Red blood cell carrier loaded with multi-mode nano-particles as well as preparation method and application of red blood cell carrier

Abstract

The invention belongs to the technical field of red blood cell carriers, and relates to a preparation method and application of a red blood cell carrier loaded with multi-mode nanoparticles. The invention discloses an erythrocyte carrier loaded with multi-mode nano-particles. The erythrocyte carrier comprises an erythrocyte skeleton and the multi-mode nano-particles loaded in the erythrocyte skeleton, in an extracellular solution with the osmotic pressure of 50-200mOsm / kg, the erythrocyte skeleton is loaded with multi-mode nano-particles with the average particle size of 10-200nm. The invention also discloses a preparation method of the erythrocyte carrier loaded with the multi-modal nanoparticles, which comprises the following steps: adding a multi-modal nanoparticle resuspension into an extracellular solution, then adding a 100% HCT erythrocyte skeleton, and carrying out low-speed shaking incubation; then adding a NaCl solution and carrying out closed incubation; and centrifuging, washing and resuspending to obtain the erythrocyte carrier loaded with the multi-mode nanoparticles. The erythrocyte carrier loaded with the multi-mode nano-particles keeps a complete biconcave disc-shaped structure, and maintains long-acting circulation capability in vivo for more than 30 days.

Description

Technical Field

[0001] The present invention belongs to the technical field of red blood cell carriers, and relates to a preparation method and application of a red blood cell carrier loaded with multimodal nanoparticles. Background Art

[0002] Red blood cell carriers refer to natural red blood cells transformed into lesion-targeted or systemic drug delivery carriers through physical, chemical or bioengineering methods. Due to the advantages of red blood cells such as low immunogenicity, large number and long circulation life in the body, red blood cell carriers have great application potential in clinical transfusion medicine and basic nanomedicine.

[0003] Hypotonic loading is an important technique for encapsulating nanoparticles by changing the osmotic pressure of the extracellular solution to cause transient pores in the erythrocyte membrane. However, the scale range of erythrocyte hypotonic loading nanoparticles and the matching hypotonic loading conditions are still unclear, and the method of loading different model nanoparticles into erythrocytes lacks a paradigm, resulting in low nanoparticle loading efficiency and erythrocyte carrier yield. Therefore, further clarifying the universal hypotonic loading osmotic pressure conditions for erythrocyte loading of multimodal nanoparticles is a key scientific issue facing erythrocyte carriers in biomedical applications. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and to propose a red blood cell carrier loaded with multimodal nanoparticles with high multimodal nanoparticle loading efficiency. The preparation method of the present invention can improve the yield of red blood cell carriers loaded with multimodal nanoparticles.

[0005] One object of the present invention can be achieved by the following technical solutions:

[0006] A red blood cell carrier loaded with multimodal nanoparticles, comprising a red blood cell skeleton and multimodal nanoparticles loaded therein;

[0007] In an extracellular solution with an osmotic pressure of 50-200 mOsm / kg, the erythrocyte skeleton is loaded with multimodal nanoparticles with an average particle size of 10-200 nm.

[0008] Preferably, the multimodal nanoparticles include one or more of gold nanoparticles, silicon dioxide nanoparticles, ferroferric oxide nanoparticles, and gold nanorod particles.

[0009] Preferably, when the average particle size of the multimodal nanoparticles is 16 to 136 nm, the loading osmotic pressure is 150 to 50 mOsm / kg.

[0010] Further preferably, when the average particle size of the multimodal nanoparticles is 16 to 33 nm, the loading osmotic pressure is 150 mOsm / kg;

[0011] When the average particle size of the multimodal nanoparticles is 33 to 100 nm, the loading osmotic pressure is 150 to 100 mOsm / kg;

[0012] When the average particle size of the multimodal nanoparticles is 100-136 nm, the loading osmotic pressure is 100-50 mOsm / kg.

[0013] Preferably, the yield of the erythrocyte carrier loaded with multimodal nanoparticles is ≥ 10%.

[0014] Further preferably, the yield of the erythrocyte carrier loaded with multimodal nanoparticles is ≥30%.

[0015] Preferably, for 136 nm nanoparticles, the yield of erythrocyte carriers loaded with multimodal nanoparticles is ≥ 10% in an extracellular solution with an extracellular osmotic pressure of 50 mOsm / kg.

[0016] Preferably, for 66 nm nanoparticles, the yield of erythrocyte carriers loaded with multimodal nanoparticles is ≥ 20% in an extracellular solution with an extracellular osmotic pressure of 100 mOsm / kg.

[0017] Preferably, for 33 nm nanoparticles, the yield of erythrocyte carriers loaded with multimodal nanoparticles is ≥30% in an extracellular solution with an extracellular osmotic pressure of 150 mOsm / kg.

[0018] The second object of the present invention is to provide a method for preparing an erythrocyte carrier loaded with multimodal nanoparticles, comprising: adding a multimodal nanoparticle resuspension to an extracellular solution, then adding an erythrocyte skeleton with 100% HCT (hematocrit), and incubating at 4°C with low-speed shaking; then adding a NaCl solution, and incubating at 37°C in a closed state; and then centrifuging, washing, and resuspending to obtain an erythrocyte carrier loaded with multimodal nanoparticles with an average particle size of 10 to 200 nm.

[0019] Preferably, the preparation method comprises: adding 0.1 ml of a multimodal nanoparticle resuspension to 0.8 ml of an extracellular solution of 50 to 200 mOsm / kg, and then adding 0.1 ml of a 100% HCT (hematocrit) erythrocyte skeleton, and incubating at 4° C. with low-speed shaking for 1 to 60 min; then adding 0.1 ml of a 9% NaCl solution, and incubating at 37° C. for 1 to 60 min in a sealed manner; and then centrifuging, washing, and resuspending to obtain an erythrocyte carrier loaded with multimodal nanoparticles.

[0020] Preferably, NaCl solution is used as the blocking solution, and its osmotic pressure is 3080-4110 mOsm / kg.

[0021] Preferably, the method for preparing the erythrocyte skeleton comprises: placing freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin, placing it in a centrifuge precooled at 4°C, and centrifuging it at 500-5000 rpm for 1-60 min; washing with PBS, resuspending it evenly with 100% HCT to obtain the erythrocyte skeleton, and storing it at 4°C for use.

[0022] Preferably, the molar concentration of the multimodal nanoparticles in the multimodal nanoparticle resuspension is 7.5-30 mM.

[0023] Preferably, the multimodal nanoparticles include one or more of gold nanoparticles, silicon dioxide nanoparticles, ferrosoferric oxide nanoparticles, and gold nanorods.

[0024] Further preferably, the preparation method of the gold nanoparticles comprises: (1) heating a sodium citrate aqueous solution to 100-150° C. under condensation reflux, then adding a HAuCl4 solution and boiling for 1-240 min, wherein the color of the solution changes to colorless, gray, purple, and wine red in sequence, to obtain a solution containing gold seeds;

[0025] (2) Adding HAuCl4 solution to the solution containing gold seeds twice and boiling them for 1 to 240 min respectively, then adding sodium citrate solution, repeating the process 1 to 20 times to obtain gold nanoparticles.

[0026] Further preferably, the preparation method of silica nanoparticles includes: mixing ethanol and H2O and then adding ammonia water, stirring in a water bath to obtain a mixed solution; slowly adding a mixture of TEOS and anhydrous ethanol to the mixed solution, stirring for reaction, centrifuging, washing, and resuspending in water for storage.

[0027] More preferably, the volume ratio of ethanol, H2O and aqueous ammonia is (1-20):1:(0.01-1).

[0028] More preferably, the water bath temperature is 20-50° C. and the time is 1-60 min.

[0029] More preferably, the volume ratio of TEOS to anhydrous ethanol is (1-5):(1-5).

[0030] The third object of the present invention is to provide an application of a red blood cell carrier loaded with multimodal nanoparticles in fluorescence imaging.

[0031] Preferably, the erythrocyte carrier loaded with multimodal nanoparticles is applied to in vivo fluorescence imaging and circulates in vivo for more than 30 days.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention uses red blood cells as building blocks, clarifies the advantageous extracellular osmotic pressure conditions when loading multimodal nanoparticles, and achieves high loading efficiency of multimodal nanoparticles in red blood cell carriers loaded with multimodal nanoparticles, and high yield of red blood cell carriers loaded with multimodal nanoparticles.

[0034] 2. The erythrocyte carrier loaded with multimodal nanoparticles of the present invention can be loaded with multimodal nanoparticles of different components with an average particle size of 10 to 200 nm, and there is little restriction on the types of multimodal nanoparticles.

[0035] 3. The preparation method of the erythrocyte carrier loaded with multimodal nanoparticles of the present invention is universal. In an extracellular solution with an osmotic pressure of 50 to 200 mOsm / kg, the erythrocyte skeleton can be loaded with multimodal nanoparticles with an average particle size of 10 to 200 nm.

[0036] 4. The erythrocyte carrier loaded with multimodal nanoparticles of the present invention maintains a complete biconcave disc-shaped structure, and the normal expression of the membrane protein CD47 can maintain its long-term circulation ability in the body for more than 30 days.

[0037] 5. The erythrocyte carrier loaded with multimodal nanoparticles of the present invention can be applied to in vivo fluorescence imaging and has long-term circulation, which expands the application potential of erythrocyte carriers in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the preparation process of the red blood cell carrier loaded with multimodal nanoparticles of the present invention.

[0039] Figure 2 Transmission electron microscopy images of gold nanoparticles of different particle sizes in Examples 1 to 3 of the present invention.

[0040] Figure 3 This is a graph showing the loading efficiency of the red blood cell carrier loaded with multimodal nanoparticles in Examples 1 to 3 and 6 of the present invention.

[0041] Figure 4 It is a quantitative graph of the yield of red blood cell carriers loaded with multimodal nanoparticles in Examples 1 to 3 and 6 of the present invention.

[0042] Figure 5 These are laser confocal microscopy images of red blood cell carriers loaded with multimodal nanoparticles in Examples 1 to 3 of the present invention.

[0043] Figure 6 Transmission electron microscopy images, loading efficiency images, and laser confocal microscopy images of the red blood cell carrier loaded with multimodal nanoparticles in Example 4 of the present invention.

[0044] Figure 7Transmission electron microscopy images, loading efficiency images, and laser confocal microscopy images of the red blood cell carrier loaded with multimodal nanoparticles in Example 5 of the present invention.

[0045] Figure 8 This is a protein immunoblot of the membrane protein CD47 of the erythrocyte carrier loaded with multimodal nanoparticles in Examples 1 to 3 in Application Example 1 of the present invention.

[0046] Fig. 9 These are in vivo imaging images of small animals circulating red blood cell carriers loaded with multimodal nanoparticles in Examples 1 to 3 of the present invention. DETAILED DESCRIPTION

[0047] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0048] Unless otherwise specified, the materials used in the present invention are conventional commercial products, and the methods used are conventional technical means.

[0049] In this paper, within a certain range, as the extracellular osmotic pressure decreases, the loading amount of erythrocytes loaded with multimodal nanoparticles increases (fluorescence increases).

[0050] In this paper, within a certain range, as the average particle size of multimodal nanoparticles increases, the extracellular osmotic pressure required for the optimal loading of the erythrocyte skeleton decreases and the loading efficiency decreases.

[0051] In this article, the preparation method of the erythrocyte skeleton includes: putting freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin, placing it in a centrifuge precooled at 4°C, and centrifuging it at 500-5000 rpm for 1-60 minutes; washing with PBS, resuspending it evenly with 80-100% HCT to obtain the erythrocyte skeleton, and storing it at 4°C for use.

[0052] In this article, multimodal nanoparticles include nanoparticles in the prior art, and the average particle size is 10 to 200 nm.

[0053] Herein, the method for preparing gold nanoparticles includes:

[0054] (1) Under condensation reflux, 100-300 mL of H2O and 0.1-10 mL of 100-300 mM sodium citrate solution are mixed and heated to 100-130°C, and then 0.1-10 ml of 10-50 mM HAuCl4 solution is added and boiled for 1-240 min. The color of the solution changes to colorless, gray, purple, and wine red in sequence, and a solution containing gold seeds is obtained;

[0055] (2) Add 0.1-10 ml of 10-50 Mm HAuCl4 solution to the solution containing gold seeds twice and boil them for 1-240 min respectively, then add 0.1-10 ml of 10-100 Mm sodium citrate solution, repeat 1-20 times to obtain gold nanoparticles.

[0056] In this article, the preparation method of silica nanoparticles includes: mixing ethanol and H2O, adding ammonia water, and stirring in a water bath to obtain a mixed solution; slowly dropping a mixture of TEOS and anhydrous ethanol into the mixed solution, stirring for reaction, centrifuging, and washing to obtain the silica nanoparticles.

[0057] In this article, the schematic diagram of the preparation process of the red blood cell carrier loaded with multimodal nanoparticles of the present invention is shown in Figure 1 .

[0058] Example 1

[0059] (1) Separation of erythrocyte skeleton;

[0060] Put the freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin to prevent blood coagulation, put it into a precooled centrifuge at 4°C, set at 1000 rpm, and centrifuge for 10 minutes, wash it 3 times with 1X PBS, resuspend it evenly with 100% HCT to obtain the erythrocyte skeleton, and store it at 4°C for use.

[0061] (2) Synthesize nanoparticles;

[0062] Preparation of gold nanoparticles according to the gold seed growth method:

[0063] (2.1) Add 150 mL of H2O and 1 mL of 200 mM sodium citrate solution (final concentration of 1.33 mM) to a 250 mL three-necked flask (stir at 1000 rpm), and heat the oil bath to 100°C under condensation reflux. After steady boiling, add 1.5 mL of 25 mM HAuCl4 solution and continue boiling for 30 min (the color changes from colorless to gray, then to purple and finally to wine red) to obtain gold seeds.

[0064] (2.2) Continue to grow gold nanoparticles on the gold seeds: Add 1 mL of 25 mM HAuCl4 solution to the solution and continue to boil for 30 min. Add 1 mL of 25 mM HAuCl4 solution again and continue to boil for 30 min. Add 2 mL of 60 mM sodium citrate solution, pipette 53 mL of the original solution and add 55 mL of H2O and stir for 5 min.

[0065] (2.3) Repeat (2.2) three times to obtain a resuspension containing 33 nm Au NPs.

[0066] (3) preparing extracellular solutions with different osmotic pressures, and loading the multimodal nanoparticles described in step (2) into the red blood cell skeleton described in step (1);

[0067] Prepare extracellular solutions with different osmotic pressures. The osmotic pressure of 1X PBS is 300mOsm / kg. Use H2O and 1X PBS to prepare extracellular solutions with different osmotic pressures of 300, 200, 150, 100, and 50mOsm / kg.

[0068] Add 0.8 mL of the above-configured extracellular solution with different osmotic pressures, 0.1 mL of the resuspension containing 33 nm Au NPs in step (2) (concentration of 24.4 mM) to a 1.5 mL centrifuge tube, mix well, and add 0.1 mL of 100% HCT red blood cells. Shake at 45 rpm for 20 min at 4 ° C. Add 0.1 mL of 9% NaCl solution (osmotic pressure of 3080 mOsm / kg), and transfer the centrifuge tube to 37 ° C for 30 min. After completion, centrifuge the centrifuge tube at 1000 rpm for 10 min, wash 3 times with 1X PBS, and obtain a red blood cell carrier loaded with multimodal nanoparticles. Resuspend with 1X PBS and store at 4 ° C for use.

[0069] Figure 2 Transmission electron microscopy image of 33nm Au NPs (step 3); Figure 3 is a graph showing the loading efficiency of erythrocyte carriers loaded with multimodal nanoparticles; Figure 4 Quantitative graph of the yield of erythrocyte carriers loaded with multimodal nanoparticles; Figure 5 Laser confocal microscopy image of red blood cell carrier loaded with multimodal nanoparticles.

[0070] Figure 3 , 4 All data are expressed as mean ± standard deviation. Statistical analysis between designated groups was performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. ns (nonsignificant) indicates no statistically significant difference, *p<0.05, **p<0.01, ***p<0.001. Data represent the normalized results of three independent experiments.

[0071] according to Figure 3 , 4 , the optimal extracellular osmotic pressure of 33 nm Au NPs was determined to be 150 mOsm / kg, denoted as 33 nm Au-RBC (150 mOsm / kg).

[0072] Example 2

[0073] Compared with Example 1, the difference is that the multimodal nanoparticles are 66 nm Au NPs.

[0074] (1) Separation of erythrocyte skeleton;

[0075] Put the freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin to prevent blood coagulation, put it into a precooled centrifuge at 4°C, set at 1000 rpm, and centrifuge for 10 minutes, wash it 3 times with 1X PBS, resuspend it evenly with 100% HCT to obtain the erythrocyte skeleton, and store it at 4°C for use.

[0076] (2) Synthesize nanoparticles;

[0077] Preparation of gold nanoparticles according to the gold seed growth method:

[0078] (2.1) Add 150 mL of H2O and 1 mL of 200 mM sodium citrate solution (stirring at 1000 rpm) to a 250 mL three-necked flask and heat the mixture to 100°C in an oil bath under reflux. After steady boiling, add 1.5 mL of 25 mM HAuCl4 solution and continue boiling for 30 min (the color changes from colorless to gray, then to purple and finally to wine red) to obtain gold seeds.

[0079] (2.2) Continue to grow gold nanoparticles on the gold seeds: Add 1 mL of 25 mM HAuCl4 solution to the solution and continue to boil for 30 min. Add 1 mL of 25 mM HAuCl4 solution again and continue to boil for 30 min. Add 2 mL of 60 mM sodium citrate solution, pipette 53 mL of the original solution and add 55 mL of H2O and stir for 5 min.

[0080] (2.3) Repeat (2.2) 6 times to obtain a resuspension containing 66 nm Au NPs.

[0081] (3) preparing extracellular solutions with different osmotic pressures, and loading the multimodal nanoparticles described in step (2) into the red blood cell skeleton described in step (1);

[0082] Prepare extracellular solutions with different osmotic pressures. The osmotic pressure of 1X PBS is 300mOsm / kg. Use H2O and 1X PBS to prepare extracellular solutions with different osmotic pressures of 300, 200, 150, 100, and 50mOsm / kg.

[0083] Add 0.8 mL of the above-configured extracellular solution with different osmotic pressures, 0.1 mL of the resuspension containing 66 nm Au NPs in step (2) (concentration of 28.3 mM) to a 1.5 mL centrifuge tube, mix well, and add 0.1 mL of 100% HCT red blood cells. Shake at 45 rpm for 20 min at 4 ° C. Add 0.1 mL of 9% NaCl solution and transfer the centrifuge tube to 37 ° C for 30 min. After completion, centrifuge the centrifuge tube at 1000 rpm for 10 min, wash 3 times with 1X PBS, and obtain a red blood cell carrier loaded with multimodal nanoparticles. Resuspend with 1X PBS and store at 4 ° C for use.

[0084] Figure 2 Transmission electron microscopy image of 66nm Au NPs (step 6); Figure 3 is a graph showing the loading efficiency of erythrocyte carriers loaded with multimodal nanoparticles; Figure 4 Quantitative graph of the yield of erythrocyte carriers loaded with multimodal nanoparticles; Figure 5 Laser confocal microscopy image of red blood cell carrier loaded with multimodal nanoparticles.

[0085] according to Figure 3 , 4 , the optimal extracellular osmotic pressure of 66 nm Au NPs was determined to be, denoted as 66 nm Au-RBC (100 mOsm / kg).

[0086] Example 3

[0087] Compared with Example 1, the difference is that the multimodal nanoparticles are 136 nm Au NPs.

[0088] (1) Separation of erythrocyte skeleton;

[0089] Put the freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin to prevent blood coagulation, put it into a precooled centrifuge at 4°C, set at 1000 rpm, and centrifuge for 10 minutes, wash it 3 times with 1X PBS, resuspend it evenly with 100% HCT to obtain the erythrocyte skeleton, and store it at 4°C for use.

[0090] (2) Synthesize nanoparticles;

[0091] Preparation of gold nanoparticles according to the gold seed growth method:

[0092] (2.1) Add 150 mL of H2O and 1 mL of 200 mM sodium citrate solution (stirring at 1000 rpm) to a 250 mL three-necked flask and heat the mixture to 100°C in an oil bath under reflux. After steady boiling, add 1.5 mL of 25 mM HAuCl4 solution and continue boiling for 30 min (the color changes from colorless to gray, then to purple and finally to wine red) to obtain gold seeds.

[0093] (2.2) Continue to grow gold nanoparticles on the gold seeds: Add 1 mL of 25 mM HAuCl4 solution to the solution and continue to boil for 30 min. Add 1 mL of 25 mM HAuCl4 solution again and continue to boil for 30 min. Add 2 mL of 60 mM sodium citrate solution, pipette 53 mL of the original solution and add 55 mL of H2O and stir for 5 min.

[0094] (2.3) Repeat (2.2) 13 times to obtain a resuspension containing 136 nm Au NPs.

[0095] (3) preparing extracellular solutions with different osmotic pressures, and loading the multimodal nanoparticles described in step (2) into the red blood cell skeleton described in step (1);

[0096] Prepare extracellular solutions with different osmotic pressures. The osmotic pressure of 1X PBS is 300mOsm / kg. Use H2O and 1X PBS to prepare extracellular solutions with different osmotic pressures of 300, 200, 150, 100, and 50mOsm / kg.

[0097] Add 0.8 mL of the above-configured extracellular solution with different osmotic pressures, 0.1 mL of the resuspension containing 136 nm Au NPs in step (2) (concentration of 29.9 mM) to a 1.5 mL centrifuge tube, mix well, and add 0.1 mL of 100% HCT red blood cells. Shake at 45 rpm for 20 min at 4 ° C. Add 0.1 mL of 9% NaCl solution and transfer the centrifuge tube to 37 ° C for 30 min. After completion, centrifuge the centrifuge tube at 1000 rpm for 10 min, wash 3 times with 1X PBS, and obtain a red blood cell carrier loaded with multimodal nanoparticles. Resuspend with 1X PBS and store at 4 ° C for use.

[0098] Figure 2 Transmission electron microscopy image of 136nm Au NPs (step 13); Figure 3 is a graph showing the loading efficiency of erythrocyte carriers loaded with multimodal nanoparticles; Figure 4 Quantitative graph of the yield of erythrocyte carriers loaded with multimodal nanoparticles; Figure 5 Laser confocal microscopy image of red blood cell carrier loaded with multimodal nanoparticles.

[0099] according to Figure 3 , 4 , the optimal extracellular osmotic pressure of 136 nm Au NPs was determined to be 50 mOsm / kg, denoted as 136 nm Au-RBC (50 mOsm / kg).

[0100] according to Figure 5 It can be seen that the results of laser confocal imaging show that there is uniform spherical green fluorescence (three gold nanoparticles labeled with FITC) within the complete circular red fluorescence (red blood cells labeled with DiD), indicating that the three gold nanoparticles are completely loaded into the red blood cells under their respective matching optimal osmotic pressure conditions.

[0101] Example 4

[0102] Compared with Example 1, the difference is that the multimodal nanoparticles are 35 nm SiO2 NPs.

[0103] (1) Separation of erythrocyte skeleton;

[0104] Put the freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin to prevent blood coagulation, put it into a precooled centrifuge at 4°C, set at 1000 rpm, and centrifuge for 10 minutes, wash it 3 times with 1X PBS, resuspend it evenly with 100% HCT to obtain the erythrocyte skeleton, and store it at 4°C for use.

[0105] (2) Synthesize nanoparticles;

[0106] Add 35 mL of ethanol and 5 mL of H2O to a round-bottom flask and mix them, then add 0.5 mL of aqueous ammonia and stir for 15 min in a water bath at 35°C; ultrasonically mix 1 mL of TEOS and 1 mL of anhydrous ethanol, slowly add them dropwise to the mixed solution, stir at 500 rpm for 2 h, centrifuge the resulting product at 13000 rpm for 20 min, wash with water 3 times to obtain 35 nm SiO2 NPs, resuspend with water to obtain a resuspension containing 35 nm SiO2 NPs, and store at 4°C for use.

[0107] (3) preparing extracellular solutions with different osmotic pressures, and loading the multimodal nanoparticles described in step (2) into the red blood cell skeleton described in step (1);

[0108] Prepare extracellular solutions with different osmotic pressures. The osmotic pressure of 1X PBS is 300mOsm / kg. Use H2O and 1X PBS to prepare extracellular solutions with different osmotic pressures of 300, 200, 150, 100, and 50mOsm / kg.

[0109] Add 0.8 mL of the above-prepared extracellular solution with different osmotic pressures and 0.1 mL of the resuspension containing 35 nm SiO2 NPs in step (2) to a 1.5 mL centrifuge tube, mix well, and add 0.1 mL of 100% HCT red blood cells. Shake at 45 rpm for 20 min at 4 ° C. Add 0.1 mL of 9% NaCl solution and transfer the centrifuge tube to 37 ° C for 30 min. After completion, centrifuge the centrifuge tube at 1000 rpm for 10 min, wash 3 times with 1X PBS, and obtain the red blood cell carrier loaded with multimodal nanoparticles. Resuspend with 1X PBS and store at 4 ° C for use.

[0110] The transmission electron microscope image, loading efficiency image, and laser confocal microscope image of the red blood cell carrier loaded with multimodal nanoparticles of this embodiment are shown in Figure 6 , the optimal extracellular osmotic pressure of 35nm SiO2 NPs was determined to be 150mOsm / kg.

[0111] Example 5

[0112] Compared with Example 1, the difference is that the multimodal nanoparticles are 100 nm SiO2 NPs.

[0113] (1) Separation of erythrocyte skeleton;

[0114] Put the freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin to prevent blood coagulation, put it into a precooled centrifuge at 4°C, set at 1000 rpm, and centrifuge for 10 minutes, wash it 3 times with 1X PBS, resuspend it evenly with 100% HCT to obtain the erythrocyte skeleton, and store it at 4°C for use.

[0115] (2) Synthesize nanoparticles;

[0116] Add 35 mL of ethanol and 5 mL of H2O to a round-bottom flask and mix them. Then add 0.7 mL of aqueous ammonia and stir for 15 min in a water bath at 35 °C. Ultrasonic mix 1 mL of TEOS and 1 mL of anhydrous ethanol and slowly add them dropwise to the mixed solution. Stir and react at 500 rpm for 2 h. Centrifuge the product at 13,000 rpm for 20 min, wash with water three times to obtain 35 nm SiO2NPs. Resuspend with water to obtain a resuspension containing 100 nm SiO2NPs, which is stored at 4 °C for use.

[0117] (3) preparing extracellular solutions with different osmotic pressures, and loading the multimodal nanoparticles described in step (2) into the red blood cell skeleton described in step (1);

[0118] Prepare extracellular solutions with different osmotic pressures. The osmotic pressure of 1X PBS is 300mOsm / kg. Use H2O and 1X PBS to prepare extracellular solutions with different osmotic pressures of 300, 200, 150, 100, and 50mOsm / kg.

[0119] Add 0.8 mL of the above-configured extracellular solution with different osmotic pressures and 0.1 mL of the resuspension containing 100 nm SiO2 NPs in step (2) to a 1.5 mL centrifuge tube, mix well, and add 0.1 mL of 100% HCT red blood cells. Shake at 45 rpm for 20 min at 4 ° C. Add 0.1 mL of 9% NaCl solution and transfer the centrifuge tube to 37 ° C for 30 min. After completion, centrifuge the centrifuge tube at 1000 rpm for 10 min, wash 3 times with 1X PBS, and obtain the red blood cell carrier loaded with multimodal nanoparticles. Resuspend with 1X PBS and store at 4 ° C for use.

[0120] The transmission electron microscope image, loading efficiency image, and laser confocal microscope image of the red blood cell carrier loaded with multimodal nanoparticles of this embodiment are shown in Figure 7 , the optimal extracellular osmotic pressure of 100nm SiO2 NPs was determined to be 100mOsm / kg.

[0121] Example 6

[0122] Compared with Example 1, the difference is that the multimodal nanoparticles are 16 nm Au NPs.

[0123] (1) Separation of erythrocyte skeleton;

[0124] Put the freshly collected blood into an anticoagulant centrifuge tube containing sodium heparin to prevent blood coagulation, put it into a precooled centrifuge at 4°C, set at 1000 rpm, and centrifuge for 10 minutes, wash it 3 times with 1X PBS, resuspend it evenly with 100% HCT to obtain the erythrocyte skeleton, and store it at 4°C for use.

[0125] (2) Synthesize nanoparticles;

[0126] Preparation of gold nanoparticles according to the gold seed growth method:

[0127] (2.1) Add 150 mL of H2O and 1 mL of 200 mM sodium citrate solution (stirring at 1000 rpm) to a 250 mL three-necked flask and heat the mixture to 100°C in an oil bath under reflux. After steady boiling, add 1.5 mL of 25 mM HAuCl4 solution and continue boiling for 30 min (the color changes from colorless to gray, then to purple and finally to wine red) to obtain gold seeds.

[0128] (2.2) Pipette 53 mL of the original solution and add 55 mL of H2O and stir for 5 min; a resuspension containing 16 nm Au NPs is obtained.

[0129] (3) preparing extracellular solutions with different osmotic pressures, and loading the multimodal nanoparticles described in step (2) into the red blood cell skeleton described in step (1);

[0130] Prepare extracellular solutions with different osmotic pressures. The osmotic pressure of 1X PBS is 300mOsm / kg. Use H2O and 1X PBS to prepare extracellular solutions with different osmotic pressures of 300, 200, 150, 100, and 50mOsm / kg.

[0131] Add 0.8 mL of the above-configured extracellular solution with different osmotic pressures and 0.1 mL of the resuspension containing 16 nm Au NPs in step (2) to a 1.5 mL centrifuge tube, mix well, and add 0.1 mL of 100% HCT red blood cells. Shake at 45 rpm for 20 min at 4 ° C. Add 0.1 mL of 9% NaCl solution and transfer the centrifuge tube to 37 ° C for 30 min. After completion, centrifuge the centrifuge tube at 1000 rpm for 10 min, wash 3 times with 1X PBS, and obtain a red blood cell carrier loaded with multimodal nanoparticles. Resuspend with 1X PBS and store at 4 ° C for use.

[0132] The transmission electron microscopy image of the red blood cell carrier loaded with multimodal nanoparticles in this example (step 1) is shown in Figure 2 The test results of loading efficiency and carrier yield are shown in Figure 3 and Figure 4 , the optimal extracellular osmotic pressure of 16nm Au NPs was determined to be 150mOsm / kg.

[0133] Application Example 1

[0134] Cell level testing:

[0135] Expression of the membrane protein CD47 (integrin-associated protein) on mouse erythrocytes functions as a self-marker to avoid recognition as foreign by the immune system. CD47 on normal erythrocytes prevents this elimination by binding to the inhibitory receptor signal regulatory protein (SIRPa), and the presence or absence of CD47 is used to distinguish between self and foreign.

[0136] Testing process and testing methods:

[0137] (1) Sample preparation:

[0138] After washing with PBS buffer, 300 μL of RIPA lysis buffer containing protease inhibitors (PMSF) and phosphatase inhibitors was added to lyse the cells on ice for 30 minutes. Centrifugation was performed at 4°C, and the supernatant was the cell protein. The protein concentration was determined by UV-vis method (30 mg / mL).

[0139] The multimodal nanoparticle-loaded erythrocyte carriers 33 nm Au-RBC (150 mOsm / kg), 66 nm Au-RBC (100 mOsm / kg), and 136 nm Au-RBC (50 mOsm / kg) in Examples 1 to 3 were mixed with the supernatant buffer at a volume ratio of 4:1 and heated at 70°C in a metal bath for 10 minutes to fully denature the protein.

[0140] (2) Gel electrophoresis: Prepare 10% separation gel and stacking gel. Use 120V constant voltage electrophoresis until the bromophenol blue indicator reaches the bottom edge of the gel.

[0141] (3) Transfer: Use a constant current of 400 mA for 30 min.

[0142] (4) Blocking: Prepare a blocking solution of 5% BSA (bovine serum albumin) solution in TBST buffer and block the PVDF membrane with shaking at room temperature for 2 h.

[0143] (5) CD47 primary antibody incubation: Dilute the CD47 primary antibody solution (blocking solution 5% BSA diluted 1000 times), incubate the PVDF membrane with slow shaking at 4°C for 15 h, and rinse with TBST buffer at room temperature.

[0144] (6) Secondary antibody incubation: Horseradish peroxidase was diluted 4000 times in TBST buffer and incubated at room temperature with slow shaking for 1 hour, followed by rinsing with TBST buffer at room temperature.

[0145] (7) Detection: Developed by an all-purpose imaging analyzer, followed by grayscale analysis and other subsequent processing.

[0146] Figure 8 The protein immunoblot images of the membrane protein CD47 of the erythrocyte carrier loaded with multimodal nanoparticles in Examples 1 to 3 of the present invention are shown. It can be seen that compared with normal erythrocytes, the normal expression of the erythrocyte membrane protein CD47 of the erythrocyte carrier loaded with multimodal nanoparticles provides a basis for achieving long circulation in vivo.

[0147] Application Example 2

[0148] Animal testing:

[0149] In vivo imaging of small animals:

[0150] (1) The multimodal nanoparticle-loaded erythrocyte carriers 33 nm Au-RBC (150 mOsm / kg), 66 nm Au-RBC (100 mOsm / kg), and 136 nm Au-RBC (50 mOsm / kg) in Examples 1 to 3 were resuspended in 1X PBS buffer and stored at 4°C for later use.

[0151] (2) 5 mL of 40 μM DiR (MCE) in PBS solution and 5 mL of 4% HCT red blood cells were uniformly mixed and incubated for staining for 30 min. The cells were washed with 1X PBS buffer and resuspended into 50% HCT red blood cell suspension and stored at 4°C for later use.

[0152] (3) Healthy BALB / c nude mice (female, 6 weeks old) were selected as experimental mice and divided into four groups: blank group, experimental group 1, experimental group 2, and experimental group 3.

[0153] Blank group: After isoflurane anesthesia, each experimental mouse was injected with 100 μL of 50% HCT red blood cell suspension through the tail vein;

[0154] Experimental group 1: After isoflurane anesthesia, each experimental mouse was injected with 100 μL of 50% HCT 33nm Au-RBC (150 mOsm / kg) red blood cell suspension via the tail vein;

[0155] Experimental group 2: After isoflurane anesthesia, each experimental mouse was injected with 100 μL of 50% HCT 66nm Au-RBC (100 mOsm / kg) red blood cell suspension via the tail vein;

[0156] Experimental group 3: After isoflurane anesthesia, each experimental mouse was injected with 100 μL of 50% HCT 136nm Au-RBC (50 mOsm / kg) red blood cell suspension via the tail vein.

[0157] In vivo fluorescence imaging of small animals was recorded before injection and 1, 3, 7, 15, 22, and 30 days after injection under Ex: 740nm and Em: 790nm lasers.

[0158] like Fig. 9 As shown, the erythrocyte carriers loaded with nanoparticles under the optimal extracellular loading osmotic pressure conditions in Examples 1 to 3 can be circulated throughout the body after tail vein injection, and have obvious aggregation in the liver, spleen, cervical lymph nodes and bone marrow (blood cell precursors). Compared with normal erythrocytes, the three erythrocyte carriers loaded with nanoparticles can circulate in the body for more than 30 days, and have good in vivo circulation stability.

[0159] In summary, the present invention clarifies for the first time the osmotic pressure conditions for the advantageous loading of nanoparticles of different particle sizes by hypotonic loading of erythrocytes; the present invention uses gold nanoparticles of different particle sizes as multimodal nanoparticles, and designs hypotonic solutions with different osmotic pressure gradients, which clarifies the advantageous extracellular hypotonic loading conditions for erythrocytes to load particles of different sizes; silica nanoparticles can also be loaded into erythrocytes according to the law of osmotic pressure gradient, proving the universality of the preparation method of the erythrocyte carrier loaded with multimodal nanoparticles of the present invention. Among them, as the extracellular osmotic pressure decreases, the average particle size of the nanoparticles encapsulated by the erythrocyte skeleton gradually increases, and the yield of the erythrocyte carrier gradually decreases. In addition, the erythrocyte carrier loaded with multimodal nanoparticles of the present invention still maintains a complete biconcave disc-shaped structure, and the normal expression of the membrane protein CD47 can maintain its long-term circulation ability in the body for more than 30 days. The erythrocyte carrier loaded with multimodal nanoparticles and the preparation method of the present invention provide a feasible solution for the engineering transformation of erythrocytes, and are expected to promote their application in clinical transfusion medicine.

[0160] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A red blood cell carrier loaded with multimodal nanoparticles, characterized in that: It includes the erythrocyte skeleton and multimodal nanoparticles loaded inside it; In an extracellular solution with an osmotic pressure of 50-200 mOsm / kg, the erythrocyte skeleton is loaded with multimodal nanoparticles with an average particle size of 10-200 nm.

2. The erythrocyte carrier loaded with multimodal nanoparticles according to claim 1, characterized in that: The multimodal nanoparticles include one or more of gold nanoparticles, silicon dioxide nanoparticles, ferroferric oxide nanoparticles, and gold nanorod particles.

3. The erythrocyte carrier loaded with multimodal nanoparticles according to claim 1, characterized in that: When the average particle size of the multimodal nanoparticles is 16 to 33 nm, the loading osmotic pressure is 150 mOsm / kg; when the average particle size of the multimodal nanoparticles is 33 to 100 nm, the loading osmotic pressure is 150 to 100 mOsm / kg; when the average particle size of the multimodal nanoparticles is 100 to 136 nm, the loading osmotic pressure is 100 to 50 mOsm / kg.

4. A method for preparing a red blood cell carrier loaded with multimodal nanoparticles as claimed in claim 1, characterized in that: The preparation method comprises: adding a multimodal nanoparticle resuspension into an extracellular solution, then adding a 100% HCT erythrocyte skeleton, and incubating at 4°C with low-speed shaking; then adding a NaCl solution, and incubating at 37°C for insulation and sealing; and then centrifuging, washing, and resuspending to obtain an erythrocyte carrier loaded with multimodal nanoparticles with an average particle size of 10 to 200 nm.

5. The method for preparing the erythrocyte carrier loaded with multimodal nanoparticles according to claim 4, characterized in that: The osmotic pressure of the NaCl solution is 3080-4110 mOsm / kg.

6. The method for preparing the erythrocyte carrier loaded with multimodal nanoparticles according to claim 4, characterized in that: The molar concentration of the multimodal nanoparticles in the multimodal nanoparticle resuspension is 7.5-30 mM.

7. The method for preparing the erythrocyte carrier loaded with multimodal nanoparticles according to claim 4, characterized in that: The preparation method comprises: adding 0.1 ml of a multimodal nanoparticle resuspension to 0.8 ml of an extracellular solution of 50 to 200 mOsm / kg, then adding 0.1 ml of a 100% HCT erythrocyte skeleton, incubating at 4° C. with low-speed shaking for 1 to 60 min; then adding 0.1 ml of a 9% NaCl solution, incubating at 37° C. with insulation and sealing for 1 to 60 min; and then centrifuging, washing, and resuspending to obtain an erythrocyte carrier loaded with multimodal nanoparticles.

8. The method for preparing the erythrocyte carrier loaded with multimodal nanoparticles according to claim 4, characterized in that: The preparation method of the erythrocyte skeleton comprises: putting blood into an anticoagulant centrifuge tube containing sodium heparin, placing it in a centrifuge precooled at 4°C, and centrifuging it at 500-5000 rpm for 1-60 minutes; washing with PBS, resuspending it evenly with 100% HCT to obtain the erythrocyte skeleton, and storing it at 4°C for use.

9. Use of a red blood cell carrier loaded with multimodal nanoparticles as claimed in claim 1 in fluorescence imaging.

10. The use of the erythrocyte carrier loaded with multimodal nanoparticles in fluorescence imaging according to claim 9, characterized in that: The erythrocyte carrier loaded with multimodal nanoparticles is applied to in vivo fluorescence imaging and circulates in vivo for more than 30 days.

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