Preparation method of negatively-charged melittin nanocapsule targeting tumor microenvironment

Abstract

The invention discloses a preparation method of negatively-charged melittin nanocapsules of a targeted tumor microenvironment, and belongs to the field of targeted tumor drugs. The preparation method comprises the following steps: enriching a monomer and a cross-linking agent around melittin by using hydrophobic interaction force and hydrogen-bond interaction force of melittin, adding phenylboronic acid capable of targeting a sialic acid receptor on the surface of a tumor cell, and then adding an initiator to induce polymerization of the monomer and the cross-linking agent, so as to form a cross-linked network to encapsulate melittin; the tumor targeting property of the medicine after systemic circulation is greatly improved, and the toxic and side effects of the medicine on normal tissues are reduced. The in-vivo circulation time of melittin is greatly prolonged.

Description

Technical Field

[0001] The invention relates to a method for preparing negatively charged melittin nanocapsules capable of targeting tumor cells, and belongs to the field of tumor targeting drugs. Background Art

[0002] Melittin (MEL) is a bioactive substance extracted from the venom of honey bees (Apis mellifera). It is the component with the highest content in bee venom, accounting for about 40%-60% of the total protein in bee venom. As a small molecule peptide, melittin is composed of 26 amino acids. It can achieve effective anti-tumor effects by destroying tumor cell membranes, inhibiting tumor cell growth and division, inducing tumor cell apoptosis, inhibiting tumor angiogenesis, and regulating immune responses. Among them, the physical destruction of the tumor cell membrane structure is the basis for melittin to treat tumors, and cancer cells will not develop resistance to membrane pores. So far, studies have found that melittin can inhibit the proliferation of various types of cancer cells such as breast cancer, lung cancer and melanoma. Melittin, which has no drug resistance, has good prospects as an anticancer agent in preclinical studies.

[0003] As a toxic substance, melittin has the main mechanism of action of destroying cell membranes, so it has effects on both normal cells and tumor cells, which may cause serious side effects. Therefore, how to ensure that melittin can accurately target tumor tissues after undergoing systemic circulation, rather than being dispersed throughout the body, is a difficult problem that needs to be solved urgently for the current clinical application of melittin anti-cancer. In order to achieve the purpose of targeted and efficient treatment of tumors with melittin and reduce the possible toxic side effects of melittin on normal tissues of organisms, it is necessary to prepare suitable nano drug delivery carriers. Liposomes, cell penetrating peptides, cationic polymers, zwitterionic polymers, extracellular vesicles and other drug delivery carriers have been widely used in the field of anticancer drugs. Among them, amphiphilic ionic polymers, as an emerging nanomaterial, have made important contributions to the realization of long circulation of anticancer drugs in vivo. Amphiphilic ionic polymers such as polyethylene glycol, polymethacrylate, polylactic acid-co-glycolic acid, polysuccinic acid-glycerate have been used to meet different types of drug delivery needs. They can stably encapsulate drugs in vivo and achieve controlled release by changing chemical structure and composition.

[0004] In addition, drug targeting is also a key factor affecting the efficacy of anticancer treatment. Receptor antagonists, RGD groups, deoxyglucose groups, boric acid groups, epidermal growth factor receptors, insulin-like growth factor-1 receptors, tumor necrosis factor-related apoptosis-inducing ligands, etc. can all be used for targeted modification of anticancer drugs. One of the prominent characteristics of cancer is abnormal cell surface glycosylation. The cell surface sialylation, that is, the sialic acid content is higher than that of normal tissues, and the modified structure changes significantly. Phenylboronic acid (PBA) derivatives are synthetic mimics of lectins that can specifically recognize sialic acid residues on the surface of tumors. Some studies have reported that under the conditions of the acidic microenvironment of tumors, phenylboronic acid-functionalized anticancer drug delivery carriers can efficiently bind to sialic acid residues on the surface of tumor cells, significantly improving the biosafety, targeting and therapeutic effects of nanomedicines.

[0005] Based on the existing research data, how to reduce the biological toxic side effects of bee venom peptide during systemic circulation and enhance its targeting effect at tumor lesions remains an important problem for the clinical transformation of bee venom peptide as an anticancer drug. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing negatively charged melittin nanocapsules (nMEL) targeting tumor cells.

[0007] The present invention is based on an in situ free radical polymerization method, which utilizes the hydrophobic force and hydrogen bonding force of melittin to enrich monomers and cross-linking agents around melittin, and simultaneously adds phenylboronic acid that can target sialic acid receptors on the surface of tumor cells, and then adds an initiator to induce the polymerization of the monomers and the cross-linking agent to form a cross-linked network to encapsulate melittin, thereby greatly improving the tumor targeting of the drug after systemic circulation and reducing its toxic side effects on normal tissues.

[0008] The method for preparing melittin nanocapsules with negative charge and targeting tumor cells is characterized in that melittin, 2-methacryloyloxyethyl phosphorylcholine, 2-acrylamide-2-methylpropanesulfonic acid, N,N-bis(acryl)cystamine, and 3-acrylamidephenylboronic acid are added into a reactor, and after stirring, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine are added to initiate free radical in-situ polymerization to form an encapsulation shell around melittin, and the mixture is reacted in an ice bath for 2 hours or placed in a refrigerator at 4 degrees for 12 hours to obtain melittin nanocapsules with negative charge and targeting tumor cells.

[0009] 2-Methacryloyloxyethylphosphorylcholine (MPC) and 2-acrylamide-2-methylpropanesulfonic acid (AMPS) were added in the form of being dissolved in phosphate buffered saline (PBS), and N,N-bis(acryl)cystamine (BAC) and 3-acrylamidophenylboronic acid (AAPBA) were added in the form of being dissolved in ultra-dry dimethyl sulfoxide (DMSO).

[0010] The molar ratio of melittin (MEL), 2-methacryloyloxyethyl phosphorylcholine (MPC), 2-acrylamide-2-methylpropanesulfonic acid (AMPS), N,N-bis(acryl)cystamine (BAC) and 3-acrylamidophenylboronic acid (AAPBA) is 1:200-600:40-200:50:40, preferably 1:400:40:50:40; the molar ratio of melittin (MEL) to free radical initiator ammonium persulfate (APS) and catalyst N,N,N',N'-tetramethylethylenediamine (TEMED) is 1:50:100.

[0011] The monomer selected is 2-methacryloyloxyethyl phosphorylcholine (MPC), which includes a hydrophilic phosphorylcholine head group and a hydrophilic methacryloyl group. It is usually used in the preparation of coatings, nanoparticles and biomedical materials to improve their biocompatibility and anti-biological adhesion, especially for situations where adverse reactions when in contact with organisms need to be reduced. By adding a small amount of 2-acrylamide-2-methylpropanesulfonic acid (AMPS) monomer, the encapsulation rate of the monoclonal antibody is increased and the polymer shell is made negatively charged. The phenylboronic acid selected is 3-acrylamidophenylboronic acid (AAPBA), which can be modified to the nanocapsule under the action of an initiator through double bond action. The cross-linking agent selected is N,N-bis(acryloyl)cystamine (BAC), which can respond to the degradation of excessive glutathione (GSH) in the tumor microenvironment, and realize the release of bee venom peptide at the lesion site and the isolation at the normal tissue site.

[0012] The capsule encapsulates the highly toxic melittin and modifies the surface with phenylboronic acid groups that target the sialic acid receptors on the surface of tumor cells, enhancing its anti-tumor effect, improving its biocompatibility and anti-adhesion, reducing toxicity to normal tissues, and greatly increasing the circulation time of melittin in the body. MEL mainly accumulates in organs such as the liver and kidneys, with relatively few or no tumor sites. Under the same conditions, nMEL mainly accumulates in tumor sites and less in organs such as the liver and kidneys. In other words, nMEL has good targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the synthesis of bee venom peptide nanocapsules

[0014] Figure 2 The standard curve of bee venom peptide UV absorbance and concentration

[0015] Figure 3 Agarose gel electrophoresis analysis of melittin nanocapsules

[0016] Figure 4 Zeta potential analysis of melittin nanocapsules

[0017] Figure 5 Circular dichroism analysis of melittin nanocapsules

[0018] Figure 6 Analysis of cytotoxicity results of beet venom peptide nanocapsules

[0019] Figure 7 Live imaging of mice after injection of MEL, nBSA and nMEL (yellow circles represent tumor areas)

[0020] Figure 8 Fluorescence images of tissue distribution of MEL (A) and nMEL (B), and MEL (C) and nMEL (D)

[0021] In various organizations

[0022] Fluorescence quantification in. He, Li, Sp, Lu, Ki and Tu represent heart, liver, spleen, lung, kidney and tumor respectively.

[0023] Unless otherwise specified, the methods in the examples are all conventional methods.

[0024] Melittin (MEL) was purchased from Chengdu Yunxi Chemical Co., Ltd., 2-acrylamide-2-methylpropanesulfonic acid (AMPS) was purchased from Tianjin Xiensi Biochemical Technology Co., Ltd., and the remaining reagents were purchased from Sigma-Aldrich.

[0025] 1. Synthesis and purification of melittin nanocapsules

[0026] Melittin (MEL) was encapsulated to form negatively charged, targetable nanocapsules through in-situ polymerization of monomers (MPC, AMPS) and crosslinkers (BAC), as well as double bond modification of phenylboronic acid (AAPBA) on the nanocapsules. Before synthesis, the required reagents were prepared into stock solutions of certain concentrations (MPC, 400 mg / mL dissolved in phosphate buffer (PBS), AMPS 100 mg / mL dissolved in PBS, BAC 100 mg / mL dissolved in ultra-dry dimethyl sulfoxide (DMSO), AAPBA 100 mg / mL dissolved in DMSO). Among them, the molar ratio of MEL, MPC, and AMPS in the range of 1:200:40-1:600:200 can achieve encapsulation, and BAC and APS are added at a molar ratio of 12.5% ​​of MPC. Among them, the encapsulation effect is best in the ratio shown in Table 1. Therefore, the required reagents are added to the reaction tube in sequence according to the ratio shown in Table 1, and then ammonium persulfate (APS, 100 mg / mL dissolved in PBS, with a molar ratio of 50:1 to MEL) and N, N, N', N'-tetramethylethylenediamine (TEMED, with a mass ratio of 2:1 to APS) are added to initiate free radical polymerization on the surface of MEL, and PBS is added to make up the required volume. The preparation system in the present invention is MEL1mg / ml. The reaction is carried out in an ice bath for 2 hours, or placed in a refrigerator at 4 degrees for 12 hours, and then the sample is purified to remove unreacted free monomers, initiators and unencapsulated MEL. The sample is added to a 50,000 molecular weight ultrafiltration centrifuge tube and centrifuged at 4°C and 5000r / min for 20 minutes. Then, an appropriate amount of PBS is added to restore the original volume and continue centrifugation. After repeating 5 times, purification is completed to obtain nMEL. See the schematic diagram of the synthesis. Figure 1 .

[0027] Table 1 Synthesis ratio of bee venom peptide nanocapsules

[0028]

[0029] 2. Determination of the encapsulation efficiency of bee venom peptide nanocapsules

[0030] The encapsulation efficiency was determined by the BCA protein concentration determination method. First, MEL was dissolved into concentrations of 0.25, 0.5, 0.75, and 1 mg / mL to prepare a standard curve, and then the sample to be tested was diluted to within the standard curve range (0-1 mg / mL). Then, the Cu reagent and the BCA reagent were mixed at a ratio of 1:50 to prepare the BCA working solution. Then, the sample and the BCA working solution were mixed at a ratio of 1:10, incubated in a constant temperature metal bath at 60°C for 15 minutes, and then the absorbance values ​​of the sample to be tested and the standard curve were measured at 562nm using nanodrop.

[0031] Encapsulation efficiency of bee venom peptide (%) = m1 / m0×100%

[0032] Among them, m1 is the mass of melittin loaded into the nanocapsule, and m0 is the mass of melittin fed.

[0033] Therefore, a standard curve was drawn with different concentrations of MEL, and the encapsulation efficiency of nMEL was determined based on the standard curve. Figure 2 As shown, the formula of MEL standard curve is: Y = 2.440X-0.03067, and the R 2 =0.9981. The absorbance of the sample nMEL was measured, and the concentration of nMEL was calculated using the MEL standard curve equation. The encapsulation efficiency of MEL was calculated to be 60.2% according to the encapsulation efficiency formula.

[0034] 3. Agarose gel electrophoresis

[0035] To confirm that bee venom peptide nanocapsules are negatively charged, bee venom peptide was labeled with fluorescein isothiocyanate (FITC), and then bee venom peptide nanocapsules were prepared using the same method, followed by agarose gel electrophoresis test. Melittin and FITC (molar ratio of 1:1) were mixed in PBS solution and reacted at room temperature for 2 hours, with a bee venom peptide concentration of 20 mg / mL. Subsequently, 1% agarose gel was prepared, 300 μL of sample was added to each well and electrophoresis analysis was performed in 1×TAE solution at a voltage of 120 V for 15 minutes, and then the agarose gel was exposed to ultraviolet light (excitation wavelength 488 nm, emission wavelength 520 nm) to capture the electrophoresis image. Figure 3 As shown, band ① is negatively charged MEL, and band ② is negatively charged nMEL. Because the molecular weight increases after nanocapsule encapsulation, although band ① and band ② both move toward the electrophoresis positive electrode, band ① moves a longer distance due to the smaller molecular weight of MEL, while band ② moves a very short distance due to the larger molecular weight of nMEL, which proves the successful preparation of nMEL.

[0036] 4. Zeta potential

[0037] The bee venom peptide nanocapsules were diluted with PB to a final concentration of 100 μg / mL, and then the potential of the nanocapsules was detected using a ZETAPALS / BI-200SM instrument produced by Brook Heaven, USA. The results are shown in Figure 4 As shown, it further proves that the prepared melittin nanocapsules are negatively charged.

[0038] 5. Circular dichroism analysis

[0039] In order to verify that the encapsulation process did not change the secondary structure of melittin, that is, the encapsulated melittin still had an α-helical structure, the circular dichroism spectrum was measured. 1 mL of MEL and nMEL solution were prepared, and the circular dichroism spectra (CD) of the samples at 200-260 nm were measured using a J815 circular dichroism spectrometer produced by JASCO. The results are shown in Figure 2. Figure 5 Both MEL and nMEL have negative peaks at 208nm and 222nm, that is, both MEL and nMEL have α-helical structures, indicating that nMEL does not destroy the α-helical structure of melittin after encapsulation, and still ensures its biological activity.

[0040] VI. Cytotoxicity Analysis

[0041] The purpose of this analysis is to verify the biosafety of melittin nanocapsules and the killing effect of melittin on tumor cells through CCK-8 cytotoxicity assay. This assay is based on evaluating the metabolic activity of cells by measuring the absorbance of the orange product produced by the reduction reaction of CCK8 reagent, thereby determining the effect of compounds or drugs on cells.

[0042] The specific operation process is described as follows. In the cell culture stage, mouse breast cancer cell line 4T1 cells were cultured until the cell density reached 80%-90%, and then the cells were digested and counted. The cell solution was diluted to 10 / ml using DMEM complete medium. 5 The cells were plated and evenly distributed using a pipette. The cell suspension was then added to a 96-well plate, 100 μL per well. 100 μL of 1× PBS solution was added to each well around the periphery of the 96-well plate to maintain the cell growth environment. The 96-well plate with cells was placed in an incubator and cultured for 24 hours.

[0043] During the drug addition phase, serum-free DMEM medium was prepared using 99% DMEM medium and 1% PS. The nMEL, MEL, and nBSA samples were diluted to different concentrations, 3.13, 6.25, 12.5, 25, 50, and 100 μg / mL, respectively. Among them, in order to simulate the microenvironment of the tumor site, GSH was added to the corresponding samples; in order to evaluate the effect of the nanoshell material on cell activity, inactive BSA model protein nanocapsules (nBSA) were added as blank controls. Finally, the experiment was divided into four groups: nMEL with added GSH (i.e., nMELw / GSH), nMEL without added GSH (i.e., nMEL w / oGSH), nBSA, and MEL.

[0044] During the cytotoxicity detection phase, discard the old culture medium in the 96-well plate, add sample solutions of different concentrations to the well plate, and add three replicate wells for each sample concentration, 100 μL per well. The 96-well plate after administration was placed in an incubator and cultured for 12 hours. Prepare the CCK-8 working solution according to the CCK8 reagent instructions, and add the corresponding drugs to the 96-well plate according to the standard process indicated in the instructions. After placing it in the incubator for 4 hours, use an enzyme reader to measure the absorbance value at a wavelength of 450 nm. The cell survival rate was evaluated by the following formula:

[0045]

[0046] Wherein, Oda is the absorbance of the well with cells, OD is the absorbance of the well without cells, and ODc is the absorbance of the well with cells only without serum medium.

[0047] The cell survival rate and cell activity were detected by the above experiment. Figure 6 As shown, the nMEL w / oGSH group is the group of bee venom peptide nanocapsules incubated without GSH, the nMEL w / GSH group is the group of bee venom peptide nanocapsules incubated with GSH, and the other two groups are as explained in the initial experiment. It can be seen that free MEL has very strong cytotoxicity. At a concentration of only 3.13μg / mL, the survival rate of 4T1 cells is less than 50%; when the concentration of nMEL with GSH added reaches 12.5μg / mL, the cell survival rate is less than 50%; in contrast, even at a concentration of 100μg / mL for nMEL without GSH, the cell survival rate is still very high. This result not only shows the good in vitro cytotoxicity of nMEL, but also the significant difference in cytotoxicity between those with and without GSH, which indirectly illustrates the responsive release of nMEL. At the same time, in order to prove that the cytotoxicity of nMEL is not caused by the encapsulated nanoshell material, the nanocapsule nBSA encapsulating the inactive model protein BS was used as a blank and the cytotoxicity test was performed simultaneously with nMEL. The results showed that nBSA did not affect the cell activity, indicating that the cytotoxicity of nMEL comes from the toxicity of the released MEL, and its own biosafety is good.

[0048] VII. Drug Targeting Analysis

[0049] The targeting effect of nMEL was verified by tumor-bearing mouse model.

[0050] Fifteen tumor-bearing mice were randomly divided into three groups, with 5 mice in each group, and were used to evaluate the tumor targeting of nMEL. First, the animal model was established. All animal experiments were conducted in compliance with the principles of the Animal Ethics Committee of Beijing University of Chemical Technology. The feeding and care of the animals were carried out in accordance with the guidelines. The animal model was balb / c mice bearing 4T1 subcutaneous tumors. First, the purchased balb / c mice (6-8 weeks) were normally maintained for 3-5 days. The bedding of the mice was changed every two days, and the mice were kept with sufficient food and pure water every day. The day before the experiment, the hair on the back of the mouse was removed with a shaver and depilatory cream, and the depilatory area was cleaned with a cotton ball dipped in warm water to avoid the residue of depilatory cream. The 4T1 cells were digested from the culture flask with trypsin and diluted to 5×10 per ml with complete culture medium. 7 After dilution, the solution was placed in a centrifuge tube and placed on ice. Aspirate the fully mixed cell solution with a 1mL sterile syringe, and then inject 100μL subcutaneously into the back of the mouse. Observe the growth of the mouse tumor every day. When the tumor grows to 100mm 3 Experiment with around.

[0051] Subsequently, drug injection was performed, and MEL, nBSA, and nMEL solutions labeled with anthocyanin fluorescent dye Cy5.5 (Cyanine 5.5) were injected into the body of tumor-bearing mice through the tail vein. The drug concentration was 0.5 mg / mL, filtered with a 0.22 μm filter membrane before injection, and the injection dose was 2.5 mg / kg. The time points after injection included 0.5 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 48 hours, and then the tumor-bearing mice were imaged in vivo at different time points after injection. The mice were anesthetized with isoflurane before imaging, and the mice were imaged after anesthesia. The excitation and emission wavelengths were both 710 nm to ensure consistency.

[0052] After the 48-hour in vivo imaging experiment, the mice were anesthetized and the five major organs and tumors of the heart, liver, spleen, lungs and kidneys were removed. The mice were anesthetized and their limbs were fixed before removal. The mice were under anesthesia during the entire removal operation. In order to avoid the influence of blood in each organ on the quantitative tissue drug, each mouse was perfused with 50 mL of normal saline to ensure that there was no or very little blood residual in each organ. After removing the major organs (heart, liver, spleen, lungs and kidneys) and tumors, ensure that the tissues are intact and no fat tissue is residual. Clean them in normal saline and absorb excess water with absorbent paper. Using an in vivo imager, the tissue fluorescence of organs and tumors was measured at the same excitation and emission wavelengths to evaluate the tumor targeting of nMEL and its situation at different time points. The specific results Figure 7 shown.

[0053] Figure 7The results showed that the MEL group mice had a strong fluorescence intensity in the liver shortly after injection, and the fluorescence intensity of the liver gradually weakened over time, indicating that MEL was quickly metabolized by the liver, which was consistent with the pharmacokinetic performance; at the same time, the fluorescence intensity of the tumor site of the MEL group mice was negligible. The mice in the nanoformulation group (BSA group and nMEL group) also showed a strong liver fluorescence intensity shortly after injection, and the fluorescence intensity gradually weakened over time. The fluorescence intensity at the tumor site gradually increased, and there was still obvious fluorescence at 48 hours, indicating that the nanoformulation drug can achieve tumor aggregation.

[0054] Figure 8 The results showed that the fluorescence of the MEL group was mainly concentrated in the kidneys, followed by the liver, and the content in other tissues was very small. The fluorescence intensity of the kidneys was about one order of magnitude higher than that of the liver, and much higher than that of the tumor. In the nMEL group, the tissue with the highest fluorescence intensity was the kidney, followed by the liver; although the fluorescence intensity of the tumor tissue was lower than that of the kidney and liver, the difference was only two or three times, which was much lower than that of the MEL group. The liver is the main metabolic organ of the drug, and the kidney is the main excretion organ. The relative content of the drug in these two organs of the nMEL group was much lower than that of the MEL group, and the relative content in the tumor was higher than that of the MEL group, indicating that nMEL has better tumor targeting aggregation than MEL, verifying the effective targeting of the drug.

[0055] from Figure 7 and Figure 8 Comprehensive analysis: MEL mainly accumulates in organs such as the liver and kidneys, with relatively few or no MEL in tumor sites. Under the same conditions, nMEL mainly accumulates in tumor sites and accumulates less in organs such as the liver and kidneys. In other words, nMEL has good targeting.

Claims

1. A method for preparing negatively charged melittin nanocapsules targeting tumor microenvironment, characterized in that: Melittin, 2-methacryloyloxyethyl phosphorylcholine, 2-acrylamide-2-methylpropanesulfonic acid, N,N-bis(acryl)cystamine, and 3-acrylamidephenylboronic acid are added into a reactor, and after stirring, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine are added to initiate free radical in situ polymerization to form an encapsulation shell layer around the melittin. The reaction is carried out in an ice bath for 2 hours or placed in a refrigerator at 4 degrees for 12 hours to obtain negatively charged melittin nanocapsules targeting tumor cells.

2. The method according to claim 1, characterized in that 2-Methacryloyloxyethylphosphorylcholine (MPC) and 2-acrylamide-2-methylpropanesulfonic acid (AMPS) were added in the form of being dissolved in phosphate buffered saline (PBS), and N,N-bis(acryl)cystamine (BAC) and 3-acrylamidophenylboronic acid (AAPBA) were added in the form of being dissolved in ultra-dry dimethyl sulfoxide (DMSO).

3. The method according to claim 1, characterized in that The molar ratio of melittin (MEL), 2-methacryloyloxyethyl phosphorylcholine (MPC), 2-acrylamide-2-methylpropanesulfonic acid (AMPS), N,N-bis(acryl)cystamine (BAC) and 3-acrylamidophenylboronic acid (AAPBA) is 1:200-600:40-200:50:40, preferably 1:400:40:50:40; the molar ratio of melittin (MEL) to free radical initiator ammonium persulfate (APS) and catalyst N,N,N',N'-tetramethylethylenediamine (TEMED) is 1:50:

100.

4. Negatively charged melittin nanocapsules targeting tumor microenvironment prepared according to the method of any one of claims 1 to 3.

5. Use of the negatively charged melittin nanocapsules targeting tumor microenvironment prepared by the method according to any one of claims 1 to 3 as a drug for preparing tumor-targeted drugs to reduce liver and kidney aggregation.

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