Monoclonal modification and drug loading of a polycysteine polypeptide

Abstract

The application provides an antibody modification and drug delivery system of a polycysteine polypeptide, which comprises two general drug loading strategies of preparing drug-loaded micelles by covalently connecting amphiphilic polypeptide carrier naphthyl-S-((2-carboxyethyl)thio)-L-cysteine (P30) through an ester bond to load hydroxyl-containing drugs and by self-assembly of micelles to embed hydrophobic drugs, and two general modification targeting antibody ways of covalently connecting modification of amino-containing antibodies through an amide bond and modification of positively charged and stable antibodies under pH 7.4 conditions through electrostatic adsorption. The P30 carrier, the general drug-loaded micelles and the antibody modification products prepared by the application have excellent biocompatibility, the final products have the characteristics of GSH / pH dual-response drug release and active targeting delivery, have high drug loading efficiency, low toxic side effects, can be suitable for precise treatment of different molecular subtype breast cancers such as Luminal A type, Luminal B type and HER2 positive type, and have good application prospects in the fields of nano-drug delivery and individualized tumor treatment.

Description

Technical Field

[0001] This application relates to the fields of biomedical materials and nanomedicine delivery technology, specifically to the preparation, drug loading, and monoclonal antibody modification methods of a polycysteine ​​amphiphilic polypeptide carrier with dual GSH / pH responsiveness, and its application in the precision treatment of breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women. The high heterogeneity of its molecular subtypes leads to key challenges in clinical treatment, such as insufficient targeting, significant toxic side effects, and drug resistance. Traditional chemotherapy drugs, endocrine therapy drugs, and targeted drugs have limitations such as poor water solubility, strong off-target toxicity, and low delivery efficiency. For example, tamoxifen, a classic endocrine therapy drug, is highly hydrophobic and easily damages normal tissues, while trastuzumab, as a HER2-targeting drug, suffers from insufficient in vivo circulation stability and limited targeted enrichment efficiency.

[0003] Nanoparticle drug delivery systems offer an effective approach to addressing the aforementioned challenges. Amphiphilic peptides, due to their excellent biocompatibility, biodegradability, and self-assembly properties, have become ideal drug carriers. However, existing peptide carriers often lack precise tumor microenvironment responsiveness, making it difficult to simultaneously achieve drug loading efficiency and targeting, thus failing to meet the individualized treatment needs of different breast cancer subtypes. Therefore, developing peptide nanocarriers that combine dual-response drug release characteristics, high-efficiency drug loading capacity, and targeted delivery function is of great significance for improving the therapeutic effect of breast cancer and reducing toxic side effects. Summary of the Invention

[0004] One aspect of this application discloses a GSH / pH dual-responsive amphiphilic polypeptide carrier P30, characterized by containing a hydrophobic naphthyl group, a hydrophilic carboxyl group, and a reduction-responsive disulfide bond, which can self-assemble to form core-shell micelles, providing a structural basis for drug loading and monoclonal antibody modification.

[0005] Another aspect of this application discloses two general and efficient drug delivery strategies: loading hydroxyl-containing drugs via ester bond covalent linkage and loading hydrophobic drugs via micelle self-assembly. These strategies address the problems of drug leakage, low loading efficiency, and narrow applicability in traditional drug delivery methods. The drug-loaded micelles can respond to the tumor microenvironment to achieve precise drug release.

[0006] Another aspect of this application discloses two mild and universal targeting modification methods that, while preserving antibody activity and drug loading capacity, achieve active targeting of target-positive tumor cells and improve the drug enrichment efficiency at the tumor site.

[0007] Another aspect of this application discloses the excellent biocompatibility and broad-spectrum antitumor activity of the nanomedicine delivery system, which can be adapted to the treatment needs of different molecular subtypes of breast cancer, especially showing a synergistic therapeutic effect on Luminal B double-positive breast cancer.

[0008] Another aspect of this application discloses the preparation and characterization methods of the system, verifying the product structure through various spectroscopic and microscopic characterization methods, and verifying the biosafety and antitumor activity through cytotoxicity experiments, thereby ensuring the reliability and practicality of the system. Detailed Implementation

[0009] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0010] The features and performance of this application will be further described in detail below with reference to the embodiments. Example 1

[0011] As used herein and unless otherwise specified, “P30” represents naphthyl-S-((2-carboxyethyl)thio)-L-cysteine, “TAM-P30” represents polycysteine ​​micelles covalently loaded with tamoxifen via ester bonds, “P30@TAM” represents micelles self-assembled with tamoxifen, the “Tra-” prefix represents trastuzumab amide bond modified products, and “Tra / ” represents trastuzumab electrostatic adsorption modified products. 1. Synthesis of the amphiphilic polypeptide carrier P30

[0012] (1) Preparation of S-phenylsulfonyl-L-cysteine: Under ice bath and nitrogen protection, 49g of L-cysteine ​​hydrochloride monohydrate was dissolved in 140mL of 2M hydrochloric acid, and 19.32g of sodium nitrite aqueous solution was added dropwise. After stirring for 2h, 91g of sodium benzenesulfinate aqueous solution was added and stirred until a white solid precipitated. After recrystallization and purification, it was dried for later use.

[0013] (2) Preparation of S-phenylsulfonyl-L-cysteine ​​intracyclic anhydride: 7.64 g of the above product was dissolved in 120 mL of anhydrous tetrahydrofuran under nitrogen protection in a water bath at 50 °C. 4.92 g of triphosgene tetrahydrofuran solution was injected and stirred for 4 h. The product was then purified by precipitation with hexane to obtain a pale yellow solid.

[0014] (3) Preparation of naphthyl-poly(S-phenylsulfonyl-L-cysteine): 5.4 g of intracyclic anhydride was dissolved in 50 mL of DMF under the protection of nitrogen in an ice bath at 5 °C. 0.23 g or 0.09 g of 1-naphthylamine was added and stirred for 72 h. The precursor polymer with a degree of polymerization of 12 or 30 was obtained by dialysis (1000 Da) and freeze drying.

[0015] (4) Preparation of P30: At room temperature, 1 mmol of the precursor polymer was dissolved in DMF, and 12 mmol or 30 mmol of 3-mercaptopropionic acid was added. After stirring for 4 h, P12 or P30 solid powder was obtained by dialysis (1000 Da) and freeze drying. 2. Preparation of drug-loaded micelles

[0016] (1) Preparation of TAM-P30: 20 mg P30 was dissolved in anhydrous dimethyl sulfoxide (DMSO), 2 mg EDC and 2.5 mg DMAP were added, and the mixture was stirred for 4 h. Then 4 mg 4-hydroxytamoxifen was added and the reaction was continued for 24 h. The ester-bonded drug micelles were obtained by dialysis (1000 Da) and freeze-drying. They were dispersed in PBS (pH 7.4) and self-assembled to form a 1 mg / mL micelle solution.

[0017] (2) Preparation of P30@TAM: 4 mg tamoxifen and 20 mg P30 were dissolved in 20 mL DMSO and stirred vigorously for 24 h. After dialysis (1000 Da) and freeze drying, self-assembled drug-loaded micelles were obtained and dispersed in PBS (pH 7.4) to form a 1 mg / mL micelle solution. 3. Preparation of trastuzumab modified products

[0018] (1) Preparation of amide bond-linked products: 10 mg P30, TAM-P30 or P30@TAM were dispersed in 10 mL PBS (pH 7.4), 0.22 mg EDC and 0.16 mg NHS were added, and the mixture was stirred for 4 h. Then 1.16 mg trastuzumab was added and the mixture was reacted at room temperature for 24 h. Tra-P30, Tra-TAM-P30 or Tra-P30@TAM were obtained by dialysis (1000 Da) and freeze-drying.

[0019] (2) Preparation of electrostatic adsorption products: 10 mg TAM-P30 was dispersed in 10 mL PBS (pH 7.4), and 1.16 mg trastuzumab was added. Electrostatic adsorption was achieved by using the negative charge generated by the ionization of the carboxyl group of the carrier and the positive charge of trastuzumab to obtain Tra / TAM-P30. 4. Product characterization and performance verification

[0020] (1) Structural characterization: The chemical structures of the carrier, drug loading and modified product were verified by ¹H NMR, the characteristic functional groups were verified by FTIR, the trastuzumab binding efficiency was verified by MALDI-TOF MS, the morphology and particle size were observed by TEM, and the CMC value and Zeta potential were determined by DLS.

[0021] (2) Drug loading performance verification: The standard curve of tamoxifen was determined by ultraviolet spectrophotometry (278nm), and the encapsulation efficiency and drug loading rate of drug-loaded micelles were calculated.

[0022] (3) Response drug release verification: In vitro drug release experiments were conducted in different dialysis media (pH 7.4, pH 6.2, with / without 10mM GSH) at 37℃ and 100rpm, and the amount of drug released was detected at regular intervals.

[0023] (4) Biocompatibility and tumor suppressor activity verification: MCF10A, MCF7, T47D and SKBR3 cells were selected, and cell viability after treatment with different concentrations of samples for 72 hours was determined by CCK-8 method to evaluate biocompatibility and tumor suppressor effect. Attached Figure Description

[0024] Figure 1 Flowchart of P30 synthesis Figure 2 Schematic diagram of ester covalent linkage of drug-loaded micelles Figure 3 Schematic diagram of micelle self-assembly and embedding of drug-loaded micelles Figure 4 Schematic diagram of amide bond linkage of trastuzumab Figure 5 Schematic diagram of the electrostatic adsorption of trastuzumab. Figure 6 ¹H NMR spectra of TAM, P30 and TAM-P30 Figure 7 ¹H NMR spectra of P30, Tra-P30 and Tra-TAM-P30 Figure 8 UV absorbance of tamoxifen at different concentrations (a) and tamoxifen standard curve (b). Figure 9 In vitro drug release profiles of drug-loaded micelles (under different environmental conditions). Figure 10 The critical micelle concentrations for each sample are: (a) P30 micelles, (b) P30@TAM micelles, (c) TAM-P30 micelles, (d) Tra-TAM-P30 micelles, (e) Tra-P30@TAM micelles, (f) Tra / TAM-P30 micelles, and (g) Tra-P30 micelles. Figure 11TEM images of each sample: (a) P30 micelles, (b) P30@TAM micelles, (c) TAM-P30 micelles, (d) Tra-TAM-P30 micelles, (e) Tra-P30@TAM micelles, (f) Tra / TAM-P30 micelles, (g) Tra-P30 micelles Figure 12 Cell viability images of various samples against normal breast MCF10A cells, Luminal A MCF7 breast cancer cells, Luminal B T47D cells, and HER2-positive SKBR3 cells. Samples included: 1. P30; 2. Free TAM; 3. P30@TAM; 4. TAM-P30; 5. Tra-TAM-P30; 6. Tra-P30@TAM; 7. Tra / TAM-P30; 8. Tra-P30 Experimental verification results

[0025] (1) Structural verification:¹H NMR and FTIR characterization showed that the naphthyl, carboxyl and disulfide bond characteristic peaks of P30 were clear, TAM-P30 showed the characteristic peak of tamoxifen benzene ring, and Tra-P30 showed the characteristic peak of trastuzumab, confirming that the product was successfully synthesized.

[0026] (2) Drug loading performance: TAM-P30 has an encapsulation efficiency of 90.06% and a drug loading efficiency of 15.26%, while P30@TAM has an encapsulation efficiency of 66.09% and a drug loading efficiency of 11.67%, which meet the performance indicators of claim 4.

[0027] (3) Responsive drug release: The release rate of both drug-loaded micelles reached over 90% within 15 hours in a pH 6.2 + GSH environment, while the release amount was extremely low in a pH 7.4 GSH-free environment and a pH 6.2 GSH-free environment, achieving dual-response drug release.

[0028] (4) Biocompatibility: The blank vector, drug-loaded micelles and trastuzumab modified products had no significant effect on the viability of normal breast cells MCF10A, which met the biocompatibility requirements.

[0029] (5) Antitumor activity: Drug-loaded micelles showed antitumor effects on all three types of breast cancer cells. The antitumor activity of trastuzumab modified products on SKBR3 cells was significantly enhanced, and cell viability decreased to 29%-30% at 50x concentration.

[0030] The above embodiments are merely preferred embodiments of this application and are not intended to limit the scope of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application based on the core technical solutions of this application should be included within the protection scope of this application.

Claims

1. A GSH / pH dual-responsive nanomedicine delivery system for tumor therapy, characterized in that, The product includes an amphiphilic peptide carrier naphthyl-S-((2-carboxyethyl)thio)-L-cysteine ​​(P30), universal drug-loaded micelles, and a monoclonal antibody modified product. The carrier contains a hydrophobic naphthyl group, a hydrophilic carboxyl group, and a reduction-responsive disulfide bond. The drug-loaded micelles are constructed using two universal strategies, and the monoclonal antibody modified product is prepared using two mild and universal methods.

2. The nanomedicine delivery system according to claim 1, characterized in that, The synthesis methods of P30 and related products include: (1) Using L-cysteine ​​hydrochloride as raw material, the amphiphilic polypeptide P30 containing disulfide bonds was synthesized by intracyclic anhydride synthesis and ring-opening polymerization. The polymerization degree was controlled by adjusting the amount of 1-naphthylamine (preferably 30). (2) Two general drug loading strategies were used to prepare drug-loaded micelles: one was to use EDC / DMAP catalysis to form ester bonds between the carboxyl group of P30 and the hydroxyl group of the hydroxyl-containing drug to achieve covalent drug loading (hydroxyl drug-P30), wherein the hydroxyl-containing drug included 4-hydroxytamoxifen, docetaxel, paclitaxel, epirubicin, irinotecan, and curcumin; the other was to use the amphiphilic self-assembly of P30 to form micelles and embed hydrophobic drugs through hydrophobic interactions (P30@hydrophobic drug), wherein the hydrophobic drugs included tamoxifen, paclitaxel, docetaxel, doxorubicin, camptothecin, sorafenib, and imatinib; (3) Two general targeted modification methods are adopted: one is to use EDC / NHS catalysis to form amide bonds between the carboxyl group of P30 or drug-loaded micelles and amino-containing monoclonal antibodies (Ab-P30, Ab-hydroxy drug-P30, Ab-P30@hydrophobic drug), wherein the amino-containing monoclonal antibodies include trastuzumab, pertuzumab, cetuximab, rituximab, bevacizumab, and nivolumab; the other is to achieve electrostatic adsorption of positively charged antibodies that are not easily oxidized and decomposed in water with hydroxy drug-P30 (Ab / hydroxy drug-P30) through charge matching under pH 7.4 conditions, wherein the positively charged stable antibodies include trastuzumab, pertuzumab, cetuximab, and rituximab.

3. The nanomedicine delivery system according to claim 1, characterized in that, The P30 has a naphthyl hydrophobic group and a carboxyl hydrophilic group. The disulfide bond can be broken in response to the high glutathione (GSH) in the tumor microenvironment, and the carboxyl group can regulate the hydrophilicity and hydrophobicity of the carrier in response to the acidic tumor microenvironment (pH 6.2), thus achieving dual GSH / pH response drug release.

4. The nanomedicine delivery system according to claim 1, characterized in that, In the drug-loaded micelles, taking tamoxifen as an example, the ester bond covalently loaded 4-hydroxytamoxifen TAM-P30 has an encapsulation rate of ≥90% and a drug loading rate of ≥15%, while the micelle self-assembled loaded tamoxifen P30@TAM has an encapsulation rate of ≥66% and a drug loading rate of ≥11%. Both drug loading systems have extremely low drug release in normal tissue environment (pH 7.4, no GSH), but in tumor microenvironment (pH 6.2, high GSH), the drug release rate is ≥90% within 15 hours.

5. The nanomedicine delivery system according to claim 1, characterized in that, In the monoclonal antibody modification products, taking trastuzumab as an example, the critical micelle concentration (CMC) of the amide-linked trastuzumab product (Tra-P30) increased from 0.2 mg / mL to 0.363 mg / mL compared to P30, while the CMC of the electrostatically adsorbed trastuzumab product (Tra / TAM-P30) remained at 0.3 mg / mL compared to (TAM-P30). The modification did not affect the drug loading and responsive drug release, and could enhance the targeting of HER2-positive breast cancer cells.

6. The nanomedicine delivery system according to claim 1, characterized in that, The P30 blank carrier, drug-loaded micelles, and trastuzumab-modified products maintain a relative cell viability of 80%-120% in normal mammary epithelial cells (MCF10A), exhibiting excellent biocompatibility and reducing the off-target toxicity of tamoxifen.

7. The nanomedicine delivery system according to claim 1, characterized in that, The drug-loaded micelles and trastuzumab-modified products all exhibited antitumor activity against Luminal A (MCF7), Luminal B (T47D), and HER2-positive (SKBR3) breast cancer cells. Among them, the trastuzumab-modified product showed significantly better antitumor effect on HER2-positive cells than the drug-loaded micelles without monoclonal antibody.

8. The synthesis method according to claim 2, characterized in that, The synthesis of P30 requires dialysis purification (molecular weight cutoff of 1000 Da) and freeze drying. The drug-loaded micelles and trastuzumab-modified products need to be characterized and verified by means of nuclear magnetic resonance hydrogen spectroscopy (¹H NMR), infrared spectroscopy (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), etc.

9. The nanomedicine delivery system according to claim 1, characterized in that, The average particle size of the drug-loaded micelles and trastuzumab-modified products is 40-100 nm, and they have a spherical or near-spherical structure. The trastuzumab amide bond-modified products exhibit irregular shapes due to steric hindrance, but still maintain good self-assembly stability.

10. The nanomedicine delivery system according to any one of claims 1-9, characterized in that, It can be used for precision treatment of different molecular subtypes of breast cancer, especially for the synergistic treatment of Luminal B breast cancer that is double-positive for estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2).

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