CN115919776A - Mixed lipid material nano preparation of pig spleen extract and application thereof - Google Patents

Abstract

The mixed lipid nano preparation obtained by the entrapment of the method has the advantages of good stability, high delivery efficiency, strong cell-entering capability, high bioactivity, low toxicity and the like, and has important potential application value in the aspect of antitumor treatment.

Description

Mixed lipid nano preparation of pig spleen extract and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a mixed lipid nanometer preparation of a pig spleen extract.

Background

The spleen aminopeptide oral freeze-dried powder (hereinafter referred to as 'Fuketuo') is polypeptide and nucleotide substances extracted from fresh pig spleen, has a molecular weight of less than 10000 and an average molecular weight of about 3500, and contains more than 10 trace elements and multiple immunoregulatory factors necessary for human body. The traditional Chinese medicine composition is clinically used for treating low cellular immune function, immunodeficiency, autoimmune dysfunction diseases, radiotherapy and chemotherapy of malignant tumor patients, postoperative improvement of life quality and the like.

The compound cotor is a polypeptide and nucleotide mixture, is delivered by a carrier, and has better transmembrane absorption effect. Most of traditional drug carriers are cationic liposome and cationic polymer, and can be combined with drugs through electrostatic interaction due to the existence of positive charges on the surface, but can be combined with negatively charged serum protein after intravenous injection, so that the targeting property is poor and the long-term circulation in vivo cannot be realized; and can be combined with biological membranes in cells to destroy the normal membrane structure and generate stronger in vivo toxicity.

Therefore, reducing the toxicity of the compound cotyl preparation is one of the main research and development directions.

Disclosure of Invention

The first purpose of the invention is to provide a mixed lipid nanometer preparation of pig spleen extract, which has high efficiency and low toxicity, so as to improve the pharmaceutical property of the medicine.

The inventor previously designed and synthesized cytidine acetamide glycerol ether molecule DNCA (CN 108059619A), which can combine and entrap nucleic acid drugs through hydrogen bonds and pi-pi stacking (CN 1084478807A). Combining DNCA and cationic lipid material CLD which is synthesized by previous design of the inventor and takes cystine as a framework, 3' -dipeptide-siRNA conjugate is transfected with high efficiency and has been successfully applied at a cellular level while reducing toxicity of the cationic lipid material (MolPharm, 2019, 16, 4920). The delivery system is further optimized, an optimal DNCA/CLD prescription is newly formulated and explored, a PEG2000-DSPE is used for assistance, a high-efficiency low-toxicity neutral/cation mixed lipid delivery system (a Mix delivery system for short) is obtained, the high-efficiency low-toxicity neutral/cation mixed lipid delivery system can be delivered to cells repeatedly, the growth of tumor cells is inhibited by targeted silencing of key genes for tumor occurrence and development, and the anti-tumor treatment effect is played. The CCK-8 experiment is used for detecting the proliferation inhibition effect of the liposome-coated carbendazim on ten kinds of tumor cells, and the Mix-coated carbendazim can show obvious inhibition effect (about 70%) on A549 (human lung adenocarcinoma cell line) and BCPAP (human thyroid cancer cell line) cell lines at1 mg/mL. The delivery system provides a new strategy and a new idea for preparation of the compound cotyl for resisting human lung adenocarcinoma and thyroid cancer. PAT/Mix can obviously inhibit the tumor growth of A549 tumor-bearing mice, and has better in-vivo safety. Mix entrapment provides possibility for PAT treatment of human lung adenocarcinoma and thyroid cancer, and expands potential indications of the drug.

In-vitro BMDCs activation experiments show that the expression quantity of CD40 on the surface of BMDCs is remarkably improved and the percentage of CD40+ cells is remarkably increased after PAT and PAT/Mix treatment, so that the maturation of BMDCs cells is promoted, the antigen processing and presenting capacity of the cells is enhanced, and the PAT has a certain tumor immunotherapy effect. The nano preparation has the advantages of good stability, high delivery efficiency, strong cell-entering capability, low toxicity, good biological activity and the like, and has important potential application value in the aspect of anti-tumor treatment.

The technical purpose of the invention is realized by the following technical scheme:

a mixed lipid nanometer preparation of pig spleen extract comprises neutral cytidine lipid DNCA, cationic lipid CLD, PEG2000-DSPE, genOpti buffer system and pig spleen extract.

Preferably, the structural formula of the neutral cytidine lipid DNCA is shown as formula I:

the structural formula of the cationic lipid CLD is shown as formula II:

the structural formula of the PEG2000-DSPE is shown as the formula III:

preferably, the pig spleen extract is spleen aminopeptide oral freeze-dried powder.

Preferably, the mixed lipid nano preparation is prepared by the following method: dissolving neutral cytidine lipid materials DNCA, cationic lipid materials CLD and PEG2000-DSPE in an ethanol solution, dissolving spleen aminopeptide oral lyophilized powder in enzyme-free water, mixing the substances by using prepared Genopti, and performing ultrasonic treatment to obtain the mixed lipid material nano preparation, namely PAT/Mix.

Preferably, the dosage of the lipid material in the preparation is DNCA: CLD = 10/10. Mu.g/ml, pig spleen extract 1mg/ml, PEG2000-DSPE incorporated at 0.1mol% of all lipids, and Genopti were used to mix these substances.

The second purpose of the invention is to provide the application of the mixed lipid nanometer preparation of the pig spleen extract.

The technical purpose of the invention is realized by the following technical scheme:

the application of the mixed lipid material nano preparation of the pig spleen extract in the preparation of antitumor drugs or reagents by using the mixed lipid material nano preparation alone or in combination with other drugs.

Preferably, the tumors include, but are not limited to, lung cancer and thyroid cancer.

Preferably, the drug or agent comprises unit preparations with different specifications, and the prevention or treatment of the disease is carried out by one or more administration routes of oral administration, intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection and the like through clinically feasible administration routes.

The technical effects of the invention are mainly reflected in the following aspects:

1. the neutral/cation mixed entrapment method innovatively designs the neutral/cation mixed lipid nano preparation with high efficiency and low toxicity, and realizes high-efficiency cell transfection and in-vivo delivery of the pig spleen extract. The preparation can realize the anti-tumor treatment effect in various tumors (including but not limited to lung cancer and thyroid cancer) by administration, has the advantages of good stability, high delivery efficiency, strong cell-entering capability, high biological activity, low toxicity and the like, and has important potential application value in the aspect of anti-tumor treatment.

2. The base head of the neutral nucleoside lipid can be combined with the drug through hydrogen bond action and pi-pi stacking action, the dosage of the cationic lipid is reduced after the neutral nucleoside lipid is doped, the surface of the nanoparticle is electronegative, charged particles or proteins are not easy to adsorb in the circulation process, and the neutral nucleoside lipid is more stable in vivo application.

Drawings

FIG. 1 is a formulation characterization of PAT/Mix lipid complex and empty carrier;

A. particle size of empty carrier and PAT/Mix; B. potentials of empty vector and PAT/Mix; transmission Electron Microscopy (TEM) pictures of empty vector and PAT/Mix.

FIG. 2 is PAT/Mix formulation optimization and lipid toxicity investigation;

A. cell viability of 48hA549 and BCPAP cells administered with DNCA and CLD-entrapped PAT (1 mg/ml) at different ratios; B. illustrates the material toxicity of two ratios of empty lipid in A549, BCPAP, MC-38, HT29 and LLC cells; C. the proliferation inhibition effect of different mol% PEG (DNCA/CLD 10/10 mu g/mL, PAT1mg/mL,48 h) on A549 cell line by adding PAT into liposome; D. the survival rates of A549 cells and BCPAP cells after 48 hours of administration of PAT/Mix nano-composite with different concentrations.

FIG. 3 is a study of the effect of PAT/Mix preparations on A549 cell cycle and apoptosis;

A-B, representative scattergram (A) of PAT (1 mg/mL)/Mix dosing 24hA549 cell apoptosis and mean histogram (B) of three independent experiments; C. the cycle-blocking effect of PAT (1 mg/mL)/Mix administered for 24h on A549 cells was examined by flow cytometry.

FIG. 4 is a graph showing the effect of PAT/Mix on in vitro activation of BMDCs;

flow cytometry detection of PAT/Mix dosing 24hBMDCs: A. surface CD40 mean fluorescence intensity value (MFI); percentage of cd40 positive cells; C. surface MHC II MFI; D. surface CD80MFI.

FIG. 5 shows the anti-tumor effect of PAT/Mix in A549 tumor-bearing mice;

A. tumor-bearing mice modeling and dosing schematic; B. tumor growth profile of tumor-bearing mice; C. mean tumor weight in treatment endpoint mice; D. tumor schematic of tumor-bearing mice; E. the change curve of the body weight of the tumor-bearing mice; (n =5, P < 0.05).

Detailed Description

The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description of the specific embodiments proceeds. The examples are illustrative only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

EXAMPLE 1 encapsulation of PAT drugs by neutral/cationic Mixed lipid Material to form PATPATPAT lipid Complex

Mixing the PAT medicine with lipid material at a dose of 1mg/mL, and mixing the lipid material with DNCA (chemical formula I): CLD (chemical formula II) = 10: 10 (mu g/ml), the doping amount of PEG2000-DSPE accounts for 0.1mol% of all lipid materials, and the solution environment of the preparation is Genopti. PAT was extracted according to literature (a method for efficiently extracting splenopeptide), DNCA was synthesized according to literature (CN 108059619A), CLD was synthesized according to literature (NewJChem, 2014, 38 (10), 4952-4962), PEG2000-DSPE was purchased from Yuanye, and Genopti was purchased from Beijing Meyer.

Briefly, lipid material was dissolved in ethanol solution, PAT was dissolved in enzyme-free water, these materials were mixed using Genopti, and then sonicated at 40 ℃ for 20min at a sonication frequency of 150w,40khz to obtain a nanoliposome complex, PAT/Mix.

The specific method comprises the following steps: preparing fresh lipid working solution in ethanol, weighing 5mg of DNCA and CLD, and dissolving in 673 μ l and 438 μ l of ethanol respectively to form 10mM lipid stock solution; 1mg of PEG2000-DSPE was dissolved in 13.5ml of ethanol to form a 10. Mu.M stock solution, and sonicated at 37 ℃ for 5 minutes to form a homogeneous lipid mixture. Solution A was prepared by dissolving 2.15. Mu.l DNCA, 1.4. Mu.l CLD and 3.55. Mu.l PEG2000-DSPE in 76.4. Mu.l Genopti buffer. 5mg of the dry drug powder of PAT was dissolved in 143. Mu.l of enzyme-free water to form a 10mM working solution, and 22.86. Mu.l of the working solution was added to 57. Mu.l of Genopti buffer to form solution B. And uniformly mixing the solution A and the solution B, and carrying out ultrasonic treatment at 40 ℃ for 20min to form the lipid complex PAT/Mix.

FIG. 1 is an exemplary Transmission Electron Micrograph (TEM) of PATPAT and empty vector prepared by the above method, showing that PAT/Mix are uniform dense spherical particles with a diameter of about 100nm; dynamic Light Scattering (DLS) results showed a particle surface charge of-9 mV and a diameter of 220nm. Since the sample is dehydrated and dried before TEM photography, the particles shrink due to water loss, and DLS measures the hydrated particle size of the nanoparticles, the particle size measured by TEM is smaller than that measured by DLS.

Example 2CCK-8 method to examine the tumor cell proliferation inhibiting ability of PAT/Mix with different preparation ratios and concentrations in A549 and BCPAP cells

1. Sample preparation: genopti and PAT encapsulated in different lipid ratios were used to prepare corresponding AT/Mix formulations as described in example 1.

2. The method comprises the following steps:

adding appropriate amount of pancreatin solution into tumor cells in logarithmic phase for digestion to prepare single cell suspension. Cell concentration was adjusted to inoculate 96-well plates. The A549 plating density was 4000/well and BCPAP was 8000/well. At 37 ℃ C, 5% CO ₂ After culturing for 18-24h under the condition, the corresponding preparation shown in the figure is dripped into the cell sap, and Genopti is set as a blank control group. Culturing at 37 ℃ for 48h, and treating the cells: the medium was discarded, and 10. Mu.L of LCCK-8 reagent and 90. Mu.L of the culture medium were added to each well, and after incubation at 37 ℃ for 1 hour, the light absorption value was measured with a microplate reader.

3. As a result:

when PAT was administered at a concentration of 1mg/mL, all of the five formulations showed significant inhibition (-70-80%) of A549 (human lung adenocarcinoma cell line) and BCPAP (human thyroid cancer cell line) in the five formulations (FIG. 2A), and the lipid material was essentially non-toxic in a variety of tumor cell lines (FIG. 2B). The best effect is achieved when DNCA/CLD is 10/10 mug/ml (FIG. 2A) by combining the dosage and toxicity of lipid materials. Moderate gradients decreased the dose of PAT administered, controlled DNCA/CLD at 10/10. Mu.g/mL, with a substantially concentration-dependent trend of inhibition, with optimal efficacy at1mg/mL and substantial inefficiency at 0.1mg/mL (FIG. 2C).

Polyethylene glycol (PEG) can enhance the systemic stability of liposomes of cations and mixtures thereof, protect the lipid complexes from interaction with blood components and capture by macrophages, and thus prolong their residence time in the blood circulation. DSPEPEG-2000 is incorporated into DNCA/CLD liposomes to attempt to prolong the future residence time of the drug in the animal without altering the efficacy of the drug. The results show that when the content of PEG is 0.1 percent of the sum of the DNCA/CLD substances, a better entrapment effect can be displayed, and the killing capacity of the tumor is equivalent to that of the unincorporated substance.

Example 3 examination of the effects of PAT/Mix preparations on a549 cell cycle and apoptosis by flow cytometry;

representative scatter plots (a) and mean histograms (B) of three independent experiments for flow cytometry detection of apoptosis in PAT (1 mg/mL)/Mix administered 24hA549 cells; C. the cycle-blocking effect of PAT (1 mg/mL)/Mix administered for 24h on A549 cells was examined by flow cytometry.

1. Sample preparation: genopti, mix Liposomes, PAT/Mix, the corresponding PAT/Mix formulations were prepared as described in example 1.

2. The method comprises the following steps:

adding a proper amount of pancreatin solution into tumor cells in logarithmic growth phase for digestion to prepare single cell suspension, adjusting the cell concentration and inoculating the single cell suspension into a 12-hole plate, wherein the A549 plate laying density is 9 ten thousand per hole. After culturing for 18-24h at 37 ℃ and 5% CO2, the sample was added dropwise to the cell fluid, and Genopti was set as a blank control. After incubation at 37 ℃ for 24h, plates were removed and medium was transferred to 1.5ml EP tubes for each well.

Apoptosis assay: add 1ml Genopti to each well of the plate for rinsing once, add 200. Mu.l of pancreatin to digest the cells, take 800. Mu.l of the original culture medium in the EP tube to inactivate the pancreatin in the corresponding well, gently blow off the cells, transfer back to 1.5ml EP tube, and centrifuge at 4 ℃ 1000rpm for 3min. Discarding the supernatant, washing the cells 1-2 times with the culture medium, staining according to the apoptosis kit instruction, sieving, and detecting by flow cytometry.

Periodic experiment: the pancreatin in the corresponding wells was inactivated with the original medium in the EP tube, the cells were gently blown off and transferred back to 1.5ml EP tube, centrifuged at 1000rpm at 4 ℃ for 3min, and the supernatant carefully aspirated. Cells were resuspended using precooled Genopti, centrifuged at 1000rpm for 3min at 4 ℃ and the supernatant carefully aspirated. Resuspend the cells with 300. Mu.l precooled Genopti, add 700. Mu.l absolute ethanol to another EP tube, drop the cell suspension drop by drop into the absolute ethanol, blow and beat uniformly to prevent the cells from agglomerating, and finally fix the cells in 70% ethanol overnight (the EP tube is placed in a 4 ℃ refrigerator during fixation). After the cells were fixed, 500. Mu.l of precooled Genopti were added to the EP tube, centrifuged at 1000rpm at 4 ℃ for 3min and the supernatant carefully aspirated. Resuspend the cells with 1ml of precooled Genopti, centrifuge at 4 ℃ 1000rpm for 3min, carefully aspirate the supernatant and leave about 50. Mu.l of liquid to avoid aspiration of the cells. After staining according to the cell cycle kit instructions, sieving, flow cytometry detection.

3. As a result:

the apoptosis results show that: PAT (1 mg/mL)/Mix administration for 24h significantly promoted the apoptotic progression of a549 cells with 13.47%, 8.04% and 1.01% of cells that withered early, withered late and necrotic, respectively (fig. 3A and B).

The leftmost peak in the cycle test results plot represents the G0/G1 phase (dark purple, FIG. 3C): the G1 phase is the phase with the least DNA content, and DNA replication has not yet begun; the G0 phase is a cell quiescent phase, does not replicate DNA, and cannot be separated from the G1 phase. The second, not tall but very broad peak is the S phase (red): the period from the beginning of replication to the completion of replication is a process from one-fold DNA to two-fold DNA. The third peak (light purple) of the detection result graph is in a G2/M period: the G2 phase is the period of time from the completion of DNA replication to division, when the cell contains twice as much DNA; the M phase is a process of cell division, in which the cell is twice as much DNA and cannot be separated from the G2 phase.

The results show (fig. 3C) that the percentage of cells with different DNA content did not change significantly after PAT/Mix administration, and PAT/Mix had no significant cycle-arrest effect on a549 cells. PAT/Mix exerts an anti-tumor effect mainly through pro-apoptosis rather than cycle block.

EXAMPLE 4 examination of the Effect of PAT/Mix on in vitro activation of BMDCs

1. Sample preparation: PBS, mix, PAT (1 mg/mL) and PAT (1 mg/mL)/Mix groups, the corresponding PAT/Mix formulations were prepared as in example 1.

2. The method comprises the following steps:

bone marrow of tibia and femur of healthy C57bl/6 mouse was taken, immature DC cells were collected after hemolysis, cultured until the sixth day for plating, and subjected to 24h to detect expression of maturation markers (CD 80, CD40 and MHC II) on BMDCs cell surface by flow cytometry.

3. As a result:

the results show that the expression level of CD40 on the surface of BMDCs is obviously improved and the percentage of CD40+ cells is obviously increased after PAT and PAT/Mix treatment (figures 4A and B), the maturation of BMDCs cells is promoted, the antigen processing and presenting capacity of the cells is enhanced, and the PAT has certain tumor immunotherapy effect. No significant effect was seen on the other two mature markerCD80 and MHC II (fig. 4C and D).

Example 5 examination of the efficacy of PAT/Mix in A549 tumor-bearing mice

1. Sample preparation: genopti group, mix lipid material group, PAT6 mg/body group and PAT (6 mg)/Mix group, the corresponding PAT/Mix formulations were prepared as in example 1.

2. The method comprises the following steps:

an A549 subcutaneous transplantation tumor model is constructed in Balb/c-nude mice, female nude mice with the age of 4 weeks are selected, 300 ten thousand A549 cells are inoculated to each right axillary area for modeling, tumor bulges with the size of rice grains can be touched on the surface of the skin of the mice 7 days after inoculation, the tumors grow to 50mm < 3 > and are divided into four groups, the four groups comprise a Genopti group, a Mix lipid material group, a PAT6 mg/Mix group and a PAT (6 mg)/Mix group, 5 mice in each group start to be administrated, and the modeling process and the administration frequency are shown in figure 4A. During the treatment period, the differences in drug effects of the PAT/Mix, nude and Genopti groups were observed as tumor volumes (V =0.5 xl × W2) were measured in the different administration groups, and mice were sacrificed and dissected 12 days after the treatment, tumors were taken out and photographed by weighing, and the differences in tumor weights of the different treatment groups were compared.

3. As a result:

the results showed that PAT/Mix significantly inhibited tumor growth in tumor-bearing mice (FIG. 4B-D, P < 0.05), tumors were weighed in 12-day dissected mice, and tumor weights were significantly lower in the PAT/Mix group than in the Genopti group (P = 0.021)

And Mix group (P =0.007, fig. 5C). The weight of the mice slowly increased during the treatment period, the weight loss and the death of the mice caused by the excessive tumor burden or the drug toxicity are not seen, and the in vivo safety of the preparation is better (figure 5E).

The information shown and described in detail herein is sufficient to achieve the above-mentioned objects of the present invention, and therefore the preferred embodiments of the present invention represent the subject matter of the present invention, which is broadly encompassed by the present invention. The scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art, and is therefore not limited by anything other than the appended claims, in which the singular forms of an element used are not intended to mean "one and only" unless explicitly so stated, but rather "one or more. All structural, compositional, and functional equivalents to the elements of the above-described preferred embodiments and additional embodiments that are known to those of ordinary skill in the art are therefore incorporated herein by reference and are intended to be encompassed by the present claims.

Moreover, no apparatus or method is required to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. It will be apparent, however, to one skilled in the art that various changes and modifications in form, reagents and synthetic details may be made without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims (8)

1. A mixed lipid nanometer preparation of a pig spleen extract is characterized by comprising neutral cytidine lipid DNCA, cationic lipid CLD, PEG2000-DSPE, a GenOpti buffer system and a pig spleen extract.

2. The pig spleen extract mixed lipid nanometer preparation as claimed in claim 1, wherein the structure formula of the neutral cytidine lipid DNCA is shown as formula I:

I；

the structural formula of the cationic lipid CLD is shown as formula II:

II；

the structural formula of the PEG2000-DSPE is shown as the formula III:

，/>

III。

3. the mixed lipid nanometer preparation of the pig spleen extract as claimed in claim 1, wherein the pig spleen extract is spleen aminopeptide oral lyophilized powder.

4. The nano preparation of mixed lipid material of pig spleen extract as claimed in claim 1, wherein the nano preparation of mixed lipid material is prepared by the following steps: dissolving neutral cytidine lipid DNCA, cationic lipid CLD and PEG2000-DSPE in ethanol solution, dissolving spleen aminopeptide oral lyophilized powder in enzyme-free water, mixing the substances by using prepared Genopti, and performing ultrasonic treatment to obtain the mixed lipid nano preparation, namely PAT/Mix.

5. The pig spleen extract mixed lipid nano-preparation according to claim 4, wherein the dosage of the lipid in the preparation is DNCA: CLD = 10/10. Mu.g/ml, pig spleen extract 1mg/ml, PEG2000-DSPE incorporated at 0.1mol% of all lipids, and Genopti were used to mix these substances.

6. The use of the pig spleen extract mixed lipid material nano-preparation as claimed in any one of claims 1-5 in the preparation of anti-tumor drugs or reagents, either alone or in combination with other drugs.

7. The use of claim 6, wherein the tumor includes, but is not limited to, lung cancer and thyroid cancer.

8. The use according to claim 6, wherein the medicament or agent comprises unit preparations of different specifications, and the prevention or treatment of the disease is carried out by one or more administration routes of oral administration, intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection and the like through clinically feasible administration routes.

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