CN107952072A - Carry the preparation method of medicine oxygen carrier hybrid protein nanoparticle, carry medicine oxygen carrier hybrid protein nanoparticle and application - Google Patents
Carry the preparation method of medicine oxygen carrier hybrid protein nanoparticle, carry medicine oxygen carrier hybrid protein nanoparticle and application Download PDFInfo
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- 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
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Abstract
The present invention provides a kind of preparation method for carrying medicine oxygen carrier hybrid protein nanoparticle, medicine oxygen carrier hybrid protein nanoparticle and application are carried, is related to biopharmaceutical technology, includes the following steps：First by reducing agent and seralbumin mixing homogeneous, reduction reaction is carried out, obtains reduced form seralbumin；Again by reduced form seralbumin, hemoglobin and medicine mix homogeneous under aerobic conditions, it is made and carries medicine oxygen carrier hybrid protein nanoparticle, existing suction hyperbaric oxygen mode is alleviated to supply oxygen, oxygen can not be transported to inside tumor, the oxygen of high concentration can also make lung and central nervous system that the technical problem of oxygen poisoning occur at the same time, reached carry the targeting of medicine oxygen carrier hybrid protein nanoparticle by oxygen and drug delivery to inside tumor, improve drug-rich, and can be according to the oxygen concentration under residing microenvironment, carry out the reversible release of oxygen molecule, it is and without side-effects, it is integrated with medicine delivery to realize tumour oxygenation, improve the technique effect of the bioavailability of medicine.
Description
Technical field
The present invention relates to biopharmaceutical technology, more particularly, to a kind of preparation for carrying medicine oxygen carrier hybrid protein nanoparticle
Method, carry medicine oxygen carrier hybrid protein nanoparticle and application.
Background technology
Entity tumor shows the micro-loop of anoxic due to chaotic and complicated blood vessel structure and vigorous cell metabolism
Border feature, research have shown that tumor hypoxia causes the special microenvironment of tumour, have impact on angiogenesis, the cell of tumor tissues
Division etc. process, cause it is insensitive to chemotherapeutics and radiotherapy, or even also result in oncogene mutation and metastases.
In photodynamic therapy, oxygen has certain influence as reactant for photodynamic therapy effect, and tumor hypoxia limits
The generation of optical dynamic therapy active oxygen, have impact on therapeutic effect.
Clinical treatment uses the mode of suction hyperbaric oxygen to increase the oxygen content of tumor region at present, and obtains tumour to putting
Penetrate the effect of enhanced sensitivity for the treatment of, chemotherapy and optical therapeutic.Hyperbaric oxygen ation is supplied oxygen by red blood cell in blood, but red blood cell without
Method penetrates blood vessel and oxygen is transported to inside tumor, and tumor vascular irregular growth limits oxygen and medicine reaches tumour.
Meanwhile the oxygen of high concentration can also make lung and central nervous system that oxygen poisoning occur.Therefore, those skilled in the art need development one
Kind there is cancer target and oxygen carrier, can be and without side-effects by the inside tumor of oxygen and high-efficiency delivery of medicament to anoxic
Therapeutic scheme, to meet the needs of oncotherapy.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the present invention to providing a kind of preparation method for carrying medicine oxygen carrier hybrid protein nanoparticle, to alleviate mesh
Preclinical therapy is supplied oxygen by the way of hyperbaric oxygen is sucked by the red blood cell in blood, but red blood cell can not penetrate blood vessel and incite somebody to action
Oxygen is transported to inside tumor, and tumor vascular irregular restriction of production oxygen and medicine reach tumour, while the oxygen of high concentration
It can also make lung and central nervous system that the technical problem of oxygen poisoning occur.
The present invention provides a kind of preparation method for carrying medicine oxygen carrier hybrid protein nanoparticle, include the following steps：
(a) by reducing agent and seralbumin mixing homogeneous, reduction reaction is carried out, obtains reduced form seralbumin；
(b) reduced form seralbumin, hemoglobin and medicine are mixed into homogeneous under aerobic conditions, that is, is made and carries medicine load
Oxa- hands over protein nano grain.
Further, the hemoglobin derives from animal, it is preferable that the hemoglobin derives from people, ox or pig；
Preferably, the seralbumin is obtained from animal or using biofermentation mode, it is further preferred that institute
State seralbumin and derive from people or ox, it is further preferred that the seralbumin is the weight that biological fermentation method obtains
Group human serum albumins.
Further, the reducing agent is in glutathione, dithiothreitol (DTT), cysteine or homocysteine
It is at least one.
Further, the medicine is chemotherapeutics and/or photosensitizer；
Preferably, the chemotherapeutics is selected from least one of doxorubicin hydrochloride, methotrexate (MTX) or hypericin；
Preferably, the photosensitizer in indocyanine green, haematoporphyrin, protoporphyrin or chlorin-e6 at least one
Kind.
Further, hemoglobin and sero-abluminous mass ratio are 1：(2-50), is preferably 1：(3-15), more preferably
For 1：(5-10)；
Preferably, the mass ratio of the seralbumin and reducing agent is 1：(0.1-1), is preferably 1：(0.15-0.5),
More preferably 1：(0.15-0.3).
Further, in step (b), the pH value of the mixed solution of reduced form seralbumin, hemoglobin and medicine
For 7-9；
Preferably, it is 10-180 minutes to mix homogenizing time；
Preferably, the sero-abluminous concentration of reduced form is 0.5-3%.
Further, in step (b), first by reduced form seralbumin and hemoglobin and medicine under aerobic conditions
Homogeneous is mixed, then addition precipitant mix is uniform, finally by vacuum drying, ultrafiltration or dialyses precipitating reagent and floating preteins
And medicine removes, you can is made and carries medicine oxygen carrier hybrid protein nanoparticle；Preferably, the precipitating reagent is anhydrous low-carbon alcohol, more excellent
Elect absolute ethyl alcohol as.
Further, the mixed solution of reduced form seralbumin, hemoglobin and medicine and the volume ratio of precipitating reagent are
1：(1-2.5)；
Preferably, reduced form seralbumin, hemoglobin and the incorporation time of medicine mixed solution and precipitating reagent are
When 0.5-12 is small.
The second object of the present invention is to provide a kind of preparation method according to above-mentioned load medicine oxygen carrier hybrid protein nanoparticle
The load medicine oxygen carrier hybrid protein nanoparticle being prepared.
The third object of the present invention is that providing above-mentioned load medicine oxygen carrier hybrid protein nanoparticle is preparing anti-tumor medicine
In application.
The preparation method provided by the invention for carrying medicine oxygen carrier hybrid protein nanoparticle, it is simple and easy to do, it is easy to utilize, fit
For preparing on a large scale.
Load medicine oxygen carrier hybrid protein nanoparticle provided by the invention, passes through disulfide bond by hemoglobin and seralbumin
Covalent bond, contains medicine, with reference to oxygen, it is provided simultaneously with the load of sero-abluminous tumor-targeting function and hemoglobin
Oxygen function, can target by oxygen and drug delivery to inside tumor, improve enrichment of the medicine in inside tumor, and being capable of root
According to the oxygen concentration under residing microenvironment, the reversible release of oxygen molecule is carried out, does not interfere with the tissue of normal oxygen concentratio, no secondary work
With can be effectively improved tumor hypoxia, strengthen the therapeutic effect of tumour, realize that tumour oxygenation is integrated with medicine delivery, carry
The high bioavailability of medicine.In addition load medicine oxygen carrier hybrid protein nanoparticle structure provided by the invention is stablized, storage time
It is long, it is easy to be extended and applied.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
Put, other attached drawings can also be obtained according to these attached drawings.
Fig. 1 is the grain size distribution for the load medicine oxygen carrier hybridization nanometer grain that the embodiment of the present invention 7 provides；
Fig. 2 is the oxygen-containing hemoglobin changes of contents of tumor locus after tumor bearing nude mice injection load medicine oxygen carrier hybrid protein nanoparticle
Photoacoustic imaging monitoring figure；
Fig. 3 be injection carry medicine oxygen carrier hybrid protein nanoparticle 6 it is small when after tumor bearing nude mice and the free chlorin-e6 of injection
The fluorescence imaging comparison diagram of tumor bearing nude mice after when solution 6 is small；
Fig. 4 is that the tumour cell after load medicine oxygen carrier hybrid protein nanoparticle and free hydrochloric acid Doxorubicin solution progress chemotherapy is deposited
Motility rate contrasts block diagram；
Fig. 5 is increasing to carry medicine oxygen carrier hybrid protein nanoparticle solution and free adriamycin and chlorin-e6 mixed solutions
The survival rate contrast block diagram with tumour cell after oxygenation light power link treatment is treated in oxidation.
Embodiment
Technical scheme will be clearly and completely described below, it is clear that described embodiment is this hair
Bright part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
All other embodiments obtained under the premise of creative work are made, belong to the scope of protection of the invention.
According to the first aspect of the invention, the present invention provides a kind of preparation side for carrying medicine oxygen carrier hybrid protein nanoparticle
Method, includes the following steps：
(a) by reducing agent and seralbumin mixing homogeneous, reduction reaction is carried out, obtains reduced form seralbumin；
(b) reduced form seralbumin, hemoglobin and medicine are mixed into homogeneous under aerobic conditions, that is, is made and carries medicine load
Oxa- hands over protein nano grain.
The preparation method provided by the invention for carrying medicine oxygen carrier hybrid protein nanoparticle, it is simple and easy to do, it is easy to utilize, fit
For preparing on a large scale.
In step (a), reduction reaction is carried out by reducing agent and seralbumin, seralbumin is intramolecular
Disulfide bond disconnects, and obtains the reduced form seralbumin molecule of disorganized form.In step (b), reduced form seralbumin point
Son is crosslinked with the free sulfhydryl groups of hemoglobin, is reconstructed into intermolecular disulfide bond, formation hybrid protein nano-carrier, and
Disulfide bond reconstruction stage, hybridization nanometer carrier can contain medicine and combine oxygen, formed and carry medicine oxygen carrier hybrid protein nanoparticle.
In the present invention, mixing homogeneous carries out homogeneous again after referring to mixing.
In the preferred embodiment of the present invention, hemoglobin derives from animal, it is preferable that the hemoglobin comes
Come from people, ox or pig；Preferably, seralbumin is obtained from animal or using biofermentation mode, it is preferable that the blood
Pure albumen source is in people and Niu, it is highly preferred that the seralbumin is the recombinant human serum albumin that biological fermentation method obtains
Albumen.
Hemoglobin derives from animal, can extract and obtain from animal blood, when hemoglobin is dynamic from people, ox and pig etc.
When being extracted in thing blood, made carries the stability of medicine oxygen carrier hybrid protein nanoparticle in vivo more preferably.Hemoglobin
Including hemoglobin and the like molecule, the hemoglobin raw material hypotype such as including HbA, HbA2, HbF.
Seralbumin is extracted from animal blood to be obtained or is obtained by biofermentation mode.When seralbumin be from
When the seralbumin extracted in animal blood, made carries the internal of medicine oxygen carrier hybrid protein nanoparticle for people, ox
Stability is more preferable, when seralbumin is the recombination human serum albumin obtained using biofermentation mode, made load
The internal stability of medicine oxygen carrier hybrid protein nanoparticle is more preferably.
In a kind of typical but non-limiting embodiment of the present invention, reducing agent is selected from glutathione, two sulphur threoses
At least one of alcohol, cysteine or homocysteine.
In the typical but non-limiting embodiment of invention, reducing agent can be glutathione, dithiothreitol (DTT), half
One kind in cystine and homocysteine, or glutathione, half Guang ammonia of dithiothreitol (DTT), cysteine and homotype
Any two kinds of mixture in acid, such as mixing for the mixture of glutathione and dithiothreitol (DTT), dithiothreitol (DTT) and cysteine
Mixture of compound or cysteine and homocysteine etc., can also be glutathione, dithiothreitol (DTT), cysteine and
The mixture or paddy Guang of any three kinds of mixture in homocysteine, such as sweet peptide of Gu Guang, dithiothreitol (DTT) and cysteine
Mixture of sweet peptide, second-rate threitol and homocysteine etc., or glutathione, dithiothreitol (DTT), cysteine
With the mixture of four kinds of materials of homocysteine.
In the preferred embodiment of the present invention, medicine is chemotherapeutics and/or photosensitizer；
Preferably, chemotherapeutics is selected from least one of doxorubicin hydrochloride, methotrexate (MTX) or hypericin；
Preferably, photosensitizer is selected from least one of indocyanine green, haematoporphyrin, protoporphyrin or chlorin-e6.
Load medicine oxygen carrier hybrid protein nanoparticle provided by the invention is by carrying chemotherapeutics and/or photosensitizer and oxygen
Gas, to discharge oxygen, chemotherapeutics and/or photosensitizer inside hypoxic tumor, improve tumor hypoxia and improve chemotherapeutics and/
Or photosensitizer is in the enrichment of inside tumor, so as to strengthen the therapeutic effect of tumour.
The present invention typical but non-limiting embodiment in, chemotherapeutics for doxorubicin hydrochloride, methotrexate (MTX) or
Hypericin, or any two kinds of mixture in doxorubicin hydrochloride, methotrexate (MTX) and hypericin, can also be hydrochloric acid
The mixture of adriamycin, methotrexate (MTX) and hypericin.
The present invention typical but non-limiting embodiment in, photosensitizer for indocyanine green, haematoporphyrin, protoporphyrin or
Chlorin-e6, or any two kinds of mixture in indocyanine green, haematoporphyrin, protoporphyrin and chlorin-e6, also
It can be the mixture of indocyanine green, haematoporphyrin, protoporphyrin and chlorin-e6.
In the preferred embodiment of the present invention, hemoglobin and sero-abluminous mass ratio are 1：(2-50),
Preferably 1：(3-15), more preferably 1：(5-10)；
Preferably, the mass ratio of seralbumin and reducing agent is 1：(0.1-1), is preferably 1：(0.15-0.5), it is more excellent
Elect 1 as：(0.15-0.3).
It is 1 by controlling hemoglobin and sero-abluminous mass ratio：(2-50), seralbumin carries for hemoglobin
For sufficiently protection, the stability of medicine oxygen carrier hybrid protein nanoparticle in vivo is carried so as to improve, extends and carries medicine oxygen carrier hybridization egg
The white circulation time of nanoparticle in vivo, when hemoglobin and sero-abluminous mass ratio are 1：It is made when (3-15)
It is good to carry the stability of medicine oxygen carrier hybrid protein nanoparticle not only in vivo, circulation time length, and oxygen carrier amount and drugloading rate compared with
Greatly, especially when being hemoglobin and sero-abluminous mass ratio is 1：When between (0.15-0.3), its internal stability is more
Good, circulation time is longer, oxygen carrier amount and drugloading rate bigger.
It is 1 by controlling the mass ratio of seralbumin and reducing agent：(0.1-1), so that reducing agent and the white egg of serum
When white hair gives birth to reduction reaction, abundant, the intramolecular disulfide bond of seralbumin of seralbumin molecule reduction reaction progress
It can disconnect, the reduced form serum of generation is white；Especially the mass ratio of seralbumin and reducing agent is 1：When (0.15-0.5),
Seralbumin reduction reaction carry out more fully, the intramolecular disulfide bond of seralbumin disconnects more, works as serum
The mass ratio of albumin and reducing agent is 1：When (0.15-3), its seralbumin reduction reaction carries out more abundant, generation
Free sulfhydryl groups contained by reduced form seralbumin molecule are more.
In the preferred embodiment of the present invention, in step (b), reduced form seralbumin and hemoglobin
The pH value of mixed solution is 7-9；
Preferably, it is 10-180 minutes to mix homogenizing time；
Preferably, the sero-abluminous concentration of reduced form is 0.5-3%.
It is 7-9 by controlling the pH value of mixed solution of reduced form seralbumin and hemoglobin in step (b),
To promote to carry the generation of medicine oxygen carrier hybrid protein nanoparticle, mix it is homogeneous during, sodium acid carbonate tune can be passed through
PH value is saved, its pH value is maintained at 7-9.
Time by controlling mixing homogeneous is 10-180 minutes, so that reduced form seralbumin and hemoglobin are anti-
More fully it should improve the yield for carrying medicine oxygen carrier hybrid protein nanoparticle.
The above-mentioned sero-abluminous concentration of reduced form refers to reduced form seralbumin in the pure egg of reduced blood and blood red
Concentration in the mixed solution of albumen.It is 0.5-3% by controlling the sero-abluminous concentration of reduced form, medicine oxygen carrier is carried to improve
The preparation efficiency of hybrid protein nanoparticle.
In the preferred embodiment of the present invention, in step (b), first by reduced form seralbumin and blood red egg
Bletilla medicine mixes homogeneous under aerobic conditions, and then addition precipitant mix is uniform, finally by vacuum drying, ultrafiltration or saturating
Analysis removes precipitating reagent and floating preteins and medicine, you can is made and carries medicine oxygen carrier hybrid protein nanoparticle.
In step (b), precipitating reagent is used to reduce hemoglobin and the sero-abluminous solubility of reduced form, is made blood red
Albumen carries out more abundant, the yield of raising load medicine oxygen carrier hybrid protein nanoparticle with reduced form seralbumin cross-linking reaction.
Precipitating reagent and floating preteins and medicine are removed finally by vacuum drying, ultrafiltration or dialysis, with to carrying medicine oxygen carrier
Hybrid protein nanoparticle is purified, and above-mentioned floating preteins includes not carrying out the free seralbumin of cross-linking reaction and free blood
Lactoferrin.
In the typical but non-limiting preferred embodiment of the present invention, precipitating reagent is anhydrous low-carbon alcohol, described anhydrous
Low-carbon alcohols refer to the low-carbon alcohols of C1-C4, it is preferable that the precipitating reagent is absolute ethyl alcohol.
In present invention further optimization embodiment, using ultrafiltration or dialyse precipitating reagent and floating preteins and medicine
During removal, the molecular weight of ultrafiltration or dialysis retention is 100-300kDa.
In the preferred embodiment of the present invention, the mixing of reduced form seralbumin, hemoglobin and medicine is molten
The volume ratio of liquid and precipitating reagent is 1：(1-2.5)；
Preferably, reduced form seralbumin, hemoglobin and the incorporation time of medicine mixed solution and precipitating reagent are
When 0.5-12 is small.
It is 1 by controlling the mixed solution of reduced form seralbumin, hemoglobin and medicine and the volume ratio of precipitating reagent：
(1-2.5), so that the mixed solution of precipitating reagent and seralbumin, hemoglobin and medicine is sufficiently mixed, reduces reduced form
The solubility of the white egg of serum and hemoglobin, carries out two kinds of albumen cross-linking reactions more abundant.
By controlling reduced form seralbumin, hemoglobin and the incorporation time of medicine mixed solution and precipitating reagent to be
When 0.5-12 is small, so that precipitating reagent can be uniformly mixed with reduced form seralbumin and hemoglobin mixed solution, reduce
The solubility of reduced form seralbumin and hemoglobin, carries out two kinds of albumen cross-linking reactions more abundant.
According to the second aspect of the invention, the present invention provides prepared according to above-mentioned load medicine oxygen carrier hybrid protein nanoparticle
The load medicine oxygen carrier hybrid protein nanoparticle formed.
Load medicine oxygen carrier hybrid protein nanoparticle provided by the invention, passes through disulfide bond by hemoglobin and seralbumin
Covalent bond, contains medicine, with reference to oxygen, it is provided simultaneously with the load of sero-abluminous tumor-targeting function and hemoglobin
Oxygen function, can target by oxygen and drug delivery to inside tumor, improve enrichment of the medicine in inside tumor, and being capable of root
According to the oxygen concentration under residing microenvironment, the reversible release of oxygen molecule is carried out, does not interfere with the tissue of normal oxygen concentratio, no secondary work
With can be effectively improved tumor hypoxia, strengthen the therapeutic effect of tumour, realize that tumour oxygenation is integrated with medicine delivery, carry
The high bioavailability of medicine.In addition load medicine oxygen carrier hybrid protein nanoparticle structure provided by the invention is stablized, storage time
It is long, it is easy to be extended and applied.
According to the third aspect of the invention we, the present invention provides the application of above-mentioned load medicine oxygen carrier hybrid protein nanoparticle, its
It is used to prepare anti-tumor medicine.
It is provided by the invention load medicine oxygen carrier hybrid protein nanoparticle be provided simultaneously with sero-abluminous tumor-targeting function and
The oxygen carrier function of hemoglobin, and contained medicine, is combined with oxygen, can target by oxygen and drug delivery to intra-tumor
Portion, oxygen and medicine are discharged inside hypoxic tumor, drug-rich is improved while tumor hypoxia is improved, strengthens controlling for tumour
Therapeutic effect.
Technical solution provided by the invention is further described with reference to embodiment and comparative example.
Embodiment 1
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg bovine serum albumin(BSA)s, 30mg dithiothreitol (DTT)s are codissolved in 5mL deionized waters, shake 1 at room temperature
Hour, solution pours into bag filter (3kD), when dialysis 12 is small in anaerobic deionized water, obtains reduced form serum albumin solution,
Reduced form serum albumin solution is settled to 6mL with deionized water, obtains the reduced form seralbumin that concentration is 50mg/mL
Solution；
(b) by 20mg reduced forms serum albumin solution, 10mg hemoglobins and 0.8mg doxorubicin hydrochlorides in aerobic bar
Homogeneous 10 minutes in 2mL pure water are codissolved under part, are 7 with sodium acid carbonate tune pH value, under vigorous stirring, with 1mL/min's
Absolute ethyl alcohol 2mL is added dropwise to mixed solution in speed, when stirring 0.5 is small after, it is 100kD that mixed solution is loaded molecular cut off
Bag filter, when dialysis 12 is small, obtains containing the load medicine oxygen carrier hybrid protein nanoparticle solution of doxorubicin hydrochloride.
Embodiment 2
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg human serum albumins, 300mg homocysteines are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 0.4mg hemoglobins and 0.8mg doxorubicin hydrochlorides in aerobic bar
Homogeneous 180 minutes in 2mL pure water are codissolved under part, are 9 with sodium acid carbonate tune pH value, under vigorous stirring, with 1mL/min's
Speed to mixed solution be added dropwise absolute ethyl alcohol 5mL, stirring 12 it is small when after, by solution load molecular cut off be 300 kD dialysis
Bag, when dialysis 12 is small, obtains containing the load medicine oxygen carrier hybrid protein nanoparticle solution of doxorubicin hydrochloride.
Embodiment 3
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg recombination human serum albumins, 45mg glutathione are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 6.7mg hemoglobins and 0.8mg doxorubicin hydrochlorides in aerobic bar
Homogeneous 20 minutes in 2mL pure water are codissolved under part, are 7.5 with sodium acid carbonate tune pH value, under vigorous stirring, with 1mL/min
Speed to mixed solution be added dropwise absolute ethyl alcohol 4mL, stirring 1 it is small when after, by solution load molecular cut off be 300kD it is saturating
Bag is analysed, when dialysis 12 is small, obtains containing the load medicine oxygen carrier hybrid protein nanoparticle solution of doxorubicin hydrochloride.
Embodiment 4
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg recombination human serum albumins, 150mg glutathione are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 1.3mg hemoglobins and 0.8mg doxorubicin hydrochlorides in aerobic bar
Homogeneous 120 minutes in 2mL pure water are codissolved under part, are 8.5 with sodium acid carbonate tune pH value, under vigorous stirring, with 1mL/min
Speed to mixed solution be added dropwise absolute ethyl alcohol 3mL, stirring 6 it is small when after, by solution load molecular cut off be 300 kD it is saturating
Bag is analysed, when dialysis 12 is small, obtains containing the load medicine oxygen carrier hybrid protein nanoparticle solution of doxorubicin hydrochloride.
Embodiment 5
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg recombination human serum albumins, 45mg cysteines are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 4mg hemoglobins and 0.8mg doxorubicin hydrochlorides in aerobic conditions
Under be codissolved in homogeneous 60 minutes in 2mL pure water, with sodium acid carbonate tune pH value be 8, under vigorous stirring, with the speed of 1mL/min
Spend to mixed solution be added dropwise absolute ethyl alcohol 3mL, stirring 6 it is small when after, by solution load molecular cut off be 300kD bag filter,
Dialyse 12 it is small when, obtain containing the load medicine oxygen carrier hybrid protein nanoparticle solution of doxorubicin hydrochloride.
Embodiment 6
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg recombination human serum albumins, 90mg glutathione are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 3mg hemoglobins and 0.8mg doxorubicin hydrochlorides in aerobic conditions
Under be codissolved in homogeneous 45 minutes in 2mL pure water, with sodium acid carbonate tune pH value be 8, under vigorous stirring, with the speed of 1mL/min
Spend to mixed solution be added dropwise absolute ethyl alcohol 3mL, stirring 6 it is small when after, by solution load molecular cut off be 300kD bag filter,
Dialyse 12 it is small when, obtain containing the load medicine oxygen carrier hybrid protein nanoparticle solution of doxorubicin hydrochloride.
Embodiment 7
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg recombination human serum albumins, 75mg glutathione are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 3.2mg hemoglobins and 0.8mg doxorubicin hydrochlorides in aerobic bar
Under part, homogeneous 30 minutes in 2mL pure water are codissolved in, are 8 with sodium acid carbonate tune pH value, under vigorous stirring, with 1mL/min's
Absolute ethyl alcohol 3mL is added dropwise to mixed solution in speed, when stirring 5 is small after, it is 100kD that mixed solution is loaded molecular cut off
Bag filter, when dialysis 12 is small, obtains containing the load medicine oxygen carrier hybrid protein nanoparticle of doxorubicin hydrochloride.
Embodiment 8
Present embodiments provide a kind of difference for carrying medicine oxygen carrier hybrid protein nanoparticle, the present embodiment and embodiment 7
It is, in step (a), the dosage of glutathione is 10mg.
Embodiment 9
Present embodiments provide a kind of difference for carrying medicine oxygen carrier hybrid protein nanoparticle, the present embodiment and embodiment 7
It is, in step (b), the dosage of hemoglobin is 0.2mg.
Embodiment 10
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg recombination human serum albumins, 75mg glutathione are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 3.2mg hemoglobins and 0.8mg chlorin-e6 aerobic
Under the conditions of be codissolved in homogeneous 30 minutes in 2mL pure water, with sodium acid carbonate tune pH value be 8, under vigorous stirring, with 1mL/min
Speed to mixed solution be added dropwise absolute ethyl alcohol 3mL, stirring 3 it is small when, solution load molecular cut off be 100kD bag filter,
Dialyse 12 it is small when, obtain containing the load medicine oxygen carrier hybrid protein nanoparticle of chlorin-e6.
Embodiment 11
A kind of load medicine oxygen carrier hybrid protein nanoparticle is present embodiments provided, is prepared as follows：
(a) 300mg recombination human serum albumins, 75mg glutathione are codissolved in 5mL deionized waters, shaken at room temperature
Swing 1 it is small when, solution pours into bag filter (3kD), in anaerobic deionized water dialyse 12 it is small when, it is molten to obtain reduced form seralbumin
Liquid, 6mL is settled to by reduced form serum albumin solution with deionized water, and it is white to obtain the reduced form serum that concentration is 50mg/mL
Protein solution；
(b) by 20mg reduced forms serum albumin solution, 3.2mg hemoglobins, 0.4mg doxorubicin hydrochlorides and 0.4mg bis-
Hydrogen porphines-e6 is codissolved in homogeneous 30 minutes in 2mL pure water under aerobic conditions, is 8 with sodium acid carbonate tune pH value, is stirring strongly
Mix down, with the speed of 1mL/min to mixed solution be added dropwise absolute ethyl alcohol 3mL, stirring 4 it is small when after, by mixed solution load retain
Molecular weight is the bag filter of 100kD, when dialysis 12 is small, obtains containing doxorubicin hydrochloride and the load medicine oxygen carrier of chlorin-e6
Hybrid protein nanoparticle.
Test example 1
The load medicine oxygen carrier hybrid protein nanoparticle that embodiment 1-12 is provided is measured into grain by dynamic light scattering analysis
Footpath is distributed, and the particle diameter for the load medicine oxygen carrier hybrid protein nanoparticle that the results show embodiment 1-12 is provided is at 20-200 nanometers.Fig. 1
The grain size distribution of the load medicine oxygen carrier hybridization nanometer grain for containing doxorubicin hydrochloride provided for embodiment 7, can from Fig. 1
Go out, the particle diameter for containing doxorubicin hydrochloride load medicine oxygen carrier hybrid protein nanoparticle of the offer of embodiment 7 is 20-50 nanometers.
Test example 2
The load medicine oxygen carrier hybrid protein nanoparticle that embodiment 10 provides is injected in the nude mouse of lotus knurl, by optoacoustic into
As the photoacoustic signal of equipment (850 nanometers of exciting light) the monitoring oxygen-containing hemoglobin of tumor locus (HbO2) is to judge tumour oxygenation feelings
Condition.Fig. 2 is the light that tumor bearing nude mice injection carries the oxygen-containing hemoglobin changes of contents of tumor locus after medicine oxygen carrier hybrid protein nanoparticle
Acoustic imaging monitoring figure；Figure it is seen that after when tumor bearing nude mice injection load medicine oxygen carrier hybrid protein nanoparticle 2 is small, tumor locus
Oxygen-containing content of hemoglobin significantly increase, can maintain always 6 it is small when oxygen environment, 24 it is small when after increase return to anoxic shape
State, the load medicine oxygen carrier hybrid protein nanoparticle for containing chlorin-e6 that this explanation embodiment of the present invention 10 provides have target
Tropism, can penetrate blood vessel, be enriched with tumor locus, and oxygen is discharged inside hypoxic tumor, strengthen oncotherapy effect.
Test example 3
Tumor bearing nude mice two is taken, wherein the load medicine oxygen carrier for containing chlorin-e6 that an injection embodiment 10 provides
Hybrid protein nanoparticle, in addition an injection at the same time dissociate chlorin-e6 solution with dosage.By fluorescence imaging device to two
Nude mice carries out fluorescence monitoring, Fig. 3 be injection carry medicine oxygen carrier hybrid protein nanoparticle 6 it is small when after tumor bearing nude mice and injection swim
The fluorescence imaging comparison diagram of tumor bearing nude mice after when small from chlorin-e6 solution 6, wherein, hybrid protein Nano medication refers to
It is the load medicine oxygen carrier hybrid protein nanoparticle for containing chlorin-e6 that embodiment 10 provides；From figure 3, it can be seen that injection 6
After hour, the chlorin-e6 of the tumor bearing nude mice tumor section enrichment for the load medicine oxygen carrier nanoparticle that injection embodiment 10 provides is shown
The tumor bearing nude mice higher than the free chlorin-e6 solution of injection is write, the load medicine oxygen carrier hybridization that this explanation embodiment of the present invention 10 supplies
Protein nano grain has targeting, can penetrate blood vessel, is enriched with tumor locus, its Targeting Performance is significantly higher than free drug,
The therapeutic effect of tumour can be strengthened.
Test example 4
The doxorubicin hydrochloride that contained that embodiment 7 is provided carries medicine oxygen carrier hybrid protein nanoparticle and free hydrochloric acid adriamycin
It is 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 solution to be configured to doxorubicin hydrochloride strengths respectively, so
Above-mentioned solution is incubated altogether with the human breast cancer cell (MCF-7) cultivated under anoxia condition respectively afterwards 2 it is small when, after incubation,
Be cultivated for above-mentioned tumour cell 24 it is small when, measure cell survival rate.Fig. 4 is load medicine oxygen carrier hybrid protein nanoparticle and trip
The tumor cell survival after chemotherapy is carried out from doxorubicin hydrochloride solution and contrasts block diagram, wherein, hybrid protein Nano medication refers to
Be embodiment 7 provide the load medicine oxygen carrier hybrid protein nanoparticle for containing doxorubicin hydrochloride；From fig. 4, it can be seen that implement
The load medicine oxygen carrier hybrid protein nanoparticle for doxorubicin hydrochloride that what example 7 provided contain can effective killing tumor cell, reduce swollen
The survival rate of oncocyte, and the raising of the doxorubicin hydrochloride strengths with package-contained, its fragmentation effect to tumour cell is more
Substantially, when it is 1 μ g/mL to carry the concentration of doxorubicin hydrochloride of medicine oxygen carrier hybrid protein nanoparticle package-contained, tumour cell is deposited
Motility rate is less than 5%, and free hydrochloric acid Doxorubicin solution does not have tumour cell obvious fragmentation effect, even if free hydrochloric acid Ah mould
When the concentration of plain solution is 1 μ g/mL, its tumor cell survival is still more than 95%.What this explanation embodiment of the present invention 7 provided
The oxygen carrier load medicine hybrid protein nanoparticle for containing doxorubicin hydrochloride passes through the targeted therapy to tumour cell and oxygenation chemotherapy, energy
Enough effective killing tumor cells, reduce the survival rate of tumour cell, enhance the therapeutic effect of tumour.
Test example 5
Doxorubicin hydrochloride and the load medicine oxygen carrier hybrid protein nanoparticle of chlorin-e6 are contained by what embodiment 11 provided
Adriamycin/chlorin-e6 concentration is configured to as 0.2/0.21 μ g/mL, 0.4/0.42 μ g/mL, 0.6/0.64 μ g/mL, 0.8/
The load medicine oxygen carrier hybrid protein nanoparticle solution of 0.85 μ g/mL and 1/1.05 μ g/mL, and above-mentioned load medicine is carried into hybrid protein nanometer
Grain solution with anoxia condition culture human breast cancer cell (MCF-7) is incubated altogether 2 it is small when, while use 660 nanometer lasers
(power 100mW/cm2) irradiated tumor cell, then proceed to cultivate above-mentioned tumour cell 24 it is small when, measure tumor cell survival
Rate, test result are as shown in Figure 5.
Doxorubicin hydrochloride and chlorin-e6 are mixedly configured into doxorubicin hydrochloride/chlorin-e6 concentration as 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 dissociate adriamycin and
Chlorin-e6 mixed solutions, by above-mentioned free adriamycin and chlorin-e6 mixed solutions and the people in anoxia condition culture
Breast cancer cell (MCF-7) be incubated altogether 2 it is small when, while use 660 nanometer lasers (power 100mW/cm2) irradiation tumour it is thin
Born of the same parents, then proceed to cultivate above-mentioned tumour cell 24 it is small when, measure tumor cell survival.Test result is as shown in Figure 5.
Fig. 5 is increasing to carry medicine oxygen carrier hybrid protein nanoparticle solution and free adriamycin and chlorin-e6 mixed solutions
The survival rate contrast block diagram with tumour cell after oxygenation light power link treatment is treated in oxidation, wherein, hybrid protein Nano medication
Refer to the offer of embodiment 11 contains doxorubicin hydrochloride and the load medicine oxygen carrier hybrid protein nanoparticle of chlorin-e6；From
Fig. 5 can be with, and what the embodiment of the present invention 11 provided contain doxorubicin hydrochloride and the load medicine oxygen carrier hybrid protein of chlorin-e6 is received
The grain of rice is significantly improved, carried with the raising of doxorubicin hydrochloride/chlorin-e6 concentration, its fragmentation effect to tumour cell
When the concentration for doxorubicin hydrochloride/chlorin-e6 that medicine oxygen carrier hybrid protein nanoparticle contains is 0.2/0.21 μ g/mL, tumour
The survival rate of cell is less than 5%, and in free hydrochloric acid adriamycin and chlorin-e6 mixed solutions, doxorubicin hydrochloride/bis-
When the concentration of hydrogen porphines-e6 is 1/1.06 μ g/mL, the survival rate of its tumour cell remains above 50%, this explanation present invention is implemented
Example 11 provide contain doxorubicin hydrochloride and the load medicine oxygen carrier hybrid protein nanoparticle of chlorin-e6 passes through to tumour cell
Oxygenation treatment and oxygenation optical dynamic therapy, the lethality to tumour cell can not only be significantly improved, reduce tumour cell
Survival rate, and the fragmentation effect very strong to tumour cell can be reached when drug concentration is relatively low, strengthen the treatment effect of tumour
Fruit.
Test example 6
The load medicine oxygen carrier hybrid protein nanoparticle for containing doxorubicin hydrochloride that embodiment 1-9 is provided carries out drug encapsulation
Rate and oxygen affinity P50 values measure, measurement result are as shown in table 1 below：
Table 1 carries medicine oxygen carrier hybrid protein nanoparticle performance data table
As it can be seen from table 1 by the contrast of embodiment 1-7 and embodiment 8-9 can be seen that carry out carry medicine oxygen carrier it is miscellaneous
When handing over the preparation of protein nano grain, the mass ratio of seralbumin and reducing agent is 1：(0.1-1), hemoglobin and seralbumin
Mass ratio be 1：When (2-50), the oxygen affinity P50 values of made load medicine oxygen carrier hybrid protein nanoparticle significantly improve, and carry
Dose dramatically increases, and oxygen affinity P50 values are above 13mmHg, and drugloading rate is above 2.5%
The oxygen affinity P50 value highests of embodiment 5-7 can be seen that by the contrast of embodiment 1-7, embodiment 3-4's
Oxygen carrier amount and drugloading rate are below embodiment 5-7 but are higher than embodiment 1-2.This explanation, when hemoglobin with it is sero-abluminous
Mass ratio is 1：(3-15), and the mass ratio of seralbumin and reducing agent is 1：When (0.15-0.5), manufactured load medicine oxygen carrier
The oxygen affinity P50 values of hybrid protein nanoparticle are stronger, and drugloading rate is higher, when hemoglobin and sero-abluminous mass ratio are
1：(5-7), and the mass ratio of seralbumin and reducing agent is 1：(0.15-0.3), manufactured load medicine oxygen carrier hybrid protein are received
The oxygen affinity P50 value highers of the grain of rice, drugloading rate higher.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, it will be understood by those of ordinary skill in the art that：Its according to
Can so modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic into
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
Claims (10)
1. a kind of preparation method for carrying medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that include the following steps：
(a) by reducing agent and seralbumin mixing homogeneous, reduction reaction is carried out, obtains reduced form seralbumin；
(b) reduced form seralbumin, hemoglobin and medicine are mixed into homogeneous under aerobic conditions, that is, it is miscellaneous that load medicine oxygen carrier is made
Hand over protein nano grain.
2. the preparation method according to claim 1 for carrying medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that described blood red
Albumen source is in animal, it is preferable that the hemoglobin derives from people, ox or pig；
Preferably, the seralbumin is obtained from animal or using biofermentation mode, it is further preferred that the blood
Pure albumen source is in people or ox, it is further preferred that the seralbumin is the recombined human that biological fermentation method obtains
Seralbumin.
3. the preparation method according to claim 1 for carrying medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that the reduction
Agent is selected from least one of glutathione, dithiothreitol (DTT), cysteine or homocysteine.
4. the preparation method according to claim 1 for carrying medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that the medicine
For chemotherapeutics and/or photosensitizer；
Preferably, the chemotherapeutics is selected from least one of doxorubicin hydrochloride, methotrexate (MTX) or hypericin；
Preferably, the photosensitizer is selected from least one of indocyanine green, haematoporphyrin, protoporphyrin or chlorin-e6.
5. the preparation method according to claim 1 for carrying medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that hemoglobin
It is 1 with sero-abluminous mass ratio：(2-50), is preferably 1：(3-15), more preferably 1：(5-10)；
Preferably, the mass ratio of seralbumin and reducing agent is 1：(0.1-1), is preferably 1：(0.15-0.5), more preferably
1：(0.15-0.3).
6. the preparation method according to claim 1 for carrying medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that in step
(b) in, the pH value of the mixed solution of reduced form seralbumin, hemoglobin and medicine is 7-9；
Preferably, it is 10-180 minutes to mix homogenizing time；
Preferably, the sero-abluminous concentration of reduced form is 0.5-3%.
7. the preparation method of medicine oxygen carrier hybrid protein nanoparticle is carried according to claim 1-6 any one of them, it is characterised in that
In step (b), reduced form seralbumin is first mixed into homogeneous under aerobic conditions with hemoglobin and medicine, is then added
Precipitant mix is uniform, removes precipitating reagent and floating preteins and medicine finally by vacuum drying, ultrafiltration or dialysis, that is, is made
Carry medicine oxygen carrier hybrid protein nanoparticle；
Preferably, the precipitating reagent is anhydrous low-carbon alcohol, more preferably absolute ethyl alcohol.
8. the preparation method according to claim 7 for carrying medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that reduced form blood
The volume ratio of pure albumen, the mixed solution of hemoglobin and medicine and precipitating reagent is 1：(1-2.5)；
Preferably, reduced form seralbumin, hemoglobin and the incorporation time of medicine mixed solution and precipitating reagent are 0.5-12
Hour.
9. one kind carries medicine oxygen carrier hybrid protein nanoparticle, it is characterised in that carries medicine according to claim 1-8 any one of them and carries
Oxa- hands over protein nano grain to be prepared.
10. the application according to claim 9 for carrying medicine oxygen carrier hybrid protein nanoparticle in anti-tumor medicine is prepared.
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| CN112263679B (en) * | 2020-10-26 | 2023-11-21 | 深圳先进技术研究院 | Targeted oxygen-carrying nano sound-sensitive agent and preparation method thereof |
| CN112546220A (en) * | 2020-12-09 | 2021-03-26 | 广东医科大学 | Preparation method and application of oxygen-carrying hybrid protein-loaded metal complex nano system |
| CN113456612A (en) * | 2021-06-21 | 2021-10-01 | 上海市胸科医院 | Albumin drug delivery system for treating sepsis myocardial injury |
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