Tumor-targeted activatable nano photosensitizer as well as preparation method and application thereof

Abstract

The invention discloses a tumor targeting activatable nano photosensitizer as well as a preparation method and application thereof. The tumor targeting activatable nano photosensitizer comprises a cationic polymer, an anionic polymer and an aromatic structure photosensitizer. Cationic-pi interaction is formed between a cationic side chain in the cationic polymer and the photosensitizer with an aromatic structure, the nano photosensitizer is formed through self-assembly under the interaction, and the photosensitive activity of the nano photosensitizer in blood circulation is in a completely inactivated state; and no light toxic or side effect is generated on normal light exposure tissues. When selectively enriched in tumor tissues, the photosensitizer is structurally degraded in tumor cells in a specific environment, and the photosensitive activity of the photosensitizer is quickly activated and recovered, so that efficient tumor inhibition treatment is realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a tumor-targeted activatable nano photosensitizer and a preparation method and application thereof. Background Art

[0002] Compared with the two most conventional chemotherapy and surgical treatments in current cancer treatment, photodynamic therapy has gained wide attention in the treatment of cancer and other diseases due to its high spatiotemporal selectivity and non-invasiveness. (Karges J, et al. Angew. Chem., Int. Ed. 2019, 58, 14334). So far, more and more photosensitizers have been commercialized or used in clinical trials, such as porfimer sodium, verteporfin or and 5-aminolevulinic acid (Zhao X, et al. Chem. Soc. Rev. 2021, 50, 4185). Although current research has made significant progress in improving the effectiveness of photodynamic therapy, the defect of poor in vivo selectivity of photosensitizers often leads to inevitable side effects during treatment. The low efficiency of tumor-specific delivery and the photosensitizing activity of traditional photosensitizers have been in an activated state, which usually leads to poor excitation and significant skin phototoxicity (Li YS, et al. ACS Nano 2022, 16, 17298). In addition, traditional photosensitizers have a slow metabolism rate, and in order to avoid phototoxic side effects, patients need to avoid sunlight exposure for several weeks after treatment. This limitation not only hinders the practicality of photodynamic therapy, but also reduces the quality of life of patients (Xie ZJ, et al. Chem. Soc. Rev. 2020, 49, 8065). Obviously, the insufficient efficiency of tumor-selective delivery and the defects of current photosensitizers significantly limit their clinical application.

[0003] Enhancing the specific delivery to the tumor site and improving the specific activation of photosensitizers at the tumor site are two effective methods to solve the above problems. Constructing nanophotosensitizers with tumor targeting capabilities can enhance the accumulation of photosensitizers at the tumor site (Pham TC, et al. Chem. Rev. 2021, 121, 13454). However, despite the rapid development of nanomedicines, existing drug delivery systems still have the problem of poor delivery efficiency (about 0.7%), which is far from enough to reduce the skin phototoxicity of photosensitizers (Li M, et al. J. Am. Chem. Soc. 2018, 140, 15820). Therefore, it is very necessary to develop photosensitizers that selectively activate tumor sites to achieve targeted and on-demand tumor photodynamic therapy.

[0004] In general, the design of activatable photosensitizers is based on the principle of inhibiting the fluorescence emission of photosensitizers and blocking the energy transfer pathway between photosensitizers and oxygen molecules after light irradiation (Gao J, et al. J. Am. Chem. Soc. 2018, 140, 4945). At present, many studies combine photosensitizers with energy / electron transfer processes, such as fluorescence resonance energy transfer and light-induced electron transfer. These two strategies are widely used and are considered to be more effective construction strategies. Specifically, these activatable photosensitizer systems usually covalently connect the quenching group and the photosensitizer through a cleavable linker, wherein the linker can be destroyed under pathological and physiological stimuli, such as various tumor-specific enzymes, weak acid environments, high redox environments, etc., which can eventually lead to the release of photosensitizers and the activation of photosensitizing activity. In theory, inhibiting the singlet oxygen production ability of photosensitizers in the blood circulation and non-tumor tissues helps to reduce the phototoxic side effects of the skin. On the contrary, the activation of photosensitizers in tumor tissues can restore their singlet oxygen production ability and restore their anti-tumor effects (Liu Y, et al. Biomaterials 2022, 284, 121467). Based on the above strategies, many activatable photosensitizer systems have been reported and have been shown to reduce the phototoxic side effects of the skin during photodynamic therapy. However, challenges still exist, such as the complex molecular structure design, time-consuming synthesis process, high cost and potential toxicity of the newly introduced quenching group of most activated photosensitizers (Li X, et al. J. Am. Chem. Soc. 2019, 141, 1366). In addition, the incomplete inhibition of photosensitization activity has side effects on normal tissues that cannot be ignored. Most importantly, only a few photosensitizers with modification sites in their chemical structures can be developed into activated photosensitizers, which directly leads to the limitations of constructing this type of activated photosensitizer. Summary of the invention

[0005] The present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, the present invention aims to provide a tumor-targeted activatable nano-photosensitizer and a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] In the first aspect of the present invention, there is provided a compound of formula I or a compound of formula II:

[0008]

[0009] Wherein, R is a polymer block formed by polymerization of a hydrophilic monomer;

[0010] A is a CAT fragment; B is H, -CH3 or absent;

[0011] L1 and L2 are each independently selected from -O-, -NH-, -response key-, -O-response key-, -NH-response key-, or are absent;

[0012] R1 and R3 are each independently selected from H, -CH3;

[0013] R2 is selected from -C1-C6 alkylene-basic cationic group, -ester group-C1-C6 alkylene-basic cationic group, -C1-C6 iminoester group-C1-C6 alkylene-basic cationic group; the basic cationic group includes any one of positively charged amino group, primary amine group, secondary amine group, tertiary amine group, quaternary amine group, guanidine group;

[0014] R4 is selected from -OH, -NH2, -NH-C1-C6 alkyl, -O-C1-C6 alkylene-N(C1-C6 alkylene)2, -O-C0-C6 alkylene-PEG-O-C0-C6 alkyl;

[0015] 0 < x ≤ 1; m and y are natural numbers between 2 and 200 (such as 10, 20, 30, 40, 50, 80, 100, 120, 150, 160, 180, etc.); z is a natural number between 0 and 200 (such as 5, 10, 20, 30, 40, 50, 80, 100, 120, 150, 160, 180, etc.);

[0016] The degree of polymerization of the polymer block is a natural number between 0 and 200 (such as 5, 10, 20, 30, 40, 50, 80, 100, 120, 150, 160, 180, etc.).

[0017] In some embodiments of the present invention, the hydrophilic monomer includes at least one of ethylene glycol, methoxypolyethylene glycol methacrylate, acrylic acid, methacrylic acid, acrylamide, N-isopropylacrylamide, or N,N-dimethylaminoethyl methacrylate.

[0018] In some embodiments of the present invention, the CAT fragment refers to a reversible addition fragmentation chain transfer polymerization (RAFT) chain transfer agent (CAT) used in a polymerization reaction, and the CAT fragment is retained as a polymer end group or linker. The chain transfer agents include 3,5-bis(2-dodecylthiothiocarbonylthio-1-oxopropoxy)benzoic acid, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid 3-butenyl ester, 2-cyanobutane-2-yl 4-chloro-3,5-dimethyl-1H-pyrazole-1-dithiocarboxylate, 2-cyanobutyl-2-yl 3,5-dimethyl-1H-pyrazole-1-dithiocarboxylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanol, 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanol, cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithiocarboxylate, cyanomethyl dodecyl trithiocarbonate, [3-(trimethoxysilyl)propyl]trithiocarboxylate, Thiocarbonic acid cyanomethyl ester, trithiocarbonic acid 2-cyano-2-propyl dodecyl ester, trithiocarbonic acid S, S-dibenzyl ester, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid N-hydroxysuccinimide ester, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiothiocarbonylthio) propionic acid, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid methyl ester, pentaerythritol tetrakis[2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid ester], phthalimidomethyl butyl trithiocarbonate, 1,1,1-Tris[(dodecylthiothiocarbonylthio)-2-methylpropionate]ethane, 1H-pyrrole-1-dithiocarbamate phenyl, diphenyl dithiocarbamate cyanomethyl ester, methyl (phenyl) dithiocarbamate cyanomethyl ester, methyl (4-pyridyl) dithiocarbamate cyanomethyl ester, N-methyl-N-(pyridin-4-yl) dithiocarbamate 2-cyanopropane-2-yl ester, 2-[methyl (4-pyridyl) thiocarbamate thio] propionate methyl Ester, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)thiocarbamic acid thio] pentanoate, benzyl dithiobenzoate, cyanomethyl dithiobenzoate, 4-cyano-4-(phenylthiocarbonylthio) pentanoic acid, 4-cyano-4-(phenylthiocarbonylthio) pentanoic acid N-succinimidyl ester, 2-cyano-2-propyl dithiobenzoate, 2-cyano-2-propyl 4-cyanodithiobenzoate, 2-(4-methoxy ethyl 2-methyl-2-(phenylthiocarbonylthio)propanoate, ethyl 2-(phenylthiocarbonylthio)-2-phenylacetate, ethyl 2-(phenylthiocarbonylthio)propanoate, ethyl 2-(phenylthiocarbonylthio)propanoate, 1-(methoxycarbonyl)ethyl dithiobenzoate, 2-(4-methoxyphenylthiocarbonylthio)acetic acid, 2-nitro-5-(2-propynyloxy)benzyl 4-cyano-4-(phenylthiocarbonylthio)pentanoate, 2-(phenylthiocarbonylthio)pentanoate methyl (4-pyridyl) dithiocarbamate, 2-cyanopropane-2-yl N-methyl-N-(pyridin-4-yl) dithiocarbamate, methyl 2-[methyl (4-pyridyl) thiocarbamate thio] propionate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl) thiocarbamate thio] pentanoate, etc.

[0019] The fragmentation of CTA and the resulting fragment structures are well established in the art, and thus the structure of the CAT fragment can be determined by utilizing existing techniques without using inventive techniques.

[0020] Common CAT fragments are selected from wait.

[0021] In some embodiments of the present invention, when L1 or L2 is present, -C(O)-L1- or -C(O)-L2- is each independently selected from -C(O)-O-, -C(O)-NH-, -C(O)-O-responsive bond-, and -C(O)-NH-responsive bond-.

[0022] In some embodiments of the present invention, the responsive bond includes a disulfide bond, a hydrazone bond, a thioketal bond, and an ester bond. The disulfide bond is GSH responsive, the hydrazone bond is weak acid responsive, the thioketal bond is active oxygen responsive, and the ester bond is esterase responsive.

[0023] In some embodiments of the invention, the basic cationic group comprises a positively charged primary amine group. Secondary amine group Tertiary amine group Quaternary amine group Guanidine Any one of .

[0024] In some embodiments of the present invention, R2 is selected from

[0025] In some embodiments of the present invention, the compound of formula I or the compound of formula II is selected from the following compounds:

[0026]

[0027] In some embodiments of the present invention, the compound of Formula I or the compound of Formula II is a cationic polymer.

[0028] The second aspect of the present invention provides a method for preparing a compound of formula I or a compound of formula II, comprising the following steps:

[0029]

[0030] When x is 1, the compound of formula I-1 is polymerized under the action of a chain transfer agent and an initiator modified by R to obtain a compound of formula I;

[0031]

[0032] When x is not 1, the compound of formula I-1, formula I-1, and the compound of formula I-2, formula I-2, are polymerized under the action of a chain transfer agent and an initiator modified by R to obtain a compound of formula I;

[0033]

[0034] When z is 0, the compound of formula I-1 is polymerized under the action of a chain transfer agent and an initiator to obtain a compound of formula II;

[0035]

[0036] When z is not 0, the compound of formula I-1 and the compound of formula I-2 are polymerized under the action of a chain transfer agent and an initiator to obtain a compound of formula II;

[0037] Wherein, R, A, B, R1-R4, L1, L2, x, and z are defined as above.

[0038] The third aspect of the present invention provides a cationic polymer comprising

[0039] (I) a homopolymer, a (meth)acrylate monomer containing an R2 group, or a (meth)acrylamide monomer unit containing an R2 group; and / or,

[0040] (II) a block copolymer comprising a polymer block formed by polymerization of a hydrophilic monomer and a (meth)acrylate monomer containing an R2 group and / or a (meth)acrylamide monomer unit containing an R2 group; and / or

[0041] (III) a random copolymer comprising a hydrophilic monomer unit and a (meth)acrylate monomer containing an R2 group and / or a (meth)acrylamide monomer unit containing an R2 group;

[0042] Wherein, the definition of R2 is as described above.

[0043] In some embodiments of the present invention, the cationic polymer further comprises (meth)acrylate monomers and / or (meth)acrylamide monomer units, wherein the (meth)acrylate monomers and / or (meth)acrylamide monomers are optionally unsubstituted or substituted by C1-C6 alkyl, polyethylene glycol chain, -N(C1-C6 alkylene)2; the degree of polymerization of the polyethylene glycol chain is 2-200.

[0044] In some embodiments of the present invention, the degree of polymerization of the monomer units in the cationic polymer is 2-200.

[0045] In some embodiments of the present invention, the method for preparing the cationic polymer comprises subjecting the corresponding monomer units to polymerization reaction under the action of a chain transfer agent and an initiator.

[0046] The fourth aspect of the present invention provides a compound of formula III:

[0047]

[0048] Wherein, T is a tumor targeting group;

[0049] The definitions of A and B are as mentioned above;

[0050] L3 is selected from -O-, -NH-;

[0051] R5 is selected from H, -CH3;

[0052] R6 is selected from negative charge, -C1-C6 alkyl-sulfonate;

[0053] k is a natural number between 1 and 200 (such as 5, 10, 20, 30, 40, 50, 80, 100, 120, 150, 160, 180, etc.).

[0054] In some embodiments of the present invention, the tumor targeting group is derived from at least one of cRGD, folic acid (FA), hyaluronic acid (HA) and transferrin (TRf).

[0055] In some embodiments of the present invention, the compound of formula III is selected from the following compounds:

[0056]

[0057] Wherein, k is defined as described above.

[0058] The fifth aspect of the present invention provides a method for preparing the compound of formula III, comprising the following steps:

[0059]

[0060] The compound of formula III-1 is polymerized under the action of a chain transfer agent, and then coupled with the compound providing the tumor targeting group to obtain the compound of formula III.

[0061] In some embodiments of the present invention, the polymerization reaction is carried out at a temperature of 50 to 75° C. and for a time of 10 to 20 hours.

[0062] In some embodiments of the present invention, the polymerization reaction is carried out under the action of an initiator; the initiator includes any one of azobisisobutyronitrile, azobisisoheptanenitrile, cyclohexanone peroxide, dibenzoyl peroxide, and tert-butyl hydroperoxide.

[0063] In some embodiments of the present invention, the catalyst for the coupling reaction is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS).

[0064] The sixth aspect of the present invention provides a tumor-targeted activatable nano-photosensitizer, comprising:

[0065] (I) the compound of formula I or formula II or the cationic polymer, and

[0066] (II) the anionic polymer, and

[0067] (III) Aromatic photosensitizers.

[0068] In the present invention, compared with conventional control group materials (such as Tween 80, polyoxyethylene castor oil, liposomes and designed control group materials), the introduction of cation-π interaction induces complete quenching of the fluorescence and singlet oxygen generation ability of the photosensitizer, and can be used as an ultra-low background activated nano photosensitizer for safe and non-toxic tumor photodynamic therapy; while the conventional control group materials all retain the photosensitizer's photosensitizer activity to varying degrees after being loaded with the photosensitizer, which can cause different degrees of phototoxic side effects, and therefore cannot be used as a good activatable photosensitizer.

[0069] In some embodiments of the present invention, the mass ratio of the aromatic structure photosensitizer to the cationic polymer is 1:5-30.

[0070] In some embodiments of the present invention, the mass ratio of the anionic polymer to the cationic polymer is 1:5-30.

[0071] In some embodiments of the present invention, the aromatic structure photosensitizer includes metal phthalocyanine photosensitizers such as zinc phthalocyanine, aluminum phthalocyanine, silicon phthalocyanine, etc.; porphyrin photosensitizers such as protoporphyrin, hematoporphyrin derivatives, verteporfin, temoporphin, tetraaminophenylporphyrin, dihydrochlorin E6, etc.; IR780, Bengal rose red, methylene blue, indocyanine green, etc. At least one.

[0072] In some embodiments of the present invention, the aromatic structure photosensitizer includes the following compounds:

[0073]

[0074] The seventh aspect of the present invention provides a method for preparing the tumor-targeting activatable nano-photosensitizer, comprising the following steps:

[0075] The compound of formula I or formula II or the cationic polymer, the aromatic structure photosensitizer and the anionic polymer are self-assembled in an organic solvent to prepare the tumor-targeting activatable nano photosensitizer.

[0076] In some embodiments of the present invention, the organic solvent is at least one of N,N-dimethylformamide, dimethyl sulfoxide, ethanol and methanol.

[0077] In some embodiments of the present invention, the preparation method further comprises dialyzing the self-assembled product to obtain the tumor-targeted activatable nano-photosensitizer.

[0078] The eighth aspect of the present invention provides a pharmaceutical composition comprising the tumor-targeting activatable nano-photosensitizer.

[0079] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable medium or excipient.

[0080] In some embodiments of the present invention, the medium comprises an aqueous solution; the tumor-targeted activatable nano-photosensitizer is dispersed in the aqueous solution and stored in liquid form until needed, or can be dried and introduced and dispersed in the aqueous solution before administration to the subject.

[0081] Pharmaceutically acceptable media or excipients include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; astragalus powder; malt; gelatin; lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); esters, such as ethyl oleate, ethyl laurate; agar; buffers, such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates and / or polyanhydrides; fillers, such as polypeptides and amino acid serum components, such as serum albumin, HDL and LDL; C2-C 12 Alcohols, such as ethanol; and other non-toxic compatible substances used in pharmaceutical preparations.

[0082] Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, aromas, preservatives and antioxidants may also be present in the pharmaceutical composition.

[0083] The aqueous solution contains, for example, water, cell culture medium, buffer (e.g., phosphate buffered saline), polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), or a suitable mixture thereof. In some embodiments, the aqueous solution can be a buffered solution (e.g., PBS).

[0084] The ninth aspect of the present invention proposes the use of at least one selected from the compound of formula I or the compound of formula II or the cationic polymer, the anionic polymer, the tumor-targeting activatable nano-photosensitizer, and the pharmaceutical composition in the preparation of an anti-tumor drug.

[0085] In some embodiments of the present invention, the tumor includes melanoma, prostate cancer, brain cancer, colon cancer, ovarian cancer, breast cancer, head cancer, eye cancer, squamous cell carcinoma, meibomian gland cancer; at least one of the neck and chest wall tumors caused by breast cancer, skin cancer, lung cancer, esophageal cancer and bladder cancer.

[0086] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～bAlkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C 1～4 The term "alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0087] The "alkyl" described in the present invention refers to a saturated hydrocarbon group having a specified number of carbon atoms. For example, C1-8 alkyl refers to an alkyl group having 1 to 8 carbon atoms, for example, preferably 1 to 4 carbon atoms. The alkyl group may be linear or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may also be optionally substituted with one or more substituents as defined herein. Specific examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0088] The term "alkylene" as used herein refers to a divalent saturated aliphatic hydrocarbon group having a specified number of carbon atoms. a ~ b "Alkylene" refers to an alkylene group having a to b carbon atoms. The alkylene group can be straight chain or branched. For example, "C1-6 alkylene" is intended to include methylene, ethylene, propylene, 2-methylpropylene, dimethylethylene, pentylene, and the like. Thus, the term "propylene" can be exemplified by the following structures: Likewise, the term "dimethylpropylene" can be exemplified, for example, by any of the following structures:

[0089] The beneficial effects of the present invention are:

[0090] The present invention forms a nano photosensitizer by copolymerizing or homopolymerizing cationic monomers into cationic polymers and forming a strong cation-π interaction with an aromatic photosensitizer. Most importantly, the cation-π interaction can induce complete quenching of the photosensitizer, and the quenching effect is better than the π-π stacking caused by the photosensitizer loaded by the commonly used clinical carrier materials. By using this feature, the construction of ultra-low background activated nano photosensitizers is conducive to greatly reducing the phototoxicity of the photosensitizer to light-exposed (such as skin and eyes) tissues. Since the photosensitizers are almost all aromatic molecules, this construction strategy has universal applicability. At the same time, there is no need to chemically modify the photosensitizer, avoiding cumbersome chemical synthesis and purification steps, and greatly simplifying the preparation process of conventional activatable photosensitizers.

[0091] In a preferred embodiment, the polyethylene glycol block in the polymer is exposed outside the nano-photosensitizer, which is beneficial to improve the stability of the particles and prolong the blood circulation time of the nano-photosensitizer.

[0092] The present invention also modifies the surface of the nano-photosensitizer with anionic polymers covalently linked to tumor targeting groups through electrostatic interaction, so that the nano-photosensitizer has excellent tumor targeting effect, greatly reduces the accumulation of the nano-photosensitizer in non-tumor sites, and further eliminates the phototoxicity of the nano-photosensitizer to light-exposed tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the polycationic block polymer prepared in Example 1 of the present invention.

[0094] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the cRGD covalently coupled anionic polymer prepared in Example 3 of the present invention.

[0095] Figure 3 It is the particle size distribution diagram of the nano photosensitizer prepared in Examples 4 to 6 of the present invention.

[0096] Figure 4 It is a transmission electron microscope image of the nano photosensitizer prepared in Examples 4 to 6 of the present invention.

[0097] Figure 5 These are the ultraviolet absorption graphs (a-c) and fluorescence spectra (d-f) of the nano-photosensitizers prepared in Examples 4-6 of the present invention.

[0098] Figure 6 It is a statistical graph of the in vitro singlet oxygen production capacity of the nano photosensitizers prepared in Examples 4 to 6 of the present invention.

[0099] Figure 7 This is a graph showing the recovery of the photosensitizing activity of the nano photosensitizer prepared in Example 4 of the present invention in 4T1 cells.

[0100] Figure 8 This is a statistical graph of the toxicity of the nano-photosensitizer prepared in Example 4 of the present invention to 4T1 cells.

[0101] Fig. 9 This is the in vivo tumor inhibition effect evaluation of the nano photosensitizer prepared in Example 4 of the present invention; wherein, a is a schematic diagram of the experimental process, b is the tumor volume inhibition effect, c is the tumor size inhibition effect, and d is the tumor section analysis result.

[0102] Fig.10 This is a skin toxicity study of the nano photosensitizer prepared in Example 4 of the present invention; wherein a is a fluorescence imaging diagram, b is a fluorescence intensity statistical diagram, c is a physical diagram of the phototoxic reaction of experimental animals, and d is a slice analysis diagram. DETAILED DESCRIPTION

[0103] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0104] 4T1 cells were purchased from Shanghai ATCC Cell Bank; N,N-dimethylethanolamine and isocyanoethyl methacrylate were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; iodomethane; polyethylene glycol methyl ether acrylate and dibutyltin dilaurate were purchased from Sigma-Aldrich.

[0105] Example 1

[0106] This example prepares a polycationic block polymer, the structural formula of which is as follows:

[0107]

[0108] The preparation method comprises the following steps:

[0109] 1) PEG 45 -b-PTAMA 30 The preparation route is as follows:

[0110]

[0111] TAMA (400 mg, 1.03 mmol), PEG 45 -CTA (78 mg, 0.034 mmol) and azobisisobutyronitrile (1.5 mg, 0.009 mmol) were dissolved in 500 μL N, N-dimethylformamide and then transferred to a polymerization tube, degassed through three freeze-thaw cycles, and finally sealed under vacuum. The polymerization tube was then immersed in a preheated 65°C oil bath. After stirring for 12 hours, the polymerization tube was placed in liquid nitrogen for a while, and the lid was opened to terminate the polymerization. 3 mL of pure water was added to the reaction mixture and transferred to a dialysis bag, which was dialyzed in water for 8 hours, and the dialysis external solution was changed every two hours. The solution in the dialysis bag was then freeze-dried to obtain 379 mg PEO. 45 -b-PTAMA 30 (Yield: 79%).

[0112] Example 2

[0113] This example prepares a polycationic polymer, the structural formula of which is as follows:

[0114]

[0115] The preparation method comprises the following steps:

[0116]

[0117] TAMA (400 mg, 1.03 mmol), poly(ethylene glycol) methacrylate 300 (206 mg 0.69 mmol), CTA (9.4 mg, 0.034 mmol) and azobisisobutyronitrile (1.5 mg, 0.009 mmol) were dissolved in 500 μL N, N-dimethylformamide and then transferred to a polymerization tube, degassed by three freeze-thaw cycles, and finally sealed under vacuum. The polymerization tube was then immersed in a preheated 65°C oil bath. After stirring for 12 h, the polymerization tube was placed in liquid nitrogen for a while, and the lid was opened to terminate the polymerization. 3 mL of pure water was added to the reaction mixture, and it was transferred to a dialysis bag and dialyzed in water for 8 h. The dialysis external solution was changed every two hours, and then the solution in the dialysis bag was lyophilized to obtain 523 mg P(TAMA 0.6 -co-OEGMA 0.4 ) 50 (Yield: 85%).

[0118] Example 3

[0119] This example prepares a cRGD-modified anionic polymer, and the specific process is as follows:

[0120]

[0121] SPMA (300 mg, 1.22 mmol), CTA-COOH (23 mg, 0.08 mmol) and azobisisobutyronitrile (1.5 mg, 0.009 mmol) were dissolved in 500 μL N, N-dimethylformamide, then transferred to a polymerization tube, degassed by three freeze-thaw cycles, and finally sealed under vacuum. The polymerization tube was then immersed in a preheated 65°C oil bath. After stirring for 16 h, the polymerization tube was placed in liquid nitrogen for a while, and the lid was opened to terminate the polymerization. 3 mL of pure water was added to the reaction mixture, and it was transferred to a dialysis bag and dialyzed in water for 8 h. The dialysis external solution was changed every two hours, and then the solution in the dialysis bag was freeze-dried to obtain 203 mg PSPMA8-COOH (yield: 63%).

[0122] For cRGD coupling, PSPMA8-COOH (30 mg, 13.2 μmol), Sulfo-NHS (18.2 mg, 84 μmol) and EDCl (48 mg, 250 μmol) were dissolved in 2 mL MES buffer and stirred at room temperature for 4 h. Then cRGD (9.7 mg, 16 μmol) was added to the above mixture and stirred for 12 h at room temperature. The product was dialyzed and purified in pure water and finally lyophilized. The polymer was recorded as PSPMA8-cRGD.

[0123] Example 4

[0124] This example prepares a tumor-targeted activatable nano-photosensitizer, and the specific process is as follows:

[0125] 10 mg of PEO prepared in Example 1 45 -b-PTAMA 30 The mixture was co-dissolved with zinc phthalocyanine (ZnPc) in 1 mL of N,N-dimethylformamide, and quickly added into 9 mL of vigorously stirred pure water (containing 1 mg of PSPMA8-cRGD in Example 3). After stirring for 10 min, the sample was transferred to a dialysis bag and dialyzed in water to remove the organic solvent and unloaded drug.

[0126] Example 5

[0127] In this example, a tumor-targeted activatable nano-photosensitizer is prepared. The preparation method is the same as that of Example 4, except that the zinc phthalocyanine (ZnPc) in Example 4 is replaced by the photosensitizer 4-aminophenylporphyrin (TAPP).

[0128] Example 6

[0129] In this example, a tumor-targeted activatable nano-photosensitizer is prepared. The preparation method is the same as that of Example 4, except that the zinc phthalocyanine (ZnPc) in Example 4 is replaced by photosensitizer IR780.

[0130] Test Example 1

[0131] This test example characterizes the performance of the product obtained in the embodiment, specifically:

[0132] The cationic polymer PEG prepared in Example 1 45 -b-PTAMA x The H NMR spectra were confirmed. Figure 1 ,from Figure 1 The degree of polymerization of TAMA was calculated based on the peak areas of the characteristic peak of polyethylene glycol (near 3.6 ppm) and the characteristic peak of TAMA (near 3.4 ppm chemical shift of the methyl hydrogen of the quaternary ammonium group), and the polymer composition was determined to be PEG. 45-b-PTAMA 30 .

[0133] The cRGD-modified anionic polymer PSPMA8-cRGD prepared in Example 3 was confirmed by hydrogen nuclear magnetic resonance spectroscopy. Figure 2 .from Figure 2 The degree of polymerization of SPMA was calculated based on the peak areas of the characteristic methylene peak near the amide bond in the chain transfer agent (around 2.3 ppm) and the characteristic peak of SPMA (around 4.1 ppm of the methylene chemical shift near the ester bond); at the same time, the successful connection of cRGD can be proved based on the signal of aromatic hydrogen.

[0134] The transmission electron microscopy and particle size determination of the tumor-targeted activatable nano-photosensitizers of Examples 4 to 6 were performed as follows:

[0135] 1 mL of the tumor-targeted activatable nano-photosensitizer aqueous solution prepared in Examples 4 to 6 was placed in an unopened dynamic light scattering culture dish and the particle size of the solution was tested; a small amount of the nano-photosensitizer was taken for conventional transmission electron microscopy testing.

[0136] from Figure 3 It can be seen that the tumor-targeted activatable nanophotosensitizer is a nanoparticle with a hydrated particle size of about 60 to 100 nm and a small PDI (less than 0.2), indicating that the size of the nanoparticles is relatively uniform. From the electron microscopy results of representative nanophotosensitizers ( Figure 4 ), the nano-photosensitizer appears as solid nanoparticles of uniform size under an electron microscope.

[0137] The ultraviolet absorption, fluorescence spectrum and photosensitization activity of the tumor-targeted activatable nano-photosensitizers of Examples 4 to 6 were measured, and the specific process was as follows:

[0138] The UV-visible absorption spectrum and fluorescence spectrum of each group of nano-photosensitizers at a specific concentration were collected respectively, and the UV-visible absorption spectrum and fluorescence spectrum of the corresponding free photosensitizer were also detected.

[0139] For the ZnPc group and TAPP group nanophotosensitizers, 2 mL of the nanophotosensitizer aqueous dispersion of Examples 4 to 6 was taken into a cuvette, and singlet oxygen detection probe ABDA (50 μM) was added. After mixing, the mixture was irradiated with an LED light (660 nm, 20 mW / cm 2 ), after irradiation for a specific time, the ultraviolet absorption of ABDA was detected, and the absorbance was used as the ordinate and the irradiation time was used as the abscissa to draw a graph; for the IR780 group of nano-photosensitizers, DCFH was used as the singlet oxygen detection probe, 2 mL of the nano-photosensitizer aqueous dispersion of Examples 4 to 6 was taken in a cuvette, 10 μM of DCFH singlet oxygen detection was added, and after mixing, laser irradiation (808 nm, 20 mW / cm 2After irradiation for a specific time, the fluorescence spectrum of DCFH is detected and plotted with the fluorescence intensity as the ordinate and the irradiation time as the abscissa.

[0140] from Figure 5 It can be seen that compared with the control group material, the absorbance of the ultraviolet absorption spectrum of each nanophotosensitizer group (CE) at its characteristic absorption peak has decreased, and the fluorescence is in a quenched state.

[0141] from Figure 6 It can be seen that in the in vitro test tube experiment, after illumination, almost no singlet oxygen is generated, and its photosensitivity is in an unactivated state; compared with commonly used clinical pharmaceutical excipients such as Tween 80 (Tw80), polyoxyethylene castor oil (CE) and liposomes (Lipids), the tumor-targeted activatable nano-photosensitizers prepared in Examples 4 to 6 have significantly better quenching of photosensitivity at non-active sites in vitro, which indicates that when the nano-photosensitizer is used in vivo, when the nano-photosensitizer circulates with the blood to light-exposed tissues such as skin, eyes, etc., it will not cause phototoxic side effects after illumination during treatment or adverse exposure to daily sunlight after treatment.

[0142] Example 6

[0143] In this example, the tumor-targeted activatable nano photosensitizer prepared in Example 4 was applied to 4T1 cells to activate the photosensitivity and test its toxicity. The specific process is as follows:

[0144] The targeted group TNP1 / ZnPc and the non-targeted group NP1 / ZnPc prepared in Example 4 were respectively subjected to the photosensitivity detection at the cellular level. The commonly used DCFH-DA probe was used to detect reactive oxygen species, and the cells used were 4T1 cells. First, 2x10 5 4T1 cells were cultured for 24 hours to allow them to adhere to the wall. The upper culture medium was then discarded and the culture medium containing the targeted group TNP1 / ZnPc and the non-targeted group NP1 / ZnPc (converted to the same free ZnPc concentration) was added. After incubation for 2 hours, the drug-containing culture medium was discarded, washed twice with PBS, and fresh serum-free culture medium containing 10μM DCFH-DA reactive oxygen probe was added. After incubation for 30 minutes, the supernatant was discarded, washed twice with PBS, and fresh culture medium was added, followed by LED light irradiation (660nm, 20mW / cm 2 , 5min), followed by confocal imaging. Figure 7 shown.

[0145] Cytotoxicity test: The standard MTT test method was used, and the cells used were 4T1 cells. First, 4T1 cells (5000 cells / well) were planted on a 96-well cell culture plate and cultured for 24 hours to allow them to adhere to the wall. The upper culture medium was then discarded and cell culture medium containing the targeted group TNP1 / ZnPc and the non-targeted group NP1 / ZnPc was added to make the drug concentration in the 4T1 cell culture medium reach the corresponding concentration. After incubation for 2 hours, LED light irradiation treatment (660nm, 20mW / cm 2 , 5min), then discard the drug-containing complete medium, add fresh medium and continue incubation for 22h, then take out the 96-well cell culture plate and perform cytotoxicity detection according to the MTT standard operation method. The results are as follows Figure 8 shown.

[0146] The results showed that in the detection of reactive oxygen species at the in vitro cellular level, after cell uptake and LED light irradiation, both the targeted group TNP1 / ZnPc and the non-targeted group NP1 / ZnPc produced a large amount of reactive oxygen species, which indicates that the targeted group TNP1 / ZnPc and the non-targeted group NP1 / ZnPc activated photosensitivity in cells and subsequently produced cell killing effects.

[0147] In the cytotoxicity experiment, at the designed highest concentration (10 μM), the cell survival rate of the targeted group TNP1 / ZnPc after irradiation with LED light was 28.8%, while that of the non-targeted group was 37.8%, showing a significant toxicity enhancement phenomenon. This indicates that the targeted group TNP1 / ZnPc has better targeted uptake ability, greatly improving its killing effect on tumor cells.

[0148] Example 7

[0149] In this example, the tumor-targeted activatable nano-photosensitizer prepared in Example 4 is applied to in vivo tumor inhibition, and the specific process is as follows:

[0150] In order to verify the tumor killing effect of this “on / off” type nano-photosensitizer, Fig. 9 As shown in the figure, a corresponding mouse subcutaneous transplant tumor model was constructed at the animal level to evaluate its in vivo anti-tumor effect, wherein the animals were divided into the following groups: PBS blank control group (no irradiation), nano-photosensitizer experimental group (no irradiation), PBS blank control group (irradiation) and nano-photosensitizer experimental group (irradiation); when the tumor volume grew to 100-150mm 3At 14:00, the animals were injected with tail vein according to the experimental groups, with each injection volume of 0.6 μmol / kg. The relevant groups were subjected to local laser irradiation for a period of time after the injection, and the above operation was repeated at specific time intervals. During the experimental period, the weight, behavior and tumor volume of the animals were monitored. After the treatment period, the animals were blooded for blood index analysis, and the tumors and major organs were collected for routine section analysis (H&E, TUNEL and other immunohistochemical experiments).

[0151] The results showed that during the 14-day treatment cycle, the TNP1 / ZnPc group without LED light irradiation had no tumor inhibitory effect, and the tumor volume growth was almost similar to that of the PBS group. The TNP1 / ZnPc group irradiated with LED light showed a stronger tumor growth inhibitory ability. The tumor tissues of each group were sliced ​​and analyzed. Compared with other groups, the TNP1 / ZnPc group irradiated with LED light had more cell necrosis areas.

[0152] Example 8

[0153] In this example, the tumor-targeted activatable nano-photosensitizer prepared in Example 4 was applied to the study of skin phototoxicity. The specific process is as follows:

[0154] In order to simulate the skin damage caused by sunlight exposure after photosensitizer treatment, first, TNP1 / ZnPc was administered to small animals by intravenous injection, and PBS and ZnPc loaded with common drug carrier materials (such as Tween 80, polyoxyethylene castor oil, lipid nanoparticles, etc.) were used as controls. The enrichment in parts such as skin and eyes was monitored by in vivo imaging or fluorescence imaging of ex vivo tissues within a specific time, and the clearance of photosensitizers was tracked for a long time; the small animals were further placed under a solar simulator for light exposure treatment to study the toxic reactions of photosensitizers on animal skin (rash, swelling, scabs and necrosis, etc.); the pathological changes of ex vivo tissues (skin and eyes, etc.) after light exposure were studied by section analysis (H&E and immunohistochemistry analysis, etc.) of light-exposed tissues and organs (skin and eyes, etc.).

[0155] The results showed that compared with the polyoxyethylene castor oil photosensitizer group, after irradiation with the solar simulator, the targeted group TNP1 / ZnPc had almost no fluorescence signal in the skin, indicating that it was very little enriched in the skin and was almost not activated. Under long-term monitoring, no skin phototoxic side effects were produced, while the polyoxyethylene castor oil photosensitizer group produced obvious skin phototoxicity.

[0156] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A compound of formula I or a compound of formula II: in, R is a polymer block formed by polymerization of hydrophilic monomers; A is a CAT fragment; B is H, -CH3 or absent; L1 and L2 are each independently selected from -O-, -NH-, -responsive bond-, -O-responsive bond-, -NH-responsive bond- or absent; R1 and R3 are each independently selected from H, -CH3; R2 is selected from -C1-C6 alkylene-basic cationic group, -ester group-C1-C6 alkylene-basic cationic group, -C1-C6 iminoester group-C1-C6 alkylene-basic cationic group; the basic cationic group includes any one of a positively charged amino group, a primary amine group, a secondary amine group, a tertiary amine group, a quaternary amine group, and a guanidine group; R4 is selected from -OH, -NH2, -NH-C1-C6 alkyl, -O-C1-C6 alkylene-N(C1-C6 alkylene)2, -O-C1-C6 alkylene-PEG-O-C1-C6 alkyl; 0 < x ≤ 1; m and y are natural numbers between 2 and 200; z is a natural number between 0 and 200; The degree of polymerization of the polymer block is a natural number between 0 and 200.

2. The compound of formula I or formula II according to claim 1, characterized in that: The compound of formula I or the compound of formula II satisfies at least one of the following conditions: (a) The hydrophilic monomer includes at least one of ethylene glycol, methoxypolyethylene glycol methacrylate, acrylic acid, methacrylic acid, acrylamide, N-isopropylacrylamide, or N,N-dimethylaminoethyl methacrylate; (b) CAT fragment is selected from (c) When L1 or L2 is present, -C(O)-L1- or -C(O)-L2- are each independently selected from -C(O)-O-, -C(O)-NH-, -C(O)-O-responsive bond-, -C(O)-NH-responsive bond-; (d) The responsive bond includes a disulfide bond, a hydrazone bond, a thioacetal bond, and an ester bond; (e) The basic cationic group includes a positively charged primary amine group Secondary amine group Tertiary amine group Quaternary amine group Guanidine Any of the following: (f) R2 is selected from 3. The compound of formula I or formula II according to claim 1, characterized in that: The compound of formula I or the compound of formula II is selected from the following compounds:

4. A cationic polymer, characterized in that: Including (I) a homopolymer, a (meth)acrylate monomer containing an R2 group, or a (meth)acrylamide monomer unit containing an R2 group; and / or, (II) a block copolymer including a polymer block formed by polymerization of hydrophilic monomers and a (meth)acrylate monomer containing an R2 group and / or a (meth)acrylamide monomer unit containing an R2 group; and / or, (III) a random copolymer including a hydrophilic monomer unit and a (meth)acrylate monomer containing an R2 group and / or a (meth)acrylamide monomer unit containing an R2 group; wherein the definition of R2 is as described in any one of claims 1 to 3.

5. A compound of formula III: in, T is a tumor targeting group; The definitions of A and B are as described in any one of claims 1 to 3; L3 is selected from -O-, -NH-; R5 is selected from H, -CH3; R6 is selected from a negative charge, -C1-C6 alkyl-sulfonate; k is a natural number between 1 and 200.

6. A tumor-targeted activatable nano-photosensitizer, characterized in that: Including (I) the compound of formula I or the compound of formula II described in any one of claims 1 to 3 or the cationic polymer described in claim 4, and (II) the anionic polymer described in claim 5, and (III) an aromatic structure photosensitizer.

7. The tumor-targeted activatable nano-photosensitizer according to claim 6, characterized in that: The tumor-targeted activatable nano-photosensitizer meets at least one of the following conditions: (a) the mass ratio of the aromatic structure photosensitizer to the cationic polymer is 1:5 to 30; (b) the mass ratio of the anionic polymer to the cationic polymer is 1:5 to 30; (c) The aromatic structure photosensitizer includes at least one of metal phthalocyanine photosensitizers, porphyrin photosensitizers, IR780, Bengal rose red, methylene blue, and indocyanine green.

8. A method for preparing the tumor-targeting activatable nano-photosensitizer according to claim 6 or 7, comprising the following steps: The tumor-targeted activatable nano photosensitizer is prepared by self-assembling the compound of formula I or formula II described in any one of claims 1 to 3 or the cationic polymer, aromatic structure photosensitizer and anionic polymer described in claim 5 in an organic solvent.

9. A pharmaceutical composition comprising the tumor-targeting activatable nano-photosensitizer according to claim 6 or 7.

10. Use of at least one of the compounds of formula I or II according to any one of claims 1 to 3, the cationic polymer according to claim 4, the anionic polymer according to claim 5, the tumor-targeted activatable nanophotosensitizer according to claim 6 or 7, and the pharmaceutical composition according to claim 9 in the preparation of anti-tumor drugs.

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