Polylysine nanoparticle for delivering oxaliplatin and preparation method thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to polylysine nanoparticles for delivering oxaliplatin and a preparation method of the polylysine nanoparticles. The invention discloses a preparation method of polylysine nanoparticles for delivering oxaliplatin. The polylysine nanoparticles are applied to preparation of a tumor targeting drug mPssPC-OXA NPs with small particle size, positive surface charge and excellent stability. The mPssPC-OXA NPs can respond to a tumor microenvironment with high GSH concentration to quickly release a drug, and shows an excellent HCT-116 cell uptake effect and an effective RAW264.7 cell immune escape effect in an in-vitro anti-colon cancer cell test. In addition, the mPssPC-OXA NPs also shows more remarkable cytotoxicity, cell migration inhibition, cell proliferation inhibition and HCT-116 cell apoptosis induction effects compared with free OXA.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a polylysine nanoparticle for delivering oxaliplatin and a preparation method thereof. Background Art

[0002] As one of the common malignant tumors, the treatment strategy of colorectal cancer (CRC) has made remarkable progress in recent years. After surgical resection of the tumor, chemotherapy has become an important means to consolidate the curative effect and prevent recurrence. As a third-generation platinum-based anti-cancer drug, oxaliplatin (OXA) occupies an important position in the chemotherapy of CRC due to its unique mechanism of interfering with DNA replication. However, the clinical application of OXA also faces many challenges, including poor targeting, significant toxic effects (such as peripheral neuropathy, diarrhea, vomiting, myelosuppression, etc.), drug resistance, and drug degradation, which limit the therapeutic efficiency and wide application of OXA.

[0003] To overcome these challenges, researchers have begun to explore new drug delivery strategies to improve the therapeutic efficiency of OXA and reduce its toxic side effects. The nanodrug delivery system provides new ideas for cancer treatment due to its good biocompatibility, low side effects, targeting, and controlled release characteristics. However, traditional nanodelivery systems still face many problems in practical applications, such as difficulty in effectively accumulating at the target site and being easily cleared by the immune system. The root cause of these problems is that traditional nanoparticles rely too much on the enhanced permeability and retention (EPR) effect, and the stability of the EPR effect varies greatly among different individuals and tumors; at the same time, traditional nanoparticles also lack sufficient immune escape ability and are easily cleared by macrophages.

[0004] In response to the problems of in vivo distribution and immune escape of nanoparticles, researchers have proposed various optimization strategies. Among them, designing new nanoparticles that target and aggregate in tumor tissues by using the characteristics of the tumor tissue microenvironment is an innovative idea. The tumor tissue microenvironment usually shows characteristics such as hypoxia, acidity, and chronic inflammation, and the concentration of reduced glutathione (GSH) is much higher than that of the blood circulation system, which provides the possibility of constructing passively targeted nanoparticles using sensitive bonds such as disulfide bonds. In addition, nanoparticles modified with polyethylene glycol (PEG) can reduce immunogenicity and prolong the systemic circulation time, thus avoiding being phagocytosed by macrophages.

[0005] Polylysine (PLLs), as a natural cationic polymer, has become an ideal choice for constructing nanoparticles due to its good water solubility, biocompatibility, and a large number of amino groups. The large amount of positive charges carried by PLLs helps the nanoparticles to be taken up by tumor cells, thereby improving the bioavailability of drugs. There have been no reports on the application of polylysine nanoparticles to oxaliplatin. The present invention aims to explore the construction of a novel nano-drug delivery system and its potential in drug delivery and tumor treatment to improve the targeting and therapeutic efficiency of oxaliplatin. Summary of the Invention

[0006] To solve the above technical problems, the present invention constructs a novel nano-drug delivery system for oxaliplatin, aiming to overcome the problems of poor targeting and limited therapeutic efficiency of current oxaliplatin, and to provide a new drug delivery strategy for the clinical treatment of colorectal cancer.

[0007] On the one hand, the present invention provides a preparation method for polylysine nanoparticles for delivering oxaliplatin, and the method includes the following steps:

[0008] Step 1: Activate 3,3'-dithiobispropionic acid;

[0009] Step 2: Add mPEG to the activated product of Step 1, react, and after the reaction ends, dialyze and lyophilize to obtain the product mPEG-s-s-COOH;

[0010] Step 3: Activate cholesterol succinate, add poly-L-lysine to the activated cholesterol succinate, react, and after the reaction ends, dialyze and lyophilize to obtain the product PLLS-CHS;

[0011] Step 4: Place the mPEG-s-s-COOH prepared in Step 2 in a reaction vessel, activate it, and then add the PLLS-CHS prepared in Step 3 to react. After the reaction ends, dialyze and lyophilize to obtain mPEG-s-s-PLLS-CHS, which is the polylysine nanoparticles for delivering oxaliplatin.

[0012] Further, in the preparation method, the mass ratio of mPEG: poly-L-lysine: cholesterol succinate is 2:1:4 - 2:1:10.

[0013] Further, in the preparation method, the mass ratio of mPEG: poly-L-lysine: cholesterol succinate is 2:1:6.

[0014] On the other hand, the present invention also provides the polylysine nanoparticles for delivering oxaliplatin prepared by the above preparation method.

[0015] On yet another hand, the present invention also provides the application of the polylysine nanoparticles for delivering oxaliplatin in the preparation of tumor-targeted therapeutic drugs.

[0016] Further, in the application, the tumor-targeting therapeutic drug contains oxaliplatin.

[0017] Further, in the application, the tumor is colorectal cancer.

[0018] In addition, the present invention also provides a preparation method of a tumor-targeting therapeutic drug using the polylysine nanoparticles for delivering oxaliplatin, which is characterized by comprising dissolving the polylysine nanoparticles for delivering oxaliplatin and oxaliplatin and then performing dialysis to obtain mPssPC-OXA NPs.

[0019] In addition, the present invention also provides a tumor-targeting drug prepared by the preparation method of the tumor-targeting therapeutic drug using the polylysine nanoparticles for delivering oxaliplatin. The tumor-targeting drug is mPssPC-OXA NPs, and the mPssPC-OXA NPs have tumor targeting property.

[0020] Further, for the tumor-targeting drug, mPssPC-OXANPs improves the delivery stability of oxaliplatin in vivo and the apoptosis rate of oxaliplatin on colorectal cancer cells.

[0021] Compared with the prior art, the present invention has at least the following advantages or beneficial effects:

[0022] The present invention has studied and prepared mPssPC-OXA NPs with small particle size, positive surface charge and excellent stability. mPssPC-OXANPs can respond to the tumor microenvironment with high GSH concentration to rapidly release drugs, and show excellent HCT-116 cell uptake effect and effective RAW264.7 cell immune escape effect in the in vitro anti-colorectal cancer cell experiment. In addition, mPssPC-OXA NPs also show more significant cytotoxicity, cell migration inhibition, cell proliferation inhibition and induction of HCT-116 cell apoptosis effects compared with free OXA. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the synthesis route of mPssPC.

[0024] Figure 2 It is the infrared spectrum diagram of PLLS, s-s, mPEG-s-s-COOH, PLLS-CHS, mPEG-s-s-PLLS-CHS.

[0025] Figure 3 It is the hydrogen spectrum diagram of PLLS, s-s, mPEG-s-s-COOH, PLLS-CHS, mPEG-s-s-PLLS-CHS.

[0026] Figure 4 Particle size distribution and potential diagrams of mPssPC and mPssPC-OXA nanoparticles. Among them, A is the particle size distribution diagram of mPssPC nanoparticles; B is the polydispersity coefficient (PDI value) of mPssPC nanoparticles; D is the zeta potential diagram of mPssPC nanoparticles; E is the average potential of mPssPC nanoparticles; C is the particle size distribution diagram of mPssPC-OXA nanoparticles; F is the zeta potential diagram of mPssPC-OXA nanoparticles.

[0027] Figure 5 Size and morphology of TEM of mPssPC-OXA NPs nanoparticles under TEM transmission electron microscope.

[0028] Figure 6 Stability test results of mPssPC-OXA. Among them, A is the diagram of the change in the particle size of nanoparticles; B is the diagram of the change in the PDI value; C is the diagram of the change in the zeta potential.

[0029] Figure 7 In vitro drug release test results of mPssPC-OXA NPs.

[0030] Figure 8 Cytotoxicity test results of mPssPC-OXANPs. Among them, A is the survival rate of HCT-116 cells treated with different concentrations of OXA; B is the survival rate of HCT-116 cells treated with different concentrations of PC NPs and mPssPC NPs; C is the cytotoxicity test results of OXA, PC-OXANPs, and mPssPC-OXANPs on HCT-116 cells.

[0031] Figure 9 Results diagram of inhibiting cell proliferation with different treatments.

[0032] Figure 10 Results diagram of in vitro cellular uptake experiment of nanoparticles. Among them, A is the cellular uptake diagram of HCT-116 cells for free FITC, mPssPC-FITC NPs, and mPssPC-OXA-FITC within 6 hours; B is the uptake rate of free FITC, mPssPC-FITC NPs, and mPssPC-OXA-FITC groups of cells.

[0033] Figure 11It is the result graph of the in vitro immune escape test of nanoparticles. Among them, A is the result graph of the uptake of PC-OXA-FITC NPs by RAW264.7 cells at 3, 6, and 9 hours; B is the result graph of the uptake of mPssPC-OXA-FITC NPs by RAW264.7 cells at 3, 6, and 9 hours; C is the average fluorescence intensity of cells in the PC-OXA-FITC NPs group and the mPssPC-OXA-FITC NPs group at 3, 6, and 9 hours.

[0034] Figure 12 It is the test result of the inhibitory effect of mPssPC-OXANPs on the migration of HCT-116 cells. Among them, A is the effect of mPssPCNPs, OXA, and mPssPC-OXANPs on cell scratch healing; B is the cell migration rate of mPssPC NPs, OXA, and mPssPC-OXA NPs at 12 hours; C is the cell migration rate of mPssPC NPs, OXA, and mPssPC-OXA NPs at 24 hours.

[0035] Figure 13 It is the result of AO / EB fluorescence staining.

[0036] Figure 14 It is the test result of flow cytometry. Detailed implementation manners

[0037] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments.

[0038] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings, but the embodiments cited are not intended to limit the present invention.

[0039] In the following embodiments, the experimental methods and detection methods, unless otherwise specified, are all conventional methods; the reagents and materials, unless otherwise specified, can be purchased on the market. The purchase information of some reagents in the embodiments is as follows:

[0040] 3,3'-Dithiodipropionic acid (s-s), methoxypolyethylene glycol (mPEG), 4-dimethylaminopyridine (DMAP), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI), cholesterol succinate (CHS), N-hydroxysuccinimide (NHS) are purchased from Shanghai Macklin Biochemical Co., Ltd.

[0041] Poly-L-lysine (PLLS), oxaliplatin (OXA) are purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0042] N,N-Dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0043] Example 1

[0044] This example is for the synthesis of mPssPC polymer.

[0045] Weigh 0.54 g of 3,3'-dithiobispropionic acid, 0.98 g of DMAP, and 1.53 g of EDCI, add DMSO to dissolve, and heat and activate for 2 hours to obtain an activated mixture solution.

[0046] Weigh 1.00 g of mPEG, add DMSO to dissolve and pour the solution into the activated mixture solution, and heat and react for 48 hours. After the reaction is complete, dialyze and lyophilize to obtain the product mPEG-s-s-COOH.

[0047] Weigh 0.98 g of CHS, 0.57 g of EDCI, and 0.34 g of NHS, dissolve and activate for 2 hours, then weigh 3.55 g of PLLS, dissolve and add it to the above-activated product, and react in a round-bottom flask for 48 hours. Dialyze and lyophilize to obtain the product PLLS-CHS (PLLS:CHS = 1:2).

[0048] Weigh 1.96 g of CHS, 1.14 g of EDCI, and 0.68 g of NHS, dissolve and activate for 2 hours, then weigh 3.55 g of PLLS, dissolve and add it to the above-activated product, and react in a round-bottom flask for 48 hours. Dialyze and lyophilize to obtain the product PLLS-CHS (PLLS:CHS = 1:4).

[0049] Weigh 2.94 g of CHS, 1.70 g of EDCI, and 1.00 g of NHS, dissolve and activate for 2 hours, then weigh 3.55 g of PLLS, dissolve and add it to the above-activated product, and react in a round-bottom flask for 48 hours. Dialyze and lyophilize to obtain the product PLLS-CHS (PLLS:CHS = 1:6).

[0050] Weigh 3.92 g of CHS, 2.28 g of EDCI, and 1.36 g of NHS, dissolve and activate for 2 hours, then weigh 3.55 g of PLLS, dissolve and add it to the above-activated product, and react in a round-bottom flask for 48 hours. Dialyze and lyophilize to obtain the product PLLS-CHS (PLLS:CHS = 1:8).

[0051] Place mPEG-s-s-COOH in a clean beaker, add 0.170 g of EDCI and 0.100 g of NHS, dissolve and activate for 2 hours, then add PLLS-CHS (PLLS:CHS = 1:2; 1:4; 1:6; 1:8) respectively and react for 48 hours. Then, dialyze and lyophilize to obtain the products mPEG-s-s-PLLS-CHS (mPssPC) with feed ratios of 2:1:2; 2:1:4; 2:1:6; 2:1:8 (mPEG:PLLS:CHS). The schematic synthesis route of mPssPC is as shown in Figure 1 shown.

[0052] Example 2

[0053] This example is for the identification of mPssPC polymer.

[0054] Take 3 mg of dry sample powders of s-s, PLLS, mPEG-s-s-COOH, PLLS-CHS, and mPssPC respectively, mix them evenly with 15 mg of potassium bromide powder, grind and press them into transparent sheet-like solids, and obtain the infrared absorption spectra of the samples by scanning in the range of 400 - 4000 cm -1 using an infrared spectrometer (FTIR).

[0055] Take 8 mg of sample powders of s-s, PLLS, mPEG-s-s-COOH, PLLS-CHS, and mPssPC respectively, dissolve them in 0.6 mL of deuterated DMSO, load them into NMR sample tubes, and in 1 The 1H NMR spectrum is obtained by scanning the absorption from 0 ppm to 15 ppm to get the hydrogen spectra of each sample.

[0056] The FITR results of PLLS, s-s, mPEG-s-s-COOH, PLLS-CHS, and mPEG-s-s-PLLS-CHS (mPssPC) are as shown in Figure 2 shown. The absorption peaks at 614.5 cm -1 , 535.8 cm -1 are the stretching vibration peaks of the disulfide bond (-s-s-); there are two absorption bands at 1325.1 - 1220.8 cm -1 , which are the absorption bands of the ester bond ν C＝O and ν C-O-C respectively, proving that s-s is successfully connected to mPEG and grafted onto the PLLS structure; the absorption peak at 1606.3 cm -1 is the stretching vibration absorption peak of the carbon-carbon double bond in the CHS structure; the absorption band at 3552.6 - 3135.4 cm -1 , the absorption peak at 1668.3 cm -1 , and the absorption bands at 1475.8 - 1420.1 cm -1The absorption bands respectively correspond to the absorption bands and peaks of the amide bond ν NH , ν C＝O , ν C━N , indicating that CHS has been successfully grafted onto the PLLS structure. The characteristic absorption bands and peaks of the above functional groups prove the successful synthesis of the polymer mPssPC.

[0057] 1 The 1H NMR results are as Figure 3 shown. The chemical shift of the H on the amino group without a linking group on PLLS is δ H 2.01; the chemical shift of the H of the amide bond is δ H 8.06, and the chemical shift of the H on the double bond carbon of the six-membered ring is δ H 5.34, indicating that CHS has been successfully linked to the PLLS structure by forming an amide bond; the chemical shift of the hydrogen atom on 15C is shifted to a lower field and appears at δ H 3.05 due to the strong electron-withdrawing effects of s-s and the carbonyl group of the ester bond, indicating that mPEG-s-s-COOH has been successfully linked to the PLLS structure by an amide bond. The above results show the successful synthesis of the mPssPC polymer.

[0058] Example 3

[0059] This example is for the preparation of mPssPC NPs and mPssPC-OXA NPs.

[0060] Weigh 6 mg of the mPssPC polymer, add DMSO and dissolve it thoroughly. Transfer the solution into a dialysis bag with a molecular weight cut-off of 8000 - 14000 kDa, and dialyze it with primary water, changing the primary water every hour. After complete dialysis, blank mPssPC NPs are prepared.

[0061] Weigh 6 mg of mPssPC and 10 mg of OXA, add 10 mL of DMF and dissolve them thoroughly. Transfer the solution into a dialysis bag with a molecular weight cut-off of 8000 - 14000 kDa, and dialyze it with primary water, changing the primary water every hour. After complete dialysis, mPssPC-OXA NPs are prepared.

[0062] Example 4

[0063] This example is for the performance test of mPssPC-OXA NPs.

[0064] (1) Measurement of the particle size and zeta potential of mPssPC-OXA NPs

[0065] The particle size distribution and surface potential of mPssPC NPs with four feed ratios (2:1:4, 2:1:6, 2:1:8, 2:1:10) and mPssPC-OXA NPs (2:1:6) were measured using a dynamic light scattering instrument (DLS). Colloidal solutions of mPssPC NPs and mPssPC-OXA NPs with a concentration of 1 mg / mL were taken and placed in the DLS instrument for detection, and each sample was tested three times.

[0066] The results are as Figure 4 shown. Figure 4 As shown in A and B in [reference], the particle sizes and polydispersity indices (PDI values) of the nanoparticles with feed ratios of 2:1:4, 2:1:6, 2:1:8, and 2:1:10 were 247.77 ± 2.70 nm, 0.200 ± 0.043; 203.67 ± 1.08 nm, 0.100 ± 0.012; 190.77 ± 2.91 nm, 0.102 ± 0.013; 152.40 ± 3.15 nm, 0.165 ± 0.015, respectively. The particle size distributions of the four feed ratio nanoparticles were uniform, and with the increase of the feed of the hydrophobic part (CHS), the particle sizes of the nanoparticles showed a decreasing trend. Figure 4 As shown in D and E in [reference], the Zeta potentials of the nanoparticles with feed ratios of 2:1:4, 2:1:6, 2:1:8, and 2:1:10 were 48.40 ± 1.00 mV, 46.97 ± 0.32 mV, 43.20 ± 0.72 mV, and 38.40 ± 1.25 mV, respectively. It indicates that the surfaces of mPssPC NPs prepared with different feed ratios all carry a large amount of positive charges.

[0067] Considering the particle size, potential, and stability of mPssPC NPs with four feed ratios comprehensively, mPssPC NPs prepared with a feed ratio of 2:1:6 were preferred.

[0068] Figure 4 As shown in C and F in [reference], the particle size of mPssPC-OXA NPs (2:1:6) was 274.47 ± 1.07 nm, the PDI value was 0.127 ± 0.051, and the Zeta potential was 46.77 ± 0.681 mV.

[0069] (2) Morphology observation of mPssPC-OXA NPs

[0070] Take 10 μL of the colloidal solution sample of mPssPC-OXA NPs (2:1:6) and drop it on a copper mesh coated with a carbon support film. After natural drying, observe the nanoparticle size and morphology under a TEM transmission electron microscope.

[0071] The results are as Figure 5As shown in A and B, the TEM images showed that mPssPC-OXA NPs were spherical and had a uniform particle size distribution. The particle size of the drug-loaded nanoparticles in the field of view was about 200 nm, slightly lower than the result obtained by DLS measurement (274.47 ± 1.07 nm), which was presumably related to the different measurement conditions. The nanoparticles observed by TEM had no solvent, while DLS measurement was performed on a colloidal solution. The mPEG attached to the outside of the nanoparticles was prone to form hydrogen bonds with water molecules, resulting in a slightly higher particle size of mPssPC-OXA NPs measured by DLS than that of the nanoparticles in the TEM field of view.

[0072] (3) Stability test of mPssPC NPs

[0073] Take 1 mg / mL mPssPC NPs (2:1:4, 2:1:6, 2:1:8, 2:1:10) colloidal solution and let it stand at room temperature. At 0, 10, 30, and 50 days, use a DLS instrument to measure the changes in the particle size, zeta potential, and PDI value of the nanoparticles to analyze the stability of the nanoparticles.

[0074] The results are as Figure 6 shown, where Figure 6 A shows that with the increase of time, the particle sizes of mPssPC NPs all increased to varying degrees. Among them, the particle size of mPssPC NPs with a feed ratio of 2:1:6 increased slightly within 50 days but basically remained stable. The PDI value reflects the concentration and dispersion of the nanoparticle size distribution. Figure 6 B shows that the PDI value of 2:1:6 mPssPC NPs was basically stable within 50 days compared with other feed ratio nanoparticles. Figure 6 C shows that the zeta potential of 2:1:6 mPssPC NPs was more stable compared with 2:1:4, 2:1:8, 2:1:10 mPssPC NPs.

[0075] In summary, the particle size, zeta potential, and PDI value of 2:1:6 mPssPC NPs were more stable compared with other feed ratio nanoparticles.

[0076] Example 5

[0077] This example is for the determination of the drug loading of mPssPC-OXA NPs.

[0078] Prepare a 2.52 mM OXA solution and dilute it with primary water to obtain OXA solutions with concentrations of 1.26 mM, 0.63 mM, 0.315 mM, 0.1575 mM, 0.07875 mM, 0.039375 mM, and 0.019688 mM. Detect the ultraviolet absorbance of OXA at 249 nm and draw the standard curve of OXA.

[0079] Dilute the colloidal solution of mPssPC-OXA NPs (2:1:6) to an ultraviolet absorbance between 1.0 and 2.0, and calculate the drug loading of the drug-loaded nanoparticles by substituting into the following formula:

[0080]

[0081] The standard curve equation of OXA is: y = 2.1776x + 0.0053, and the correlation coefficient R 2 = 0.9993, which proves that in the concentration range of 1.26 - 0.019688 mM, the concentration of OXA has a good linear correlation with the absorbance. Measure the absorbance of the mPssPC-OXA NPs colloidal solution at 249 nm using an ultraviolet spectrophotometer, and substitute it into the standard curve equation to calculate that the drug loading of the drug-loaded nanoparticles is 20.33 ± 0.71%.

[0082] Example 6

[0083] This example is an in vitro drug release test of mPssPC-OXA NPs.

[0084] Weigh three portions of 10 mg each of mPssPC-OXA NPs, dissolve them using PBS with pH 7.4, and transfer the colloidal solution into a 3000 kDa dialysis bag. Place the dialysis bag in PBS with pH 7.4, PBS with pH 7.4 and a GSH concentration of 10 mM, and PBS with pH 5.5 and a GSH concentration of 10 mM for dialysis, and oscillate on a shaker (37 °C, 75 rpm). Replace the PBS in the dialysis bag at different time points, measure the volume Vt, detect the absorbance of PBS at a wavelength of 249 nm, calculate the content of released OXA, and calculate the drug release rate according to the following formula:

[0085]

[0086] where m drug is the total mass of OXA in the dialysis bag, and t is the time point for replacing PBS (t = 0, 1, 2, 4, 8, 12, 24, 48 hours, V0 = 0, C0 = 0).

[0087] The results are as Figure 7As shown, free OXA was rapidly released within 8 hours, and the release rates of free OXA under the three conditions were 81.92±1.62%, 80.32±1.18%, and 78.60±1.06% respectively. Under the same three treatment conditions, the total release rates of OXA from mPssPC-OXA NPs were 70.02±1.96%, 62.84±1.54%, and 43.67±1.91% respectively. The results showed that the drug release rate of the drug-loaded nanoparticle group was significantly slower than that of the free drug group, indicating the sustained-release effect of mPssPC-OXA NPs. Under the same condition of pH 7.4, the drug-loaded nanoparticles in the group with the addition of GSH had a faster release rate and a higher total release rate at 48 hours, indicating that the release of OXA from mPssPC-OXA NPs had the characteristic of GSH sensitivity. At pH 5.5, the drug-loaded nanoparticles in the group with the addition of GSH had a higher total release rate and a faster drug release rate compared to the drug-loaded nanoparticles in the group with the addition of GSH at pH 7.4, indicating that the mPssPC-OXA NPs prepared in the present invention could respond to the tumor microenvironment and release OXA, thereby achieving the purpose of increasing the drug concentration in tumor tissues.

[0088] Example 7

[0089] This example was a test for the cytotoxicity and colony formation of mPssPC-OXA NPs.

[0090] HCT-116 cells were seeded in a 96-well plate at a density of 6000 cells per well. After the cells adhered and grew for 24 hours, they were divided into 7 groups, with 4 wells in each group. Except for the first group without cells as the blank control group, different concentrations of mPssPC, OXA, and mPssPC-OXA NPs were added to the other groups. After incubation for 48 hours, 10% CCK-8 reagent was added and treated for 2 hours, and the absorbance was detected at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0091] HCT-116 cells were added to a 24-well plate at a quantity of 1000 cells per well. After adhering and growing for 24 hours, free OXA and mPssPC-OXA NPs with the same concentrations of mPssPC and OXA were added. On the 8th day of culture, the cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet.

[0092] The inhibitory effects of free OXA, mPssPC NPs, PC-OXA NPs, and mPssPC-OXA NPs on the cell viability of HCT-116 cells were detected by the CCK-8 cytotoxicity assay.

[0093] The results were as Figure 8 shown, in which Figure 8 A in showed that the inhibitory effect of OXA on the viability of HCT-116 cells was concentration-dependent, and the IC of OXA against HCT-116 was calculated.50 was 17.82 ± 3.29 μM.

[0094] Figure 8 As shown in B, the blank nanomaterials (mPssPC NPs) had no obvious cytotoxicity to HCT-116 cells.

[0095] Figure 8 As shown in C, the cytotoxicities of free OXA, PC-OXA NPs, and mPssPC-OXA NPs all showed concentration dependence. And at the same OXA concentration, PC-OXA NPs and mPssPC-OXA NPs had more significant cytotoxicity than free OXA.

[0096] Figure 9 As shown, after co-culturing for 8 days, the relative colony formation rates of the cells in the blank control (Control), mPssPC NPs, free OXA, and mPssPC-OXANPs groups were 100.08 ± 5.81%, 103.62 ± 20.21%, 63.20 ± 2.60%, and 28.84 ± 2.84%, respectively. It can be observed that there was no obvious difference in the relative colony formation rate between the blank nanomaterial group (mPssPC NPs) and the blank control group, indicating that the blank material (mPssPC NPs) had no effective ability to inhibit the proliferation of HCT-116 cells. The relative colony formation rates of the cells in the OXA and mPssPC-OXA NPs groups were significantly reduced compared with the blank control group, and the relative colony formation rate of the cells in the mPssPC-OXA NPs group was significantly lower than that in the free OXA group. It shows that at the same OXA concentration, mPssPC-OXA NPs showed a more significant inhibitory effect on the proliferation of HCT-116 cells than free OXA.

[0097] Example 8

[0098] This example was an in vitro cell uptake test of nanoparticles.

[0099] mPssPC-FITC NPs and mPssPC-OXA-FITC NPs were labeled with fluorescein isothiocyanate (FITC). HCT-116 cells were seeded in a six-well plate at a density of 5 × 10 4 per well, and free FITC, FITC-labeled mPssPC-FITC NPs, and mPssPC-OXA-FITC NPs were added respectively. After incubating for 6 hours, the cells were fixed with 4% paraformaldehyde for 10 minutes, and then the cell nuclei were stained with DAPI dye for 10 minutes. The excess dye was washed with PBS, and the six-well plate was observed using an inverted fluorescence microscope.

[0100] The results were as Figure 10 shown.Figure 10 As shown in A and B, taking the free FITC group as the control, the cellular uptake rates of the mPssPC-FITC NPs group and the mPssPC-OXA-FITC NPs group were 622.65±35.30% and 570.63±0.56% respectively. This indicates that mPssPC-FITC NPs and mPssPC-OXA-FITC NPs have a more obvious endocytosis effect compared to free FITC. In addition, the average fluorescence intensity and cellular uptake rate of the mPssPC-OXA-FITC NPs group were significantly higher than those of the free FITC group, indicating that encapsulating OXA does not affect the promotion of HCT-116 cell uptake by mPssPC NPs.

[0101] Example 9

[0102] This example is an in vitro immune escape test of nanoparticles.

[0103] RAW264.7 cells were seeded in 6-well plates at a density of 5×10 4 per well. After incubating with PC-OXA-FITC NPs and mPssPC-OXA-FITC NPs with the same FITC concentration for 3, 6, and 9 hours, the cells were fixed with 4% paraformaldehyde for 10 minutes, and then the cell nuclei were stained with DAPI dye for 10 minutes. Excess dye was washed with PBS, and the 6-well plates were observed using an inverted fluorescence microscope.

[0104] The results are as Figure 11 shown. Figure 11 As shown in A and C, the average fluorescence intensities of the cells in the PC-OXA-FITC NPs group treated for 3, 6, and 9 hours were 27.95±0.36, 29.28±0.15, and 31.93±1.22 respectively. Figure 11As shown in B and C, the average fluorescence intensities of cells treated with mPssPC-OXA-FITC NPs for 3, 6, and 9 hours were 13.82±0.24, 17.00±1.08, and 14.80±1.17, respectively. It indicates that when RAW264.7 cells were treated with PC-OXA-FITC NPs and mPssPC-OXA-FITC NPs for the same time, the average fluorescence intensity of cells in the PC-OXA-FITC NPs group was significantly greater than that in the mPssPC-OXA-FITC NPs group. In addition, the maximum average fluorescence intensity of cells in the mPssPC-OXA-FITC NPs group appeared at 6 h, and then the fluorescence intensity began to decrease. On the contrary, in the PC-OXA-FITC NPs group, the average fluorescence intensity of cells showed an increasing trend with the extension of time. The experimental results show that modifying PC-OXA NPs with mPEG can effectively reduce the recognition and clearance of drug-loaded nanoparticles by macrophages, thereby enhancing the stability of drug-loaded nanoparticles during the delivery process.

[0105] Example 10

[0106] This example is a test for the inhibitory effect of mPssPC-OXA NPs on the migration of HCT-116 cells.

[0107] Seed HCT-116 cells in a six-well plate at a density of 1×10 5 per well. When the cell density proliferates to 90%, use a 200 μL pipette tip to scratch the cells and replace the medium with serum-free medium for culture. Add mPssPC NPs, free OXA at a concentration of IC 50 and mPssPC-OXA NPs with the concentration of free OXA and co-culture for 24 hours. Observe the wound healing at 0 h, 12 h, and 24 h using a microscope. Use Image J software to calculate the area of the scratched part and perform data analysis.

[0108]

[0109] T is the time point (0, 12, 24).

[0110] As shown in Figure 12, Figure 12As shown in A, B, and C, in the presence of obvious scratches, cell migration in the control group was significant at 12 h and 24 h, and the cell migration rates were approximately 21.29 ± 0.69% and 32.75 ± 0.18% respectively. The cell migration rates of mPssPC NPs at 12 h and 24 h were 16.72 ± 3.36% and 32.27 ± 0.97% respectively, indicating that the blank nanomaterials had no obvious effect on the migration ability of HCT-116 cells. The cell migration rates of the free OXA group at 12 h and 24 h were 18.40 ± 4.46% and 28.41 ± 1.31% respectively. The result at 12 h was not significantly reduced compared with the control group, but was significantly less than the control group at 24 h, presumably related to the low drug uptake within a short period. The cell migration rates of the mPssPC-OXA NPs group at 12 h and 24 h were 12.53 ± 0.46% and 18.92 ± 0.32% respectively. This result was significantly lower than that of the control group, and the cell migration rate at 24 h was significantly lower than that of the free OXA group, indicating that mPssPC-OXA NPs could enhance the inhibitory effect of OXA on the migration of HCT-116.

[0111] Example 11

[0112] This example was a test on the ability of mPssPC-OXA NPs to induce apoptosis in HCT-116 cells.

[0113] (1) AO / EB staining test

[0114] HCT-116 cells were seeded in six-well plates at a density of 5×10 4 per well. After adherent growth for 16 hours, mPssPC NPs, free OXA at a concentration of IC 50 concentration (17.82 μM) of free OXA and mPssPC-OXA NPs (where the concentration of OXA was 17.82 μM) were added respectively and co-cultured for 24 hours. HCT-116 cells without any agent were used as the blank control (control). The cells were digested with 0.25% trypsin and resuspended with PBS to wash the remaining culture medium. After washing, the staining solution was prepared using an AO / EB kit according to Acridine Orange:Ethidium Bromide:Dilution Buffer = 1:1:8. The cells were resuspended with 100 μL of Dilution Buffer, 4 μL of the prepared staining solution was added to each group of cell suspensions, and stained for 15 minutes. 20 μL of the cell suspension was dropped onto the glass slide, and apoptotic fluorescence staining was observed under an inverted fluorescence microscope.

[0115] The results were as Figure 13As shown, the cells in the control group showed uniform green fluorescence; there was no obvious difference between the cells in the mPssPC NPs group and the control group. The cells in the free OXA group showed green, partial yellowish-green, and orange-red fluorescence, and early apoptosis occurred in some cells. Almost all the cells in the mPssPC-OXA NPs group were yellow and orange-red. There was a tendency for more cells in the mPssPC-OXA NPs group to enter the apoptotic and necrotic states. The data indicate that mPssPC-OXA NPs have a more significant potential to induce apoptosis in HCT-116 cells compared with free OXA.

[0116] (2) Flow cytometry assay

[0117] HCT-116 cells were seeded in six-well plates at a density of 5×10 4 per well. After adherent growth for 16 hours, mPssPC NPs, free OXA at the IC50 concentration (17.82 μM), and mPssPC-OXA NPs (34.63 μg / mL, with the OXA concentration being 17.82 μM) were added respectively and co-cultured for 24 hours. The cells were digested with 0.25% trypsin and resuspended with PBS to wash away the remaining culture medium. After washing, the Annexin V-FITC / PI Apoptosis Kit was used. 500 μL of diluted Annexin V Binding Buffer was added to each group to resuspend the cells. 5 μL of Annexin V-FITC Reagent and PI Reagent (50 μg / ml) were added to the cell suspension, and the cells were incubated at room temperature in the dark for 15 minutes. Flow cytometry was used to detect the apoptosis of the cells.

[0118] The detection results are as shown in Figure 14 A and B below. The total apoptosis rates of the cells in the control group and the mPssPC NPs group were 7.19±0.98% and 7.11±0.35% respectively, and there was no obvious difference in the apoptosis rates of the two groups of cells, indicating that the blank nanomaterials (mPssPC NPs) had no effect on inducing apoptosis in HCT-116 cells. The total apoptosis rates of the cells in the free OXA and mPssPC-OXA NPs groups were 16.05±0.96% and 24.52±1.15% respectively. It shows that treating HCT-116 cells with mPssPC-OXA NPs can significantly increase the apoptosis rate of cancer cells compared with free OXA.

[0119] As described above, the present invention can be preferably implemented. The above embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the present invention.

Claims

1. Preparation method of polylysine nanoparticles for delivering oxaliplatin, characterized in that, It includes the following steps: Step 1: Activate 3,3'-dithiobispropionic acid; Step 2: Add mPEG to the activated product of Step 1, react, after the reaction is completed, dialyze and lyophilize to obtain the product mPEG-s-s-COOH; Step 3: Activate cholesterol succinate, add poly-L-lysine to the activated cholesterol succinate, react, after the reaction is completed, dialyze and lyophilize to obtain the product PLLS-CHS; Step 4: Place the mPEG-s-s-COOH prepared in Step 2 in a reaction vessel, activate it, then add the PLLS-CHS prepared in Step 3 to react, after the reaction is completed, dialyze and lyophilize to obtain mPEG-s-s-PLLS-CHS, which is the polylysine nanoparticles for delivering oxaliplatin.

2. The preparation method according to claim 1, wherein, The mass ratio of mPEG: poly-L-lysine: cholesterol succinate is 2:1:4 - 2:1:

10.

3. The preparation method according to claim 2, wherein The mass ratio of mPEG: poly-L-lysine: cholesterol succinate is 2:1:

6.

4. Polylysine nanoparticles for delivering oxaliplatin prepared by the method according to any one of claims 1 - 3.

5. Use of the polylysine nanoparticles for delivering oxaliplatin according to claim 4 in the preparation of a tumor-targeted therapeutic drug.

6. The application according to claim 5, characterized in that, The tumor-targeted therapeutic drug contains oxaliplatin.

7. The application according to claim 6, wherein The tumor is colorectal cancer.

8. The preparation method of a tumor-targeted therapeutic drug using the polylysine nanoparticles for delivering oxaliplatin according to claim 4, characterized in that, It includes dissolving the polylysine nanoparticles for delivering oxaliplatin and oxaliplatin and then dialyzing to obtain mPssPC-OXA NPs.

9. The tumor-targeted drug prepared by the method according to claim 8, characterized in that, The tumor-targeted drug is mPssPC-OXA NPs, and the mPssPC-OXA NPs have tumor-targeting property.

10. The tumor-targeting drug according to claim 9, wherein mPssPC-OXA NPs improve the delivery stability of oxaliplatin in vivo and improve the apoptosis rate of oxaliplatin on cancer cells, and the cancer cells are colorectal cancer cells.

Images