A polymer for encapsulating protein drugs, redox-responsive nanoparticles made therefrom and applications thereof

Abstract

This application provides a polymer for encapsulating protein drugs, redox-responsive nanoparticles made from the polymer, and their applications. The structure of the polymer is shown in the following general formula I. Protein drug-encapsulated nanoparticles can be prepared by encapsulating protein drugs with the polymer. These nanoparticles can efficiently load protein drugs, disassemble in the redox microenvironment of tumors, and release the drug, thus providing a new technical means for the targeted delivery of protein drugs.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine, and more specifically, relates to a polymer for encapsulating protein drugs, redox-responsive nanoparticles made therefrom, and their applications. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens the lives and health of Chinese residents. Traditional chemotherapy is prone to causing drug resistance and serious adverse reactions, necessitating the search for new strategies and technologies for tumor treatment. Immunotherapy has revolutionized clinical tumor treatment, with immune checkpoint blockade therapy and cytokine therapy significantly improving clinical benefits for some patients. Protein drugs, such as immune checkpoint antibodies and cytokines, are widely used in immunotherapy; however, protein drugs have poor stability and readily bind specifically to targets in normal tissues, causing serious adverse reactions and affecting treatment duration and efficacy. Therefore, the development of new tumor-specific delivery technologies for protein drugs remains extremely urgent.

[0003] Tumor tissue possesses an inflammatory microenvironment and oxidative stress, exhibiting high concentrations of reactive oxygen species (ROS). Simultaneously, tumor cells, due to metabolic abnormalities, produce large amounts of reduced glutathione (GSH), creating a unique redox microenvironment within the tumor. This characteristic allows oxidation- and reduction-sensitive substances to undergo corresponding chemical reactions in the tumor microenvironment, resulting in responsive denaturation and cleavage processes. Constructing redox-responsive nanoparticles to deliver protein drugs can improve tumor-specific drug delivery, achieve tumor microenvironment-responsive drug release, shield protein drugs from interactions with normal tissues, reduce adverse reactions, and ultimately improve therapeutic efficacy against various tumors. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing traditional drug treatments for tumors and provide a drug delivery system for targeted therapy of tumors (such as colorectal cancer, melanoma, breast cancer, and liver cancer). Traditional drugs often suffer from low targeting, high side effects, and damage to normal cells and tissues. The inventors of this application have discovered that boronic esters undergo chemical bond breakage upon oxidation, enabling a response under the high ROS conditions of the tumor microenvironment; and that the exchange between disulfide bonds and thiol groups in reduced GSH enables a response under the high GSH conditions within tumor cells. By introducing these two chemical structures—boronic esters and disulfide bonds—into a polymer carrier, redox-responsive nanoparticles for delivering protein drugs are constructed. By synthesizing redox-responsive polymers and encapsulating protein drugs in nanoparticles, the aim is to enrich and release protein drugs at the tumor site.

[0005] Another object of the present invention is to provide redox-responsive nanoparticles constructed using the above-described drug delivery system.

[0006] Another object of the present invention is to provide the use of the above-mentioned redox-responsive nanoparticles in the preparation of antitumor drugs.

[0007] To achieve the above objectives, in one respect, the present invention provides a polymer having the structure shown in the following general formula I:

[0008]

[0009] In the above general formula I, n = 45 to 340, L is selected from -CH2-CH2- or -CH2-CH2-CONH(CH2)5-, and R1 is hydrogen or hydroxyl. express or

[0010] On the other hand, the present invention provides a method for preparing the above-mentioned polymer, the method comprising the following steps:

[0011] (1) In the presence of a weak organic base, CH3O-PEG-NH2 is reacted with N-hydroxysuccinimide ester of 3-(2-pyridinyl dithio)propionic acid (SPDP) or succinimide-6-(3-[2-pyridinyl dithio]-propionylamino)hexanoate (LC-SPDP) in an organic solvent to prepare PEG-SPDP / LC-SPDP.

[0012] (2) React the PEG-SPDP / LC-SPDP obtained in step (1) with 4-mercaptophenylboronic acid (PBA) in an organic solvent to obtain the product PEG-SPDP / LC-SPDP-PBA.

[0013] (3) The PEG-SPDP / LC-SPDP-PBA obtained in step (2) is reacted with epigallocatechin gallate (EGCG) or its analogues in an organic solvent to obtain the polymer, wherein the analogues of EGCG include epigallocatechin gallate (ECG) or gallatechin gallate (GCG).

[0014] In a specific embodiment, the organic solvents in steps (1), (2), and (3) are each independently selected from chloroform, methanol, ethanol, and acetone.

[0015] In a specific embodiment, in step (1), the molecular weight range of CH3O-PEG-NH2 is 2000 to 15000 Da, for example 5000 Da.

[0016] In a specific embodiment, in step (1), the mass ratio of CH3O-PEG-NH2∶SPDP / LC-SPDP is (10~20)∶1;

[0017] In step (2), the mass ratio of PEG-SPDP / LC-SPDP:PBA is (15-45):1;

[0018] In step (3), the mass ratio of PEG-SPDP / LC-SPDP-PBA:EGCG or its analogues is (1-16):1.

[0019] In a specific embodiment, in step (1), the organic weak base may be selected from triethylamine, cyclohexylamine and aniline.

[0020] In a specific embodiment, in step (1), the amount of the organic weak base relative to SPDP / LC-SPDP can be 0.5-50 μL / 1 mg SPDP / LC-SPDP.

[0021] In a specific embodiment, the reaction temperature in step (1) is room temperature, and the reaction time is 4 to 8 hours;

[0022] The reaction temperature in step (2) is room temperature, and the reaction time is 4 to 8 hours;

[0023] The reaction temperature in step (3) is 30-40°C, preferably 37°C overnight.

[0024] In a specific embodiment, in step (2), PEG-SPDP / LC-SPDP and PBA are dissolved in an organic solvent, and the PBA solution is slowly added to the PEG-SPDP / LC-SPDP solution while stirring to react.

[0025] In specific embodiments, after steps (1) and (2), the operation of removing organic solvents is included. For example, in step (1), the organic solvent is removed by rotary evaporation at 20-60°C, and the resulting solid is dried in an oven at 20-60°C for 0.5-4 hours; for example, in step (2), the organic solvent is removed by rotary evaporation at room temperature, and the precipitated solid is washed multiple times with diethyl ether or methyl tert-ethyl ether until the yellow color is basically removed, the supernatant is removed, and the solid is dried in an oven at 20-60°C for 0.5-4 hours.

[0026] On the other hand, the present invention provides a method for preparing nanoparticles encapsulating protein drugs, the method comprising:

[0027] The protein drug is dissolved in deionized water, and the polymer solution in an organic solvent is added during ultrasonic homogenization to obtain the protein drug-loaded nanoparticles.

[0028] In a specific embodiment, the organic solvent is selected from chloroform, methanol, ethanol and acetone.

[0029] In specific embodiments, the protein drugs include, but are not limited to, immune checkpoint antibody drugs (such as Opdivo (nivolumab) and Keytruda (pembrolizumab)), cytokines (such as interferon series (IFN) and interleukin series (IL)), and other monoclonal antibody drugs (such as Erbitux (cetuximab) and Herceptin (trastuzumab)).

[0030] In a specific embodiment, the mass ratio of the protein drug to the polymer is (0.5-50):1.

[0031] In another aspect, the present invention provides nanoparticles carrying protein drugs prepared by the above method.

[0032] In another aspect, the present invention provides the use of the above-mentioned protein-loaded nanoparticles in the preparation of antitumor drugs.

[0033] In a specific embodiment, the tumor includes all tumors with an oxidative and / or reducing microenvironment, including but not limited to colorectal cancer, melanoma, breast cancer, liver cancer, pancreatic cancer, and gastric cancer.

[0034] Beneficial effects

[0035] This invention develops a novel polymer material for encapsulating protein drugs and redox-responsive nanoparticles prepared using the same, which can be used for tumor-specific delivery and controlled release of protein drugs. These nanoparticles can efficiently load protein drugs and disassemble within the tumor's redox microenvironment (ROS and GSH), releasing the drug and enabling various tumor treatments, including immune checkpoint blockade, vaccines, and cytokine therapy. These nanoparticles provide a new technology for protein drug delivery and have significant clinical implications. Attached Figure Description

[0036] Figure 1 This is a mass-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) image of PEG-SPDP-PBA-EGCG prepared in Example 1.

[0037] Figure 2 This is a transmission electron microscope image of the protein drug-loaded nanoparticles prepared in Example 2.

[0038] Figure 3 This is a transmission electron microscope image of the protein drug-loaded nanoparticles prepared in Example 2 undergoing oxidative cleavage in 1% hydrogen peroxide.

[0039] Figure 4 This is a transmission electron microscope image of the reduction and cleavage of the protein drug-loaded nanoparticles prepared in Example 2 in a 10 nM reducing GSH solution.

[0040] Figure 5The stability test results of the protein-loaded drug nanoparticles prepared in Example 2 are shown.

[0041] Figure 6 The distribution of nanoparticles prepared by the method of the present invention in tumor-bearing nude mice was shown 24 hours after administration via tail vein injection; wherein the aforementioned nanoparticles were loaded with an anti-EGFR antibody linked to the fluorescent probe Cy5. Detailed Implementation

[0042] The technical solution of the present invention will be described in detail below through specific embodiments, so that those skilled in the art can better understand the present invention; however, the scope of protection of the present invention is not limited thereto.

[0043] In this application, the logical symbol " / " in "SPDP / LC-SPDP" represents the relation "OR".

[0044] Example 1: Synthesis and Characterization of a Polymer for Encapsulating Protein Drugs (PEG-SPDP-PBA-EGCG)

[0045] Accurately weigh PEG-NH2 and SPDP with a molecular weight of 4800 Da in a mass ratio of PEG-NH2 : SPDP = 12 : 1, and dissolve them in chloroform. Add ethylamine at a ratio of 5 μL / 1 mg SPDP, and stir the mixture at room temperature for 4 hours. Remove the chloroform by rotary evaporation at room temperature, precipitating an oily liquid. Add n-hexane to precipitate solid PEG-SPDP. Remove the supernatant, and dry the solid in an oven at 40°C for 0.5 hours to remove the organic solvent.

[0046] Accurately weigh PEG-SPDP and PBA at a mass ratio of PEG-SPDP:PBA = 33:1, and dissolve them separately in chloroform. Slowly add the PBA solution to the PEG-SPDP solution using a syringe, and stir the reaction at room temperature for 4 hours. Remove the organic solvent by rotary evaporation at room temperature, and wash the precipitated solid repeatedly with diethyl ether until the yellow color is mostly gone. Remove the supernatant, and dry the solid in a 40°C oven for 0.5 hours to remove the organic solvent.

[0047] Accurately weigh PEG-SPDP-PBA and EGCG in a mass ratio of PEG-SPDP-PBA : EGCG = 12 : 1, dissolve them in methanol, and incubate overnight at 37°C.

[0048] The resulting solution was evaporated to dryness and then analyzed by MALDI-TOF-MS. The results are as follows: Figure 1 As shown.

[0049] Example 2: Preparation and Characterization of Redox-Responsive Nanoparticles

[0050] Accurately weigh the anti-epidermal growth factor receptor protein (anti-EGFR) antibody (MedChemExpress) and dissolve it in deionized water. Slowly add the PEG-EGCG methanol solution prepared in Example 1 to an ultrasonic homogenizer (JYD-650L ultrasonic cell disruptor, Shanghai Zhixin Instrument Co., Ltd.), until the final mass ratio of anti-EGFR antibody to PEG-SPDP-PBA-CG is 17:1. The obtained PEG-SPDP-PBA-CG nanoparticles were observed using transmission electron microscopy, and the results are as follows: Figure 2 As shown in the figure, it is a spherical nanoparticle with a particle size of ~200nm.

[0051] Example 3: Oxidation responsiveness of redox-responsive nanoparticles

[0052] 1 mL of the nanoparticle aqueous solution prepared in Example 2 was precisely measured and added to a 30% H2O2 aqueous solution to a final concentration of 100 μmol / L. The solution was then placed on a shaker at 37°C and shaken for 0.5 hours. Dynamic light scattering (Zetasizer Nano ZS 90 laser particle size analyzer, Malvern Instruments Ltd., UK) revealed that the particle size had changed to approximately 500 nm (see [link to relevant documentation]). Figure 3 ).

[0053] Example 4: Reduction responsiveness of redox-responsive nanoparticles

[0054] 1 mL of the nanoparticle aqueous solution prepared in Example 2 was precisely measured, and GSH was added to a final concentration of 10 μmol / L. The solution was then placed on a shaker at 37°C and shaken for 0.5 hours. Dynamic light scattering analysis revealed that the particle size had changed to approximately 500 nm (see [link to sample image]). Figure 4 ).

[0055] Example 5: Protein stability of redox-responsive nanoparticles

[0056] 1 mL of the nanoparticle aqueous solution prepared in Example 2 was added to 9 mL of 10% fetal bovine serum (FBS) aqueous solution. After mixing, the particle size was measured by dynamic light scattering (DLS), and the particle size change trend was detected by DLS at 12 hours, 24 hours, 48 ​​hours, 5 days, and 9 days. The results are as follows. Figure 5 As shown in the figure, the particle size of the nanoparticles remained basically unchanged within 9 days, proving that the nanoparticles prepared by this invention have good stability and therefore have the potential to be prepared into drugs.

[0057] Example 6: Distribution of redox-responsive nanoparticles in tumor-bearing mice

[0058] Accurately measure 75 μg of water-soluble Cy5 and 3 mg of anti-EGFR antibody and dissolve them in 10 mL of deionized water. Add 10 μL of triethylamine and incubate at room temperature for 4 hours. Then, slowly add the PEG-SPDP-PBA-CG methanol solution prepared in Example 1 in an ultrasonic homogenizer. The final solution has a mass ratio of anti-EGFR antibody to PEG-SPDP-PBA-CG of 17:1.

[0059] Nude mice with a CT26 tumor model (CT26 tumor cells were obtained from the Cell Bank of the Chinese Academy of Sciences, and 4-week-old female BALB / c nude mice were obtained from the Shanghai Laboratory Animal Center) were prepared, and 100 μL of the above solution was injected via the tail vein. The distribution of nanoparticles was observed using in vivo imaging of the small animals 24 hours later. The results are as follows: Figure 6 As shown, from Figure 6 As can be seen, most of the nanoparticles were found to be enriched at the tumor site.

[0060] In summary, this invention develops a novel polymer material for encapsulating protein drugs. Nanoparticles made from this encapsulated protein drug exhibit sensitive redox responsiveness, making them a promising delivery material for targeted delivery of protein drugs, particularly antitumor drugs.

Claims

1. A polymer having the structure shown in the following general formula I: I In the above general formula I, n = 45~340, L is -CH2-CH2-, and R1 is a hydroxyl group. The polymer is prepared by the following method: (1) In the presence of a weak organic base, CH3O-PEG-NH2 is reacted with 3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester (SPDP) in an organic solvent to prepare PEG-SPDP. (2) React the PEG-SPDP obtained in step (1) with 4-mercaptophenylboronic acid (PBA) in an organic solvent to obtain the product PEG-SPDP-PBA. (3) The polymer is prepared by reacting the PEG-SPDP-PBA obtained in step (2) with epigallocatechin gallate (EGCG) in an organic solvent.

2. A method for preparing the polymer as described in claim 1, the method comprising the following steps: (1) In the presence of a weak organic base, CH3O-PEG-NH2 is reacted with 3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester (SPDP) in an organic solvent to prepare PEG-SPDP. (2) React the PEG-SPDP obtained in step (1) with 4-mercaptophenylboronic acid (PBA) in an organic solvent to obtain the product PEG-SPDP-PBA. (3) The polymer is prepared by reacting the PEG-SPDP-PBA obtained in step (2) with epigallocatechin gallate (EGCG) in an organic solvent.

3. The method according to claim 2, wherein, The organic solvents in steps (1), (2), and (3) are each independently selected from chloroform, methanol, ethanol, and acetone.

4. The method according to claim 2, wherein, In step (1), the molecular weight of CH3O-PEG-NH2 ranges from 2000 to 15000 Da.

5. The method according to claim 4, wherein, The molecular weight of CH3O-PEG-NH2 is 5000 Da.

6. The method according to claim 2, wherein, In step (1), the mass ratio of CH3O-PEG-NH2∶SPDP is (10~20):1; and / or In step (2), the mass ratio of PEG-SPDP to PBA is (15~45):1; and / or In step (3), the mass ratio of PEG-SPDP-PBA to EGCG is (1~16):

1.

7. The method according to claim 2, wherein, In step (1), the organic weak base is selected from triethylamine, cyclohexylamine and aniline, and the amount of the organic weak base relative to SPDP is 0.5-50 μL / 1 mg SPDP.

8. The method according to claim 2, wherein, After steps (1) and (2), the removal of organic solvents is performed.

9. A method for preparing nanoparticles encapsulating protein drugs, the method comprising: The protein drug is dissolved in deionized water, and a solution of the polymer in an organic solvent as described in claim 1 is added during ultrasonic homogenization to obtain the nanoparticles encapsulating the protein drug.

10. The method according to claim 9, wherein, The organic solvent is selected from chloroform, methanol, ethanol, and acetone, and / or The protein drug includes immune checkpoint antibody drugs, cytokines, or other monoclonal antibody drugs, wherein the immune checkpoint antibody drug is selected from nivolumab and pembrolizumab, the cytokines are selected from interferon series and interleukin series, and the other monoclonal antibody drugs are selected from cetuximab and trastuzumab; and / or The mass ratio of the protein drug to the polymer is (0.5~50):

1.

11. A nanoparticle carrying a protein drug, prepared by the method of claim 9 or 10.

12. The use of the protein-loaded nanoparticles as described in claim 11 in the preparation of antitumor drugs, wherein, The tumor is colorectal cancer.

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