A core-shell structured thermosensitive carrier, its preparation method and application

Abstract

This invention provides a core-shell structured thermosensitive carrier, comprising a core and a shell at least partially covering the core. The core is composed of an LCST polymer; the shell is composed of a UCST polymer; the mass fraction of the shell is 40-60%, the mass fraction of the core is 40-60%, and the total mass of the thermosensitive carrier based on the core-shell structure is [not specified]. This core-shell structured thermosensitive carrier can serve as a carrier for various mitochondrial repair drugs.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine carrier technology, specifically to a core-shell structured thermosensitive carrier, its preparation method, and its application. Background Technology

[0002] Mitochondria are not only the cell's "energy factories" (synthesizing ATP through oxidative phosphorylation), but also key centers regulating cellular metabolism, reactive oxygen species (ROS) balance, calcium homeostasis, and apoptosis. Therefore, mitochondrial health is the cornerstone of maintaining tissue and organ function. Damage to mitochondrial function can lead to a variety of serious diseases, such as neurodegenerative diseases, cardiovascular diseases, metabolic diseases, cancer, and aging. Traditional antioxidants (such as MitoQ and SkQ1) or drugs are difficult to effectively target the mitochondrial matrix and cannot achieve controlled release, resulting in limited therapeutic effects and significant side effects.

[0003] Recent studies have shown that normally functioning mitochondria can have an internal temperature 6-10°C higher than other parts of the cytoplasm due to their highly efficient energy metabolism. However, when mitochondria are damaged, this temperature difference weakens or even disappears. This provides a novel, endogenous physical signal—temperature—for targeted therapy.

[0004] Currently, some thermosensitive materials are used for drug delivery, such as polymers with low critical solution temperature (LCST) characteristics, such as poly(N-isopropylacrylamide) (PNIPAM). However, carriers with only a single response to LCST or upper critical solution temperature (UCST) have the following inherent drawbacks: wide release window and insufficient precision: a single thermosensitive behavior can only trigger drug release when the temperature exceeds or falls below a certain critical point, resulting in a flat release curve and difficulty in achieving "burst" precise release at the lesion site. Inability to adapt to complex pathological temperature microenvironments: temperature changes in the lesion area may be subtle, and a single response mechanism may not be able to effectively distinguish between pathological and normal physiological states, leading to a low signal-to-noise ratio. The contradiction between stability and release efficiency: in systemic circulation, the carrier needs to be highly stable to prevent drug leakage; while at the target site, rapid and complete release is required. A single thermosensitive mechanism cannot simultaneously optimize these two properties.

[0005] Therefore, there is an urgent need in this field for a novel intelligent nanosystem capable of precisely delivering and releasing drugs by utilizing the pathological characteristics of mitochondria themselves (such as temperature changes). Summary of the Invention

[0006] The purpose of this invention is to provide a core-shell structured thermosensitive carrier that can be used as a carrier for various mitochondrial repair drugs.

[0007] In a first aspect, the present invention provides a core-shell structured thermosensitive carrier, comprising a core and a shell layer that at least partially covers the core. The core is made of LCST polymer; the shell is made of UCST polymer; The shell has a mass fraction of 40-60%, preferably 45-55%; the core has a mass fraction of 40-60%, preferably 45-55%, and the total mass of the temperature-sensitive carrier based on the core-shell structure is measured.

[0008] In some embodiments, the LCST polymer is selected from one or more of poly(N-isopropylacrylamide) and its derivatives, polyethylene glycol methyl ether methacrylate and its derivatives, poly(N-3-aminopropyl methacrylamide) and its derivatives, polyepoxyalkane and its derivatives, polymethyl vinyl ether and its derivatives, poly-N-vinylcaprolactam and its derivatives, polysiloxane and its derivatives, copolymers of poly(N-isopropylacrylamide) with ethylene glycol methyl ether methacrylate and N-3-aminopropyl methacrylamide and their derivatives, preferably copolymers of poly(N-isopropylacrylamide) with ethylene glycol methyl ether methacrylate and N-3-aminopropyl methacrylamide and their derivatives; The UCST polymer is selected from one or more of polyacrylamide, polyacrylonitrile, polyacrylamide-acrylonitrile copolymer, polystyrene, polystyrene copolymer and polyethylene oxide copolymer, preferably polyacrylamide-acrylonitrile copolymer.

[0009] In some embodiments, the lower critical dissolution temperature of the LCST polymer is 37~48°C, preferably 39~45°C; The upper critical dissolution temperature of the UCST polymer is 40~52℃, preferably 42~50℃; and / or The thickness of the shell layer is 50~70nm, preferably 55~65nm; the particle size of the core is 210~250nm, preferably 225~232nm; the particle size of the core-shell structured thermosensitive carrier is 265~320nm, preferably 275~310nm.

[0010] Secondly, the present invention provides a method for preparing the aforementioned core-shell structured thermosensitive carrier, comprising: Provides core particles formed from LCST polymer; The UCST polymer is used to form a shell layer on the surface of the core particles to obtain a core-shell structured thermosensitive carrier. The mass ratio of LCST polymer to UCST polymer is (0.5~2): (0.5~2), preferably (0.5~1.5):1, and more preferably 1:1.

[0011] In some embodiments, the UCST polymer comprises the structural units -CH2-CH(CONH2)- and -CH2-CH(CN)-; and / or The LCST polymer comprises the structural units -CH2-CH(CONH-iPr)-, -CH2-C(CH3)(COO-PEG)-, and -CH2-C(CH3)(CO-NH-(CH2)3-NH2)-.

[0012] In some embodiments, the UCST polymer is prepared by polymerizing polymer monomers in the presence of a chain extender to obtain the UCST polymer. The polymer monomer is one or more of olefin amide monomers and olefin acrylonitrile monomers; Preferably, the acrylonitrile monomer is C2-6 acrylonitrile, wherein the C2-6 acrylonitrile is selected from one or more of acrylonitrile, methacrylonitrile, 2-butenonitrile, and 4-pentenonitrile, and more preferably acrylonitrile; Preferably, the amide monomer is a C2-6 amide, wherein the C2-6 amide is selected from acrylamide, methacrylamide, N-isopropylacrylamide, N,N-dimethylacrylamide or N-hydroxyethylacrylamide, and more preferably acrylamide; Preferably, the molar ratio of the amide monomer to the acrylonitrile monomer is (1~20):1; more preferably (2~10):1; and even more preferably (3~6):1. Preferably, the chain extender is a macromolecular chain extender containing PEG.

[0013] In some embodiments, the LCST polymer is prepared by the following method: The polymer raw materials are subjected to a polymerization reaction in the presence of a crosslinking agent to obtain LCST polymer; The polymerization raw material comprises N-alkylacrylamide compounds, hydrophilic comonomers, and functional monomers containing reactive functional groups; the polymerization raw material further comprises bisacrylamide compounds; Preferably, the crosslinking agent is a macromolecular crosslinking agent containing acrylate units; more preferably, it is a polyβ-amino ester with acrylate groups as end groups. The N-alkylacrylamide compound is selected from one or more of N-isopropylacrylamide, N-isopropylmethacrylamide, N-ethylacrylamide, N-tert-butylacrylamide, and N-hydroxyethylacrylamide; preferably N-isopropylacrylamide. The hydrophilic comonomer is selected from one or more of polyethylene glycol methyl ether (meth) acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, N,N-dimethylacrylamide, and vinylpyrrolidone, preferably polyethylene glycol methyl ether methacrylate; The functional monomer containing the reactive functional group is selected from one or more of N-(3-aminopropyl)methacrylamide, acrylic acid, acrolein, propargyl (meth)acrylamide, and azidoethyl (meth)acrylamide, preferably N-(3-aminopropyl)methacrylamide hydrochloride; The bisacrylamide compounds are selected from N,N′-methylenebisacrylamide.

[0014] Thirdly, the present invention provides a core-shell thermosensitive drug, comprising the aforementioned core-shell thermosensitive carrier and a drug loaded on the core-shell thermosensitive carrier; preferably, the drug is CoQ10 or doxorubicin.

[0015] Fourthly, the present invention provides a pharmaceutical composition comprising the aforementioned core-shell structured thermosensitive drug and pharmaceutical excipients.

[0016] Fifthly, the present invention provides the application of the aforementioned core-shell structure thermosensitive drug or the aforementioned pharmaceutical composition in the preparation of a drug for treating diseases related to mitochondrial dysfunction; Preferably, the mitochondrial dysfunction-related diseases are Parkinson's disease, Alzheimer's disease, senile dementia, Huntington's disease, amyotrophic lateral sclerosis, or frontotemporal dementia.

[0017] The core-shell thermosensitive carrier of this invention constructs a "temperature-gated" system by using UCST polymer as the shell and LCST polymer as the core. As a drug carrier, this core-shell structure is structurally stable at normal body temperature (37°C) with minimal drug leakage. Within a specific pathological temperature window (e.g., 42°C), both the UCST polymer shell and the LCST polymer core undergo a hydrophilic transition (the shell changes from hydrophobic to hydrophilic, and the core changes from hydrophilic to hydrophobic), generating internal stress and maximizing, explosive drug release. At higher temperatures (e.g., 48°C), the hydrophobic LCST core inhibits drug release, thus forming an intelligent "on-off" release curve.

[0018] The core-shell structured thermosensitive carrier of this invention serves as a drug carrier, capable of simultaneously loading drugs with different properties (such as hydrophilic doxorubicin and hydrophobic CoQ10). It utilizes the temperature difference between diseased and normal mitochondria as a trigger signal. When the carrier reaches the vicinity of the damaged mitochondria at a lower temperature, its unique thermosensitive properties are activated, preferentially releasing the drug there, thus achieving "treatment where the damage is." Attached Figure Description

[0019] Figure 1 UCST polymers 1 H NMR spectrum; Figure 2FT-IR spectra of UCST polymers, LCST polymers, and core-shell thermosensitive supports; Figure 3 Phase transition temperature profiles of UCST polymers; Figure 4 Phase transition temperature profiles of LCST polymers; Figure 5 Release curves of CoQ10 from the CoQ10-core-shell thermosensitive carrier in 100 µM DTT buffer at different temperatures; Figure 6 Release curves of DOX from a DOX-core-shell thermosensitive vector in 100 µM DTT buffer at different temperatures; Figure 7 Confocal microscopy images show drug release from the DOX-core-shell thermosensitive carrier in a rotenone-treated Parkinson's disease cell model. Figure 8 Mito Thermo Yellow probe was used to test the effects of CoQ10 and CoQ10-core-shell thermosensitive carrier on mitochondrial temperature repair. Figure 9 Mito-SOX probe assays showed that the CoQ10-core-shell thermosensitive carrier reduced mitochondrial ROS levels. Figure 10 Parkinson's disease model cells were exposed to different concentrations of CoQ10 and CoQ10-core-shell thermosensitive carrier for 24 hours to improve cell viability. Figure 11 Cell viability of SH-SY5Y cells after 24 hours of exposure to different concentrations of CoQ10-core-shell thermosensitive carrier.

[0020] Note: In the attached diagram, CoQ10@UL refers to CoQ10-core-shell structured thermosensitive carrier.

[0021] abbreviations AAm: Acrylamide; AN: Acrylonitrile; AIBN: Azobisisobutyronitrile; DOX: Dorothy Star; EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS: N-hydroxysuccinimide; DMSO: Dimethyl sulfoxide; CPTP: 4-Cyano-4-(phenylcarbothioiminothio)pentanoic acid. Detailed Implementation

[0022] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the operations involved in the embodiments are conventional techniques in the art.

[0023] <Core-shell structured thermosensitive carrier> This invention provides a core-shell structured thermosensitive carrier, comprising a core and a shell that at least partially covers the core. The core is made of LCST polymer; the shell is made of UCST polymer; The shell has a mass fraction of 40-60%, preferably 45-55%, more preferably 47-53%; the core has a mass fraction of 40-60%, preferably 45-55%, more preferably 47-53%, and the total mass of the temperature-sensitive carrier based on the core-shell structure is measured.

[0024] In some embodiments, the LCST polymer is selected from one or more of poly(N-isopropylacrylamide) and its derivatives, polyethylene glycol methyl ether methacrylate and its derivatives, poly(N-3-aminopropyl methacrylamide) and its derivatives, polyepoxyalkane and its derivatives, polymethyl vinyl ether and its derivatives, poly-N-vinylcaprolactam and its derivatives, polysiloxane and its derivatives, copolymers of poly(N-isopropylacrylamide) with ethylene glycol methyl ether methacrylate and N-3-aminopropyl methacrylamide and their derivatives, preferably copolymers of poly(N-isopropylacrylamide) with ethylene glycol methyl ether methacrylate and N-3-aminopropyl methacrylamide and their derivatives.

[0025] In some embodiments, the UCST polymer is selected from one or more of polyacrylamide, polyacrylonitrile, polyacrylamide-acrylonitrile copolymer, polystyrene, polystyrene copolymer and polyethylene oxide copolymer, preferably polyacrylamide-acrylonitrile copolymer.

[0026] In some embodiments, the UCST polymer comprises the structural units -CH2-CH(CONH2)- and -CH2-CH(CN)-. In some embodiments, the UCST polymer comprises the structural units -CH2-CH(CONH2)-, -CH2-CH(CN)-, and -CH2-CH2-O-.

[0027] In some embodiments, the molecular weight of the UCST polymer is 7-20 kDa, preferably 7-15 kDa, and more preferably 7-9 kDa. For example, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, and 19 kDa.

[0028] In some embodiments, the LCST polymer comprises the structural unit -CH2-CH(CONH-iPr)-, the structural unit -CH2-C(CH3)(COO-PEG)-, and the structural unit -CH2-C(CH3)(CO-NH-(CH2)3-NH2)-; preferably, the number average molecular weight of PEG in the structural unit -CH2-C(CH3)(COO-PEG)- is 500~2000, more preferably 800~1500, for example 600, 900, 950, 1100, 1300, 1600, 1800.

[0029] In some embodiments, the lower critical dissolution temperature of the LCST polymer is 37~48°C, preferably 39~45°C, for example 38°C, 40°C, 41°C, 42°C, 43°C, 44°C, 46°C or 47°C.

[0030] In some embodiments, the upper critical dissolution temperature of the UCST polymer is 40~52°C, preferably 42~50°C, for example 41°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C or 51°C.

[0031] In some embodiments, the thickness of the shell layer is 50-70 nm, preferably 55-65 nm, such as 52 nm, 54 nm, 56 nm, 58 nm, 60 nm, 62 nm, 64 nm, 66 nm, or 68 nm. In some embodiments, the particle size of the core is 210-250 nm, preferably 225-232 nm, such as 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, or 245 nm. In some embodiments, the particle size of the core-shell structured thermosensitive carrier is 265-320 nm, preferably 275-310 nm, such as 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, or 315 nm.

[0032] <Preparation Method> This invention provides a method for preparing a core-shell structured thermosensitive carrier as described above, comprising: (1) Provide core particles formed from LCST polymer; (2) The UCST polymer is used to form a shell on the surface of the core particles to obtain a core-shell structured thermosensitive carrier; The mass ratio of LCST polymer to UCST polymer is (0.5~2): (0.5~2), preferably (0.5~1.5):1, and more preferably 1:1.

[0033] The preparation methods for UCST polymers and LCST polymers are described below: Preparation method of UCST polymer The UCST polymer was prepared by the following method: The polymer monomers are polymerized in the presence of a chain extender to obtain the UCST polymer.

[0034] The polymer monomer is one or more of olefin amide monomers and olefin acrylonitrile monomers.

[0035] In some embodiments, the acrylonitrile monomer is a C2-6 acrylonitrile, wherein the C2-6 acrylonitrile is selected from one or more of acrylonitrile, methacrylonitrile, 2-butenylonitrile, and 4-pentenylonitrile, preferably selected from acrylonitrile. In some embodiments, the acrylamide monomer is a C2-6 acrylamide, wherein the C2-6 acrylamide is selected from acrylamide, methacrylamide, N-isopropylacrylamide, N,N-dimethylacrylamide, or N-hydroxyethylacrylamide, preferably selected from acrylamide.

[0036] The UCST polymer is obtained by polymerizing amide monomers and nitrile monomers in the presence of a chain extender.

[0037] In some embodiments, the amide monomer and the acrylonitrile monomer are polymerized in the presence of a chain extender and a free radical initiator to obtain the UCST polymer.

[0038] In some embodiments, an enamide monomer, an enacrylonitrile monomer, a free radical initiator, and a chain extender are added to a first polar solvent to obtain a first precursor solution. The first precursor solution is degassed under an inert atmosphere and then reacted for 12 to 36 hours to obtain the UCST polymer.

[0039] In some embodiments, the molar ratio of the enamide monomer to the enacrylonitrile monomer is (1~20):1; preferably (2~10):1; more preferably (3~6):1; In some embodiments, the free radical initiator is selected from one or more of azobisisobutyronitrile, azobis(4-cyanovaleric acid), azobis(2-methylbutyronitrile), benzoyl peroxide, di-tert-butyl peroxide, or ammonium persulfate / sodium bisulfite redox pair; preferably azobisisobutyronitrile or azobis(4-cyanovaleric acid); more preferably azobisisobutyronitrile.

[0040] In some embodiments, the first polar solvent is selected from one or more of dimethyl sulfoxide, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), acetonitrile, acetone, and an ethanol / water mixture, preferably selected from DMSO or DMF, and more preferably selected from DMSO.

[0041] In some embodiments, the total concentration of the enamide monomer and the enacrylonitrile monomer in the first precursor solution is 0.5~5.0 mol / L, preferably 1.0~3.0 mol / L, and more preferably 1.5~2.5 mol / L.

[0042] In some embodiments, the molar ratio of the free radical initiator to the chain extender is (0.1~1):1; preferably (0.2~0.6):1; more preferably (0.3~0.5):1. In some embodiments, the molar ratio of (enamide monomer, nitrile monomer) to the free radical initiator is (10~1000):1; preferably (50~500):1; more preferably (100~300):1.

[0043] In some embodiments, the chain extender is a macromolecular chain extender containing PEG.

[0044] In some embodiments, the number average molecular weight of the PEG containing the macromolecular chain extender is 2000-8000, preferably 4000-6000, for example 3000, 4000, 5000 or 7000.

[0045] In some embodiments, the PEG-containing macromolecular chain extender is terminally composed of a thiocarbonyl group, a trithioester, a dithioester, or a xanthate. In some embodiments, the PEG-containing macromolecular chain extender is a CPTP-PEG conjugate. In the preparation method of the UCST polymer, using a PEG-containing macromolecular chain extender ensures the introduction of PEG and controllable reaction.

[0046] The first precursor solution is degassed under an inert atmosphere, and then reacted for 12-36 hours to obtain the UCST polymer. Preferably, the first precursor solution is degassed under an inert atmosphere, and then reacted for 12-36 hours. After the reaction is complete, the solution is dialyzed and dried to obtain the UCST polymer. In some embodiments, the inert atmosphere is selected from nitrogen, argon, helium, or a mixture thereof; preferably argon or nitrogen; more preferably argon. In some embodiments, the degassed method is selected from bubbling, ultrasonic degassed, and membrane degassed; preferably bubbling; more preferably bubbling. In some embodiments, the degassed time is 5-30 min, preferably 7-20 min, more preferably 8-15 min. In some embodiments, the reaction temperature is 50-90 °C; preferably 60-80 °C; more preferably 70 °C. In some embodiments, the reaction time is 12-30 h; preferably 16-28 h; more preferably 20-24 h. In some embodiments, the reaction is carried out under an inert atmosphere.

[0047] In some embodiments, the molecular weight cutoff during dialysis is 3-20 kDa; preferably 4-15 kDa; more preferably 5-10 kDa. In some embodiments, the dialysis time is 6-48 h; preferably 12-36 h; more preferably 24 h. In this application, the dialysis medium is selected from water, physiological saline, or phosphate-buffered saline (PBS), preferably water.

[0048] In this application, the drying method is selected from freeze drying, spray drying, vacuum drying, and room temperature air drying; freeze drying is preferred.

[0049] Preparation method of CPTP-PEG conjugate The preparation method of the CPTP-PEG conjugate is as follows: A carboxyl-containing RAFT chain transfer agent, a carbodiimide condensing agent, and an N-hydroxysuccinimide derivative were mixed and activated, and then incubated with NH2-PEG-COOH to obtain a CPTP-PEG conjugate.

[0050] In some embodiments, the CPTP-PEG conjugate is prepared as follows: A carboxyl-containing RAFT chain transfer agent, a carbodiimide condensing agent, and an N-hydroxysuccinimide derivative were added to a polar organic solvent for activation to obtain a first mixed solution. The first mixed solution was mixed with NH2-PEG-COOH and incubated to obtain the CPTP-PEG conjugate.

[0051] In some embodiments, the carboxyl-containing RAFT chain transfer agent is one or more of 4-cyano-4-(phenylcarbonthioniminothion)pentanoic acid, 4-cyano-4-(dodecylcarbonthioniminothion)pentanoic acid, and 2-cyano-2-propylbenzodisulfide; preferably 4-cyano-4-(phenylcarbonthioniminothion)pentanoic acid.

[0052] In some embodiments, the concentration of the carboxyl-containing RAFT chain transfer agent is 1 to 100 mmol / L; preferably 5 to 50 mmol / L; more preferably 8 to 20 mmol / L.

[0053] In this application, the carbodiimide condensing agent is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), EDC·HCl, N,N'-dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC), preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; In this application, the N-hydroxysuccinimide derivative is one or more of NHS, sulfonic acid-NHS, and NHS-PEG-NHS, preferably NHS or sulfonic acid-NHS.

[0054] In some embodiments, the molar ratio of the carboxyl-containing RAFT chain transfer agent to the carbodiimide condensing agent is (0.1~5):1; preferably (1.2~3):1:1; more preferably (1.5~2):1. In some embodiments, the molar ratio of the N-hydroxysuccinimide derivative to the carboxyl-containing RAFT chain transfer agent is (1~5):1; preferably (1.2~3):1; more preferably (1.3~1.7):1.

[0055] In this application, the polar organic solvent is one or more of DMSO, DMF, DMAc, NMP, acetonitrile, and acetone, preferably DMSO or DMF; more preferably DMSO.

[0056] In some embodiments, the activation time is 0.2 to 4 hours; preferably 0.5 to 2 hours; more preferably 1 hour.

[0057] In some embodiments, the molar ratio of the carboxyl-containing RAFT chain transfer agent to NH2-PEG-COOH is (0.2~2):1; preferably (0.3~1):1; more preferably (0.4~0.6):1.

[0058] In this application, the incubation time is 2 to 48 hours; preferably 8 to 30 hours; more preferably 20 to 24 hours.

[0059] In some embodiments, the first mixed solution is mixed with NH2-PEG-COOH, incubated, dialyzed, and dried to obtain the CPTP-PEG conjugate.

[0060] In some embodiments, the number average molecular weight of PEG in the NH2-PEG-COOH is 2000-8000, preferably 4000-6000, for example 3000, 4000, 5000 or 7000.

[0061] In some embodiments, the dialysis time is 6–48 h, preferably 12–36 h, more preferably 20–30 h. In some embodiments, the molecular weight cutoff for dialysis is 1–50 kDa; preferably 3–20 kDa; more preferably 7–10 kDa. In some embodiments, the drying method is one or more of freeze-drying, spray drying, and vacuum drying; freeze-drying is preferred.

[0062] Preparation method of LCST polymer The LCST polymer was prepared by the following method: The polymer raw materials are subjected to a polymerization reaction in the presence of a crosslinking agent to obtain LCST polymer.

[0063] The polymerization raw materials are N-alkylacrylamide compounds, hydrophilic comonomers, and functional monomers containing reactive functional groups.

[0064] The polymeric raw materials containing N-alkylacrylamide compounds, hydrophilic comonomers, and functional monomers containing reactive functional groups are subjected to polymerization in the presence of a crosslinking agent to obtain LCST polymer.

[0065] In some embodiments, the crosslinking agent is a macromolecular crosslinking agent containing acrylate units; preferably, it is a polyβ-amino ester with acrylate groups as end groups. The addition of a crosslinking agent in the preparation of the LCST polymer helps to form a stable core and provides drug storage space.

[0066] In some embodiments, the polymerization raw material further comprises a bisacrylamide compound; In some embodiments, the N-alkylacrylamide compound is selected from one or more of N-isopropylacrylamide, N-isopropylmethylacrylamide, N-ethylacrylamide, N-tert-butylacrylamide, and N-hydroxyethylacrylamide; preferably N-isopropylacrylamide.

[0067] In some embodiments, the hydrophilic comonomer is selected from one or more of polyethylene glycol methyl ether (meth)acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, N,N-dimethylacrylamide, and vinylpyrrolidone, preferably polyethylene glycol methyl ether methacrylate. The PEG number-average molecular weight of the polyethylene glycol methyl ether methacrylate is 400-1500, preferably 600-1200, for example 500, 700, 800, 900, 950, 1000, 1100, 1200, 1300, and 1400.

[0068] In some embodiments, the functional monomer containing the reactive functional group is selected from one or more of N-(3-aminopropyl)methacrylamide, acrylic acid, acrolein, propargyl (meth)acrylamide, and azidoethyl (meth)acrylamide, preferably N-(3-aminopropyl)methacrylamide hydrochloride.

[0069] In some embodiments, the bisacrylamide compound is selected from N,N′-methylenebisacrylamide.

[0070] The molar ratio of N-alkylacrylamide compounds to hydrophilic comonomers is 1:(0.05~0.5), preferably 1:(0.08~0.3), and more preferably 1:(0.09~0.2). The molar ratio of N-alkylacrylamide compounds to functional monomers containing reactive functional groups is 1:(0.01~0.5), preferably 1:(0.02~0.3), and more preferably 1:(0.03~0.1). The molar ratio of N-alkylacrylamide compounds to bisacrylamide compounds is 1:(0.001~0.1), preferably 1:(0.002~0.05), and more preferably 1:(0.005~0.02). The molar ratio of N-alkylacrylamide compound to crosslinking agent is 1:(0.01~0.5), preferably 1:(0.02~0.3), and more preferably 1:(0.03~0.1).

[0071] The polymeric raw materials containing N-alkylacrylamide compounds, hydrophilic comonomers, and functional monomers containing reactive functional groups are subjected to polymerization in the presence of a crosslinking agent and a surfactant to obtain LCST polymer.

[0072] In some embodiments, the surfactant is selected from sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, dioctyl sulfosuccinate, Tween-20, Brij-35, Pluronic F-127 or CTAB, preferably sodium dodecyl sulfate or Tween-20, and more preferably sodium dodecyl sulfate.

[0073] In some embodiments, the molar ratio of N-alkylacrylamide compound to surfactant is 1:(0.001~0.5), preferably 1:(0.01~0.3), and more preferably 1:(0.03~0.1).

[0074] In some embodiments, the LCST polymer is prepared as follows: An N-alkylacrylamide compound, a hydrophilic comonomer, a functional monomer containing a reactive functional group, a bisacrylamide compound, a crosslinking agent, and a surfactant are added to a second polar solvent to obtain a second precursor solution. The second precursor solution was heated to a temperature sufficient to initiate free radical polymerization, a thermal initiator was added, and the reaction was carried out under an inert atmosphere to obtain the LCST polymer.

[0075] In some embodiments, an N-alkylacrylamide compound, a hydrophilic comonomer, a functional monomer containing a reactive functional group, a bisacrylamide compound, a crosslinking agent, and a surfactant are added to a second polar solvent under stirring and inert gas protection to obtain a second precursor solution. In some embodiments, the stirring is vigorous stirring.

[0076] In some embodiments, the second polar solvent is selected from one or more of water, DMSO, DMF, ethanol, isopropanol, and ethylene glycol, preferably DMSO / water, DMF / water, or ethanol / water, more preferably DMSO / water, wherein the volume ratio of DMSO to water is (1~9):(1~9), preferably (1~4):(1~4), more preferably (1~2):(1~2), and even more preferably 1:1.

[0077] In some embodiments, the concentration of the N-alkylacrylamide compound in the second precursor solution is 0.001~1.2 mol / L, preferably 0.02~1 mol / L, more preferably 0.03~0.5 mol / L, and even more preferably 0.03~0.1 mol / L.

[0078] In some embodiments, the second precursor solution is heated to a temperature sufficient to initiate free radical polymerization, a thermal initiator is added, the reaction is carried out under an inert atmosphere, the solution is cooled, dialyzed, and dried to obtain the LCST polymer.

[0079] In some embodiments, the temperature sufficient to initiate free radical polymerization is 60-90 °C, preferably 65-75 °C, and more preferably 70 °C.

[0080] In some embodiments, the thermal initiator is selected from ammonium persulfate, potassium persulfate, sodium persulfate, VA-044, AIBN, V-501, BPO, TBHP, and preferably ammonium persulfate.

[0081] In some embodiments, the molar ratio of N-isopropylacrylamide in the thermal initiator and the second precursor solution is (0.01~1):1, preferably (0.03~0.5):1, more preferably (0.05~0.2):1, and even more preferably (0.08~0.15):1. In some embodiments, the inert atmosphere is at least one of nitrogen, argon, and helium, preferably argon. In some embodiments, the reaction time is 1~12 h, preferably 2~6 h, more preferably 4 h. In some embodiments, cooling is performed after the reaction is completed, cooling to room temperature.

[0082] In some embodiments, the molecular weight cutoff during dialysis is 1-50 kDa, preferably 3-10 kDa, and more preferably 7 kDa. In some embodiments, the dialysis solvent is water or PBS. In some embodiments, the dialysis time is 12 hours to 5 days, preferably 2-4 days, and more preferably 3 days. In some embodiments, the drying method is freeze-drying.

[0083] Preparation method of poly-β-amino ester The preparation method of polyβ-amino ester with acrylate group as end group is to react acrylated cystamine compound and 4-(aminomethyl)piperidine in a polar organic solvent.

[0084] In some embodiments, the acrylated cystamine compound is selected from N,N′-bis(acryloyl)cystamine.

[0085] In some embodiments, the molar ratio of acrylamide cysteine ​​compound to 4-(aminomethyl)piperidine is (1~3):1, preferably (1.5~2.5):1, and more preferably (1.8~2.2):1.

[0086] In some embodiments, the polar organic solvent is one or more of DMSO, DMF, DMAc, and NMP, preferably DMSO.

[0087] In some embodiments, the reaction temperature is 50~70°C, preferably 60°C. The reaction time is 2~4 days, preferably 3 days.

[0088] In some embodiments, the reaction is carried out under the protection of an inert gas, which is argon or nitrogen; preferably, the inert gas is argon.

[0089] In some embodiments, the resulting polyβ-amino ester has acrylate groups as end groups. In some embodiments, the polyβ-amino ester is stored in the dark at -25°C to -15°C, preferably at -20°C.

[0090] Preparation method of core-shell structured thermosensitive carrier The preparation method of the core-shell structured thermosensitive carrier as described above includes: (1) Provide core particles formed from LCST polymer; (2) The UCST polymer is used to form a shell on the surface of the core particles to obtain a core-shell structured thermosensitive carrier.

[0091] In some embodiments, the UCST polymer is incubated on the surface of the core particles to obtain a core-shell structured thermosensitive carrier. In some embodiments, the core particles are LCST polymers.

[0092] In some embodiments, the core-shell structured thermosensitive carrier is prepared by the following steps: The UCST polymer, carbodiimide condensing agent, and N-hydroxysuccinimide derivative were mixed and activated, then LCST polymer was added and incubated to obtain a core-shell structured thermosensitive carrier.

[0093] More preferably, the core-shell structured thermosensitive carrier is prepared by the following steps: The UCST polymer, carbodiimide condensing agent, and N-hydroxysuccinimide derivative were added to a third polar solvent to obtain a third precursor solution; the LCST polymer was added to the third precursor solution and incubated to obtain a core-shell structured thermosensitive carrier.

[0094] In some embodiments, the UCST polymer, carbodiimide condensing agent, and N-hydroxysuccinimide derivative are added to a third polar solvent and activated to obtain a third precursor solution.

[0095] In some embodiments, the carbodiimide condensing agent is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), EDC·HCl, N,N'-dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC), preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the N-hydroxysuccinimide derivative is one or more of NHS, sulfonic acid-NHS, and NHS-PEG-NHS, preferably NHS or sulfonic acid-NHS. In some embodiments, the third polar solvent is one or more of DMSO, DMF, DMAc, and NMP, preferably DMSO.

[0096] In some embodiments, the concentration of the UCST polymer in the third polar solvent is 2-20 mg / mL, preferably 5-15 mg / mL, even more preferably 8-12 mg / mL, and more preferably 10 mg / mL. In some embodiments, the molar ratio of the carbodiimide condensing agent to the N-hydroxysuccinimide derivative is 1:(0.8-1.5), preferably 1:1. In some embodiments, the concentration of the carbodiimide condensing agent is 0.005-0.05 mol / L, preferably 0.015 mol / L.

[0097] In this application, the activation is carboxyl activation. In some embodiments, the activation time is 0.5 to 3 hours, preferably 1 hour.

[0098] In some embodiments, LCST polymer is added to a third precursor solution, incubated, dialyzed, and dried to obtain a core-shell structured thermosensitive carrier.

[0099] In some embodiments, the mass ratio of the LCST polymer to the UCST polymer in the third precursor solution is (0.5~2):(0.5~2), preferably (0.5~1.5):1, and more preferably 1:1.

[0100] In some embodiments, the incubation temperature is 15-40°C, preferably 20-30°C. In some embodiments, the incubation time is 12-48 hours, preferably 24 hours. In some embodiments, the molecular weight cutoff for dialysis is 1-50 kDa; preferably 3-20 kDa; more preferably 7-10 kDa. In some embodiments, the dialysis medium is water. In this application, the dialysis time is 6-48 hours, preferably 12-36 hours, more preferably 20-30 hours, and even more preferably 24 hours. In some embodiments, the drying method is one or more of freeze-drying, spray drying, and vacuum drying; freeze-drying is preferred.

[0101] <Core-shell structured thermosensitive drugs> The present invention provides a core-shell thermosensitive drug, which includes a core-shell thermosensitive carrier as described above and a drug loaded on the core-shell thermosensitive carrier.

[0102] In some embodiments, the drug is a drug capable of repairing damaged mitochondria. In some embodiments, the drug can be hydrophilic or lipophilic. In some embodiments, the drug is CoQ10 or doxorubicin.

[0103] In some embodiments, core-shell thermosensitive drugs are prepared by solvent substitution.

[0104] In some embodiments, the core-shell thermosensitive drug is obtained by the following preparation method: The core-shell thermosensitive carrier and the drug, as described above, were mixed in a polar organic solvent, and water was added to obtain a mixed solution. The mixed solution was dialyzed to obtain the core-shell thermosensitive drug.

[0105] In some embodiments, the mass ratio of the core-shell thermosensitive carrier to the drug is (1~50):1, preferably (3~30):1, even more preferably (4~20):1, more preferably (5~15):1, and even more preferably (8~12):1. In some embodiments, the polar organic solvent is selected from DMSO.

[0106] The core-shell thermosensitive drug of the present invention can increase the temperature of damaged mitochondria; reduce mitochondrial ROS levels; and significantly improve the survival rate of damaged neurons. The core-shell thermosensitive drug of the present invention has a significantly better repair effect on damaged mitochondria than unloaded drugs.

[0107] The core-shell structured thermosensitive drug of the present invention has an upper critical dissolution temperature in the range of 42~50°C and a lower critical dissolution temperature in the range of 39~45°C.

[0108] <Composition> The present invention provides a pharmaceutical composition comprising a core-shell thermosensitive drug and pharmaceutical excipients as described above.

[0109] <Application> This invention provides the use of a core-shell structured thermosensitive drug or a pharmaceutical composition as described above for the preparation of a drug for treating diseases related to mitochondrial dysfunction.

[0110] In some embodiments, the mitochondrial dysfunction-related diseases are Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia.

[0111] The following embodiments are examples to illustrate the technical solution of the present invention: Preparation Example 1: Preparation of UCST Polymer Step 1: Synthesis of CPTP-PEG conjugates 4-Cyano-4-(phenylcarbothioiminothio)pentanoic acid (14 mg, 0.05 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (14.3 mg, 0.075 mmol), and N-hydroxysuccinimide (8.6 mg, 0.075 mmol) were dissolved in 5 mL of DMSO, and the carboxyl groups were activated for 1 hour. Then, NH2-PEG-COOH (500 mg, 0.1 mmol) was added, and the mixture was incubated in the above solution for 24 hours. Subsequently, the reaction solution was dialyzed against double-distilled water for 24 hours (molecular weight cutoff 7 kDa), and then freeze-dried to obtain the CPTP-PEG conjugate. The number-average molecular weight of PEG in NH2-PEG-COOH was 5000.

[0112] Step 2: Synthesis of UCST polymer The CPTP-PEG conjugate (130 mg, 0.025 mmol), azobisisobutyronitrile (1.2 mg, 0.007 mmol), acrylamide (427 mg, 6 mmol), and acrylonitrile (96 μL, 1.5 mmol) were dissolved in 5 mL DMSO to obtain the first precursor solution. The first precursor solution was degassed under argon bubbling for about 10 minutes, and then placed in a 70°C oil bath and reacted under argon protection for 24 hours. Subsequently, it was dialyzed with double-distilled water for 24 hours (molecular weight cutoff 7 kDa), and then freeze-dried to obtain a powder, which is the UCST polymer.

[0113] The 1H NMR spectrum of the UCST polymer was obtained as follows: Figure 1 As shown; the Fourier transform infrared spectrum of the UCST polymer is shown below. Figure 2 As shown.

[0114] Preparation Example 2: Preparation of LCST Polymer Step 1: Synthesis of poly-β-amino esters N,N'-bis(acryloyl)cysteamine (520 mg, 2 mmol) and 4-(aminomethyl)piperidine (114.2 mg, 1 mmol) were mixed in 5 mL of DMSO. The solution was stirred at 60 °C for 3 days under argon protection. This yielded poly-β-amino esters with acrylate end groups, which were stored at -20 °C protected from light.

[0115] Step 2: Synthesis of LCST polymer N-Isopropylacrylamide (113 mg, 1 mmol), polyβ-amino ester (30 mg, 0.005 mmol), polyethylene glycol methyl ether methacrylate (120 mg, 0.125 mmol), N-(3-aminopropyl)methacrylamide hydrochloride (8.9 mg, 0.05 mmol), N,N'-methylenebisacrylamide (1.5 mg, 0.01 mmol), and sodium dodecyl sulfate (20 mg, 0.069 mmol) were dissolved in 20 mL of a DMSO / H₂O mixed solvent (1 / 1 v / v) under vigorous stirring and argon purging to obtain a second precursor solution. When the solution temperature reached 70 °C, ammonium persulfate (22.4 mg, 0.1 mmol) was rapidly added as an initiator, and the reaction was carried out at 70 °C for 4 hours under argon protection. The solution was cooled to room temperature, dialyzed against ddH₂O for 3 days (molecular weight cutoff 7 kDa), and freeze-dried to obtain the LCST polymer. The number-average molecular weight of polyethylene glycol methyl ether methacrylate is 950.

[0116] The Fourier transform infrared spectrum of the LCST polymer was obtained as follows: Figure 2 As shown.

[0117] Example 1: Preparation of a core-shell structured thermosensitive carrier UCST polymer (50 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (14.3 mg, 0.075 mmol), and N-hydroxysuccinimide (8.6 mg, 0.075 mmol) were dissolved in 5 mL of DMSO, and the carboxyl groups were activated for 1 hour to obtain the third precursor solution. LCST polymer (50 mg) was added to the third precursor solution, and the mixture was incubated for 24 hours. The solution was then dialyzed with ddH2O for 24 hours (molecular weight cutoff 7 kDa), and freeze-dried to obtain the core-shell thermosensitive carrier.

[0118] The Fourier transform infrared spectrum of the core-shell thermosensitive carrier was obtained as follows: Figure 2 As shown.

[0119] Based on the total mass of the temperature-sensitive carrier with the core-shell structure, the shell has a mass fraction of 50%, and the core has a mass fraction of 50%.

[0120] The shell thickness of the core-shell structured thermosensitive carrier is 59 nm, and the core particle size is 228 nm. The particle size of the core-shell structured thermosensitive carrier is 287 nm.

[0121] Example 2: Preparation of CoQ10-core-shell thermosensitive carrier and DOX-core-shell thermosensitive carrier The CoQ10-core-shell thermosensitive carrier was prepared using a solvent substitution method: 20 mg of the core-shell thermosensitive carrier and 2.5 mg of CoQ10 were mixed in 1 mL of DMSO, and 9 mL of deionized water was added under sonication. The solution was dialyzed against ddH2O for 24 hours to obtain the CoQ10-core-shell thermosensitive carrier.

[0122] The same method was used to prepare DOX-core-shell structured thermosensitive carriers, except that CoQ10 was replaced with DOX.

[0123] Test Example 1: Phase Transition Temperature Determination The UCST and LCST of the UCST polymer and LCST polymer were determined by pyrene fluorescence method.

[0124] Add 20 μL of pyrene acetonitrile solution (20 μg / mL) to a 20 mL brown bottle and air-dry the bottle until the acetonitrile evaporates. Add 4 mL of aqueous solution containing 2 mg / mL UCST polymer or LCST polymer to the bottle. After equilibration at different water bath temperatures for 1 hour, record the excitation spectrum of the solution at an excitation wavelength of 300-360 nm and an emission wavelength of 395 nm (both excitation and emission bandwidths are set to 5 nm).

[0125] The results are as follows Figure 3 and Figure 4 As shown.

[0126] Figure 3 The results show that when the temperature rises from 42°C to 50°C, the UCST polymer undergoes a phase transition, during which the UCST polymer can change from a highly hydrophobic form to a hydrophilic form. Figure 4 The results showed that when the temperature increased from 39°C to 45°C, the LCST polymer also underwent a phase transition, changing from a hydrophilic form to a hydrophobic form.

[0127] Test Example 2: In vitro release experiment The CoQ10-core-shell thermosensitive vector was diluted with PBS solution containing 1% ethanol. This solution was then mixed with 1 mM DTT (dithiothreitol) and incubated at 37°C, 42°C, and 48°C for specific times. The mixture was then centrifuged, and the supernatant was collected. The supernatant was analyzed using a UV-Vis spectrophotometer.

[0128] A DOX-core-shell thermosensitive carrier was added to a dialysis bag, which was then immersed in 20 mL of phosphate-buffered saline (PBS) solution containing 1 mM DTT. Dialysis was performed at 37°C, 42°C, and 48°C. At regular time intervals, 4 mL of dialysate was removed, and an equal volume of fresh buffer was added to the dialysis tube. DOX release was recorded using a fluorescence spectrophotometer. Results are as follows: Figure 5 and Figure 6 As shown.

[0129] As can be seen, approximately 27% of CoQ10 was released after 24 hours at 37°C. This is because the hydrophobicity of the outer UCST polymer prevented DTT from entering, thus reducing the reduction effect on the inner LCST polymer. However, at 42°C, the release of CoQ10 increased to 40%, attributed to the outer UCST polymer increasing the amount of DTT, resulting in a higher reduction effect on the inner LCST polymer. The release at 48°C was lower than at 42°C because the inner LCST polymer prevented DTT from attacking disulfide bonds.

[0130] The release behavior of DOX is similar to that of CoQ10.

[0131] Test Example 3: Intracellular Drug Release Experiment To investigate the intracellular release behavior of a core-shell thermosensitive vector, SH-SY5Y cells were initially stocked at a density of 1×10⁻⁶ cells. 4 Cells were seeded per well in confocal culture dishes and cultured for 24 hours. Then, they were co-incubated with 50 nM and 100 nM rotenone for 2 hours each to establish a Parkinson's disease cell model. Next, the Parkinson's model cells were treated with a DOX-nuclear-shell thermosensitive carrier for 12 hours. The cells were then washed three times with PBS. The nuclei were stained with Hoechst 33342 for 15 minutes, and then observed under a confocal laser scanning microscope. Drug release from the nucleus-shell thermosensitive carrier in the cells was assessed using confocal microscopy. Results are as follows: Figure 7 As shown.

[0132] It is evident that the fluorescence intensity of cells treated with rotenone is stronger than that of normal cells, which means that when mitochondria are damaged, the release effect of DOX-nuclear-shell thermosensitive carrier is better, and the release amount is also greater as the degree of damage increases.

[0133] Test Example 4: Experiment on the Repair of Mitochondrial Temperature Normal mitochondria have a higher temperature, while damaged mitochondria have a lower temperature. In this experiment, the Mito thermoyellow probe was used to monitor mitochondrial temperature. Parkinson's cell models were treated with CoQ10 and CoQ10-nuclear-shell thermosensitive carriers for 24 hours. After washing the cells three times with PBS, they were incubated with mitochondrial probes for 15 minutes, followed by incubation with Hoechst 33342 for 15 minutes. The cells were then observed under a confocal laser scanning microscope.

[0134] The results are as follows Figure 8 As shown, the probe exhibited the strongest fluorescence intensity in cells treated with rotenone. Since the fluorescence of the Mitothermo yellow probe decreases with increasing temperature, the highest fluorescence intensity in rotenone-treated cells indicates a lower temperature in cells containing damaged mitochondria. In contrast, cells further treated with a CoQ10-core-shell thermosensitive carrier after rotenone treatment showed relatively higher temperatures. It is evident that the CoQ10-core-shell thermosensitive carrier can release CoQ10 at damaged mitochondria and repair them.

[0135] Test Example 5: Changes in Reactive Oxygen Species in Mitochondria The level of CoQ10 in the mitochondria of Parkinson's disease patients is relatively low. Supplementing with exogenous CoQ10 can improve the CoQ10 deficiency in diseased tissues, thereby restoring mitochondria to a normal state. Therefore, this experiment investigated whether the CoQ10-nuclear-shell thermosensitive carrier could repair mitochondria by altering mitochondrial reactive oxygen species (ROS). To investigate the content of ROS in mitochondria, Parkinson's model cells were treated with CoQ10 and the CoQ10-nuclear-shell thermosensitive carrier for 24 hours. After washing the cells three times with PBS, they were incubated with Mito-SOX for 10 minutes, then with Hoechst 33342 for 15 minutes, and then observed under a confocal laser scanning microscope. The results are as follows: Figure 9 As shown.

[0136] It is evident that the production of mitochondrial reactive oxygen species (ROS) in the Parkinson's disease cell model is higher than normal. The levels of mitochondrial ROS decreased after the addition of CoQ10 and a CoQ10-core-shell thermosensitive carrier (also known as CoQ10@UL), respectively. These results indicate that the CoQ10-core-shell thermosensitive carrier can attach to damaged mitochondria and release CoQ10 to repair them.

[0137] Test Example 6: In vitro cytotoxicity experiment The cytotoxicity of the CoQ10-nuclear-shell thermosensitive vector was evaluated in vitro using the MTT assay on SH-SY5Y cells, a Parkinson's disease model. Results are as follows: Figure 10 and Figure 11 As shown.

[0138] like Figure 10 As shown, when free CoQ10 was co-incubated with SH-SY5Y cells of the Parkinson's disease model, cell proliferation did not change significantly. When the concentration of free CoQ10 increased from 0 to 25 µM, cell viability increased from 62% to 75%, an increase of only about 10%.

[0139] In contrast, when the CoQ10-core-shell thermosensitive carrier was co-incubated with SH-SY5Y cells of a Parkinson's disease model, cell proliferation was significant. After 24 hours of treatment with 25 µM CoQ10, cell viability reached as high as 87%. Furthermore, as... Figure 11 As shown, the CoQ10-nuclear-shell thermosensitive carrier did not have a significant inhibitory or promoting effect on normal SH-SY5Y cells.

[0140] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0141] literature 1. Chrétien, D. et al. Mitochondria are physiologically maintained atclose to 50℃. PLOS Biology (2018), 16(1), e2003992. 2. Wang, D. et al. A thermoresponsive nanocarrier for mitochondria-targeted drug delivery, Chemical Conmmunications, 2019, 55(28), 4051-4054.

Claims

1. A core-shell structured thermosensitive carrier, characterized in that, Includes a kernel and a shell that at least partially covers the kernel. The core is made of LCST polymer; the shell is made of UCST polymer; The shell has a mass fraction of 40-60%, preferably 45-55%; the core has a mass fraction of 40-60%, preferably 45-55%, and the total mass of the temperature-sensitive carrier based on the core-shell structure is measured.

2. The core-shell structured thermosensitive carrier according to claim 1, characterized in that, The LCST polymer is selected from one or more of poly(N-isopropylacrylamide) and its derivatives, polyethylene glycol methyl ether methacrylate and its derivatives, poly(N-3-aminopropyl methacrylamide) and its derivatives, polyepoxyalkane and its derivatives, polymethyl vinyl ether and its derivatives, poly-N-vinylcaprolactam and its derivatives, polysiloxane and its derivatives, and copolymers of poly(N-isopropylacrylamide) with ethylene glycol methyl ether methacrylate and N-3-aminopropyl methacrylamide and their derivatives, preferably copolymers of poly(N-isopropylacrylamide) with ethylene glycol methyl ether methacrylate and N-3-aminopropyl methacrylamide and their derivatives; The UCST polymer is selected from one or more of polyacrylamide, polyacrylonitrile, polyacrylamide-acrylonitrile copolymer, polystyrene, polystyrene copolymer and polyethylene oxide copolymer, preferably polyacrylamide-acrylonitrile copolymer.

3. The core-shell structured thermosensitive carrier according to claim 1, characterized in that, The lower critical dissolution temperature of the LCST polymer is 37~48℃, preferably 39~45℃; The upper critical dissolution temperature of the UCST polymer is 40~52℃, preferably 42~50℃; and / or The thickness of the shell layer is 50~70nm, preferably 55~65nm; the particle size of the core is 210~250nm, preferably 225~232nm; the particle size of the core-shell structured thermosensitive carrier is 265~320nm, preferably 275~310nm.

4. A method for preparing the core-shell structured thermosensitive carrier according to any one of claims 1-3, comprising: Provides core particles formed from LCST polymer; The UCST polymer is used to form a shell layer on the surface of the core particles to obtain a core-shell structured thermosensitive carrier. The mass ratio of LCST polymer to UCST polymer is (0.5~2): (0.5~2), preferably (0.5~1.5):1, and more preferably 1:

1.

5. The preparation method according to claim 4, characterized in that, The UCST polymer comprises the structural units -CH2-CH(CONH2)- and -CH2-CH(CN)-; and / or The LCST polymer comprises the structural units -CH2-CH(CONH-iPr)-, -CH2-C(CH3)(COO-PEG)-, and -CH2-C(CH3)(CO-NH-(CH2)3-NH2)-.

6. The preparation method according to claim 4, characterized in that, The UCST polymer was prepared by the following method: polymer monomers were polymerized in the presence of a chain extender to obtain the UCST polymer. The polymer monomer is one or more of olefin amide monomers and olefin acrylonitrile monomers; Preferably, the acrylonitrile monomer is C2-6 acrylonitrile, wherein the C2-6 acrylonitrile is selected from one or more of acrylonitrile, methacrylonitrile, 2-butenonitrile, and 4-pentenonitrile, and more preferably acrylonitrile; Preferably, the amide monomer is a C2-6 amide, wherein the C2-6 amide is selected from acrylamide, methacrylamide, N-isopropylacrylamide, N,N-dimethylacrylamide or N-hydroxyethylacrylamide, and more preferably acrylamide; Preferably, the molar ratio of the amide monomer to the acrylonitrile monomer is (1~20):1; more preferably (2~10):1; and even more preferably (3~6):

1. Preferably, the chain extender is a macromolecular chain extender containing PEG.

7. The preparation method according to claim 6, characterized in that, The LCST polymer was prepared by the following method: The polymer raw materials are subjected to a polymerization reaction in the presence of a crosslinking agent to obtain LCST polymer; The polymerization raw material comprises N-alkylacrylamide compounds, hydrophilic comonomers, and functional monomers containing reactive functional groups; the polymerization raw material further comprises bisacrylamide compounds; Preferably, the crosslinking agent is a macromolecular crosslinking agent containing acrylate units; more preferably, it is a polyβ-amino ester with acrylate groups as end groups. The N-alkylacrylamide compound is selected from one or more of N-isopropylacrylamide, N-isopropylmethacrylamide, N-ethylacrylamide, N-tert-butylacrylamide, and N-hydroxyethylacrylamide; preferably N-isopropylacrylamide. The hydrophilic comonomer is selected from one or more of polyethylene glycol methyl ether (meth) acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, N,N-dimethylacrylamide, and vinylpyrrolidone, preferably polyethylene glycol methyl ether methacrylate; The functional monomer containing the reactive functional group is selected from one or more of N-(3-aminopropyl)methacrylamide, acrylic acid, acrolein, propargyl (meth)acrylamide, and azidoethyl (meth)acrylamide, preferably N-(3-aminopropyl)methacrylamide hydrochloride; The bisacrylamide compounds are selected from N,N′-methylenebisacrylamide.

8. A core-shell thermosensitive drug comprising the core-shell thermosensitive carrier as described in any one of claims 1 to 7 and a drug loaded on the core-shell thermosensitive carrier; preferably, the drug is CoQ10 or doxorubicin.

9. A pharmaceutical composition comprising the core-shell thermosensitive drug of claim 8 and pharmaceutical excipients.

10. The use of a core-shell thermosensitive medicament as described in claim 8 or a pharmaceutical composition as described in claim 9 in the preparation of a medicament for treating diseases related to mitochondrial dysfunction; Preferably, the mitochondrial dysfunction-related disease is Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia.

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