CN107982544B - Hybrid protein nano oxygen carrier and preparation method and application thereof - Google Patents
Hybrid protein nano oxygen carrier and preparation method and application thereof Download PDFInfo
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- CN107982544B CN107982544B CN201711215596.7A CN201711215596A CN107982544B CN 107982544 B CN107982544 B CN 107982544B CN 201711215596 A CN201711215596 A CN 201711215596A CN 107982544 B CN107982544 B CN 107982544B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
- A61K38/42—Haemoglobins; Myoglobins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Abstract
The invention provides a hybrid protein nano oxygen carrier and a preparation method and application thereof, and relates to the technical field of oxygen carriers, wherein the hybrid protein nano oxygen carrier is mainly formed by covalently combining hemoglobin and serum albumin through disulfide bonds, the size of the hybrid protein nano oxygen carrier is 20-200 nanometers, the technical problems of poor stability in vivo and possible side effects of the existing artificial oxygen carrier are solved, the hemoglobin is protected by covalently combining the serum albumin and the hemoglobin through disulfide bonds, the hybrid protein nano oxygen carrier has good biocompatibility and oxygen carrying stability in vivo, can efficiently convey oxygen to organisms, does not cause cross infection and agglutination reaction, and has the technical effect of no side effect. In addition, the hybrid protein nano oxygen carrier provided by the invention has a stable structure, can be synthesized on a large scale, has storage time of more than two years, and is convenient to popularize and apply.
Description
Technical Field
The invention relates to the technical field of oxygen carriers, in particular to a hybrid protein nano oxygen carrier and a preparation method and application thereof.
Background
Clinical blood transfusion is an important means for life treatment and is widely applied to clinical operation, disaster resistance and war wound treatment, but at present, the blood transfusion still has a plurality of problems: 1. the blood may carry pathogens (hepatitis virus, HIV, etc.), some diseases may be transmitted by blood transfusion; 2. the blood type mismatch causes agglutination effect, which can threaten the life of the transfused person; 3. shortage of blood source; 4. the short storage time of blood limits its use in the treatment of critically ill patients and in emergency time settings.
In recent years, nanotechnology has brought about rapid development of artificial oxygen carriers, which are nano/micron-sized particles having an oxygen carrier function, which can be synthesized on a large scale without causing cross-infection and agglutination, and have attracted much attention. At present, various artificial oxygen carrier products are clinically applied as blood substitutes, and the artificial oxygen carriers comprise perfluorocarbons, polyhemoglobin, cross-linked hemoglobin, hemoglobin liposome and the like, but the perfluorocarbons need to be stored in a refrigeration mode, high oxygen with the concentration of more than 95% must be inhaled simultaneously during infusion, the biological half-life period is short, and serious side effects exist. The poly-hemoglobin and cross-linked hemoglobin are not protected by other molecules, so that the functional stability of hemoglobin in vivo is not easy to maintain, and renal toxicity is easy to cause. The hemoglobin liposome has poor structural stability in vivo, and encapsulated hemoglobin is easy to leak.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a hybrid protein nano oxygen carrier, which solves the technical problems that the existing artificial oxygen carrier has poor stability in vivo and may have side effects.
The hybrid protein nano oxygen carrier provided by the invention is mainly formed by covalently combining hemoglobin and serum albumin through disulfide bonds, and the size of the hybrid protein nano oxygen carrier is 20-200 nanometers.
Further, the hemoglobin is derived from animals, preferably, the hemoglobin is derived from human, cattle or pig;
preferably, the serum albumin is derived from animals or obtained by biological fermentation, further preferably, the serum albumin is derived from human or cattle, and further preferably, the serum albumin is recombinant human serum albumin obtained by biological fermentation.
The invention also aims to provide a preparation method of the hybrid protein nano oxygen carrier, which comprises the following steps:
(a) mixing a reducing agent with serum albumin, and carrying out reduction reaction to obtain reduced serum albumin;
(b) and mixing and homogenizing the reduced serum albumin and the hemoglobin to obtain the hybrid protein nano oxygen carrier.
Further, the reducing agent is selected from at least one of glutathione, dithiothreitol, cysteine or homocysteine.
Further, the mass ratio of the hemoglobin to the serum albumin is 1: (2-50), preferably 1: (3-15), more preferably 1: (5-10).
Further, the mass ratio of the serum albumin to the reducing agent is 1: (0.1-1), preferably 1: (0.15-0.5), more preferably 1: (0.15-0.3).
Further, in the step (b), the pH value of the mixed solution of the reduced serum albumin and the hemoglobin is 7-9;
preferably, the mixing and homogenizing time is 10-180 minutes;
preferably, the concentration of reduced serum albumin is 0.5-3%.
Further, in the step (b), the reduced serum albumin and the hemoglobin are mixed and homogenized, then the precipitator is added and mixed uniformly, and finally the precipitator and the free protein are removed through vacuum drying, ultrafiltration or dialysis, so that the hybrid protein nano oxygen carrier can be prepared;
preferably, the precipitant is an anhydrous lower alcohol, and more preferably, the precipitant is anhydrous ethanol.
Further, the volume ratio of the reduced serum albumin and hemoglobin mixed solution to the precipitator is 1: (1-2.5);
preferably, the mixing time of the precipitant and the mixed solution of reduced serum albumin and red blood cells is 0.5 to 12 hours.
The third purpose of the 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 invention has the advantages that the serum albumin and the hemoglobin are covalently combined through the disulfide bond, so that the hemoglobin is protected, the biocompatibility and the oxygen carrying stability are good in vivo, the oxygen can be efficiently conveyed to an organism, the cross infection and the agglutination reaction cannot be caused, and the side effect is avoided. In addition, the hybrid protein nano oxygen carrier provided by the invention has a stable structure, can be synthesized on a large scale, has storage time of more than two years, and is convenient to popularize and apply.
The preparation method of the hybrid protein nano oxygen carrier provided by the invention is simple and easy to implement, is convenient to operate and popularize, and is suitable for large-scale preparation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram of the state of a hybrid protein nano oxygen carrier solution in a test tube according to example 7 of the present invention;
FIG. 2 is a graph showing the distribution of the particle size of the hybrid protein nano-oxygen carrier solution provided in example 7;
FIG. 3 is the oxygen release curve of hybrid protein nano oxygen carrier and hemoglobin in oxygen-free water.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to the first aspect of the invention, the invention provides a hybrid protein nano oxygen carrier which is mainly formed by covalently combining hemoglobin and serum albumin through a disulfide bond, and the size of the hybrid protein nano oxygen carrier is 20-200 nanometers.
The hybrid protein nano oxygen carrier provided by the invention has the advantages that the serum albumin and the hemoglobin are covalently combined through the disulfide bond, so that the hemoglobin is protected, the biocompatibility and the oxygen carrying stability are good in vivo, the oxygen can be efficiently conveyed to an organism, the cross infection and the agglutination reaction cannot be caused, and the side effect is avoided. In addition, the hybrid protein nano oxygen carrier provided by the invention has a stable structure, can be synthesized on a large scale, has storage time of more than two years, and is convenient to popularize and apply.
The size of the hybrid protein nano oxygen carrier provided by the invention is 20-200 nm, so that the hybrid protein nano oxygen carrier is beneficial to stable transmission in vivo and prolongs the 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, has no erythrocyte surface antigenic determinant, can eliminate the trouble of blood type adaptation, avoids agglutination effect, simultaneously avoids pathogenic microorganisms from polluting blood sources and further cross infection, does not depend on blood supply crowds, has wide sources and convenient material taking, can ensure sufficient supply, can be produced in a large scale, and meets the requirements of people on oxygen supply blood substitutes.
In a preferred embodiment of the invention, the hemoglobin is of animal origin, preferably the hemoglobin is of human, bovine or porcine origin;
preferably, the serum albumin is derived from animals or obtained by biological fermentation, more preferably, the serum albumin is derived from human and cattle, and even more preferably, the serum albumin is recombinant human serum albumin obtained by biological fermentation.
The hemoglobin source animal can be extracted from animal blood, and when the hemoglobin is extracted from animal blood such as human, cattle, pig and the like, the prepared hybrid protein nano oxygen carrier has better stability in vivo. Hemoglobin includes hemoglobin and its analog molecules, and hemoglobin raw materials include HbA, HbA2, HbF, etc. subtypes. Serum albumin is extracted from animal blood or obtained by biological fermentation. When the serum albumin is extracted from blood of animals such as human and cattle, the in vivo stability of the prepared hybrid protein nano oxygen carrier is better, and when the serum albumin is recombinant human serum albumin obtained by adopting a biological fermentation mode, the in vivo stability of the prepared hybrid protein nano oxygen carrier is better.
According to a second aspect of the present invention, the present invention provides a method for preparing the hybrid protein nano oxygen carrier, comprising the following steps:
(a) mixing a reducing agent with serum albumin, and carrying out reduction reaction to obtain reduced serum albumin;
(b) and mixing and homogenizing the reduced serum albumin and the hemoglobin to obtain the hybrid protein nano oxygen carrier.
The preparation method of the hybrid protein nano oxygen carrier provided by the invention is simple and easy to implement, is convenient to operate and popularize, and is suitable for large-scale preparation.
In the step (a), a reducing agent and serum albumin undergo a reduction reaction to cleave disulfide bonds within the serum albumin molecules, thereby obtaining reduced serum albumin molecules in a random state. In the step (b), the reduced serum albumin molecules and free sulfydryl of the hemoglobin are crosslinked to form intermolecular disulfide bonds, and the hybrid protein nano oxygen carrier is formed.
In the present invention, the term "mixing and homogenizing" means mixing and then homogenizing.
In a typical but non-limiting embodiment of the 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 may be one of glutathione, dithiothreitol, cysteine and homocysteine, or a mixture of any two of glutathione, 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, or a mixture of any three of glutathione, dithiothreitol, cysteine and homocysteine, such as a mixture of glutathione, dithiothreitol and cysteine, or a mixture of glutathione, dithiothreitol, cysteine and homocysteine, or the like, or a mixture of four of glutathione, dithiothreitol, cysteine and homocysteine.
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 the hemoglobin to the serum albumin to be 1: (2-50), the serum albumin provides sufficient protection for the hemoglobin, so that the stability of the hybrid protein nano oxygen carrier in vivo is improved, the circulation time of the hybrid protein nano oxygen carrier in vivo is prolonged, and when the mass ratio of the hemoglobin to the serum albumin is 1: (3-15), the prepared hybrid protein nano oxygen carrier not only has good stability in vivo and long circulation time, but also has larger oxygen carrying amount, and particularly when the mass ratio of the hemoglobin to the serum albumin is 1: (0.15-0.3), the composition has better in vivo stability, longer circulation time and larger oxygen carrying capacity.
In a preferred embodiment of the present invention, the mass ratio of the serum albumin to the 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 the serum albumin to the reducing agent to be 1: (0.1-1) so that when the reducing agent and the serum albumin are subjected to reduction reaction, the reduction reaction of serum albumin molecules is sufficiently carried out, disulfide bonds in the serum albumin molecules can be broken, and reduced serum albumin is generated; in particular, the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.5), the reduction reaction of serum albumin is more fully performed, the disulfide bonds in the serum albumin molecules are more broken, and when the mass ratio of the serum albumin to the reducing agent is 1: (0.15-3), the reduction reaction of serum albumin proceeds most sufficiently, and the produced reduced serum albumin molecules contain more free thiol groups.
In a preferred embodiment of the present invention, in the step (b), the pH of the mixed solution of reduced serum albumin and hemoglobin is 7 to 9;
preferably, the mixing and homogenizing time is 10-180 minutes;
preferably, the concentration of reduced serum albumin is 0.5-3%.
In the step (b), the generation of the hybrid protein nano oxygen carrier is promoted by controlling the pH value of the mixed solution of the reduced serum albumin and the hemoglobin to be 7-9, and the pH value can be adjusted by sodium bicarbonate to keep the pH value of the mixed solution at 7-9 in the process of carrying out mixing homogenization.
The time for mixing and homogenizing is controlled to be 10-180 minutes, so that the reaction of the reduced serum albumin and the hemoglobin is more complete, and the yield of the hybrid protein nano oxygen carrier is improved.
The concentration of reduced serum albumin is the concentration of reduced serum albumin in a mixed solution of reduced serum albumin and hemoglobin. The preparation efficiency of the hybrid protein nano oxygen carrier is improved by controlling the concentration of the reduced serum albumin to be 0.5-3%.
In a preferred embodiment of the invention, in the step (b), the reduced serum albumin and the hemoglobin are mixed and homogenized, then the precipitant is added and mixed uniformly, and finally the precipitant and the free protein are removed through vacuum drying, ultrafiltration or dialysis, so as to prepare the hybrid protein nano oxygen carrier; preferably, the precipitant is an anhydrous lower alcohol, and more preferably, the precipitant is anhydrous ethanol.
In the step (b), a precipitator is adopted to reduce the solubility of the hemoglobin and the reduced serum albumin, so that the cross-linking reaction of the hemoglobin and the reduced serum albumin is more fully carried out, and the yield of the hybrid protein nano oxygen carrier is improved.
And finally, removing the precipitant and the free protein by vacuum drying, ultrafiltration or dialysis to purify the cross-linked protein nano oxygen carrier, and removing the precipitant and the free protein, wherein the free protein comprises free serum albumin and free hemoglobin which are not subjected to cross-linking reaction.
In a typical but non-limiting preferred embodiment of the present invention, the precipitating agent is an anhydrous lower alcohol, which refers to a lower alcohol of C1-C4, preferably, the precipitating agent is anhydrous ethanol.
In a further preferred embodiment of the invention, the molecular weight cut-off by ultrafiltration or dialysis is 100-300kDa when the precipitant and free proteins are removed by ultrafiltration or dialysis.
In a preferred embodiment of the present invention, the volume ratio of the reduced serum albumin and hemoglobin mixed solution to the precipitant is 1: (1-2.5);
preferably, the mixing time of the precipitant and the mixed solution of reduced serum albumin and red blood cells is 0.5 to 12 hours.
By controlling the volume ratio of the mixed solution of the reduced serum albumin and the hemoglobin to the precipitator to be 1: (1-2.5) so that the precipitator is fully mixed with the mixed solution of the reduced serum albumin and the hemoglobin, the solubility of the reduced serum albumin and the hemoglobin is reduced, and the crosslinking reaction of the two proteins is fully performed.
The mixing time of the mixed solution of the reduced serum albumin and the hemoglobin and the precipitator is controlled to be 0.5-12 hours, so that the precipitator can be uniformly mixed with the mixed solution of the reduced serum albumin and the hemoglobin, the solubility of the reduced serum albumin and the hemoglobin is reduced, and the crosslinking reaction of the two proteins is more sufficient.
According to a third aspect of the invention, the invention provides the use of the hybrid protein nano oxygen carrier in the preparation of a blood substitute.
The hybrid protein nano oxygen carrier provided by the invention is used as a nano-sized oxygen carrier, has no erythrocyte surface antigenic determinant, can eliminate the trouble of blood group adaptation, avoids agglutination effect, can avoid pathogenic microorganisms from polluting blood sources and further cross infection, can be combined with oxygen molecules with high affinity, and can reversibly release oxygen in vivo to replace the function of blood for conveying oxygen.
The technical solution provided by the present invention is further described with reference to the following examples and embodiments.
Example 1
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of bovine serum albumin and 30mg of dithiothreitol in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using deionized water to obtain a reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 10mg of hemoglobin in 2mL of purified water together, homogenizing for 10 minutes, adjusting the pH value to 7 by using sodium bicarbonate, dropwise adding 2mL of absolute ethanol into the mixed solution at the speed of 1mL/min under strong stirring, stirring and homogenizing for 0.5 hour, filling the mixed solution into a dialysis bag with the molecular weight cutoff of 100kD, and dialyzing for 12 hours to obtain the hybrid protein nano oxygen carrier solution.
Example 2
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of human serum albumin and 300mg of homocysteine in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using the deionized water to obtain the reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 0.4mg of hemoglobin in 2mL of purified water together, homogenizing for 180 minutes, adjusting the pH value to 9 by using sodium bicarbonate, dropwise adding 5mL of absolute ethanol into the mixed solution at the speed of 1mL/min under strong stirring, stirring and homogenizing for 12 hours, filling the solution into a dialysis bag with the molecular weight cutoff of 300kD, and dialyzing for 12 hours to obtain the hybrid protein nano oxygen carrier solution.
Example 3
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of recombinant human serum albumin and 45mg of glutathione in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using the deionized water to obtain the reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 6.7mg of hemoglobin in 2mL of purified water together, homogenizing for 20 minutes, adjusting the pH value to 7.5 by using sodium bicarbonate, dropwise adding 4mL of absolute ethanol into the mixed solution at the speed of 1mL/min under strong stirring, stirring for 1 hour, filling the solution into a dialysis bag with the molecular weight cutoff of 300kD, and dialyzing for 12 hours to obtain the hybrid protein nano oxygen carrier solution.
Example 4
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of recombinant human serum albumin and 150mg of glutathione in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using the deionized water to obtain the reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 1.3mg of hemoglobin in 2mL of purified water together, homogenizing for 120 minutes, adjusting the pH value to 8.5 by using sodium bicarbonate, dropwise adding 3mL of absolute ethanol into the mixed solution at the speed of 1mL/min under strong stirring, stirring for 6 hours, filling the solution into a dialysis bag with the molecular weight cutoff of 300kD, and dialyzing for 12 hours to obtain the hybrid protein nano oxygen carrier solution.
Example 5
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of recombinant human serum albumin and 45mg of cysteine in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using the deionized water to obtain the reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 4mg of hemoglobin in 2mL of purified water together, homogenizing for 60 minutes, adjusting the pH value to 8 by using sodium bicarbonate, dropwise adding 3mL of absolute ethanol into the mixed solution at the speed of 1mL/min under strong stirring, stirring for 6 hours, filling the solution into a dialysis bag with the molecular weight cutoff of 300kD, and dialyzing for 12 hours to obtain the hybrid protein nano oxygen carrier solution.
Example 6
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of recombinant human serum albumin and 90mg of glutathione in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using the deionized water to obtain the reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 3mg of hemoglobin in 2mL of purified water together, homogenizing for 45 minutes, adjusting the pH value to 8 by using sodium bicarbonate, dropwise adding 3mL of absolute ethanol into the mixed solution at the speed of 1mL/min under strong stirring, stirring for 6 hours, filling the solution into a dialysis bag with the molecular weight cutoff of 300kD, and dialyzing for 12 hours to obtain the hybrid protein nano oxygen carrier solution.
Example 7
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of recombinant human serum albumin and 75mg of glutathione in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using the deionized water to obtain the reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 3.2mg of hemoglobin in 2mL of purified water together, homogenizing for 30 minutes, adjusting the pH value to 8 by using sodium bicarbonate, dropwise adding 3mL of absolute ethanol into the mixed solution at the speed of 1mL/min under strong stirring, stirring for 3 hours, and removing ethanol and free protein through vacuum drying to obtain the hybrid protein nano oxygen carrier solution.
Example 8
The embodiment provides a hybrid protein nano oxygen carrier, which is prepared by the following steps:
(a) dissolving 300mg of recombinant human serum albumin and 50mg of cysteine in 5mL of deionized water, oscillating for 1 hour at room temperature, pouring the solution into a dialysis bag (3kD), dialyzing for 12 hours in oxygen-free deionized water to obtain a reduced serum albumin solution, and fixing the volume of the reduced serum albumin solution to 6mL by using the deionized water to obtain the reduced serum albumin solution with the concentration of 50 mg/mL;
(b) dissolving 20mg of reduced serum albumin solution and 3.2mg of hemoglobin in 2mL of purified water together, homogenizing for 50 minutes, adjusting the pH value to 8.5 by using sodium bicarbonate, dropwise adding 3.6mL of absolute ethyl alcohol into the mixed solution at the speed of 1mL/min under strong stirring, stirring for 3 hours, filling the solution into a dialysis bag with the molecular weight cutoff of 100kD, and dialyzing for 12 hours to obtain the hybrid protein nano oxygen carrier solution.
Example 9
This example provides a hybrid protein nano oxygen carrier, and the difference between this example and example 7 is that, in step (a), the amount of cysteine is 10 mg.
Example 10
This example provides a hybrid protein nano oxygen carrier, and differs from example 7 in that hemoglobin is used in an amount of 0.2mg in step (b).
Test example 1
The hybrid protein nano oxygen carrier solutions provided in examples 1-10 were all observed by naked eyes, and the hybrid protein nano oxygen carriers provided in examples 1-10 were all clear and transparent and free of precipitate 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 fig. 1, and as can be seen from fig. 1, the hybrid protein nano oxygen carrier solution provided in example 7 is clear and transparent, and has no impurities or precipitates.
Test example 2
The solution of the hybrid protein nano oxygen carrier provided in the examples 1 to 10 was measured for particle size distribution by dynamic light scattering analysis, and the results showed that the hybrid protein nano oxygen carrier provided in the examples 1 to 10 had particle sizes of 20 to 200 nm. FIG. 2 is a graph showing the distribution of the particle size of the hybrid protein nano-oxygen carrier solution provided in example 7; as can be seen from FIG. 2, the hybrid protein nano oxygen carrier provided in example 7 has a particle size of 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 determined separately and the results are shown in table 1 below:
TABLE 1 data sheet of oxygen carrier oxygen affinity of hybrid protein nano-scale P50
As can be seen from Table 1, the comparison between examples 1-8 and examples 9-10 shows that the mass ratio of serum albumin to the reducing agent is 1: (0.1-1), wherein the mass ratio of the hemoglobin to the serum albumin is 1: (2-50), the oxygen affinity P50 value of the prepared hybrid protein nano oxygen carrier is obviously improved, and both are higher than 13mmHg, and the oxygen carrying capacity is good.
As can be seen by comparing examples 1-8, examples 5-8 have the highest value of oxygen affinity, P50, and examples 3-4 have oxygen carrying capacity lower than examples 5-8 but higher than examples 1-2. This indicates that when the mass ratio of hemoglobin to serum albumin is 1: (3-15), and the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.5), the prepared hybrid protein nano oxygen carrier has stronger oxygen affinity P50 value and better in vivo stability, and when the mass ratio of the hemoglobin to the serum albumin is 1: (5-7), and the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.3), the prepared hybrid protein nano oxygen carrier has higher oxygen affinity P50 value, better in vivo stability and better oxygen carrying capacity.
Test example 4
Introducing pure oxygen into the hybrid protein nano oxygen carrier solution and the free hemoglobin solution provided in the embodiment 8 respectively to a saturated state, and then injecting 1mL of the hybrid protein nano oxygen carrier solution and the free hemoglobin solution into 10mL of oxygen-free pure water in a closed environment, wherein the hemoglobin contents of the two solutions are the same. And respectively detecting the rise of 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 by adopting an oxygen dissolving probe. FIG. 3 is an oxygen release profile of an oxygen-saturated hybrid protein nanooxygen carrier solution and a free hemoglobin solution in oxygen-free water; as can be seen from fig. 3, the oxygen release amount of the oxygen-saturated hybrid protein nano-oxygen carrier solution is significantly higher than that of the saturated free hemoglobin solution at the same concentration, which indicates that the hybrid protein nano-oxygen carrier provided in example 8 of the present invention provides protection for hemoglobin by hybridization between serum albumin and hemoglobin, improves the stability of hemoglobin, and increases the oxygen carrying amount.
Test example 5
The hybrid protein nano oxygen carriers provided by the embodiments 1 to 10 are respectively stored for 2 years at room temperature, and then performance detection is carried out, and the results show that the performance of the hybrid protein nano oxygen carriers provided by the embodiments 1 to 10 is not obviously changed, which indicates that the hybrid protein nano oxygen carriers provided by the invention have 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 solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (17)
1. The hybrid protein nano oxygen carrier is characterized by being mainly formed by covalently combining hemoglobin and serum albumin through disulfide bonds, wherein the size of the carrier is 20-200 nanometers, and the mass ratio of the hemoglobin to the serum albumin is 1: (2-50).
2. The hybrid protein nanooxygen carrier of claim 1, wherein the hemoglobin is derived from an animal and the serum albumin is derived from an animal.
3. The hybrid protein nanooxygen carrier according to claim 2, wherein the hemoglobin is derived from human, bovine or porcine; the serum albumin is derived from human or bovine.
4. The hybrid protein nano-oxygen carrier according to claim 3, wherein the serum albumin is recombinant human serum albumin obtained by biological fermentation.
5. The method for preparing the hybrid protein nano oxygen carrier according to any one of claims 1 to 4, which is characterized by comprising the following steps:
(a) mixing a reducing agent with serum albumin, and carrying out reduction reaction to obtain reduced serum albumin;
(b) and (2) mixing and homogenizing the reduced serum albumin and the hemoglobin to obtain the hybrid protein nano oxygen carrier, wherein the mass ratio of the hemoglobin to the serum albumin is 1: (2-50).
6. The method for preparing the hybrid protein nano oxygen carrier according to claim 5, wherein the reducing agent is at least one selected from glutathione, dithiothreitol, cysteine or homocysteine.
7. The method for preparing the hybrid protein nano oxygen carrier according to claim 5, wherein the mass ratio of the hemoglobin to the serum albumin is 1: (2-50).
8. The method for preparing the hybrid protein nano oxygen carrier according to claim 7, wherein the mass ratio of the hemoglobin to the serum albumin is 1: (3-15).
9. The method for preparing the hybrid protein nano oxygen carrier according to claim 7, wherein the mass ratio of the hemoglobin to the serum albumin is 1: (5-10).
10. The method for preparing the hybrid protein nano oxygen carrier according to claim 5, wherein the mass ratio of the serum albumin to the reducing agent is 1: (0.1-1).
11. The method for preparing the hybrid protein nano oxygen carrier according to claim 10, wherein the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.5).
12. The method for preparing the hybrid protein nano oxygen carrier according to claim 10, wherein the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.3).
13. The method for preparing the hybrid protein nano oxygen carrier according to claim 5, wherein in the step (b), the pH value of the mixed solution of the reduced serum albumin and the hemoglobin is 7-9;
mixing and homogenizing for 10-180 min;
the concentration of reduced serum albumin is 0.5-3%.
14. The method for preparing the hybrid protein nano oxygen carrier according to any one of claims 5 to 13, wherein in the step (b), the reduced serum albumin and the hemoglobin are mixed and homogenized, then the precipitant is added and mixed uniformly, and finally the precipitant and the free protein are removed through vacuum drying, ultrafiltration or dialysis, so as to prepare the hybrid protein nano oxygen carrier;
the precipitant is anhydrous low-carbon alcohol.
15. The method for preparing the hybrid protein nano oxygen carrier according to claim 14, wherein the precipitant is absolute ethyl alcohol.
16. The method for preparing the hybrid protein nano oxygen carrier according to claim 14, wherein the volume ratio of the reduced serum albumin and hemoglobin mixed solution to the precipitant is 1: (1-2.5);
the mixing time of the precipitator, the reduced serum albumin and the red blood cell mixed solution is 0.5-12 hours.
17. Use of the hybrid protein nanooxygen carrier according to any one of claims 1 to 4 for the preparation of a blood substitute.
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| CN1767851A (en) * | 2003-04-09 | 2006-05-03 | 苏志国 | Hemoglobin conjugate and its preparing method and use |
| EP1419171B1 (en) * | 2001-08-21 | 2010-12-22 | Apex Bioscience, Inc. | Methods for the synthesis of a modified hemoglobin solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1419171B1 (en) * | 2001-08-21 | 2010-12-22 | Apex Bioscience, Inc. | Methods for the synthesis of a modified hemoglobin solution |
| CN1767851A (en) * | 2003-04-09 | 2006-05-03 | 苏志国 | Hemoglobin conjugate and its preparing method and use |
Non-Patent Citations (1)
| Title |
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| "Reactivity of 42 disulfides with thiol group of human haemoglobin and human serum albumin";Jean-Pierre Mahieueu et al.;《International Journal of Biological Macromolecules》;19930831;第15卷(第4期);第233-240页 * |
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