CN115634211A - Nanoparticles for delivering lotus plumule alkaloid by using human serum albumin as carrier, and preparation method and application thereof - Google Patents
Nanoparticles for delivering lotus plumule alkaloid by using human serum albumin as carrier, and preparation method and application thereof Download PDFInfo
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Images
Abstract
The invention discloses a nanoparticle for delivering lotus plumule alkaloid by using human serum albumin as a carrier, and a preparation method and application thereof. The preparation method has the advantages of simple preparation process, mild conditions and low cost, and the prepared nanoparticles have the advantages of high endogenous level, high targeting property, complementary treatment mechanism, biological safety and the like. The nano-drug delivery platform can realize the delivery of various hydrophobic traditional Chinese medicine monomers, realizes the cooperative target treatment of complex progressive diseases such as tumors and the like, and has good application prospect.
Description
Technical Field
The invention relates to a nano preparation, a preparation method and application thereof, in particular to a nano particle for delivering lotus plumule alkaloid by taking human serum albumin as a carrier, and a preparation method and application thereof.
Background
Tumors are progressive diseases which have great harm to human health, and the treatment difficulty is great. The clinical common cancer treatment methods, including surgical resection, radiotherapy, chemotherapy and the like, have the limitations of strong toxic and side effects, limited treatment effect and the like. Chemotherapy is the main means for treating breast cancer at present, but the clinical treatment effect is poor, the proliferation and the metastasis of tumors cannot be effectively inhibited, and in addition, the toxic and side effects of chemotherapy cause great pain to patients.
The lotus plumule is green embryo of mature seed of lotus of Nymphaeaceae, and is also an important traditional Chinese medicine with homology of medicine and food. The medicinal plant part represented by the lotus plumule is the basic stone of the traditional Chinese medicine in China. The lotus plumule has been recorded in the book of edible herbal medicine for the first time, "raw material is taken as powder, three coins are regulated by rice water, and blood thirst diseases and postpartum thirst diseases are treated". The compendium of materia medica also says "the lotus plumule can clear heart and remove heat". The lotus plumule is rich in alkaloid compounds, nearly 20 kinds of alkaloids which are separated and identified at present are obtained, and the methyl liensinine, liensinine and isoliensinine are important isoquinoline alkaloids. In recent years, lotus plumule alkaloid is proved to have treatment effect on partial tumors and great potential in treatment of diseases such as tumors, but is not applied to clinical treatment of cancers until now. The reasons for this are mainly as follows: 1. the water solubility is very poor, and the particle size is very large (more than 800 nm) after the traditional medicine carrying means such as liposome is carried. 2. When the single drug is used, the cytotoxicity is generally not high. 3. Has no targeting ability and insufficient drug accumulation concentration at tumor sites. Therefore, the targeted delivery of the lotus plumule alkaloid to the tumor part, the multi-target effect characteristic of the traditional Chinese medicine, and the search of the combined treatment scheme matched with the lotus plumule alkaloid is the key point for clinical application.
In recent years, with the rapid development of nanotechnology, the characteristics of high permeability and retention effect of a nano carrier on a tumor part are utilized, and a way for applying the lotus plumule alkaloid to clinic is expected to be opened up. Although the nano-drug has achieved a series of encouraging progress in tumor immunotherapy, the average enrichment of the nano-drug in tumor tissues is only about 0.7%, the targeting ability is seriously insufficient, and the nano-drug faces a plurality of problems such as premature ejaculation. Therefore, a delivery system and a technical means for realizing the high-efficiency delivery of the hydrophobic drug and the accurate targeted delivery to the pathological part and exerting the accurate targeted therapy of the traditional Chinese medicine monomer are in urgent need of development.
Albumin is a main component of serum protein, is a multifunctional drug delivery nano-carrier, and has good biocompatibility, gene editability and preparation stability due to the natural structural characteristics. HSA-PTX nano-drug (trade name) formed by combining Paclitaxel (PTX) and HSA
) Has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic breast cancer, non-small cell lung cancer and other diseases. In that
In clinical studies of (a), albumin-based nanoparticles have good tolerability. HSA can be used as an ideal carrier for loading hydrophobic drugs such as lotus plumule alkaloid.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide nanoparticles for delivering lotus plumule alkaloids by taking human serum albumin as a carrier so as to realize targeted delivery. The other purpose is to provide the preparation method of the nanoparticle for delivering the lotus plumule alkaloid by taking the human serum albumin as the carrier, which has the advantages of simple process, mild condition, low cost and high entrapment efficiency.
The technical scheme is as follows: the invention discloses a method for preparing nano-particles by using human serum albumin as a carrier to deliver lotus plumule alkaloids, which comprises the steps of utilizing two hydrophobic pockets of the human serum albumin to encapsulate the hydrophobic lotus plumule alkaloids and preparing the nano-particles by a mechanical stirring method.
The human serum albumin is nanoparticles for delivering the lotus plumule alkaloid as a carrier, and the hydrophobic lotus plumule alkaloid is selected from liensinine, neferine and isoliensinine.
The preparation method of the nanoparticle for delivering the lotus plumule alkaloid by taking the human serum albumin as the carrier comprises the following steps:
(1) Preparing an organic solution A containing lotus plumule alkaloid and a phosphoric acid buffer solution B containing human serum albumin;
(2) Taking a phosphoric acid buffer solution B, and slowly dropwise adding the solution A under the mild continuous stirring condition;
(3) Dialyzing in dialysis bag overnight to remove free drug.
The preparation method of the nano-particle for delivering the lotus plumule alkaloid by using the human serum albumin as the carrier comprises the following steps of (1) preparing a lotus plumule alkaloid solution and a human serum albumin solution, wherein the mass concentration ratio of the lotus plumule alkaloid solution to the human serum albumin solution is 40: 1-10: 1; preferably 20: 1 to 10: 1.
According to the preparation method of the nanoparticle for delivering the lotus plumule alkaloid by taking the human serum albumin as the carrier, in the step (2), the dropping speed is not more than 0.5mL/min.
The preparation method of the nanoparticle for delivering the lotus plumule alkaloid by using the human serum albumin as the carrier comprises the following steps of (2), wherein the mass ratio of the human serum albumin to the lotus plumule alkaloid is 1: 40-1: 10 after the dripping is finished; preferably 1: 20 to 1: 10.
The human serum albumin is used as a carrier to deliver the nano-particles of the lotus plumule alkaloid to the application of the nano-particles in the preparation of the antitumor drugs.
The tumor comprises breast cancer and leukemia.
The particle size of the nanoparticle for delivering the lotus plumule alkaloid by taking the human serum albumin as the carrier is preferably 130-150nm.
The invention exerts the bioactivity of the lotus plumule alkaloid, exerts the targeting performance of the carrier human serum albumin, carries out entrapment on the hydrophobic drug, fully utilizes the hydrophobic space in the carrier, delivers the drug to a tumor part in a targeting way, stimulates an immune system and realizes the antitumor activity. According to the invention, human serum albumin is used as a carrier to encapsulate lotus plumule alkaloid, and the obtained nanoparticles of the human serum albumin delivery lotus plumule alkaloid are prepared by a mechanical stirring method, so that the problems of low bioavailability, weak targeting capability, single treatment mechanism, limited carrier loading capability and the like of hydrophobic alkaloid can be effectively improved. It has the following advantages:
(1) Highly endogenous: human serum albumin which is natural source of human serum is used as a carrier, and the carrier has high endogenous property.
(2) High targeting property: the preparation with the particle size of less than 200nm has passive targeting property and is easy to gather at the tumor tissue part; human serum albumin can bind to glycoprotein 60 (gp 60) in the tumor microenvironment, enhancing tumor targeting ability through receptor-mediated endocytosis.
(3) The biological safety is as follows: the plumula Nelumbinis alkaloid is natural plant extract, has no obvious toxicity to normal cells, and has high endogenous level of human serum albumin.
Has the advantages that: compared with the prior art, the invention has the following advantages: (1) The invention adopts a simple mechanical stirring method to prepare the nano-particles for delivering the lotus plumule alkaloid by the human serum albumin in one step, and has simple method, low cost and mild condition; (2) The nanoparticle for delivering the lotus plumule alkaloid by the human serum albumin provided by the method has high endogenous and biological safety; (3) The nanoparticles for delivering the lotus plumule alkaloid by the human serum albumin provided by the invention can provide a simple and feasible entrapment method for delivery of hydrophobic traditional Chinese medicine monomers by loading different fat-soluble lotus plumule alkaloids. (4) The human serum albumin nanoparticle drug loading space for delivering the lotus plumule alkaloid provided by the invention is large, the entrapment rate is high, and compared with other entrapment means such as exosomes and the like, the human serum albumin nanoparticle drug loading space can be used for loading more therapeutic drugs. The bionic nanoparticle for homologous recombination exosome multi-drug delivery provided by the invention can complete delivery of various lotus plumule alkaloids, the nano preparation has the characteristics of high endogenous property, high targeting property, biological safety and the like, a delivery system of human serum albumin entrapped hydrophobic drugs is successfully constructed, a model is provided for construction of efficient and safe targeted therapy of tumors and other neurodegenerative diseases, and the bionic nanoparticle has a wide application prospect and clinical transformation potential.
Drawings
FIG. 1 is a graph showing the determination of the optimum charge ratio of Nef @ HSA, lien @ HSA and Iso @ HSA in examples 1 to 3;
FIG. 2 is TEM electron micrograph of optimum charge ratio of Iso @ HSA (A), nef @ HSA (B), lien @ HSA (C) in examples 1-3;
FIG. 3 is the cytotoxicity test of Nef and Nef @ HSA (A), iso and Iso @ HSA (B), lien and Lien @ HSA (C) on normal cells in 1.1 of example 4;
FIG. 4 is the cytotoxicity assay of Nef and Nef @ HSA on cancer cells in 1.2 of example 4;
FIG. 5 is the apoptosis assay of Nef, lien, iso and the corresponding nanoparticles in 1.3 of example 4;
FIG. 6 is an experiment on the generation of active oxygen by Nef and Nef @ HSA in 1.4 of example 4 on cancer cells;
FIG. 7 is a water solubility study of Nef and Nef @ HSA in 1.5 of example 4;
FIG. 8 shows the results of the molecular docking of Nef (A, B), iso (C, D) and Lien (E, F) with the A and B chains of HSA, respectively, in example 4, 1.6.
Detailed Description
Example 1: preparation process of Human Serum Albumin (HSA) nano-particles carrying Nef
Taking 20mg of Nef powder, and adding 1mL of methanol solution to prepare 20mg/mL of Nef mother solution; HSA powder (10 mg) was taken and 10mL of phosphate buffer solution (PBS, pH7.4, 0.01M) was added to prepare a 1mg/mL HSA stock solution. Slowly dripping the Nef mother liquor into the HSA mother liquor under the condition of soft stirring, stirring for 20min until the system is uniform, collecting the reaction liquid, putting the reaction liquid into a dialysis bag, dialyzing overnight, and removing free medicines to obtain the product.
The dosage of Nef and HSA used for preparing the nanoparticles is inspected, the formula is specifically inspected and shown in a table 1-1, and a proper amount of the preparation is taken, and the particle size and PDI of the preparation are measured by a Malvern dynamic light scattering instrument. Sucking 200 μ L of the prepared nanoparticle solution, adding 10 times volume of acetonitrile, vigorously vortexing for 10min, centrifuging to obtain supernatant, filtering with 0.22 μm organic filter membrane, and determining Nef Encapsulation Efficiency (EE) in the preparation by High Performance Liquid Chromatography (HPLC).
Table 1-1 dosage finding prescription
In the preparations prepared by the three prescriptions, the preparation particle size of the prescription 3 is larger, the preparation particle sizes prepared by the prescription 1 and the prescription 2 are in a proper range, but the drug-loading rate of the lotus plumule alkaloid in the prescription 1 is lower than 10 percent, so the preparation prescription is selected as the preparation with the molar ratio of HSA to Nef of 1: 20.
Example 2: preparation process of Lien-loaded Human Serum Albumin (HSA) nanoparticles
Taking 20mg of Lien powder, and adding 1mL of methanol solution to prepare 20mg/mL of Lien mother solution; HSA powder (10 mg) was taken and 10mL of phosphate buffer solution (PBS, pH7.4, 0.01M) was added to prepare a 1mg/mL HSA stock solution. Slowly dripping the Lien mother liquor into the HSA mother liquor under the condition of soft stirring, stirring for 20min until the system is uniform, collecting the reaction liquid, putting the reaction liquid into a dialysis bag, dialyzing overnight, and removing free medicines to obtain the final product.
The dosage of Lien and HSA used for preparing the nanoparticles is inspected, the formula is specifically inspected in a table 1-2, and a proper amount of preparation is taken, and the particle size and PDI of the preparation are measured by a Malvern dynamic light scattering instrument. Sucking 200 μ L of the prepared nanoparticle solution, adding 10 times volume of acetonitrile, vigorously vortexing for 10min, centrifuging to obtain supernatant, filtering with 0.22 μm organic filter membrane, and determining the Encapsulation Efficiency (EE) of Lien in the preparation by High Performance Liquid Chromatography (HPLC).
Table 1-2 dosage finding prescription
In the preparations prepared by the three formulas, the prepared particle sizes of the formula 2 and the formula 3 are both larger, so that the molar ratio of HSA to Lien is 1: 10.
Example 3: preparation process of isoparagonine (Iso) -loaded Human Serum Albumin (HSA) nanoparticles
20mg of Iso powder is taken and added with 1mL of methanol solution to prepare 20mg/mL of Lien mother solution; HSA powder (10 mg) was taken and 10mL of phosphate buffer solution (PBS, pH7.4, 0.01M) was added to prepare a 1mg/mL HSA stock solution. Slowly dripping Iso mother liquor into HSA mother liquor under the condition of soft stirring, stirring for 20min until the system is uniform, collecting reaction liquid, putting into a dialysis bag, dialyzing overnight, and removing free drug to obtain the final product.
The dosage of Iso and HSA used for preparing the nanoparticles is inspected, the prescription is specifically inspected in tables 1-3, and a proper amount of the preparation is taken, and the particle size and PDI of the preparation are measured by a Malvern dynamic light scattering instrument. Sucking 200 mu L of the prepared nanoparticle solution, adding 10 times of acetonitrile, violently whirling for 10min, centrifuging to obtain a supernatant, filtering by using a 0.22 mu m organic system filter membrane, and measuring the Encapsulation Efficiency (EE) of Iso in the preparation by using a High Performance Liquid Chromatography (HPLC).
Table 1-3 dosage visit recipe
In the preparation prepared by the three prescriptions, the prepared particle sizes of the prescription 2 and the prescription 3 are both larger, so that the molar ratio of HSA to Iso is 1: 10.
The particle size of the nanoparticles was measured at the optimum charge ratio obtained in examples 1-3, and the results are shown in FIG. 1. And the nanoparticles with the optimal charge ratio obtained in examples 1-3 were placed under an electron microscope, and the results are shown in FIG. 2.
Example 4: property investigation of delivery of multiple lotus plumule alkaloid nanoparticles by using human serum albumin as carrier
Selecting an optimal prescription to prepare the nanoparticles, and performing the following investigation:
1.1 MTT method for investigating toxicity of nanoparticles on normal human mononuclear cells (PBMCs)
Separating PBMCs from human whole blood, transferring 10mL of whole blood into a centrifuge tube, adding 10mL of PBS solution, diluting and mixing uniformly. Adding 5mL of Ficoll solution into a centrifuge tube, slowly adding diluted blood into the upper layer of the Ficoll solution, centrifuging at 2,000rpm for 20min, sucking white cell layer liquid containing PBMCs, adding PBS to 15mLCentrifuging at 1,500 rpm for 10min, removing supernatant, adding culture medium, cleaning, and suspending cells with 5-10ml of culture medium to obtain PBMCs cell suspension; taking PBMCs cells in logarithmic growth phase, and completely suspending the PBMCs cells in culture medium (5 x 10) 4 One/well) were seeded in 96-well plates at 100. Mu.L per well. Standing at 37 deg.C, 5% CO 2 And incubated overnight in the incubator. After the cells are attached to the wall, the culture medium is sucked off, 100 mu L of serum-free culture medium is added into a blank hole, and Nef, lien and Iso with gradient alkaloid concentration and corresponding nanoparticles are added into an administration hole. 24h later, 10. Mu.L of MTT (5 mg/mL, PBS dissolved), 37 ℃,5% 2 Incubate for 4h. Removing the solution from each well by suction, adding DMSO 100 μ L, shaking in constant temperature shaking table at 37 deg.C for 10min, measuring absorbance (A) with microplate reader at 490nm, and calculating cell survival rate.
Cell viability (cell viability) = [ a = [) Administration of drugs -A Blank space /(A Control -A Blank space )]×100％
The results are shown in FIG. 3. 64 μmol/L of free Nef, lien and Iso reduced cell viability to 27.54%,26.53% and 25.13%, respectively. However, nanoparticles prepared by packaging Nef, lien and Iso separately into HSA have a weak effect on attenuating cell activity. Shows that the nano-particle loaded lotus plumule alkaloid can reduce the toxicity of the lotus plumule alkaloid on normal cells and improve the biological safety and biocompatibility.
1.2CCK-8 method to investigate the toxicity of nanoparticles on triple negative breast cancer cells (4T 1)
4T1 cells from the logarithmic growth phase were taken and resuspended in complete medium (8X 10) 4 One/well) were seeded in 96-well plates at 100. Mu.L per well. Standing at 37 deg.C, 5% CO 2 Was incubated overnight in the incubator of (1). After the cells were attached to the wall, the medium was aspirated off, 100. Mu.L of serum-free medium was added to the blank wells, and free Nef and Nef @ HSA were added to the administration wells at the same Nef concentration. After 24h, removing the solution from each well by suction, adding 100 μ L of CCK-8 working solution (10 μ L of CCK-8 is added into 1mL of DMEM medium, mixing uniformly and then being used as prepared), incubating at 37 ℃ for 2h, measuring absorbance (A) under 450nm of an enzyme-labeling instrument, and calculating the cell survival rate.
Cell viability (= a) = a Administration of drugs -A Blank space /(A Control -A Blank space )]×100％
Results As shown in FIG. 4, IC of Nef after 24h of treatment 50 IC at 16.65. Mu.M, nef @ HSA 50 3.81 mu M, and the toxicity of the HSA-coated nanoparticles to tumor cells is obviously improved.
1.3 In-vitro cytotoxicity of nanoparticles inspected by Annexin V/PI staining method
Jurkat cells were harvested at logarithmic growth phase and resuspended in complete medium (5X 10) 3 One/well) were seeded in 96-well plates at 100. Mu.L per well. Standing at 37 deg.C, 5% CO 2 And incubated overnight in the incubator. After the cells are attached to the wall, the culture medium is removed by suction, 100 mu L of serum-free culture medium is added into a blank hole, and the monomer drug and the HSA-coated nanoparticles with the same concentration are added into an administration hole. After 24h, removing the solution from each well by suction, adding 100. Mu.L of Annexin V/PI fluorescence working solution, incubating at 37 ℃ for 20min, and detecting the apoptosis of each group of cells by flow cytometry.
The results are shown in fig. 5, after 24h treatment, nef, lien and Iso had negligible killing effect on tumor cells, while the corresponding nanoparticle groups had good killing effect on tumor cells.
1.4 flow cytometry investigation of influence of nanoparticles on production of active oxygen by tumor cells
4T1 cells from the logarithmic growth phase were taken and resuspended in complete medium (5X 10) 4 One/well) were seeded in 6-well plates, 2mL per well. Standing at 37 deg.C, 5% CO 2 And incubated overnight in the incubator. After the cells are attached to the wall, the culture medium is sucked off, 2mL of serum-free culture medium is added into a blank hole, and Nef @ HSA with the same concentration are added into an administration hole. After the administration of the drugs for 0h, 2h,4h,6h,8h and 12h, the solution in each hole is sucked, DCFH-DA fluorescence working solution is added, the mixture is incubated at 37 ℃ for 20min, and the apoptosis condition of each group of cells is detected by flow cytometry.
As shown in FIG. 6, with the increase of the administration time, the level of active oxygen produced by 4T1 cells under Nef @ HSA stimulation was much higher than that of Nef group, probably due to the increased targeting of HSA vector to cells.
1.5 Water solubility of Nef and Nef @ HSA
Taking 20mg of Nef powder, and adding 1mL of methanol solution to prepare 20mg/mL of Nef mother solution; HSA powder (10 mg) was taken and 10mL of phosphate buffer solution (PBS, pH7.4, 0.01M) was added to prepare a 1mg/mL HSA stock solution. Slowly dripping the Nef mother liquor into HSA mother liquor and PBS respectively under soft stirring, and stirring for 20min until the system is uniform to obtain Nef @ HSA aqueous solution (FIG. 7, left) and Nef aqueous solution (FIG. 7, right). It was observed that the Nef aqueous solution had significant precipitation and particles, while the Nef @ HSA solution was uniformly clear. HSA loading increased the aqueous solubility of Nef.
1.6 Nef, iso, lien and HSA molecular docking investigation on nanoparticle stability
Nef, iso and Lien are respectively butted with two hydrophobic pockets of HSA, and the stability of the HSA loaded with three alkaloids to form the nano-particles is inspected. The binding energies of the three alkaloids to the HSAA and B chains are shown in table 2.
TABLE 2 binding energy of Nef, iso, lien to HSAA and B chains
The results of Nef docking with HSAA chain and B chain molecules are shown in FIG. 8A, B, the results of Iso docking with HSAA chain and B chain molecules are shown in FIG. 8C, D, and the results of Lien docking with HSA chain and B chain molecules are shown in FIG. 8E, F. It can be seen that drug molecules are combined with hydrophobic interaction between hydrophobic domains on HSA, and the lotus plumule alkaloid monomer can be encapsulated by the hydrophobic pocket of HSA to form nanoparticles.
Claims (8)
1. A nanoparticle for delivering lotus plumule alkaloids by using human serum albumin as a carrier is characterized in that two hydrophobic pockets of the human serum albumin are used for encapsulating the hydrophobic lotus plumule alkaloids, and the nanoparticle is prepared by a mechanical stirring method.
2. The human serum albumin nanoparticle for delivering the liensinine alkaloid by using the carrier according to claim 1, wherein the hydrophobic liensinine alkaloid is selected from liensinine, neferine and isoliensinine.
3. The method for preparing nanoparticles for delivering lotus plumule alkaloids by using human serum albumin as a carrier according to claim 1, comprising the following steps:
(1) Preparing an organic solution A containing lotus plumule alkaloid and a phosphoric acid buffer solution B containing human serum albumin;
(2) Taking a phosphoric acid buffer solution B, and slowly dropwise adding the solution A under the mild and continuous stirring condition;
(3) Dialyzing in dialysis bag overnight to remove free drug.
4. The method for preparing nanoparticles for delivering lotus plumule alkaloids by using human serum albumin as a carrier according to claim 3, wherein the mass concentration ratio of the lotus plumule alkaloid solution prepared in the step (1) to the human serum albumin solution is 40: 1-10: 1.
5. The method for preparing nanoparticles of human serum albumin as a carrier to deliver the liensinine alkaloid according to claim 3, wherein in the step (2), the dropping speed is not more than 0.5mL/min.
6. The method for preparing nanoparticles for delivering lotus plumule alkaloids by using human serum albumin as a carrier according to claim 3, wherein in the step (2), the mass ratio of the human serum albumin to the lotus plumule alkaloids after the dripping is finished is 1: 40-1: 10.
7. The use of nanoparticles of human serum albumin as a carrier for delivery of plumula Nelumbinis alkaloids in the preparation of anti-tumor drugs according to claim 1.
8. The use of claim 7, wherein the tumor comprises breast cancer, leukemia.
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| CN102048695A (en) * | 2009-08-11 | 2011-05-11 | 南京大学 | Preparation method of protein nanoparticle for in vivo delivery of pharmacologically active agent |
| CN112933052A (en) * | 2021-01-25 | 2021-06-11 | 西安交通大学医学院第一附属医院 | Nano drug delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy |
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| CN102048695A (en) * | 2009-08-11 | 2011-05-11 | 南京大学 | Preparation method of protein nanoparticle for in vivo delivery of pharmacologically active agent |
| CN112933052A (en) * | 2021-01-25 | 2021-06-11 | 西安交通大学医学院第一附属医院 | Nano drug delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy |
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