Preparation method of metal organic framework armored red blood cells and application of metal organic framework armored red blood cells in cryopreservation

Abstract

The invention discloses a preparation method of metal organic framework armored red blood cells and an application of the red blood cells in cryopreservation, an MOF armor coated on the surfaces of the red blood cells in situ in one step is prepared in a physiological environment, the red blood cells can be effectively prevented from being damaged by freezing induction, and meanwhile, the natural oxygen carrying function of the red blood cells is kept. The unique ice control capacity of the MOF armored red blood cells is explored, including inhibition of recrystallization of ice, induction of smoothness of the ice front, reduction of formation of sodium chloride eutectic and acceleration of ice melting, a novel design strategy is provided for an MOF-based ice control material, and the future prospect of the MOF-based ice control material in the field of efficient ice inhibition is possibly widened.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cryogenic technology, and particularly relates to a metal-organic framework armor capable of being actively attached to the surface of red blood cells and capable of rapid degradation, which is used to reduce ice crystal damage and improve the recovery rate of red blood cells in cryopreservation and maintain the natural oxygen-carrying function of red blood cells. BACKGROUND

[0002] Cryopreservation can suspend cell metabolism at low temperature (-80℃ or -196℃), and plays a crucial role in maintaining the function of red blood cells in vitro and achieving long-term storage. However, the freezing and thawing process will bring significant physiological stress, which is mainly due to the damage caused by ice crystals. Ice nucleation, growth and recrystallization can cause irreversible freezing damage, damage cell integrity and reduce preservation efficiency. Cryoprotective agents (CPA) have made great progress in cryobiology. High concentrations of cell-permeable CPAs, such as dimethyl sulfoxide and glycerol, help reduce ice formation. However, their solvent toxicity and challenges associated with removal constitute major obstacles for clinical applications. Inspired by the antifreeze proteins (AFPs) naturally produced in extreme cold environments, various synthetic compounds and nanomaterials that mimic the function of AFPs have been explored for cryopreservation. For example, poly (vinyl alcohol) and zwitterionic betaine can perform cell-friendly regulation on ice crystals, while nanoparticles such as graphene oxide and carbon nitride quantum dots exhibit ice recrystallization inhibition (IRI) properties, making them promising candidates for cell cryopreservation. Despite these advances, the development of new nanomaterials with strong IRI activity and effective enhancement of red blood cell cryopreservation recovery rate remains a very desirable goal. SUMMARY

[0003] The present application aims to provide a preparation method of metal-organic framework armored red blood cells and its application in cryopreservation. A new strategy for cryopreservation of red blood cells using zinc (Zn) based MOF, which can effectively encapsulate red blood cells into MOF armor in a physiological environment. In the sheep red blood cell cryopreservation test, MOF@RBC showed excellent cell recovery efficiency (~51%) at a low concentration of 1 mg·mL -1 , which was comparable to the performance of commercial cryoprotective agent hydroxyethyl starch (HES, 200 mg·mL -1 ). In addition, MOF SA particles capable of self-forming protective shells were also synthesized. Although MOF SAThe MOF armored RBCs do not exhibit IRI activity by themselves, but the introduction of RBCs activates this property, highlighting the importance of the MOF armored RBC structure for ice control. These MOF armored red blood cells not only inhibit the growth of ice crystals by limiting the movement of free water molecules, but also act as "catalysts" to smooth the ice front, reduce the formation of sodium chloride eutectics, and promote the melting of ice crystals, thereby improving the efficiency of cryopreservation. This armored strategy highlights the potential of MOF-based shell structures in cryopreservation and opens up avenues for the wider application of MOF-inspired spore-like structures in the biological field.

[0004] A method for preparing metal organic framework (MOF) armored red blood cells (RBCs), comprising an in-situ synthesis method or a physical mixing method;

[0005] The in-situ synthesis method is that red blood cells are mixed with raw materials for preparing MOF particles when the MOF particles are not synthesized, to generate a MOF armored red blood cell structure, thereby obtaining metal organic framework armored red blood cells.

[0006] The physical mixing method is that red blood cells are added after the synthesis of MOF particles, so that the MOF particles and the red blood cells are physically mixed, thereby obtaining metal organic framework armored red blood cells.

[0007] The present application constructs MOF particles that can be autonomously coated on the surface of red blood cells in-situ. This unique MOF armor has superior ice control ability, can reduce cryoinjury and improve cell recovery rate, and at the same time maintains its natural oxygen-carrying function.

[0008] As a preferred embodiment, the metal organic framework is a zinc-based metal organic framework.

[0009] As a preferred embodiment, the synthesis method of the MOF particles is that zinc ions (Zn 2 +) are coordinated with imidazole to prepare MOF particles in a water system containing sodium chloride, the synthesis temperature range is 4-37℃, and the synthesis time range is 5-30min.

[0010] As a preferred embodiment, the molar ratio of the zinc ions to the imidazole is 1:1 to 1:20, preferably 1:4.

[0011] As a preferred embodiment, the zinc ions are selected from zinc nitrate hexahydrate and zinc acetate.

[0012] As a preferred embodiment, the imidazole is selected from imidazole-2-carboxaldehyde and 2-methylimidazole.

[0013] As a preferred embodiment, the molar concentration of sodium chloride in the water system containing sodium chloride ranges from 0 to 154mmol / L, and is not 0.

[0014] As a preferred embodiment, the water containing sodium chloride is physiological saline.

[0015] As preferred, the Zn 2 + the concentration in the reaction system ranges from 10.8 to 54.0 μmol / mL, preferably 10.8 μmol / mL.

[0016] As preferred, the synthesis time of the in-situ synthesis method ranges from 5 to 30 min, and the temperature ranges from 4 to 37℃.

[0017] As preferred, the mixing time of the physical mixing method ranges from 1 to 20 min, and the temperature ranges from 4 to 37℃.

[0018] As preferred, the amount of red blood cells added ranges from 1 to 900 million per milliliter.

[0019] As preferred, the in-situ synthesis method comprises the following steps:

[0020] Dissolve polyvinylpyrrolidone (PVP) and imidazole in water containing sodium chloride, then add red blood cells and zinc ion solution, react to obtain metal organic framework (MOF) armored red blood cells.

[0021] Among them, the concentration of polyvinylpyrrolidone ranges from 0.5 to 5 mg / mL, and the molecular weight of PVP ranges from 40,000 to 54,000.

[0022] As preferred, in the in-situ synthesis method, the temperature of the reaction ranges from 4 to 37℃, and the time ranges from 5 to 30 min.

[0023] As preferred, in the in-situ synthesis method, the reaction is carried out under stirring, and the stirring speed ranges from 100 to 1,000 rpm.

[0024] As preferred, the in-situ synthesis method for synthesizing MOF@RBC specifically comprises the following steps:

[0025] 1. Add 0.5 mg of polyvinylpyrrolidone (PVP) and 4.8 mg of imidazole-2-formaldehyde to 980 μL of physiological saline, dissolve, then add 25 μL of red blood cells (5×10) and mix to obtain a mixture.

[0026] 2. Add 3.7 mg of zinc nitrate hexahydrate to 150 μL of physiological saline, then add the mixture obtained in step 1, stir the resulting mixture at room temperature for 10 minutes to obtain metal organic framework armored red blood cells prepared by in-situ synthesis, and name the resulting product as MOF@RBC.

[0027] As preferred, the physical mixing method comprises the following steps:

[0028] Polyvinylpyrrolidone (PVP) and imidazole are dissolved in water containing sodium chloride, then zinc ion solution is added, reacted, mixed with red blood cells after completion, and metal organic framework (MOF) armored red blood cells are obtained.

[0029] The concentration of polyvinylpyrrolidone is 0.5-5 mg / mL, and the molecular weight of PVP is 40,000-54,000.

[0030] Preferably, in the physical mixing method, the reaction temperature is 4-37℃, and the reaction time is 5-30 min; the mixing temperature is 4-37℃, and the mixing time is 1-20 min.

[0031] Preferably, in the physical mixing method, the reaction and mixing are carried out under stirring, and the stirring speed is 100-1000 rpm.

[0032] Preferably, in the physical mixing method, the MOF SA +RBC is synthesized.

[0033] 1. 0.5 mg of polyvinylpyrrolidone (PVP) and 4.8 mg of imidazole-2-formaldehyde are added to 980 μL of physiological saline to obtain a mixture.

[0034] 2. 3.7 mg of zinc nitrate hexahydrate is added to 150 μL of physiological saline, and then the mixture obtained in step 1 is added. The resulting mixture is stirred at room temperature for 10 minutes. Then, the supernatant is discarded by centrifugation, and the remaining precipitate is washed with pure water. Finally, they are redispersed in 1130 μL of physiological saline to obtain a dispersion.

[0035] 3. 25 μL of red blood cells (5×10) are added to the dispersion prepared in step 2 above, and mixed for 10 min to obtain metal organic framework armored red blood cells prepared by physical mixing, and the product is named MOF SA +RBC.

[0036] The metal organic framework (MOF) armored red blood cells (RBC) obtained by the above preparation method.

[0037] Preferably, the MOF armored red blood cells can be degraded in a citric acid buffer to unload the red blood cells, and the pH of the citric acid buffer is 5-6.5, and the unloading time required is 20-30 min.

[0038] The above metal organic framework (MOF) armored red blood cells (RBC) are applied in cryopreservation.

[0039] As a preference, the conditions of the cryopreservation are: freezing at -80°C for 24h followed by thawing at 37°C, or immersion in liquid nitrogen for 2h followed by thawing at 4°C for 150min.

[0040] The metal-organic framework (MOF) armored red blood cells (RBCs) have superior ice-controlling abilities including inhibiting ice recrystallization (IRI) activity, smoothing ice front morphology, inhibiting sodium chloride eutectic formation, and promoting ice melting.

[0041] The present application can effectively protect red blood cells from freeze-induced damage while maintaining their natural oxygen-carrying function by preparing MOF armor in situ on the surface of red blood cells in a physiological environment. The unique ice-controlling ability of MOF armored red blood cells is explored, providing a novel design strategy for MOF-based ice-controlling materials, which may broaden their future prospects in the field of efficient ice inhibition.

[0042] Advantages:

[0043] MOF armored red blood cells not only inhibit ice recrystallization by limiting the movement of water molecules within the porous MOF structure, but also act as "catalysts" to induce the smoothing of ice fronts during growth, reduce the formation of sodium chloride eutectics, and accelerate ice melting. These findings highlight the potential of MOF-derived structures in the cryopreservation of various cell types and tissue samples, paving the way for innovative preservation techniques. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 are schematic diagrams and SEM characterization of MOF armored red blood cells. Among them, A) synthesis schematic diagram of MOF armored red blood cells. B) MOF WA , C) MOF SA , D) MOF@RBC and E) MOF SA +RBC representative SEM images. The scale bar is 1 μm.

[0045] Figure 2 are the characterization of the synthesized MOF armored red blood cells. Among them, A) conceptual diagram of MOF armored red blood cells formed in water or salt-based systems, using zinc ions as central ions and imidazole as organic ligands. Carbon atoms are marked as purple balls, nitrogen atoms are marked as yellow balls, zinc atoms are marked as blue balls, oxygen atoms are marked as red balls, and hydrogen atoms are marked as white balls. MOF WA , MOF SA , MOF@RBC and MOF SA +RBC characterization spectrum B) DLS, C) XRD, D) TGA curve and E) FTIR spectrum.

[0046] Figure 3Ice inhibition mechanism of MOF armored red blood cells. Wherein, A) MOF WA , MOF SA , MOF@RBC and MOF SA +RBC added respectively. Scale bar, 100 μm. (B) Quantitative analysis of ice crystal maximum size (MGS). Statistical analysis was performed using Tukey's test. Different letters indicate statistically significant differences between groups, p<0.05. C) Directional ice growth microscopy (DIGM) images. The microscope captured the ice growth front at 150 μm intervals (marked with vertical dashed lines) and recorded its time progression. Scale bar, 50 μm. (D) DSC melting curves of frozen dispersion droplets with a sample mass control of 10 mg. (E) Melting temperature (Tm-max) corresponding to the highest peak of the melting curve of frozen droplets containing different samples. *p<0.05. -1

[0047] Figure 4 Evaluation of MOF armored red blood cells' cryopreservation capacity. Wherein, A) Recovery rate of sheep red blood cells cryopreserved in physiological saline dispersions added with different MOF materials or hydroxyethyl starch (HES, Aladdin). The diagonal-filled freeze-thaw condition is freezing at -80℃ for 24 h followed by thawing at 37℃, and the fast immersion in liquid nitrogen for 2 h followed by slow thawing at 4℃. B) Time-dependent luminescence curves of native RBC and MOF armored red blood cells after adding a luminol-perborate mixture. The red line indicates that the sample has not been freeze-thawed, and the blue line indicates that the sample has been freeze-thawed. Wherein, -f indicates that the sample has been freeze-thawed, and -fd indicates that the sample has been freeze-thawed and degraded with MOF armor.

[0048] Figure 5 Optical microscope images of MOF armored red blood cells. Wherein, -f indicates that the sample has been frozen at -80℃ for 24 h and thawed at 37℃, and -fd indicates that the sample has been freeze-thawed and degraded with MOF armor. Scale bar, 5 μm.

[0049] Figure 6 SEM images of MOF armored red blood cells, -fd indicates that the sample has been freeze-thawed and degraded with MOF armor. Wherein, A) MOF SA +RBC, B) MOF@RBC. DETAILED DESCRIPTION

[0050] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but in no way limit the present application.

[0051] ​In the following examples, the red blood cells were sheep red blood cells, purchased from Hunan Bickman.

[0052] Comparative Example 1

[0053] 4.8 mg imidazole-2-carboxaldehyde was dissolved in 1005 μL of deionized water. Then, 150 μL of deionized water containing 3.7 mg zinc nitrate hexahydrate was added under vortexing at 1000 rpm. MOF was synthesized at room temperature and vortexed at 1000 rpm for 10 min. WA , to obtain MOF WA The dispersion.

[0054] Comparative Example 2

[0055] Similar to Comparative Example 1, except that deionized water was replaced with physiological saline (0.9%) to synthesize MOF. SA , to obtain MOF SA The dispersion. Other raw material parameters are similar to MOF. WA The synthesis methods are consistent.

[0056] Comparative Example 3

[0057] Add 25 μL of red blood cells (5 × 10⁻⁶) to 1130 μL of physiological saline. 8 (1), to obtain a dispersion containing RBC.

[0058] Comparative Example 4

[0059] Add 51 mg of hydroxyethyl starch (HES, Aladdin) to 255 μL of physiological saline to prepare a 200 mg / mL HES-200 solution, then add 25 μL of red blood cells (5 × 10⁻⁶). 8 (1), to obtain a dispersion of red blood cells containing HES, which was used for the cryopreservation experiment in Example 7.

[0060] Comparative Example 5

[0061] (1) Dissolve 4.8 mg imidazole-2-carboxaldehyde in 1005 μL of deionized water, and add 150 μL of deionized water containing 3.7 mg zinc nitrate hexahydrate under vortexing at 1000 rpm. The mixture is then vortexed at 1000 rpm for 10 min at room temperature to obtain a solution containing MOF. WA The dispersion was then centrifuged at 6000 rpm for 10 minutes and washed with 2 mL of pure water. Finally, the resulting solid was redispersed in 1130 μL of physiological saline to obtain the dispersion.

[0062] (2) Add 25 μL of red blood cells (5 × 10⁻⁶) to the dispersion obtained in (1). 8 (number), to obtain MOF-containing WA +RBC dispersion.

[0063] Comparative Example 6

[0064] (1) 0.5 mg of polyvinylpyrrolidone (PVP, M.W. ~40000, K30) and 4.8 mg of imidazole-2-carboxaldehyde (98%, Macron) were added into 980 μL of normal saline under vortex at 1000 rpm to obtain solution A.

[0065] (2) 3.7 mg of zinc nitrate hexahydrate was added into 150 μL of normal saline, and then mixed with solution A. The resulting mixture was stirred at room temperature for 10 minutes. Subsequently, it was centrifuged at 6000 rpm for 10 minutes and washed with 2 mL of pure water. Finally, the resulting solid was re-dispersed in 1130 μL of normal saline to obtain a dispersion.

[0066] (3) 25 μL of red blood cells (5 x 10 8 were added into the above prepared dispersion, and stirred at room temperature under vortex (1000 rpm) for 10 seconds to obtain a dispersion containing MOF SA + RBC-S, which was immediately used for the cryopreservation experiment of Example 7.

[0067] Example 1

[0068] The method for preparing metal organic framework (MOF) armored red blood cells (RBC) is prepared by an in-situ method, comprising the following steps:

[0069] (1) 0.5 mg of polyvinylpyrrolidone (PVP, M.W. ~40000, K30) and 4.8 mg of imidazole-2-carboxaldehyde (98%, Macron) were added into 980 μL of normal saline under vortex at 1000 rpm to obtain a mixture.

[0070] (2) 3.7 mg of zinc nitrate hexahydrate was dissolved into 150 μL of normal saline to obtain solution B.

[0071] (3) 25 μL of red blood cells (5 x 10 8 were added into the mixture obtained in step (1), and then solution B was added. The resulting mixture was stirred at room temperature under vortex (1000 rpm) for 10 minutes to obtain a dispersion containing MOF@RBC. This protection strategy can quickly and efficiently create spore-like structures using the MOF platform.

[0072] Example 2

[0073] The method for preparing metal organic framework (MOF) armored red blood cells (RBC) is prepared by a physical mixing method, comprising the following steps:

[0074] (1) To 980 μL of normal saline, 0.5 mg of polyvinylpyrrolidone (PVP, M.W. ~40000, K30) and 4.8 mg of l-imidazole-2-carboxaldehyde (98%, Macron) were added to obtain solution A under vortex at 1000 rpm.

[0075] (2) 3.7 mg of zinc nitrate hexahydrate was added to normal saline 150 μL, then mixed with solution A, the resulting mixture was stirred at room temperature for 10 minutes. Subsequently, by centrifugation at 6000 rpm for 10 minutes, and washed with 2 mL of pure water. Finally, the resulting solid was redispersed in 1130 μL of normal saline to obtain a dispersion.

[0076] (3) To the above prepared dispersion, 25 μL of red blood cells (5 x 10 8 ) were added, stirred at room temperature under vortex (1000 rpm) (1000 rpm) for 10 minutes to obtain a dispersion containing MOF SA + RBC.

[0077] Scanning electron microscopy (SEM) was used to analyze and characterize the detailed morphology of the synthesized materials. MOF WA was characterized by flake-based helixes, forming hollow spheres with a diameter of about 500 nm. This hollow structure is attributed to the Ostwald ripening Figure 1 B) during the synthesis process. Meanwhile, in the saline synthesis system, MOF SA exhibited the morphology of larger solid spheres with a diameter of 2-3 μm, which was due to the self-assembly process during the polymerization process Figure 1 C). In the in-situ coating strategy (MOF@RBC), a uniform MOF protective shell was constructed as a continuous nanobiointerface, covering the entire surface of the red blood cell Figure 1 D). Compared with the in-situ coating, the physical mixing coating method obtained MOF SA + RBC by directly mixing the pre-synthesized MOF SA with RBC, while stirring for 10 minutes. The physical mixing method could only lead to a discontinuous coating of MOF SA with partially exposed RBC Figure 1 E).

[0078] The successful preparation of MOF armored red blood cells by the reaction of zinc ions and imidazole-2-carboxaldehyde was further confirmed Figure 2 . Dynamic light scattering (DLS) analysis was used to evaluate the hydrodynamic size and distribution of particles in solution Figure 2 B). MOF WA exhibited good size uniformity, with a hydrodynamic size of 670 ± 5 nm, which corresponded well with the SEM images. MOF SAA large peak at 2.6 μm was observed, confirming the formation of solid spherical assemblies. MOF@RBC and MOF SA Both +RBC exhibited a similar bimodal distribution, with the peak at 299 nm of MOF@RBC being smaller than that of MOF. SA The peak at 595 nm of the RBC is attributed to the interference of RBC on the self-assembly of free MOF nanoparticles. Their main peak around 3.8 μm corresponds to MOF-armored RBC composite particles. X-ray diffraction (XRD) analysis indicates that MOF... SA MOF@RBC and MOF SA +RBC has a good crystal structure and exhibits similarities to MOF. WA Same diffraction peaks ( Figure 2 C). Thermal properties of MOF particles obtained using thermogravimetric analysis (TGA) Figure 2 D). Initial weight loss around 100°C can be attributed to retained water within the pores. Rapid weight loss above 300°C is likely due to frame deformation. MOF@RBC and MOF SA +RBC exhibits a comparable maximum decomposition temperature at 339℃, but its weight loss rate is lower than that of MOF. SA More significantly, this can be attributed to the carbonization of the erythrocytes. Fourier transform infrared spectroscopy (FTIR) is used to identify functional groups ( Figure 2 E). At 1660cm -1 A characteristic peak was observed at a wavenumber corresponding to the C=O bond of the aldehyde group present in the ligand. The band at 545 cm⁻¹ indicates Zn-N stretching, confirming coordination of the zinc ion with the nitrogen (N) in the imidazole. Furthermore, with MOF… WA and MOF SA In comparison, MOF@RBC and MOF SA The FTIR spectra of +RBC all show a shift of the characteristic peak of C=O to a lower wavenumber, which may be due to the interaction between MOF and RBC.

[0079] Example 4

[0080] Ice recrystallization inhibition (IRI) activity was quantitatively assessed using the "splat" assay. 20 μL of dispersion at room temperature (25°C) (the final dispersions obtained in Comparative Examples 1-3 and Examples 1-2, with physiological saline as a control) was dropped onto a glass slide pre-cooled to -60°C at a depth of 1.5 m to form a thin film of solid ice. The film was then incubated at 10°C / min. -1 The cold stage (Lankam, BCS196) temperature was raised to -9°C at a certain rate, and annealed at this temperature for 30 min to evaluate the material's activity in inhibiting ice recrystallization. Polarized optical microscopy images of ice crystals after MOF@RBC addition showed the smallest grain size compared to the negative control (physiological saline).Figure 3 A) Quantitative analysis showed that the maximum ice crystal size (MGS) in MOF@RBC was about 58% of that in the physiological saline group, MOF SA +RBC group was about 78%( Figure 3 B) These findings suggest that MOF armored red blood cells effectively inhibit ice crystal recrystallization. Compared with MOF SA +RBC, MOF@RBC has higher IRI activity, which can be attributed to the more uniform MOF protective shell of the latter.

[0081] Example 5

[0082] In addition to IRI, the morphology of ice crystals also affects the efficiency of cell cryopreservation. The observation method for smoothing ice peak morphology is to use a micro-pool to determine the critical velocity associated with the morphological transition of the growing ice front. The right side of the device is located at the center of the cold stage to locally reduce the temperature, while the other side is kept at an ambient temperature of 15°C. This setup spatially induces a temperature gradient of 2.5 cm in length. The dispersion of MOF armored red blood cells (Comparative Example 2, the dispersion finally obtained in Examples 1 and 2, and physiological saline as a control) confined in the channel of the above device undergoes gradual growth from the cold end to the room temperature region, and the growth of ice gradually slows down away from the cold source. The morphology of the ice front is recorded every 150 μm, the curvature transition of the ice peak from sharp to smooth is observed, and the time interval is measured to calculate the critical transition velocity to characterize the ability of the material to smooth the ice peak morphology.

[0083] The sharp front of the ice peak in the control physiological saline transitions to a smoother one, and the critical velocity is calculated to be 2.0 ± 0.9 μm / s. The introduction of MOF SA , MOF SA +RBC and MOF@RBC increases the critical velocity of ice front transition to 4.0 ± 0.3, 4.7 ± 0.4 and 6.6 ± 0.3 μm / s, respectively, promoting the curvature transition of the ice growth front Figure 3 C) For red blood cell cryopreservation, smooth ice crystal morphology is superior to needle-shaped ice crystal morphology because they reduce the possibility of cell membrane damage caused by sharp ice protrusions during the freezing storage process.

[0084] Example 6

[0085] The observation method for inhibiting eutectic formation and promoting melting is to examine the effect of incorporating MOF armored red blood cells on the thermal conductivity of the system using differential scanning calorimetry (DSC). Liquid samples (Comparative Examples 1-3, the dispersions finally obtained in Examples 1-2, and physiological saline as a control) (10 mg) are cooled from 25°C to -60°C at a rate of 20°C·min -1 , held for 3 minutes, and then heated at a rate of 2°C·min -1The MOF was slowly reheated to 20 °C. The results showed that... SA MOF SA +RBC and MOF@RBC frozen dispersions compared to natural RBC and MOF WA The dispersion melts faster. Figure 3 (D and E). Unlike IRI, this property is MOF. SA The particles are inherent and do not require the formation of spore-like structures. Furthermore, DSC analysis showed that MOFs... SA MOF SA +RBC and MOF@RBC significantly inhibited the formation of NaCl eutectic at -22℃, which may help improve the cryopreservation recovery of RBC.

[0086] Example 7

[0087] The cryoprotective effect of the armor structure was evaluated through freeze-thaw cycles to demonstrate the potential of the synthesized MOF material in enhancing red blood cell cryopreservation. This was achieved using MOF@RBC and MOF... SA In the unique structure of RBCs, they are protected by self-assembled MOFs. Therefore, to determine the cell recovery rate after freeze-thaw cycles, 100 μL of citrate buffer (pH 6.25) was added every 5 min until the amount of original MOF was reached. WA MOF SA MOF@RBC and MOF SA The MOF degradation and removal of the protective MOF shell were achieved by increasing the volume of the RBC dispersion to twice its volume. Two different freeze-thaw protocols were used for the red blood cell cryopreservation. The first method involved cooling cryovials containing 1 mL of the prepared sample (the final dispersions obtained in Comparative Examples 4-6 and Examples 1-2) to -80°C and holding them frozen for 24 hours, followed by thawing in a water bath at 37°C. The second method involved immersing 1 mL of each sample (the final dispersions obtained in Comparative Examples 4-6 and Examples 1-2) in liquid nitrogen for 2 hours. Subsequently, the samples were thawed at 4°C for 150 minutes. MOF@RBC and MOF were removed via a stepwise degradation method using citric acid. SA MOF armor was extracted from RBCs, and the remaining sample was adjusted to an equal volume by adding physiological saline. All samples were centrifuged at 300g for 3 minutes to remove intact cells. Subsequently, the absorbance of the supernatant was measured at 541nm using a microplate reader to assess the degree of hemolysis and measure cell recovery rate. Notably, under mild testing conditions, MOF@RBCs showed good hemolysis at 1 mg / mL. -1 A peak cell recovery rate of 51% was achieved at a low concentration, matching the performance of the commercial cryoprotectant HES, which requires a much higher concentration (200 mg / mL). -1Only then can the same effect be achieved. Even under strict freeze-thaw conditions, MOF@RBCs exhibited superior preservation capabilities with a recovery rate of 32%, outperforming HES, highlighting the cryoprotection potential of this one-step MOF-based erythrocyte loading strategy. Figure 4 A). In contrast, MOF WA It failed to provide protection under mild freeze-thaw conditions, and the protection increased with prolonged mixing time (from MOF). SA +RBC-S to MOF SA +RBC), MOF SA The incorporation increased the recovery rate from 9% to 35%. This indicates that the formation of spore-like structures in MOF-armored erythrocytes is crucial for effective cryopreservation. Optical microscopy further confirmed the presence of intact erythrocytes after freeze-thaw protection within the MOF shell. Figure 5 It is worth noting that after removing the MOF shell, MOF@RBC and MOF SA +RBC retains its original biconcave disk shape. Figure 6 In summary, these results highlight MOF@RBC as an effective cryoprotectant that offers superior preservation compared to conventional HES, while requiring a significantly lower concentration.

[0088] Example 8

[0089] Luminol-based chemiluminescence assays were used to assess hemoglobin-related behavior in red blood cells (RBCs). The method for detecting erythrocyte oxygen-carrying capacity involved dissolving 250 mg sodium carbonate, 35 mg sodium perborate, and 5 mg luminol in 5 mL of water. The luminol solution was allowed to stand undisturbed in a dark room for 5 minutes. Subsequently, 150 μL of sample (the final dispersions obtained in Comparative Examples 3, 5, and Examples 1-2) was added to a black 96-well plate at a concentration of 100 million red blood cells per mL. Then, 30 μL of luminol solution was rapidly added to each well using a pipette, and after mixing in the dark for 30 seconds, the luminescence intensity trend at OD425 nm was immediately measured using a microplate reader. (RBC, MOF) WA MOF@RBC and MOF SA +RBCs exhibited chemiluminescence, with intensity increasing within 20 minutes. This indicates that, due to the inherent porosity of MOFs, the armor shell does not hinder the entry of small molecules such as luminol and peroxides into the RBCs. Notably, unprotected RBCs and MOFs after freeze-thaw cycles... WA The decreased luminescence intensity of +RBCs indicates that freezing impaired the iron-catalytic properties of intracellular hemoglobin. (In MOF) SA +RBCs, red blood cells and MOF SACombination mitigated the decrease in luminescence intensity due to freezing. Even after freeze-thaw cycles, MOF@RBCs still maintained increased luminescence intensity, demonstrating the effectiveness of MOF armor in reducing freeze damage Figure 4 B).

[0090] For any skilled person familiar with the art, many possible variations and modifications to the technical solutions of the present application can be made using the technical content disclosed above, or modified as equivalent embodiments of equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the content of the technical solutions of the present application, should still belong to the scope of protection of the technical solutions of the present application.

Claims

1. A method of preparing metal organic framework armored red blood cells, characterized in that, The in-situ synthesis method or the physical mixing method; The in-situ synthesis method is that red blood cells are mixed with raw materials for preparing MOF particles to obtain MOF armored red blood cells when the MOF particles are not synthesized; The physical mixing method is that red blood cells are added after the synthesis of MOF particles to physically mix the MOF particles with the red blood cells to obtain metal organic framework armored red blood cells.

2. The production method according to claim 1, characterized by, The synthesis method of the MOF particles is that zinc ions are coordinated with imidazole in a water system containing sodium chloride to prepare MOF particles, the synthesis temperature is 4-37℃, and the synthesis time is 5-30 min.

3. The production method according to claim 2, characterized by, The molar ratio of zinc ion to imidazole is 1:1 to 1:20; the zinc ion is selected from zinc nitrate hexahydrate, zinc acetate; the imidazole is selected from imidazole-2-carboxaldehyde, 2-methylimidazole; the molar concentration of sodium chloride in the water system containing sodium chloride ranges from 0 to 154 mmol / L, and is not 0; the Zn 2 + is in the reaction system at a concentration of 10.8-54.0 μmol / mL.

4. The method of claim 1, wherein, The synthesis time of the in-situ synthesis method is 5-30 min, and the temperature is 4-37℃; the mixing time of the physical mixing method is 1-20 min, and the temperature is 4-37℃; the amount of red blood cells added is 1-9 billion per milliliter.

5. The preparation method according to claim 1, characterized in that, The in-situ synthesis method comprises the following steps: Polyvinylpyrrolidone and imidazole are dissolved in water containing sodium chloride, and then red blood cells and zinc ion solution are added to obtain metal organic framework armored red blood cells after reaction; The physical mixing method comprises the following steps: Polyvinylpyrrolidone and imidazole are dissolved in water containing sodium chloride, and then zinc ion solution is added to react, and after the reaction is completed, red blood cells are mixed to obtain metal organic framework armored red blood cells.

6. The preparation method according to claim 5, characterized in that, The concentration of polyvinylpyrrolidone is 0.5-5 mg / mL, and the molecular weight is 40,000-54,000.

7. The metal organic framework armored red blood cells obtained by the preparation method of any one of claims 1-6.

8. The MOF- armored RBC of claim 7, wherein, The metal organic framework armored red blood cells are degraded in a citric acid buffer to unload the red blood cells.

9. The MOF- armored RBC of claim 8, wherein, The pH of the citric acid buffer ranges from 5 to 6.5, and the required time for unloading is 20-30 min.

10. The metal organic framework armored red blood cells of claim 7 are used in cryopreservation.

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