CN107982544A - Hybrid protein nanometer carrier of oxygen and its preparation method and application - Google Patents

Abstract

The invention provides a hybrid protein nano-oxygen carrier and its preparation method and application, which relate to the technical field of oxygen carrier, mainly composed of hemoglobin and serum albumin covalently bonded through disulfide bonds, and its size is 20-200 nanometers, which relieves The existing artificial oxygen carrier has poor stability in the body and may have technical problems of side effects. Through the covalent combination of serum albumin and hemoglobin through a disulfide bond, the hemoglobin is protected and has good biocompatibility and oxygen-carrying properties in the body. Stability, can efficiently transport oxygen to the body, and will not cause cross-infection and agglutination reaction, and has no technical effect of side effects. In addition, the hybrid protein nano-oxygen carrier provided by the invention has a stable structure, can be synthesized on a large scale, and has a storage time of more than two years, which is convenient for popularization and application.

Description

Hybrid protein nano oxygen carrier and its preparation method and application

technical field

The invention relates to the technical field of oxygen carriers, in particular to a hybrid protein nano-oxygen carrier and its preparation method and application.

Background technique

As an important means of life-saving, clinical blood transfusion is widely used in clinical operations, disaster relief and war wound treatment, but there are still many problems in blood transfusion: 1. Blood may carry pathogens (hepatitis virus, HIV, etc.), and some diseases may pass through Transfusion transmission; 2. Blood type mismatch leads to agglutination effect, which can threaten the life of the transfused person; 3. Shortage of blood sources; 4. The short storage time of blood limits its treatment of acute and critically ill patients and in emergency situations under the application.

In recent years, nanotechnology has enabled the rapid development of artificial oxygen carriers, which has attracted widespread attention. Artificial oxygen carriers are nano/micro-sized particles with oxygen-carrying functions, which can be synthesized on a large scale without causing cross-infection and agglutination reactions. At present, a variety of artificial oxygen carrier products have been used clinically as blood substitutes. These artificial oxygen carriers include perfluorocarbons, polyhemoglobin, cross-linked hemoglobin, hemoglobin liposomes, etc., but perfluorocarbons need to be refrigerated. , must be inhaled at the same time hyperoxia with a concentration of more than 95% during infusion, and the biological half-life is relatively short, and there are serious side effects. Polyhemoglobin and cross-linked hemoglobin, because the hemoglobin molecule is not protected by other molecules, it is difficult to maintain the functional stability of hemoglobin in the body, and it is easy to cause nephrotoxicity. Hemoglobin liposomes have poor structural stability in vivo, and the encapsulated hemoglobin is easy to leak.

In view of this, the present invention is proposed.

Contents of the invention

One of the objectives of the present invention is to provide a hybrid protein nano-oxygen carrier to alleviate the technical problems of poor stability in vivo and possible side effects of existing artificial oxygen carriers.

The hybrid protein nano-oxygen carrier provided by the invention is mainly composed of hemoglobin and serum albumin covalently combined through disulfide bonds, and its size is 20-200 nanometers.

Further, the hemoglobin is derived from animals, preferably, the hemoglobin is derived from humans, cattle or pigs;

Preferably, the serum albumin is derived from animals or obtained by biological fermentation, further preferably, the serum albumin is derived from humans or cattle, and even more preferably, the serum albumin is recombinant human serum obtained by biological fermentation albumin.

The second object of the present invention is to provide a method for preparing the hybrid protein nano oxygen carrier, comprising the following steps:

(a) mixing the reducing agent with serum albumin to perform a reduction reaction to obtain reduced serum albumin;

(b) Homogenously mixing reduced serum albumin and hemoglobin to prepare a hybrid protein nano-oxygen carrier.

Further, the reducing agent is at least one selected from glutathione, dithiothreitol, cysteine or homocysteine.

Further, the mass ratio of hemoglobin to serum albumin is 1:(2-50), preferably 1:(3-15), more preferably 1:(5-10).

Further, the mass ratio of serum albumin to reducing agent is 1:(0.1-1), preferably 1:(0.15-0.5), more preferably 1:(0.15-0.3).

Further, in step (b), the pH value of the mixed solution of reduced serum albumin and hemoglobin is 7-9;

Preferably, the mixing homogenization time is 10-180 minutes;

Preferably, the concentration of reduced serum albumin is 0.5-3%.

Further, in step (b), first mix the reduced serum albumin and hemoglobin homogeneously, then add the precipitating agent and mix evenly, and finally remove the precipitating agent and free protein by vacuum drying, ultrafiltration or dialysis, and then the preparation Obtain hybrid protein nano oxygen carrier;

Preferably, the precipitating agent is anhydrous low-carbon alcohol, more preferably, the precipitating agent is absolute ethanol.

Further, the volume ratio of the mixed solution of reduced serum albumin and hemoglobin to the precipitating agent is 1: (1-2.5);

Preferably, the mixing time of the precipitation agent and the mixed solution of reduced serum albumin and red blood cells is 0.5-12 hours.

The third object of the present invention is to provide the application of the hybrid protein nano oxygen carrier in the preparation of blood substitutes.

The hybrid protein nano-oxygen carrier provided by the present invention is covalently combined with serum albumin and hemoglobin through disulfide bonds, so that the hemoglobin is protected, has good biocompatibility and oxygen-carrying stability in the body, and can be efficiently transported to the body Oxygen, and will not cause cross-infection and agglutination reaction, no side effects. In addition, the hybrid protein nano-oxygen carrier provided by the invention has a stable structure, can be synthesized on a large scale, and has a storage time of more than two years, which is convenient for popularization and application.

The preparation method of the hybrid protein nano-oxygen carrier provided by the invention is simple and easy to operate, easy to operate and popularize, and suitable for large-scale preparation.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Fig. 1 is the state diagram of the hybrid protein nano-oxygen carrier solution in a test tube provided by Example 7 of the present invention;

Fig. 2 is the particle size distribution figure of the hybrid protein nano oxygen carrier solution provided by embodiment 7;

Figure 3 is the oxygen release curve of the oxygen-saturated hybrid protein nano-oxygen carrier and hemoglobin in anaerobic water.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below, and obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

According to the first aspect of the present invention, the present invention provides a hybrid protein nano-oxygen carrier, which is mainly composed of hemoglobin and serum albumin covalently bonded through disulfide bonds, and its size is 20-200 nanometers.

The hybrid protein nano-oxygen carrier provided by the present invention is covalently combined with serum albumin and hemoglobin through disulfide bonds, so that the hemoglobin is protected, has good biocompatibility and oxygen-carrying stability in the body, and can be efficiently transported to the body Oxygen, and will not cause cross-infection and agglutination reaction, no side effects. In addition, the hybrid protein nano-oxygen carrier provided by the invention has a stable structure, can be synthesized on a large scale, and has a storage time of more than two years, which is convenient for popularization and application.

The size of the hybrid protein nano-oxygen carrier provided by the invention is 20-200 nanometers, so as to facilitate its stable transmission in vivo and prolong its circulation time in vivo.

The hybrid protein nano-oxygen carrier provided by the invention is formed by covalently combining serum albumin and hemoglobin through disulfide bonds, and has no antigenic determinants on the surface of red blood cells, which can eliminate the trouble of matching blood types, avoid agglutination effects, and avoid pathogenic microorganisms at the same time Pollution of blood sources and further cross-infection, without relying on blood supply groups, wide sources, convenient materials, sufficient supply can be guaranteed, large-scale production can be carried out, and people's demand for oxygen-supplied blood substitutes can be met.

In a preferred embodiment of the present invention, the hemoglobin is derived from animals, preferably, the hemoglobin is derived from humans, cattle or pigs;

Preferably, the serum albumin is derived from animals or obtained by biological fermentation, more preferably, the serum albumin is derived from humans and cattle, and even more preferably, the serum albumin is recombinant human serum albumin obtained by biological fermentation protein.

The hemoglobin is derived from animals, which can be extracted from animal blood. When the hemoglobin is extracted from the blood of animals such as humans, cattle, and pigs, the hybrid protein nano-oxygen carrier produced has better stability in the body. Hemoglobin includes hemoglobin and its analog molecules, and hemoglobin raw materials include subtypes such as HbA, HbA2, and HbF. Serum albumin is extracted from animal blood or obtained through biological fermentation. When the serum albumin is extracted from the blood of human, bovine and other animals, the in vivo stability of the prepared hybrid protein nano oxygen carrier is better; when the serum albumin is the recombinant human obtained by biological fermentation When serum albumin is present, the in vivo stability of the prepared hybrid protein nano-oxygen carrier is better.

According to the second aspect of the present invention, the present invention provides a method for preparing the above hybrid protein nano oxygen carrier, comprising the following steps:

(a) mixing the reducing agent with serum albumin to perform a reduction reaction to obtain reduced serum albumin;

(b) Homogenously mixing reduced serum albumin and hemoglobin to prepare a hybrid protein nano-oxygen carrier.

The preparation method of the hybrid protein nano-oxygen carrier provided by the invention is simple and easy to operate, easy to operate and popularize, and suitable for large-scale preparation.

In step (a), the reduction reaction between the reducing agent and the serum albumin breaks the disulfide bond inside the serum albumin molecule to obtain the reduced serum albumin molecule in a random state. In step (b), the reduced serum albumin molecule cross-links with the free sulfhydryl group of hemoglobin, reconstitutes an intermolecular disulfide bond, and forms a hybrid protein nano-oxygen carrier.

In the present invention, mixing and homogenizing means mixing and then homogenizing.

In a typical but non-limiting embodiment of the present invention, the reducing agent is selected from at least one of glutathione, dithiothreitol, cysteine or homocysteine.

In a typical but non-limiting embodiment of the invention, the reducing agent can be one of glutathione, dithiothreitol, cysteine and homocysteine, or glutathione , a mixture of any two of dithiothreitol, cysteine and homocysteine, such as a mixture of glutathione and dithiothreitol, a mixture of dithiothreitol and cysteine, or A mixture of cysteine and homocysteine, etc., can also be a mixture of any three of glutathione, dithiothreitol, cysteine and homocysteine, such as glutathione , a mixture of dithiothreitol and cysteine or a mixture of glutathione, dithiothreitol and homocysteine, etc., or glutathione, dithiothreitol, cysteine and Homocysteine A mixture of four substances.

In a preferred embodiment of the present invention, the mass ratio of hemoglobin to serum albumin is 1:(2-50), preferably 1:(3-15), more preferably 1:(5-7).

By controlling the mass ratio of hemoglobin and serum albumin to 1: (2-50), serum albumin provides sufficient protection for hemoglobin, thereby improving the stability of the hybrid protein nano-oxygen carrier in vivo and prolonging the hybrid protein nano-oxygen carrier in vivo When the mass ratio of hemoglobin to serum albumin is 1: (3-15), the hybrid protein nano-oxygen carrier not only has good stability in the body, but also has a long circulation time and a large oxygen carrying capacity. , especially when the mass ratio of hemoglobin to serum albumin is between 1: (0.15-0.3), the in vivo stability is better, the circulation time is longer, and the oxygen carrying capacity is larger.

In a preferred embodiment of the present invention, the mass ratio of serum albumin to reducing agent is 1:(0.1-1), preferably 1:(0.15-0.5), more preferably 1:(0.15-0.3).

By controlling the mass ratio of serum albumin and reducing agent to 1: (0.1-1), so that when the reducing agent and serum albumin undergo a reduction reaction, the reduction reaction of serum albumin molecules is relatively sufficient, and the internal molecules of serum albumin Disulfide bonds can be broken to generate reduced serum albumin; especially when the mass ratio of serum albumin to reducing agent is 1: (0.15-0.5), the reduction reaction of serum albumin is more fully carried out, and serum albumin The disulfide bonds inside the molecule are broken more. When the mass ratio of serum albumin to reducing agent is 1: (0.15-3), the reduction reaction of serum albumin is the most complete, and the resulting reduced serum albumin The molecule contains more free sulfhydryl groups.

In a preferred embodiment of the present invention, in step (b), the pH value of the mixed solution of reduced serum albumin and hemoglobin is 7-9;

Preferably, the mixing homogenization time is 10-180 minutes;

Preferably, the concentration of reduced serum albumin is 0.5-3%.

In step (b), by controlling the pH value of the mixed solution of reduced serum albumin and hemoglobin to be 7-9, to promote the generation of hybrid protein nano-oxygen carrier, in the process of mixing and homogenizing, bicarbonate Sodium adjusts the pH, keeping it at a pH of 7-9.

By controlling the time for mixing and homogenizing to 10-180 minutes, the reduced serum albumin and hemoglobin react more fully, and the yield of the hybrid protein nano oxygen carrier is improved.

The above concentration of reduced serum albumin refers to the concentration of reduced serum albumin in a mixed solution of reduced serum albumin and hemoglobin. By controlling the concentration of the reduced serum albumin to 0.5-3%, the preparation efficiency of the hybrid protein nano-oxygen carrier is improved.

In a preferred embodiment of the present invention, in step (b), the reduced serum albumin and hemoglobin are firstly mixed homogeneously, then the precipitating agent is added and mixed uniformly, and finally the precipitating agent is removed by vacuum drying, ultrafiltration or dialysis and free protein removal, the hybrid protein nano-oxygen carrier can be prepared; preferably, the precipitating agent is anhydrous low-carbon alcohol, more preferably, the precipitating agent is absolute ethanol.

In step (b), a precipitating agent is used to reduce the solubility of hemoglobin and reduced serum albumin, so that the cross-linking reaction between hemoglobin and reduced serum albumin can be carried out more fully, and the yield of the hybrid protein nano-oxygen carrier can be increased.

Finally, the precipitant and free protein are removed by vacuum drying, ultrafiltration or dialysis, so as to purify the cross-protein nano-oxygen carrier, and the precipitant and free protein are removed. The above-mentioned free protein includes free serum albumin and free hemoglobin.

In a typical but non-limiting preferred embodiment of the present invention, the precipitating agent is anhydrous low-carbon alcohol, and the anhydrous low-carbon alcohol refers to C1-C4 low-carbon alcohol. Preferably, the precipitating agent is absolute ethanol.

In a further preferred embodiment of the present invention, when the precipitant and free protein are removed by ultrafiltration or dialysis, the molecular weight cut off by ultrafiltration or dialysis is 100-300 kDa.

In a preferred embodiment of the present invention, the volume ratio of the mixed solution of reduced serum albumin and hemoglobin to the precipitating agent is 1: (1-2.5);

Preferably, the mixing time of the precipitation agent and the mixed solution of reduced serum albumin and red blood cells is 0.5-12 hours.

By controlling the volume ratio of the mixed solution of reduced serum albumin and hemoglobin to the precipitating agent to be 1: (1-2.5), so that the precipitating agent can be fully mixed with the mixed solution of reduced serum albumin and hemoglobin to reduce reduced serum albumin The solubility with hemoglobin enables the cross-linking reaction of the two proteins to proceed more fully.

By controlling the mixing time of the mixed solution of reduced serum albumin and hemoglobin and the precipitant to 0.5-12 hours, so that the precipitant can be mixed evenly with the mixed solution of reduced serum albumin and hemoglobin, thereby reducing the reduced serum albumin and hemoglobin The solubility makes the cross-linking reaction of the two proteins more fully.

According to the third aspect of the present invention, the present invention provides the application of the above-mentioned hybrid protein nano-oxygen carrier in the preparation of blood substitutes.

As a nanometer-sized oxygen carrier, the hybrid protein nano-oxygen carrier provided by the present invention not only has no antigenic determinants on the surface of erythrocytes, can eliminate the trouble of matching blood types, avoid agglutination effects, and can also avoid contamination of blood sources by pathogenic microorganisms and further cross-infection. At the same time, it can also bind oxygen molecules with high affinity, reversibly release oxygen in the body, and replace the oxygen transport function of blood.

The technical solutions provided by the present invention will be further described below in conjunction with examples and examples.

Example 1

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Co-dissolve 300mg bovine serum albumin and 30mg dithiothreitol in 5mL deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 10 mg of hemoglobin in 2 mL of pure water and homogenize for 10 minutes, adjust the pH value to 7 with sodium bicarbonate, and drop into the mixed solution at a rate of 1 mL/min under strong stirring Add 2 mL of absolute ethanol, stir and homogenize for 0.5 hours, put the mixed solution into a dialysis bag with a molecular weight cut-off of 100 kD, and dialyze for 12 hours to obtain a hybrid protein nano-oxygen carrier solution.

Example 2

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Dissolve 300mg of human serum albumin and 300mg of homocysteine in 5mL of deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 0.4 mg of hemoglobin in 2 mL of pure water and homogenize for 180 minutes, adjust the pH value to 9 with sodium bicarbonate, and add to the mixed solution at a speed of 1 mL/min under vigorous stirring. Add 5 mL of absolute ethanol dropwise, stir and homogenize for 12 hours, put the solution into a dialysis bag with a molecular weight cut-off of 300 kD, and dialyze for 12 hours to obtain a hybrid protein nano-oxygen carrier solution.

Example 3

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Dissolve 300mg recombinant human serum albumin and 45mg glutathione in 5mL deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 6.7 mg of hemoglobin in 2 mL of pure water and homogenize for 20 minutes, adjust the pH value to 7.5 with sodium bicarbonate, and add to the mixed solution at a speed of 1 mL/min under vigorous stirring Add 4 mL of absolute ethanol dropwise, stir for 1 hour, put the solution into a dialysis bag with a molecular weight cut-off of 300 kD, and dialyze for 12 hours to obtain a hybrid protein nano-oxygen carrier solution.

Example 4

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Dissolve 300mg recombinant human serum albumin and 150mg glutathione in 5mL deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 1.3 mg of hemoglobin in 2 mL of pure water and homogenize for 120 minutes, adjust the pH value to 8.5 with sodium bicarbonate, and add to the mixed solution at a speed of 1 mL/min under strong stirring Add 3 mL of absolute ethanol dropwise, stir for 6 hours, put the solution into a dialysis bag with a molecular weight cut-off of 300 kD, and dialyze for 12 hours to obtain a hybrid protein nano-oxygen carrier solution.

Example 5

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Dissolve 300mg recombinant human serum albumin and 45mg cysteine in 5mL deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 4 mg of hemoglobin in 2 mL of pure water and homogenize for 60 minutes, adjust the pH value to 8 with sodium bicarbonate, and drop into the mixed solution at a speed of 1 mL/min under strong stirring Add 3 mL of absolute ethanol, stir for 6 hours, put the solution into a dialysis bag with a molecular weight cut-off of 300 kD, and dialyze for 12 hours to obtain a hybrid protein nano-oxygen carrier solution.

Example 6

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Dissolve 300mg recombinant human serum albumin and 90mg glutathione in 5mL deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 3 mg of hemoglobin in 2 mL of pure water and homogenize for 45 minutes, adjust the pH value to 8 with sodium bicarbonate, and drop into the mixed solution at a speed of 1 mL/min under strong stirring Add 3 mL of absolute ethanol, stir for 6 hours, put the solution into a dialysis bag with a molecular weight cut-off of 300 kD, and dialyze for 12 hours to obtain a hybrid protein nano-oxygen carrier solution.

Example 7

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Dissolve 300mg recombinant human serum albumin and 75mg glutathione in 5mL deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 3.2 mg of hemoglobin in 2 mL of pure water and homogenize for 30 minutes, adjust the pH to 8 with sodium bicarbonate, and add to the mixed solution at a speed of 1 mL/min under strong stirring 3 mL of absolute ethanol was added dropwise, stirred for 3 hours, and ethanol and free protein were removed by vacuum drying to obtain a hybrid protein nano-oxygen carrier solution.

Example 8

This example provides a hybrid protein nano-oxygen carrier, which is prepared according to the following steps:

(a) Dissolve 300mg of recombinant human serum albumin and 50mg of cysteine in 5mL of deionized water, shake at room temperature for 1 hour, pour the solution into a dialysis bag (3kD), and dialyze in anaerobic deionized water for 12 hours to obtain Reduced serum albumin solution, dilute the reduced serum albumin solution to 6 mL with deionized water to obtain a reduced serum albumin solution with a concentration of 50 mg/mL;

(b) Dissolve 20 mg of reduced serum albumin solution and 3.2 mg of hemoglobin in 2 mL of pure water and homogenize for 50 minutes, adjust the pH value to 8.5 with sodium bicarbonate, and add to the mixed solution at a speed of 1 mL/min under strong stirring Add 3.6 mL of absolute ethanol dropwise, stir for 3 hours, put the solution into a dialysis bag with a molecular weight cut-off of 100 kD, and dialyze for 12 hours to obtain a hybrid protein nano-oxygen carrier solution.

Example 9

This example provides a hybrid protein nano-oxygen carrier. The difference between this example and Example 7 is that in step (a), the amount of cysteine used is 10 mg.

Example 10

This example provides a hybrid protein nano-oxygen carrier. The difference between this example and Example 7 is that in step (b), the amount of hemoglobin is 0.2 mg.

Test example 1

The hybrid protein nano-oxygen carrier solutions provided in Examples 1-10 were all visually observed, and it was found that the hybrid protein nano-oxygen carriers provided in Examples 1-10 were all clear and transparent, without precipitation and impurities. The state of the hybrid protein nano oxygen carrier solution provided in Example 7 in the test tube is photographed, as shown in Figure 1, as can be seen from Figure 1, the hybrid protein nano oxygen carrier solution provided in Example 7 is clear and transparent, free of impurities and precipitation.

Test example 2

The particle size distribution of the hybrid protein nano-oxygen carrier solutions provided in Examples 1-10 was measured by dynamic light scattering analysis, and the results showed that the particle sizes of the hybrid protein nano-oxygen carriers provided in Examples 1-10 were all 20-200 nm. Figure 2 is a particle size distribution diagram of the hybrid protein nano-oxygen carrier solution provided in Example 7; it can be seen from Figure 2 that the particle size of the hybrid protein nano-oxygen carrier provided in Example 7 is 20-50 nm.

Test example 3

The oxygen affinity P50 values of the hybrid protein nano oxygen carrier solutions provided in Examples 1-10 were measured respectively, and the test results are shown in Table 1 below:

Table 1 Hybrid protein nano oxygen carrier oxygen affinity P50 value data table

As can be seen from Table 1, as can be seen from the comparison of Examples 1-8 and Examples 9-10, when the hybrid protein nano-oxygen carrier is prepared, the mass ratio of serum albumin to reducing agent is 1: (0.1- 1), when the mass ratio of hemoglobin to serum albumin is 1: (2-50), the oxygen affinity P50 value of the prepared hybrid protein nano-oxygen carrier is significantly improved, all higher than 13mmHg, and the oxygen-carrying capacity is good.

It can be seen from the comparison of Examples 1-8 that the oxygen affinity P50 value of Example 5-8 is the highest, and the oxygen loading capacity of Example 3-4 is lower than that of Example 5-8 but higher than that of Example 1-2. This shows that when the mass ratio of hemoglobin and serum albumin is 1: (3-15), and the mass ratio of serum albumin and reducing agent is 1: (0.15-0.5), the hybrid protein nano oxygen carrier made The oxygen affinity P50 value is strong, and the stability in the body is good. When the mass ratio of hemoglobin to serum albumin is 1: (5-7), and the mass ratio of serum albumin to reducing agent is 1: (0.15-0.3) , the prepared hybrid protein nano-oxygen carrier has a higher oxygen affinity P50 value, better stability in vivo, and better oxygen-carrying capacity.

Test example 4

The hybrid protein nano-oxygen carrier solution and the free hemoglobin solution provided in Example 8 were passed through pure oxygen to a saturated state, and then 1 mL of the hybrid protein nano-oxygen carrier solution and the free hemoglobin solution were respectively injected into 10 mL of oxygen-free pure water in a closed environment, wherein , the hemoglobin content of the above two solutions is the same. Dissolved oxygen probes were used to detect the increase in the oxygen concentration of the solution caused by the release of oxygen from the hybrid protein nano-oxygen carrier solution and the free hemoglobin solution. Figure 3 is the oxygen release curve of oxygen-saturated hybrid protein nano-oxygen carrier solution and free hemoglobin solution in anaerobic water; as can be seen from Figure 3, the oxygen release of oxygen-saturated hybrid protein nano-oxygen carrier solution is significantly higher than the same concentration The oxygen release rate of the saturated free hemoglobin solution shows that the hybrid protein nano-oxygen carrier provided by Example 8 of the present invention hybridizes with hemoglobin through serum albumin, provides protection for hemoglobin, improves the stability of hemoglobin, and increases the oxygen-carrying capacity. quantity.

Test example 5

The hybrid protein nano-oxygen carriers provided in the above examples 1-10 were stored at room temperature for 2 years, and then performance testing was performed. The results showed that the performance of the hybrid protein nano-oxygen carriers provided in Examples 1-10 did not change significantly. This shows that the hybrid protein nano-oxygen carrier provided by the invention has a long storage time, which can reach more than 2 years.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (10)

1. a kind of hybrid protein nanometer carrier of oxygen, it is characterised in that disulfide bond is mainly passed through by hemoglobin and seralbumin Covalent bond forms, its size is 20-200 nanometers.

2. the hybrid protein nanometer carrier of oxygen according to claim 1, it is characterised in that the hemoglobin derives from dynamic Thing, it is preferable that the hemoglobin derives from people, ox or pig；

Preferably, the haemocyanin derives from animal, it is further preferred that the seralbumin derives from people or ox, more It is further preferred that the seralbumin is the recombination human serum albumin that biological fermentation method obtains.

3. the preparation method of the hybrid protein nanometer carrier of oxygen according to claim 1 or 2, it is characterised in that including as follows Step：

(a) reducing agent is mixed with seralbumin, carries out reduction reaction, obtain reduced form seralbumin；

(b) by reduced form seralbumin and hemoglobin mixing homogeneous, that is, the hybrid protein nanometer carrier of oxygen is made.

4. the preparation method of the hybrid protein nanometer carrier of oxygen according to claim 3, it is characterised in that the reducing agent choosing From at least one of glutathione, dithiothreitol (DTT), cysteine or homocysteine.

5. the preparation method of the hybrid protein nanometer carrier of oxygen according to claim 3, it is characterised in that hemoglobin and blood The mass ratio of pure albumen is 1：(2-50), is preferably 1：(3-15), more preferably 1：(5-10).

6. the preparation method of the hybrid protein nanometer carrier of oxygen according to claim 3, it is characterised in that seralbumin with The mass ratio of reducing agent is 1：(0.1-1), is preferably 1：(0.15-0.5), more preferably 1：(0.15-0.3).

7. the preparation method of the hybrid protein nanometer carrier of oxygen according to claim 3, it is characterised in that in step (b), The pH value of the mixed solution of reduced form seralbumin and hemoglobin is 7-9；

Preferably, it is 10-180 minutes to mix homogenizing time；

Preferably, the sero-abluminous concentration of reduced form is 0.5-3%.

8. according to the preparation method of claim 3-7 any one of them hybrid protein nanometer carriers of oxygen, it is characterised in that in step Suddenly in (b), first by reduced form seralbumin and hemoglobin mixing homogeneous, then addition precipitant mix is uniform, finally leads to Cross vacuum drying, ultrafiltration or dialysis to remove precipitating reagent and floating preteins, you can the hybrid protein nanometer carrier of oxygen is made；

Preferably, the precipitating reagent is anhydrous low-carbon alcohol, it is highly preferred that the precipitating reagent is absolute ethyl alcohol.

9. the preparation method of the hybrid protein nanometer carrier of oxygen according to claim 8, it is characterised in that reduced form serum is white Albumen and the volume ratio of hemoglobin mixed solution and precipitating reagent are 1：(1-2.5)；

Preferably, when the incorporation time of precipitating reagent and reduced form seralbumin and erythrocyte mixed solution is 0.5-12 small.

10. application of the hybrid protein nanometer carrier of oxygen according to claim 1 or 2 in blood substitute is prepared.