CN108822267B - preparation and application of pH-responsive protein macromolecule amphiphile - Google Patents
preparation and application of pH-responsive protein macromolecule amphiphile Download PDFInfo
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- CN108822267B CN108822267B CN201810247741.8A CN201810247741A CN108822267B CN 108822267 B CN108822267 B CN 108822267B CN 201810247741 A CN201810247741 A CN 201810247741A CN 108822267 B CN108822267 B CN 108822267B
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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Abstract
The invention provides a method for preparing protein-macromolecule amphiphile, which comprises the following steps: (1) Covalently linking an initiator to a protein to obtain a protein-initiator combination; (2) Carrying out in-situ polymerization on the protein-initiator combination and a monomer compound so as to obtain a protein-macromolecule amphiphile, wherein the macromolecule has pH responsiveness within the range of pH 5-10, and the pKa of the macromolecule is 5-10; the method has simple operation, definite chemical structure and high yield, and the protein-macromolecule amphiphile prepared by the method has high protein activity and high pH response sensitivity, can be used as a delivery carrier of chemotherapeutic drugs or diagnostic reagents, improves the targeting property of small molecule drugs and reduces the side effect; meanwhile, the protein can select some medicinal proteins, and the stability and the targeting property of the medicinal proteins can be improved by virtue of the targeting effect of the pH-responsive macromolecule, so that the application of the protein medicament in tumor diagnosis and treatment is expected to be greatly expanded.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to preparation and application of a pH-responsive protein macromolecule amphiphile.
Background
The protein self-assembly has been widely used in the fields of drug delivery, tumor diagnosis and treatment, etc. as an important biomaterial. The protein and the hydrophobic polymer are connected together to form protein polymer amphiphile, and the protein polymer amphiphile can be self-assembled into nano particles with different shapes.
Both small molecule drugs and protein drugs have the problems of poor stability, short half-life and poor targeting, and the application of the small molecule drugs and the protein drugs in tumor diagnosis and treatment is restricted.
In tumor tissue, active glycolysis produces a large amount of lactic acid, and the extracellular pH of the tumor is acidic, about 6.5, compared to the weakly basic extracellular pH of normal tissue.
Therefore, the development of a drug delivery system with pH responsiveness is particularly important by utilizing the tumor acidic microenvironment.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
based on the discovery of the problems, the inventor develops a protein-polymer amphiphile with pH responsiveness and a preparation method thereof, wherein the preparation method is simple to operate and high in yield, and the protein-polymer amphiphile has targeting property and can generate high-sensitivity reaction to specific pH. The protein-macromolecule amphiphile can be used as a delivery carrier of chemotherapeutic drugs or diagnostic reagents, so that the targeting property of small molecule drugs is improved, and the side effect of the small molecule drugs is reduced; on the other hand, the protein can select some medicinal proteins, and the stability and the targeting property of the medicinal proteins can be improved by virtue of the targeting effect of the pH-responsive macromolecule, so that the application of the protein medicament in tumor diagnosis and treatment is expected to be greatly expanded.
In a first aspect of the invention, the invention provides a method for preparing a protein-macromolecule amphiphile. According to an embodiment of the invention, the method comprises: (1) Covalently linking an initiator to a protein to obtain a protein-initiator combination; (2) And carrying out in-situ polymerization on the protein-initiator combination and a monomer compound to obtain a protein-macromolecule amphiphilic substance, wherein the macromolecule has pH responsiveness within the pH range of 5-10, and the pKa of the macromolecule is 5-10. In situ polymerization can be carried out by Atom Transfer Radical Polymerization (ATRP) or reversible addition fragmentation chain transfer polymerization (RAFT). According to the method provided by the embodiment of the invention, the whole polymerization reaction is carried out in the aqueous solution, so that the contact between the protein and an organic solvent is effectively avoided, and the activity of the protein is ensured. In addition, the inventors succeeded in binding a polymer having pH responsiveness to a protein, and the resulting protein-polymer amphiphile has more sensitive pH responsiveness, exists in a micellar form under the ambient pH > amphiphile pKa condition, and disaggregates under the ambient pH < amphiphile pKa condition. The protein-polymer amphiphile obtained by the method according to the embodiment of the invention is particularly sensitive to a tumor microenvironment, and the pH of the tumor microenvironment is less than the pKa of the protein-polymer amphiphile according to the embodiment of the invention, so that the protein-polymer amphiphile obtained by the method according to the embodiment of the invention has the efficacy of targeting the tumor to disintegrate, and the site-specific release of protein drugs or other loadable molecules in the tumor is realized. The method provided by the embodiment of the invention is simple to operate and high in yield, and meanwhile, the protein-polymer amphiphile prepared by the method provided by the embodiment of the invention has high pH responsiveness, especially has more sensitive response to a tumor microenvironment, and the obtained protein-polymer amphiphile has high protein activity, clear chemical structure, good cell entering effect and high biological safety.
According to an embodiment of the present invention, the method further includes at least one of the following additional technical features:
according to an embodiment of the invention, the initiator comprises an initiator of atom transfer radical polymerization, a chain transfer agent of reversible addition-fragmentation chain transfer polymerization.
According to an embodiment of the present invention, the initiator of atom transfer radical polymerization comprises at least one selected from polyhalides, halogenated esters, halogenated ketones, halogenated nitriles.
According to an embodiment of the present invention, the initiator of the reversible addition-fragmentation chain transfer polymerization includes a compound selected from the group consisting of thiocarbonylthio group and thiophosphinylthio group.
According to an embodiment of the present invention, the chain transfer agent initiator of the reversible addition-fragmentation chain transfer polymerization comprises at least one selected from dithioesters, dithiocarbamates, xanthates, trithiocarbonates.
According to embodiments of the invention, the protein is covalently linked to the initiator at the N-or C-terminus of the protein and at any other site remote from and/or not interfering with the activity of the protein. The protein-initiator conjugate thus obtained is stable in properties without impairing the activity of the protein.
According to embodiments of the present invention, the proteins include proteins, small peptides and antibodies associated with pharmaceutical, agricultural, scientific and other industrial fields, including in particular therapeutic proteins such as insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, therapeutic vaccines, calcitonin, tumor Necrosis Factor (TNF) and enzymes, and the like. Specific examples include, but are not limited to: <xnotran> , , , , , , , , , (EGF), (IGF), (TGF), (NGF), (PDGF), (BMP), , , , , , , , (CSF), , , , , , (VIP), (CCK), , , , , , , , , (TRH), , , (LHRH), , -1, -15, (IL-1 RA), -1 (GLP-1), , , (GM-CSF), -2 (IL-2), , , , , , , , </xnotran> Heparin, atrial natriuretic peptides, hemoglobin, retroviral vectors, relaxin, cyclosporine, oxytocin, vaccines, monoclonal antibodies, single chain antibodies, ankyrin repeat proteins, affibodies, and the like.
According to a particular embodiment of the invention, the protein is human serum albumin. Further, a range of pharmaceutical proteins may be employed;
according to an embodiment of the invention, the attachment site is cysteine 34 on human serum albumin.
The initiator is a compound shown as a formula (I)
According to an embodiment of the invention, the protein, previously dissolved in a buffer solution, is reacted with the initiator at room temperature for 5 minutes to 24 hours.
According to an embodiment of the invention, the coupling ratio of the protein to the initiator is 1. According to the specific embodiment of the invention, the cysteine at position 34 of the HSA is modified, the human serum albumin has only one free cysteine, and the protein and the initiator can realize that the ratio of 1:1, so as to realize site-directed modification and ensure the definite chemical structure and the homogeneity of the product.
According to a specific embodiment of the present invention, the in situ polymerization is an atom transfer radical polymerization.
According to an embodiment of the present invention, the in-situ polymerization is performed under a low oxygen or inert gas atmosphere at a temperature of 0 to 80 ℃ for 5 minutes to 24 hours. Further, the obtained high molecular weight by polymerization reaction has narrow molecular weight distribution and high polymerization efficiency.
According to embodiments of the present invention, the in situ polymerization includes atom transfer radical polymerization and reversible addition-fragmentation chain transfer polymerization.
According to an embodiment of the present invention, the atom transfer radical polymerization is carried out under catalysis of copper and ligand, the copper is CuCl or CuCl 2 、CuBr、CuBr 2 The ligand is a nitrogen-containing complexing agent.
According to an embodiment of the invention, the ligand is 2, 2-bipyridine, pentamethyldiethylenetriamine, tris (2-dimethylaminoethyl) amine or 1,4,7, 10-hexamethyltriethylenetetramine.
According to an embodiment of the present invention, the in-situ polymerization is carried out under catalysis of CuCl and 1,4,7, 10-hexamethyltriethylenetetramine. Further, the efficiency of in-situ bonding is further improved.
According to the embodiment of the invention, the molar ratio of the protein-initiator combination to the monomer compound to the CuCl to the 1,4,7, 10-hexamethyl triethylene tetramine is 1 (200-3000) to 10-500 to 10-4000. Further, the efficiency of in-situ polymerization is further improved.
According to an embodiment of the present invention, the molar ratio of the protein-initiator combination, the monomeric compound, the CuCl, the 1,4,7, 10-hexamethyltriethylenetetramine is 1. Further, the efficiency of in-situ polymerization is further improved.
According to an embodiment of the present invention, the monomer compound has a structure represented by formula (II):
wherein R is
The protein-polymer amphiphile formed by the monomer compound and the protein has pH responsiveness.
According to a particular embodiment of the invention, the monomer compound is 2- (diisopropylamino) ethyl methacrylate (DPA). Further, the pH-responsive sensitivity of the protein-polymer amphiphile thus obtained is further improved.
In a second aspect of the invention, the invention provides a protein-macromolecule amphiphile. According to an embodiment of the present invention, the protein-polymer amphiphile is obtained according to the method described above. The protein-polymer amphiphiles according to the embodiments of the present invention have a more sensitive pH responsiveness, i.e., exist in a micelle form under a pH > pKa condition, and disaggregate under a pH < pKa condition. The protein-macromolecule amphiphile according to the embodiment of the invention is particularly sensitive to a tumor microenvironment, and the pH of the tumor microenvironment is less than the pKa of the protein-macromolecule amphiphile according to the embodiment of the invention, so that the protein-macromolecule amphiphile according to the embodiment of the invention has the efficacy of targeting a tumor to be disintegrated, and the fixed-point release of a protein drug or other entrapable molecules in the tumor is realized. The protein-polymer amphiphile structure of the embodiment of the invention is definite, the product uniformity is good, the biological safety is high, and the protein activity is high.
In a third aspect of the invention, a protein-macromolecule amphiphile is presented. According to an embodiment of the invention, the protein-macromolecule amphiphile comprises: a hydrophilic region comprising a protein; and a hydrophobic region comprising a macromolecule having a pH responsiveness in a pH range of 5 to 10, the macromolecule having a pKa between 5 and 10. The protein-polymer amphiphiles according to the embodiments of the present invention have a more sensitive pH responsiveness, i.e., exist in a micelle form under a pH > pKa condition, and disaggregate under a pH < pKa condition. The protein-macromolecule amphiphile according to the embodiment of the invention is particularly sensitive to a tumor microenvironment, and the pH of the tumor microenvironment is less than the pKa of the protein-macromolecule amphiphile according to the embodiment of the invention, so that the protein-macromolecule amphiphile according to the embodiment of the invention has the efficacy of tumor targeting and disintegration, and the site-specific release of protein drugs or other loadable molecules in tumors is realized. The protein-polymer amphiphilic substance has a definite structure, good product uniformity, high biological safety and high protein activity.
In a fourth aspect of the invention, a nanoparticle is presented. According to an embodiment of the present invention, the nanoparticle is formed by self-assembly of the protein-polymer as described above, and comprises: a hydrophobic core consisting of hydrophobic regions of the protein-macromolecule amphiphile; and a hydrophilic shell disposed on an outer surface of the hydrophobic core and composed of hydrophilic regions of the protein-polymer amphiphiles. The nanoparticles according to embodiments of the present invention have pH responsiveness, exist in a micellar form under pH > pKa conditions, and disaggregate under pH < pKa conditions. The nanoparticles according to the embodiments of the present invention are particularly sensitive to the tumor microenvironment, and the pH of the tumor microenvironment is less than the pKa of the nanoparticles according to the embodiments of the present invention, so that the nanoparticles according to the embodiments of the present invention have the efficacy of targeting the tumor to disintegrate, and the site-specific release of protein drugs or other entrapable molecules in the tumor is realized. The nanoparticles provided by the embodiment of the invention are easy to enter cells, and have high biological safety, high protein activity, high entrapment rate and high drug loading.
According to an embodiment of the present invention, the nanoparticle further comprises at least one of the following additional technical features:
according to an embodiment of the invention, further comprising loading the compound. The loading mode is chemical coupling or physical embedding, namely, the small molecular compound can be connected to the macromolecule through a chemical bond, and can also be loaded in the micelle inner core through a simple physical adsorption mode. Physical embedding is used in embodiments according to the invention. The protein-macromolecule amphiphile according to the embodiment of the invention and the compound to be loaded can be loaded in the core of the nanoparticle through hydrophilic and hydrophobic effects in a buffer solution. The nanoparticles coated with the compound exist in a micelle form in certain pH conditions such as blood circulation, and are depolymerized under other pH conditions such as tumor subacid environment to release the coated compound, so that targeted disintegration is realized, and the coated compound is released at a targeted position. The nanoparticles provided by the embodiment of the invention have the advantages of good targeting property, high entrapment rate and high drug loading.
According to an embodiment of the invention, the compound comprises a hydrophobic small molecule drug and a fluorescent molecule. For example, the small molecule drug may be a chemotherapeutic drug.
According to an embodiment of the invention, the small molecule drug comprises at least one selected from gemcitabine, paclitaxel, doxorubicin, gefitinib, methotrexate. The micro-molecular drugs are mostly chemotherapy drugs, the nanoparticles encapsulating the micro-molecular drugs exist in a micelle form under the condition that the pH is greater than pKa (pKa), such as blood circulation, and are depolymerized under the condition that the pH is less than pKa, such as the slightly acidic environment of tumors, so that the encapsulated micro-molecular drugs are released, and the targeted delivery of the micro-molecular drugs is realized.
According to an embodiment of the invention, the fluorescent molecule comprises at least one selected from sulforhodamine B, indocyanine green, fluorescein isothiocyanate, ethidium bromide, water-soluble fluorescent blue, a thean-based water-soluble fluorescent dye, a water-soluble cyanine dye succinimide ester dye, phycoerythrin coumarin, a cell membrane red fluorescent probe, rhodamine 200 and fluorescein ester. The nanoparticles encapsulating the fluorescent molecules exist in a micelle form under the condition of pH > pKa (pKa), such as blood circulation, and are depolymerized under the condition of pH < pKa, such as a slightly acidic environment of a tumor, so that the encapsulated fluorescent molecules are released, and imaging in a living tumor is realized.
According to an embodiment of the invention, the compound is indocyanine green. Indocyanine green is the only clinically-used near-infrared imaging contrast agent approved by food and drug administration at present, but the indocyanine green has short half-life and poor targeting property, and the use of the indocyanine green in the field of tumor imaging is restricted. After the indocyanine green is encapsulated by the nano particles, the indocyanine green exists in a micelle form in blood circulation, and the micelle depolymerizes under a tumor microenvironment to release the indocyanine green, so that the targeted release of the indocyanine green is realized, and the half-life period of the indocyanine green is improved.
In a fifth aspect of the present invention, the present invention provides a method of preparing a nanoprobe. According to an embodiment of the present invention, the method comprises contacting the protein-macromolecule amphiphile as described above with a fluorescent molecule to be encapsulated so as to obtain the nanoprobe. The protein-macromolecule amphiphile entraps the compound to be entrapped in the middle of the micelle of the protein-macromolecule amphiphile through the hydrophilic and hydrophobic effect. According to the method provided by the embodiment of the invention, the entrapment rate of the protein-macromolecule amphiphile is high, and the drug loading capacity is high.
According to an embodiment of the present invention, the method further includes at least one of the following additional technical features:
according to the embodiment of the invention, the fluorescent molecule to be encapsulated comprises at least one selected from sulforhodamine B, indocyanine green, fluorescein isothiocyanate, ethidium bromide, water-soluble fluorescent blue, theanhydride water-soluble fluorescent dye, water-soluble cyanine dye succinimide ester dye, phycoerythrin coumarin, cell membrane red fluorescent probe, rhodamine 200 and fluorescein ester.
According to an embodiment of the present invention, the contacting is performed under dark treatment conditions at room temperature for 5 minutes to 24 hours. Further avoiding fluorescence quenching and further improving entrapment rate.
According to the embodiment of the invention, the molar ratio of the protein-macromolecule amphiphile to the fluorescent molecule to be encapsulated is 1 (0.5-20). The present inventors have found that the protein-polymer amphiphile entrapment efficiency is high and the drug loading is high under the above molar ratio conditions.
In a sixth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the present invention, the pharmaceutical composition comprises the protein-macromolecule amphiphile or comprises the nanoparticle, and the protein-macromolecule amphiphile and/or the molecule and/or the protein carried by the nanoparticle are drug molecules. The pharmaceutical composition provided by the embodiment of the invention can deliver chemotherapeutic drugs or diagnostic reagents in a targeted manner, improve the targeting property of small molecule drugs and reduce the side effects of the small molecule drugs; meanwhile, the protein can select some medicinal proteins, and the stability and the targeting property of the medicinal proteins can be improved by virtue of the targeting effect of the pH-responsive macromolecule, so that the application of the protein medicament in tumor diagnosis and treatment is expected to be greatly expanded.
In a seventh aspect of the invention, a fluorescence imaging method is provided. According to an embodiment of the present invention, the protein-macromolecule amphiphile or the nanoparticle including the protein-macromolecule amphiphile and/or the nanoparticle is introduced into a microenvironment to be imaged, and the molecule carried by the protein-macromolecule amphiphile and/or the nanoparticle is a fluorescent molecule. It should be noted that the imaging microenvironment includes both ex vivo and in vivo cells and tissues. According to the method provided by the embodiment of the invention, after the protein-polymer amphiphile or the nanoparticle is introduced into an imaging microenvironment, the protein-polymer amphiphile or the nanoparticle is depolymerized under the condition that the pH is less than pKa, and a fluorescent molecule is released, so that imaging is performed in the microenvironment.
In yet another aspect of the invention, the invention provides a method for synthesizing pH-responsive protein macromolecule amphiphiles and entrapped compounds and a general method for characterization. According to a specific embodiment of the invention, the method comprises:
(1) Synthesis of protein-initiator conjugate Human Serum Albumin (HSA) -bromine-containing initiator (Br): the initiator is first synthesized and then covalently linked to HSA.
Wherein, taking an initiator EBMP as an example, the initiator has the following synthetic route:
(2) Synthesis of pH-responsive protein-macromolecule amphiphile HSA-PDPA: initiating with protein-initiator combination HSA-Br, and polymerizing DPA monomer by atom transfer radical polymerization to form protein-polymer amphiphile HSA-PDPA;
(3) Encapsulation of indocyanine green (ICG) to form HSA-PDPA/ICG (HDI) nanoprobes: ICG is dissolved in a small amount of DMSO, and is directly dripped into the PBS solution of HSA-PDPA, and the ICG is wrapped and loaded into the HSA-PDPA micelle core by utilizing the hydrophilic and hydrophobic effects to form an HSA-PDPA/ICG nano probe;
(4) Characterizing a series of physicochemical properties such as molecular weight, radius, absorption spectrum and fluorescence spectrum of HSA-PDPA and HSA-PDPA/ICG by using analytical means such as Gel Permeation Chromatography (GPC), enzyme mark instrument, dynamic Light Scattering (DLS) and Transmission Electron Microscope (TEM);
(5) Selecting a C8161 cell line to test the cell-entering condition of HSA-PDPA/ICG, and selecting C8161, L929, MCF-10 and HEMC cell lines to test the cytotoxicity of the HSA-PDPA nano-particles;
(6) Establishing a nude mouse tumor model, and testing the tumor imaging effect and the drug distribution of the HSA-PDPA/ICG.
Drawings
FIG. 1 is a schematic diagram of a synthetic route and its application of a pH-responsive amphiphilic HSA-PDPA according to an embodiment of the present invention;
FIG. 2 is a SDS-PAGE gel image result before and after HSA-PDPA purification according to an embodiment of the present invention;
FIG. 3 is a GPC chart of PDPA after hydrolysis according to an embodiment of the present invention;
FIG. 4 is a GPC chart of hydrolyzed PHPMA according to an embodiment of the present invention;
FIG. 5 is a characterization of the physicochemical properties of HSA-PDPA and HDI according to an embodiment of the present invention;
FIG. 6 is a topographical characterization of HSA-PHPMA and HHI according to embodiments of the present invention;
FIG. 7 is a pH responsive characterization of HSA-PHPMA and HHI according to embodiments of the present invention;
FIG. 8 is a graph showing the critical micelle concentration of HSA-PDPA and HSA-PHPMA according to an embodiment of the present invention;
FIG. 9 is a pH responsive optical property characterization of HDI according to an embodiment of the present invention;
FIG. 10 shows the effect of HDI and HHI at different pH for endocytosis according to the embodiment of the present invention;
FIG. 11 is a biosafety characterization of HSA-PDPA according to embodiments of the invention; and
FIG. 12 shows the in vivo tumor imaging effect and biodistribution of HSA-PDPA/ICG according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The C8161 melanoma cells (transfected with GFP) in the following examples were donated by the academy of military medical sciences, and L929, HEMC and MCF-10 were purchased from the tumor cell bank of the academy of Chinese sciences.
The cell culture medium in the following examples is a product of Gibco.
The female athymic (Nude) Nude mice in the following examples are products of experimental animal technology ltd, viton, beijing. Female athymic (Nude) Nude mice are hereinafter abbreviated Nude mice.
In the following examples, SDS-PAGE was used to qualitatively analyze the effect of ATRP polymerization and product purification; quantitatively analyzing the molecular weight of the product by Gel Permeation Chromatography (GPC); observing the appearance of HSA-PDPA/ICG under different pH values or different molecular weights by using a Transmission Electron Microscope (TEM); measuring changes of particle size and zeta potential under different pH values by using a dynamic light scattering particle sizer (DLS); the change of the fluorescence characteristics of HSA-PDPA/ICG at different pH values was measured using a microplate reader. Testing the cell-entering effect of HSA-PDPA/ICG and the biological safety of HSA-PDPA by utilizing an endocytosis experiment and a cytotoxicity experiment; establishing a nude mouse tumor model, and testing the tumor imaging effect and the biodistribution of the HSA-PDPA/ICG.
In the following examples, three replicates were set up unless otherwise specified, and the results averaged.
Example 1 method for preparing pH-responsive protein-Polymer amphiphile HSA-PDPA
1. Preparation of initiator HSA-Br for macromolecular atom transfer radical polymerization
1) Synthesis of small molecular initiator EBMP
Firstly, maleimide and tetraethylene glycol are subjected to a mitsunobu reaction to obtain maleimide tetraethylene glycol, and then the maleimide tetraethylene glycol and bromine isobutyryl bromide are subjected to an esterification reaction to obtain a small molecular initiator EBMP with the yield of 54%. 1 H NMR(400MHz,CDCl 3 ):δ6.702(s,2H),4.324(m,2H),3.604-3.749(m,14H),1.938(s,6H).ESI-mass m/z:444.1([M+Na] + ),446.1([M+Na] + ).
2) Synthesis of HSA-Br
Dissolving human serum albumin HSA (2mL, 100. Mu.M) in Tris-HCl buffer, adding a thiol-modified initiator EBMP (20. Mu.L, 50 mM) using the free thiol of cysteine at position 34 of HSA, mixing, standing at room temperature, and reacting for 3 hours. After the reaction is finished, filtering the mixture by using a 0.22 mu m filter membrane, desalting the mixture by using an AKTA protein purifier, and removing residual EBMP to obtain purified human serum albumin (HSA-Br) connected with an initiator, wherein the modification efficiency is 45 percent.
2. Synthesis of pH-responsive protein-polymer amphiphile HSA-PDPA
Taking 1ml of 50 mu M HSA-Br solution obtained in the previous step, adding monomer DPA (200-4000 times of equivalent), introducing N into a reaction tube 2 The oxygen in the system was removed by magnetic stirring for 30min. Another container was charged with CuCl powder (40 equivalent times), and HMTETA (100 equivalent times), also N 2 After magnetically stirring for 30min, the mixture was poured into a reaction tube, reacted overnight under a closed condition, and then exposed to air to terminate the reaction, thereby obtaining a reaction mixture.
The reaction mixture was dialyzed to remove unreacted monomers and catalyst, and then HSA-PDPA was isolated and purified by molecular sieve (Hiload 26/600Superdex75pg, GE Healthcare) at a yield of 70%, using 1 mM PBS, pH7.4 as a buffer.
Example 2 method for preparing non-pH-responsive protein-macromolecule amphiphile HSA-PHPMA
Human serum albumin-polyhydroxypropylmethacrylate (HSA-PHPMA) without pH responsiveness was synthesized as a control in the same procedure as in example 1, as follows:
1ml of the 50. Mu.M HSA-Br solution obtained in the previous step was taken, monomer HPMA (200-4000 times equivalent) was added, and N was passed through the reaction tube 2 The oxygen in the system was removed by magnetic stirring for 30min. Another container was charged with CuCl powder (40 equivalent times), and HMTETA (100 equivalent times), also N 2 After stirring magnetically for 30min, the mixture was poured into a reaction tube, reacted overnight under a closed condition, and then exposed to air to terminate the reaction, thereby obtaining a reaction mixture.
The reaction mixture was dialyzed to remove unreacted monomers and catalyst, and then HSA-PHPMA was separated and purified by molecular sieve (Hiload 26/600Superdex75pg, GE Healthcare) at a yield of 75%, using a buffer of 10mMPBS, pH 7.4.
Example 3 encapsulation of indocyanine Green with pH-responsive HSA-PDPA
0.38mg of indocyanine green was dissolved in DMSO and then added dropwise to 1ml of the above-mentioned HSA-DPA solution at 5mg/ml, the solution was reacted overnight at room temperature in the dark, and the product was dialyzed to remove DMSO and free ICG, thereby obtaining a loaded HSA-PDPA/ICG (HDI) fluorescent probe.
HSA-PHPMA/ICG (HHI) fluorescent probe was synthesized in the same procedure as a control.
Example 4 physicochemical characterization of HSA-PDPA
SDS-PAGE is used for representing the polymerization and purification of HSA-PDPA, and FIG. 2 shows the SDS-PAGE result, channel 1 is a protein standard sample, channel 2 is HSA-Br, channel 3 is unpurified HSA-PDPA, channel 4 is purified HSA-PDPA, compared with HSA-Br, it is shown that channel 3 and channel 4 have a diffuse zone to prove that the polymerization is successful, and compared with channel 3, channel 4 shows that the unpolymerized HSA-Br and the monomer are successfully purified from the product.
The molecular weight of the high molecular chain segment in HSA-PDPA is measured by Gel Permeation Chromatography (GPC), and before the molecular weight is measured, the protein in HSA-PDPA needs to be hydrolyzed, and the method is as follows: 12mg of HSA-PDPA was placed in a hydrolysis tube, while adding 3ml of 6M hydrochloric acid, vacuum-pumping and reacting overnight at 110 ℃, after the reaction was completed, the cleaved polymer was obtained by dialysis, and its molecular weight was characterized by GPC preparation, as shown in FIG. 3, and the number average molecular weight was 10.1kDa and the molecular weight distribution was 1.68.
In the same procedure, the molecular weight of the polymer chain segment in the control HSA-PHPMA was determined by GPC, and the number average molecular weight was 40kDa, and the molecular weight distribution was 1.2, as shown in FIG. 4.
The hydration radii and zeta potentials of HSA-PDPA and HSA-PDPA/ICG were determined by Dynamic Light Scattering (DLS) on a Malvern Zetasizer Nano-zs 90. Samples were diluted in PBS buffer and filtered through a 0.22 μm pore filter before testing. The hydrated diameter of HSA-PDPA was 92nm and that of HSA-PDPA/ICG after ICG encapsulation was 101nm as measured by DLS. Fig. 5a shows the DLS results. The morphology of the protein nanoparticles was observed with a Transmission Electron Microscope (TEM). The protein was diluted to 0.1mg/ml, dropped onto a carbon-covered copper mesh, allowed to stand for 5min, after which the solution was aspirated, stained with a 2% (w/v) phosphotungstic acid solution, air-dried at room temperature, and then the sample was observed with a Hitachi H-7650B transmission electron microscope. TEM showed that both HSA-PDPA and HSA-PDPA/ICG nanoparticles were about 30nm in diameter, and TEM results are shown in FIG. 5 b. To characterize the pH responsiveness of the samples, HSA-PDPA and HDI were dissolved in buffers of different pH and their hydration radii and zeta potentials were determined by DLS, and FIGS. 5c and 5d show that, at a pH change from 6.6 to 6.4, the HSA-PDPA and HDI hydration radii changed from about 86nm and about 120nm to about 10nm, and correspondingly, the zeta potential also changed from negative to positive, indicating that the PDPA was protonated and from hydrophobic to hydrophilic, resulting in disassembly of micelles, while the zeta potential changed to positive due to the protonation system. FIG. 6 shows the distribution of hydrated diameters of HSA-PHPMA and HHI measured by DLS as 81nm and 82nm, and TEM shows that HSA-PHPMA and HHI are micellar in morphology with diameters of 51.6. + -. 6.8nm and 50.88. + -. 9.9nm, respectively. FIG. 7 shows that the particle size at different pH values determined by DLS indicates that HSA-PHPMA and HHI have no pH responsiveness.
According to the embodiment of the present invention, the pKa of PDPA is about 6.5 and that of HSA-PDPA is also 6.5.HSA-PHPMA is not pH responsive between pH 5-8.
The critical concentration of HSA-PDPA and HSA-PHPMA maximum micelles was determined by Nile Red staining method. Nile red dye was dissolved in PBS to a final concentration of 1.25 μ M, and the sample was diluted 1-fold with nile red from 8.3 μ M to 0.03 μ M1. The fluorescence intensity was then measured on a SpectraMax M3Microplate Reader (Molecular Devices) using an excitation light of 550nm and an emission light of 630 nm. FIG. 8 shows that the lowest micelle critical concentrations of HSA-PDPA and HSA-PHPMA are about 0.5. Mu.M and 0.1. Mu.M.
The encapsulation efficiency and drug loading of HSA-PDPA were determined, as determined by a standard curve of ICG concentration ICG, and the concentration of HSA was determined by BCA. The specific steps of the ICG standard curve establishment are as follows: first, a series of concentrations of ICG solution were prepared in PBS containing 30% serum, and the absorbance at 805nm was measured with a microplate reader, and a standard curve was plotted with the concentration as the vertical axis and the absorbance as the horizontal axis. The encapsulation efficiency is defined as the ratio of the mass of ICG loaded in the micelle to the initial mass of ICG added, and the drug loading is defined as the ratio of the mass of ICG loaded in the micelle to the total mass of the system. Table 1 shows the results of their measurements, by which we screened the charge ratios of ICG: HSA = 1.
TABLE 1
The fluorescence and absorption curves of HSA-PDPA/ICG in PBS buffer at different pH were measured using a microplate reader, all samples containing 10. Mu.g/ml ICG. FIG. 9a shows the absorption spectra of ICG, HSA-ICG and HDI at pH7.4 or 6.4, showing that the maximum absorption peak (807 nm) of HDI is much greater than that of HSA-ICG (798 nm) and ICG (774), indicating that most of the ICG is loaded into the micellar core of HSA-PDPA, whose structure is shown in FIG. 9 b. Thus, the fluorescence of HDI was quenched due to aggregation-induced quenching effects, whereas ICG and HHI were absent, as shown in FIG. 9 c. When the pH was changed from 6.6 to 6.4, the fluorescence intensity of HDI suddenly increased as shown in the fluorescence spectrum of FIG. 9d, while the fluorescence intensity at 810nm hardly changed when excited at an excitation wavelength of 740nm in the range of pH5.5-7.4 for ICG and HHI. This also indicates that the pH responsiveness of HSA-PDPA also brings pH responsiveness to the fluorescence properties of ICG.
Example 5 Biosafety and cellular Effect of HSA-PDPA
The entrance effect of HSA-PDPA encapsulated ICG was determined using C8161 melanoma cells transfected with Green Fluorescent Protein (GFP). After culturing the cells in DMEM/F-12 containing 10% FBS, 50U/mL penicillin and 50. Mu.g/mL streptomycin for a certain period of time, a cell suspension (50000 cells) was inoculated at a certain concentration in a 35mm glass-bottomed culture dish, and HSA-PDPA/ICG samples (20. Mu.g/mL ICG) at pH7.4 and pH6.4 were added to the cells for culture for 30min. The culture medium was then discarded, washed 2 times with pre-cooled PBS, then fixed with 4% paraformaldehyde, then washed again with PBS, nucleated with 200. Mu.l Hoechst, then washed again with PBS, and observed with an LSM710 laser scanning confocal microscope (Carl Zeiss) with excitation emission wavelengths of Hoechst, GFP and ICG set at 346/460nm, 488/544nm and 633/664nm. For further confirmation, C8161 was seeded in 6-well plates, similarly added HSA-PDPA/ICG at the same ICG concentration at different pH for half an hour, cells were collected and then assayed by BD FACS Aria III flow cytometer analysis. As shown in FIG. 10, both the confocal microscope and the flow cytometer showed that HHI was not changed in the effect of cell-entry under the conditions of pH7.4 and 6.4, while HDI was more easily incorporated by being electrostatically adsorbed to the cell surface due to protonation of HSA-PDPA to positive charge under acidic conditions, and its intracellular fluorescence intensity was seven times that of HHI under the condition of pH 6.4.
The biological safety of HSA-PDPA was determined by MTT. C8161, L929, MCF-10A and HMECs cell lines were selected. Culturing the cells in DMEM/F-12 (DMEM for L929 and MCF-10A, RPMI-1640 for HMECs) containing 10 FBS, 50U/mL penicillin and 50. Mu.g/mL streptomycin for a period of time, diluting the cells into 96-well plates (20. Mu.l, 5000 wells per well), setting a negative control (not containing HSA-PDPA) and a blank control (containing culture solution only), 37 ℃,5 ℃ CO 2 Culturing for 48h, adding 20 μ L/well of MTT dissolving solution (Promega), measuring the absorbance value of 490nm wavelength of each well by a microplate reader after 3h, and comparing the cell proliferation degree after different samples are treated. FIG. 11 shows that HSA-PDPA has good biosafety at protein concentrations below 300. Mu.g/ml.
Example 6 tumor-targeted delivery Effect and biodistribution test of HSA-PDPA
All of the following animal experiments were performed under the guidelines of the various regulations of the university of Qinghua regarding animal experiments. In the invention, a C8161 melanoma tumor model is established by utilizing a BABL/C athymic female nude mouse model, and when the tumor grows to 150-200mm 3 In this case, mice were randomly divided into three groups, 1.5mg/kg of ICG, HSA-PHPMA/ICG and HSA-PDPA/ICG having no pH response were injected, fluorescence images at 1, 8, 24 and 32 hours were collected using the IVIS Lumina II in vivo imaging System (Perkinelmer), and thereafter the mice were dissected and the fluorescence intensities of heart, liver, spleen, lung, kidney and tumor were measured. As shown in FIG. 12, the fluorescence was distributed throughout the body 1h after injection and concentrated mainly in the liver, and the HHI and HDI nanoprobes began to accumulate on the tumor after 8h due to the enhanced osmotic retention Effect (EPR). At 24 and 32 hours, only HDI fluoresced strongly at the tumor site, with an intensity of tumor fluorescence resolved 3 times that of HHI, 6 times that of ICG, the control group, indicating that HDI can respond to the acidic microenvironment of the tumor, facilitating its tumor imaging.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (21)
1. A method of preparing a protein-polymer amphiphile, comprising:
in a Tris-HCl buffer solution, adding a buffer solution,
(1) Covalently linking an initiator to a protein, said protein being human serum albumin, so as to obtain a protein-initiator conjugate;
(2) Carrying out in-situ polymerization on the protein-initiator combination and a monomer compound, wherein the monomer compound is 2- (diisopropylamino) ethyl methacrylate so as to obtain a protein-macromolecule amphiphile, the macromolecule has pH responsiveness within the pH range of 5-10, and the pKa of the macromolecule is 5-10;
the in-situ polymerization is carried out for 5 minutes to 24 hours under the conditions of low oxygen or inert gas atmosphere, temperature of 0 to 80 ℃ and magnetic stirring;
the in-situ polymerization is carried out under the catalysis of CuCl and 1,4,7, 10-hexamethyl triethylene tetramine;
the molar ratio of the protein-initiator combination to the monomer compound to the CuCl to the 1,4,7, 10-hexamethyl triethylene tetramine is 1 (200-3000) to 10-500 to 10-4000;
wherein the initiator is an initiator for atom transfer radical polymerization reaction.
2. The method of claim 1, wherein the initiator of atom transfer radical polymerization is selected from at least one of polyhalides, halogenated esters, halogenated ketones, and halogenated nitriles.
3. The method of claim 1, wherein the protein and the initiator are covalently linked at a site that is at the N-or C-terminus of the protein and at other sites that do not interfere with the activity of the protein.
4. The method of claim 3, wherein the attachment site is cysteine 34 of human serum albumin.
5. The method of claim 1, wherein the initiator is a compound of formula (I),
6. the method according to claim 1, characterized in that the protein pre-dissolved in a buffer solution is subjected to a linking treatment with the initiator at room temperature for 5 minutes to 24 hours.
7. The method according to claim 1, wherein the coupling ratio of the protein to the initiator is 1.
8. The method of claim 1, wherein the molar ratio of the protein-initiator combination, the monomeric compound, the CuCl, the 1,4,7, 10-hexamethyltriethylene tetramine is 1.
9. A protein-polymer amphiphile obtained by the method according to any one of claims 1 to 8.
10. A nanoparticle formed by the self-assembly of the protein-macromolecule amphiphile of claim 9, comprising:
a hydrophobic core consisting of hydrophobic regions of the protein-macromolecule amphiphile; and
a hydrophilic shell disposed on an outer surface of the hydrophobic core, the hydrophilic shell being comprised of hydrophilic regions of the protein-macromolecule amphiphile.
11. The nanoparticle of claim 10, further comprising a loading compound.
12. The nanoparticle of claim 11, wherein the compound comprises a hydrophobic small molecule drug and a fluorescent molecule.
13. The nanoparticle of claim 12, wherein the small molecule drug is selected from at least one of gemcitabine, paclitaxel, doxorubicin, gefitinib, and methotrexate.
14. The nanoparticle of claim 12, wherein the fluorescent molecule is selected from at least one of sulforhodamine B, indocyanine green, fluorescein isothiocyanate, ethidium bromide, water-soluble fluorescent blue, a theanhydride water-soluble fluorescent dye, a water-soluble cyanine dye succinimide ester dye, phycoerythrin coumarin, a cell membrane red fluorescent probe, rhodamine 200, and fluorescein ester.
15. The nanoparticle of claim 11, wherein the compound is indocyanine green.
16. A method for preparing a nanoprobe, characterized in that the protein-macromolecule amphiphile of claim 9 is contacted with a fluorescent molecule to be entrapped, so as to obtain the nanoprobe.
17. The method according to claim 16, wherein the fluorescent molecule to be encapsulated is at least one selected from the group consisting of sulforhodamine B, indocyanine green, fluorescein isothiocyanate, ethidium bromide, water-soluble fluorescent blue, a thean-based water-soluble fluorescent dye, a water-soluble cyanine dye succinimide ester dye, phycoerythrin coumarin, a cell membrane red fluorescent probe, rhodamine 200, and fluorescein ester.
18. The method of claim 16, wherein the contacting is performed at room temperature under dark conditions for 5 minutes to 24 hours.
19. The method of claim 16, wherein the molar ratio of the protein-macromolecule amphiphile to the fluorescent molecule to be encapsulated is 1 (0.5-20).
20. A pharmaceutical composition comprising the protein-macromolecule amphiphile of claim 9 or comprising the nanoparticle of any one of claims 10 to 15, wherein the protein-macromolecule amphiphile and/or the molecule and/or the protein encapsulated by the nanoparticle is a drug molecule.
21. A method of fluorescence imaging wherein the protein-macromolecule amphiphile of claim 9 or the nanoparticle comprising any one of claims 10 to 15 is delivered to the microenvironment to be imaged, and wherein the protein-macromolecule amphiphile and/or the nanoparticle entrapped molecule is a fluorescent molecule.
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| CA3141707A1 (en) * | 2019-05-24 | 2020-12-03 | Glytech, Inc. | Novel artificial protein catalyst |
| CN110201191B (en) * | 2019-07-10 | 2022-03-25 | 莎穆(上海)生物科技有限公司 | Compound of functional protein and cyanine dye molecule and preparation method and application thereof |
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| CN106674325B (en) * | 2015-11-04 | 2020-07-14 | 清华大学 | Method for preparing interferon high-molecular conjugate IFN-POEGMA |
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| CN105056240A (en) * | 2015-07-21 | 2015-11-18 | 中国药科大学 | PH (potential of hydrogen)-responsive integral dual-energy nano-carrier, method for preparing same and application |
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