A reduced glutathione-responsive cationic polymer and its preparation method and application

Abstract

The present invention discloses a reduced glutathione-responsive cationic polymer, its preparation method, and application. The cationic polymer has the following structure: #imgabs0#, where m is 5 to 500 and n is 5 to 500. Compared to commercial PEI 25kDa, the reduced glutathione-responsive cationic polymer of the present invention exhibits lower cytotoxicity and higher uptake efficiency, demonstrating potential practical value in gene therapy.

Description

Technical Field

[0001] The invention belongs to the technical field of cationic polymers, and in particular relates to a reduced glutathione-responsive cationic polymer and a preparation method and application thereof. Background Art

[0002] With the continuous advancement of biotechnology, gene therapy, a new generation of disease treatments, is gaining increasing attention in the treatment of chronic diseases, tumors, and genetic disorders. Gene therapy artificially delivers genes containing specific genetic information to target cells, where they express target proteins, thereby modulating, treating, and even curing conditions caused by congenital or acquired genetic defects. While viral vectors have been widely used due to their high transfection and expression efficiencies, their development is severely hampered by inherent shortcomings such as immunogenicity, potential tumorigenicity, insufficient gene loading capacity, complex preparation processes, and low yields.

[0003] Polycations, as synthetic carriers, compress DNA / RNA into nanocomplexes through electrostatic interactions between positive and negative charges, protecting genes from enzymatic degradation and other factors within the body while facilitating their cellular uptake, thereby achieving the therapeutic efficacy of gene therapy. Various synthetic polymers, such as polyethyleneimine (PEI), poly-L-lysine (PLL), and poly-β-amino esters, have been widely reported for gene delivery. PEI 25 kDa has generally been used as the gold standard for evaluating gene transfection. Although PEI is one of the most effective cationic polymers and is widely used in preclinical and clinical research, its high positive charge density and biotoxicity to cell membranes limit its clinical application. While high molecular weight and long chain structure facilitate transfection, polycationic carriers offer high delivery efficiency, limited by their biotoxicity, making their clinical application difficult. Summary of the Invention

[0004] The main purpose of the present invention is to provide a reduced glutathione-responsive cationic polymer and its preparation method and application, so as to overcome the shortcomings of the prior art.

[0005] To achieve the aforementioned object of the invention, the technical solutions adopted by the present invention include:

[0006] The embodiment of the present invention provides a reduced glutathione-responsive cationic polymer, wherein the cationic polymer has a structure as shown in formula (I):

[0007]

[0008]

[0009] Here, m is 5 to 500, and n is 5 to 500.

[0010] The present invention also provides a method for preparing a reduced glutathione-responsive cationic polymer, which comprises:

[0011] Caprolactone is subjected to a ring-opening condensation reaction with aminothiolactone protected by tert-butyloxycarbonyl, and then subjected to a deprotection reaction to prepare a reduced glutathione-responsive cationic polymer.

[0012] The present invention also provides a reduced glutathione-responsive cationic polymer prepared by the aforementioned preparation method, wherein the cationic polymer has a structure as shown in formula (I):

[0013]

[0014] Here, m is 5 to 500, and n is 5 to 500.

[0015] The embodiments of the present invention also provide the use of the aforementioned reduced glutathione-responsive cationic polymer in preparing a gene transfection reagent or in gene transfection.

[0016] An embodiment of the present invention further provides a gene transfection reagent, which includes the aforementioned reduced glutathione-responsive cationic polymer.

[0017] The present invention also provides a gene transfection method, which comprises:

[0018] mixing a transfection reagent with a gene drug and incubating the mixture to prepare a gene drug nanocomplex;

[0019] Wherein, the transfection reagent and the gene drug form the gene drug nanocomplex at least through electrostatic interaction; the transfection reagent includes the aforementioned reduced glutathione-responsive cationic polymer and / or the aforementioned gene transfection reagent.

[0020] The embodiment of the present invention also provides a gene-drug nanocomplex prepared by the aforementioned gene transfection method.

[0021] Compared with the prior art, the present invention has the following advantages: the present invention provides a GSH-responsive cationic polymer (thiolactone-functionalized cationic polymer carrier) with efficient gene delivery capability; at the same time, the thiolactone-functionalized cationic polymer carrier is less cytotoxic and has higher uptake efficiency than commercial PEI25kDa, and has potential practical value for gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is the H NMR spectrum of the thiolactone-functionalized cationic polymer carrier prepared in Example 1 of the present invention;

[0024] Figure 2 This is a graph showing the cytotoxicity of the thiolactone-functionalized cationic polymer carrier to huh-7 cells in Example 2 of the present invention;

[0025] Figure 3 is a graph showing the hydrated particle size of the DNA nanocomplex in Example 3 of the present invention as determined by dynamic light scattering (DLS);

[0026] Figure 4 is a graph showing the hydrated particle size of the DNA nanocomplex in Example 3 of the present invention as determined by dynamic light scattering (DLS);

[0027] Figure 5a-5b This is a graph showing the gel retardation results of DNA or RNA nanocomplexes under different GSH concentration conditions in Example 4 of the present invention;

[0028] Figure 6a-6c This is an in vitro cellular uptake diagram of fluorescently labeled DNA transfected with the thioester-functionalized carrier and PEI 25Kda in Example 5 of the present invention;

[0029] Figure 7a-7c This is the cell uptake pathway for the fluorescently labeled RNA transfected with the thioester functionalized carrier and PEI25Kda in Example 5 of the present invention. DETAILED DESCRIPTION

[0030] In light of the shortcomings of the existing technology, the inventors of this case, after extensive research and extensive practice, were able to propose the technical solution of the present invention. The main point is that introducing GSH-responsive thioester groups into cationic polymers is an effective means for achieving efficient gene delivery. Under physiological conditions, the exchange reaction between thioesters and thiols can degrade the molecular structure of cationic polymers. The carrier can successfully load DNA or RNA and undergo stimulus-responsive degradation within tumor cells, resulting in targeted and efficient release of gene drugs, achieving high transfection efficiency, biocompatibility, and cellular uptake efficiency.

[0031] The technical solution of the present invention will be described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0032] Specifically, as one aspect of the technical solution of the present invention, a reduced glutathione-responsive cationic polymer has a structure as shown in formula (I):

[0033]

[0034] Here, m is 5 to 500, and n is 5 to 500.

[0035] Another aspect of the embodiments of the present invention further provides a method for preparing a reduced glutathione-responsive cationic polymer, comprising:

[0036] Caprolactone is subjected to a ring-opening condensation reaction with aminothiolactone protected by tert-butyloxycarbonyl, and then subjected to a deprotection reaction to prepare a reduced glutathione-responsive cationic polymer.

[0037] In some preferred embodiments, the preparation method specifically comprises:

[0038] A first mixed reaction system comprising caprolactone, tert-butyloxycarbonyl-protected aminothiolactone and a catalyst is subjected to a ring-opening condensation reaction at 30 to 200° C. to obtain a first intermediate product;

[0039] Furthermore, the first intermediate product is mixed with trifluoroacetic acid and subjected to a deprotection reaction at 30-200° C. to obtain the reduced glutathione-responsive cationic polymer.

[0040] Further, the catalyst includes 1,8-diazabicyclo[5.4.0]undec-7-ene and / or 1,57-triazidobicyclo(4.4.0)dec-5-ene, but is not limited thereto.

[0041] Furthermore, the first intermediate product has a structure as shown in formula (II):

[0042]

[0043] Here, m is 5 to 500, and n is 5 to 500.

[0044] Furthermore, the molar ratio of the caprolactone, the tert-butyloxycarbonyl-protected aminothiolactone and the catalyst is 5-95:95-5:0.1-10.

[0045] Furthermore, the molar ratio of the first intermediate product to trifluoroacetic acid is 50-95:1-30.

[0046] Furthermore, the ring-opening condensation reaction time is 0.5 to 48 hours.

[0047] Furthermore, the deprotection reaction time is 0.5 to 48 hours.

[0048] In some preferred embodiments, the preparation method further comprises: first reacting di-tert-butyl dicarbonate with aminothiolactone to obtain the tert-butyloxycarbonyl-protected aminothiolactone.

[0049] Furthermore, the aminothiolactone has a structure as shown in formula (III):

[0050]

[0051] In some preferred embodiments, the preparation method of the reduced glutathione-responsive cationic polymer (also referred to as a thiolactone-functionalized cationic polymer carrier) comprises:

[0052] Caprolactone and tert-butyloxycarbonyl protected aminothiolactone were catalyzed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and then deprotected with trifluoroacetic acid (TFA) to obtain the thiolactone functionalized cationic polymer support.

[0053] The specific reaction equation is shown below:

[0054]

[0055] Another aspect of the present invention further provides a reduced glutathione-responsive cationic polymer prepared by the aforementioned preparation method, wherein the cationic polymer has a structure as shown in formula (I):

[0056]

[0057] Here, m is 5 to 500, and n is 5 to 500.

[0058] Another aspect of the embodiments of the present invention further provides use of the aforementioned reduced glutathione (GHS)-responsive cationic polymer in preparing a gene transfection reagent or in gene transfection.

[0059] Another aspect of the embodiments of the present invention further provides a gene transfection reagent, which includes the aforementioned reduced glutathione-responsive cationic polymer.

[0060] Another aspect of the embodiments of the present invention further provides a gene transfection method, which comprises:

[0061] mixing a transfection reagent with a gene drug and incubating the mixture to prepare a gene drug nanocomplex;

[0062] Wherein, the transfection reagent and the gene drug form the gene drug nanocomplex at least through electrostatic interaction; the transfection reagent includes the reduced glutathione-responsive cationic polymer according to claim 1 or 6 and / or the aforementioned gene transfection reagent.

[0063] Furthermore, the gene medicine includes any one of DNA, RNA, and protein, or a combination of two or more thereof, but is not limited thereto.

[0064] Furthermore, the molecular weight of the gene drug is 2,000 to 500,000.

[0065] Furthermore, the incubation temperature is 30-40° C., and the incubation time is 1-120 min.

[0066] In some more specific embodiments, the gene transfection method includes: dispersing the thiolactone functionalized cationic polymer and the gene drug in water at a certain concentration, mixing and co-incubating, and forming a gene drug nanocomplex through electrostatic interaction.

[0067] Another aspect of the embodiments of the present invention further provides a gene-drug nanocomplex prepared by the aforementioned gene transfection method.

[0068] The present invention utilizes stimulus-responsive cationic polymers to achieve high transfection efficiency while effectively reducing cytotoxicity.

[0069] The reduced glutathione (GHS)-responsive cationic polymer of the present invention is an intelligent degradable cationic polymer with sufficient charge density, molecular weight and molecular chain length. It can effectively complex gene drugs through electrostatic interaction and can be degraded into small fragments in response to stimulation in a physiological environment, thereby having the potential to achieve high gene transfection efficiency and low cytotoxicity.

[0070] The concentration of reduced glutathione (GSH) in tumor cells is approximately 10 times that of normal cells. The present invention introduces GSH-responsive thioester groups into cationic polymers, proving an effective method for efficient gene delivery. Under physiological conditions, the exchange reaction between thioesters and thiols degrades the molecular structure of cationic polymers. These carriers can successfully load DNA or RNA and undergo stimulus-responsive degradation within tumor cells, enabling targeted and efficient release of gene drugs, achieving high transfection efficiency, biocompatibility, and cellular uptake.

[0071] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0072] The applicant in this case verified the transfection efficiency and biocompatibility in vitro through cell experiments, and specifically conducted gel retardation, cell uptake and transfection efficiency experiments to comprehensively evaluate the in vitro transfection effect of the gene-drug nanocomplex.

[0073] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0074] The reagents used in the present invention include: aminothiolactone, BOC anhydride, caprolactone, dichloromethane, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,57-triazidobicyclo(4.4.0)dec-5-ene, and sterile water.

[0075] Testing instruments: dynamic light scattering instrument, microplate reader, gel imager, flow cytometer, fluorescence microscope, etc.

[0076] Example 1

[0077] BOC protection of aminothiolactone; using BOC-protected aminothiolactone, caprolactone, and 1,8-diazabicyclo[5.4.0]undec-7-ene as raw materials (the molar ratio of the three is 10:10:0.2), heating with dichloromethane as solvent and carrying out ring-opening condensation at 80°C for 10 hours, further deprotection reaction is carried out at 80°C in the presence of TFA for 10 hours to prepare a thiolactone-functionalized cationic polymer carrier, that is, a reduced glutathione-responsive cationic polymer. Figure 1 This is the H-NMR spectrum of the prepared thiolactone-functionalized cationic polymer carrier. The characteristic peaks of the polymer structure can be found in the figure.

[0078] Table 1. Molecular weight and distribution of thiolactone-functionalized cationic polymer carriers

[0079] <![CDATA[[Thiolactone]0:[Caprolactone]0]]> Molecular weight Molecular weight distribution 3：1 31000 1.75

[0080] Example 2

[0081] Cytotoxicity of Thiolactone-functionalized Cationic Polymer Carriers in Example 1

[0082] Huh-7 cells were seeded into 96-well plates at a cell density of 1.0 × 10 4Cells were cultured at 400 μL per well in complete DMEM medium (10% FBS and 1% penicillin / streptomycin) for 24 hours. The supernatant was aspirated, the medium was replaced, and 10 μL of polymer solution at different concentrations was added. The cells were then incubated for 48 hours. The cells were washed with PBS, 100 μL of fresh medium was added, and 10 μL of CCK-8 solution was added to each well. The cells were incubated for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. Figure 2 This is the cytotoxicity test result of the thioester carrier on huh-7 cells. When the carrier concentration is as high as 3.13 μg / ml, the cytotoxicity is still above 80%, and is significantly lower than the corresponding concentration of PEI 25kDa.

[0083] Example 3

[0084] Preparation of Thiolactone-functionalized Cationic Polymer Carrier and DNA or RNA Nanocomplex in Example 1

[0085] The thiolactone functionalized cationic polymer carrier is dispersed in sterile water and gently mixed with the DNA or RNA solution at the desired weight ratio (cationic polymer to nucleic acid). Finally, the mixture is vortexed for 5 seconds and incubated in a 37°C incubator for 30 minutes to prepare a gene nanocomplex. Dynamic light scattering measurements show that under certain carrier and gene mass ratio conditions, DNA (160nm) or RNA (110nm) can be effectively compressed into nanomedicines within 200nm. Nanomedicines in this size range are conducive to cellular uptake. Among them, when the thiolactone functionalized cationic polymer carrier / DNA mass ratio w / w=3, the hydrated particle size of the prepared DNA nanocomplex is as follows: Figure 3 As shown; when the mass ratio of thiolactone functionalized cationic polymer carrier to DNA is w / w=15, the hydrated particle size of the prepared DNA nanocomplex is as follows: Figure 4 As shown;

[0086] Example 4

[0087] Agarose gel electrophoresis to evaluate the reduction responsiveness of gene nanocomplexes

[0088] Gene-complexed nanocomplexes were prepared using a thiolactone-functionalized cationic polymer carrier / DNA = 15 or a thiolactone-functionalized cationic polymer carrier / RNA = 3 (w / w) and subjected to agarose gel electrophoresis. The GSH responsiveness of the complexes was determined by electrophoresis at a gradient concentration for 30 minutes, followed by electrophoresis at 90V for 25 minutes, and then visualization using a gel imager. Agarose gel electrophoresis images are available at Figure 5a 、 Figure 5b The carrier can effectively block DNA or RNA and can controllably release it under GSH concentration conditions within 10mM.

[0089] Example 5

[0090] Evaluation of cellular uptake and transfection efficiency of DNA or RNA nanocomplexes

[0091] Fluorescently labeled pEGFP and cy3siRNA were used as representatives of DNA and RNA, respectively, to be complexed with thiolactone-functionalized cationic polymer carriers (abbreviated as thioester-functionalized carriers). Huh-7 cells were seeded into 48-well plates and cultured overnight. Each well contained 1.5×10 4 cells and 200 μL DMEM. Before transfection, the old culture medium in the well plate was replaced with fresh serum-free culture medium containing the vector of the present invention or PEI25kDa complexed DNA or RNA nanoparticles (pEGFP 200ng per well, w / w ratio of 3; cy3siRNA 100nM per well, w / w ratio of 15). Untransfected cells served as blank controls. After 6 hours of culture, the transfection results were observed using a fluorescence microscope. Figure 6a-6c and Figure 7a-7c As shown, the vectors showed better cell uptake and transfection efficiency than PEI25kDa.

[0092] Example 6

[0093] BOC protection of aminothiolactone; using BOC-protected aminothiolactone, caprolactone, and 1 5 7-triazidobicyclo(4.4.0)dec-5-ene as raw materials (the molar ratio of the three is 5:95:0.1), heating with dichloromethane as solvent and carrying out ring-opening condensation at 30°C for 48 hours, further deprotection reaction is carried out in the presence of TFA at 30°C for 48 hours to prepare a thiolactone-functionalized cationic polymer carrier, that is, a reduced glutathione-responsive cationic polymer.

[0094] Example 7

[0095] BOC protection of aminothiolactone; using BOC-protected aminothiolactone, caprolactone, and 1,8-diazabicyclo[5.4.0]undec-7-ene as raw materials (the molar ratio of the three is 95:5:5), heating with dichloromethane as solvent and carrying out ring-opening condensation at 200°C for 0.5h, and further deprotection reaction at 200°C for 0.5h in the presence of TFA to prepare a thiolactone-functionalized cationic polymer carrier, i.e., a reduced glutathione-responsive cationic polymer.

[0096] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments using other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0097] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical variations made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the scope of protection of the present invention.

Claims

1. A reduced glutathione-responsive cationic polymer, characterized in that The cationic polymer has a structure as shown in formula (I): Formula (I) Among them, m is 5~500 and n is 5~500.

2. A method for preparing a reduced glutathione-responsive cationic polymer, characterized in that: include: Caprolactone is subjected to a ring-opening condensation reaction with aminothiolactone protected by tert-butyloxycarbonyl, and then subjected to a deprotection reaction to prepare a reduced glutathione-responsive cationic polymer.

3. The preparation method according to claim 2, characterized in that Specifically include: subjecting a first mixed reaction system comprising caprolactone, tert-butyloxycarbonyl-protected aminothiolactone, and a catalyst to a ring-opening condensation reaction at 30-200° C. to obtain a first intermediate product; Furthermore, the first intermediate product is mixed with trifluoroacetic acid and subjected to a deprotection reaction at 30-200° C. to obtain the reduced glutathione-responsive cationic polymer.

4. The preparation method according to claim 3, characterized in that The catalyst includes 1,8-diazabicyclo[5.4.0]undec-7-ene and / or 1,57-triazidobicyclo(4.4.0)dec-5-ene; And / or, the first intermediate product has a structure as shown in formula (II): Formula (II) Among them, m is 5~500, n is 5~500; and / or, the molar ratio of caprolactone, tert-butyloxycarbonyl-protected aminothiolactone, and catalyst is 5-95:95-5:0.1-10; and / or, the molar ratio of the first intermediate product to trifluoroacetic acid is 50-95:1-30; And / or, the ring-opening condensation reaction time is 0.5 to 48 h; And / or, the deprotection reaction time is 0.5 to 48 h.

5. The preparation method according to claim 2, characterized in that Also includes: First, di-tert-butyl dicarbonate is reacted with aminothiolactone to obtain the tert-butyloxycarbonyl-protected aminothiolactone.

6. A reduced glutathione-responsive cationic polymer prepared by the method according to any one of claims 2 to 5, wherein the cationic polymer has a structure as shown in formula (I): Formula (I) in, m is 5~500, n is 5~500.

7. Use of the reduced glutathione-responsive cationic polymer according to claim 1 or 6 in preparing a gene transfection reagent or in gene transfection.

8. A gene transfection reagent, characterized in that The invention comprises the reduced glutathione-responsive cationic polymer according to claim 1 or 6.

9. A gene transfection method, characterized in that: include: mixing a transfection reagent with a gene drug and incubating the mixture to prepare a gene drug nanocomplex; Wherein, the transfection reagent and the gene drug form the gene drug nanocomplex at least through electrostatic interaction; the transfection reagent comprises the reduced glutathione-responsive cationic polymer according to claim 1 or 6 and / or the gene transfection reagent according to claim 8; The gene drug includes any one of DNA, RNA, and protein, or a combination of two or more thereof; the molecular weight of the gene drug is 2,000 to 500,000; The incubation temperature is 30-40° C., and the incubation time is 1-120 min.

10. The gene-drug nanocomplex prepared by the gene transfection method according to claim 9.

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