Novel esterase-responsive polypeptide vesicle drug delivery system

Abstract

The invention provides a compound as shown in a figure (1), under the action of esterase, the permeability of vesicles of the compound is remarkably improved, drug release is accelerated, meanwhile, lysine side groups are cross-linked with other modified side groups through an amide reaction, the vesicle structure is maintained, rapid drug release is avoided, and the drug release rate is increased. Therefore, the toxic and side effects on adjacent normal cells are reduced. The stimuli-responsive drug delivery material has higher biological safety, and provides a new thought for designing safe and efficient anti-cancer drugs.

Description

Technical Field

[0001] The present invention relates to an esterase-responsive polyamino acid nano drug delivery system. Specifically, under the action of esterase, the permeability of the vesicle is significantly improved to accelerate the release of the drug. At the same time, the lysine side groups are cross-linked with other modified side groups through amide reaction, maintaining the vesicle structure, avoiding the rapid release of the drug, and thus reducing the toxic side effects on adjacent normal cells. This stimulus-responsive drug delivery material has higher biosafety. The technology belongs to the field of nanomaterials. Background Art

[0002] Polypeptide-based nanoassemblies have shown great application potential in drug delivery systems due to their excellent biocompatibility and biodegradability. These properties enable them to effectively deliver drugs to target sites without affecting the biological system, and gradually degrade in the body, reducing the potential risk of long-term toxicity. In recent years, peptide nanoassemblies loaded with paclitaxel (NK105), doxorubicin (DOX, NK911) and cisplatin (NC6004) have entered Phase II and Phase III clinical trials, indicating that drug delivery systems based on polypeptides are gradually moving towards clinical application.

[0003] Among the many peptide nanoassemblies, peptide vesicles, as a spherical nanostructure, are particularly eye-catching. They can not only load hydrophilic and hydrophobic drugs, but also continuously release drugs after reaching the target site, thereby achieving long-term treatment. Compared with lipid vesicles, peptide vesicles show advantages in stability and toughness, which makes them more suitable as carriers for drug delivery.

[0004] However, the challenges of drug release rate of peptide vesicles cannot be ignored. Their release rate is usually slow, which may reduce the efficacy of drugs. To overcome this problem, researchers have developed stimulus-responsive peptide vesicles. These vesicles are not only sensitive to external stimuli (such as temperature, light or ultrasound), but also respond to physiological or pathological changes in the diseased site (such as enzymes, pH, redox or hypoxia, etc.), thereby achieving triggered drug release. Among them, enzyme-responsive co-peptide vesicles have attracted much attention due to their highly selective enzymatic reaction properties. These peptide vesicles are able to adopt specific conformations (such as α-helices and β-sheets) to self-assemble into biomimetic structures, which undergo conformational changes or phase transitions under the action of enzymes, thereby triggering drug release. However, compared with chemical stimulus-responsive polymer vesicles, enzyme-responsive peptide vesicles have a slower stimulus response to drug release, mainly because the diffusion of enzymes into solid polymer vesicles is a rate-limiting step. In addition, the enzyme may be consumed during the reaction, further limiting the rapid release of free drugs and the maintenance of effective concentrations.

[0005] Nevertheless, once the enzyme reaction is fast enough, the polypeptide vesicles can achieve exclusive rupture, releasing almost all the encapsulated drugs. These released drugs can induce apoptosis of cancer cells and package cancer cells into small bag membranes for "garbage recycling" through phagocytosis. However, this sudden release of free drugs is more likely to penetrate the membranes of dead or dying cells and the extracellular matrix of tumor cells, which may increase the risk of acute toxicity to neighboring cells.

[0006] In order to balance the contradiction between rapid enzyme reaction and burst release induced by rupture, researchers are exploring new strategies. Recently, the development of chemical cross-linking methods has provided new possibilities for regulating the stability of nanostructures and the transport of drug molecules. Through carefully designed chemical cross-linking, the structure and properties of polypeptide vesicles can be regulated, thereby achieving more precise drug release and reducing the risk of toxicity to neighboring cells. These advances provide new directions for solving the challenges of polypeptide vesicles in drug delivery systems. Summary of the invention

[0007] PEG-b-PLLNA self-assembles into nanovesicles in aqueous solution, which can generate unstable phenolic substances under the action of esterase, and then undergo elimination reaction to expose the amino group of lysine. At this time, the hydrophilicity of the vesicle is greatly increased, which improves the permeability and allows the drug to be released. On the other hand, the exposed amino group undergoes amidation reaction with other undeprotected lysine side groups to form a cross-linking system, which maintains the stability of the vesicle structure, which effectively avoids the collapse caused by the change of vesicle hydrophilicity and rapid release of drugs, thereby reducing the impact on neighboring cells. At the same time, the block co-peptide was characterized by spectral data and tandem gel permeation chromatography (GPC), the drug loading capacity (DLC) and drug loading efficiency (DLE) were determined by UV-visible absorption spectroscopy, and the cell fluorescence was observed by spectral confocal microscopy (Nikon C2+). Through in vivo and in vitro experiments in mice, the optical images of mouse ex vivo tumor sections were recorded, which proved the anti-cancer effect of PEG-b-PLLNA polypeptide vesicles loaded with DOX. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Structural design and esterase response mechanism of PEG-b-PLLNA vesicles;

[0009] Figure 2 1HNMR spectra of PEG-b-PLL in D2O and PEG-b-PLLNA in CDCl3 (a); FT-IR spectra of PEG-b-PLL and PEG-b-PLLNA (b);

[0010] Figure 3 TEM image of the vesicle (a), DLS measurement results (b), CMC (c) and circular dichroism results (d);

[0011] Figure 4 Polymer before and after esterase 1 H NMR spectra (a), release curves of Nile Red (b) and DOX (c);

[0012] Figure 5 Esterase reaction in L929 fibroblasts and HeLa cells. Confocal fluorescence images of DOX-loaded peptide vesicles. Cell nuclei were stained with Hoechst 33342 (blue).

[0013] Figure 6 Confocal fluorescence images of DOX-loaded vesicles after esterase reaction in L929 fibroblasts and HeLa cells. Lysosomes were stained with LysoTracker (shown in green);

[0014] Figure 7 The killing effects of DOX and vesicles on HeLa and L929 cells;

[0015] Figure 8 (a) Representative photos of tumor-bearing mice treated with PBS, free DOX, or DOX-loaded polypeptide vesicles for 27 days, with tumors represented by ovals. (b) Changes in tumor volume and (c) weight of mice in each group. (d) Representative optical images of ex vivo tumor sections stained with H&E. DETAILED DESCRIPTION

[0016] The methods and techniques of the present invention are generally performed according to conventional methods known in the art, unless otherwise indicated. The nomenclature, experimental methods and techniques related to biology, pharmacology, medicine and medicinal chemistry described herein are those known and commonly used in the art. Chemical syntheses, chemical analyses, pharmaceutical preparations, formulations and delivery methods, and detection or testing methods all employ standard techniques.

[0017] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by one of ordinary skill in the art.

[0018] Examples of the compounds of the present invention are the following compounds I:

[0019]

[0020] like Figure 1 As shown, the synthesis of compound I comprises the following steps:

[0021] 1) Synthesis of Compound 2: Acetic anhydride (10.9 ml) was added to a mixed solution of 4-hydroxybenzyl alcohol (compound 1, 19.75 g) and trimethylamine (7.7 mL) in THF (250 mL) under N2 atmosphere at 0°C for more than 5 minutes. The reaction was stirred at room temperature overnight and the solvent was removed by rotary evaporation. The oily product was dissolved in ethyl acetate, washed with brine, and dried over MgSO4. It was purified by flash column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain 21.0 g (yield 79.0%) of compound 2 as a transparent oily liquid. 1HNMR (400 MHz, CDCl3): δ (ppm): 7.36 (d, J = 6.8 Hz, 1H), 7.06 (d, J = 6.8 Hz, 1H), 4.65 (d, J = 4 Hz, 2H), 2.30 (s, 3H).

[0022] 2) Synthesis of compound 4: A solution of 4-(hydroxymethyl)phenyl acetate (compound 2, 7.38 g) and trimethylamine (7.39 mL) in anhydrous acetonitrile (60 mL), 10.74 g of 4-nitrobenzene carbonyl chloride (compound 3) was added with a constant pressure funnel and dissolved in 30 ml of anhydrous acetonitrile. The reaction was monitored by thin layer chromatography. After the reaction was completed, the solvent was removed under vacuum. The crude product was dissolved in a minimum amount of ethyl acetate, washed three times with ice water, and dried over MgSO4. A yellow solid was obtained by recrystallization from petroleum ether with a yield of 10.96 g (74.5%). 1HNMR (400 Hz, CDCl3): δ (ppm): 8.28 (dJ

[0023] =7.6Hz, 2H), 7.47 (d J = 6.8Hz, 2H), 7.33 (d J = 7.6Hz, 2H), 7.13 (d J = 6.8Hz, 2H), 5.28 (s, 2H), 2.32 (s, 3H).

[0024] 3) Synthesis of PEG-b-PLLN block copolymer: PEG-b-PLLNA co-peptide was prepared by post-modification of preformed peptide (PEG-b-PLL). Triethylamine was added to PEG-b-PLL (250 mg) and 4-

[0025] To a solution of ((((4-nitrophenoxycarbonyloxymethylphenylacetate (compound 4, 63 mg) was added 15 mL of anhydrous DMF and stirred for 2 days. The resulting polypeptide was precipitated with ether and dried in vacuo (250 mg, yield: 52.1%).

[0026] The chemical structure of the co-peptide synthesized simultaneously was confirmed by 1HNMR and FT-IR spectroscopy ( Figure 2-ab). The new peaks at 6.9, 5.1, and 2.3 ppm correspond to the protons of the LLNA moiety, indicating that 4-acetoxybenzyl carbamate was successfully grafted onto the amine of PEG-b-PLL. The FT-IR spectrum showed that the peaks at 1715 and 1456 cm -1 The characteristic peaks of νN-COO- and ν4-acetoxybenzyl of PLLNA are shown in the figure. By comparing the integral of the 4-acetoxybenzyl methyl peak (f, CH3-COO-) and the polyethylene glycol methylene peak (e, -CH2-CH2-), the amine of PEG-b-PLL was nearly quantitatively converted to LLNA. PEG-b-PLLNA vesicle formation and characterization

[0027] To prepare vesicles, 10 mg of the co-peptide was dissolved in 2 mL of THF and stirred for 3 h. An equal volume of deionized water was added using a microsyringe at a rate of 1 mL / h. The mixture was stirred at room temperature until most of the THF evaporated. Then, the mixture was placed in a dialysis bag (MWCO = 2000 Da) and dialyzed against deionized water for 24 h. The deionized water was replaced every 4 hours, and the resulting suspension was then removed from the dye or DOX premixed in THF or deionized water to prepare DOX-loaded vesicles. Characterization: The PEG-b-PLLNA co-peptide was first dissolved in THF, and then an equal volume of water was added, which was then dialyzed against the water. TEM observations confirmed that the PEG-b-PLLNA co-peptide could self-assemble into vesicles by the solvent switch method ( Figure 3 -a) During this process, THF diffuses rapidly into the aqueous phase, causing the hydrophobic chains to aggregate and drive the assembly process. After removing THF, DLS analysis showed that the hydrodynamic radius value of the blank vesicles (0.2 mg / mL) was 49.8 ± 207 nm ( Figure 3 -b), which is in good agreement with the TEM observation value (9.8-58.9nm). Over time, the vesicles formed a stable suspension without aggregation or precipitation. The CMC of the co-peptide was determined by fluorescence spectroscopy using lipophilic Nile red as a probe. The CMC value of PEG-b-PLLNA was calculated to be approximately 20μg / mL ( Figure 3 -c). We further use CD spectroscopy ( Figure 3 -d) The conformation of PEG-b-PLLNA (0.5 mg / mL) in aqueous solution was studied. Figure 3 -d), showing a classic α-helical conformation with double negative peaks at 208 and 222 nm. This indicates that the secondary structure of the peptide is affected by the side chain structure. After esterase treatment, the initial CD pattern of the peptide almost disappears ( Figure 3 -d), indicating a conformational change from helical to disordered. The unwinding of the α-helix is ​​considered a prerequisite for regulating cell membrane permeability.

[0028] Esterase responsiveness of poly(peptide) vesicles

[0029] First, the enzymatic reactivity of the poly(peptide) vesicles was measured by incubating with pig liver esterase (10 U / mL) at 37°C for 8 h. A 1 mL sample was taken from the solution and analyzed by spectrometer. The solution was freeze-dried and the resulting dry powder was diluted in D2O for 1H NMR measurement ( Figure 4 -a). The growth peaks (δ: 4.56, 2H; 6.92, 2H; 7.31, 2H) can be attributed to the by-product 4-hydroxybenzyl alcohol from the self-cleavage reaction.

[0030] The enzymatic reactivity of the polypeptide vesicles was studied. The vesicles containing Nile red were diluted in a PBS solution (pH 7.4) containing 0.05 (wt%) NaN3, and the final vesicle concentration was adjusted to 200 μg / mL. Esterase stock solution (150 units / mL) was added to the solution below 37°C, and the fluorescence changes of the polypeptide solution at 620nm were observed over time. DLS analysis was performed every 24 hours to monitor the changes in vesicle size in the esterase reaction ( Figure 4 -b).

[0031] In vitro release profile

[0032] The in vitro release behavior of DOX in polypeptide vesicles was evaluated by dialysis using PBS (pH 7.4) as the release medium. Briefly, 2 ml of DOX-loaded polypeptide preparation (0.2 mg / mL) was added to a dialysis tube (MWCO 2000 Da) with or without 4 μL of esterase. The mixture was fully immersed in PBS (pH 7.4) at 37°C and continuously shaken. At predetermined time points, the external buffer solution was removed and replaced with fresh culture medium. Figure 4 The release of DOX was quantified by measuring the fluorescence at an emission wavelength of 593 nm (excitation wavelength of 480 nm) on a standard curve (Fig.

[0033] Cell culture and in vitro cytotoxicity assays

[0034] HeLa (human cervical cancer cells) and L929 (mouse fibroblasts) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin in an environment of 5% CO2 and 95% air at 37°C. To characterize the interaction between cells and DOX-loaded vesicles, HeLa and L929 cells were incubated with DOX-loaded vesicles for 4 hours. The cells were washed twice with PBS buffer, treated with Hoechst 33342 and LysoTracker Green for 15 minutes, and then analyzed by confocal microscopy ( Figure 5-6 ).

[0035] The in vitro cytotoxicity of DOX-loaded vesicles on HeLa and L929 cells was evaluated by MTT assay and live / dead staining. Briefly, 1*104 cells were seeded in 96-well plates and maintained in 100 μL DMEM supplemented with 10% fetal bovine serum and 1% antibiotics. After 24 h, the cells were treated with new medium containing different concentrations (3.2, 6.4, 12.8, 25.6 mg / L DOX equivalents) of free DOX or DOX-loaded polypeptide vesicles for 24 h, and the drug-containing medium was discarded and replaced with 100 μL serum-free medium. Then, MTT reagent (20 μL) was added to each well and incubated with the cells for 4 h. The medium was removed and 100 μL DMSO was added. The absorbance of the solution at 492 nm was measured on a Tecan Infinite 200PRO microplate reader. Each experimental condition was divided into four groups, and the data were expressed as the mean plus standard deviation (± SD). To further observe cell viability, cells treated with free DOX or peptide vesicles were stained with the LIVE / DEAD detection kit. Fluorescence imaging of cells was assessed and recorded using an inverted fluorescence microscope ( Figure 7 ).

[0036] In vivo antitumor efficacy

[0037] All animal experiments were approved by the Shandong Provincial Science and Technology Department and the Experimental Animal Center of Qingdao Hao Bioengineering Co., Ltd. HeLa cells (3*106 cells, PBS 100μL) were injected subcutaneously on the left side of the mouse to establish a human cervical cancer cell xenograft tumor model. When the tumor volume reached 30mm 3 The mice were randomly divided into 3 groups and injected with PBS (group 1), free DOX (5 mg / kg, group 2) and DOX-loaded polypeptide vesicles (5 mg / kg DOX, group 3) through the tail vein on days 0, 4, 8 and 12, respectively. The tumor status of the mice was recorded every 2 days ( Figure 8 -a). At the same time, the tumor volume and body weight should be measured every 2 days to evaluate the anti-tumor activity and systemic toxicity of the vesicles. According to the formula (V = (a × b 2 / 2)), calculate the tumor volume ( Figure 8 -bc), where a and b represent the longest and shortest diameters of the tumor, respectively. On day 27, all mice died, and the tumors were photographed and recorded, then embedded in formalin. 5 μm thick sections were placed on polylysine-coated slides and stained with hematoxylin and eosin (H&E), and optical images of mouse ex vivo tumor sections were observed under a microscope ( Figure 8 -d).

[0038] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An application of an esterase-responsive polypeptide vesicle as a nano drug delivery system, characterized in that: The vesicles are designed through an enzyme-regulated cross-linking strategy, which can achieve rapid, sustained and controllable release of drugs in a specific esterase environment.

2. The esterase-responsive polypeptide vesicle according to claim 1, characterized in that The cross-linked structure of the vesicle contains esterase-sensitive groups. When the vesicle enters the interior of cells with high cytoplasmic esterase expression, the esterase can act on specific groups on the surface of the vesicle, causing the vesicle structure to change, thereby releasing the drug.

3. The esterase-responsive polypeptide vesicle according to claim 1 or 2, characterized in that: The vesicle maintains a stable structure in normal cells with low esterase activity and does not release drugs, thereby reducing side effects on normal cells.

4. The esterase-responsive polypeptide vesicle according to any of the above claims, characterized in that Its size is at the nano level and it can easily pass through cell membranes and tissue gaps to achieve deep penetration of drugs.

5. The esterase-responsive polypeptide vesicle according to any of the above claims, characterized in that The vesicles are biodegradable and can be naturally metabolized by the body after completing the drug delivery mission.

6. A drug delivery system, comprising the esterase-responsive polypeptide vesicles according to any one of claims 1 to 5, and a drug loaded in the vesicles, wherein the system can be used for cancer treatment to achieve precise delivery and controlled release of the drug.

7. The drug delivery system according to claim 6, characterized in that The system may also include one or more auxiliary components for monitoring drug release and / or vesicle distribution.

8. A drug delivery system according to any preceding claim, characterized in that The system also includes one or more external stimuli for regulating the rate of drug release.

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