CN112778421B

CN112778421B - Polydopamine hemoglobin-loaded micro-nano particle and preparation method and application thereof

Info

Publication number: CN112778421B
Application number: CN201911059467.2A
Authority: CN
Inventors: 赵莲; 王权; 周虹; 王瑛; 尤国兴; 胡吉林
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2019-11-01
Filing date: 2019-11-01
Publication date: 2022-10-18
Anticipated expiration: 2039-11-01
Also published as: CN112778421A

Abstract

The invention discloses a polydopamine hemoglobin-loaded micro-nano particle and a preparation method and application thereof. The polydopamine hemoglobin-loaded micro-nano particle has a complete chemical structure, good oxygen carrying capacity, blood compatibility, biological safety and oxidation resistance due to a simple synthesis process, has a wide blood substitute application prospect, and can play an important role in the field of medicines.

Description

Polydopamine hemoglobin-loaded micro-nano particle and preparation method and application thereof

Technical Field

The invention relates to the technical field of hemoglobin oxygen carrier blood substitutes, in particular to a polydopamine hemoglobin-loaded micro-nano particle and a preparation method and application thereof.

Background

Timely and effective blood transfusion is an important and common clinical treatment and cure means, and is widely used in the aspects of surgical operation, treatment of internal anemia, war wound treatment and the like. However, the blood has increasingly serious limitations of blood-borne disease transmission, difficult storage and cross matching before treatment, so that the blood which is in short supply is difficult to meet the increasing clinical requirement of blood transfusion, which causes the traditional blood transfusion treatment to face a significant challenge and provides an important opportunity for the research of blood substitutes.

The blood substitute realizes partial replacement of blood by simulating the structure and function of blood components, can be used as necessary supplement of clinical blood mainly due to the advantages of sufficient sources, convenient storage and transportation, no need of cross matching and the like, and can also become important logistics guarantee in military battles. Although the current technical level for preparing a true 'whole blood' substitute cannot be realized, blood component substitutes with certain functions, especially red blood cell substitutes with oxygen carrying/releasing functions, are the key points and difficulties of the current research in various countries. Hemoglobin is an oxygen carrying/releasing function in red blood cells, however, free hemoglobin easily permeates vascular endothelium, is combined with vasodilation factors, causes vasoconstriction, has the risk of glomerular filtration, and cannot be directly used for infusion. Therefore, the erythrocyte substitute with the Oxygen carrying/releasing function is mainly prepared by chemically modifying, polymerizing, crosslinking and the like the stroma-free Hemoglobin or wrapping the stroma-free Hemoglobin in a high-molecular microcapsule to enable the stroma-free Hemoglobin to simulate the Oxygen carrying/releasing capacity of the Hemoglobin in natural erythrocytes and be safely and effectively used for human bodies, and is also called Hemoglobin Oxygen Carriers (HBOCs). The research and development of hemoglobin oxygen carriers have subversive scientific significance for transfusion medicine and blood guarantee.

It can be seen that the hemoglobin oxygen carrier needs to have the following characteristics: 1) Hemoglobin is widely available and readily available; 2) Surface antigens of the red blood cells are removed, and cross matching is not needed during use; 3) Long preservation time; 4) Particle size is much smaller than that of natural red blood cells, and microcirculation oxygen supply can be improved through narrowing blood vessels.

The first work in developing hemoglobin-based oxygen carriers was to chemically modify and crosslink matrix-free hemoglobin, with chemically modified and crosslinked HBOCs representing the most species. Among them, HBOC-201 developed by Biopure corporation of America, which cross-linked bovine hemoglobin with glutaraldehyde, was marketed successively in south Africa and Russia, was the only HBOCs product successfully used in clinic so far, but further application thereof was limited due to more clinical side reactions.

In recent years, the template self-assembly technology has the advantages of simple process, high hemoglobin entrapment amount, controllable morphology and particle size and the like in the aspect of constructing hemoglobin oxygen carriers, and is widely concerned. Relevant studies have shown that: the microspheres formed by alternate layer-by-layer assembly based on glutaraldehyde and hemoglobin can obviously improve the content of hemoglobin in the microspheres, so that the concentration of hemoglobin can reach 1.36g/cm ³ (Duan, L.; yan, X.; wang, A.; jia, Y.; li, J., high ply charged heparin spheres as promoting aromatic oxygen carriers. Acs Nano2012,6, 6897-6904.); the hemoglobin-loaded micro-nano particles with the particle size of about 800nm can be prepared by selecting manganese carbonate as an adsorption template of hemoglobin, and the hemoglobin-loaded micro-nano particles can remarkably reduce the vasoactivity of the micro-nano particles so as to avoid side effects such as vasoconstriction and hypertension (Xiong, Y.; liu, Z.; georgieva, R.; smuda, K.; steffen, A.; sendski, M.; voigt, A.; patzak, A.;

nonsoconstrictive hemoglobin as oxygen carriers, acs Nano 2013,7, 7454-61.); however, on one hand, the hemoglobin micro-nano particles prepared based on the template self-assembly technology still need to form Schiff base bonds between glutaraldehyde and hemoglobin for cross-linking assembly (the schematic microstructure thereof is shown in FIG. 16A (Yu C; huang X; qian D; han F; xu L; tang Y; bao N; gu H, diagnosis and evaluation of hemoglobin-based hemoglobin micro capsules as oxygen carriers, 2018,54, 4136-4139), wherein the round balls in the figure represent hemoglobin, the short sticks between the round balls represent the Schiff base bonds, and the hemoglobin micro-nano particles are hollow and have reticular surfaces, and the Schiff base bonds between the two are easy to break during use, have insufficient chemical stability, so that the glutaraldehyde is free and easily causes biotoxicity after infusion. Secondly, the micro-nano particlesThe surface lacks a cell-like membrane as a protective barrier, and hemoglobin is naked, is easy to be captured and identified by a reticuloendothelial system and is difficult to stably exist in blood.

In addition, mitragortri et al flexibly select PLGA (poly (lactic acid-co-glycolide)) or PS (polystyrene) microspheres as a preparation template, so that the prepared micro-nano particles are closer to the three-dimensional structure and deformation characteristics of erythrocytes, and are beneficial to passing narrow blood vessel parts and improving microcirculation. (Doshi, N.; zahr, A.S.; bhaskar, S.; lahann, J.; mitrapotri, S., red blood cell-simulating synthetic biological components. Proceedings of the National Academy of Sciences of the United States of America 2009,106, 21495-21499.). The micro-nano particles also have the defects.

It can be seen that the above insufficient stability is an important reason for limiting further development of the template self-assembly-like HBOCs. In addition, due to the lack of a reductase system contained in native red blood cells, hemoglobin undergoes increased autoxidation under off-physiological conditions, changes in conformation, and suffers from impaired oxygen-carrying capacity, i.e., hemoglobin is structurally and functionally unstable.

Disclosure of Invention

The invention aims to solve the technical defects in the prior art, and provides a high-stability polydopamine-loaded hemoglobin micro-nano particle which is obtained by wrapping hemoglobin with polydopamine.

The micro-nano particles are in a regular spherical shape, polydopamine is adhered to the outer surface of the spherical shape to form an adhesion layer as a protective film of hemoglobin, and the Zeta potential of the adhesion layer is-22.33 to-21.81 mV.

The average hydrated particle size distribution of the micro-nano particles is 814-866 nm, and the polydispersity coefficient PDI is 0.11-0.29.

In a second aspect, the invention provides a method for preparing the polydopamine-loaded hemoglobin micro-nano particle, which sequentially comprises the steps of forming a primary particle by taking manganese carbonate as an adsorption template to immobilize hemoglobin in a coprecipitation reaction system, constructing a polydopamine adhesion layer on the surface of the primary particle, and removing a manganese carbonate adsorption template.

The method for forming the primary particles by taking manganese carbonate as an adsorption template to immobilize hemoglobin specifically comprises the following steps: to contain MnCl ₂ Adding Na into the mixed solution of 2.4-12mg/mL hemoglobin ₂ CO ₃ Stirring the solution until the reaction system is brick red and no precipitate is generated, and centrifuging at 6000-10000g for 5-15min to obtain precipitate as formed primary particles.

The method for constructing the polydopamine adhesion layer on the surface of the primary particle specifically comprises the following steps: adding a Tris-HCl buffer solution into the primary particles to ensure that the pH of a reaction system is =8.5, adding 1-10mg/mL of dopamine solution, and continuously stirring until the amount of dopamine is not reduced any more, which indicates that a polydopamine adhesion layer is constructed on the surfaces of the primary particles; preferably, the ratio of the added mass of dopamine to the amount of hemoglobin is 1: (1-5).

The adsorption template for removing manganese carbonate specifically comprises the following steps: the reaction system in which the poly-dopamine adhesion layer was constructed was centrifuged, the supernatant was discarded, and after washing with PBS (pH = 7.4), na was added ₂ Suspending in EDTA (pH = 7.4) solution, centrifuging and discarding supernatant, and precipitating to obtain the polydopamine-loaded hemoglobin micro-nano particles; preferably, the centrifugation condition is 6000-10000g centrifugation for 5-15min.

The concentration of the hemoglobin in the mixed solution is 2.4-12.0mg/mL.

Said MnCl-containing compound ₂ And 2.4-12mg/mL of hemoglobin in MnCl ₂ The concentration of (A) is 0.1-0.3M.

In a third aspect, the invention provides an application of the polydopamine-loaded hemoglobin micro-nano particle or the polydopamine-loaded hemoglobin micro-nano particle prepared by the method in a blood substitute, wherein the micro-nano particle partially or completely replaces red blood cells to play an oxygen carrying/releasing function in a blood transfusion process (caused by trauma, blood loss, anemia, surgery and the like).

Compared with the prior art, the invention has the beneficial effects that:

(1) The polydopamine hemoglobin-loaded micro-nano particle can maintain good stability under two conditions of standing and a flow field, the particle size change is small, and the hemoglobin is not released suddenly.

(2) The polydopamine hemoglobin-loaded micro-nano particle has the advantages of simple and mild preparation conditions and strong controllability, is suitable for large-scale production, and does not need the fussy operation of removing an organic solvent in the traditional preparation process of microcapsule-coated HBOCs.

(3) The polydopamine hemoglobin micro-nano particle does not need to adopt glutaraldehyde to crosslink hemoglobin, so that biotoxicity caused by free glutaraldehyde can be avoided.

(4) The polydopamine hemoglobin-loaded micro-nano particle takes polydopamine as a protective barrier of hemoglobin, so that stable existence of hemoglobin in blood is facilitated, and leakage risk of hemoglobin is reduced.

(5) The polydopamine hemoglobin-loaded micro-nano particle is in a regular spherical shape, can keep complete chemical structures of polydopamine and hemoglobin, and has good blood compatibility and lower cytotoxicity.

(6) The polydopamine hemoglobin-loaded micro-nano particle can effectively carry/release oxygen, can efficiently remove free radicals, and provides protection for hemoglobin against oxidative damage.

Drawings

FIG. 1 shows a scanning electron micrograph of Hb-PDA MPs according to the present invention;

FIG. 2 shows a transmission electron micrograph of Hb-PDA MPs according to the present invention;

FIG. 3 is a graph showing the particle size distribution of Hb-PDA MPs of the present invention;

FIG. 4 is a chart showing UV-Vis spectra of Hb-PDA MPs of the present invention;

FIG. 5 shows an X-ray photoelectron spectrum (full spectrum) of Hb-PDA MPs of the present invention;

FIG. 6 shows an X-ray photoelectron spectrum (700-740 nm) of the Hb-PDA MPs of the present invention;

FIG. 7 is a bar graph showing the hemolysis experiment of Hb-PDA MPs according to the present invention;

FIG. 8 is a bar graph showing the coagulation assay of Hb-PDA MPs of the present invention;

FIG. 9 is a graph showing experimental viscosity profiles of Hb-PDA MPs of the present invention;

FIG. 10 is a graph showing the particle size stability of Hb-PDA MPs of the present invention;

FIG. 11 is a graph showing the hemoglobin leakage of Hb-PDA MPs of the present invention;

FIG. 12 is a bar graph showing the particle size stability of Hb-PDA MPs of the present invention in a shear system;

FIG. 13 is a bar graph showing hemoglobin leakage of Hb-PDA MPs of the present invention under a shear system;

FIG. 14 is a bar graph showing the oxygen release of Hb-PDA MPs of the present invention;

FIG. 15 is a graph showing experimental hydroxyl radical scavenging of Hb-PDA MPs of the present invention;

fig. 16A and 16B are schematic views of microstructures of hemoglobin micro-nano particles of the prior art and the present invention, respectively.

Detailed Description

Polydopamine is a novel functional polymer, and is widely applied to the research fields of biomedicine, material chemistry and the like due to good adhesion property and biocompatibility. At present, the polydopamine-based hemoglobin oxygen carrier is mainly formed by adhesion of polydopamine on the surface of a single hemoglobin molecule, the particle size of the polydopamine is too small, is only 6-8nm, and is likely to penetrate through vascular endothelium and combine with vasodilation factors in the blood circulation process, so that the polydopamine-based hemoglobin oxygen carrier is not suitable for large-dose infusion.

On the basis of a template self-assembly technology, manganese carbonate is selected as an adsorption template, hemoglobin is immobilized on the manganese carbonate through coprecipitation of the Hemoglobin and the manganese carbonate to form primary particles, an adhesion layer is constructed on the surface of the primary particles by utilizing the adhesion characteristic of Polydopamine to completely wrap the primary particles in the adhesion layer, and finally the manganese carbonate adsorption template is removed to obtain Polydopamine-loaded (adhered) Hemoglobin micro-nano particles (Hb-PDA MPs), wherein the microstructure of the Polydopamine-loaded (adhered) Hemoglobin micro-nano particles is shown in figure 16B, a dot in the figure is Hemoglobin, and the outermost layer is the Polydopamine adhesion layer; therefore, the micro-nano particles are spherical, a plurality of hemoglobins are distributed in the whole sphere, and the outer surface of the sphere is provided with a polydopamine adhesion layer which wraps the plurality of hemoglobins in the sphere. The micro-nano particle can overcome the defect that the stability of the existing template self-assembly HBOCs is insufficient. In addition, the polydopamine adhesion layer can prevent the hemoglobin from directly contacting other components in blood, provide a protective barrier for the hemoglobin and reduce the generation of free hemoglobin. Meanwhile, the antioxidant property of polydopamine enables the polydopamine to be capable of eliminating oxygen free radicals, inhibiting the autoxidation process of hemoglobin and maintaining the oxygen carrying function of the hemoglobin. The process of modifying the substrate substance by the polydopamine is simple and mild, and the polyquinone groups in the molecules of the polydopamine enable the polydopamine to have high-efficiency free radical scavenging capacity. Polydopamine has good stability as a modifying carrier of hemoglobin, and the spectral characteristics of the micro-nano particles are not changed after the micro-nano particles are stored for 24 hours, so that the colloidal stability can be maintained for a long time.

The present invention will be described in more detail with reference to specific examples and will be further described below, but the present invention is not limited to these examples.

Example (b): preparation of polydopamine hemoglobin-loaded micro-nano particles

The method comprises the following steps:

(1) 10ml of MnCl solution containing 0.1-0.3M is prepared ₂ And 2.4-12.0mg/ml hemoglobin, and rapidly adding 0.1-0.3M Na at 4 deg.C ₂ CO ₃ Stirring the solution until the reaction system is brick red and no precipitate is generated, wherein the reaction time is generally 5-10min; mn (Mn) ²⁺ Combined with hemoglobin and then with CO ₃ ^2- Combined to form MnCO ₃ Precipitation with Mn ²⁺ Hemoglobin with interaction with Mn ²⁺ With CO ₃ ^2- Is entrapped in MnCO ₃ In the middle, hb-MnCO is formed ₃ (ii) MPs; centrifuging at 6000-10000g for 5-15min, discarding supernatant, and precipitating to obtain Hb-MnCO ₃ MPs (i.e. primary particles).

(2) Adding a Tris-HCl buffer (pH = 8.0-8.8) to the precipitate obtained in step (1) so that the pH of the reaction system =8.0-8.8; then adding 1-10mg/mL dopamine solution, wherein the adding ratio of dopamine to hemoglobin is 1: (1-5), stirring is continued(ii) a Based on the characteristic absorption of polydopamine at 320nm, sampling from the reaction system every 30min, centrifuging at 6500g for 5min, discarding the precipitate, and measuring the absorption value A of the supernatant at 320nm with a spectrophotometer ₃₂₀ When A is ₃₂₀ When the change is not changed, the poly-dopamine is not consumed by the adhesion process, namely the adhesion of the poly-dopamine on the surface of the primary particles reaches saturation, and the reaction is stopped at the moment, generally for 2 to 6 hours. Dopamine introduced in the process can rapidly undergo auto-polymerization on the surface of Hb-MnCO3 MPs to form polydopamine, and covalent oxidative polymerization and non-covalent self-assembly of the dopamine should play a dominant role in the polymerization process. Polydopamine adheres to the surface of the primary particles by intermolecular forces such as hydrogen bonding with hemoglobin.

(3) Centrifuging the reaction system of step (2) at 6000-10000g for 5-15min, discarding the supernatant, washing with PBS buffer (pH = 7.4) for 3 times, and then washing with 20ml of 0.25M Na ₂ The EDTA (pH = 7.4) solution was resuspended and shaken for 30min to give resuspension i.

(4) Centrifuging the heavy suspension I obtained in the step (3) according to the conditions of the step (3), discarding the supernatant, and washing for 3 times by using a PBS (pH = 7.4) buffer solution to obtain the polydopamine-loaded hemoglobin micro-nano particles (Hb-PDA MPs for short); then re-suspending with 4ml distilled water to obtain Hb-PDA MPs water re-suspension, and storing at 4 deg.C for use.

A series of Hb-PDA MPs of the examples were prepared according to the above-described method, the preparation parameters of which are shown in Table 1.

TABLE 1 preparation parameters of Hb-PDA MPs of inventive example

Experiment one: method for measuring hemoglobin encapsulation efficiency in Hb-PDA MPs by coprecipitation method

In the encapsulation efficiency determination experiment, the reaction solution obtained in step (1) is centrifuged at 8000g for 10min, the supernatant is collected, and a trace hemoglobin kit (purchased from Nanjing bioengineering institute) is used to detect the Hb concentration (marked as C) in the supernatant _t And the volume of the reaction supernatant is denoted by V _t ) Hb and MnCl in step (1) ₂ Hb concentration in the liquid mixture (denoted as C) ₀ Volume of the mixed stock solution is denoted as V ₀ ) Envelope ratio = (C) ₀ V ₀ -C _t V _t )/C ₀ V ₀ . The final volume of the Hb-PDA MPs aqueous resuspension in step (4) was recorded as V _f Hemoglobin density in suspension = (C) ₀ V ₀ -C _t V _t )/V _f . The results are shown in Table 2.

TABLE 2 measurement of hemoglobin encapsulation efficiency

Table 2 the results show that: hb-PDA MPs, mnCO, were prepared from a Hb stock solution (i.e., the reaction system of example step (1)) at 2.4-12mg/ml ₃ The encapsulation efficiency of the template to Hb is between 36.6% and 85.6%, and the encapsulation efficiency is reduced along with the increase of Hb concentration. The Hb density in the Hb-PDA MPs suspension (i.e., the distilled water resuspension in example step (4)) was increased to 11.3 to 24.2mg/mL as the amount of Hb used in the reaction system of step (1) was increased, and the Hb content in MPs was further increased by increasing the Hb concentration in the preparation solution. As shown in Table 2, the hemoglobin density in the Hb-PDA MPs suspension increased with the initial concentration of hemoglobin in the preparation solution, but when the initial concentration of hemoglobin exceeded 7.2mg/mL, the initial concentration of hemoglobin continued to increase, and the increase in the hemoglobin density in the Hb-PDA MPs suspension was less pronounced, so the present invention selected 7.2mg/mL as the optimum preparation concentration.

Experiment two: characterization of properties

The morphology characteristics of Hb-PDA MPs are observed by adopting a scanning electron microscope and a transmission electron microscope, the element composition of the Hb-PDA MPs is determined by an accessory X-ray energy dispersion spectrometer of the scanning electron microscope, and the particle size distribution of the Hb-PDA MPs and the Zeta potentials of the Hb, blank-PDA MPs and Hb-PDA MPs are determined by adopting a laser particle sizer. Taking Hb-PDA MPs of example 3 as an example, the scanning electron microscope results are shown in FIG. 1, the transmission electron microscope results are shown in FIG. 2, the particle size distribution is shown in FIG. 3, the elemental composition is shown in Table 3, and the Zeta potential is shown in Table 4. Wherein Hb is the mixed solution containing hemoglobin in step (1); blank-PDA MP is an unloaded polydopamine particle, i.e. Hb-free MnCl is selected for use in the co-precipitation step ₂ And Na ₂ CO ₃ As a preparation raw material, the other steps are the same as the preparation of the invention; hb-PDA MPs are aqueous resuspension of Hb-PDA MPs obtained by resuspension after adding distilled water in step (4).

TABLE 3 elemental composition of Hb-PDA MPs

Element(s)	Content/%)
		C	66.43
N	8.90
		O	24.58
Mn	0.04

TABLE 4 Zeta potentials of Hb, DA and Hb-PDA MPs

Sample name	Zeta potential (mV)
		Hb	-20.13±1.93
Blank-PDA MPs	-26.07±1.32
		Hb-PDA MPs	-22.07±0.26

Fig. 1 and 2 show that Hb-PDA MPs have a spherical shape, which means that the structure is not collapsed after the template is removed, and the size of the micro-nano particles in a dry state is about 400 to 600nm. FIG. 3 shows that the average hydrated particle size of Hb-PDA MPs is 840.9 +/-26.3 nm, and the particle size distribution is uniform and concentrated. Table 3 shows that the Mn content in Hb-PDA MPs is only 0.04%, indicating that the template has been sufficiently removed. The Hb-PDA MPs of the invention have negative charges on the surface, and the Zeta potential is-22.07 +/-0.26 mV, which is caused by deprotonation of a large amount of phenolic hydroxyl groups on the surface of polydopamine under a neutral condition. The negative surface charge of the Hb-PDA MPs is beneficial to reducing the interaction with other components in blood and stably existing in the blood.

Experiment three: chemical Structure characterization

Measuring ultraviolet-visible spectra of Hb, PDA and Hb-PDA MPs by using a spectrophotometer (bandwidth: 2nm; wavelength range: 190-850nm; sample cell specification: 1 cm), wherein Hb is mixed solution containing hemoglobin in the step (1); PDA is polydopamine; hb-PDA MPs are the polydopamine hemoglobin micro-nano particles obtained by adding distilled water and then re-suspending in the step (4). Hb, DA and Hb-MnCO determination by adopting X-ray photoelectron spectrometer ₃ MPs and Hb-MnCO ₃ Photoelectron spectroscopy of PDA MPs and analysis of the surface elemental composition, EDTA dissolution of MnCO due to the strong chelating capacity of the PDA molecules on the metal elements ₃ May result in the binding of Mn to PDA ²⁺ Thus influencing the measurement result, hb-MnCO is measured ₃ -surface element content of PDA MPs, i.e. surface element content of Hb-PDA MPs; hb is a mixed solution containing hemoglobin in step (1); DA is a commercially available dopamine monomer(available from Sigma-Aldrich, USA); hb-MnCO ₃ MPs are manganese carbonate hemoglobin-loaded micro-nano particles obtained by a coprecipitation method in the step (1); hb-MnCO ₃ PDA MPs are polydopamine coated in step (3), but MnCO ₃ Hemoglobin-loaded micro-nano particles of which the templates are not removed yet. Hb and Hb-MnCO determination by adopting X-ray photoelectron spectrometer ₃ MPs and Hb-MnCO ₃ Photoelectron spectroscopy (700-740 eV) of PDA MPs, this interval being able to reflect the characteristic signals of the Fe element, which is a characteristic element of the haemoglobin subunit. The results are shown in fig. 4, fig. 5, fig. 6 and table 5, taking the preparation parameters of example 3 as an example.

TABLE 5 Hb, DA, hb-MnCO ₃ MPs and Hb-MnCO ₃ Surface element content of PDA MPs

Element(s)	Hb/％	DA/％	Hb-MnCO ₃ MPs/％	Hb-MnCO ₃ -PDA MPs/％
					C	61.19	71.15	46.48	58.70
N	18.45	10.08	16.36	20.09
					O	16.49	12.89	33.03	19.10
Fe	0.18	0	0.21	0
					Mn	0	0	2.77	0.22
N/O	1.12	0.78	0.50	1.05

FIG. 4 shows that Hb-PDA MPs retain the characteristic peak of oxyHb at 412nm, indicating that the process of preparation does not destroy the chemical structure of Hb, and Hb-PDA MPs have oxygen carrying capacity. As the PDA has stronger absorption at 500-700nm, the characteristic peaks of Hb at 540 and 575nm are covered, which indicates that Hb and PDA are effectively loaded, and the structural characteristics of Hb and PDA are not damaged.

FIG. 5 and Table 5 show that Hb-MnCO compares to the unpolymerized DA (dopamine) ₃ The surface of PDA MPs had both increased elements N and O, but the ratio of N/O increased from 0.78 to 1.05. Because the molecular surfaces of Hb and PDA are bothSince a large number of phenolic hydroxyl groups are present, it is presumed that the formation of the PDA layer is mainly based on a hydrogen bond by the action of both hydroxyl groups, and the amino group of PDA is exposed on the outer side of the PDA layer. FIG. 6 shows that the content of porphyrin Fe element is reduced from 0.18% to 0% after the PDA is coated on the surface of MPs, which indicates that the coating effect of PDA is more sufficient and the hemoglobin is not exposed.

FIG. 6 shows Hb-MnCO ₃ No Fe element signal was detected on the surface of PDA MPs, indicating that the primary particles are sufficiently coated with PDA and the hemoglobin is not exposed.

Experiment four: evaluation of blood compatibility

HBOCs are administered in large doses and are involved in systemic blood circulation, and therefore their hemocompatibility is not negligible. 200 μ l of Hb-PDA MPs with the concentrations of 200 μ g/mL, 400 μ g/mL and 600 μ g/mL respectively and 1mL of erythrocyte suspension containing 2% (volume percentage) of erythrocytes (2% rat erythrocytes +98% physiological saline) are uniformly mixed to obtain a mixed solution, the mixed solution is incubated for 1h at 37 ℃, the mixed solution is centrifuged at 3000rpm for 10min, the precipitate is discarded, and the supernatant is used as an experimental group. 1mL of a suspension of erythrocytes containing 2% (volume percentage) of erythrocytes was mixed with 200. Mu.L of physiological saline, and the supernatant obtained in the same manner as above was used as a negative control group, and 1mL of a suspension of erythrocytes containing 2% (volume percentage) of erythrocytes was mixed with 200. Mu.L of distilled water, and the supernatant obtained in the same manner as above was used as a positive control group. Measuring absorbance at 545nm of the supernatant obtained from the experimental group, the negative control group and the positive control group respectively, and recording as D _t 、D _- 、D ₊ Then hemolyzation Ratio) = (D) _t -D _- )/(D ₊ -D _- ). The results are shown in FIG. 7, using the Hb-PDA MPs obtained in example 3 as an example.

The blood coagulation time is an important index for reflecting the blood coagulation function of the body. Among them, the Activated Partial Thromboplastin Time (APTT) mainly reflects the condition of the intrinsic coagulation system, the Prothrombin Time (PT) mainly reflects the condition of the extrinsic coagulation system, and the Thrombin Time (TT) mainly reflects the time for converting fibrinogen into fibrin. 200 mu.L of 1.6mg/ml Hb-PDA MPs aqueous suspension and 1ml of rat Platelet-Poor Plasma (PPP) are respectively mixed and incubated for 1h, the PPP without adding MPs is used as a control group, and the coagulation indexes PT, APTT and TT of each group are measured and are subjected to statistical analysis. The results are shown in FIG. 8.

Viscosity is an important index reflecting the rheological properties of blood, and changes in blood viscosity will affect the flow rate and quantity of blood and the normal perfusion of tissues and organs. Mixing Hb-PDA MPs and whole blood in a volume ratio of 3:7, incubated at 37 ℃ for 1h, and measured at a shear rate of 1S ^-1 、10S ^-1 、100S ^-1 The effects of Hb-PDA MPs on the rheological characteristics of whole blood were examined by using whole blood, a mixture of whole blood and physiological saline (physiological saline and whole blood mixed at a volume ratio of 3. The results are shown in FIG. 9.

FIG. 7 shows that the haemolysis rate of 200, 400 and 600. Mu.g/ml Hb-PDA MPs is not more than 5% after acting on the erythrocyte suspension, which indicates that the Hb-PDA MPs do not cause erythrocyte hemolysis.

FIG. 8 shows that there was a slight change in the APTT and TT values of the PPP + Hb-PDA MPs group compared to the control group (PPP without Hb-PDA MPs added), but there was no statistical difference (P > 0.05). The result shows that the Hb-PDA MPs can not cause abnormal change of the blood coagulation time after entering the blood and have no influence on the blood coagulation function.

FIG. 9 shows that the viscosity is reduced at each shear rate for the whole blood + Hb-PDA MPs system compared to whole blood at the same shear rate, although the rheological properties of whole blood are shown to be affected after the Hb-PDA MPs are applied to whole blood. However, the viscosity-shear rate curve of the whole blood + Hb-PDA MPs system is closer to that of the whole blood + physiological saline group, which indicates that the influence degree of the whole blood viscosity of the Hb-PDA MPs does not exceed the physiological saline, so that the abnormal change of the whole blood viscosity cannot be caused excessively after the infusion.

Experiment five: stability survey

The aqueous suspension of Hb-PDA MPs was left at 4 ℃ for two weeks, and the hydrated particle size distribution was measured every two days with a laser particle sizer. Mixing Hb-PDA MPs with Hb-MnCO ₃ MPs suspension (manganese carbonate loaded hemoglobin micro-nano particles obtained by coprecipitation method in step (1)) is stored at 4 ℃ for 42 days at 2 nd, 4 th, 6 th, 8 th, 10 th, 14 th, 18 th, 22 th and 2 nd6. Hb leakage was measured on 30, 36 and 42 days. The Hb leakage amount was measured by the following method: hb concentration in Hb-PDA MPs corresponding to an encapsulation efficiency of 52.0 + -4.7% as calculated by the encapsulation efficiency is C _total . At the time of measuring days, taking Hb-PDA MPs water suspension, centrifuging at 8000rpm for 10min, measuring Hb concentration in supernatant by potassium ferricyanide method, and recording as C _super Hb leakage = C _super /C _total . The results are shown in FIGS. 10 and 11.

HBOCs are required to participate in blood circulation to supply oxygen to aerobic tissues and parts of the body, so that the tolerance to the flow shear environment is an important basis for predicting the safety and effectiveness of the HBOCs in vivo. Shear conditions at different vascular sites were simulated by establishing a parallel plate flow cell based flow shear system. Respectively putting Hb-PDA MPs at 200S ^-1 、400S ^-1 、800S ^-1 、1600S ^-1 Respectively circulating for 10, 20 and 30min at the shear rate of (i.e. 200S) ^-1 400S at a shear rate of 10, 20, 30min ^-1 、800S ^-1 、1600S ^-1 Also circulating for 10, 20 and 30 min), respectively, hb leakage and average particle size of Hb-PDA MPs were measured. The results are shown in FIGS. 12 and 13.

FIG. 10 shows that the particle size of Hb-PDA MPs after preparation was 820.7 + -26.0 nm, the particle size did not change significantly in two weeks, the average particle size was 807.5 + -19.0 nm on day 13, and there was no statistical difference compared to day 1, indicating that Hb-PDA MPs have better particle size stability.

FIG. 11 shows that no burst of Hb in aqueous Hb-PDA MPs suspension was detected during the 42 day storage period. Hb-PDA MPs and Hb-MnCO ₃ Hb leakage at day 42 of MPs was 3.9 + -0.4% and 37.5 + -0.3%, respectively (statistically significant differences), indicating MnCO ₃ The template is not effective in preventing hemoglobin leakage, and the structure is more stable after PDA and Hb are combined, which may be caused by hydrogen bonding interaction between the two.

Fig. 12 and 13 show that: hb leakage of Hb-PDA MPs at each shear rate does not exceed 4%, and the average particle size is maintained at 810 +/-10 nm. This result indicates that the Hb-PDA MPs have good stability in the flow shear state, and the PDA membrane layer does not collapse or swell.

Experiment six: evaluation of oxygen supply Capacity

The experiment was carried out using a relative evaluation apparatus and a relative evaluation method for the oxygen carrying-releasing ability of the oxygen carrier. The relative evaluation device is disclosed in patent application No. CN107228947A, and comprises flow cells A and B. 2ml0.9wt% normal saline is added into the flow cell A, and nitrogen is filled until the oxygen partial pressure is reduced to 0 so as to simulate an anoxic environment. And adding an equal amount of sample to be detected (Hb-PDA MPs) into the flow cell B, and filling oxygen to enable the sample to reach an oxygen saturation state. And stopping inflating after the pressure values of the two flow cells are stable. And mixing the sample in the pool B into the pool A, wherein the anoxic environment promotes the sample to continuously release oxygen into the environment, and measuring the oxygen partial pressure after mixing. The oxygen partial pressures before and after mixing in the B pool are respectively marked as P ₀ 、P ₁ Oxygen partial pressure difference Δ P = P ₁ -P ₀ And the delta P can qualitatively reflect the oxygen carrying-releasing capacity of the sample. Hemoglobin stock solution, 0.9wt% NaCl Solution (NS), BSA-PDA MPs as controls. Wherein BSA-PDA MPs are micro-nano particles of poly-dopamine-loaded bovine hemoglobin, namely MnCl containing BSA (the concentration of a stock solution of Hb is the same in the preparation process of BSA and Hb-PDA MPs) is selected in the coprecipitation step ₂ And Na ₂ CO ₃ The raw materials are prepared by the same method as the invention in other steps. The results are shown in FIG. 14, using the Hb-PDA MPs of example 3 as an example. The preparation method of the hemoglobin stock solution comprises the following steps: centrifuging fresh whole blood of cattle at 4 deg.C and 4000rpm for 10min, and removing supernatant; resuspending the precipitated erythrocytes with a 0.9% nacl solution, washing with the same centrifugation parameters until the supernatant is clear and transparent; resuspending red blood cells with 0.9% NaCl solution, adding 5 times volume of hypotonic NaCl solution and chromatographic balance solution, stirring at 100rpm for 60min, and filtering with 0.22 μm microfiltration membrane to obtain hemoglobin crude product; 13.2g/dL of hemoglobin stock solution is extracted from the hemoglobin primary product by anion exchange chromatography.

FIG. 14 shows that Δ P for the NS group, BSA-PDA MPs group, hb group, and Hb-PDA MPs group are: 52.1 +/-2.9mmHg, 54.5 +/-0.4mmHg, 67.2 +/-1.1mmHg and 78.7 +/-0.7 mmHg. This result indicates that NS and BSA-PDA MPs do not have oxygen carrying-releasing ability, while Hb increases the partial pressure level of oxygen in the system and has a certain oxygen carrying-releasing ability. Compared with Hb, the higher value of the delta P of the Hb-PDA MPs group shows that the oxygen release effect of the Hb-PDA MPs group is better under the hypoxia environment, and the oxygen supply under the acute anemia condition is more favorable.

Experiment seven: evaluation of radical scavenging ability

Hydroxyl radical is an important index for evaluating the antioxidant capacity of bioactive substances. H ₂ O ₂ With Fe ²⁺ Can generate hydroxyl free radical through Fenton reaction to react with phenanthrene-Fe ²⁺ Fe in aqueous solution ²⁺ Oxidation to Fe ³⁺ Leading to the decrease of the absorbance of the system at 536nm, and the inhibition degree of the Hb-PDA MPs on the decrease rate of the absorbance of the test system at 536nm can reflect the capability of the test system in clearing hydroxyl radicals. The measurement is carried out by adopting a hydroxyl radical scavenging capacity measurement kit (purchased from Beijing Solaibao science and technology Co., ltd.), and the measurement method is as follows: adding hydroxyl radical test solution 550 μ l into experimental group, control group and blank group, respectively, adding Hb-

PDA MPs

25, 500, 75, 100, 125, 150, 175, 200 μ g and hydrogen peroxide 100 μ l (H) into experimental group ₂ O ₂ 3% by volume) of the control group, 250. Mu.l of distilled water and 100. Mu.l of H were added to the control group ₂ O ₂ The blank was added 350. Mu.l of distilled water. After incubation at 37 ℃ for 1h, the experimental groups were centrifuged at 10000rpm for 10min and the supernatant was collected. Double distilled water was zeroed and the absorbance at 536nm was immediately determined for each group. The measured values of the experimental group, the control group and the blank group were respectively designated as A _t 、A _c 、A ₀ Hydroxyl radical clearance rate = (a) _t -A _c )/(A ₀ -A _c ). Hydroxyl radical scavenging ability of Trolox (water-soluble vitamin E) and Hb (same as experiment six) at different concentrations was measured in the same manner, and compared with Hb-PDA MPs for analysis. The results are shown in FIG. 15.

FIG. 15 shows that Hb-PDA MPs can remove hydroxyl radicals up to 83.1%, trolox can remove hydroxyl radicals up to 98.1% as a known antioxidant, and Hb-PDA MPs can remove hydroxyl radicals up to 85% of Trolox. Hb-PDA MPs help reduce the attacking effect of free radicals on Hb in vivo, thereby maintaining the structural and functional stability thereof. The highest clearance rate of Hb on hydroxyl free radicals is only 9.2%, which shows that Hb does not have anti-oxidation property and can not resist the attack of free radicals.

The foregoing is only a preferred embodiment of the invention and it should be noted that modifications and embellishments could be made by those skilled in the art without departing from the principle of the invention and should also be considered as the content of the invention.

Claims

1. The method for preparing the polydopamine-loaded hemoglobin micro-nano particles is characterized by sequentially comprising the steps of forming primary particles by taking manganese carbonate as an adsorption template to immobilize hemoglobin in a coprecipitation reaction system, constructing a polydopamine adhesion layer on the surfaces of the primary particles, and removing a manganese carbonate adsorption template;

the method for forming the primary particles in the coprecipitation reaction system by taking manganese carbonate as an adsorption template to immobilize hemoglobin specifically comprises the following steps: to contain MnCl ₂ Adding Na into the mixed solution of 2.4-12mg/mL hemoglobin ₂ CO ₃ Stirring the solution until the reaction system is brick red and no precipitate is generated, and centrifuging at 6000-10000g for 5-15min to obtain precipitate as formed primary particles;

the method for constructing the polydopamine adhesion layer on the surface of the primary particle specifically comprises the following steps: adding a Tris-HCl buffer solution into the primary particles to ensure that the pH of a reaction system is =8.5, adding 1-10mg/mL of dopamine solution, and continuing stirring until the amount of dopamine is not reduced any more, which indicates that a polydopamine adhesion layer is constructed on the surfaces of the primary particles; the ratio of the added mass of dopamine to the amount of hemoglobin is 1: (1-5);

2. The method according to claim 1, wherein the removing of the manganese carbonate adsorption template is specifically: the reaction system for constructing the poly dopamine adhesion layer was centrifuged, the supernatant was discarded, and after washing with PBS having a pH =7.4, na having a pH =7.4 was added ₂ Resuspending in EDTA solution, centrifuging, and discarding supernatant to obtain precipitateThe polydopamine hemoglobin-loaded micro-nano particle.

3. The method according to claim 2, wherein the centrifugation is performed under conditions of 6000 to 10000g for 5 to 15min.

4. The method according to any one of claims 1 to 3, wherein the concentration of hemoglobin in the mixed solution is 7.2mg/mL, mnCl ₂ Is 0.15M, and the concentration of the dopamine solution added in the step of constructing a polydopamine adhesion layer on the surface of the primary particles is 5 mg/mL.

5. The polydopamine hemoglobin-loaded micro-nano particle prepared by the method of claim 4, which is characterized in that the micro-nano particle is in a regular spherical shape, polydopamine is adhered to the outer surface of the spherical shape to form an adhesion layer as a hemoglobin protective film, and the Zeta potential of the polydopamine-loaded hemoglobin micro-nano particle is-22.33 to-21.81 mV.

6. The polydopamine-loaded hemoglobin micro-nano particle according to claim 5, wherein the average hydrated particle size distribution of the micro-nano particle is 814nm to 866nm, and the polydispersity PDI is 0.11 to 0.29.

7. Use of the polydopamine-loaded hemoglobin micro-nano particle according to claim 5 or 6 or the polydopamine-loaded hemoglobin micro-nano particle prepared by the method according to any one of claims 1 to 4 in preparation of a blood substitute, wherein the micro-nano particle partially or completely replaces red blood cells to play a role in oxygen carrying and releasing during blood transfusion.

8. The use of claim 7, wherein said blood transfusion is caused by blood loss.

9. The use of claim 7, wherein the blood transfusion is caused by trauma, anemia or surgery.