In-situ dePEGylation nano-drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses an in-situ dePEGylation nano drug delivery system as well as a preparation method and application of the in-situ dePEGylation nano drug delivery system. The preparation method comprises the following steps: adding EDC.HCl and NHS into DMF (Dimethyl Formamide) containing mPEG-COOH, and stirring at room temperature; dropwise adding the activated solution into a DMF (Dimethyl Formamide) solution containing MMP2 sensitive peptide, stirring overnight at room temperature, dialyzing and freeze-drying to obtain MMP2 sensitive peptide-PEG-COOH; continuously activating terminal carboxyl, adding MDM2 targeting peptide, stirring at room temperature, dialyzing and freeze-drying to obtain a PEG-MMP2 peptide-MDM2 peptide compound; dissolving the compound in chloroform / methanol, performing ultrasonic dissolution, sequentially adding Dox and ATO nanoparticles, and stirring to form an oil phase solution; and carrying out rotary evaporation to form a film, carrying out PBS resuspension and ultrasonic hydration, and carrying out dialysis and purification to obtain the nano drug delivery system. Peeling of the PEG layer is specifically triggered through a tumor microenvironment, and precise drug release and synergistic anti-tumor treatment are realized in combination with a multi-stage response mechanism.

Description

Technical Field

[0001] The present invention relates to the field of nanomedicine technology, and particularly relates to an in-situ dePEGylated nanodrug delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] As a key tumor suppressor factor, the p53 protein plays a central role in maintaining genomic stability, regulating the cell cycle, and inducing apoptosis. However, p53 gene mutations or functional inactivation exist in approximately 50% of malignant tumors, resulting in the loss of its tumor suppressor function. In p53 wild-type tumors, the overexpression of the MDM2 protein mediates the degradation of p53 through ubiquitination, inhibiting its activity; while in p53 mutant tumors, the mutant p53 protein loses its DNA-binding ability, thus being unable to inhibit tumor growth. Traditional p53-targeted strategies, such as MDM2 inhibitors, although can partially restore the function of wild-type p53, are ineffective against mutant p53 and are prone to cause drug resistance. Therefore, there is an urgent need to develop a synergistic treatment plan that can simultaneously solve the problems of p53 inhibition and mutation.

[0003] The polyethylene glycol (PEG)ylation technology significantly improves the physicochemical properties and in-vivo behavior of drugs by covalently modifying PEG molecules, and has advantages such as prolonging blood circulation time, enhancing stability, and reducing immunogenicity. It has been widely used in anti-tumor drug delivery. However, this technology still has many limitations. First, the risk of immunogenicity cannot be ignored. Repeated injection of PEGylated drugs may induce the production of anti-PEG antibodies (APA), thereby accelerating drug clearance and triggering allergic reactions. According to statistics, pre-existing APA exists in approximately 7% of healthy people and up to 15% - 20% of cancer patients, and there have even been cases where treatment was aborted due to too high APA titers in phase III clinical trials. Second, due to the steric hindrance effect of PEG molecules, it may shield the drug active sites and interfere with the interaction between the drug and the target.

[0004] Therefore, designing a dynamic dePEGylation mechanism that can smoothly strip PEG at the action site while retaining the original advantages of PEGylation and avoiding immunogenicity is a major challenge currently faced. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an in-situ dePEGylated nanodrug delivery system, a preparation method thereof, and an application thereof. Based on the in-situ dePEGylation design, the present invention adopts a dual mechanism of active targeting and environmental response, and uses the thin film hydration method to prepare the nanodrug delivery system.

[0006] In the first aspect, the present invention provides a preparation method of an in-situ dePEGylated nanodrug delivery system, which is achieved by adopting the following technical solutions.

[0007] A preparation method of an in-situ de-PEGylated nano-drug delivery system, comprising the following steps: S1. Dissolve mPEG-COOH in N,N-dimethylformamide (DMF), add the carboxyl activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and the stabilizer N-hydroxysuccinimide (NHS), and stir at room temperature for 0.5 - 2 hours to activate the carboxyl group; drop the activation solution into the N,N-dimethylformamide solution containing the MMP2-sensitive peptide, stir at room temperature overnight, dialyze and freeze-dry to obtain MMP2-sensitive peptide-PEG-COOH; wherein, the molar ratio of mPEG-COOH to EDC·HCl, NHS and the MMP2-sensitive peptide is 1: (1 - 1.5):(1 - 1.2):1; S2. Dissolve the product of step S1 in N,N-dimethylformamide, add the condensing agent O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and the organic base catalyst N,N-diisopropylethylamine (DIPEA), stir at room temperature for 15 - 30 minutes to activate the terminal carboxyl group; add the MDM2-targeting peptide, stir for 12 - 24 hours, dialyze and freeze-dry to obtain the PEG-MMP2 peptide-MDM2 peptide complex; wherein, the molar ratio of MMP2-sensitive peptide-PEG-COOH to HATU, DIPEA and the MDM2-targeting peptide is 1:(1 - 1.5):(1 - 1.5):1; S3. Dissolve the product of step S2 in a mixed solution of chloroform and methanol, ultrasonically dissolve it, then add doxorubicin (Dox) and arsenic trioxide (ATO) nanoparticles in sequence, and stir for 12 - 24 hours to form an oil-phase solution; rotate and evaporate to form a film, resuspend with PBS and ultrasonically hydrate, and dialyze and purify to obtain the nano-drug delivery system.

[0008] Further, in step S1, the MMP2-sensitive peptide sequence is as shown in SEQ ID NO.1.

[0009] Further, in step S2, the MDM2-targeting peptide is Mtrap-2.

[0010] Further, in step S3, the molar ratio of methanol to chloroform is 1:(4 - 5).

[0011] Further, in step S3, the mass of both doxorubicin and arsenic trioxide accounts for 0 - 50% of the total mass of doxorubicin, arsenic trioxide and the product of step S2, and the sum of the masses of doxorubicin and arsenic trioxide does not exceed 50% of the total mass of doxorubicin, arsenic trioxide and the product of step S2.

[0012] Further, in step S3, the rotation evaporation temperature is 30 - 50 °C.

[0013] In a second aspect, the present invention provides an in-situ dePEGylated nano-drug delivery system, which is achieved by the following technical solutions.

[0014] An in-situ dePEGylated nano-drug delivery system prepared by the above preparation method.

[0015] In a third aspect, the present invention provides the use of an in-situ dePEGylated nano-drug delivery system, which is achieved by the following technical solutions.

[0016] The application of the above in-situ dePEGylated nano-drug delivery system in the preparation of anti-tumor products.

[0017] This application has the following beneficial effects.

[0018] (1) The in-situ dePEGylation design of the present invention specifically triggers the peeling of the PEG layer through the tumor microenvironment, dynamically relieves the steric hindrance effect of PEG, retains the advantage of PEGylation in prolonging the circulation time, and at the same time avoids the accelerated blood clearance effect (ABC effect) caused by long-term PEG modification and the shielding effect on the targeting ligand. It enables the precise exposure of the targeting ligand at the lesion site, significantly enhances the selective binding ability of tumor cells, effectively solves the immunogenicity and targeting inhibition problems brought by traditional PEGylation, and provides a new idea for the precise delivery of nano-drugs.

[0019] (2) The present invention adopts a dual mechanism of active targeting and tumor microenvironment response, and simultaneously utilizes MMP2 and MDM2 overexpressed in the tumor microenvironment to increase the targeting property, so that the tumor targeting ligand is only exposed and delivers drugs at the lesion site, increases the drug concentration at the tumor site, and reduces the damage to normal tissues. For the two drugs Dox and ATO, which have strong cardiac damage, the dual targeting mechanism of the present invention greatly reduces their off-target toxicity.

[0020] (3) The present invention simultaneously loads chemotherapy drugs and p53 pathway regulators through a single nano-carrier, realizing "chemotherapy + gene regulation" synergistic therapy, reducing the types of carriers and the number of drug administrations required for multi-drug combination, and improving patient compliance.

[0021] (4) The present invention greatly improves the drug loading capacity through self-assembled nanostructures, and significantly improves patient compliance compared with the frequent drug administration of traditional chemotherapy.

[0022] (5) Relying on the innovative dual-targeting - enzyme-responsive synergistic delivery system, the present invention is expected to promote the global precise treatment of tumor chemotherapy, achieve targeted enrichment, drug release on demand and synergistic enhancement, and meet the dual requirements of improving clinical efficacy and reducing side effects. Description of the Drawings

[0023] Figure 1This is the construction flowchart of the in-situ de-PEGylated nano-drug delivery system of the present invention; Figure 2 This is the particle size result graph of MMDA and PMMDA of the present invention; Figure 3 This is the particle size distribution graph of the nanoparticles after MMP2 treatment of the present invention; Figure 4 This is the release curve graph of Dox of the present invention; Figure 5 This is the release curve graph of ATO of the present invention; Figure 6 This is the tumor weight result graph of each group after treating 4T1 tumor-bearing mice with different formulations of the present invention; Figure 7 This is the body weight change result graph of each group after treating 4T1 tumor-bearing mice with different formulations of the present invention. Detailed implementation manners

[0024] The in-situ de-PEGylated nano-drug delivery system (PMMDA) of the present invention simultaneously loads chemotherapeutic drugs and p53 pathway regulators, realizing the synergistic treatment of "chemotherapy + gene regulation". Photothermal imaging experiments have proved that PMMDA has good heating effects, and multiple rounds of fluorescence irradiation experiments have proved its good photothermal stability. Finally, in the anti-tumor experiment, PMMDA achieved good tumor suppression effects through multi-level response precision drug release and synergistic anti-tumor treatment in the mouse 4T1 tumor model. At the same time, through safety evaluation, PMMDA has good biocompatibility and no obvious toxicity.

[0025] The present invention will be further described below in conjunction with embodiments. Unless otherwise specified, the materials used in the embodiments of the present application are all commercially available products.

[0026] The mPEG-COOH used in the following embodiments of the present application is purchased from Shanghai Macklin Biochemical Co., Ltd.; The GPLGLAG (SEQ ID NO.1) polypeptide sequence used in the following embodiments of the present application is custom synthesized by Tianjin Aladdin Chemistry Co., Ltd.; Mtrap-2 used in the following examples of this application can be found in MDM2-Targeting Reassembly Peptide (TRAP) Nanoparticles for p53-Based Cancer Therapy. Fangzhou Li, Delin Chen, Qianqian Sun, Jiale Wu, Yaling Gan, Kam W. Leong, and Xing-Jie Liang. Advanced Materials 2023, 35, 2305164, which was custom synthesized by Beijing Saibaisheng Gene Technology Co., Ltd. The structure is as follows:

[0027] Example A method for preparing an in-situ de-PEGylated nanodrug delivery system, comprising the following steps: 1) Dissolve 100 mg of mPEG-COOH in 10 mL of anhydrous DMF, add EDC·HCl (14 mg) and NHS (14 mg) with a molar ratio of carboxylic acid to 1:1.2, and stir at room temperature for 2 hours to activate the carboxyl group. Dropwise add the activated solution into the DMF solution (5 mL) containing 30 mg of GPLGLAG, and stir at room temperature overnight (12 hours) to connect the amino group of the peptide with the PEG carboxyl group through an amide bond. Transfer the reaction solution to a dialysis bag (MWCO 3500 Da) and dialyze in deionized water for 48 hours. After freeze-drying, a white solid product GPLGLAG-PEG-COOH is obtained.

[0028] 2) Dissolve GPLGLAG-PEG-COOH (40 mg) in 10 mL of anhydrous DMF, add HATU (9 mg) and DIPEA (1 mmol / ml, 60 μL), and stir at room temperature for 30 minutes to activate the terminal carboxyl group. Dissolve Mtrap-2 (90 mg) in 2 mL of DMF, slowly drop it into the above activated solution, and stir at room temperature for 12 hours. Dialyze and purify (MWCO 3500 Da, deionized water), and after freeze-drying, a PEG-MMP2 peptide-MDM2 peptide complex is obtained.

[0029] 3) Dissolve the product of step 2 (30 mg) in chloroform / methanol (2:1, 10 mL), and sonicate for 10 minutes until completely dissolved to form a clear solution. Slowly drip the Dox solution (5 mg / mL, 2 mL) into the above solution, and stir for 30 minutes to ensure uniform dispersion of the drug. Subsequently, add the pretreated ATO nanoparticle suspension (10 mg / mL, 1 mL) (the pretreatment method is as follows: dissolve ATO (10 mg) and poloxamer 188 (10 mg) in absolute ethanol (2 mL), and vortex to mix. Preheat the PBS buffer (3 mL) to 40 °C. Slowly drip the organic phase into the aqueous phase (1 mL / min) while stirring (500 rpm, room temperature). Immediately sonicate (200 W, ice bath, 10 minutes) to form a uniform emulsion (avoid bubbles). Continue to stir (300 rpm, room temperature) for 4 hours to completely evaporate the ethanol and form an ATO nanoparticle suspension. Centrifuge at 12,000 rpm for 10 minutes (4 °C) to remove the unencapsulated free ATO. Resuspend the precipitate with 1 mL of PBS and repeat centrifugation twice to finally obtain 1 mL of purified ATO nanoparticle suspension.), continue to stir for 2 hours to form a uniform oil-phase solution. Transfer the mixture to a rotary evaporator and evaporate under reduced pressure at 40 °C to remove the organic solvent until a dry transparent film is formed. Resuspend the film in 10 mL of PBS buffer and sonicate in a water bath at 40 °C (300 W, 15 minutes) to rapidly hydrate and rupture the film to obtain a coarsely dispersed nanoparticle. After dialysis purification (MWCO 3500 Da, PBS buffer), a nanoparticle drug delivery system loaded with Dox and ATO is obtained.

[0030] Control group Preparation method of Matrix Metalloproteinase 2 peptide-Mouse Double Minute 2 Homolog (MDM2) peptide complex-Doxorubicin-Arsenic Trioxide Nanoparticle Drug Delivery System (MMDA), comprising the following steps: Prepare PMMDA (obtained from the examples) solution (final concentration 100 μg / mL) and MMP2 solution (final concentration 1 μg / mL) with PBS (pH 7.4). Gently mix the PMMDA solution (1 mL) with the MMP2 solution (1 mL) (vortex for 10 seconds), avoiding the generation of bubbles. Immediately transfer it to an incubator shaker for incubation. Set the shaker parameters: 37°C, 150 rpm. After incubating for 4 hours, immediately cool the sample on ice to terminate the enzyme activity. Collect the nanoparticles by high-speed centrifugation (12,000 rpm, 4°C, 10 minutes), and wash them twice with pre-cooled PBS to remove free MMP2 and reaction by-products to obtain MMDA.

[0031] Perform the following tests on the in-situ de-PEGylated nano-drug delivery system prepared by the present invention: 1. Determination of particle size and potential Detect the particle size distribution and zeta potential of MMDA and PMMDA by a dynamic light scattering particle size analyzer. Dilute the sample to be measured with ultrapure water to a concentration of 0.1 mg / mL, and ultrasonically treat it for 5 minutes to disperse it evenly. Adjust the instrument parameters to: 25 ± 0.1°C; 173° backscattering mode. After measurement, use software to calculate the particle size distribution (polydispersity index PDI) and Zeta potential, and measure each sample in parallel 3 times.

[0032] It can be seen from Figure 2 that the average particle size of MMDA is about 90 nm, and the average particle size of PMMDA is about 120 nm. The surface charges of all materials are negative.

[0033] 2. Verification of in-situ de-PEGylation Mix PMMDA with MMP2 (1 μg / mL) in PBS (pH 7.4), and incubate it in an incubator shaker at 37°C for 4 hours at a rotation speed of 150 rpm. Then detect the particle size change by a dynamic light scattering particle size analyzer to verify the shedding of the PEG layer. Take 100 μL of the sample before and after incubation respectively, dilute it to 1 mL and then perform DLS detection. Observe the morphological changes by transmission electron microscopy.

[0034] It can be seen from Figure 3 that after treatment with MMP2, the particle size of the nanoparticles decreases by about 30 nm, and a dense core structure without PEG coating is presented. It can be analyzed that the PEG layer has shed and the MDM2 targeting peptide is exposed.

[0035] 3. Drug release test The dialysis bag method (MWCO 3.5 kDa) was used to simulate the in vitro drug release behavior. PMMDA (containing 100 μg of drug) was loaded into a dialysis bag and immersed in 5 mL of PBS buffer (pH 6.8, simulating the tumor microenvironment) containing MMP2 (1 μg / mL). It was oscillated at a constant temperature of 37 °C (100 rpm). 1 mL of the release solution was taken out at regular intervals, and an equal amount of fresh medium was added at the same time. The concentration of Dox was detected by HPLC, and the concentration of ATO was detected by UV-Vis.

[0036] It can be seen from Figure 4 , Figure 5 that in the PBS buffer containing MMP2 (1 μg / mL), the cumulative release rates of Dox and ATO reached 78% and 65% respectively within 24 hours.

[0037] 4. Evaluation of anti-tumor effect 1×10 7 4T1 cells were subcutaneously inoculated into the right femur of female BALB / c mice. After the tumor volume reached 100 mm³, drug administration began. The tumor-bearing mice were randomly divided into five groups: G1: PBS; G2: ATO; G3: Dox; G4: MMDA; G5: PMMDA. Tail vein injection (5 mg / kg Dox equivalent) was performed every 7 days, and the drug was administered twice. The mice were sacrificed on the 28th day. The tumor weight is as Figure 6 shown.

[0038] It can be seen from Figure 6 that compared with the ATO group and Dox group of the simple drug administration groups, the tumor weights of the mice in the MMDA group and PMMDA group were significantly reduced, and the tumor weight of the PMMDA group was the smallest and the effect was the best. It can be concluded that PMMDA can effectively inhibit tumor growth.

[0039] 5. Safety evaluation The body weights of the mice in each group were weighed regularly after the start of drug administration. It can be seen from Figure 7 that the mice treated with free Dox and free ATO both showed severe weight loss; in contrast, the body weights of the two groups of mice injected with PMMDA and MMDA did not change significantly. It can be obtained that PMMDA and MMDA have good biocompatibility.

[0040] The embodiments of this specific implementation manner are all preferred embodiments of the present invention, and do not limit the protection scope of the present invention accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A preparation method of an in-situ de-PEGylated nano-drug delivery system, characterized in that: It includes the following steps: S1. Dissolve mPEG-COOH in N,N-dimethylformamide, add the carboxyl activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the stabilizer N-hydroxysuccinimide, and stir at room temperature for 0.5 - 2 hours to activate the carboxyl group; drop the activation solution into the N,N-dimethylformamide solution containing the MMP2-sensitive peptide, stir at room temperature overnight, dialyze and freeze-dry to obtain MMP2-sensitive peptide-PEG-COOH; wherein, the molar ratio of mPEG-COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and MMP2-sensitive peptide is 1: (1 - 1.5):(1 - 1.2):1; S2. Dissolve the product of step S1 in N,N-dimethylformamide, add the condensing agent O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and the organic base catalyst N,N-diisopropylethylamine, stir at room temperature for 15 - 30 minutes to activate the terminal carboxyl group; add the MDM2-targeting peptide, stir for 12 - 24 hours, dialyze and freeze-dry to obtain the PEG-MMP2 peptide-MDM2 peptide complex; wherein, the molar ratio of MMP2-sensitive peptide-PEG-COOH, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, and MDM2-targeting peptide is 1:(1 - 1.5):(1 - 1.5):1; S3. Dissolve the product of step S2 in a solution of chloroform and methanol, ultrasonically dissolve it, then add doxorubicin and arsenic trioxide nanoparticles, stir for 12 - 24 hours to form an oil-phase solution; rotary evaporate to form a film, resuspend with PBS and ultrasonically hydrate, dialyze and purify to obtain the nano-drug delivery system.

2. The preparation method of an in-situ de-PEGylated nano drug delivery system according to claim 1, characterized in that: In step S1, the MMP2-sensitive peptide sequence is as shown in SEQ ID NO.

1.

3. The preparation method of an in-situ de-PEGylated nano drug delivery system according to claim 1, wherein: In step S2, the MDM2-targeting peptide is Mtrap-2.

4. The preparation method of an in-situ de-PEGylated nano-drug delivery system according to claim 1, characterized in that: In step S3, the molar ratio of methanol to chloroform is 1:(4 - 5).

5. The preparation method of an in-situ de-PEGylated nano-drug delivery system according to claim 1, wherein: In step S3, the mass of both doxorubicin and arsenic trioxide accounts for 0 - 50% of the total mass of doxorubicin, arsenic trioxide, and the product of step S2, and the sum of the masses of doxorubicin and arsenic trioxide does not exceed 50% of the total mass of doxorubicin, arsenic trioxide, and the product of step S2.

6. The preparation method of an in-situ de-PEGylated nano drug delivery system according to claim 1, wherein: In step S3, the rotary evaporation temperature is 30 - 50 °C.

7. An in-situ de-PEGylated nano-drug delivery system prepared by the preparation method according to any one of claims 1 - 6.

8. Use of the in-situ de-PEGylated nano-drug delivery system according to claim 7 in the preparation of anti-tumor products.

Images