A biotin or avidin labeled lipid body and a method for preparing the same, a nanocarrier system

Abstract

A kind of biotin or avidin labeled fat body and its preparation method, nano-carrier system. Biotin or avidin labeled fat body is prepared using specific amount of Bio-phospholipid or avidin-phospholipid, and the prepared fat body has the advantages of high purity, small particle size, good uniformity and high stability. Based on the biotin-avidin system, a nano-carrier system is constructed, which includes the biotin labeled fat body and the avidin modified target component. The system enables the fat body to carry any interested substance, such as viral recombinant protein, antibody and other protein drugs, nucleic acid drugs or small molecule drugs, etc., so as to be used for the treatment of diseases such as vaccines, cancer, infectious diseases and metabolic diseases.

Description

Biotin or avidin labeled fat body and preparation method thereof, and nanocarrier system

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on November 14, 2023, with application number 202311512319.8 and invention name “A biotin or avidin labeled fat body and its preparation method, nanocarrier system”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to a biotin- or avidin-labeled fat body, a preparation method thereof, and a nanocarrier system. Background Art

[0003] Nanoparticles, defined as particles with a diameter between 1 and 1000 nanometers, are a new type of drug carrier with unique advantages in improving drug delivery efficiency, reducing side effects, and enhancing targeting. As a new type of nanoparticle, liposomes are nanospheres composed of a neutral lipid core encapsulated by a monomolecular phospholipid membrane. Similar in structure to naturally occurring lipid droplets and lipoproteins, they possess significant biomedical applications. However, universal methods and strategies for imparting specific targeting and multifunctionality to liposomes remain in urgent need of solutions.

[0004] Biotin-avidin technology is a non-covalent interaction, and the two have extremely high affinity and specificity. d =10 -14 to 10 -15 Currently, it is widely used in protein affinity purification, cell labeling and tracking, immunoassay and diagnosis and other fields.

[0005] Currently, there are no reports linking fat bodies with the biotin-avidin system and applying it to targeted disease treatment with nano-delivery systems. This invention, based on biotin-avidin, constructs a universal fat body linker technology. The nano-carrier system can be used to carry any substance of interest, such as viral recombinant proteins for vaccine development, antibodies for targeted drug delivery, or nucleic acid drugs. This can be used for vaccines, cancer, infectious diseases, metabolic diseases, and other diseases, with extremely significant medical applications.

[0006] Summary of the Invention

[0007] The present invention provides a biotin- or avidin-labeled fat body, a preparation method thereof, and a nanocarrier system. The nanocarrier is a biotin- or avidin-labeled fat body with high purity, small particle size, good uniformity, and high stability. It can also carry any substance of interest and has a wide range of medical applications.

[0008] The present invention provides a method for preparing a biotin- or avidin-labeled fat body, wherein the method for preparing a biotin- or avidin-labeled fat body comprises:

[0009] The biotin or avidin labeled phospholipids, unlabeled phospholipids and neutral lipids are mixed in a solvent and reacted to obtain the biotin or avidin labeled fat body.

[0010] In the present invention, when preparing avidin or biotin-labeled fat bodies, the fat bodies can be prepared first and then labeled with avidin or biotin. The labeling can be performed using a commonly used method in the art, such as chemical coupling method, incubation method, etc., according to the groups on the surface of the fat bodies.

[0011] In the present invention, the mass fraction of the biotin or avidin-labeled phospholipids in the total amount of phospholipids is X, and 0<X≤100%. Research results show that when 0<X<40%, no additional purification steps are required after the reaction to obtain labeled fat bodies with high purity and good uniformity. When 10%≤X≤20%, the labeled fat bodies obtained can be better used to recruit target proteins (such as GFP-avidin), the green ring structure formed is more uniform, and the morphology of the fat bodies is more complete. When the amount of Bio-PE added is greater than or equal to 40%, that is, 40%≤X≤100%, the labeled fat body membrane obtained has more impurities and the PDI is greater than 0.2. However, adding an additional purification step after the reaction can improve the purity and uniformity of the fat body, and obtain fat bodies with high purity and good uniformity. In addition, the present invention has found that by controlling X, fat bodies of different particle sizes can be obtained to meet different application requirements. As X increases, the particle size of the formed fat bodies gradually decreases, and the average particle size decreases from about 120 nm to about 70 nm. In some embodiments, 0 < X ​​< 40%. Specifically, X can be selected from the following numerical ranges or specific values: 0 < X ​​< 1.25%, 1.25% ≤ X ≤ 5%, 5% < X < 10%, 10% ≤ X ≤ 20%, 20% < X < 40%, 1.25%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, 38%, or 39%. In other embodiments, 40%≤X≤100%, specifically, 40%≤X<60%, 60%≤X<80% or 80%≤X≤100%, or X is selected from 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0012] In the present invention, the purification comprises:

[0013] First, centrifuge at 15000-2300g for 5-10 minutes, remove the precipitate, and resuspend. Then centrifuge at 500-1500g for 5-10 minutes, remove the upper white band, and resuspend. Repeat this step 3 times.

[0014] In some specific embodiments, the purification includes: first centrifuging at 21000g for 6 minutes, removing the precipitate, resuspending, then centrifuging at 1000g for 6 minutes, removing the upper white band, and resuspending; repeating this step 3 times.

[0015] In the present invention, the resuspension solution is phosphate buffer, 15mM-25mM HEPES buffer, NaCl solution, KCl solution, MgCl2 solution. In some specific embodiments, the resuspension buffer is phosphate buffer.

[0016] PDI stands for Polydispersity Index, an indicator used to measure the uniformity of nanoparticles. The smaller the PDI, the more uniform the particle size distribution. When the PDI is less than 0.2, it is generally considered that the uniformity is good, while when it is greater than 0.2, the uniformity of the nanoparticles is relatively poor. The results showed that when the amount of added Bio-phospholipids was 1.25% to 20%, the average particle size gradually decreased, and the PDI was less than 0.2. When the amount of Bio-PE added was 20%, the average particle size reached 75.6nm, and the PDI was less than 0.2; when Bio-phospholipids were added to 40%, optical microscopy showed many membrane impurities with non-fat body-like structures, and the PDI was greater than 0.2, significantly exceeding the fat body labeled with 20% Bio-phospholipids. However, after additional purification, the purity and uniformity were improved, and the PDI was below 0.2. The results showed that the amount of added Bio-phospholipids was one of the important factors in the formation of biotin or avidin labeled fat bodies. When the added amount reached 40% or more, additional purification steps were required to obtain fat bodies with high purity and good uniformity.

[0017] The present invention has no special requirements for the method of mixing the labeled and unlabeled phospholipids. They can be dissolved in chloroform separately and then mixed, or they can be mixed first and then dissolved in chloroform. In some embodiments of the present invention, they are first dissolved in chloroform separately and then mixed. Specifically, the biotin- or avidin-labeled phospholipid and the unlabeled biotin- or avidin-labeled phospholipid are each dissolved in chloroform to a final concentration of 25 mg / ml, and then the two are mixed in a volume ratio of (0-80):(0-80) so that the mass fraction of the biotin- or avidin-labeled phospholipid in the total phospholipids is 0-100%.

[0018] In the present invention, the neutral lipid is at least one of triglycerides, cholesterol esters, retinol esters, ether esters, sterol esters, and polyhydroxyalkanoates. In a specific embodiment, the neutral lipid is triglycerides; the phospholipid is at least one of phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). In a specific embodiment, the phospholipid is DOPC (dioleoylphosphatidylcholine) and / or PE. In some specific embodiments, the unlabeled phospholipid is unlabeled DOPC, and the biotin-labeled phospholipid is biotin-labeled PE; the mass ratio of the unlabeled biotin phospholipid, the biotin-labeled phospholipid and the neutral lipid is (0-2): (0-2): 3-10. In some embodiments, the mass ratio is (0-2): (0-2): 3, (0-2): (0-2): 4, (0-2): (0-2): 5, (0-2): (0-2): 6, (0-2): (0-2): 7, (0-2): (0-2): 8, (0-2): (0-2): 9, (0-2): (0-2): 10. ~2):6, (0~2):(0~2):7, (0~2):(0~2):8, (0~2):(0~2):9 or (0~2):(0~2):10, preferably (0~2):(0~2):5; further, the mass ratio is (1.975~2):(0~0.025):5, (1.2~1.975):(0.025~0.8):5, (1.6~1.975):(0.025~0.4):5, (1 .6~1.8):(0.2~0.4):0.5, (1.2~1.6):(0.4~0.6):5, (0~1.2):(0.8~2):5, (0.4~1.2):(0.8~1.6):5 or (0~0.4):(1.6~2):5; specifically 1.975:0.025:5, 1.9:0.1:5, 1.8:0.2:5, 1.7:0.3:5, 1.6:0.4:5, 1.5:0.5 :5, 1.4:0.6:5, 1.3:0.7:5, 1.2:0.8:5, 1.1:0.9:5, 1:1:5, 0.9:1.1:5, 0.8:1.2:5, 0.7:1.3:5, 0.6:1.4:5, 0.5:1.5:5, 0.4:1.6:5, 0.3:1.7:5, 0.2:1.8:5, 0.1:1.9:5 or 0:2:5, preferably 1.8:0.2:5 or 1.6:0.4:5.

[0019] In the present invention, the solvent is at least one of phosphate buffer, HEPES buffer, sucrose solution, NaCl solution, KCl solution, and MgCl2 solution. In a specific embodiment of the present invention, the solvent is phosphate buffer.

[0020] In the present invention, the mixing includes vortex mixing, ultrasonic mixing, stirring mixing, or high-pressure homogenizer mixing. In some embodiments, the mixing is vortex mixing; in some specific embodiments, the vortexing is: vortexing force of 3000-4000 rpm, vortexing for 8-15 seconds per cycle, stopping for 5-15 seconds, and vortexing for a total of 3-7 minutes. In one specific embodiment, the vortexing is: vortexing force of 4000 rpm, vortexing for 10 seconds per cycle, stopping for 10 seconds, and vortexing for a total of 5 minutes.

[0021] In the present invention, after the reaction is completed, the step of centrifuging and collecting the liquid phase is further included. In some embodiments, the centrifugation and collecting the liquid phase specifically include:

[0022] 1) Centrifuge the vortexed solution at 100-1000 g for 3-7 minutes to remove the upper white band; centrifuge again at 18000-22000 g for 3-7 minutes to remove the precipitate and collect the liquid emulsion;

[0023] 2) resuspending the liquid emulsion, centrifuging it for a third time at 100-1000 g for 3-7 minutes, removing the upper white band, collecting the liquid emulsion, and obtaining biotin-labeled fat bodies.

[0024] In some specific embodiments, in step 1), the first centrifugation condition is 1000g centrifugation for 5 min; the second centrifugation condition is 20000g centrifugation for 5 min; in step 2), the third centrifugation condition is 1000g third centrifugation for 5 min.

[0025] The present invention also provides biotin or avidin labeled fat bodies prepared by the preparation method.

[0026] The present invention provides a biotin or avidin-labeled fat body, wherein the phospholipid monolayer of the fat body comprises phospholipids labeled with biotin or avidin; in the phospholipid monolayer of the fat body, the average content of the biotin or avidin label is Y, 0<Y≤100%, preferably, 5≤Y≤100%.

[0027] The present invention uses TLC (thin layer chromatography) to analyze the lipid composition of fat bodies containing different amounts of biotin. The grayscale value of each sample on a silica gel plate is analyzed using Image J software to calculate the proportion Y of biotin-labeled phospholipids in the fat body phospholipid monolayer. In some embodiments, Y is selected from 5% to 20%, 10% to 20%, 15% to 30%, 20% to 40%, 35% to 65% or 60% to 100%, or is 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 75% to 80%, 85% to 90% or 95% to 100%, specifically 7.24%, 14.38%, 18.05%, 25.71%, 38.05%, 62.2% or 100%.

[0028] Experiments show that the average particle size of the biotin- or avidin-labeled fat bodies prepared by the present invention ranges from 60 to 150 nm, and the PDI is less than 0.2. Dynamic light scattering was used to measure changes in particle size and PDI after storage at 4°C for 1, 9, 15, and 22 days. The results showed that the particle size of the fat bodies containing different amounts of biotin or avidin remained constant, and the PDI remained essentially unchanged. These results demonstrate that the biotin- or avidin-labeled fat bodies prepared by the present invention exhibit small particle size, good uniformity, and high stability. Further, the average particle size is 65 to 120 nm, or 60 nm to 65 nm, 65 nm to 70 nm, 70 nm to 75 nm, 75 nm to 80 nm, 80 nm to 85 nm, 85 nm to 90 nm, 90 nm to 95 nm, 95 nm to 100 nm, 100 nm to 105 nm, 105 nm to 110 nm, 110 nm to 115 nm, 115 nm to 120 nm, 120 nm to 130 nm, 130 nm to 140 nm or 140 nm to 150 nm, and specifically may be 117.3 nm, 110.0 nm, 83.17 nm, 78.1 nm, 75.6 nm, 106.77 nm, 102.57 nm, 83.83 nm, 75.07 nm, 71.1 nm, 74.53 nm, 74.27 nm or 68.13 nm.

[0029] The present invention also provides the use of the biotin- or avidin-labeled fat body prepared by the preparation method as a nanocarrier in the preparation of nanomedicine.

[0030] The present invention also provides a nanocarrier system comprising a biotin-labeled adipocyte and an avidin-labeled target component;

[0031] or comprising an avidin-labeled fat body and a biotin-labeled target component;

[0032] The fat body and the target component are non-covalently linked via biotin-avidin.

[0033] In some embodiments, the biotin-labeled fat body is prepared by the preparation method of the present invention, or is the biotin-labeled fat body of the present invention described above; the avidin-labeled fat body is prepared by the preparation method of the present invention described above, or is the avidin-labeled fat body of the present invention described above.

[0034] In a specific example, the present invention investigated the ability of fat bodies prepared with different Bio-PE contents (0, 1.25%, 5%, 10%, and 20%) to recruit the target protein GFP-avidin. The results showed that fat bodies prepared with 10% to 20% Bio-PE were more effective at recruiting the target protein GFP-avidin, with the green ring-like structures formed being more uniform and the fat body morphology more complete. In contrast, fat bodies prepared with other Bio-PE contents (1.25% to 5%) showed that GFP-avidin was mostly green dot-like structures, which affected the fat body morphology and caused fat body aggregation. This indicates that the biotin content in biotin-labeled fat bodies affects the number and shape of recruited proteins, as well as their morphology and stability. Fat bodies prepared with 10% to 20% Bio-PE were more effective at recruiting the target protein tagged with avidin. Furthermore, fat bodies prepared with 20% Bio-PE showed the strongest recruitment capacity.

[0035] In some embodiments, the target component is a pharmaceutical active ingredient, which includes a peptide, an antibody, a nucleic acid substance, or a small molecule. The nucleic acid substance is preferably DNA or RNA, and more preferably one or more of mRNA, RNAi, siRNA, shRNA, miRNA, sgRNA, and crRNA.

[0036] In some embodiments, the target component is a marker protein, and the marker protein includes GFP protein, mCherry protein, Luciferase protein and / or horseradish peroxidase.

[0037] As shown in Figure 1, the nanocarrier system provided by the present invention utilizes biotin-modified adipose tissues, prepared from phospholipids or other polar lipids, and biotin-labeled adipose tissues. Furthermore, by expressing and purifying recombinant proteins tagged with avidin or chemically modifying other substances such as nucleic acids and small molecules with avidin, the avidin-tagged target substance non-covalently binds to the biotin on the surface of the adipose tissues. This biotin-avidin-based nanocarrier system allows adipose tissues to carry any substance of interest, such as viral recombinant proteins for vaccine development, antibodies for targeted drug delivery, or nucleic acids, thereby potentially being used to treat cancer, infectious diseases, metabolic disorders, and other diseases, possessing significant medical applications.

[0038] In some embodiments, the target component is a marker protein, and the marker protein includes GFP protein, mCherry protein, Luciferase protein or horseradish peroxidase (HRP).

[0039] In a specific embodiment, the present invention constructs a recombinant protein GFP-avidin with an avidin tag, the amino acid sequence is shown in SEQ ID NO: 1, and the nucleic acid sequence is shown in SEQ ID NO: 2. After vortexing, incubating, and centrifuging the Bio-PE prepared by the present invention and the recombinant protein GFP-avidin with an avidin tag, the liquid emulsion is resuspended and identified by laser confocal microscopy and silver staining analysis. The results show that the recombinant protein GFP-avidin can bind to the biotin-labeled fat body. Among them, when the recruited protein is GFP protein, the ring-shaped structure formed by the biotin-labeled fat body prepared with a dosage of 10% to 20% Bio-PE (when the mass percentage of Bio-PE to the total phospholipids is 10% to 20%) is more uniform, and the morphology of the fat body is more complete, indicating that the fat body has the strongest ability to recruit the target protein GFP-avidin.

[0040] The present invention provides a nanocarrier—a biotin- or avidin-labeled adipocyte. This nanocarrier can carry any substance of interest via a biotin-avidin system. Experiments have shown that the biotin-labeled adipocytes prepared by the present invention have an average particle size between 60 and 150 nm and a particle size distribution index (PDI) less than 0.2. Dynamic light scattering analysis of particle size and PDI after storage at 4°C for 1, 9, 15, and 22 days revealed that the particle size and PDI of adipocytes containing different biotin contents remained constant. These results demonstrate that the biotin-labeled adipocytes prepared by the present invention have a small average particle size, good uniformity, and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a schematic structural diagram of a nanocarrier system consisting of a fat body, a biotin-avidin linker, and a target component (e.g., a pharmaceutical active ingredient);

[0042] Figure 2 shows light microscopic images of the process of preparing biotin-labeled fat bodies; A is the optical microscopic image of the upper white band after centrifugation at 1000g for 5 minutes in step 3; B is the optical microscopic image of the precipitate after centrifugation at 20,000g for 5 minutes in step 4; C is the optical microscopic image of the final biotin-labeled fat bodies in step 5; a is the DIC result, and b is the result of fat bodies stained with the neutral lipid-specific dye LipidTox Red. The scale bar is 5 μm.

[0043] Figure 3 shows the particle size and PDI of bio-fat bodies after different storage times; fat bodies prepared with 0%, 1.25%, 5%, 10%, and 20% Bio-PE were stored at 4°C for 1 day (blue), 9 days (red), 15 days (green), and 22 days (purple), and the changes in particle size and PDI were measured.

[0044] Figure 4 shows the purity and homogeneity of fat bodies with different Bio-PE contents, where A is the fat body prepared by adding 20% ​​Bio-PE, B is the fat body prepared by adding 40% Bio-PE, and C is the PDI of the fat body;

[0045] FIG5 shows the average particle size (A) and PDI (B) of fat bodies with different biotin contents prepared by the optimized purification method when the mass ratio of Bio-PE input ranges from 0% to 100%.

[0046] FIG6 shows the lipid composition of biotin-labeled fat bodies detected by TLC analysis; lanes 1 to 5 are fat bodies prepared under conditions of adding 0%, 1.25%, 5%, 10%, and 20% Bio-PE, respectively.

[0047] Figure 7 shows the lipid composition analysis of 8 fat body samples in three independent experiments (A) and the proportion of biotin-labeled phospholipids in the fat body phospholipid monolayer (B).

[0048] Figure 8 shows the construction of recombinant protein GFP-avidin with an avidin tag; the upper part shows the staining results of each sample during the purification process, and the lower part shows the WB results;

[0049] Figure 9 shows that biotin-tagged fat bodies recruit the recombinant protein GFP-avidin. A to E are fat bodies prepared under conditions of adding 0%, 1.25%, 5%, 10%, and 20% Bio-PE, respectively, and incubated with the recombinant protein GFP-avidin tagged with avidin, followed by centrifugation and purification to observe whether there is a fluorescent signal on the fat body surface. Among them, the green signal shown in a is the recombinant protein GFP-avidin, b is a fat body stained with the neutral lipid-specific dye LipidTox Red, and c is a photo of the two channels a and b fused together. The white arrow indicates the presence of a fluorescent signal on the spherical surface, indicating that the fat bodies in this group can recruit the GFP-avidin signal. The scale bar is 5 microns.

[0050] Figure 10 shows the recruitment of recombinant GFP-avidin to biotin-tagged fat bodies. Fat bodies prepared with 0% and 10% Bio-PE were incubated with avidin-tagged recombinant GFP-avidin, then purified by centrifugation. Silver staining was used to identify the presence of recombinant GFP-avidin on the fat body surface. Lane 1 shows recombinant GFP-avidin added alone without fat bodies; Lane 2 shows recombinant GFP-avidin added without biotin; Lane 3 shows recombinant GFP-avidin added with biotin-tagged fat bodies.

[0051] FIG11 shows that biotin-labeled fat bodies carry protein horseradish peroxidase HRP and have biological activity; A, HRP activity in fat bodies of 8 samples prepared by the optimized method of Example 1; B, average absorbance of each well at 450 nm;

[0052] Figure 12 shows biotin-labeled adipocytes carrying the small molecule fluorescein isothiocyanate (FITC); A, results after incubation of adipocytes prepared with 0% Bio-PE and FITC; B, results after incubation of adipocytes prepared with 1.25% Bio-PE and FITC; B, results after incubation of adipocytes prepared with 40% Bio-PE and FITC; a is a fat body stained with the neutral lipid-specific dye LipidTox Red, b is a fat body stained with FITC labeled with avidin, and c is a fusion of a and b. Scale bar is 5 μm.

[0053] Figure 13 shows biotin-labeled fat bodies carrying nucleic acids. Fat bodies prepared with 0%, 10%, and 20% Bio-PE were incubated with avidin-tagged small nucleic acid siRNAs, purified by centrifugation, and analyzed for siRNA-avidin on the fat body surface by agarose gel electrophoresis. Lane 1 shows free siRNA-avidin; lane 2 shows siRNA-avidin in fat bodies with 0% Bio-PE; lane 3 shows siRNA-avidin in fat bodies with 10% Bio-PE; and lane 4 shows siRNA-avidin in fat bodies with 20% Bio-PE. DETAILED DESCRIPTION

[0054] Example 1 Construction of fat bodies with biotin markers

[0055] Materials: 18:1(Δ9-Cis)PC (DOPC) without biotin; 18:1Biotinyl Cap PE (Bio-PE) modified with biotin; and glyceryl trioleate (TAG) (neutral lipid) were purchased from Sigma.

[0056] (1) Dissolve the above DOPC and Bio-PE in chloroform to a final concentration of 25 mg / ml.

[0057] (2) DOPC and Bio-PE were added to a microcentrifuge tube according to the following volumes, with the mass ratio of Bio-PE being 0%, 1.25%, 5%, 10%, and 20%, respectively. The mixture was then vortexed to mix thoroughly, and the solvent was dried with high-purity nitrogen.

[0058] Table 1

[0059] (3) Add 100 μl of phosphate buffered saline (PBS) and 5 mg of triacylglycerol (TAG) or other neutral lipids to a microcentrifuge tube and vortex for 5 min (vortex for 10 s, stop for 5 s) to obtain a milky white lipid mixture 1 (i.e., the initial preparation component). Centrifuge the lipid mixture 1 at 1000 g for 5 min and remove the upper white band ( Figure 2A ).

[0060] (4) The lipid mixture 1 was centrifuged at 20,000 g for 5 min. After centrifugation, the sediment at the bottom of the tube was removed ( FIG2B ), and the remaining emulsion in the tube was resuspended to obtain a milky white lipid mixture 2.

[0061] (5) The lipid mixture 2 was centrifuged at 1000 g for 5 min. After centrifugation, the upper white band was removed and the remaining emulsion in the tube was resuspended to obtain a milky white lipid mixture 3, which was the final biotin-labeled fat body (Figure 2C).

[0062] Furthermore, we used a dynamic light scattering instrument to measure the average particle size and PDI (polymer dispersibility index) of fat bodies containing different amounts of biotin. The test results of the prepared biotin-labeled fat bodies placed at 4°C for one day are as follows:

[0063] Table 2

[0064] The results showed that when the amount of Bio-PE was 1.25% to 20%, as the content of biotin-PE in the fat body increased, its average particle size gradually decreased, but its PDI was always within 0.2, indicating that the biotin-labeled fat body constructed by the present invention had high purity and good uniformity.

[0065] We stored fat bodies containing varying amounts of biotin at 4°C for 1, 9, 15, and 22 days and then measured particle size and PDI using dynamic light scattering. The results showed that the particle size and PDI of the fat bodies remained largely unchanged, demonstrating excellent stability and uniformity (Figure 3).

[0066] In addition, the present invention used 20% Bio-PE and 40% Bio-PE to prepare biotin-labeled adipocytes according to the above method. The samples were placed at 4°C for one day for testing. The PDI and light microscopy results are shown in Figure 4. The results showed that when the amount of Bio-PE added was 20%, the average particle size reached 75.6nm. When the Bio-PE was added to 40%, the optical microscope showed many membrane impurities with non-adipocyte-like structures (Figures 4-A and 4-B), and the PDI was greater than 0.2 (Figure 4-C), significantly exceeding the adipocytes labeled with 20% Bio-PE. PDI refers to the polymer dispersibility index, an indicator used to measure the uniformity of nanoparticles. The smaller the PDI, the more uniform the particle size distribution. A PDI less than 0.2 is generally considered to indicate good uniformity, while a PDI greater than 0.2 indicates relatively poor uniformity of the nanoparticles.

[0067] According to the above preparation method, when the amount of Bio-PE added is 40%, the prepared fat body membrane contains more impurities and the PDI is greater than 0.2. To this end, we further optimized the purification method. When the amount of Bio-PE exceeds 20%, the obtained biotin-labeled fat bodies are of higher purity and better uniformity. We added a separation and purification step to the subsequent operation of step 5 of Example 1. Specifically, the lipid mixture 3 was centrifuged at 21000g for 6 minutes. After centrifugation, the sediment at the bottom of the tube was removed and the remaining emulsion in the tube was resuspended to obtain a milky white lipid mixture. The lipid mixture was centrifuged at 1000g for 6 minutes. After centrifugation, the upper white band was removed and the remaining emulsion in the tube was resuspended. The above steps were repeated three times to obtain fat bodies with different biotin contents. According to the above steps, we added DOPC (25 mg / ml) and Bio-PE (25 mg / ml) prepared in the previous step (1) into a microcentrifuge tube according to the following volumes, with the mass ratio of Bio-PE input ranging from 0% to 100%. The samples were numbered 1 to 8, respectively, to prepare fat bodies with different biotin contents.

[0068] Table 3

[0069] We further tested the average particle size and PDI (polymer dispersibility index) of the fat bodies with different Bio-PE contents using a dynamic light scattering instrument. The test results of the prepared biotin-labeled fat bodies placed at 4°C for one day are shown in Table 4 and Figure 5.

[0070] Table 4

[0071] The results showed that after the optimized method, the first five samples were consistent with the results before the method was optimized. As the biotin-PE content in the fat body increased, the average particle size gradually decreased, and its PDI remained below 0.2. However, when the amount of Bio-PE added exceeded 20%, reaching 40%, its purity and uniformity improved, and the PDI was also below 0.2. Even when the amount of Bio-PE added reached 100%, its PDI was still below 0.2. In addition, as the Bio-PE content increased, the final particle size of the biotin-labeled fat body gradually decreased, eventually stabilizing at around 70nm. These results show that through the optimized purification method, when the mass ratio of Bio-PE input ranged from 0% to 100%, the purity and uniformity of fat bodies with different biotin contents were high.

[0072] Example 2 Detection of lipid composition in biotin-labeled fat bodies

[0073] The lipid composition of fat bodies containing different amounts of biotin was detected by TLC (thin layer chromatography) analysis.

[0074] The constructed fat bodies containing different amounts of biotin were added to the same volume of methanol and 2 volumes of chloroform to extract lipids. The organic phase was collected and dried with nitrogen to obtain total lipids. The total lipids were added to 100 μl of chloroform, and 10 μl was loaded onto a silica gel plate. The plate was developed in a solvent of n-hexane: ether: glacial acetic acid (volume ratio of 80:20:1) to separate TAGs. The silica gel plate was then placed in a solvent of chloroform: methanol: glacial acetic acid: water (volume ratio of 75:13:9:3) to separate DOPC and Bio-PE.

[0075] The results showed that the lipid compositions of the five groups of fat body samples were similar, corresponding to the raw materials we added when preparing the fat bodies. The neutral lipids were all TAG, and the phospholipids were mainly DOPC. In sample No. 1, since Bio-PE was not added, the Bio-PE signal could not be detected in the fat body. In samples No. 2-5, since 1.25%, 5%, 10%, and 20% Bio-PE were added respectively, the Bio-PE signal could be detected. Moreover, as the amount of Bio-PE added increased, the biotin signal on the fat body became stronger (as shown by the arrows in Figure 6).

[0076] Similarly, we further tested the optimized purification method. When the mass ratio of Bio-PE input was 0% to 100%, the lipid composition of fat bodies with different biotin contents and the content of biotin-labeled phospholipids in the fat body phospholipid monolayer were prepared. The results are shown in Figure 7.

[0077] The results showed that the lipid composition of the eight groups of fat body samples was similar, corresponding to the raw materials added during fat body preparation. The neutral lipids were all TAG, and the phospholipids were mainly DOPC. In sample 1, no Bio-PE signal was detected in the fat body due to the lack of Bio-PE. However, in samples 2 to 8, Bio-PE signals were detected due to the addition of 1.25% to 100% Bio-PE, respectively. Furthermore, the biotin signal in the fat body increased with increasing amounts of Bio-PE added (Figure 7-A). We further analyzed the grayscale value of each sample on the silica gel plate using Image J software to calculate the proportion of biotin-labeled phospholipids in the fat body phospholipid monolayer. For example, in Sample 6 of Experiment 1, the circled points represent the Bio-PE signal, and the squared points represent the DOPC signal. The percentage of biotin-labeled phospholipids in the fat body phospholipid monolayer was calculated using the formula: grayscale of the circled points / (grayscale of the circled points + grayscale of the squared points), as shown in Figure 7-B. The Bio-PE content in the fat body of eight samples from three independent experiments is shown in the table below:

[0078] Table 6:

[0079] The above results show that when the mass ratio of Bio-PE input ranges from 0% to 100%, as the mass ratio of Bio-PE input increases, the proportion of biotin-labeled phospholipids in the formed fat body phospholipid monolayer also gradually increases, with the calculated results ranging from 0% to 100%. This shows that the present invention successfully constructs high-purity and uniform biotin-labeled fat bodies.

[0080] Example 3 Construction of recombinant protein GFP-avidin with avidin tag

[0081] Materials: E. coli Rosetta was from TIANGEN; nickel-chelated Chelating Sepharose Fast Flow was from GE Healthcare; and the column was from Thermo Fisher Scientific. Buffer A: 500 mM Tris, 1.5 M sodium chloride, 40% glycerol (v / v), adjusted to pH 7.8 with hydrochloric acid.

[0082] The gene sequence for the target protein GFP-avidin was inserted into the expression vector pET28a(+), designated pET28a-GFP-avidin. The vector was transformed into Rosetta competent cells, and single colonies were picked. The Rosetta cells containing pET28a-GFP-avidin were inoculated into LB liquid medium containing kanamycin and cultured at 37°C in a shaker at 200 rpm. When the bacterial solution reached an OD600 of 0.6, isopropylthiogalactoside (IPTG) was added to the culture system to a final concentration of 0.4 mM. The culture system was then induced at 16°C for 24 hours. The cultured bacteria were then disrupted using a high-pressure cell disruptor, and the resulting bacterial lysate was ultracentrifuged at 30,000 g for 60 minutes, and the supernatant was collected. 50 μl of the supernatant was added to an equal volume of 2x Sample Buffer, and the resulting mixture was used as Sample 1 (Input). The remaining supernatant was incubated with Chelating Sepharose FastFlow, a filler chelated with nickel ions. After incubation at 4°C for 2 hours, the supernatant was transferred to a 4 ml column, the flow-through liquid was collected, 50 μl of the flow-through liquid was added to an equal volume of 2xSample Buffer, and the resulting mixed solution was used as sample 2 (Flow1). The filler in the column was resuspended with 40 mM imidazole to wash the nonspecific bands, the outflowing wash liquid was collected, 50 μl of the wash liquid was added to an equal volume of 2xSample Buffer, and the resulting mixed solution was used as sample 3 (40 mM). The target protein was then eluted with 500 mM imidazole, the outflowing eluate was collected, 50 μl of the eluate was added to an equal volume of 2xSample Buffer, and the resulting mixed solution was used as sample 4 (500 mM). The resulting mixture was placed in a dialysis bag, which was then dialyzed overnight in a beaker containing 100 times the volume of Buffer A. 50 μl of the solution from the dialysis bag was added to an equal volume of 2x Sample Buffer, and the resulting mixture was designated as Sample 5 (Dialysis). The sample was analyzed by SDS-PAGE, followed by staining and immunoblotting (WB).

[0083] The staining results showed that there was a clear protein band at the 45 kDa position, which was consistent with the molecular weight of the GFP-avidin sequence. At the same time, the WB results also showed a clear band, indicating that the present invention successfully constructed a recombinant protein GFP-avidin with an avidin tag (Figure 8).

[0084] Example 4 Biotin-labeled fat bodies carrying target protein GFP

[0085] Take 50ul (OD600=2.0) of the fat body prepared in Example 1 with 0%, 1.25%, 5%, 10%, and 20% Bio-PE added respectively and add them to the EP tube, then add 20ul (1.0mg / ml) of the recombinant protein GFP-avidin with avidin tag prepared in Example 3, vortex mix, incubate at room temperature for 2 hours, and flick to mix every 10 minutes. After 2 hours, the mixture is centrifuged at 20000g for 5min. After centrifugation, remove the sediment at the bottom of the tube and the lower layer of emulsion, leaving only the upper white strip portion. Add phosphate buffer to resuspend the white strip portion, centrifuge at 20000g for 5min, remove the lower layer of emulsion, leaving only the upper white strip portion. Repeat the above steps twice, and the resuspended white strip portion finally obtained is the biotin-unlabeled or biotin-labeled fat body without or with GFP-avidin. Laser confocal microscopy and silver staining analysis were used to determine whether the recombinant protein GFP-avidin could be bound to fat bodies without or with biotin labeling ( FIG9 ).

[0086] The results show that Figure 9A shows the results of incubation and centrifugation purification of fat bodies (Figure 9-Ab) with recombinant protein GFP-avidin (Figure 9-Aa) in the presence of 0% Bio-PE. Since Bio-PE was not added during fat body preparation, there is no biotin on the fat bodies. The results show that the recombinant protein GFP-avidin cannot bind to the fat bodies without biotin (no green signal is observed in Figure 9-Ac).

[0087] In Figures 9B-E, different levels of Bio-PE were added during fat body preparation, resulting in different biotin content in the fat bodies. The results show that the recombinant protein GFP-avidin can bind to the biotin-labeled fat bodies (green ring-shaped signals appear on the surface of the red spheres in Figures 9-Bc, 9-Cc, 9-Dc, and 9-Ec, as indicated by white arrows). Fat bodies prepared with 10% to 20% Bio-PE are more effective at recruiting the target protein GFP-avidin, forming more uniform ring-shaped structures and a more complete fat body morphology (Figure 9E). In fat bodies prepared with other amounts of Bio-PE (1.25% to 5%), GFP-avidin is mostly punctate, affecting fat body morphology. The fat bodies aggregate (Figures 9B-9C), resulting in a reduced ability to recruit proteins. These results indicate that different levels of biotin labeling in fat bodies affect the amount and shape of recruited proteins, as well as their morphology and stability.

[0088] Consistent with the results of laser confocal microscopy, the results of silver staining showed ( FIG. 10 ) that biotin-labeled fat bodies could successfully bind to the recombinant protein GFP-avidin.

[0089] Example 5: Biotin-labeled fat body carrying protein horseradish peroxidase HRP

[0090] Horseradish peroxidase is commonly used to catalyze ECL reagents to produce chemiluminescence in Western blot experiments and to catalyze TMB to produce blue in ELISA experiments. To better demonstrate the wide application of the biotin-labeled fat body nanocarrier system provided by this patent in carrying protein substances, we further used biotin-labeled fat body to carry protein horseradish peroxidase (HRP) and investigated whether the fat body-carried HRP has biological activity.

[0091] Because varying Bio-PE content can affect the purity and homogeneity of adipocytes, we prepared adipocytes from the eight samples described above according to the optimized method of Example 1, with adipocytes containing 0% Bio-PE serving as a negative control. 50 μl of each of the prepared adipocytes (OD600 = 4.0) was added to an EP tube. Then, 10 μl of avidin-labeled horseradish peroxidase (HRP-avidin, purchased from Bio-Time) was added. After vortexing, the mixture was incubated at room temperature for 2 hours, gently flicking the tube every 10 minutes to mix thoroughly. After 2 hours, the mixture was centrifuged at 20,000 g for 5 minutes. The sediment at the bottom of the tube and the lower emulsion were removed, retaining only the upper white band. The white band was resuspended in phosphate buffer and centrifuged at 20,000 g for 5 minutes. The lower emulsion was removed, retaining only the upper white band. This step was repeated twice. The resulting resuspended white band represents the adipocytes without or with HRP-avidin. TMB (3,3′,5,5′-Tetramethylbenzidine) was used to detect horseradish peroxidase activity on fat bodies, and the absorbance of different samples at 450 nm was measured using a microplate reader. The results showed that as the proportion of biotin-labeled phospholipids in the fat body phospholipid monolayer gradually increased, the absorbance intensity also gradually increased (see Figure 11). These results indicate that biotin-labeled fat bodies can also carry avidin-labeled horseradish peroxidase (HRP), and that the HRP carried by the fat bodies is biologically active.

[0092] Example 6: Biotin-labeled fat bodies carrying small molecule fluorescein isothiocyanate (FITC)

[0093] Fluorescein isothiocyanate (FITC) is commonly used in biology to detect or track the interaction of conjugates with other biomolecules. It has a molecular weight of 389 g / mol. To better demonstrate the wide application of the biotin-labeled fat body nanocarrier system provided in this patent for carrying small substances, we used biotin-labeled fat bodies to carry fluorescein isothiocyanate (FITC).

[0094] Because different levels of Bio-PE can affect the purity and homogeneity of fat bodies, we prepared fat bodies with 1.25% and 40% Bio-PE according to the optimized method of Example 1. We also prepared fat bodies with 0% Bio-PE as a negative control. 50 μl of the above-prepared fat bodies (OD600 = 4.0) were added to EP tubes, and then 10 μl of avidin-labeled fluorescein isothiocyanate (FITC) (FITC-avidin, purchased from Solebol) was added. After vortex mixing, the mixture was incubated at room temperature for 2 hours, gently flicking every 10 minutes to mix. After 2 hours, the mixture was centrifuged at 20,000 g for 5 minutes. After centrifugation, the sediment at the bottom of the tube and the lower emulsion were removed, retaining only the upper white band. The white band was resuspended in phosphate buffer and centrifuged at 20,000 g for 5 minutes. The lower emulsion was removed, retaining only the upper white band. Repeat the above steps twice, and the resulting resuspended white bands represent the fat bodies without or with FITC-avidin. Laser confocal microscopy revealed that the fat bodies prepared with 1.25% and 40% Bio-PE (12a) and the FITC-avidin signal (12b) overlapped (12c), while no FITC-avidin signal was detected on the surface of the fat bodies with 0% Bio-PE (see Figure 12A). These results demonstrate that biotin-labeled fat bodies can carry the small molecule fluorescein isothiocyanate (FITC).

[0095] Example 7: Biotin-labeled fat bodies carrying nucleic acid substances

[0096] Nucleic acids are primarily classified into two categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They have numerous important medical applications, such as pathogen detection, gene therapy, and vaccines. To better demonstrate the broad application of the biotin-labeled adipocyte nanocarrier system provided in this patent for carrying nucleic acid molecules, we used biotin-labeled adipocytes to carry siRNA (small interfering RNA).

[0097] Because varying Bio-PE content can affect the purity and homogeneity of adipocytes, we prepared adipocytes with 10% and 20% Bio-PE according to the method of Example 1. We also prepared adipocytes with 0% Bio-PE as a control. 50 μl of each of the prepared adipocytes (OD600 = 4.0) was added to an EP tube. Then, 20 μl of avidin-labeled siRNA (synthesized by Qingke Biotechnology, used to reduce GFP protein expression in cells / tissues, sequence: SEQ ID NO: 3: 5'-GACGUAAACGGCCACAAGUTT-3') was added. After vortexing, the mixture was incubated at room temperature for 2 hours, gently flicking every 10 minutes to mix thoroughly. After 2 hours, the mixture was centrifuged at 20,000 g for 5 minutes. The sediment and lower emulsion were removed, retaining only the upper white band. The white band was resuspended in phosphate buffer and centrifuged at 20,000 g for 5 minutes. The lower emulsion was removed, retaining only the upper white band. Repeat the above steps twice. The resulting resuspended white band represents the fat bodies without or with siRNA-avidin. Agarose gel electrophoresis analysis (see Figure 13) revealed that fat bodies prepared with 10% and 20% Bio-PE can carry avidin-labeled siRNA. The siRNA signal of siRNA-avidin in the agarose gel is enriched in the sample wells and does not migrate. However, fat bodies prepared with 0% Bio-PE cannot carry avidin-labeled siRNA, and siRNA-avidin undergoes significant migration in the agarose gel. These results demonstrate that biotin-labeled fat bodies can carry nucleic acid substances.

[0098] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a biotin or avidin labeled fat body, characterized in that: include: The phospholipids labeled with biotin or avidin, unlabeled phospholipids and neutral lipids are mixed in a solvent, and the fat bodies labeled with biotin or avidin are obtained through reaction.

2. The preparation method according to claim 1, characterized in that: The mass percentage of the biotin or biotin or avidin-labeled phospholipids in the total amount of phospholipids is X, and X is selected from any of the following ranges: 0<X<40%, 40%≤X≤100%, 0<X<1.25%, 1.25%≤X≤5%, 5%<X<10%, 10%≤X≤20%, 20%<X<40%, 40%≤X<60%, 60%≤X<80% or 80%≤X≤100%.

3. The preparation method according to claim 1, characterized in that: Before mixing the unlabeled phospholipids and the phospholipids labeled with biotin or avidin, the method further comprises: dissolving the unlabeled phospholipids and the phospholipids labeled with biotin or avidin respectively with chloroform.

4. The preparation method according to claim 1, characterized in that: The neutral lipid is at least one of triglycerides, cholesterol esters, retinol esters, ether esters, sterol esters, and polyhydroxyalkanoates; the phospholipids are at least one of PC, PE, PA, PS, PE, and PI.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the unlabeled phospholipids, biotin or avidin labeled phospholipids and neutral lipids is (0-2):(0-2):3-10, preferably (0-2):(0-2):

5.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the unlabeled phospholipids, biotin or avidin labeled phospholipids and neutral lipids is (1.975-2):(0-0.025):5, (1.2-1.975):(0.025-0.8):5, (1.6-1.975):(0.025-0.4):5, (1.6-1.8):(0.2-0.4):0.5, (1.2-1.6):(0.4-0.6):5, (0-1.2):(0.8-2):5, (0.4-1.2):(0.8-1.6):5 or (0-0.4):(1.6-2):

5.

7. The method according to claim 1, characterized in that The solvent is at least one of phosphate buffer, HEPES buffer, sucrose solution, NaCl solution, KCl solution, and MgCl2 solution.

8. The preparation method according to claim 1, characterized in that: The mixing includes vortex mixing, ultrasonic mixing, stirring mixing or high-pressure homogenization mixing.

9. The preparation method according to claim 8, characterized in that: The vortex mixing is as follows: the vortex intensity is 3000-4000 rpm / min, each vortex is 8-15 seconds, and the rest time is 5-15 seconds, and the total vortex is 3-7 minutes.

10. The preparation method according to claim 1, characterized in that: After the reaction is completed, the method further comprises the steps of centrifugation and collecting the liquid phase.

11. The method according to claim 10, characterized in that The centrifugation and collection of liquid phase specifically include: 1) Take the vortexed solution and centrifuge it for the first time at 100g-1000g for 3-7min to remove the upper white band; centrifuge it for the second time at 18000-22000g for 3-7min to remove the precipitate and collect the liquid phase emulsion; 2) resuspending the liquid phase emulsion, centrifuging for a third time at 100 g to 1000 g for 3 to 7 minutes, removing the upper white band, collecting the liquid phase emulsion, and obtaining biotin-labeled fat bodies.

12. The method according to claim 10 or 11, characterized in that: After the centrifugation and collection of the liquid phase, a purification step is also included, and the purification includes: First, centrifuge at 15000-2300g for 5-10 minutes, remove the precipitate, and resuspend. Then, centrifuge at 500-1500g for 5-10 minutes, remove the upper white band, and resuspend. Repeat this step 3 times.

13. The preparation method according to claim 11 or 12, characterized in that: The resuspending solution is at least one of phosphate buffer, HEPES buffer, sucrose solution, NaCl solution, KCl solution, and MgCl2 solution.

14. The fat body of biotin or avidin obtained by the preparation method according to any one of claims 1 to 13.

15. A biotin or avidin labeled fat body, characterized in that: The phospholipid monolayer of the fat body includes phospholipids labeled with biotin or avidin; In the phospholipid monolayer of the fat body, the average content of biotin or avidin marker accounts for Y, 0<Y≤100%, preferably 5≤Y≤100%.

16. The biotin or avidin labeled fat body according to claim 14, characterized in that The Y is selected from 5% to 20%, 10% to 20%, 15% to 30%, 20% to 40%, 35% to 65% or 60 to 100%.

17. The biotin or avidin labeled fat body according to any one of claims 14 to 16, characterized in that: The average particle size of the biotin or avidin labeled fat body is 60nm to 150nm, preferably 65 to 120nm.

18. The biotin or avidin labeled fat body according to claim 17, characterized in that The average particle size of the biotin or avidin labeled fat body is 60nm-75nm, 70nm-75nm, 75nm-85nm, 80-105nm, 100nm-110nm, 105nm-115nm, 110nm-120nm or 120nm-150nm.

19. The biotin or avidin labeled fat body according to any one of claims 15 to 18, wherein the preparation method of the biotin or avidin labeled fat body comprises: The phospholipids labeled with biotin or avidin, unlabeled phospholipids and neutral lipids are mixed in a solvent, and the biotin or avidin labeled fat bodies are obtained by reaction; or include: Fat bodies are prepared, and lipid droplet resident proteins labeled with avidin or biotin are added and incubated to obtain avidin-labeled fat bodies.

20. Use of the biotin or avidin labeled fat body prepared by the preparation method of any one of claims 1 to 13 or the biotin or avidin labeled fat body of any one of claims 14 to 19 as a nanocarrier in the preparation of drugs.

21. A nanocarrier system, characterized in that: including a biotin-labeled adipocyte and an avidin-labeled target component; or comprising avidin-labeled adipocytes and biotin-labeled target components; The fat body and the target component are non-covalently linked via biotin-avidin.

22. The nanocarrier system according to claim 21, characterized in that The biotin or avidin labeled fat body is prepared by the preparation method according to any one of claims 1 to 13, or is the biotin or avidin labeled fat body according to any one of claims 14 to 19.

23. The nanocarrier system according to claim 21, characterized in that The target component is a pharmaceutical active ingredient, which includes a peptide segment, an antibody, a nucleic acid substance or a small molecule substance.

24. The nanocarrier system according to any one of claims 21 to 23, characterized in that The target component is a marker protein, and the marker protein includes GFP protein, mCherry protein, Luciferase protein and / or horseradish peroxidase.

25. The nanocarrier system according to any one of claims 21 to 23, characterized in that The target component is a nucleic acid substance, preferably DNA or RNA, and more preferably one or more of mRNA, RNAi, siRNA, shRNA, miRNA, sgRNA, and crRNA.

Images