CN113121826A - Freeze synthesis and use of polydisulfide conjugates - Google Patents
Freeze synthesis and use of polydisulfide conjugates Download PDFInfo
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- CN113121826A CN113121826A CN202010026036.2A CN202010026036A CN113121826A CN 113121826 A CN113121826 A CN 113121826A CN 202010026036 A CN202010026036 A CN 202010026036A CN 113121826 A CN113121826 A CN 113121826A
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Abstract
The present application relates to the refrigerated synthesis and use of poly disulfide conjugates. Methods for preparing the polymers, methods for isolating substances, kits for isolating proteins, and protein-multimeric drug conjugates and methods for preparing the same are provided.
Description
Technical Field
The present application relates to the field of biomedicine, and more particularly, to a method of preparing a polymer, a method of isolating a substance using the same, a kit for isolating a protein, and a protein-multimeric drug conjugate and a method of preparing the same.
Background
The grafting (grafting) method is an effective chemical modification method, and physical, mechanical and chemical properties of a modified substance can be selectively improved after grafting modification.
For example, the most common and earliest studied grafting method of proteins to polymers is "grafting to", which grafts the synthetic polymer to the protein through ligand-protein interactions or formation of covalent bonds with amino acid residues on the protein. In this manner, temperature-sensitive, pH-sensitive, and light-sensitive polymers have been successfully incorporated into proteins to produce proteins with active "switches". However, there are still many limitations in this way, for example, the steric hindrance effect of the macromolecular polymer, the difficulty of reacting the terminal active group with the specific functional group on the protein, and the low grafting density; the prepared product often contains free protein which is not combined with the polymer, and the separation is difficult.
In view of this, researchers have proposed a grafting method of "grafting from … …", which means that polymerization is initiated directly from a protein after an initiator is grafted onto the protein. Because the steric hindrance effect of the small molecular initiator is small, the initiator can be efficiently combined with protein, and residual monomers and the small molecular initiator after the polymerization is finished are easy to remove. The polymerization methods currently used for "grafting from" are mainly atom-transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization (RAFT). Atom transfer radical polymerization can synthesize polymers with narrow molecular weight distribution, but the contamination is serious because of the use of transition metal catalyst and poor cleaning in the process. Reversible addition-fragmentation chain transfer polymerization generally employs dithioester derivatives as transfer reagents, which allow the polymerization reaction to be effectively controlled. RAFT, however, also has its own limitations, for example, the reagents used are difficult to obtain directly and need to be obtained synthetically; the use of the bisthioester derivative may increase the toxicity of the polymer, etc.
In view of the above, it is imperative to find a new grafting method that is simple and easy to implement, uses reagents that are readily available, has a high grafting density, produces products that are readily separable, and is environmentally friendly.
Disclosure of Invention
In one aspect of the present disclosure, there is provided a method of preparing a polymer, comprising ring-opening polymerizing a substance represented by formula I with a compound represented by formula II at a ring-opening temperature, and then quenching the reaction using an end-capping agent to obtain a polymer represented by formula III:
wherein:
X-L1h represents a substance having mercapto or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, or a substituted or unsubstituted azideSubstituted or unsubstituted carboxylic acid groups, substituted or unsubstituted guanidine groups, substituted or unsubstituted hydroxyl groups, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cationic groups, and wherein R is1To R3Not hydrogen at the same time;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000; and
the ring opening temperature is a temperature of 0 ℃ or lower.
In one embodiment, X is selected from the group consisting of nanoparticles, small molecules, proteins, and polymeric compounds. In one embodiment, X is selected from gold nanoparticles, silver nanoparticles, quantum dots, self-assembled nanoparticles, sodium ethanesulfonate, dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, gene-edited green fluorescent protein, mutase, azoreductase, antibodies.
In one embodiment, Z is a haloamide or anhydride. In one embodiment, Z is iodoacetamide or succinic anhydride.
In one embodiment, the ring opening temperature is from 0 ℃ to-80 ℃. In one embodiment, the ring opening temperature is from-10 ℃ to-30 ℃.
In one embodiment, R4Is selected from
Wherein k is an integer from 1 to 100.
In another aspect of the present disclosure, there is provided a method for separating a substance containing a reactive thiol or selenol, comprising:
a) subjecting a mixture comprising a reactive thiol-or selenol-containing substance represented by formula I and a compound represented by formula II to ring-opening polymerization at a ring-opening temperature, followed by quenching with a capping agent to obtain a product:
wherein:
X-L1h represents a substance having mercapto or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cation groups, and wherein R is a hydrogen atom, a hydroxyl group, a carboxyl group1To R3Not hydrogen at the same time;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000; and
the ring opening temperature is below 0 ℃;
b) separating the polymer represented by formula III from the product; and
c) mixing the polymer represented by formula III with a reducing agent to release the substance represented by formula I in situ.
In one embodiment, the separation is performed by an ion exchange column.
In one embodiment, the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine, and glutathione.
In another aspect of the present disclosure, there is provided a method for separating a protein containing an active thiol or selenol from a protein mixture, comprising:
a) subjecting the protein mixture comprising a protein containing an active thiol or selenol represented by formula IV to ring-opening polymerization with a compound represented by formula II at a ring-opening temperature, followed by quenching the reaction with a capping agent to prepare a conjugate represented by formula V:
wherein:
POI-L1h represents a protein having thiol or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cation, and at least one R is selected from the group consisting of4Is a charged group;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000;
the ring opening temperature is 0 ℃ to-80 ℃;
b) isolating the conjugate represented by formula V; and
c) mixing the conjugate represented by formula V with a reducing agent to release the protein represented by formula IV in situ.
In one embodiment, the protein mixture is selected from the group consisting of cell lysate, serum, plasma, body fluid, and urine.
In one embodiment, the POI is selected from the group consisting of dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, gene-edited green fluorescent protein, mutase, azoreductase, antibody.
In one embodiment, the at least one R is4Is composed of
In one embodiment, Z is selected from a haloamide or an anhydride. In one embodiment, Z is iodoacetamide or succinic anhydride.
In one embodiment, the ring opening temperature is from-10 ℃ to-30 ℃. In one embodiment, the ring opening temperature is-10 ℃ or-30 ℃.
In one embodiment, the isolating comprises isolating the conjugate represented by formula V using an ion exchange column.
In one embodiment, the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine, and glutathione.
In yet another aspect of the present disclosure, there is provided a kit for isolating a protein containing a reactive thiol or selenol, comprising:
a reaction unit configured to perform a ring-opening polymerization reaction between a protein mixture comprising a reactive thiol-or selenol-containing protein represented by formula IV and a compound represented by formula II:
wherein:
POI-L1h represents a protein having thiol or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cation, and at least one R is selected from the group consisting of4Is a charged group;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000;
a separation unit configured to separate a conjugate represented by formula V from the protein mixture; and
a temperature control unit configured to control and maintain a temperature of the reaction unit and/or the separation unit.
In one embodiment, the protein mixture is selected from the group consisting of cell lysate, serum, plasma, body fluid, and urine.
In one embodiment, the POI is selected from the group consisting of dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, gene-edited green fluorescent protein, mutase, azoreductase, antibody.
In one embodiment, the at least one R is4Is composed of
In one embodiment, when at least one R is4Is positively chargedRadicals, e.g.
When the isolated conjugate represented by formula V is further used to effect intracellular delivery.
In one embodiment, the ring opening temperature is from 0 ℃ to-80 ℃. In one embodiment, the ring opening temperature is from-10 ℃ to-30 ℃. In one embodiment, the ring opening temperature is-10 ℃ or-30 ℃.
In one embodiment, the separation unit is configured as an ion exchange column.
In one embodiment, the kit further comprises: a release unit configured to contact the isolated conjugate represented by formula V with a reducing agent, thereby releasing the protein represented by formula IV in situ.
In one embodiment, the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine, and glutathione.
In another aspect of the present disclosure, there is provided a method of preparing a protein-multimeric drug conjugate, comprising:
a) performing ring-opening polymerization of a protein represented by formula IV and a compound represented by formula II at a ring-opening temperature, and then quenching the reaction using a capping agent to prepare a conjugate represented by formula V:
wherein:
POI-L1h represents a protein having thiol or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted orUnsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cation groups, and wherein R is a hydrogen atom, a hydroxyl group, a carboxyl group1To R3Not hydrogen at the same time;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000; and
the ring opening temperature is below 0 ℃;
b) further reacting the conjugate represented by formula V obtained in step a) with a drug to obtain the protein-multimeric drug conjugate.
In one embodiment, the POI is selected from the group consisting of dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, gene-edited green fluorescent protein, mutant enzyme and azoreductase, antibody.
In one embodiment, when said at least one R is4In the case of a hydrazide group, the drug may be doxorubicin.
In one embodiment, the ring opening temperature is from-10 ℃ to-30 ℃.
In yet another aspect of the present disclosure, there is provided a protein-multimeric drug conjugate prepared by the above method.
Drawings
In the following, embodiments illustrated herein will be described in further detail with reference to the accompanying drawings, but it will be understood by those skilled in the art that the drawings are only intended to provide a better understanding of the present disclosure, and are not intended to limit the scope thereof.
FIG. 1 is a graph of protein gel electrophoresis using Tev-Cys-EGFP to initiate in situ polymerization of M1 at different temperatures, according to an embodiment of the present disclosure;
FIG. 2 is a graph of protein gel electrophoresis using Tev-Cys-EGFP to initiate in situ polymerization of M1 at various monomer concentrations, according to an embodiment of the present disclosure;
FIG. 3 is a graph of protein gel electrophoresis using Tev-Cys-EGFP to initiate in situ polymerization of M1 at various pHs, according to an embodiment of the present disclosure;
FIG. 4 is a graph of protein gel electrophoresis using Tev-Cys-EGFP to initiate in situ polymerization of M1 at various times, according to an embodiment of the present disclosure;
fig. 5 is a protein gel electrophoresis image of in situ polymerization of M1 initiated using different proteins, according to an embodiment of the present disclosure;
FIG. 6 is a graph of protein gel electrophoresis using Tev-Cys-EGFP to initiate in situ polymerization of different monomers, according to an embodiment of the present disclosure;
FIG. 7 is a graph of protein gel electrophoresis of in situ released proteins depolymerized with different reducing agents, according to an embodiment of the present disclosure;
FIG. 8 is a mass spectrum of depolymerized in situ released proteins with different reducing agents, according to an embodiment of the disclosure;
fig. 9 is a protein gel electrophoresis image of EGFP isolated from EGFP/azo reductase according to an embodiment of the present disclosure;
fig. 10 is a diagram of the endocytosis of EGFP-poly disulfide conjugates, in accordance with an embodiment of the disclosure. (ii) a
FIG. 11 schematically illustrates a process for preparing a polydisulfide-polyaminoacidified interferon;
figure 12 schematically shows a process for the preparation of doxorubicin @ polydisulfide-polyamino acid interferon;
figure 13 is a drug release profile of doxorubicin @ polydisulfide-polyamino acid interferon, in accordance with an embodiment of the present disclosure;
figure 14A shows cell viability at different concentrations of polyamino-acidified interferon and doxorubicin @ polydisulfide-polyamino-acidified interferon, according to embodiments of the present disclosure; FIG. 14B shows a plot of cell viability versus log doxorubicin concentration plotted by GraphPad Prism 5.0 software analysis and fitting data;
figure 15 shows the cellular uptake effect of doxorubicin and doxorubicin @ polydisulfide-polyamino acid-interferon, according to embodiments of the present disclosure;
figures 16A and 16B show blood concentrations of doxorubicin, polyamino-acidified interferon, and doxorubicin @ polydisulfide-polyamino-acidified interferon, respectively, versus time, according to embodiments of the present disclosure; and
figure 17 shows tumor volume versus time after treatment with various test substances, according to embodiments of the present disclosure.
Detailed Description
Hereinafter, the content of the present application will be further explained according to specific embodiments. However, the particular embodiments listed are for illustrative purposes only and are not intended to limit the content of the present application. Those skilled in the art will recognize that specific features in any of the following embodiments may be used in any of the other embodiments as long as they do not depart from the spirit of the present application.
Definition of
Hereinafter, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, the modifier "C1-C30"refers to a group having any integer value of carbon atoms in the backbone ranging from 1 to 30, for example, 1,2,3,4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 30 carbon atoms. Furthermore, "C" is also known to those skilled in the art1-C30"includes any subrange consisting of a number in the range from 1 to 30, e.g. C2-C30、C6-C30、C1-C20、C2-C20、C6-C20、C2-C10、C6-C18、C6-C12And the like. In addition, "C1-C10”、“C2-C30”、“C3-C30”、“C6-C30"and" C5-C30"is also defined identically and includes any subrange consisting of numerical values within the ranges of" 1 to 10 "," 2 to 30 "," 3 to 30 "," 6 to 30 "," 5 to 30 ", respectively.
As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group having a straight chain or a branched chain. Non-limiting examples thereof include methyl, ethyl, propyl, n-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.
As used herein, the term "alkenyl" refers to a hydrocarbyl group having at least one carbon-carbon double bond at one or more positions along the carbon chain of the alkyl group. Non-limiting examples thereof include ethenyl, propenyl, butenyl and the like.
As used herein, the term "alkynyl" refers to a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the carbon chain of the alkyl group. Non-limiting examples thereof include ethynyl, propynyl, and the like.
As used herein, the terms "heteroalkyl," "heteroalkenyl," "heteroalkynyl" refer to alkyl, alkenyl, alkynyl groups, respectively, that contain at least one heteroatom (e.g., 1 to 5 heteroatoms, such as 1,2, or 3 heteroatoms) selected from N, O, Si, P, and S.
As used herein, the term "cycloalkyl" refers to a monocyclic saturated hydrocarbon group. Non-limiting examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. As used herein, the term "heterocycloalkyl" refers to a monocyclic group containing, as ring-forming atoms, at least one heteroatom selected from N, O, Si, P and S. Non-limiting examples thereof include tetrahydrofuranyl and tetrahydrothienyl.
As used herein, the term "cycloalkenyl" refers to a monocyclic group having carbon atoms and at least one double bond in its ring, and which is not aromatic. Non-limiting examples thereof include cyclopentenyl, cyclohexenyl, and cycloheptenyl. As used herein, the term "heterocycloalkenyl" refers to a monocyclic group including, as ring-forming atoms, at least one heteroatom selected from N, O, Si, P, and S, and at least one double bond in its ring. Non-limiting examples thereof include 4, 5-dihydro-1, 2,3, 4-oxatriazolyl, 2, 3-dihydrofuranyl, and 2, 3-dihydrothienyl.
As used herein, the term "aryl" refers to a group comprising a carbocyclic aromatic system. Non-limiting examples thereof include phenyl, naphthyl, anthryl, phenanthryl, pyrenyl, and the like. When the aryl group includes a plurality of rings, the respective rings may be fused to each other.
As used herein, the term "heteroaryl" refers to a group having a carbocyclic aromatic system containing as ring-forming atoms at least one heteroatom selected from N, O, Si, P and S (e.g., 1 to 5 heteroatoms, such as 1,2 or 3 heteroatoms). Non-limiting examples thereof include pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolinyl, isoquinolinyl, and the like. When the heteroaryl group includes a plurality of rings, the respective rings may be fused to each other.
The term "substituted or unsubstituted" as used herein corresponds to substituted or unsubstituted with at least one substituent selected from the group consisting of a halogen atom, a hydroxyl group, a sulfonic group, a cyano group, a nitro group, an amino group, a silyl group, an amide group, an ester group, a carboxylic acid group, a guanidine group, ammoniumCationic group, C1-C20Alkyl radical, C2-C20Alkenyl radical, C6-C22Aryl radical, C3-C20Heteroaryl, polyethylene glycol (e.g., polyethylene glycol comprising 1 to 20 ethylene glycols). Further, each of the substituents exemplified above may also be substituted or unsubstituted. For example, a biphenyl group can be interpreted as an aryl group or a phenyl group substituted with a phenyl group.
As used herein, the term "protein" refers to a wild-type protein or a genetically engineered protein having medical uses. Non-limiting examples thereof include various types of antibodies such as infliximab, adalimumab, rituximab, and other anti-PD-1/PD-L1, anti-CTLA-4, anti-EGFR, anti-HER 2, anti-TNF α, anti-CD 19, anti-CD 33, anti-CD 30, anti-CD 20, anti-CD 25 antibodies; enzymes such as dihydrofolate reductase, transpeptidase, mutase, azo reductase, uricase, arginase, carboxypeptidase, phenylalanine ammonia lyase; growth factors and cytokines such as growth hormone, G-CSF, cytokines and chemokines (IL-2, interferon-. alpha.2a, interferon-. alpha.2b, interferon-2 a, factor VIII, factor IX, interferon-. beta.1a, interferon-. gamma.); bovine serum albumin; gene-edited green fluorescent protein, and the like.
As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Terms in the singular may include the plural unless the context clearly dictates otherwise.
Process for preparing polymers
In one embodiment of the present disclosure, a method of making a polymer is provided, comprising ring-opening polymerizing a material represented by formula I with a compound represented by formula II at a ring-opening temperature, followed by quenching the reaction with an endcapping agent to obtain a polymer represented by formula III:
wherein:
X-L1H、R1to R3Z, Y, n and the ring opening temperature are as defined above.
In embodiments of the present disclosure, X may be a substance having medical use. As used herein, the term "medical use" is not limited to therapeutic, palliative and prophylactic uses, but also includes auxiliary medical uses, such as auxiliary drug delivery.
In embodiments of the present disclosure, X may be a nanomaterial with medical uses, such as gold nanoparticles, silver nanoparticles, quantum dots, nanowires, nanotubes, self-assembled nanoparticles, and the like. X may also be a protein with medical use, for example antibodies such as infliximab, adalimumab, rituximab, and other anti-PD-1/PD-L1, anti-CTLA-4, anti-EGFR, anti-HER 2, anti-TNF α, anti-CD 19, anti-CD 33, anti-CD 30, anti-CD 20, anti-CD 25 antibodies; enzymes such as dihydrofolate reductase, transpeptidase, mutase, azo reductase, uricase, arginase, carboxypeptidase, phenylalanine ammonia lyase; growth factors and cytokines such as growth hormone, G-CSF, cytokines and chemokines (IL-2, interferon-. alpha.2a, interferon-. alpha.2b, interferon-2 a, factor VIII, factor IX, interferon-. beta.1a, interferon-. gamma.); bovine serum albumin; gene-edited green fluorescent protein, and the like. However, embodiments of the present disclosure are not limited thereto.
According to embodiments of the present disclosure, enzymes useful in the present disclosure include, but are not limited to: dihydrofolate reductase, transpeptidase, mutase, azoreductase, proteolytic enzyme, amylase, lipase, cellulase, trypsin, chymotrypsin, streptokinase, urokinase, plasmin, thrombin, glutaminase, arginase, serine dehydratase, phenylalanine aminolyase, leucine dehydrogenase, penicillinase, superoxide dismutase, dextranase and/or dextranase.
According to embodiments of the present disclosure, hormones that may be used in the present disclosure include, but are not limited to, hypothalamic hormones, pituitary hormones, gastrointestinal hormones, insulin, calcitonin.
According to embodiments of the present disclosure, cytokines useful in the present disclosure include, but are not limited to, interleukins, interferons, colony stimulating factors, chemokines, and/or growth factors.
According to embodiments of the present disclosure, interleukins useful in the present disclosure include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, and/or IL-32.
According to embodiments of the present disclosure, interferons useful in the present disclosure include, but are not limited to, IFN- α, IFN- β, IFN- γ α, IFN- λ, and subtypes thereof.
Colony stimulating factors useful in the present disclosure, according to embodiments of the present disclosure, include, but are not limited to: granulocyte colony stimulating factor, macrophage colony stimulating factor, granulocyte-macrophage colony stimulating factor, pluripotent colony stimulating factor, stem cell factor, leukemia inhibitory factor, and/or erythropoietin.
According to embodiments of the present disclosure, growth factors useful in the present disclosure include, but are not limited to, epidermal growth factor, transforming growth factor, insulin-like growth factor, and/or nerve growth factor.
According to embodiments of the present disclosure, monoclonal antibodies useful in the present application include, but are not limited to: trastuzumab, cetuximab, darlizumab, talnizumab, abavacizumab, aldumumab, alfuzumab, alemtuzumab, certolizumab pegol, amatuzumab, aprezumab, baveximab, betuzumab, belimumab, bevacizumab, motbivatuzumab, berrituximab-vedotti, mo-trastuzumab, la-trastuzumab, carpuzumab-pentozumab, katsutuzumab, Pogostemab, cetuzumab, Coitumumab, daclizumab, delmomab, exemestab, Egylcozumab, Egylocumab, Enscizumab, Enspelizumab, Epapuzumab, Espelizumab, Egymazumab, Eduzumab, Fatuzumab, Futuzumab, Garituzumab, Geiguzumab, Rituzumab, and Egylcob, Gemtuzumab ozogamicin, gemtuzumab-vedottin, over-itumomab, agovacizumab, lat-infliximab, inflitumumab, eculizumab-ozotacin, ipilimumab, itumumab, labezumab, lexamumab, lintuzumab, mo-lovozumab, lucatumab, ruiximab, mapatuzumab, matuzumab, milatuzumab, mitumumab, moguzumab, tanacetumab, natalizumab, nimotuzumab, nituzumab, nivolumab, ofamtuzumab, omab, omalizumab, motuzumab, panitumumab, pertuzumab, pemphilizumab, rituximab, and omalizumab, Cetuzumab, cetuximab, parp-tamitumumab, temtuzumab, temitumumab, tremelimumab, tegafuzumab, tositumomab, simukulmumab, ureuzumab, vituzumab, voluximab, voltemitumomab, and zalutumumab, including antigen-binding fragments thereof.
In embodiments of the disclosure, R1To R3May be both different from hydrogen; when R is1To R3When any one of them is hydrogen, the remaining two may not be hydrogen, and may be the same as or different from each other; when R is1To R3When two of them are both hydrogen, the other one is not hydrogen.
In an embodiment of the present disclosure, L2May be a direct bond, or a methyl, ethyl, propyl, butyl, pentyl or hexyl group; and
R4including but not limited to
Wherein k is an integer from 1 to 100.
In an embodiment of the disclosure, R1To R3May be a group having pharmaceutical activity or a prodrug group thereof. As used herein, the term "pharmaceutically active" refers to having a pharmacological or other direct effect or being capable of affecting the function and structure of the body in the diagnosis, treatment, symptom relief, management of a disease, or prevention of a disease. By "precursor group" is meant herein a group which is chemically modified to obtain a group which is inactive or less active in vitro and which releases a pharmaceutically active group in vivo by enzymatic or non-enzymatic conversion.
In embodiments of the disclosure, X and R1To R3One or more of which may have pharmaceutical activity or medical use at the same time; or, X and R1To R3One or more of which may have adjunctive medical or pharmaceutical use, while the remainder optionally have pharmaceutical activity or medical use.
In embodiments of the present disclosure, capping agents include, but are not limited to, haloamides and anhydrides, and in particular, the capping agent may be selected from C2-C10Halogenated amides and C2-C10Anhydrides, such as iodoacetamide or succinic anhydride. Alternatively, other functional groups, such as fluorophores, can also be introduced via Z.
In embodiments of the present disclosure, the ring opening temperature may be from 0 ℃ to-80 ℃. For example, the ring-opening temperature is from 0 ℃ to-70 ℃, -from 10 ℃ to-60 ℃, -from 10 ℃ to-40 ℃, -from 10 ℃ to-30 ℃. In one embodiment, the ring opening temperature may be 0 ℃, -10 ℃, -20 ℃, -30 ℃, -40 ℃, -50 ℃, -60 ℃, -70 ℃, -80 ℃, -90 ℃ or-100 ℃.
In embodiments of the present disclosure, when X is a protein, the ring-opening polymerization reaction cannot be performed at a temperature above 0 ℃ (e.g., room temperature), even at relatively high initial monomer concentrations, such as 300 mM. Moreover, higher initial monomer concentrations are prone to undesirable side reactions and precipitation of proteins.
In embodiments of the present disclosure, when X is a protein, grafting efficiencies can reach 75% and 84% at initial monomer concentrations of 50mM and 100mM, respectively.
Separation method
1) General separation method
According to another embodiment of the present disclosure, there is provided a method for separating a substance represented by formula I, comprising:
a) subjecting a mixture comprising a substance represented by formula I and a compound represented by formula II to ring opening polymerization at a ring opening temperature, followed by quenching with a capping agent to obtain a product:
wherein: X-L1H、R1To R3Z, Y, n and the ring opening temperature are as defined above;
b) separating the polymer represented by formula III from the product; and
c) mixing the polymer represented by formula III with a reducing agent to release the substance represented by formula I in situ.
In embodiments of the present disclosure, X may be a substance having a medical use as described above, but may also be a substance having other uses.
In an embodiment of the disclosure, R1To R3At least one of which may be a charged group, in which case the separation may be performed by means of an ion exchange column. However, embodiments of the present disclosure are not limited thereto.
According to the separation method of the embodiments of the present disclosure, since a substance having an active mercapto group or selenol is used and a small molecule mercapto group or selenol is used as an initiator, on the one hand, in-situ polymerization can be achieved, and since the steric hindrance effect of the initiator is small, higher grafting efficiency can be achieved, and the polymer is easily separated from the unreacted monomer after polymerization; on the other hand, traceless release of the target isolated substance can also be achieved, i.e. after reaction with a reducing agent, a substance having an active thiol or selenol represented by formula I can be released tracelessly, and used for other reactions or applications without treatment.
2) Method for separating protein
According to another embodiment of the present disclosure, there is provided a method for separating a protein represented by formula IV from a protein mixture, comprising:
a) subjecting the protein mixture comprising the protein represented by formula IV and the compound represented by formula II to ring-opening polymerization at a ring-opening temperature, and then quenching the reaction using a capping agent to prepare a conjugate represented by formula V:
wherein: POI-L1H、R1To R3Z, Y, n and the ring opening temperature are as defined above;
b) isolating the conjugate represented by formula V; and
c) mixing the conjugate represented by formula V with a reducing agent to release the protein represented by formula IV in situ.
In embodiments of the present disclosure, the protein mixture may be selected from the group consisting of cell lysate, serum, plasma, body fluid, and urine.
In embodiments of the present disclosure, the POI may be a protein having medical use, for example, antibodies such as infliximab, adalimumab, rituximab, and other anti-PD-1/PD-L1, anti-CTLA-4, anti-EGFR, anti-HER 2, anti-TNF α, anti-CD 19, anti-CD 33, anti-CD 30, anti-CD 20, anti-CD 25 antibodies; enzymes such as dihydrofolate reductase, transpeptidase, mutase, azo reductase, uricase, arginase, carboxypeptidase, phenylalanine ammonia lyase; growth factors and cytokines such as growth hormone, G-CSF, cytokines and chemokines (IL-2, interferon-. alpha.2a, interferon-. alpha.2b, interferon-2 a, factor VIII, factor IX, interferon-. beta.1a, interferon-. gamma.); bovine serum albumin; gene-edited green fluorescent protein, and the like. However, embodiments of the present disclosure are not limited thereto.
In an embodiment of the present disclosure, the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine, and glutathione. However, embodiments of the present disclosure are not limited thereto.
Reagent kit
According to still another embodiment of the present disclosure, there is provided a kit for isolating a protein represented by formula IV, including:
a reaction unit configured to perform a ring-opening polymerization reaction between a protein mixture and a compound represented by formula II:
wherein: POI-L1H、R1To R3Z, Y and n are as defined above;
a separation unit configured to separate a conjugate represented by formula V from the protein mixture; and
a temperature control unit configured to control and maintain a temperature of the reaction unit and/or the separation unit.
In embodiments of the present disclosure, the protein mixture may be selected from the group consisting of cell lysate, serum, plasma, body fluid, and urine.
In embodiments of the present disclosure, the temperature control unit may be configured to set and control only the temperature of the reaction unit, e.g., control the temperature of the reaction unit at the open loop temperature. The ring-opening temperature may be from 0 ℃ to-80 ℃, for example, from 0 ℃ to-70 ℃, -from 10 ℃ to-60 ℃, -from 10 ℃ to-40 ℃, -from 10 ℃ to-30 ℃. In one embodiment, the ring opening temperature may be 0 ℃, -10 ℃, -20 ℃, -30 ℃, -40 ℃, -50 ℃, -60 ℃, -70 ℃, -80 ℃, -90 ℃ or-100 ℃. The temperature control unit may also be configured to set and control the temperatures of the reaction unit and the separation unit, respectively. For example, the reaction unit is controlled at the ring-opening temperature as described above, and the temperature of the separation unit is set at an appropriate temperature depending on the separation method employed, for example, 10 ℃ to 50 ℃, 10 ℃ to 40 ℃, 10 ℃ to 30 ℃, 10 ℃ to 20 ℃, 20 ℃ to 40 ℃, 20 ℃ to 30 ℃, for example, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ or 50 ℃. However, embodiments of the present disclosure are not limited thereto.
In an embodiment of the disclosure, when R1To R3When at least one of the groups is a charged group, the separation unit may be configured as an ion exchange column. However, embodiments of the present disclosure are not limited thereto.
In an embodiment of the disclosure, when R1To R3At least one of which is selected from positively charged groups, i.e., at least one R4In the case of positively charged groups such as amino, N-dimethylamino or guanidino, the isolated conjugate of formula V may be further used to effect intracellular delivery by virtue of the positively charged nature of the polymer and the exchange of disulfide bonds with thiol groups of the cell membrane.
In embodiments of the present disclosure, the kit may further comprise: a release unit configured to contact the isolated conjugate represented by formula V with a reducing agent, thereby releasing the protein represented by formula IV in situ. However, embodiments of the present disclosure are not limited thereto.
Of course, according to embodiments of the present disclosure, the kit may also include other units commonly used in the art, such as an injection unit, which may be configured to hold a protein mixture; a fractionating unit, which may be configured to fractionate an unwanted protein mixture or the like from the separation unit.
Preparation of protein-multimeric drug conjugates
According to an embodiment of the present disclosure, there is provided a method of preparing a protein-multimeric drug conjugate, comprising:
a) performing ring-opening polymerization of a protein represented by formula IV and a compound represented by formula II at a ring-opening temperature, and then quenching the reaction using a capping agent to prepare a conjugate represented by formula V:
wherein: POI-L1H、R1To R3Z, Y, n and the ring opening temperature are as defined above;
b) further reacting the conjugate represented by formula V obtained in step a) with a drug to obtain the protein-multimeric drug conjugate.
In embodiments of the present disclosure, the drug may be a substance having a pharmaceutical activity, or may be a prodrug thereof. By "prodrug" is meant herein a drug which has been chemically modified to obtain a drug which is inactive or less active in vitro and which releases the drug with pharmaceutical activity in vivo by enzymatic or non-enzymatic conversion. Such drugs include, but are not limited to, doxorubicin, DOX, paclitaxel, bivalent platinum drugs, such as cisplatin, carboplatin, oxaliplatin; small interfering RNA (siRNA).
Protein-multimeric drug conjugates
According to an embodiment of the present disclosure, there is provided a protein-polymeric drug conjugate prepared by the above method.
According to embodiments of the present disclosure, the protein-polymeric drug conjugate can effectively achieve intracellular delivery of a drug, and can effectively release a pharmaceutically active ingredient via in vivo transformation, achieving sustained release of the drug.
Examples
In the following, the corresponding names of the different polymerized monomers are abbreviated as follows:
example 1: initiation of in situ polymerization of M1 monomer at different temperatures using Tev-Cys-EGFP
Lipoic acid monomer M1(206.33mg,1.0mmol) was dissolved in an equal stoichiometric 0.1M aqueous solution of sodium hydroxide (10ml) to prepare a neutral M1 monomer solution at a final concentration of 100 mM. The pH of the M1 solution was carefully adjusted to 7.0 (monitored by a pH meter) prior to use by the addition of 6M HCl or NaOH. TEV-Cys-EGFP protein solution (2.0mg/mL) was added to the freshly prepared M1 solution (50. mu.L), the mixture was incubated at room temperature, in a refrigerator at 4 ℃ and in a refrigerator at-30 ℃ for 120 minutes, and the reaction was quenched with iodoacetamide (10. mu. L x 0.1.1M) at room temperature for about 30 minutes. The reaction results were observed by non-reducing SDS-PAGE.
FIG. 1 shows the protein gel electrophoresis image using Tev-Cys-EGFP to initiate M1 polymerization in situ at different temperatures. As can be seen from FIG. 1, the formation of polymer could not be detected at the ring-opening temperature of room temperature or 4 ℃.
Example 2: Tev-Cys-EGFP initiated in situ polymerization of M1 monomer at monomer concentration
Neutral M1 monomer solutions (5, 10, 25, 50, 100mM) were prepared at various concentrations by dissolving lipoic acid monomer M1(206.33mg,1.0mmol) in an equal stoichiometric 0.1M aqueous solution of sodium hydroxide (10 ml). The pH of the M1 solution was carefully adjusted to 7.0 (monitored by a pH meter) prior to use by the addition of 6M HCl or NaOH. TEV-Cys-EGFP protein solution (2.0mg/mL) was added to a fresh M1 solution (5, 10, 25, 50, 100mM, 50. mu.L), the mixture was incubated at-30 ℃ for 120 min in a refrigerator and the reaction was quenched with iodoacetamide (10. mu. L x 0.1.1M) at room temperature for about 30 min. The reaction results were observed by non-reducing SDS-PAGE, as shown in FIG. 2.
Example 3: Tev-Cys-EGFP initiates in-situ polymerization of M1 monomer at different pH
Lipoic acid monomer M1(206.33mg,1.0mmol) was dissolved in an equal stoichiometric 0.1M aqueous solution of sodium hydroxide (10ml) to prepare a neutral M1 monomer solution at a final concentration of 100 mM. The pH of the M1 solution was carefully adjusted prior to use by the addition of 6M HCl or NaOH (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, monitored by pH meter). TEV-Cys-EGFP protein solution (2.0mg/mL) was added to a fresh M1 solution (50. mu.L), the mixture was incubated at-30 ℃ for 120 min and the reaction was quenched with iodoacetamide (10. mu. L x 0.1.1M) at room temperature for about 30 min. The reaction results were observed by non-reducing SDS-PAGE, as shown in FIG. 3.
Example 4: Tev-Cys-EGFP initiates in-situ polymerization of M1 monomer at different times
Lipoic acid monomer M1(206.33mg,1.0mmol) was dissolved in an equal stoichiometric 0.1M aqueous solution of sodium hydroxide (10ml) to prepare a neutral M1 monomer solution at a final concentration of 100 mM. The pH of the M1 solution was carefully adjusted to 7.0 (monitored by a pH meter) prior to use by the addition of 6M HCl or NaOH. TEV-Cys-EGFP protein solution (2.0mg/mL) was added to a fresh M1 solution (50. mu.L), the mixture was incubated at-30 ℃ for 15 min, 30min, 45 min, 60 min, 90 min or 120 min in a refrigerator, and the reaction was quenched with iodoacetamide (10. mu.L. x0.1M) at room temperature for about 30 min. The reaction results were observed by non-reducing SDS-PAGE, as shown in FIG. 4.
Example 5: different proteins initiate in situ polymerization of M1 monomer
Lipoic acid monomer M1(206.33mg,1.0mmol) was dissolved in an equal stoichiometric 0.1M aqueous solution of sodium hydroxide (10ml) to prepare a neutral M1 monomer solution at a final concentration of 100 mM. The pH of the M1 solution was carefully adjusted to 7.0 (monitored by a pH meter) prior to use by the addition of 6M HCl or NaOH. Different protein solutions (Tev-Cys-AzoR, wt-AzoR, BSA, DHFR, Sortase, UCHT1) (2.0mg/mL) were added to a fresh M1 solution (50. mu.L), the mixture was incubated at-30 ℃ for 120 min in a refrigerator and the reaction was quenched with iodoacetamide (10. mu. L x 0.1.1M) at room temperature for about 30 min. The results of the reaction were observed by non-reducing SDS-PAGE, and the results are shown in FIG. 5.
Example 6: Tev-Cys-EGFP initiates in-situ polymerization of different monomers
Monomer M1(206.33mg,1.0mmol) was dissolved in an equal stoichiometric 0.1M aqueous solution of sodium hydroxide (10ml) to prepare a neutral M1 monomer solution at a final concentration of 100mM, the pH of the solution being carefully adjusted to 7.0 before use by addition of 6M HCl or NaOH (monitored by a pH meter). Monomer solutions of M2 and M3 were prepared in a similar manner to the monomer solution of M1, except that 2M NaCl solution was added to prevent protein precipitation. TEV-Cys-EGFP protein solution (2.0mg/mL) was added to a fresh M1-M3 monomer solution (50. mu.L), the mixture was incubated at-30 ℃ for 120 min and the reaction was quenched with iodoacetamide (10. mu. L x0.1M) at room temperature for about 30 min. The reaction results were observed by non-reducing SDS-PAGE, as shown in FIG. 6.
Example 7: unzipping Tev-Cys-EGFP conjugates with different reducing agents for traceless release
A reducing agent, Dithiothreitol (DTT) at a concentration of 2mM, tris (2-carboxyethyl) phosphine (TCEP) at a concentration of 2mM, or Glutathione (GSH) at a concentration of 10mM, was added to the Tev-Cys-EGFP conjugate, which was incubated at 37 ℃ for 10 minutes, and the reaction results were observed by non-reducing SDS-PAGE, as shown in FIG. 7. And comparing the mass spectrum of the protein after depolymerization using different reducing agents with the mass spectrum of the protein before polymerization, as shown in fig. 8, traceless release of the Tev-Cys-EGFP conjugate could be achieved with all three reducing agents.
Example 8: in situ freeze polymerization separation in Tev-Cys-EGFP and wild-type azo reductase
Lipoic acid monomer M1(206.33mg,1.0mmol) was dissolved in an equal stoichiometric 0.1M aqueous solution of sodium hydroxide (10ml) to prepare a neutral M1 monomer solution at a final concentration of 100 mM. The pH of the M1 solution was carefully adjusted to 7.0 (monitored by a pH meter) prior to use by the addition of 6M HCl or NaOH. Tev-Cys-EGFP and wild-type azoreductase (both 2.0mg/mL) were added to a fresh solution of M1 (50. mu.L), the mixture was incubated at-30 ℃ for 120 min and the reaction was quenched with iodoacetamide (10. mu. L x 0.1.1M) at room temperature for about 30 min. And removing unreacted lipoic acid micromolecules from the obtained reaction system, and eluting by using an anion exchange column through solutions with different NaCl concentrations to obtain a clean Tev-Cys-EGFP conjugate. The reaction results were observed by non-reducing SDS-PAGE, as shown in FIG. 9.
Example 9: intracellular delivery realized by Tev-Cys-EGFP in-situ freezing polymerization
Lipoic acid monomer M2 was prepared as a neutral monomer solution with a final concentration of 100mM, and 2M NaCl solution was added to prevent protein precipitation. Tev-Cys-EGFP (2.0mg/mL) was added to a fresh solution of M2 (50. mu.L), the mixture was incubated at-30 ℃ for 120 min and the reaction was quenched with iodoacetamide (10. mu. L x 0.1.1M) at room temperature for about 30 min. Inoculating 50000 Hela cells in a cell culture dish, and culturing at 37 deg.C with 5% CO2Was incubated in the incubator of (1) for 24 hours. After aspirating the medium, the cells were incubated with Opti-MEM containing EGFP-disulfide conjugate (100nM X1.0 mL) at pH 6.8 for 1h at 37 ℃ with the same concentration of EGFP as a control. The medium was then washed three times with PBS containing 0.1mg/mL heparin sodium, and the cells were washed with Hoechst (staining nuclei) and LysoTrackerTM(lysosome) was stained for 20 minutes, the staining agent was washed off and then the medium was added again, and the endocytosis results were observed with a confocal laser scanning microscope (as shown in FIG. 10). The excitation wavelength is 488nm, and the emission wavelength is 640 nm. The free protein control group (EGFP) showed no detectable intracellular fluorescence. It is thus seen that protein poly-disulfide conjugates achieve efficient intracellular delivery.
Example 10: preparation of Adriamycin @ Poly disulfide-polyamino acid Interferon
a) Preparation of Poly-disulfide-polyamino-acidified Interferon
As shown in fig. 11, lipoic acid hydrazide (13mg, 59 μmol) was dissolved in 100 μ L DMSO to give a DMSO solution of lipoic acid hydrazide monomer at about 590 mM. Subsequently, 400. mu.L of PBS was added and sufficiently dissolved to obtain an aqueous lipoic acid hydrazide monomer solution of about 100 mM. 1mL of polyamino-acid interferon solution (1mg/mL) was added to 500. mu.L of the above lipoic acid hydrazide monomer aqueous solution, mixed well, incubated at-30 ℃ for 120 minutes in a cryostat, and quenched with iodoacetamide (20. mu. L x 0.5.5M) at room temperature for about 30 minutes. To obtain the poly disulfide-polyamino acid interferon.
b) Preparation of Adriamycin @ Poly disulfide-polyamino acid Interferon
As shown in FIG. 12, 5mg of doxorubicin hydrochloride (DOX) was dissolved in 0.4mL of DMSO, diluted with 3mL of PBS (10mM pH 7.4), followed by slow addition of 3.5mL of a polydisulfide-polyaminoacidified interferon solution (about 0.3mg/mL), and stirred at room temperature with exclusion of light for 24 h. The purification was carried out by dialysis several times with PBS (10mM pH 7.4). And (3) measuring the light absorption value of the sample solution at 495nm by using a microplate reader, and converting according to a standard curve to obtain the load capacity of the adriamycin. Drawing an adriamycin standard curve: doxorubicin was dissolved in PBS and diluted to a range of concentrations (0, 1,2, 4, 8, 16, 32, 64 μ g/mL), absorbance at 495nm was measured for various concentrations of doxorubicin solution, and a standard curve was plotted.
Example 11: drug delivery of doxorubicin @ polydisulfide-polyamino acid interferon
The indicated concentration of doxorubicin @ polydisulfide-polyaminoacidified interferon sample solution was placed in a dialysis bag and immersed in 5mL of a solution of pH 7.4, pH 6.0 and pH 7.4 containing 10mM glutathione, respectively, and incubated at 37 ℃. At different time points, the absorbance value of the buffer solution outside the dialysis bag at 495nm is measured by a microplate reader, and the concentration of the adriamycin can be converted according to a standard curve, thereby drawing a curve graph of the release percentage of the adriamycin and the time.
As can be seen in fig. 13, at the appropriate pH, drug release of doxorubicin @ polydisulfide-polyamino acid interferon occurs only in the presence of the reducing agent (glutathione).
Example 12: cytotoxicity test of Adriamycin @ Poly disulfide-polyamino acid interferon
Cytotoxicity experiments were performed using human ovarian cancer cells (SKOV 3). The cells were cultured in RMPI-1640 containing 10% fetal bovine serum, 50U/mL Penicillin (Penicillin) and 50. mu.g/mL Streptomycin (Streptomyces). Inoculating to 96-well plate (80 μ L/well, 5000 cells/well), culturing for 12 hr, and allowing adriamycin and polyamino to adhere sufficientlyThree samples of acidified interferon and doxorubicin @ polydisulfide-polyaminoacidified interferon were diluted stepwise, 20. mu.L each was added to a 96-well plate and placed in a cell culture chamber (37 ℃, 5% CO)2) And (5) incubating for 48h or 72 h. Cell proliferation was measured by CellTiter-Blue assay, adding CellTiter-Blue (20. mu.L/well), measuring the fluorescence at 590nm (excitation 560nm) in each well after 1h using a microplate reader, and comparing the inhibition of cells by different samples (see FIG. 14A). Data were analyzed and fitted by GraphPad Prism 5.0 software and the maximum semi-lethal dose IC50 was calculated (see fig. 14B).
Example 13: cellular uptake assay for doxorubicin @ polydisulfide-polyamino acid interferon
Cell culture dishes were seeded with 50000 SKOV3 cells at 37 ℃ in 5% CO2The cells were incubated in the incubator for 12 hours, and the cells were fully attached to the wall and replaced with fresh medium. Followed by incubation with 20 μ M doxorubicin or doxorubicin @ polydisulfide-polyaminoacidified interferon at 37 ℃ for various times (10 min, 30min, 1h and 2 h), rinsing three times with PBS containing 0.1mg/mL heparin sodium, staining the cells with Hoechst (stained nuclei) for 20min, adding medium again after washing off the stain, and observing the effect of uptake of doxorubicin by the cells using confocal laser scanning microscopy (as shown in FIG. 15). The excitation wavelength of the adriamycin is 488nm, and the emission wavelength is 590 nm.
As can be seen from the diffuse fluorescent signals in the figure, the adriamycin @ poly disulfide-polyamino acid interferon has high lysosome escape capacity and is effectively distributed and enriched in the cytosol.
Example 14: pharmacokinetic experiments for doxorubicin @ polydisulfide-polyamino acid interferonFemale Sprague-Duller mice (Sprague-Dawley, SD) weighing approximately 250g on average were used to evaluate the pharmacokinetic properties of doxorubicin, polyamino-interferon and doxorubicin @ polydisulfide-polyamino-acidified interferon. SD rats were randomly divided into two groups (3 per group) and were injected intraperitoneally with physically mixed doxorubicin + polyamino-acidified interferon and doxorubicin @ polydisulfide-polyamino-acidified interferon, respectively. Interferon agentsThe amount was 0.2mg IFN/kg body weight. The dose of doxorubicin was 0.6mg DOX/kg body weight. At appropriate time points (45 min, 2h, 5 h, 8h, 12h, 24h, 48h and 72 h) blood was drawn from the orbit, left at 4 ℃ for 30min and centrifuged at 4500 Xg for 20min, plasma collected and stored at-80 ℃. The concentration of IFN was determined using an ELISA kit, while the plasma background values of the mice without drug injection were subtracted. The concentration of DOX was determined using a microplate reader. Pharmacokinetic relevant parameters were analyzed by GraphPad Prism 5.0 software.
As can be seen from fig. 16A and 16B, the polyamino-acid interferon serves to assist in drug delivery. In contrast to doxorubicin, doxorubicin @ polydisulfide-polyaminoacidified interferon prepared according to the present disclosure can maintain a steady blood level of doxorubicin over a long period of time, e.g., 72 hours, i.e., achieve sustained release of the drug.
Example 15: antitumor experiment of adriamycin @ polydisulfide-polyamino acid interferon
Human ovarian carcinoma cell line SKOV3 was grown in RMPI-1640 medium containing 10% fetal bovine serum, 50U/mL penicillin and 50. mu.g/mL streptomycin, cells were collected and suspended in serum-free medium, and 1.5X 10 cells were added7A suspension of SKOV3 cells (0.1mL) was inoculated subcutaneously around the right mammary gland of 4-week-old female BALB/c nude mice. When the tumor grows to 50mm3(about two weeks is required), the mice are randomly divided into 5 groups (5-7 mice per group), and each is injected with an equal dose of saline, doxorubicin, polyamino interferon, doxorubicin + polyamino interferon or doxorubicin @ polydisulfide-polyamino interferon. The interferon was administered at a dose of 1mg/kg body weight of mice, and doxorubicin was administered at a dose of 3mg/kg body weight of mice. Once every four days, four times. Mice body weight and tumor size were measured every two days, tumor volume ═ length × width/2. Nude mice with tumor growth over 1000mm3Or the weight loss exceeds 15 percent, and the nude mice are judged to die. Data were analyzed using GraphPad Prism 5.0 software. As can be seen in FIG. 17, doxorubicin @ polydisulfide-polyamino acid interferon in accordance with the present disclosure exhibited superior resultsThe function of inhibiting tumor growth.
While the inventive concept has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the inventive concept. It is to be understood, therefore, that the above-described embodiments are illustrative and not restrictive in all respects.
Claims (10)
1. A method of preparing a polymer, comprising ring-opening polymerizing a substance represented by formula I with a compound represented by formula II at a ring-opening temperature, and then quenching the reaction with an end-capping agent to obtain a polymer represented by formula III:
wherein:
X-L1h represents a substance having mercapto or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cation groups, and wherein R is a hydrogen atom, a hydroxyl group, a carboxyl group1To R3Not hydrogen at the same time;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000; and
the ring opening temperature is a temperature of 0 ℃ or lower.
2. The method of claim 1, wherein:
x is selected from nanoparticles such as gold nanoparticles, silver nanoparticles, quantum dots, self-assembled nanoparticles; small molecules, such as sodium ethane sulfonate; proteins, for example wild-type proteins or genetically engineered proteins, such as dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, genetically edited green fluorescent protein, mutated enzymes, azoreductase, antibodies; and a high molecular compound;
R4is selected from
Wherein k is an integer from 1 to 100;
z is selected from halogenated amides or anhydrides, such as iodoacetamide, succinic anhydride; and
the ring opening temperature is from 0 ℃ to-80 ℃, for example from-10 ℃ to-30 ℃.
3. A method for separating a substance containing a reactive thiol or selenol, comprising:
a) subjecting a mixture comprising a reactive thiol-or selenol-containing substance represented by formula I and a compound represented by formula II to ring-opening polymerization at a ring-opening temperature, followed by quenching with a capping agent to obtain a product:
wherein:
X-L1h represents a substance having mercapto or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxylA substituted or unsubstituted polyethylene glycol, and a substituted or unsubstituted ammonium cationic group, and wherein R is1To R3Not hydrogen at the same time;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000; and
the ring opening temperature is below 0 ℃;
b) separating the polymer represented by formula III from the product, for example, by ion exchange column; and
c) mixing the polymer represented by formula III with a reducing agent to release the substance represented by formula I in situ; optionally, the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine, and glutathione.
4. A method for separating a protein containing reactive sulfhydryl groups or selenol groups from a protein mixture, comprising:
a) performing ring-opening polymerization of a protein mixture comprising a protein containing an active thiol or selenol represented by formula IV and a compound represented by formula II at a ring-opening temperature, and then quenching the reaction using a capping agent to prepare a conjugate represented by formula V:
wherein:
POI-L1h represents a protein having thiol or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30CycloalkenesRadical, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cation groups, and wherein at least one R is4Is a charged group;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000;
the ring opening temperature is 0 ℃ to-80 ℃;
b) isolating the conjugate represented by formula V; and
c) mixing the conjugate represented by formula V with a reducing agent to release the protein represented by formula IV in situ.
5. The method of claim 4, wherein:
the protein mixture is selected from the group consisting of cell lysate, serum, plasma, body fluid, and urine;
POI is selected from dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, gene-edited green fluorescent protein, mutase, azo reductase and antibody;
at least one R4Is composed of
Z is selected from halogenated amides or anhydrides, such as iodoacetamide, succinic anhydride;
the ring opening temperature is from-10 ℃ to-30 ℃, for example-10 ℃ or-30 ℃;
the separating comprises separating the conjugate represented by formula V using an ion exchange column; and
the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine, and glutathione.
6. A kit for isolating a protein containing reactive sulfhydryl or selenol comprising:
a reaction unit configured to perform a ring-opening polymerization reaction between a protein mixture comprising a reactive thiol-or selenol-containing protein represented by formula IV and a compound represented by formula II:
wherein:
POI-L1h represents a protein having thiol or selenol activity, L1Is S or Se;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30HeteroalkanesRadical, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cation, and at least one R is selected from the group consisting of4Is a charged group;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000;
a separation unit configured to separate a conjugate represented by formula V from the protein mixture; and
a temperature control unit configured to control and maintain a temperature of the reaction unit and/or the separation unit.
7. The kit of claim 6, wherein:
the protein mixture is selected from the group consisting of cell lysate, serum, plasma, body fluid, and urine;
POI is selected from dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, gene-edited green fluorescent protein, mutant enzyme and azo reductase, antibody;
the at least one R4Is composed of
Optionally, when said at least one R is4Being positively charged radicals, e.g.
When the isolated conjugate represented by formula V is further used to effect intracellular delivery.
The ring opening temperature is from 0 ℃ to-80 ℃, e.g., -10 ℃ to-30 ℃, e.g., -10 ℃, -30 ℃;
the separation unit is configured as an ion exchange column.
8. The kit of claim 5 or 6, further comprising:
a release unit configured to contact the isolated conjugate represented by formula V with a reducing agent, thereby releasing the protein represented by formula IV in situ;
optionally, the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine, and glutathione.
9. A method of preparing a protein-multimeric drug conjugate, comprising:
a) performing ring-opening polymerization of a protein represented by formula IV and a compound represented by formula II at a ring-opening temperature, and then quenching the reaction using a capping agent to prepare a conjugate represented by formula V:
wherein:
POI-L1h represents a protein having thiol or selenol activity, L1S or Se, e.g. dihydrofolate reductase, bovine serum albumin, transpeptidase, interferon, gene-edited green fluorescent protein, mutase and azoreductase, antibody;
R1to R3Each independently is L2R4Wherein L is2Is a direct bond or C1-C10A chain alkyl radical, and R4Selected from substituted or unsubstituted C2-C30Alkenyl, substituted or unsubstituted C2-C30Alkynyl, substituted or unsubstituted C3-C30Cycloalkyl, substituted or unsubstituted C3-C30Cycloalkenyl, substituted or unsubstituted C1-C30Alkoxy, substituted or unsubstituted C3-C30Cycloalkoxy, substituted or unsubstituted C1-C30Heteroalkyl, substituted or unsubstituted C2-C30Heteroalkenyl, substituted or unsubstituted C2-C30Heteroalkynyl, substituted or unsubstituted C1-C30Heterocycloalkyl, substituted or unsubstituted C2-C30Heterocycloalkenyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C5-C30Heteroaryl, substituted or unsubstituted azido, substituted or unsubstituted acyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonate, substituted or unsubstituted ester, substituted or unsubstituted amide, substituted or unsubstituted hydrazide, substituted or unsubstituted carboxylic acid, substituted or unsubstituted guanidine, substituted or unsubstituted hydroxyl, substituted or unsubstituted polyethylene glycol, and substituted or unsubstituted ammonium cationic groups, and wherein R is a hydroxyl group, a substituted or unsubstituted carboxyl1To R3Not hydrogen at the same time;
z represents a blocking agent;
y represents an end group introduced by the capping agent;
n is an integer of 1 to 10000; and
the ring opening temperature is a temperature below 0 ℃, for example-10 ℃ to-30 ℃;
b) further reacting the conjugate represented by formula V obtained in step a) with a drug to obtain the protein-multimeric drug conjugate,
optionally, when at least one R is4In the case of hydrazide groups, the drug is doxorubicin.
10. A protein-multimeric drug conjugate prepared by the method of claim 9.
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| WO2022227927A1 (en) * | 2021-04-30 | 2022-11-03 | 浙江大学 | Degradable polymer material, and self-assembled nano-composite and application thereof |
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| US5959071A (en) * | 1998-03-31 | 1999-09-28 | Courtaulds Aerospace, Inc. | Composition and method for producing polythioethers having pendent methyl chains |
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| US5959071A (en) * | 1998-03-31 | 1999-09-28 | Courtaulds Aerospace, Inc. | Composition and method for producing polythioethers having pendent methyl chains |
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| EUN-KYOUNG BANG，ET AL.: "Cell-penetrating poly(disulfide)s: focus on substrate-initiated co-polymerization", 《POLYMER CHEMISTRY》, vol. 5, no. 7, pages 2433 - 2441 * |
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| WO2022227927A1 (en) * | 2021-04-30 | 2022-11-03 | 浙江大学 | Degradable polymer material, and self-assembled nano-composite and application thereof |
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