal absorption experiment of rats. The better antitumor effect of the PTX / TCR-BPB micelle group may be due to the synergistic antitumor effect of rhein and PTX in the TCR conjugate; the tumor volume of the PTX / TCR-BPB micelle group was smaller than that of the PTX / TCR micelle group and there was a significant difference (P<0.05), indicating that the active targeting effect of BPB on tumors significantly improved the antitumor effect of PTX / TCR micelles.

[0224] (II) Tumor weight and tumor inhibition rate

[0225] On the 10th day after administration, the mice were killed and the tumors were removed. Fig.13 , weigh the tumor and calculate the tumor inhibition rate. The results are shown in Fig.14 and Fig.15 In terms of tumor weight, the control group Group, The tumor weights of the +TCR-BPB group, PTX / TCR micelle group, and PTX / TCR-BPB micelle group were (0.86±0.09) g, (0.62±0.05) g, (0.56±0.06) g, (0.42±0.03) g, and (0.36±0.03) g, respectively. The tumor weights of the PTX / TCR micelle group and the PTX / TCR-BPB micelle group were significantly different (P<0.05).

[0226] In terms of tumor inhibition rate, compared with the Control group, Group, The tumor inhibition rates of the +TCR-BPB group, PTX / TCR micelle group, and PTX / TCR-BPB micelle group were (28.41±5.66)%, (34.45±6.66)%, (50.96±3.57)%, and (58.43±3.16)%, respectively. There were extremely significant differences between the PTX / TCR micelle group and the PTX / TCR-BPB micelle group (P<0.001).

[0227] From the above results, it can be seen that the tumor growth of each drug administration group was significantly inhibited, and there was a significant breast cancer treatment effect. Encapsulating PTX in micelles can enhance its anti-tumor effect. In addition, the PTX / TCR-BPB mixed micelles formed by the mixed carrier formed by TCR conjugate and BPB encapsulating PTX can deliver PTX to the tumor site through active targeting and passive targeting, which has a better anti-tumor effect than PTX / TCR micelles with only passive targeting.

[0228] (III) Weight changes

[0229] The changes in body weight of mice in each group during treatment are shown in Fig.16 Compared with the average body weight on day 0, the average body weight of the Control group Group, The weight of the +TCR-BPB group, PTX / TCR micelle group and PTX / TCR-BPB micelle group increased by 2.99%, 2.82%, 4.18%, 1.50% and 6.18%, respectively. As can be seen from the figure, except for the PTX / TCR-BPB micelle group, the weight of mice in each group increased and decreased from time to time, indicating that drugs and gavage operations may affect the appetite of animals or cause fear, resulting in a trend of weight loss in mice. The overall trend of weight growth in the PTX / TCR-BPB micelle group was faster than that in other groups, and only on the 3rd and 4th days, the weight growth rate of mice was negative, which was less than that in other groups, indicating that the administration of this group had little effect on the food intake of mice and the survival state of mice was good. On the 7th and 8th days, the weight growth rate of mice in the Control group and the PTX / TCR-BPB micelle group was significantly different (P<0.05). In addition, on the other days during the treatment period, the weight changes of mice in each group were relatively gentle, with no significant differences. The experimental results showed that PTX / TCR-BPB micelles significantly reduced the toxic side effects of PTX / TCR micelles, and were close to achieving no toxic side effects, and were well accepted by mice.

[0230] (IV) Organ Index

[0231] The results of organ indexes of tumor-bearing mice in each group are shown in Fig.17 , A decrease in organ index means organ atrophy or other degenerative changes, while an increase in organ index means congestion, edema or hypertrophy. References Zhang Xiuyan, Zhan Chunlie. "Determination and comparison of blood routine, main organ mass and main organ coefficient of three types of mice: Balb / c, KM and NIH" [J]. Hubei Agricultural Science. 2012, 51(05): 962-964., The spleen organ index of Balb / c mice bearing 4T1 far exceeds the normal range of the spleen organ index of Balb / c mice (0.33±0.04)%, and there is no significant difference among the groups. There is no significant difference in the organ index of each organ between the control group and the PTX / TCR-BPB PMs group, indicating that PTX / TCR-BPB micelles have no obvious toxic side effects on each organ after administration. This result further shows that PTX / TCR-BPB micelles are suitable for targeted delivery of PTX and have good drug safety.

[0232] (V) Morphological observation of tissue sections and HE staining

[0233] To further confirm the safety of PTX / TCR-BPB micelles in vivo, histological analysis was performed on the heart, liver, spleen, lung, kidney and tumor of 4T1-bearing Balb / c mice after administration to observe whether there was inflammation or tissue lesions in each tissue. Fig.18 As shown in the figure (the black circles in the figure indicate typical locations where tumor cells grow vigorously or are broken after treatment). If the cells of each organ of each group of mice are closely arranged and the tissue structure is intact, it indicates that there is almost no toxicity to the organ. The nuclei of tumor cells in the PTX / TCR-BPB micelle group were ablated and ruptured, and the cell structure was destroyed, indicating that PTX / TCR-BPB micelles have a strong killing effect on tumor cells.

[0234] (VI) Serum biochemical index detection

[0235] The activity values ​​of ALT, AST, BUN and CRE in the serum of each group of mice were detected by enzyme-linked immunosorbent assay to investigate the degree of liver and kidney damage in mice. As shown in Table 8, among the liver function indicators, there was no significant difference in the ALT activity value of each group; the AST activity value of each medication group was low, among which the PTX / TCR micelle group (P<0.01) and the PTX / TCR-BPB micelle group (P<0.05) were significantly different from those of the Control group. Among the renal function indicators, the CRE activity value of each group was not statistically significant; the BUN activity value of the PTX / TCR micelle group (P<0.01) and the PTX / TCR-BPB micelle group (P<0.05) were significantly different from those of the Control group. The above results show that the degree of liver and kidney damage in the mice in the two micelle groups was relatively mild. Group and The slightly higher AST and BUN activity values ​​in the +TCR-BPB group may be due to the damage to the liver and kidney caused by solvent toxicity.

[0236] The CA15-3 level in the serum of each group of mice was detected by using the mouse breast cancer marker CA153 ELISA kit, and the results are shown in Table 8. As can be seen from the table, after drug treatment, the CA15-3 level of mice in each drug group decreased by 20.61% to 27.48% compared with the Control group. Group and +TCR-BPB group P<0.05, PTX / TCR micelle group and PTX / TCR-BPB micelle group P<0.01, among which the PTX / TCR-BPB micelle group decreased most significantly, with CA15-3 concentration of (0.95±0.08)U / mL, which was 72.52% of the blank group. The above results show that PTX / TCR-BPB micelle has the best therapeutic effect on breast cancer compared with other drugs.

[0237] Table 8 Effects of each group of drug treatment on breast cancer markers CA15-3, liver function (ALT, AST) and renal function (BUN, CRE) in tumor-bearing mice (n=8)

[0238]

[0239] Note: Compared with the Control group, *P<0.05; **P<0.01.

[0240] The experimental results showed that PTX / TCR-BPB micelles had a strong anti-tumor effect and could effectively inhibit the growth of mouse tumors. After 10 days of treatment, the tumor weight was (0.36±0.03) g, and the tumor inhibition rate was (58.43±3.16)%. There was a significant difference between the two groups (P<0.001).

[0241] During the treatment, the weight of mice in the PTX / TCR-BPB micelle group changed slowly. On the 10th day of treatment, the organ index showed no significant difference compared with the normal saline group. After HE staining of tissue sections, the cell structures of organs such as heart, liver, spleen, lung, and kidney were intact, and the nuclei of some tumor cells were ruptured. The serum ALT, AST, BUN, CRE and other biochemical indicators of liver and kidney function and the breast cancer marker CA15-3 were detected. The activity values ​​of AST and BUN were lower than those of the normal saline group (P<0.05), and the level of CA15-3 decreased.

[0242] The experimental results showed that PTX / TCR-BPB micelles had good anti-breast cancer effects and drug safety.

[0243] In summary, the present invention uses PTX as a model drug, encapsulates it in TCR-BPB, and prepares PTX / TCR-BPB mixed micelles. The PTX / TCR-BPB micelles have good encapsulation capacity for PTX, small particle size, uniform distribution, sustained release effect, good killing effect on tumor cells, can target tumor cells, have small toxic and side effects, can promote the absorption of PTX in the intestine, and the absorption path is unrelated to P-gp. The PTX / TCR-BPB micelles improve the oral bioavailability of PTX, have good anti-tumor effects, reduce the toxic and side effects of PTX, and are a nano drug delivery system with good development prospects.

[0244] The present invention also screened the effects of the mixing ratio of the conjugate and BPB and the drug loading ratio on the PTX / TCR-BPB micelles, determined the optimal drug loading process of the PTX / TCR-BPB micelles, and the mass ratio of Example 1 was the optimal process, that is, the mixing mass ratio of the TCR conjugate and BPB was 3:1, and the mass ratio of PTX to TCR-BPB was 1:1. The lyophilization protectant of the PTX / TCR-BPB micelles was screened, and 0.2% sucrose was selected as the optimal lyophilization protectant for the PTX / TCR-BPB micelles.

Claims

1. A carrier for drug delivery, characterized in that: The carrier is prepared by taking PEGylated biotin and TCR conjugate as raw materials, and the mass ratio of PEGylated biotin and TCR conjugate is 3:(1-9).

2. The carrier according to claim 1, characterized in that: The ratio of PEGylated biotin to TCR conjugate in the carrier is 1:

3.

3. The carrier according to claim 1, characterized in that: The mixed micelle is prepared by dissolving PEGylated biotin and TCR conjugate in water and ultrasonicating in an ice-water bath until dissolved.

4. A mixed micelle carrying a drug, characterized in that: The invention is prepared by adding a drug into the carrier described in any one of claims 1 to 3.

5. The mixed micelle according to claim 4, characterized in that: In the mixed micelles, the mass ratio of the carrier to the drug is 1:(0.8-1.6); preferably, in the mixed micelles, the mass ratio of the carrier to the drug is 1:1; preferably, the drug is paclitaxel.

6. The mixed micelle according to claim 4 or 5, characterized in that: The micelle is prepared according to the following method: dissolving the drug in a solvent, adding dropwise to the carrier solution according to any one of claims 1 to 3, stirring, ultrasonicating, dialyzing, filtering, and freeze-drying; the solvent is an organic solvent, preferably anhydrous ethanol.

7. The mixed micelle according to any one of claims 1 to 6, characterized in that: The TCR conjugate is a TPGS-modified carboxymethyl chitosan-rhein conjugate. The structure of the conjugate is shown in Formula I: wherein A and B are independently selected from rhein or TPGS; n＝436～512； In the conjugate, the molar substitution degree of rhein is 6.44±0.83%; and the molar substitution degree of TPGS is 0.28±0.06%.

8. The mixed micelle according to any one of claims 4 to 6, characterized in that: A lyoprotectant is added in the freeze-drying step; the lyoprotectant is any one or more of glucose, mannitol and sucrose; preferably, the lyoprotectant is sucrose; preferably, the lyoprotectant is 0.2% sucrose.

9. A method for preparing the mixed micelle according to any one of claims 4 to 8, characterized in that: The drug is dissolved in a solvent, and the solution is added dropwise to the carrier solution according to any one of claims 1 to 3, followed by stirring, ultrasonication, dialysis, filtration, and freeze-drying.

10. Use of the mixed micelle according to any one of claims 4 to 8 in the preparation of a drug for treating tumors; preferably, the drug for treating tumors is a drug for treating breast cancer.

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