Bionic liposome loaded with boric acid quantum dots as well as preparation method and application of bionic liposome

Abstract

The invention discloses a bionic liposome loaded with boric acid quantum dots and a preparation method and application thereof, and belongs to the technical field of biomedicine, the material is synthesized through a film hydration method, the reaction condition is mild, and the synthesis efficiency is high. The synthesized bionic liposome loaded with boric acid quantum dots is uniform in particle size and good in single dispersity. The bionic liposome loaded with boric acid quantum dots has good stability, high drug loading capacity, tumor targeting capacity and excellent biocompatibility.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a bionic liposome loaded with boric acid quantum dots, and a preparation method and application thereof. Background Art

[0002] Cancer has always been a major problem for global public health. Currently, chemotherapy is still one of the standard strategies for cancer treatment, but non-selective biodistribution and severe side effects often lead to poor treatment effects. Boron neutron capture therapy (BNCT) is a new binary radiotherapy targeted treatment technology. The principle of BNCT is to inject a radioactive substance containing the isotope boron-10 ( 10 B) is introduced into the body, causing it to rapidly accumulate in tumor cells, and then thermal neutrons slowed down by normal tissues are used to 10 B undergoes a capture reaction, producing high linear energy transfer (LET) α particles and 7 Li nuclei, these high LET particles can break the double helix of tumor cell DNA, causing irreversible damage and death of tumor cells. Since the range of the particles is only about 10 microns, which is about the diameter of a cell, it can achieve targeted killing of tumor cells at the cellular level without affecting surrounding normal cells. In addition, since the reactivity of thermal neutrons with elements rich in the human body (such as carbon, hydrogen, oxygen, and nitrogen) is much lower than that with 10 The reactivity of B avoids the severe damage to healthy body tissues caused by traditional radiotherapy, achieving the effect of precise treatment.

[0003] Whether BNCT can achieve precise destruction of tumor cells depends entirely on 10 Whether B atoms can be enriched in tumor cells with high concentration and high selectivity is the mission of boron carriers, which also shows that the development of efficient boron carriers is crucial to the development of BNCT. At present, dihydroxyboron phenylalanine (BPA) and disodium thiododecaborane (BSH) are mainly used as neutron capture agents for BNCT in clinical practice. BPA has a certain affinity for tumors, but its water solubility is poor. Generally, it needs to be dissolved with a strong base and then fructose is added to form a complex. Then the pH of the solution is adjusted to neutral. The complex has poor stability and a short storage time, and BPA is easily dissociated and separated. Although BSH has good water solubility and high boron enrichment, it does not have tumor affinity and cannot target tumors. Therefore, actively exploring a boron delivery system that combines high boron content, tumor-specific targeting, and good biocompatibility is of great significance to achieve the precise application of BNCT strategies, achieve individualized treatment goals, reduce side effects, and promote the application of BNCT.

[0004] The development of biomimetic carbon quantum dot nanomaterials based on functional nanotechnology is of great significance to improve its clinical application. Carbon quantum dots (CDs) are a type of material with a size less than 10 nm, a graphite-like structure, and significant properties such as biocompatibility and good water solubility. However, the rapid metabolism in the body caused by its ultra-small particle size prevents the matching between the boron dose in the tumor and the thermal neutron exposure time of the BNCT duration, which greatly limits its further clinical application for BNCT. Therefore, further modification is necessary.

[0005] Specifically include: (1) The small particle size of carbon quantum dots and their rapid metabolism in the body prevent the matching between the boron dose in the tumor and the thermal neutron exposure time of the BNCT duration.

[0006] (2) Insufficient targeting and functionality. Targeting to tumor tissue is the key to achieving successful BNCT. Summary of the invention

[0007] Based on the above background, the present invention has developed a bionic liposome loaded with boric acid quantum dots and its preparation method and application. Experimental results show that the nano drug particles developed by the present invention have uniform size, high drug encapsulation efficiency, and tumor-specific targeting, and can be used as a new nano drug delivery platform for boron neutron capture therapy.

[0008] The present invention is achieved through the following technical solutions: A bionic liposome loaded with boric acid quantum dots is prepared by the following steps: Step 1, preparing boric acid quantum dots; Step 2, preparing tumor cell membranes; Step 3, preparing bionic liposomes loaded with boric acid quantum dots.

[0009] A method for preparing biomimetic liposomes loaded with boric acid quantum dots comprises the following steps: Step 1, preparing boric acid quantum dots: adding glucose and boric acid into water, reacting and mixing at 170°C-200°C, centrifuging, dialysis purification, and freeze-drying to obtain boric acid quantum dots; Step 2, preparing tumor cell membranes: culturing tumor cells, washing, digesting, blowing cells, collecting cells by centrifugation, adding membrane protein extraction reagent, repeatedly freezing and thawing in liquid nitrogen and room temperature to break the cells, centrifuging cell membrane fragments, adding solvent to dilute and mix to obtain tumor cell membranes; Step 3, preparing bionic liposomes loaded with boric acid quantum dots: dissolving lipids in anhydrous ethanol, and rotary evaporating under reduced pressure to obtain a uniform translucent film, adding the boric acid quantum dots prepared in step 1 and the tumor cell membrane prepared in step 2 to dehydrate, and after hydration, the polycarbonate membrane is used to obtain bionic liposomes loaded with boric acid quantum dots.

[0010] In the above technical solution, in step 1, the reaction mixing time is 15-20 hours, the dialysis uses a dialysis bag with a molecular cutoff of 500Da, and the dialysis purification time is 20-30 hours.

[0011] In the above technical solution, in step 2, the tumor cells are 4T1 mouse breast cancer cells.

[0012] In the above technical solution, in step 2, the membrane protein extraction reagent contains phenylmethylsulfonyl fluoride with a final concentration of 1 mM.

[0013] In the above technical solution, in step 3, the pore size of the polycarbonate membrane is 100 nm.

[0014] A bionic liposome loaded with boric acid quantum dots and a preparation method thereof and its application in boron neutron capture therapy.

[0015] Beneficial effects: (1) The synthesis method is stable and has high yield.

[0016] (2) Uniform size, good monodispersity, good stability, high drug loading capacity, and tumor targeting ability.

[0017] (3) The surface is coated with homologous tumor cell membrane, which provides immune escape, improves biocompatibility, and avoids the occurrence of side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 TEM schematic diagram of biomimetic liposome nanoparticles loaded with boric acid quantum dots.

[0019] Figure 2 Fluorescence co-localization images of tumor cell membrane (DIO) and liposomes (DIR) in tumor cell membrane / liposomes.

[0020] Figure 3 SDS-PAGE protein analysis of biomimetic liposome nanoparticles loaded with boric acid quantum dots.

[0021] Figure 4 This is a curve chart showing the change in hydrated particle size of bionic liposome nanoparticles loaded with boric acid quantum dots in different systems within 7 days.

[0022] Figure 5Cell viability after 4T1 cells were co-incubated with biomimetic liposome nanoparticles loaded with boric acid quantum dots for 24 hours.

[0023] Figure 6 is the hemolysis rate of biomimetic liposome nanoparticles loaded with boric acid quantum dots.

[0024] Figure 7 Hematoxylin and eosin staining results of biomimetic liposome nanoparticles loaded with boric acid quantum dots.

[0025] Figure 8 This is the fluorescence imaging of bionic liposome nanoparticles loaded with boric acid quantum dots after being absorbed by 4T1 and HUVECs cells.

[0026] Fig. 9 Fluorescence imaging of tumor-targeted enrichment after biomimetic liposome nanoparticles loaded with boric acid quantum dots were administered through the rat tail vein (the red circle represents the subcutaneous 4T1 tumor on the right thigh).

[0027] Fig.10 This is an in vitro picture of major organs and tumors after injection of biomimetic liposome nanoparticles loaded with boric acid quantum dots.

[0028] Fig.11 This is the in vitro ROI intensity analysis diagram of major organs and tumors. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0030] Example The synthesis steps of a biomimetic liposome loaded with boric acid quantum dots are as follows: 1. Synthesis of boric acid quantum dots: Glucose and boric acid in a molar ratio of 1:6-7 are added to deionized water, and the mixture is stirred to obtain a mixed solution. Then the mixed solution of glucose and boric acid is added to a reactor, and reacted at 170°C-200°C for 15-20 hours to obtain a reaction mixture. The reaction mixture is centrifuged and then dialyzed and purified for 20-30 hours using a 500Da molecular cutoff dialysis bag. The obtained dialyzed liquid is freeze-dried to obtain a powdered product. That is, boric acid quantum dot nanoparticles, which can theoretically scale up the reaction to kilograms.

[0031] 2. Preparation of tumor cell membrane: Cultivate 4T1 cells. When the cell density is about 90%, wash the cells twice with pre-cooled PBS, add an appropriate amount of enzyme-free cell digestion solution to digest the cells so that the cells are no longer tightly attached to the wall, and blow the cells with a pipette. Centrifuge at 4℃, 600g for 5 min to precipitate and collect the cells. Gently resuspend the cell pellet with an appropriate amount of pre-cooled PBS, take a small amount for cell counting, and centrifuge the remaining cells at 4℃, 600g for 1 min to precipitate the cells. Discard the supernatant, then centrifuge at 4℃, 600g for 1 min to precipitate the residual liquid on the wall of the centrifuge tube and further precipitate the cells. Add 1 mL of membrane protein extraction reagent A (containing PMSF (Phenyl methane sulfonyl fluoride) with a final concentration of 1 mM) to 20-50 million cells, gently and fully suspend the cells, and place them in an ice bath for 10-15 min. Repeat freezing and thawing in liquid nitrogen and room temperature for about 5 times, take a small amount of cell samples and observe the degree of cell disruption under a microscope. The number of freezing and thawing can be increased until the degree of cell disruption is greater than 70%. Centrifuge at 700 g for 10 min at 4℃, and carefully collect the supernatant into a new centrifuge tube. Do not touch the precipitate when aspirating the supernatant. About 30-50 µL of supernatant liquid can be left without aspirating. Centrifuge at 14000 g for 30 min at 4℃ to precipitate cell membrane fragments. Take 1 mL1×PBS to blow the precipitate to mix it thoroughly, and transfer it to a new 1.5 mL EP tube. It can be stored at -80℃ for long-term use.

[0032] 3. Preparation of biomimetic liposomes loaded with boric acid quantum dots: The analytical balance accurately weighs the prescribed amount of lipids. The molar ratio of DOTAP, DSPC, and cholesterol in the lipids is DOTAP: DSPC: cholesterol = 5: 4: 1. Each component is placed in a round-bottom flask, and then 5 mL of anhydrous ethanol is added to completely dissolve each component. It is placed on a rotary evaporator, and the vacuum rotary evaporation is carried out at 30 ° C, with a speed of 30 rpm to obtain a uniform translucent film. Then 2 mL of boric acid quantum dot solution containing 4T1 cell membrane is added to the round-bottom flask to dehydrate the membrane. After hydration is completed, the liposomes fused with tumor cell membranes are obtained by passing through a 100 nm polycarbonate membrane.

[0033] The bionic liposome material loaded with boric acid quantum dots synthesized by this method, transmission electron microscope (TEM) images show that the bionic liposome loaded with boric acid quantum dots is spherical, and the particle size is similar to the DLS measurement result, and has good dispersibility ( Figure 1 ). Laser confocal microscopy showed that the red fluorescent probe DIR-labeled lipids and the green fluorescent probe DIO-labeled tumor cell membranes co-localized in CM / LB, indicating that the lipids and tumor cell membranes had successfully fused ( Figure 2 ). SDS-PAGE data showed that the nanomaterial retained the proteins on the tumor cell membrane ( Figure 3 ). The BA-CDs encapsulation efficiency and drug loading of CM / LBA-CDs were confirmed by ICP-MS to be 30.53 ± 1.21% and 89.13 ± 1.33%, respectively, which are much higher than the drug loading of general nanocarriers. It is worth noting that the hydrated particle size of the bionic liposomes loaded with boric acid quantum dots remained almost unchanged after incubation in different systems for 7 days, indicating that it has good stability ( Figure 4 ).

[0034] The cell and blood compatibility of the nanoparticles were evaluated by CCK-8 and hemolysis assays, respectively. After incubation with 100 ppm [B] of the boron delivery agent for 24 h, the survival rate of 4T1 cells was still above 80% ( Figure 5 ). Even when the boron content in the blood was as high as 500 ppm [B], no hemolysis was detected. This proves that it has good blood safety ( Figure 6 Hematoxylin and eosin (H&E) staining of major organs showed no significant histological variability compared with control mice 1 week after a single injection of the boron carrier ( Figure 7 ).

[0035] The biomimetic liposomes loaded with boric acid quantum dots were co-cultured with human umbilical vein endothelial cells (HUVECs) and 4T1 and observed with a confocal laser scanning microscopy (CLSM). It was found that the content of biomimetic liposomes loaded with boric acid quantum dots in 4T1 was significantly higher than that in HUVECs. This proves that it has in vitro tumor targeting ( Figure 8 The tumor-targeted accumulation of nanoparticles was studied using subcutaneous tumors of mice bearing triple-negative breast tumor 4T1 xenografts. Fluorescence imaging results Fig. 9 , Fig.10 and Fig.11 It was shown that the nanoparticles can be targeted and accumulated at the tumor site.

[0036] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A biomimetic liposome loaded with boric acid quantum dots, characterized in that: The method is prepared by the following steps: step 1, preparing boric acid quantum dots; step 2, preparing tumor cell membranes; and step 3, preparing bionic liposomes loaded with boric acid quantum dots.

2. A method for preparing biomimetic liposomes loaded with boric acid quantum dots, characterized in that: The steps include: Step 1, preparing boric acid quantum dots: adding glucose and boric acid into water, reacting and mixing at 170°C-200°C, centrifuging, dialysis purification, and freeze-drying to obtain boric acid quantum dots; Step 2, preparing tumor cell membranes: culturing tumor cells, washing, digesting, blowing cells, collecting cells by centrifugation, adding membrane protein extraction reagent, repeatedly freezing and thawing in liquid nitrogen and room temperature to break the cells, centrifuging cell membrane fragments, adding solvent to dilute and mix to obtain tumor cell membranes; Step 3, preparing bionic liposomes loaded with boric acid quantum dots: dissolving lipids in anhydrous ethanol, and rotary evaporating under reduced pressure to obtain a uniform translucent film, adding the boric acid quantum dots prepared in step 1 and the tumor cell membrane prepared in step 2 to dehydrate, and after hydration, the polycarbonate membrane is used to obtain bionic liposomes loaded with boric acid quantum dots.

3. The method for preparing a biomimetic liposome loaded with boric acid quantum dots according to claim 2, characterized in that: In step 1, the reaction mixing time is 15-20 hours, the dialysis uses a dialysis bag with a molecular cutoff of 500Da, and the dialysis purification time is 20-30 hours.

4. The method for preparing a biomimetic liposome loaded with boric acid quantum dots according to claim 2, characterized in that: In step 2, the tumor cells are 4T1 mouse breast cancer cells.

5. The method for preparing a biomimetic liposome loaded with boric acid quantum dots according to claim 2, characterized in that: In step 2, the membrane protein extraction reagent contains phenylmethylsulfonyl fluoride at a final concentration of 1 mM.

6. The method for preparing a biomimetic liposome loaded with boric acid quantum dots according to claim 2, characterized in that: In step 3, the pore size of the polycarbonate membrane is 100 nm.

7. Use of a biomimetic liposome loaded with boric acid quantum dots as claimed in any one of claims 1 to 6 in boron neutron capture therapy.

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