CN107952072B - Preparation method of drug-loaded and oxygen-loaded hybrid protein nanoparticles, drug-loaded and oxygen-loaded hybrid protein nanoparticles and application - Google Patents
Preparation method of drug-loaded and oxygen-loaded hybrid protein nanoparticles, drug-loaded and oxygen-loaded hybrid protein nanoparticles and application Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
Abstract
The invention provides a preparation method of a drug-loaded and oxygen-loaded hybrid protein nanoparticle, the drug-loaded and oxygen-loaded hybrid protein nanoparticle and application, and relates to the technical field of biological pharmacy, and the preparation method comprises the following steps: mixing and homogenizing a reducing agent and serum albumin, and carrying out reduction reaction to obtain reduced serum albumin; reduced serum albumin, hemoglobin and medicine are mixed and homogenized under the aerobic condition, so that the medicine-carrying oxygen-carrying hybrid protein nanoparticles are prepared, the technical problem that oxygen is supplied in the conventional high-pressure oxygen inhalation mode, oxygen cannot be conveyed into tumors, and meanwhile, oxygen with high concentration can also cause oxygen poisoning of the lungs and the central nervous system is solved, the aim of conveying oxygen and medicine into the tumors by the medicine-carrying oxygen-carrying hybrid protein nanoparticles is achieved, the medicine enrichment is improved, and the reversible release of oxygen molecules can be carried out according to the oxygen concentration in the microenvironment where the medicine-carrying oxygen-carrying hybrid protein nanoparticles are located, and no side effect is produced, the integration of tumor oxygenation and medicine delivery is realized, and the technical effect of the bioavailability of the medicine is improved.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a preparation method of drug-loaded and oxygen-loaded hybrid protein nanoparticles, the drug-loaded and oxygen-loaded hybrid protein nanoparticles and application.
Background
The study proves that the tumor hypoxia causes a special microenvironment of the tumor, influences processes such as angiogenesis and cell division of tumor tissues, leads to insensitivity to chemotherapeutic drugs and radiotherapy, and even leads to tumor gene mutation and tumor metastasis. In the photodynamic therapy, oxygen as a reactant has certain influence on the photodynamic therapy effect of the tumor, and the hypoxia of the tumor limits the generation of active oxygen for the photodynamic therapy, thereby influencing the treatment effect.
The current clinical treatment adopts a high-pressure oxygen inhalation mode to increase the oxygen content of a tumor area and obtain the sensitization effect of the tumor on radiotherapy, chemotherapy and optical treatment. Hyperbaric oxygen therapy supplies oxygen through red blood cells in the blood, but the red blood cells cannot penetrate blood vessels to deliver oxygen to the interior of the tumor, and the irregular growth of tumor blood vessels limits the access of oxygen and drugs to the tumor. At the same time, high concentrations of oxygen can also cause oxygen poisoning of the lungs and central nervous system. Therefore, the technical personnel in the field need to develop a treatment scheme which has tumor targeting and oxygen carrying functions, can effectively deliver oxygen and drugs into the anoxic tumor and has no side effect so as to meet the requirement of tumor treatment.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
One of the purposes of the invention is to provide a preparation method of drug-loaded oxygen-loaded hybrid protein nanoparticles, which is used for relieving the technical problems that oxygen is supplied by erythrocytes in blood by means of high-pressure oxygen inhalation in the current clinical treatment, but the erythrocytes cannot penetrate blood vessels to deliver oxygen to the inside of a tumor, the irregular production of the tumor blood vessels limits oxygen and drugs from reaching the tumor, and simultaneously, oxygen with high concentration can also cause oxygen poisoning of lung and central nervous system.
The invention provides a preparation method of drug-loaded oxygen-loaded hybrid protein nanoparticles, which comprises the following steps:
(a) mixing and homogenizing a reducing agent and serum albumin, and carrying out reduction reaction to obtain reduced serum albumin;
(b) the reduced serum albumin, the hemoglobin and the drug are mixed and homogenized under the aerobic condition, and the drug-loaded and oxygen-loaded hybrid protein nanoparticles are prepared.
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, more preferably, the serum albumin is derived from human or cattle, and even more preferably, the serum albumin is recombinant human serum albumin obtained by biological fermentation.
Further, the reducing agent is selected from at least one of glutathione, dithiothreitol, cysteine or homocysteine.
Further, the medicine is a chemotherapeutic medicine and/or a photosensitizer;
preferably, the chemotherapeutic drug is selected from at least one of doxorubicin hydrochloride, methotrexate or hypericin;
preferably, the photosensitizer is selected from at least one of indocyanine green, hematoporphyrin, protoporphyrin or chlorin-e 6.
Further, the mass ratio of the hemoglobin to the serum albumin is 1: (2-50), preferably 1: (3-15), more preferably 1: (5-10);
preferably, 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, the hemoglobin and the medicine 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, the hemoglobin and the medicament are mixed and homogenized under aerobic condition, then the precipitator is added and mixed uniformly, and finally the precipitator, the free protein and the medicament are removed through vacuum drying, ultrafiltration or dialysis, so that the medicament-carrying oxygen-carrying hybrid protein nanoparticles can be prepared; preferably, the precipitant is an anhydrous lower alcohol, more preferably anhydrous ethanol.
Further, the volume ratio of the mixed solution of the reduced serum albumin, the hemoglobin and the medicine to the precipitator is 1: (1-2.5);
preferably, the mixing time of the reduced serum albumin, hemoglobin and drug mixed solution and the precipitant is 0.5-12 hours.
The second purpose of the invention is to provide the drug-loaded oxygen-loaded hybrid protein nanoparticles prepared by the preparation method of the drug-loaded oxygen-loaded hybrid protein nanoparticles.
The invention also aims to provide the application of the drug-loaded oxygen-loaded hybrid protein nanoparticles in the preparation of tumor treatment drugs.
The preparation method of the drug-loaded oxygen-loaded hybrid protein nanoparticles provided by the invention is simple and easy to implement, is convenient to popularize and apply, and is suitable for large-scale preparation.
The drug-loaded oxygen-carrying hybrid protein nanoparticle provided by the invention has the advantages that hemoglobin and serum albumin are covalently combined through disulfide bonds, a drug is loaded, oxygen is combined, so that the drug simultaneously has the tumor targeting function of the serum albumin and the oxygen-carrying function of the hemoglobin, oxygen and the drug can be delivered to the interior of a tumor in a targeted manner, the enrichment of the drug in the interior of the tumor is improved, the reversible release of oxygen molecules can be carried out according to the oxygen concentration in a microenvironment, the tissue with normal oxygen concentration cannot be influenced, no side effect exists, the tumor hypoxia can be effectively improved, the treatment effect of the tumor is enhanced, the integration of tumor oxygenation and drug delivery is realized, and the bioavailability of the drug is improved. In addition, the drug-loaded oxygen-loaded hybrid protein nanoparticle provided by the invention has a stable structure and long storage time, and is convenient to popularize and apply.
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 showing a distribution of particle sizes of drug-loaded and oxygen-loaded hybrid nanoparticles provided in example 7 of the present invention;
FIG. 2 is a photo-acoustic imaging monitoring chart of the change of the oxygenated hemoglobin content at the tumor site after the tumor-bearing nude mouse is injected with the drug-loaded oxygen-loaded hybrid protein nanoparticles;
FIG. 3 is a comparison graph of fluorescence imaging of tumor-bearing nude mice injected with drug-loaded oxygen-carrying hybrid protein nanoparticles for 6 hours and tumor-bearing nude mice injected with free chlorin-e 6 solution for 6 hours;
FIG. 4 is a bar graph comparing the survival rates of tumor cells after chemotherapy with drug-loaded, oxygen-carrying hybrid protein nanoparticles and free doxorubicin hydrochloride solution;
FIG. 5 is a bar graph showing the survival rate of tumor cells after oxygen-enriched chemotherapy and oxygen-enriched photodynamic combined therapy of a drug-loaded oxygen-carrying hybrid protein nanoparticle solution and a mixed solution of free adriamycin and chlorin-e 6.
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 a first aspect of the invention, the invention provides a preparation method of a drug-loaded oxygen-loaded hybrid protein nanoparticle, which comprises the following steps:
(a) mixing and homogenizing a reducing agent and serum albumin, and carrying out reduction reaction to obtain reduced serum albumin;
(b) the reduced serum albumin, the hemoglobin and the drug are mixed and homogenized under the aerobic condition, and the drug-loaded and oxygen-loaded hybrid protein nanoparticles are prepared.
The preparation method of the drug-loaded oxygen-loaded hybrid protein nanoparticles provided by the invention is simple and easy to implement, is convenient to popularize and apply, 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 hemoglobin are crosslinked to form intermolecular disulfide bonds, so as to form a hybrid protein nano-carrier, and in the disulfide bond reconstruction stage, the hybrid nano-carrier can carry drugs and combine oxygen to form drug-carrying and oxygen-carrying hybrid protein nanoparticles.
In the present invention, the term "mixing and homogenizing" means mixing and then homogenizing.
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, preferably, the serum albumin is derived from human and cattle, and more preferably, the serum albumin is recombinant human serum albumin obtained by biological fermentation.
The hemoglobin is derived from animals and can be extracted from animal blood, and when the hemoglobin is extracted from the blood of animals such as human, cattle, pigs and the like, the prepared drug-loaded oxygen-loaded hybrid protein nanoparticles have 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 prepared drug-loading oxygen-carrying hybrid protein nanoparticles have better in-vivo stability, and when the serum albumin is recombinant human serum albumin obtained by adopting a biological fermentation mode, the prepared drug-loading oxygen-carrying hybrid protein nanoparticles have better in-vivo stability.
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 invention, the drug is a chemotherapeutic drug and/or a photosensitizer;
preferably, the chemotherapeutic drug is selected from at least one of doxorubicin hydrochloride, methotrexate or hypericin;
preferably, the photosensitizer is selected from at least one of indocyanine green, hematoporphyrin, protoporphyrin or chlorin-e 6.
The drug-loaded oxygen-carrying hybrid protein nanoparticles provided by the invention carry chemotherapeutic drugs and/or photosensitizers and oxygen to release oxygen, chemotherapeutic drugs and/or photosensitizers in the anoxic tumor, improve tumor hypoxia and improve the enrichment of the chemotherapeutic drugs and/or photosensitizers in the tumor, thereby enhancing the treatment effect of the tumor.
In a typical but non-limiting embodiment of the invention, the chemotherapeutic is doxorubicin, methotrexate or hypericin hydrochloride, a mixture of any two of doxorubicin, methotrexate and hypericin hydrochloride, and a mixture of doxorubicin, methotrexate and hypericin hydrochloride.
In a typical but non-limiting embodiment of the invention, the photosensitizer is indocyanine green, hematoporphyrin, protoporphyrin or chlorin-e 6, may also be a mixture of any two of indocyanine green, hematoporphyrin, protoporphyrin and chlorin-e 6, and may also be a mixture of indocyanine green, hematoporphyrin, protoporphyrin and chlorin-e 6.
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: (5-10);
preferably, 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 hemoglobin to the serum albumin to be 1: (2-50), serum albumin provides abundant protection for hemoglobin to improve the stability of medicine carrying oxygen-carrying hybrid protein nanoparticle in vivo, prolong the circulation time of medicine carrying oxygen-carrying hybrid protein nanoparticle in vivo, be 1 when the mass ratio of hemoglobin and serum albumin: (3-15), the prepared drug-loaded oxygen-loaded hybrid protein nanoparticles have good stability in vivo, long circulation time, large oxygen loading capacity and large drug loading capacity, and particularly when the mass ratio of hemoglobin to serum albumin is 1: (0.15-0.3), the stability in vivo is better, the circulation time is longer, and the oxygen carrying capacity and the drug carrying capacity are larger.
By controlling the mass ratio of the serum albumin to the reducing agent to be 1: (0.1-1) in order that the reducing reaction of the reducing agent and the serum albumin is sufficiently carried out, the disulfide bonds in the serum albumin molecules can be broken, and the generated reduced serum albumin; in particular, the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.5), the reduction reaction of the 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 serum albumin reduction reaction proceeds more 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 pH value of the mixed solution of the reduced serum albumin and the hemoglobin is controlled to be 7-9 so as to promote the generation of the drug-loaded oxygen-carrying hybrid protein nanoparticles, and the pH value can be adjusted by sodium bicarbonate in the process of mixing and homogenizing so as to keep the pH value of the mixed solution at 7-9.
The time of mixing and homogenizing is controlled to be 10-180 minutes, so that the reaction of the reduced serum albumin and the hemoglobin is more sufficient, and the yield of the drug-loaded oxygen-loaded hybrid protein nanoparticles 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 drug-loaded oxygen-loaded hybrid protein nanoparticles is improved by controlling the concentration of the reduced serum albumin to be 0.5-3%.
In a preferred embodiment of the present invention, in step (b), the reduced serum albumin, the hemoglobin and the drug are first mixed and homogenized under aerobic conditions, then the precipitant is added and mixed uniformly, and finally the precipitant, the free protein and the drug are removed through vacuum drying, ultrafiltration or dialysis, so as to obtain the drug-loaded and oxygen-loaded hybrid protein nanoparticles.
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 carried out more fully, and the yield of the drug-loaded oxygen-loaded hybrid protein nanoparticles is improved.
And finally, removing the precipitant, free protein and the medicament by vacuum drying, ultrafiltration or dialysis so as to purify the medicament-carrying oxygen-carrying hybrid protein nanoparticles, 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 and drug substances are removed by ultrafiltration or dialysis.
In a preferred embodiment of the present invention, the volume ratio of the mixed solution of reduced serum albumin, hemoglobin and drug to the precipitant is 1: (1-2.5);
preferably, the mixing time of the reduced serum albumin, hemoglobin and drug mixed solution and the precipitant is 0.5-12 hours.
By controlling the volume ratio of the mixed solution of the reduced serum albumin, the hemoglobin and the medicament to the precipitator to be 1: (1-2.5) so that the precipitant is fully mixed with the mixed solution of the serum albumin, the hemoglobin and the medicament, the solubility of the reduced serum albumin and the hemoglobin is reduced, and the crosslinking reaction of the two proteins is fully performed.
By controlling the mixing time of the reduced serum albumin, the hemoglobin and the medicine mixed solution and the precipitator to be 0.5-12 hours, the precipitator can be uniformly mixed with the reduced serum albumin and hemoglobin mixed solution, the solubility of the reduced serum albumin and the hemoglobin is reduced, and the cross-linking reaction of the two proteins is more sufficient.
According to a second aspect of the invention, the invention provides a drug-loaded and oxygen-loaded hybrid protein nanoparticle prepared according to the drug-loaded and oxygen-loaded hybrid protein nanoparticle.
The drug-loaded oxygen-carrying hybrid protein nanoparticle provided by the invention has the advantages that hemoglobin and serum albumin are covalently combined through disulfide bonds, a drug is loaded, oxygen is combined, so that the drug simultaneously has the tumor targeting function of the serum albumin and the oxygen-carrying function of the hemoglobin, oxygen and the drug can be delivered to the interior of a tumor in a targeted manner, the enrichment of the drug in the interior of the tumor is improved, the reversible release of oxygen molecules can be carried out according to the oxygen concentration in a microenvironment, the tissue with normal oxygen concentration cannot be influenced, no side effect exists, the tumor hypoxia can be effectively improved, the treatment effect of the tumor is enhanced, the integration of tumor oxygenation and drug delivery is realized, and the bioavailability of the drug is improved. In addition, the drug-loaded oxygen-loaded hybrid protein nanoparticle provided by the invention has a stable structure and long storage time, and is convenient to popularize and apply.
According to a third aspect of the invention, the invention provides application of the drug-loaded oxygen-carrying hybrid protein nanoparticles for preparing tumor treatment drugs.
The drug-loaded oxygen-carrying hybrid protein nanoparticles provided by the invention have the tumor targeting function of serum albumin and the oxygen carrying function of hemoglobin, are coated with drugs, are combined with oxygen, can deliver oxygen and drugs into tumors in a targeted manner, release oxygen and drugs in anoxic tumors, improve the tumor hypoxia, simultaneously improve the drug enrichment and enhance the treatment effect of the tumors.
The technical solution provided by the present invention is further described below with reference to examples and comparative examples.
Example 1
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 10mg of hemoglobin and 0.8mg of doxorubicin hydrochloride in 2mL of purified water under aerobic condition, 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 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 drug-loaded oxygen-loaded hybrid protein nanoparticle solution loaded with the doxorubicin hydrochloride.
Example 2
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 0.4mg of hemoglobin and 0.8mg of doxorubicin hydrochloride in 2mL of purified water under aerobic condition, 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 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 drug-loaded oxygen-loaded hybrid protein nanoparticle solution loaded with the doxorubicin hydrochloride.
Example 3
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 6.7mg of hemoglobin and 0.8mg of doxorubicin hydrochloride in 2mL of purified water under an aerobic condition, 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 drug-loaded oxygen-loaded hybrid protein nanoparticle solution loaded with the doxorubicin hydrochloride.
Example 4
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 1.3mg of hemoglobin and 0.8mg of doxorubicin hydrochloride in 2mL of purified water under aerobic condition for 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 drug-loaded oxygen-loaded hybrid protein nanoparticle solution loaded with the doxorubicin hydrochloride.
Example 5
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 4mg of hemoglobin and 0.8mg of doxorubicin hydrochloride in 2mL of purified water under aerobic condition, 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, then filling the solution into a dialysis bag with the molecular weight cutoff of 300kD, and dialyzing for 12 hours to obtain the drug-loaded oxygen-carrying hybrid protein nanoparticle solution coated with the doxorubicin hydrochloride.
Example 6
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 3mg of hemoglobin and 0.8mg of doxorubicin hydrochloride in 2mL of purified water under aerobic condition, 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, then filling the solution into a dialysis bag with the molecular weight cutoff of 300kD, and dialyzing for 12 hours to obtain the drug-loaded oxygen-carrying hybrid protein nanoparticle solution coated with the doxorubicin hydrochloride.
Example 7
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 3.2mg of hemoglobin and 0.8mg of doxorubicin hydrochloride in 2mL of purified water under aerobic condition, 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 5 hours, filling the mixed solution into a dialysis bag with the molecular weight cutoff of 100kD, and dialyzing for 12 hours to obtain the drug-loaded oxygen-loaded hybrid protein nanoparticles loaded with the doxorubicin hydrochloride.
Example 8
The present example provides a drug-loaded oxygen-carrying hybrid protein nanoparticle, and the difference between the present example and example 7 is that in step (a), the amount of glutathione is 10 mg.
Example 9
The present example provides a drug-loaded oxygen-carrying hybrid protein nanoparticle, and the difference between the present example and example 7 is that, in step (b), the amount of hemoglobin used is 0.2 mg.
Example 10
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 3.2mg of hemoglobin and 0.8mg of chlorin-e 6 in 2mL of purified water together under aerobic condition, 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, filling the solution into a dialysis bag with the molecular weight cutoff of 100kD, and dialyzing for 12 hours to obtain the medicine-carrying oxygen-carrying hybrid protein nanoparticles carrying the chlorin-e 6.
Example 11
The embodiment provides a drug-loaded oxygen-loaded hybrid protein nanoparticle, 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, 3.2mg of hemoglobin, 0.4mg of doxorubicin hydrochloride and 0.4mg of chlorin-e 6 in 2mL of purified water under aerobic condition, 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 4 hours, filling the mixed solution into a dialysis bag with the molecular weight cutoff of 100kD, and dialyzing for 12 hours to obtain the drug-loaded oxygen-loaded hybrid protein nanoparticles containing the doxorubicin hydrochloride and the chlorin-e 6.
Test example 1
The particle size distribution of the drug-loaded oxygen-loaded hybrid protein nanoparticles provided in examples 1 to 12 was determined by a dynamic light scattering analysis method, and the results show that the particle sizes of the drug-loaded oxygen-loaded hybrid protein nanoparticles provided in examples 1 to 12 were all 20 to 200 nm. Fig. 1 is a distribution diagram of the particle size of the drug-loaded oxygen-loaded hybrid nanoparticle coated with doxorubicin hydrochloride provided in example 7, and as can be seen from fig. 1, the particle size of the drug-loaded oxygen-loaded hybrid protein nanoparticle coated with doxorubicin hydrochloride provided in example 7 is 20-50 nm.
Test example 2
The drug-loaded oxygen-carrying hybrid protein nanoparticles provided in example 10 were injected into tumor-loaded nude mice, and photoacoustic signals of oxygenated hemoglobin (HbO2) at tumor sites were monitored by photoacoustic imaging equipment (excitation light 850 nm) to determine the oxygenation of tumors. FIG. 2 is a photo-acoustic imaging monitoring chart of the change of the oxygenated hemoglobin content at the tumor site after the tumor-bearing nude mouse is injected with the drug-loaded oxygen-loaded hybrid protein nanoparticles; as can be seen from fig. 2, after the drug-loaded oxygen-loaded hybrid protein nanoparticles are injected into tumor-bearing nude mice for 2 hours, the content of oxygenated hemoglobin in the tumor site is significantly increased, the hypoxic environment can be maintained for 6 hours all the time, and the tumor site is restored to the hypoxic state after 24 hours, which indicates that the drug-loaded oxygen-loaded hybrid protein nanoparticles loaded with chlorin-e 6 provided by embodiment 10 of the present invention have targeting property, can penetrate through blood vessels, are enriched in the tumor site, and release oxygen inside hypoxic tumors, so as to enhance the tumor treatment effect.
Test example 3
Two tumor-bearing nude mice were selected, one of the two tumor-bearing nude mice was injected with the drug-loaded oxygen-loaded hybrid protein nanoparticles entrapped with chlorin-e 6 provided in example 10, and the other one was injected with the same dose of free chlorin-e 6 solution. Fluorescence monitoring is carried out on two nude mice by a fluorescence imaging device, and fig. 3 is a fluorescence imaging contrast diagram of a tumor-bearing nude mouse injected with the drug-loaded oxygen-loaded hybrid protein nanoparticles for 6 hours and a tumor-bearing nude mouse injected with the free chlorin-e 6 solution for 6 hours, wherein the hybrid protein nano-drug refers to the drug-loaded oxygen-loaded hybrid protein nanoparticles encapsulated with chlorin-e 6 provided in example 10; as can be seen from fig. 3, after 6 hours of injection, the chlorin-e 6 enriched in the tumor part of the tumor-bearing nude mouse injected with the drug-loaded oxygen-carrying nanoparticle provided in example 10 is significantly higher than that of the tumor-bearing nude mouse injected with the free chlorin-e 6 solution, which indicates that the drug-loaded oxygen-carrying hybrid protein nanoparticle provided in example 10 of the present invention has targeting property, can penetrate through blood vessels, is enriched in tumor sites, has targeting property significantly higher than that of the free drug, and can enhance the therapeutic effect of tumors.
Test example 4
The doxorubicin hydrochloride-loaded oxygen-carrying hybrid protein nanoparticles and the free doxorubicin hydrochloride provided in example 7 were prepared into solutions with doxorubicin hydrochloride concentrations of 0.2 μ g/mL, 0.4 μ g/mL, 0.6 μ g/mL, 0.8 μ g/mL and 1 μ g/mL, respectively, and then the solutions were incubated with human breast cancer cells (MCF-7) cultured under anaerobic conditions for 2 hours, after the incubation was completed, the tumor cells were cultured for further 24 hours, and the cell survival rate was determined. FIG. 4 is a histogram comparing the survival rates of tumor cells after chemotherapy with drug-loaded and oxygen-loaded hybrid protein nanoparticles and free doxorubicin hydrochloride solution, wherein the hybrid protein nano-drug is the drug-loaded and oxygen-loaded hybrid protein nanoparticles coated with doxorubicin hydrochloride provided in example 7; as can be seen from fig. 4, the drug-loaded and oxygen-loaded hybrid protein nanoparticles coated with doxorubicin hydrochloride provided in example 7 can effectively kill tumor cells and reduce the survival rate of tumor cells, and the killing effect on tumor cells is more obvious with the increase of the concentration of the coated doxorubicin hydrochloride, when the concentration of the doxorubicin hydrochloride coated with the drug-loaded and oxygen-loaded hybrid protein nanoparticles is 1 μ g/mL, the survival rate of tumor cells is less than 5%, and the free doxorubicin hydrochloride solution has no obvious killing effect on tumor cells, and even when the concentration of the free doxorubicin hydrochloride solution is 1 μ g/mL, the survival rate of tumor cells is still more than 95%. This shows that the oxygen-and drug-loaded hybrid protein nanoparticles coated with doxorubicin hydrochloride provided in embodiment 7 of the present invention can effectively kill tumor cells, reduce the survival rate of tumor cells, and enhance the treatment effect of tumors by targeted therapy and oxygen-enhanced chemotherapy of tumor cells.
Test example 5
The drug-loaded oxygen-carrying hybrid protein nanoparticles loaded with doxorubicin hydrochloride and chlorin-e 6 provided in example 11 were configured into drug-loaded oxygen-carrying hybrid protein nanoparticle solutions with doxorubicin/chlorin-e 6 concentrations of 0.2/0.21. mu.g/mL, 0.4/0.42. mu.g/mL, 0.6/0.64. mu.g/mL, 0.8/0.85. mu.g/mL and 1/1.05. mu.g/mL, and the drug-loaded hybrid protein nanoparticle solutions were incubated with human breast cancer cells (MCF-7) cultured under anaerobic conditions for 2 hours while using a 660 nm laser (power of 100 mW/cm)2) Tumor cell viability was determined by irradiating tumor cells and then culturing the tumor cells for 24 hours, and the results are shown in FIG. 5.
Doxorubicin hydrochloride and chlorin-e 6 were mixed to prepare a mixed solution of doxorubicin hydrochloride/chlorin-e 6 at a concentration of 0.2/0.21 μ g/mL, 0.4/0.42 μ g/mL, 0.6/0.64 μ g/mL, 0.8/0.85 μ g/mL, and 1/1.05 μ g/mL free doxorubicin and chlorin-e 6, and the mixed solution of free doxorubicin and chlorin-e 6 was incubated with human breast cancer cells (MCF-7) cultured under hypoxic conditions for 2 hours while applying a 660 nm laser (power 100 mW/cm)2) Tumor cell viability was determined by irradiating tumor cells and then culturing the tumor cells for an additional 24 hours. The test results are shown in fig. 5.
FIG. 5 is a histogram comparing survival rates of tumor cells after oxygen-enriched chemotherapy and oxygen-enriched photodynamic combined therapy of a drug-loaded and oxygen-loaded hybrid protein nanoparticle solution and a mixed solution of free adriamycin and chlorin-e 6, wherein the hybrid protein nano-drug refers to the drug-loaded and oxygen-loaded hybrid protein nanoparticle loaded with adriamycin hydrochloride and chlorin-e 6 provided in example 11; as can be seen from fig. 5, the oxygen-carrying drug-loaded hybrid protein nanoparticles loaded with doxorubicin hydrochloride and chlorin-e 6 provided in embodiment 11 of the present invention have significantly improved killing effect on tumor cells with the increase of the concentration of doxorubicin hydrochloride/chlorin-e 6, and the survival rate of tumor cells is already less than 5% when the concentration of the doxorubicin hydrochloride/chlorin-e 6 loaded with the oxygen-carrying drug-loaded hybrid protein nanoparticles is 0.2/0.21 μ g/mL, whereas in the mixed solution of free doxorubicin hydrochloride and chlorin-e 6, the survival rate of tumor cells is still higher than 50% when the concentration of the doxorubicin hydrochloride/chlorin-e 6 is 1/1.06 μ g/mL, which indicates that the oxygen-carrying drug-loaded hybrid protein nanoparticles loaded with doxorubicin hydrochloride and chlorin-e 6 provided in embodiment 11 of the present invention are used for oxygen-enriched and photodynamic therapy on tumor cells, the killing power to the tumor cells can be obviously improved, the survival rate of the tumor cells can be reduced, and the strong killing effect to the tumor cells and the treatment effect of the tumor can be enhanced when the medicine concentration is lower.
Test example 6
The drug-loaded oxygen-carrying hybrid protein nanoparticles coated with doxorubicin hydrochloride provided in examples 1 to 9 were subjected to drug encapsulation efficiency and oxygen affinity P50 value measurement, and the measurement results are shown in table 1 below:
TABLE 1 Performance data sheet of drug-loaded oxygen-carrying hybrid protein nanoparticles
As can be seen from Table 1, the comparison between examples 1-7 and examples 8-9 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 drug-loaded oxygen-carrying hybrid protein nanoparticles is obviously improved, the drug-loaded capacity is obviously increased, the oxygen affinity P50 value is higher than 13mmHg, and the drug-loaded capacity is higher than 2.5 percent
As can be seen by comparing examples 1-7, examples 5-7 have the highest value of oxygen affinity, P50, and examples 3-4 have oxygen and drug loadings lower than those of examples 5-7 but higher than those of 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 drug-loaded oxygen-loaded hybrid protein nanoparticles have strong oxygen affinity P50 value and high drug-loading rate, and when the mass ratio of hemoglobin to 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 drug-loaded oxygen-loaded hybrid protein nanoparticles have higher oxygen affinity P50 value and higher drug-loading rate.
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. A preparation method of drug-loaded oxygen-loaded hybrid protein nanoparticles is characterized by comprising the following steps:
(a) mixing and homogenizing a reducing agent and serum albumin, carrying out reduction reaction, and breaking disulfide bonds in serum albumin molecules to obtain random reduced serum albumin;
(b) mixing reduced serum albumin, hemoglobin and a drug uniformly under an aerobic condition, wherein reduced serum albumin molecules are crosslinked with free sulfydryl of the hemoglobin to form intermolecular disulfide bonds to form a hybrid protein nano-carrier, and the hybrid protein nano-carrier can entrap the drug and combine oxygen to form drug-loaded and oxygen-loaded hybrid protein nanoparticles in a disulfide bond reconstruction stage;
the mass ratio of the hemoglobin to the serum albumin is 1: (2-50), wherein the mass ratio of the serum albumin to the reducing agent is 1: (0.1-1).
2. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 1, wherein the hemoglobin is derived from animals.
3. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 2, wherein the hemoglobin is derived from human, bovine or porcine.
4. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 1, wherein the serum albumin is derived from animals or obtained by biological fermentation.
5. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 4, wherein the serum albumin is derived from human or bovine.
6. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 4, wherein the serum albumin is recombinant human serum albumin obtained by biological fermentation.
7. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 1, wherein the reducing agent is at least one selected from glutathione, dithiothreitol, cysteine or homocysteine.
8. The preparation method of the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 1, wherein the drug is a chemotherapeutic drug and/or a photosensitizer;
the chemotherapeutic drug is selected from at least one of adriamycin hydrochloride, methotrexate or hypericin;
the photosensitizer is at least one of indocyanine green, hematoporphyrin, protoporphyrin or chlorin-e 6.
9. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 1, wherein the mass ratio of the hemoglobin to the serum albumin is 1: (3-15), the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.5).
10. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 9, wherein the mass ratio of hemoglobin to serum albumin is 1: (5-10); the mass ratio of the serum albumin to the reducing agent is 1: (0.15-0.3).
11. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 1, wherein in the step (b), the pH value of the mixed solution of reduced serum albumin, hemoglobin and drug is 7-9;
mixing and homogenizing for 10-180 min;
the concentration of reduced serum albumin is 0.5-3%.
12. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to any one of claims 1 to 11, wherein in step (b), the reduced serum albumin, the hemoglobin and the drug are first mixed and homogenized under aerobic conditions, then the precipitant is added and homogenized, and finally the precipitant, the free protein and the drug are removed by vacuum drying, ultrafiltration or dialysis, thereby obtaining the drug-loaded oxygen-carrying hybrid protein nanoparticles.
13. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 12, wherein the precipitating agent is anhydrous lower alcohol.
14. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 13, wherein the precipitating agent is absolute ethanol.
15. The method for preparing the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 12, wherein the volume ratio of the mixed solution of reduced serum albumin, hemoglobin and drug to the precipitant is 1: (1-2.5);
the mixing time of the mixed solution of the reduced serum albumin, the hemoglobin and the medicine and the precipitator is 0.5 to 12 hours.
16. A drug-loaded oxygen-loaded hybrid protein nanoparticle, which is prepared from the drug-loaded oxygen-loaded hybrid protein nanoparticle according to any one of claims 1 to 15.
17. The use of the drug-loaded oxygen-carrying hybrid protein nanoparticles according to claim 16 for the preparation of a medicament for the treatment of tumors.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1767851A (en) * | 2003-04-09 | 2006-05-03 | 苏志国 | Hemoglobin conjugate and its preparing method and use |
| WO2011069082A2 (en) * | 2009-12-04 | 2011-06-09 | The Regents Of The University Of Michigan | Red blood cell-mimetic particles and methods for making and use thereof |
| CN102988996A (en) * | 2012-12-19 | 2013-03-27 | 清华大学 | Method for preparing stable albumin nanoparticle |
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-
2017
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1767851A (en) * | 2003-04-09 | 2006-05-03 | 苏志国 | Hemoglobin conjugate and its preparing method and use |
| WO2011069082A2 (en) * | 2009-12-04 | 2011-06-09 | The Regents Of The University Of Michigan | Red blood cell-mimetic particles and methods for making and use thereof |
| CN102988996A (en) * | 2012-12-19 | 2013-03-27 | 清华大学 | Method for preparing stable albumin nanoparticle |
Non-Patent Citations (2)
| Title |
|---|
| Activatable albumin-photosensitizer nanoassemblies for triple-modal imaging and thermal-modulated photodynamic therapy of cancer;Dehong Hu，et al.;《Biomaterials》;20160331;第93卷;第10-19页 * |
| 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233-240页 * |
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