CN115975182A - Amino acid copolymer and application thereof - Google Patents
Amino acid copolymer and application thereof Download PDFInfo
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Abstract
The application discloses an amino acid copolymer and application thereof. The amino acid copolymer of the present application is a dendrimer structure having the general formula of formula (I): (PEG) n ‑(Lys) x ‑(Arg) a PEG is polyethylene glycol, the molecular weight of PEG is 2-10kDa, and n represents the polymerization degree of PEG; lys is lysine, constituting a terminal dendritic structure, as a linker for group attachment, x is an integer from 0 to 100, x is equal to the number of generations of the tree-shaped lysine linker; arg is arginine, which constitutes the terminal of the dendrimer, a is an integer of 0 to 100 as the dendron of the dendrimer, and a represents the number of terminal dendron arginines; x and a are not 0 at the same time. The amino acid copolymer has good stability, high biocompatibility and high safety, and is compatible with nucleic acidThe molecular complexing ability is strong, and the nucleic acid loading rate is high; when the nucleic acid nano-drug is used for nucleic acid nano-drugs, the nucleic acid nano-drug can be self-assembled and encapsulated in water, and the preparation process is simple and convenient.
Description
Technical Field
The application relates to the technical field of nucleic acid delivery materials, in particular to an amino acid copolymer and application thereof.
Background
Nucleic acid drugs are leading fields of biomedical development, including antisense nucleic Acids (ASO), small interfering RNAs (siRNA), micrornas (miRNA), messenger RNAs (mRNA), and the like; nucleic acid drugs are a form of gene therapy, and are a new generation of pharmaceutical technology following small molecule drugs, protein drugs, antibody drugs. The nucleic acid medicine can directly act on pathogenic target genes or target mRNA to play a role in treating diseases on the gene level; the nucleic acid medicament is subjected to gene silencing or activation treatment from a post-transcriptional level, and has the obvious advantages of high specificity, high efficiency, long acting property and the like compared with the medicament which plays a role in the traditional protein level. Innovations in nucleic acid therapy techniques and clinical trials are actively being conducted, and a number of nucleic acid therapy programs are successively approved and marketed in the united states, the european union, china, and other countries. The nucleic acid therapy has very wide application prospect in the treatment of various major diseases, such as malignant tumors, infectious diseases, cardiovascular diseases, autoimmune diseases, metabolic diseases and the like.
The core of nucleic acid therapy is nucleic acid drugs. The common nucleic acid drugs mainly include plasmid DNA (pDNA), messenger RNA (mRNA), small interfering RNA (siRNA), and the like. The DNA may carry a nucleotide sequence having a specific therapeutic action, transcribe the mRNA in the target cell of the host and translate the mRNA into a protein having a definite biological function, and treat the disease by supplementing the deficient protein or correcting the abnormal protein in vivo. The siRNA acts on mRNA of a target cell, and the expression of target protein of the siRNA is reduced by specifically shearing the mRNA of a target gene, so that diseased cells are repaired or killed, and the purpose of disease treatment is achieved. The action sites of the nucleic acid molecules are clear, and the nucleic acid molecules can be used for treating various diseases. In mRNA-based therapy, mRNA molecules synthesized in vitro or chemically modified are introduced into cytoplasm, and transcription and expression are performed using the inherent nucleotides in the cytoplasm to produce proteins required by the body. The main technical threshold of mRNA drug development lies in stability and delivery technology, and the related technical difficulties are yet to be further overcome to realize mRNA drugs as protein supplement or replacement therapy to treat related diseases. Especially with the development of technologies such as cancer gene sequencing and epitope discovery, mRNA drugs can also be used for personalized treatment of tumor patients, and have great application potential.
However, nucleic acid drugs are generally unstable and, when used in vivo, are susceptible to degradation by nucleases in the blood circulation; in addition, the nucleic acid molecules carry negative charges, and have electrostatic repulsion with cell membranes with the same negative charges, so that the difficulty of entering cells is further increased; these obstacles presented by nucleic acid drugs have largely limited their conversion to clinical use. Therefore, how to overcome the challenges caused by the physicochemical properties of nucleic acid molecules is one of the problems to be solved in need of safe and efficient delivery to the target action site.
In vivo delivery of nucleic acid drugs faces mainly 3 major difficulties: 1) The molecular weight and negative charge of the nucleic acid prevents it from passing freely through biological membranes; 2) RNA is easily degraded by RNase in plasma and tissues, rapidly cleared by liver and kidney and recognized by immune system; 3) After entering the cell, it is "trapped" in the endosome and eventually degraded by lysosomes and fails to function. Drug delivery systems are key to overcoming the technical hurdles faced by nucleic acid drug development, and currently, there are two main strategies to solve the delivery problem: firstly, the nucleic acid molecules are modified chemically, so that the stability of the nucleic acid molecules is improved and the recognition of an immune system is avoided; another strategy is to use drug delivery systems such as Lipid Nanoparticles (LNPs), galNAc (N-acetylated galactosamine) coupling technology, lipid polyplexes and polymer nanoparticles, etc.
In recent years, a variety of synthetic materials have been developed in succession for in vivo delivery of nucleic acid drugs. The dendrimer is widely applied to the field of nucleic acid drug delivery due to the accurate and controllable structure, excellent monodispersity and polyvalent synergistic effect, for example, the main component of the commercial transfection reagent SuperFectTM is the Polyamidoamine (PAMAM) dendrimer. PAMAM dendrimer has unique structural advantages: 1) The PAMAM has abundant positive charges on the surface, so that the PAMAM can effectively load nucleic acid drug molecules through electrostatic interaction, protect the PAMAM from degradation by nuclease, and is favorable for cellular uptake of nucleic acid drug preparations; 2) A large number of modifiable reaction sites on the surface of the PAMAM can introduce functional groups with different properties, and the specific multivalent synergistic amplification effect of the dendrimer can amplify the functional action and improve the efficiency of a delivery system; 3) The PAMAM has better proton buffering capacity due to rich tertiary amine groups in the PAMAM, and is beneficial to the escape of the PAMAM and nucleic acid compound from an acidic organelle through a proton sponge effect so as to effectively release a loaded nucleic acid medicament; 4) The PAMAM has a large number of amide structures in the structure, so that the PAMAM has bionic protein-like performance and good biological safety. Due to these unique structural properties, PAMAM dendrimers exhibit unique advantages and great potential in nucleic acid drug delivery.
However, the traditional spherical PAMAM dendrimer is difficult to adapt to the delivery requirements of different nucleic acid molecules; in addition, the traditional PAMAM dendrimer is difficult to degrade in vivo and has potential organism toxicity; in addition, the release specificity and delivery efficiency of the assembly of nucleic acid and dendrimer still need to be further improved and improved.
Meanwhile, the mRNA-based nucleic acid drug has a larger molecular structure and negative charges, and has higher difficulty in penetrating cell membranes; furthermore, after entering the cell, it is necessary to escape from the endosome into the cytoplasm, and the exogenous nucleic acid molecule is immunogenic and can activate the immune system of the human body. Therefore, there are many challenges in making nucleic acid drugs more stable and effectively prevented from being recognized and eliminated by the body's immune system during circulation in vivo.
The existing delivery technology patent barriers are high and have certain defects. For example, LNP technology requires multicomponent matching assembly, stability is easily affected, and allergic reactions are severe, and most target only the liver and kidney and reticuloendothelial system. The GalNAc technology can only target liver parenchymal cells, and needs to carry out complete chemical modification on a nucleic acid sequence, so that the synthesis difficulty is high. Other delivery technologies, such as PNP (polypeptide nanoparticles) are not targeting strongly; exosome delivery technologies are currently in an early stage of exploration; the shortage of suppliers and great toxicity of PEI (polyethyleneimine) delivery seriously hamper the clinical transformation requirement of nucleic acid drugs, and the development of a novel nucleic acid delivery technology is urgently needed.
Disclosure of Invention
It is an object of the present application to provide an improved amino acid copolymer and its use.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the first aspect of the application discloses an amino acid copolymer which is of a dendrimer structure and has a general formula shown in a formula (I);
formula (I): (PEG) n -(Lys) x -(Arg) a
Wherein, PEG is polyethylene glycol, the molecular weight of PEG is 2-10kDa, and n represents the polymerization degree of PEG molecules; lys is lysine, constituting the terminal dendritic structure of the amino acid copolymer, as a bond or linker for group attachment, x is an integer from 0 to 100, x is equal to the number of generations of the treelike lysine linker; arg is arginine, which constitutes the terminal of the dendrimer structure, a is an integer of 0 to 100 as the dendron of the dendrimer, and a represents the number of terminal dendron arginines; and x and a are not 0 at the same time.
It should be noted that, when the amino acid copolymer of the present application uses PEG as a polymer element and is used in nucleic acid nano-drugs, on one hand, PEG can control the particle size of nanoparticles and serve as a spatial barrier to play a stabilizing role; on the other hand, PEG can prevent aggregation of nanoparticles during storage; in addition, PEG can also extend the circulation time of nanoparticles in vivo. In addition, in the amino acid copolymer, lysine and arginine are all amino acids necessary for human bodies, and degraded amino acid products are basically nontoxic and can be used by the human bodies, so that the biocompatibility is high; in practical biomedical applications, the amino acid copolymers of the present application have a higher biosafety compared to LNP and other polymeric carriers. When the amino acid copolymer is used for nucleic acid nano-drugs, in an aqueous solution, amino groups of lysine or guanidyl groups of arginine and phosphate groups in nucleic acid molecules are combined through ionic bond interaction, and other interactions such as hydrophobic bonds among molecules are added, so that nano-particles with a certain size are formed through self-assembly. And the self-assembled nucleic acid nano-drug particles enter cells through endocytosis, and under the acidic condition of the endosome, basic amino acid is promoted to be protonated, so that the endosome is damaged, and nucleic acid molecules are released. The amino acid copolymer is adopted for packaging and delivering nucleic acid, so that the use of various lipid auxiliary materials in an LNP complex system can be avoided, the preparation process is simpler, and the problem of multi-path metabolism of the complex system in vivo is solved. In addition, compared with the traditional dendrimers PAMAM and PPI (polypropyleneimine) with the same generation number, arginine is adopted as a dendrite of the amino acid copolymer, the terminal guanidine group of the dendrite has three amino groups in a molecular plane, the complexation capacity with nucleic acid molecules is stronger, and the nucleic acid loading rate is higher.
In one implementation of the present application, the amino acid copolymer is a pharmaceutically acceptable salt, tautomer, or stereoisomer of a polymer of the general formula shown in formula (I).
It can be understood that, in addition to the polymer of the general formula shown in formula (I), the amino acid copolymer of the present application has the same or similar functions as the pharmaceutically acceptable salt, tautomer or stereoisomer thereof, can be used for nucleic acid drug delivery, and has the advantages of good stability, high biocompatibility, high safety, strong ability of complexing with nucleic acid molecules, high nucleic acid loading rate, and the like.
In one implementation of the present application, the amino acid copolymer of the present application is PEG 5k -Lys 3 、PEG 5k -Lys 2 -Arg 4 、PEG 5k -Lys 4 Or PEG 5k -Lys 3 -Arg 8 ；PEG 5k -Lys 3 The polymerization degree of PEG molecule is 5000, the generation number of lysine is 3 5k -Lys 3 Has a molecular structure shown in formula (II); PEG 5k -Lys 2 -Arg 4 The polymerization degree of PEG molecule is 5000, the generation number of lysine is 2, the number of terminal dendritic arginine is 4 5k -Lys 2 -Arg 4 Has a molecular structure shown in a formula (III); PEG 5k -Lys 4 The polymerization degree of PEG molecule is 5000, the generation number of lysine is 4 5k -Lys 4 Has a molecular structure shown in a formula (IV); PEG 5k -Lys 3 -Arg 8 The polymerization degree of PEG molecule is 5000, the generation number of lysine is 3, the number of terminal dendritic arginine is 8 5k -Lys 3 -Arg 8 Has a molecular structure shown in formula (V).
In one implementation of the present application, PEG 5k -Lys 3 The structural form of the compound is shown as a formula (2), PEG 5k -Lys 2 -Arg 4 The structural form of the compound is shown as a formula (3), PEG 5k -Lys 4 The structural form of the compound is shown as a formula (4), PEG 5k -Lys 3 -Arg 8 The structural form of (A) is shown as a formula (5).
It should be noted that the amino acid copolymers having the molecular structures represented by the formula (II), the formula (III), the formula (iv), and the formula (iv) are only the amino acid copolymers used in one embodiment of the present application; it is understood that the polymerization degree of PEG molecules, the generation number of lysine and the number of terminal dendritic arginine can be adjusted according to requirements.
A second aspect of the present application discloses the use of the amino acid copolymers of the present application in nucleic acid drug delivery.
In one implementation of the present application, the nucleic acid drug includes, but is not limited to, at least one of DNA, antisense nucleic acid, mRNA, circular RNA, siRNA, microRNA, lncRNA, and saRNA.
It is important to note that the present application is the discovery of a novel nucleic acid drug delivery material, namely the amino acid copolymer of the present application; the amino acid copolymer is adopted for nucleic acid delivery, has the advantages of good stability, high biocompatibility, high safety, strong complexing ability with nucleic acid molecules and high nucleic acid loading rate, and can better meet the clinical use requirements.
In a third aspect of the present application, a nucleic acid nanomedicine comprising the amino acid copolymer of the present application and a nucleic acid drug with a disease treatment conjugation capability is disclosed.
The nucleic acid drug in the nucleic acid nano-drug of the present application includes, but is not limited to, at least one of DNA, antisense nucleic acid, mRNA, circular RNA, siRNA, microRNA, lncRNA, and saRNA.
In one implementation of the present application, the nucleic acid nanomedicine further comprises a therapeutically relevant adjuvant and/or a pharmaceutically acceptable adjuvant.
In one implementation of the present application, the nucleic acid nanomedicine further comprises an imaging agent for monitoring the therapeutic effect.
It is noted that, the nucleic acid nano-drug of the present application, due to the adoption of the amino acid copolymer of the present application, has the advantages of good stability, high biocompatibility, high safety, strong complexing ability with nucleic acid molecules, high nucleic acid loading rate, etc., and has better clinical application prospects.
It should be further noted that the nucleic acid nano-drug of the present application can be encapsulated and delivered by using the amino acid copolymer single component of the present application, or can be encapsulated with other components according to the requirement, and is not limited specifically herein.
The fourth aspect of the application discloses a preparation method of the nucleic acid nano-drug, which comprises the steps of self-assembling the amino acid copolymer in an aqueous solution, forming a positive point group through ionization of a terminal amino group of the amino acid copolymer, encapsulating the nucleic acid drug with the electronegative nucleic acid drug through electrostatic interaction, and self-assembling the nucleic acid nano-drug to form nano-particles through amphiphilic hydrophobic interaction between dendritic molecules of the amino acid copolymer, so as to obtain the nucleic acid nano-drug.
A fifth aspect of the present application discloses a kit for nucleic acid drug delivery comprising at least one of the following components:
(a) An amino acid copolymer of the present application;
(b) A nucleic acid nano-drug of the present application.
It should be noted that the nucleic acid drug delivery kit of the present application may be a kit having a therapeutic function, in which a specific nucleic acid drug has been embedded; or the amino acid copolymer of the application, and a user designs and embeds the corresponding nucleic acid medicament according to the requirement. It will be appreciated that the key to the kits of the present application is the inclusion of the amino acid copolymers or nucleic acid nanomedicines of the present application, and that other conventional reagents required for nucleic acid delivery, such as certain lipid or polymeric materials, and the like, may be obtained by reference to the prior art or by commercial purchase. Of course, some reagents may be combined into the kit of the present application for convenience of use, and are not particularly limited herein.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the amino acid copolymer has the advantages of good stability, high biocompatibility, high safety, strong complexing ability with nucleic acid molecules and high nucleic acid loading rate; when the nucleic acid nano-drug is used for nucleic acid nano-drugs, the nucleic acid nano-drug can be self-assembled and encapsulated in water, the preparation process is simple and convenient, and the clinical use requirements can be well met.
Drawings
FIG. 1 is a MALDI-TOF spectrum of a PEG starting material (Compound 1) in an example of the present application;
FIG. 2 is a MALDI-TOF spectrum of Compound 2 in the examples of the present application;
FIG. 3 is a MALDI-TOF spectrum of Compound 3 in an example of the present application;
FIG. 4 is a MALDI-TOF spectrum of Compound 4 in the examples of the present application;
FIG. 5 is a MALDI-TOF spectrum of Compound 5 in an example of the present application;
FIG. 6 is a MALDI-TOF spectrum of Compound 6 in the examples of the present application;
FIG. 7 is a GPC spectrum of Compound 1 in the examples herein;
FIG. 8 is a GPC chart of Compound 5 in the examples herein;
FIG. 9 is a GPC spectrum of Compound 6 in the examples herein;
FIG. 10 is an agarose gel electrophoresis image of the encapsulation efficiency test in the example of the present application;
FIG. 11 shows the results of fluorescence measurements of endocytosis of fluorescently labeled mRNA loaded with amino acid copolymers in the present examples;
FIG. 12 shows the results of cytotoxicity tests of amino acid copolymers in examples of the present application;
FIG. 13 is the results of a cytotoxicity assay of an amino acid copolymer-loaded mRNA preparation in an example of the present application.
Detailed Description
The present application will be described in further detail with reference to specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the present application. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other kits, materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in this specification in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that the related operations will be fully understood from the description in the specification and the general knowledge of the art. In the following examples, the reagents or instruments used are not indicated by manufacturers, but are all conventional products available on the market.
Example 1
In this example, different schemes are respectively adopted to synthesize the dendrimer-structured amino acid copolymer as follows:
scheme (1): the reaction process and the specific parameter conditions of each step are as follows:
step 1): weighing raw material lysine Lys 1.2mM in an eggplant-shaped bottle, adding 10mL of DMF solvent for full dissolution, sequentially adding 1.2mM Oxyma racemization inhibitor and 1.2mM DIC condensing agent, and placing on ice for low-temperature activation for 10 min; then adding 0.2mM amino modified PEG raw material BocNH-PEG5k-NH 2 Reacting for 16h at room temperature; and verifying that the raw materials are completely reacted through a TLC point plate, carrying out post-reaction treatment, adding precooled methyl tert-butyl ether into an eggplant-shaped bottle, and reacting for 20min at low temperature, wherein the using amount of the methyl tert-butyl ether is 25mL of precooled methyl tert-butyl ether per 1mL of reaction liquid. After the reaction is finished, standing for 20min at room temperature, removing supernatant, centrifuging to obtain solid, washing with 25mL cold methyl tert-butyl ether for 3 times, and spin-drying to obtain compound 2, i.e. PEG 5k -Lys 1- Fomc; MALDI-TOF was used to analyze PEG starting material (compound 1 5K ) Compound 2, the results are shown in fig. 1 and 2. Fig. 1 is a graph showing the results of compound 1, and fig. 2 is a graph showing the results of compound 2. The results in FIGS. 1 and 2 show that the relative molecular weight increases after the next generation of lysine, and the mass spectrum shows a shift to the right of the peak in molecular weight, indicating the successful synthesis of Compound 2.
Step 2): dissolving all the obtained compound 2 in 20mL of a mixed solution of 4-methylpiperidine and DMF as a solvent, wherein the mixed solution is 1; adding 25mL of cold methyl tert-butyl ether, reacting for 20min, and standing for 20min; discarding supernatant, adding 25mL of cold methyl tert-butyl ether, washing for 3 times, spinning to dry, adding 2.4mM of raw material lysine and a condensing agent to continue reacting for 16 hours, wherein the condensing agent consists of 2.4mM of Oxyma racemization inhibitor and 2.4mM of DIC condensing agent, verifying that the compound 2 is completely reacted by a TCL point plate, and performing post-treatment to remove excessive unreacted lysineTo give compound 3, i.e. PEG 5k -Lys 2- And (4) Fomc. Compound 3 was analyzed by MALDI-TOF, and the results are shown in FIG. 3. The results in fig. 3 show that the relative molecular weight of compound 3 grafted with lysine generation 2 is also increased compared to the PEG starting material and compound 2, and the peak of the molecular weight is shifted to the right in the mass spectrum.
Step 3): dissolving all the obtained compounds 3 in 20mL of solution of 4-methylpiperidine, DMF is 1; adding 25mL of cold methyl tert-butyl ether, reacting for 20min, and standing for 20min; discarding supernatant, adding 25mL of cold methyl tert-butyl ether, washing for 3 times, spinning to dry, adding 4.8mM of raw material lysine and a condensing agent (namely 4.8mM of Oxyma racemization inhibitor and 4.8mM of DIC condensing agent), continuing to react for 16 hours, verifying that the compound 3 completely reacts by a TCL dot plate, and performing post-treatment to remove excessive unreacted lysine to obtain a compound 4 (namely PEG) 5k -Lys 3- And Fomc. Compound 4 was analyzed by MALDI-TOF, and the results are shown in FIG. 4. The results in FIG. 4 show that the mass spectrum peaks increase with successful synthesis of the third generation lysine molecules.
Step 4): dissolving the compound 4 obtained in the previous step into a 100mL eggplant-shaped bottle by using 20mL of a solution of 4-methylpiperidine, DMF and DMF, and carrying out deprotection treatment to react for 16h at room temperature; adding 25mL of cold methyl tert-butyl ether, reacting for 20min, and standing for 20min; discarding supernatant, adding 25mL of cold methyl tert-butyl ether, washing for 3 times, spinning to dry, adding 9.6mM arginine and condensing agent (9.6 mM Oxyma racemization inhibitor and 9.6mM DIC condensing agent), reacting for 16h, performing post-treatment after verifying that compound 4 is completely reacted by TCL dot plate, and removing excessive unreacted lysine to obtain compound 5 (PEG) 5k -Lys 3- Arg 8 -Fomc-Pbf. Compound 5 was analyzed by MALDI-TOF, and the results are shown in FIG. 5. The results in FIG. 5 show that the average molecular weight distribution of the compound is around 10000Da, indicating that the arginine-terminated dendron amino acid molecule of the 4 th generation was successfully synthesized.
Step 5): compound 5 is deprotected once and first dissolved in TFA TIS H 2 Solution of O95.52 times; dissolving the obtained primary product in 25mL of a solution of 4-methylpiperidine, DMF (1: 4) in a 100mL eggplant-shaped bottle for secondary deprotection treatment, and reacting at room temperature for 16h; adding 25mL of cold methyl tert-butyl ether, reacting for 20min, and standing for 20min; the supernatant was discarded and 25mL of cold methyl tert-butyl ether was added and the washing was repeated 3 times, after spin-drying, about 1g of the final product was frozen at-20 ℃ for storage.
Scheme (2): on the basis of the above scheme (1), the reaction process of the scheme (2) in each step is as follows:
step 6) Compound 4 (PEG) obtained in step 4) above 5k -Lys 3 -Fomc), subjecting compound 4 to Fmoc deprotection treatment on lysine, specifically, dissolving with 20mL of a solution of 4-methylpiperidine, DMF as 1; adding 25mL of cold methyl tert-butyl ether, reacting for 20min, and standing for 20min; discarding the supernatant, adding 25mL cold methyl tert-butyl ether, washing for 3 times, drying, adding 9.6mM lysine and condensing agent (9.6 mM Oxyma racemization inhibitor and 9.6mM DIC condensing agent), reacting for 16h, checking the complete reaction of compound 4 by TCL dot plate, post-treating, and removing excessive unreacted lysine to obtain compound 6 (PEG) 5k -Lys 4 -Fomc). The analysis of the product by MALDI-TOF is shown in FIG. 6. The results in FIG. 6 show that the molecular weight of the compound is increased to about 10000Da along with the synthesis of the 4 th generation lysine molecule, which indicates that the 4 th generation lysine terminal dendric amino acid molecule is successfully synthesized.
Step 7) dissolving the compound 6 in 25mL of a solution of 4-methylpiperidine and DMF (1: 4) in a 100mL eggplant-shaped bottle for Fmoc deprotection treatment on lysine, specifically comprising a room temperature reaction for 16h; adding 25mL of cold methyl tert-butyl ether, reacting for 20min, and standing for 20min; the supernatant was discarded and washed 3 times with 25mL of cold methyl tert-butyl ether, and after spin-drying, about 1g of the final product was frozen at-20 ℃ for storage.
Physical and chemical property characterization: the MALDI-TOF mass spectrum characterization of the terminal dendritic amino acid copolymer molecule is carried out by the specific operation that DMF solution with the copolymer molecule concentration of 5mg/mL is prepared, a sample is placed in an Shimadzu bottle, and MALDI-TOF detection is carried out to obtain a characterization result. Meanwhile, molecular weight characterization is carried out on the compound 1, the compound 5 and the compound 6 through gel permeation chromatography GPC, and the results of the three are sequentially shown in figure 7, figure 8 and figure 9, GPC is a common method for characterizing the average molecular weight of polymers, and shows that 4-generation amino acid molecules are successfully synthesized, the average molecular weight of the 4-generation amino acid molecules is slightly larger than that of MALDI-TOF, but the results are consistent to prove that the dendritic amino acid copolymer is successfully synthesized.
In the amino acid copolymer synthesized in this example, PEG 5k -Lys 3 、PEG 5k -Lys 2 -Arg 4 、PEG 5k -Lys 4 And PEG 5k -Lys 3 -Arg 8 The molecular structures of the four are respectively shown in formula (II), formula (III), formula (IV) and formula (V) in sequence, and the structural forms of the four are respectively shown in formula (2), formula (3), formula (4) and formula (5) in sequence.
Formula (II):
formula (III):
formula (IV):
formula (V):
formula (2):
formula (3):
formula (4):
formula (5):
example 2
The terminal dendritic amino acid copolymer prepared in example 1 was used for nucleic acid delivery, in this example specifically as an mRNA drug delivery assay.
1. mRNA/PEG 5k -Lys 3 -Arg (i.e. PEG) 5k -Lys 3 -Arg 8 ) Preparation of the Complex
2.5mg of PEG was taken 5k -Lys 3 the-Arg copolymer, the compound of example 1, was dissolved in enzyme-free water to prepare a 800. Mu.M stock solution. Preparing a compound from 10 mu L of stock solution and Luciferase mRNA according to the mass ratio of 120 to 1, enabling the final concentration of the mRNA to be 10 ng/mu L, uniformly mixing, standing at room temperature for 10min, and obtaining the mRNA/PEG 5k -Lys 3 -Arg nanocomplex.
2. mRNA/PEG 5k -Lys 3 Encapsulated Rate detection of the-Arg Complex
Step 1) Using the prepared mRNA/PEG 5k -Lys 3 -Arg Complex, with commercial PEI as a positive control, samples were prepared for agarose gelAnd (4) gel electrophoresis. The results of the electrophoretic detection are shown in FIG. 10. In FIG. 10, lane 1 is free Fluc-mRNA, lane 2 is commercial 4 lysine-loaded Fluc-mRNA, lane 3 is PEI-loaded Fluc-mRNA, lane 4 is PEG 5K -Lys 2 Loaded Fluc-mRNA (N/P = 1/5), lane 5 PEG 5K -Lys 3 Loaded Fluc-mRNA (N/P = 1/5), lane 6 PEG 5K -Lys 2 Arg loaded Fluc-mRNA (N/P = 1/5), lane 7 PEG 5K -Lys 4 Loaded Fluc-mRNA (N/P = 1/5), lane 8 PEG 5K -Lys 4 Loaded Fluc-mRNA (N/P = 1/8), lane 9 PEG 5K -Lys 4 Loaded Fluc-mRNA (N/P = 1/10), lane 10 PEG 5K -Lys 3 Arg loaded Fluc-mRNA (N/P = 1/5), lane 11 PEG 5K -Lys 3 Arg loaded Fluc-mRNA (N/P = 1/8), PEG in lane 12 5K -Lys 3 -Arg loaded Fluc-mRNA (N/P = 1/10). The results in fig. 10 show that negatively charged free mRNA runs under the gel pore after electrophoresis, and mRNA coated by positively charged dendrimers is blocked in the gel pore after forming nanoparticles with the mRNA, i.e. the more mRNA retained in the pore indicates higher encapsulation efficiency, and the results in fig. 10 demonstrate that dendrimers with terminal arginine at the 4 th generation have high nucleic acid loading efficiency.
3. mRNA/PEG 5k -Lys 3 -Arg Complex endocytosis assay
Step 1) cell culture: human embryonic kidney epithelial cells (293T) were cultured in a medium containing 10% Fetal Bovine Serum (FBS) and cultured normally at 37 ℃ in 5% carbon dioxide.
Step 2) cell plating: cells were seeded in cell culture plates prior to transfection and cultured for 24h in 100 μ L of fresh medium containing 10% fbs.
Step 3) endocytosis of cells: discarding the medium from the plate, rinsing the cells twice with a serum-free basal medium, and washing the prepared mRNA/PEG 5k -Lys 3 Adding the-Arg nano-complex into 100 mu L of culture medium, mixing uniformly, adding the-Arg nano-complex into a culture plate together, and incubating the mixture with cells, namely culturing the mixture in an incubator at 37 ℃ for 24h. Wherein the mRNA is labeled with Cy3 fluorescent dye.
Step 4) fixing cells: the culture plate was removed, the cells were rinsed 2 times with a serum-free basal medium, 100. Mu.L of 4% paraformaldehyde was added for fixation at room temperature for 10min, the fixative was removed by aspiration, 100. Mu.L of cell buffer PBS was added for rinsing 2 times, and 100. Mu.L of PBS was added.
Step 5) fluorescence imaging detection: the cells fixed in step 4) above were placed under an inverted fluorescence microscope for fluorescence imaging and images were recorded, and the results shown in fig. 11 were obtained, and the results in fig. 11 show that the dendrimer has the ability to deliver fluorescently labeled mRNA into cells similar to the commercial transfection reagents LipoMAX, PEI and the commercial 4 th generation lysine molecules, indicating that the dendrimer has the potential to transfect and deliver mRNA.
4. Cytotoxicity assays for terminal dendritic amino acid copolymers
Step 1) preparation of dendrimer stock solution: operating under aseptic condition, dissolving 10mg of the dendriform amino acid copolymer in 1mL of sterile water, performing ultrasonic treatment, standing, and preparing into stock solution;
step 2) the solution of terminal dendritic amino acid copolymer molecules was prepared to different concentrations, in this example 8 gradient concentrations were prepared, each concentration being specifically 0, 0.32. Mu.g/mL, 1.6. Mu.g/mL, 8. Mu.g/mL, 40. Mu.g/mL, 200. Mu.g/mL, 1000. Mu.g/mL, 5000. Mu.g/mL, 200. Mu.L of each concentration was added to a 293T cell seeded plate for incubation for 24h.
And 3) carrying out cell activity monitoring under the aseptic condition, diluting a CCK-8 reagent by using a basic culture medium, uniformly mixing the reagents according to the proportion of the kit specification and the culture medium to the CCK-8 reagent as 9, adding 100 mu L of mixed solution into each hole, putting the mixture into an incubator for incubation for 2h, and monitoring the cell activity under an enzyme-labeling instrument. Cell viability is shown in figure 12.
The results in FIG. 12 show that PEG 5k -Lys 2 -Arg and PEG 5k -Lys 3 The two molecules of-Arg do not generate any obvious washing toxicity to cells within the concentration range of 1mg/mL, which indicates that the two molecules have good biocompatibility, and the molecular concentration in practical clinical application is far lower than 1mg/mL, which indicates that the dendrimer is a nucleic acid delivery material with good safetyAnd (5) feeding.
5. Cytotoxicity test of mRNA/terminal dendritic amino acid molecule nano-drug
Step 1) cell plating: 293T cells were plated at 6000 cells/well in 96-well plates and cultured at 37 ℃ for 24 hours.
Step 2) adding 10ng of molecules with the mRNA content of lysine end and arginine end of third generation or fourth generation respectively, simultaneously taking commercial lysine and PEI and a transfection reagent Lipo2K as a control group, taking five multiple wells of each group of samples, incubating at 37 ℃ for 24h, and detecting the cell activity by using CCK-8 as described above. As shown in fig. 13, dendritic amino acid copolymer molecules of different generations and ends did not induce cytotoxic generation and were more viable than the commercial transfection reagent Lipo2K, suggesting that dendrimers have great advantages as nucleic acid delivery vehicles.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (10)
1. An amino acid copolymer characterized by: the amino acid copolymer is of a dendrimer structure and has a general formula shown in a formula (I);
formula (I): (PEG) n -(Lys) x -(Arg) a
Wherein, PEG is polyethylene glycol, the molecular weight of PEG is 2-10kDa, and n represents the polymerization degree of PEG molecules; lys is lysine, constituting the terminal dendritic structure of the amino acid copolymer, as a bond or linker for group attachment, x is an integer from 0 to 100, x is equal to the number of generations of the tree-shaped lysine linker; arg is arginine, which constitutes the terminal of the dendrimer structure, a is an integer of 0 to 100 as the dendron of the dendrimer, and a represents the number of terminal dendron arginines; and x and a are not 0 at the same time.
2. The amino acid copolymer of claim 1, wherein: the amino acid copolymer is a pharmaceutically acceptable salt, tautomer or stereoisomer of a polymer shown as a general formula (I).
3. The amino acid copolymer of claim 1 or 2, characterized in that: the amino acid copolymer is PEG 5k -Lys 3 、PEG 5k -Lys 2 -Arg 4 、PEG 5k -Lys 4 Or PEG 5k -Lys 3 -Arg 8 ；
PEG 5k -Lys 3 The polymerization degree of PEG molecule is 5000, the generation number of lysine is 3 5k -Lys 3 Has a molecular structure shown in formula (II);
PEG 5k -Lys 2 -Arg 4 the polymerization degree of PEG molecule is 5000, the generation number of lysine is 2, the number of terminal dendritic arginine is 4 5k -Lys 2 -Arg 4 Has a molecular structure shown in a formula (III);
PEG 5k -Lys 4 the polymerization degree of PEG molecule is 5000, the generation number of lysine is 4 5k -Lys 4 Has a molecular structure shown in a formula (IV);
PEG 5k -Lys 3 -Arg 8 the polymerization degree of PEG molecule is 5000, the generation number of lysine is 3, the number of terminal dendritic arginine is 8 5k -Lys 3 -Arg 8 Has a molecular structure represented by formula (V);
formula (II):
formula (III):
formula (IV):
formula (V):
4. the amino acid copolymer of claim 3, wherein: the PEG 5k -Lys 3 The structural form of the compound is shown as a formula (2), PEG 5k -Lys 2 -Arg 4 The structural form of (A) is shown as formula (3), PEG 5k -Lys 4 The structural form of the compound is shown as a formula (4), PEG 5k -Lys 3 -Arg 8 The structural form of the compound is shown as a formula (5);
formula (2):
formula (3):
formula (4):
formula (5):
5. use of the amino acid copolymer of any of claims 1 to 4 for nucleic acid drug delivery.
6. Use according to claim 5, characterized in that: the nucleic acid drug includes, but is not limited to, at least one of DNA, antisense nucleic acid, mRNA, circular RNA, siRNA, microRNA, lncRNA, and saRNA.
7. A nucleic acid nano-drug characterized by: the nucleic acid nano-drug comprises the amino acid copolymer of any one of claims 1 to 4 and a nucleic acid drug with conjugated energy for disease treatment.
8. The nucleic acid nanopharmaceutical of claim 7, wherein: the nucleic acid drug comprises at least one of DNA, antisense nucleic acid, mRNA, circular RNA, siRNA, microRNA, lncRNA and sarRNA;
preferably, the nucleic acid nano-medicament further comprises a treatment-related adjuvant and/or a pharmaceutically acceptable adjuvant;
preferably, the nucleic acid nano-drug further comprises an imaging agent for monitoring the therapeutic effect.
9. The method for preparing a nucleic acid nano-drug according to claim 7 or 8, characterized in that: the nucleic acid nano-drug is obtained by self-assembling the amino acid copolymer of any one of claims 1 to 4 in an aqueous solution to form a nucleic acid nano-drug by ionization of a terminal amino group of the amino acid copolymer to form a group with a positive point, encapsulating the nucleic acid drug with electronegative nucleic acid drug through electrostatic interaction, and self-assembling to form nano-particles through amphiphilic hydrophobic interaction between dendritic molecules of the amino acid copolymer.
10. A kit for nucleic acid drug delivery, characterized in that: comprises at least one of the following components,
(a) The amino acid copolymer of any one of claims 1 to 4;
(b) The nucleic acid nano-drug of claim 7 or 8.
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