Synthesis and Application of pH-Sensitive Polymers Containing Non-pH-Sensitive Monomers

Abstract

The present invention discloses the synthesis and application of pH-sensitive polymers containing non-pH-sensitive monomers. The present invention includes the synthesis of pH-sensitive block polymers, the preparation of pH-sensitive micelle formulations, and their applications in fluorescence imaging, drug delivery, immune adjuvants, tumor-targeted diagnosis, etc. The pH-sensitive block polymer described contains a hydrophilic segment and a hydrophobic segment with adjustable hydrophilicity and hydrophobicity. Among them, the hydrophilic segment is a repeating fragment composed of hydrophilic monomers, and the hydrophobic segment is a copolymer of a pH-sensitive monomer and a non-pH-sensitive monomer as shown in Formula 1 below. Among them, each marked symbol is defined as in the specification.

Description

Technical Field

[0001] The present invention relates to the fields of tumor fluorescence imaging and drug delivery, and particularly to the synthesis and preparation of pH-sensitive polymers and their applications in tumor fluorescence imaging, tumor chemotherapy drug delivery, etc. Background Art

[0002] Tumor fluorescence imaging technology refers to the technology of specifically imaging tumor tissues in an animal body with chemical molecules having fluorescence properties. Due to its rapid response and ability to achieve specific differentiation of tissues, it has been widely studied in application fields such as disease diagnosis, imaging-mediated surgery, and intraoperative rapid biopsy. However, traditional tumor fluorescence imaging mostly relies on small-molecule fluorescent probes, often unable to achieve the expected tumor specificity, with high background signals and low spatial resolution. Existing pH-sensitive polymers use one or more pH-sensitive monomers to regulate the pH responsiveness of such polymers. There are few types of pH-sensitive monomers available, the polymer structure is complex, and the pH adjustable range is relatively small.

[0003] Drug delivery to tumor sites has always been a key research area in the field of pharmacy. To achieve stable encapsulation of drugs in the body and site-specific release at tumor sites, it is necessary to design drug carriers that can achieve intelligent response to the tumor microenvironment. Currently, commonly used nano-drug carriers often have problems such as poor tumor specificity, poor blood circulation stability, incomplete drug release, and immunogenicity of the carrier itself, which restrict their clinical applications. Therefore, more optimized drug carriers should be developed to meet the various requirements of tumor drug delivery. Summary of the Invention

[0004] To solve the problems existing in the current technical field, the inventors have conducted extensive research, thus realizing the present invention. The present invention provides a pH-sensitive polymer with low pH-responsive fluorescence signal activation characteristics, and uses non-pH-sensitive monomers to regulate the pH response value and response range of the pH-sensitive polymer, thereby achieving precise and continuous control of the pH responsiveness of the pH-sensitive polymer. The present invention also provides micelles, micelle complexes, preparation methods, and biological and medical uses of the pH-sensitive polymer. For example, when a fluorescent molecule is used as a marker, in the pH-sensitive polymer of the present invention, the fluorescent molecule is covalently linked to the hydrophobic block of the pH-sensitive amphiphilic polymer. The micelles prepared from the pH-sensitive polymer linked with the fluorescent molecule can remain in the closed state in normal tissues (neutral or slightly alkaline environment), that is, without fluorescence signal; while in the slightly acidic environment of tumor tissues, the pH-sensitive micelles are dissociated, and the fluorescence signal is specifically activated, exerting the effect of tumor-related tissue fluorescence imaging. Through the design and change of the polymer structure, the present invention provides a series of polymers that can be dissociated in response to different pH values, which can efficiently and with high resolution distinguish tumor tissues from normal tissues, while reducing the systemic toxicity of the preparation, providing more options for the clinical transformation of fluorescence imaging technology.

[0005] To achieve the above object, the present invention relates to the following aspects:

[0006] [1] A pH-sensitive polymer, including two parts: a hydrophilic segment and a hydrophobic segment;

[0007] [2] The pH-sensitive polymer according to [1], wherein the hydrophilic segment can be one or more of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), poly(oligo(ethylene glycol) methacrylate) (POEG), polysaccharide, poly(betaine methacrylate) (PCB);

[0008] [3] The pH-sensitive polymer according to [1] or [2], wherein the hydrophobic segment has the structure shown in Formula 1:

[0009]

[0010] Wherein, R’, R”, R”’, X1 and X2 are respectively selected from -H, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl, C1-C12 substituted cycloalkyl, and R”” is an end group generated by the polymerization reaction. In some embodiments, R”” is -Br, and at this time, atom transfer radical polymerization (ATRP) is used for the synthesis of the polymer; in some embodiments, R”” is a sulfur-containing group, such as thiol or thioester, and at this time, reversible addition-fragmentation chain transfer polymerization (RAFT) is used for the synthesis of the polymer;

[0011] R1 and R2 may be the same or different and may be selected from C1-C16 alkyl, C1-C16 cycloalkyl, C1-C16 aromatic, C1-C16 heteroaromatic and substituted groups thereof, or R1 and R2 may be combined to form -(C1-C16)-alkyl, -(C1-C16)-alkyloxy or -(C1-C16)-alkylamino;

[0012] M1 and M2 are respectively selected from -O- and -NH-. When M1 and M2 are -O-, the polymer side chains connected thereto are connected to the polymer backbone via an ester bond; when M1 and M2 are -NH-, the polymer side chains connected thereto are connected to the polymer backbone via an amide bond;

[0013] R3 is a non-pH sensitive group. R3 is, for example, selected from one or more of the following groups: C1-C16 alkyl, C1-C16 cycloalkyl, C1-C16 aromatic, and the above groups substituted with one or more structures of one or more hydroxyl, sulfhydryl, guanidinium, halogen, ether bond, ester bond, amide bond, alkyl, aromatic, carbamate bond, or C2-C16 sulfonate, C2-C16 sulfate, glycosyl, phosphatidylcholine, phosphatidylethanolamine. In some embodiments, R3 is selected from one or more of the above groups; preferably C1-C8 alkyl, and C1-C8 alkyl substituted with hydroxyl and ether bond; more preferably C1-C4 alkyl, and C1-C4 alkyl substituted with hydroxyl and ether bond;

[0014] a and b are integers from 1 to 10 respectively;

[0015] x and y are integers respectively, and the sum of x and y is an integer between 10 and 200;

[0016] z is an integer of 1-50, and the three parts x, y, and z can be arranged in any order in the polymer hydrophobic segment; in some embodiments, the three parts x, y, and z each constitute three blocks; in some embodiments, x and z constitute a randomly arranged block, and y itself constitutes a block; in some embodiments, the three parts x, y, and z constitute a randomly arranged block;

[0017] L is a linker. In some embodiments, L is selected from a chemically stable and non-breakable structure. In some embodiments, L is selected from a structure that can break in response to a special environment, such as an acid-sensitive linker, an enzyme-sensitive linker, and a reduction-sensitive linker. F is a labeling molecule. Each L and each F can be different.

[0018] [4] The pH-sensitive polymer according to [3], wherein the labeling molecule is selected from one or more of a fluorescent molecule, a photosensitizer, a fluorescence quenching molecule, a self-luminescent molecule, a chemotherapeutic drug, an immunotherapeutic drug, and a metal chelating molecule. In some embodiments, the labeling molecule is a fluorescent molecule, selected from one or more of 5(6)-carboxytetramethylrhodamine (TMR), Cyanine 3, Cyanine3.5, Cyanine 5, Cyanine 5.5, Cyanine 7.5, indocyanine green (ICG), Bodipy FL, Bodipy 650, Pacific blue, coumarin, and structural derivatives of the above molecules; in some embodiments, the labeling molecule is a photosensitizer, selected from one or more of porphine, porphyrin, bacteriochlorophyll, phthalocyanine, cationic photosensitizer, quinone photosensitizer, curcumin photosensitizer, Bodipy photosensitizer, and structural derivatives of the above molecules; in some embodiments, the labeling molecule is a fluorescence quenching molecule, selected from one or more of DABCYL, DABSYL, QXL, QSY, BHQ, and ATTO; in some embodiments, the labeling molecule is a self-luminescent molecule, and the self-luminescent molecule is luminol; in some embodiments, the labeling molecule is a chemotherapeutic drug, selected from one or more of paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, doxorubicin, epirubicin, daunorubicin, and cisplatin; in some embodiments, the labeling molecule is an immunotherapeutic drug, selected from one or more of indoleamine-(2,3)-dioxygenase inhibitor IDOi and Toll-like receptor agonist; in some embodiments, the labeling molecule is a metal chelating group, selected from DOTA, NOTA, TETA, Diamsar, NETA, TACN-TM, and derivatives of the above structures.

[0019] [5] The pH-sensitive polymer according to any one of [1]-[4], which has the structure shown in Formula 2 below:

[0020]

[0021] Wherein, Y1 is selected from -H, -COOH, -NH2, -SH, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl, C1-C12 substituted cycloalkyl, or

[0022]

[0023] or a metal chelating group;

[0024] n is an integer from 1 to 500;

[0025] Y2 and Y3 are each independently selected from -H, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl, or C1-C12 substituted cycloalkyl;

[0026] Other symbols are defined as in [3] and [4].

[0027] [6] The pH-sensitive polymer according to any one of [1]-[5] has the structure shown in Formula 3 below:

[0028]

[0029] Wherein, M1 and M2 are -O- or -NH-;

[0030] R1’ is selected from the following structures:

[0031]

[0032] R2’ is selected from one or more of the following structures:

[0033]

[0034] The sum of x and y is an integer from 10 to 200; in some embodiments, the sum of x and y is from 40 to 150; in some embodiments, the sum of x and y is from 80 to 120; in some embodiments, the sum of x and y is 100;

[0035] The ratio of x to y can vary. In multiple embodiments, the ratio of x to y has diverse values from 1:0 to 0:1, thereby affecting the properties of the entire polymer; in some embodiments, x is 100 and y is 0; in some embodiments, x is 90 and y is 10; in some embodiments, x is 80 and y is 20; in some embodiments, x is 70 and y is 30; in some embodiments, x is 60 and y is 40; in some embodiments, x is 50 and y is 50;

[0036] z is an integer from 0 to 20. In some embodiments, z is an integer from 2 to 10; in some embodiments, z is an integer from 3 to 5;

[0037] The three parts x, y, and z can be arranged in any order in the hydrophobic segment of the polymer. In some embodiments, the three parts x, y, and z respectively form three blocks; in some embodiments, x and z form a randomly arranged block, and y forms a block by itself; in some embodiments, the three parts x, y, and z form a randomly arranged block;

[0038] F and L are respectively defined as in [3] and [4].

[0039] [7] The pH-sensitive polymer according to [6], wherein F is a fluorescent molecule selected from one or more of 5(6)-carboxytetramethylrhodamine (TMR), Cyanine 3, Cyanine 3.5, Cyanine 5, Cyanine 5.5, Cyanine 7.5, indocyanine green (ICG), Bodipy FL, Bodipy 650, Pacific blue, coumarin, and structural derivatives of the above molecules;

[0040] [8] The pH-sensitive polymer according to [1]-[7], wherein the polydispersity coefficient of the relative molecular weight of the polymer is 1.0-1.5; in some embodiments, the polydispersity coefficient of the relative molecular weight of the polymer is less than 1.2; in some embodiments, the polydispersity coefficient of the relative molecular weight of the polymer is less than 1.1;

[0041] [9] A pH-sensitive micelle formed from one or more of the pH polymers described in any one of [1]-[8]. The particle size of the micelle is 10-200 nm; in some embodiments, the particle size of the micelle is 20-100 nm; in some embodiments, the particle size of the micelle is 30-50 nm.

[0042] The pH value at which the pH-sensitive micelle exhibits a pH response is 4.0-8.0; in some embodiments, the pH value at which the pH-sensitive micelle exhibits a pH response is 5.0-6.9; in some embodiments, the pH value at which the pH-sensitive micelle exhibits a pH response is 6.3-6.9; in some embodiments, the pH value at which the pH-sensitive micelle exhibits a pH response is 5.5-6.2; in some embodiments, the pH value at which the pH-sensitive micelle exhibits a pH response is 5.0-5.5.

[0043] The pH range within which the pH-sensitive micelle completes the pH response is less than 1 pH unit; in some embodiments, the pH range within which the pH-sensitive micelle completes the pH response is less than 0.5 pH unit; in some embodiments, the pH range within which the pH-sensitive micelle completes the pH response is less than 0.25 pH unit; in some embodiments, the pH range within which the pH-sensitive micelle completes the pH response is less than 0.15 pH unit.

[0044] The fluorescence switching multiple of the pH-sensitive micelle linked with a fluorescent molecule is greater than 10; in some embodiments, the fluorescence switching multiple of the pH-sensitive micelle linked with a fluorescent molecule is greater than 50; in some embodiments, the fluorescence switching multiple of the pH-sensitive micelle linked with a fluorescent molecule is greater than 100.

[0045]

[10] A micelle complex comprising the pH-sensitive polymer micelle described in [9].

[0046]

[11] [9] The pH-sensitive polymer micelles described above are prepared for use in the following directions: diagnosis and treatment of malignant tumors, diagnosis and treatment of inflammatory diseases, autoimmune diseases, atherosclerosis, skin diseases, eye diseases, infectious diseases of pathogenic microorganisms. Description of the Drawings

[0047] Figure 1 1H NMR spectra of the three pH-sensitive polymers exemplified in Synthesis Example 2 and their respective structures. The peaks are assigned as follows:

[0048] PEG-b-P(DPA 90 -MMA 10 ) 1 1H NMR (TMS, CDCl3, ppm): 3.92 (b, COOCH2), 3.76 - 3.55 (m, OCH2CH2O), 3.52 (b, COOCH3), 3.31 (s, CH3O), 2.62 (b, COOCH2CH2N), 2.38 (b, NCH2CH2), 1.83 - 1.73 (m, CCH2C&C(CH3)2), 1.40 (m, NCH2CH2), 0.97 - 0.80 (m, CH2CH3&CCH3).

[0049] PEG-b-P(DBA 70 -HEMA 30 ) 1 1H NMR (TMS, CDCl3, ppm): 3.97 (b, COOCH2), 3.75 (b, COOCH2CH2OH), 3.63 - 3.41 (m, OCH2CH2O), 3.31 (s, CH3O), 2.62 (b, COOCH2CH2N), 2.40 (b, NCH2CH2CH2CH3), 1.88 - 1.75 (m, CCH2C&C(CH3)2), 1.36 (m, NCH2CH2CH2CH3), 1.25 (m, NCH2CH2CH2CH3), 0.99 - 0.81 (m, CH2CH3&CCH3).

[0050] PEG-b-P(EPA 50 -BMA 50 ) 11H NMR (TMS, CDCl3, ppm): 3.89 (b, COOCH2), 3.76 - 3.40 (m, OCH2CH2O), 3.31 (s, CH3O), 2.62 (b, COOCH2CH2N), 2.50 (b, NCH2CH3), 2.36 (b, NCH2CH2CH3), 1.84 - 1.74 (m, CCH2C&C(CH3)2), 1.54 (b, COOCH2CH2CH2CH3), 1.38 (m, COOCH2CH2CH2CH3&NCH2CH2CH3), 0.99 - 0.80 (m, CH2CH3&CCH3).

[0051] Figure 2 PEG copolymerized from N,N - diisopropylaminoethyl methacrylate (iDPA) and 2 - hydroxyethyl methacrylate (HEMA) 5k -P(iDPA x -HEMAy) polymer's 1H NMR spectrum.

[0052] Figure 3 PEG copolymerized from N,N - diisopropylaminoethyl methacrylate (iDPA) and n - butyl methacrylate (BMA) 5k -P(iDPA x -BMA y ) polymer's 1H NMR spectrum.

[0053] Figure 4 Particle size distribution diagrams of pH - sensitive micelles at physiological pH and acidic pH respectively. Here, a series of pH - sensitive polymers formed by copolymerizing N,N - diisopropylaminoethyl methacrylate (iDPA) with n - butyl methacrylate (BMA) and 2 - hydroxyethyl methacrylate (HEMA) are taken as examples. The results show that the pH - sensitive polymer can maintain the micelle morphology under physiological conditions (pH 7.4), while it dissociates under acidic conditions (pH 5.0).

[0054] Figure 5 PEG copolymerized from N,N - dipropylaminoethyl methacrylate (DPA) and triethylene glycol methyl ether methacrylate (TEGMA) 5k -P(DPA x -TEGMA y ) polymer's structure and 1H NMR spectrum.

[0055] Figure 6 PEG copolymerized from N,N - dipropylaminoethyl methacrylate (DPA) and 2 - hydroxyethyl methacrylate (HEMA) 5k -P(DPA x-HEMA y ) Structure of the polymer and 1H NMR spectrum

[0056] Figure 7 PEG-P(DPA-HPMA) copolymer obtained by copolymerizing N,N-dipropylaminoethyl methacrylate (DPA) and hydroxypropyl methacrylate (HPMA) 5k -P(DPA x -HPMA y ) Structure of the polymer and 1H NMR spectrum

[0057] Figure 8 PEG-P(DPA-MMA) copolymer obtained by copolymerizing N,N-dipropylaminoethyl methacrylate (DPA) and methyl methacrylate (MMA) 5k -P(DPA x -MMA y ) Structure of the polymer and 1H NMR spectrum

[0058] Figure 9 PEG-P(DPA-EMA) copolymer obtained by copolymerizing N,N-dipropylaminoethyl methacrylate (DPA) and ethyl methacrylate (EMA) 5k -P(DPA x -EMA y ) Structure of the polymer and 1H NMR spectrum

[0059] Figure 10 PEG-P(DPA-PMA) copolymer obtained by copolymerizing N,N-dipropylaminoethyl methacrylate (DPA) and propyl methacrylate (PMA) 5k -P(DPA x -PMA y ) Structure of the polymer and 1H NMR spectrum

[0060] Figure 11 PEG-P(DPA-BMA) copolymer obtained by copolymerizing N,N-dipropylaminoethyl methacrylate (DPA) and n-butyl methacrylate (BMA) 5k -P(DPA x -BMA y ) Structure of the polymer and 1H NMR spectrum

[0061] Figure 12 Fluorescence spectra of TMR-labeled PEG-P(DPA-TEGMA) polymer at different pH values 5k -P(DPA x -TEGMA y )

[0062] Figure 13 TMR-labeled PEG-P(DPA-HEMA) 5k -P(DPA x -HEMAy ) Fluorescence spectra of the polymer at different pH values.

[0063] Figure 14 is PEG labeled with the fluorescent probe TMR 5k -P(DPA x -HPMA y ) Fluorescence spectra of the polymer at different pH values.

[0064] Figure 15 is PEG labeled with the fluorescent probe TMR 5k -P(DPA x -MMA y ) Fluorescence spectra of the polymer at different pH values.

[0065] Figure 16 is PEG labeled with the fluorescent probe TMR 5k -P(DPA x -EMA y ) Fluorescence spectra of the polymer at different pH values.

[0066] Figure 17 is PEG labeled with the fluorescent probe TMR 5k -P(DPA x -PMA y ) Fluorescence spectra of the polymer at different pH values.

[0067] Figure 18 is PEG labeled with the fluorescent probe TMR 5k -P(DPA x -BMA y ) Fluorescence spectra of the polymer at different pH values.

[0068] Figure 19 is TMR-labeled PEG 5k -P(DPA-BMA), PEG 5k -P(DPA-HEMA), PEG 5k - Normalized fitting curves of the fluorescence changes of P(DPA-MMA) polymers at different pH values. The pH value corresponding to the ordinate of 0.5 in this figure is the pH transition point of the polymer.

[0069] Figure 20 On the left is Figures 10 - 16 For each specific polymer in the corresponding series of pH-sensitive polymers, the relationship between the pH transition point of the specific polymer and the proportion (R n ) of the non-pH-sensitive monomer it contains; on the right is the relationship between the slope value of each fitting line in a (used to characterize the ability of the non-pH-sensitive monomer to adjust the pH transition point of the polymer) and the logP value of the non-pH-sensitive monomer.

[0070] Figure 21 Fluorescence imaging diagrams of 8 series of pH-sensitive polymers conjugated with different fluorescent probes in different pH environments.

[0071] Figure 22 For the process of characterizing the gradual acidification of endosomes after five pH-sensitive micelles conjugated with different fluorescent probes were taken up by A549 cells.

[0072] Figure 23 In vivo tumor fluorescence imaging results of a series of pH-sensitive micelles conjugated with ICG in BALB / c mice bearing 4T1 tumors.

[0073] Figure 24 Quantitative results of the contrast-to-noise ratio of in vivo tumor fluorescence imaging in mice.

[0074] Figure 25 1H NMR spectrum of the pH-sensitive polymer-docetaxel drug conjugate.

[0075] Figure 26 Drug release curves of the enzyme-responsive polymer-docetaxel drug conjugate under papain treatment. The drug release amount of each formulation group was determined as 100% based on the enzymatically cleaved drug loading.

[0076] Figure 27 Tumor pharmacodynamic evaluation of the polymer-drug conjugate, using the size of mouse tumors as the evaluation index. The experiment was set up with three administrations, each at a dose of 10 mg / kg.

[0077] Figure 28 1H NMR spectrum of the pH-sensitive polymer-IMDQ immunostimulant conjugate.

[0078] Figure 29 Drug release curves of the enzyme-responsive polymer-IMDQ conjugate under papain treatment. The 100% value was determined based on the enzymatically cleaved drug loading. Detailed implementation mode

[0079] The present invention provides a pH-sensitive polymer, which comprises two parts: a hydrophilic segment and a hydrophobic segment.

[0080] Among them, the hydrophilic segment is selected from one or more of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC), poly (oligoethylene glycol methacrylate) (POEG), polysaccharide, poly (betaine methacrylate) (PCB), and preferably polyethylene glycol.

[0081] The hydrophobic segment has the structure shown in Formula 1:

[0082]

[0083] Among them, R’, R”, R”’, X1 and X2 are respectively selected from -H, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl, C1-C12 substituted cycloalkyl. R’, R” and R”” are preferably -H and C1-C4 alkyl, more preferably methyl; X1 and X2 are preferably -H; M1 and M2 are respectively selected from -O-, -NH-, preferably -O-.

[0084] R”” is an end group generated by the polymerization reaction, selected from halogen, mercaptan or thioester, preferably halogen, more preferably -Br.

[0085] R1 and R2 can be the same or different, and are respectively selected from C1-C16 alkyl, C1-C16 cycloalkyl, C1-C16 aryl, C1-C16 heteroaryl and the substituted groups above. Alternatively, R1 and R2 can also combine together to form -(C1-C16)-alkyl-, -(C1-C16)-alkyloxy-, or -(C1-C16)-alkylamino-. R1 and R2 are preferably C1-C5 alkyl, can be the same or different, can be straight-chain alkyls such as n-propyl, n-butyl, n-pentyl, etc., or branched-chain alkyls such as isopropyl. R1 and R2 can also combine together to form alkylenes such as pentamethylene, hexamethylene, heptamethylene, etc., for example C1-C16 alkylene, preferably C2-C8 alkylene.

[0086] R3 is a non-pH-sensitive group. For example, it can be selected from one or more of the following groups: C1-C16 alkyl, C1-C16 cycloalkyl, C1-C16 aryl, and the above groups substituted with one or more of hydroxyl, mercapto, guanidyl, halogen, ether bond, ester bond, amide bond, aryl, alkyl, carbamate bond, or C2-C16 sulfonate, C2-C16 sulfate, glycosyl, phosphatidylcholine, phosphatidylethanolamine. In some embodiments, R3 is taken from one or more of the above groups; preferably C1-C8 alkyl, and C1-C8 alkyl substituted with hydroxyl and ether bond; more preferably C1-C4 alkyl, and C1-C4 alkyl substituted with hydroxyl and ether bond;

[0087] a and b are integers from 1 to 10, preferably integers from 2 to 4, more preferably 2.

[0088] The sum of x and y is an integer from 20 to 200, preferably an integer from 40 to 150, more preferably 100.

[0089] z is an integer from 1 to 20, preferably an integer from 1 to 7, more preferably 5.

[0090] The three parts of x, y, and z can be arranged in any order, can form blocks respectively, or can be mixed with each other.

[0091] L is a linker, without special restrictions. L can be selected from chemically stable amide bonds, ester bonds, thioether bonds generated by thiol-maleimide click chemistry reactions, substituted triazoles generated by alkynyl-azide click chemistry reactions; it can also be selected from redox-sensitive disulfide bonds (including self-degradable disulfide bonds containing carbamate bonds), acid-sensitive hydrazone bonds, orthoester bonds, acetal bonds, imine bonds, and some enzyme-sensitive linkers. Among them, the enzyme-sensitive linker contains a Gly-Phe-Leu-Gly tetrapeptide sequence, a Val-Cit dipeptide sequence, a Val-Ala dipeptide sequence, and a Phe-Lys dipeptide sequence that are sensitive to cathepsin b. L is preferably an amide bond or a Gly-Phe-Leu-Gly tetrapeptide sequence. In some embodiments, L is selected from chemically stable and non-breakable structures; in some embodiments, L is selected from structures that can break in response to special environments, such as acid-sensitive linkers, enzyme-sensitive linkers, and reduction-sensitive linkers.

[0092] F is a labeling molecule, which can be selected from organic fluorescent molecules, photosensitizers, fluorescence quenchers, chemotherapeutic drugs, immunotherapeutic drugs and heavy metal chelating molecules, etc., and each F can be the same or different.

[0093] Wherein, the organic fluorescent molecule can be selected from one or more of Pacific blue, BODIPY-FL, mFluor 610, TMR, Cy 3, Cy 3.5, Cy 5, Cy 5.5, Cy 7.5, and ICG, and can be selected according to the required excitation wavelength, emission wavelength, or Stokes shift; the chemotherapy drug can be selected from paclitaxel, docetaxel, doxorubicin, camptothecin, and structural analogs, structural modifiers, and active metabolites of the above molecules.

[0094] In some specific embodiments, L is an amide bond, and F is an organic fluorescent molecule with an emission wavelength in the visible light region, such as TMR and Cy 5. In some specific embodiments, L is an amide bond, and F is an organic fluorescent molecule with an emission wavelength in the near-infrared region, such as ICG and Cy 7.5.

[0095] In some specific embodiments, the pH sensitive polymer provided by the present invention has a structure shown in Formula 2 below:

[0096]

[0097] Wherein, Y1 is selected from -H, -COOH, -NH2, -SH, C1-C18 alkyl, C1-C18 cycloalkyl, C1-C18 substituted alkyl, C1-C18 substituted cycloalkyl, or

[0098]

[0099] or a metal chelating group.

[0100] n is an integer from 1 to 500, preferably from 40 to 250, more preferably 113.

[0101] Y2 and Y3 are each independently selected from -H, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl, C1-C12 substituted cycloalkyl, and they may be the same or different; preferably both groups are C1-C6 alkyl, more preferably both groups are methyl.

[0102] Other symbols are defined as in Formula 1.

[0103] In some specific embodiments, the pH-sensitive polymer provided by the present invention has the structure shown in Formula 3 below:

[0104]

[0105] Wherein, R1’ is selected from the following structures:

[0106]

[0107] R2’ is selected from one or more of the following structures:

[0108]

[0109] M1 and M2 are each independently selected from -O-, -NH-, preferably -O-.

[0110] The sum of x and y is from 10 to 200, preferably from 60 to 150, more preferably 100. The ratio of x to y can vary. In multiple embodiments, the ratio of x to y has various values from 1:0 to 0:1, which accordingly affects the properties of the entire polymer; in some embodiments, x is 100 and y is 0; in some embodiments, x is 90 and y is 10; in some embodiments, x is 80 and y is 20; in some embodiments, x is 70 and y is 30; in some embodiments, x is 60 and y is 40; in some embodiments, x is 50 and y is 50.

[0111] Another aspect of the present invention is to provide a method for preparing the above pH-sensitive polymer, including the synthesis and purification of polymerization reaction reactants, the polymerization and purification of the pH-sensitive polymer, and the chemical coupling of functionalized labeling molecules.

[0112] For example, the pH-sensitive polymer described in the present invention can be prepared by the following method. Atom transfer radical polymerization or reversible addition-fragmentation chain transfer polymerization can be selected. The specific steps are as follows:

[0113] Step 1: Synthesis of the polymerization reaction macroinitiator

[0114]

[0115] Dissolve polyethylene glycol macromolecule with a hydroxyl group at one end and a Y1 substituent at the other end in dichloromethane. Subsequently, add 1 - 5 molar equivalents of 4 - dimethylaminopyridine and triethylamine thereto, continuously stir under an ice bath, and simultaneously add 1 - 5 molar equivalents of bromination reagent

[0116]

[0117] After reacting for 12 - 72 h, filter to obtain the filtrate, wash it 3 times with 10% NaHCO3 solution, then wash it 3 times with saturated NaCl solution, precipitate with ice - ether to obtain a white solid, and purify it several times to obtain the polymerization reaction macromolecular initiator. It can be characterized by 1H - NMR spectroscopy.

[0118] Step 2: Synthesis of pH - sensitive polymer (taking atom transfer radical polymerization as an example)

[0119]

[0120] Add the polymerization reaction macromolecular initiator obtained in Step 1 and various monomers containing acrylate structures to a polymerization reaction flask according to a specific molar ratio, and add a mixed solvent of N,N - dimethylformamide and isopropanol with a volume ratio of 1∶10 - 10∶1. After complete dissolution, add 1 - 5 equivalents of pentamethyldiethylenetriamine based on the molar amount of the macromolecular initiator. After performing 1 - 5 freeze - thaw cycles, add 1 - 1.5 equivalents of copper(I) bromide based on the molar amount of the macromolecular initiator. Under anhydrous and anaerobic conditions, react at any temperature from 30 - 90 °C for 8 - 72 h. After the reaction is completed, dilute it with tetrahydrofuran, purify it with a neutral alumina column, dialyze it with deionized water, and then lyophilize to obtain a white solid, which is the pH - sensitive polymer product.

[0121] Step 3: Linking of labeled molecules

[0122] Add the labeled molecule with a carboxyl group, dicyclohexylcarbodiimide, and N - hydroxysuccinimide to a reaction flask according to a specific molar ratio, add N,N - dimethylformamide to dissolve it completely, continuously stir and react for 2 - 12 h, then add the pH - sensitive polymer obtained in Step 2 according to a specific molar ratio, and continuously stir and react at any temperature for 2 - 12 h. The product is separated and purified by gel permeation chromatography.

[0123] On the other hand, the present invention provides micelles containing one or more pH - sensitive polymers of the present invention and a preparation method thereof.

[0124] In some embodiments, the labeling molecule of the pH-sensitive polymer is a fluorescent molecule, and the fluorescent molecule is selected from one or more of 5(6)-carboxytetramethylrhodamine (TMR), Cyanine 3, Cyanine 3.5, Cyanine 5, Cyanine 5.5, Cyanine 7.5, indocyanine green (ICG), Bodipy FL, Bodipy 650, Pacific blue, coumarin, and structural derivatives of the above molecules.

[0125] In some specific embodiments, the particle size of the pH-sensitive micelles of the present invention is 10 - 200 nm; in some embodiments, the particle size of the micelles is 20 - 100 nm; in some embodiments, the particle size of the micelles is 30 - 50 nm.

[0126] In some specific embodiments, the pH transition point at which the pH-sensitive micelles undergo a pH response is 4.0 - 8.0; in some embodiments, the pH transition point at which the pH-sensitive micelles undergo a pH response is 5.0 - 6.9; in some embodiments, the pH transition point at which the pH-sensitive micelles undergo a pH response is 6.3 - 6.9; in some embodiments, the pH transition point at which the pH-sensitive micelles undergo a pH response is 5.5 - 6.2; in some embodiments, the pH transition point at which the pH-sensitive micelles undergo a pH response is 5.0 - 5.5.

[0127] In some specific embodiments, the pH range for the pH-sensitive micelles to complete the pH response is less than 1 pH unit; in some embodiments, the pH range for the pH-sensitive micelles to complete the pH response is less than 0.5 pH unit; in some embodiments, the pH range for the pH-sensitive micelles to complete the pH response is less than 0.25 pH unit; in some embodiments, the pH range for the pH-sensitive micelles to complete the pH response is less than 0.15 pH unit.

[0128] In some specific embodiments, the fluorescence switching multiple of the pH-sensitive micelles conjugated with a fluorescent molecule is greater than 10; in some embodiments, the fluorescence switching multiple of the pH-sensitive micelles conjugated with a fluorescent molecule is greater than 50; in some embodiments, the fluorescence switching multiple of the pH-sensitive micelles conjugated with a fluorescent molecule is greater than 100.

[0129] On the other hand, the present invention provides the use of pH-sensitive micelles in cell imaging. In some specific embodiments, pH-sensitive micelles with different pH transition points can be used to characterize the processes of cell endocytosis and endosome maturation. By using several pH-sensitive micelles that dissociate in response to four different pH regions of the tumor microenvironment, early endosomes, late endosomes, and lysosomes respectively, and conjugating them with different fluorescent molecules, the stage of the endocytosis process can be detected by detecting the opening of their respective fluorescence signals.

[0130] On the other hand, the present invention provides the use of pH-sensitive micelles in animal tumor imaging. In some specific embodiments, pH-sensitive micelles with different transition points are conjugated with the near-infrared probe indocyanine green (ICG). After intravenous injection of the micelles into tumor-bearing mice, the fluorescence signal can efficiently and sensitively indicate the tumor site, and can better outline the tumor edge, achieving an ideal signal-to-noise ratio.

[0131] On the other hand, the present invention provides the use of pH-sensitive micelles in tumor chemotherapy drug delivery. In some specific embodiments, pH-sensitive polymers with different transition points are conjugated with the chemotherapy drug docetaxel through a cathepsin B-sensitive Gly-Phe-Leu-Gly (GFLG) tetrapeptide sequence. A micelle preparation is prepared from the polymer-drug conjugate and is intravenously injected into tumor-bearing mice, which can exert good anti-tumor efficacy and prolong the survival period of the mice.

[0132] On the other hand, the present invention provides the use of pH-sensitive micelles in tumor immunotherapy. In some specific embodiments, the non-pH-sensitive monomer has a guanidine group structure, which not only regulates the hydrophilicity of the polymer but also promotes endosomal escape. The pH-sensitive polymer is conjugated with the Toll-like receptor agonist IMDQ through a cathepsin B-sensitive Val-Ala dipeptide, and a micelle loaded with the drug is prepared from the polymer, which has good enzyme-sensitive drug release performance.

[0133] On the other hand, the present invention provides the use of pH-sensitive micelles in targeted diagnosis and treatment, including a) conjugating one or more of a tumor-targeting peptide, a fragment of an antibody corresponding to a tumor-associated antigen, an aptamer, a targeting protein, a small molecule (such as folic acid) through a maleimide linker, a carboxyl linker, an amino linker at one end of PEG polyethylene glycol; b) preparing a preparation from the pH-sensitive polymer with a targeting group; c) administering the preparation to the corresponding tissue or animal to exert targeted diagnostic or therapeutic functions.

[0134] On the other hand, the present invention provides the use of pH-sensitive micelles in multimodal imaging. For example, by chemical conjugation, a chemical group containing 19 F can be linked to the pH-sensitive polymer as a labeling molecule to achieve 19 F nuclear magnetic resonance imaging; by metal chelation, 64 Cu can be introduced into the labeling molecule (selected from metal chelating groups) of the pH-sensitive polymer to achieve positron emission tomography (PET) imaging.

[0135] On the other hand, the present invention provides the use of pH-sensitive micelles in research kits. The uses of the above-mentioned pH-sensitive micelles in cell imaging, animal imaging, chemotherapy drug delivery, immunotherapy, targeted diagnosis and treatment, and multimodal imaging can all be developed into research kits. The kit includes one or more suitable containers for holding the micelle preparation, and may also be equipped with necessary diluting solvents, excipients, etc.

[0136] Examples

[0137] The present invention will be described and explained by the following specific synthesis examples and embodiments, but the present invention is not limited by these specific examples.

[0138] Synthesis Example 1: Synthesis of a polyethylene glycol macroinitiator by atom transfer radical polymerization (ATRP) reaction

[0139]

[0140] Add 20 g of polyethylene glycol 5000 capped with a methoxy group at one end, 2.8 mL of triethylamine, and 2.44 g of 4-dimethylaminopyridine to a 500 mL eggplant-shaped flask, and add 120 mL of dichloromethane to completely dissolve the solid. Then dilute 2.5 mL of 2-bromo-2-methylpropionyl bromide with 50 mL of dichloromethane, and add it dropwise to the eggplant-shaped flask at a rate of 1 drop per second using a constant pressure dropping funnel. During this process, the reaction system needs to be ice-bathed and continuously stirred. After the dropwise addition is completed, react in the ice bath for 30 min, then restore to room temperature and react for 18 h. After the reaction is completed, filter with a Buchner funnel, discard the insoluble matter, concentrate by rotary evaporation, and precipitate with a 10% sodium bicarbonate solution in sufficient ice-cold diethyl ether to obtain the crude product. Subsequently, dry the diethyl ether by suction and recrystallize with absolute ethanol 3-4 times. Finally, 17.1 g of the polyethylene glycol macroinitiator product is obtained, with a yield of 83.0%.

[0141] Synthesis Example 2: ATRP polymerization method of various pH-sensitive polymers

[0142] In this example, the ATRP method is used to synthesize pH-sensitive polymers. The synthesized polymer has the following structure: Among them, the polymer has the following structure:

[0143]

[0144] As shown in the figure, the hydrophilic block of the polymer is PEG 5k , and the hydrophobic block contains pH-sensitive monomers and non-pH-sensitive monomers, where the R1' structure of the pH-sensitive monomer is as shown in Formula A

[0145]

[0146] The R2' structure of the non-pH-sensitive monomer is as shown in Formula B

[0147]

[0148] Taking three specific polymers as examples below, the synthesis method is introduced:

[0149] 1. Synthesis of pH-sensitive polymer PEG-P(DPA 90 -MMA 10 )

[0150]

[0151] Accurately weigh 250 mg of the PEG 5k -Br macroinitiator obtained in Synthesis Example 1, add it to a polymerization reaction flask, and then add 90-fold equivalents of N,N-dipropylaminoethyl methacrylate (DPA) monomer, 10-fold equivalents of methyl methacrylate (MMA) monomer, and 4-fold equivalents of aminoethyl methacrylate hydrochloride (AMA·HCl) monomer. Add 1 mL of N,N-dimethylformamide and 1 mL of isopropanol to dissolve the solid. Then add 0.2-fold equivalents of pentamethyldiethylenetriamine. After 4 freeze-thaw cycles, add 1-fold PEG 5k -Br initiator equivalent of copper bromide. Place the reaction flask in an oil bath at 40 °C and heat with stirring. After reacting for 36 h, add tetrahydrofuran to dilute the reaction solution, purify it with a neutral alumina column, then dialyze with deionized water for 12 h, and freeze-dry to obtain a white solid product. The yield is 79%. The product is characterized by 1H NMR, and the structural formula and 1H NMR spectrum of the product are as Figure 1 shown.

[0152] 2. Synthesis of pH-sensitive polymer PEG-P(DBA 70 -HEMA 30 )

[0153]

[0154] Accurately weigh 250 mg of the PEG 5k -Br macroinitiator obtained in Synthesis Example 1, add it to a polymerization reaction flask, and then add 70-fold equivalents of N,N-dibutylaminoethyl methacrylate (DBA) monomer, 30-fold equivalents of 2-hydroxyethyl methacrylate (HEMA) monomer, and 4-fold equivalents of AMA monomer. Add 1 mL of N,N-dimethylformamide and 1 mL of isopropanol to dissolve the solid. Then add 0.2-fold equivalents of pentamethyldiethylenetriamine. After 4 freeze-thaw cycles, add 1-fold PEG 5kCopper(I) bromide in an amount equivalent to the -Br initiator. Place the reaction flask in an oil bath heated to 40 °C and stir. After reacting for 36 h, add tetrahydrofuran to dilute the reaction solution, purify it using a neutral alumina column, then dialyze it against deionized water for 12 h, and freeze-dry to obtain a white solid product. The yield is 79%. The product was characterized by 1H NMR spectroscopy. The structural formula of the product and the 1H NMR spectrum are as shown in Figure 1 shown.

[0155] 3. Synthesis of pH-sensitive polymer PEG-P(EPA 50 -BMA 50 ) containing non-pH-sensitive monomers

[0156]

[0157] Accurately weigh 250 mg of the PEG 5k -Br macroinitiator obtained in Synthesis Example 1, add it to a polymerization reaction flask, and then add 50-fold equivalents of N,N-ethylpropylaminoethyl methacrylate (EPA) monomer, 50-fold equivalents of n-butyl methacrylate (BMA) monomer, and 4-fold equivalents of AMA monomer. Add 1 mL of N,N-dimethylformamide and 1 mL of isopropanol to dissolve the solid. Then add 0.2-fold equivalents of pentamethyldiethylenetriamine. After 4 freeze-thaw cycles, add 1-fold equivalent of copper(I) bromide in an amount equivalent to the PEG 5k -Br initiator. Place the reaction flask in an oil bath heated to 40 °C and stir. After reacting for 36 h, add tetrahydrofuran to dilute the reaction solution, purify it using a neutral alumina column, then dialyze it against deionized water for 12 h, and freeze-dry to obtain a white solid product. The yield is 79%. The product was characterized by 1H NMR spectroscopy. The structural formula of the product and the 1H NMR spectrum are as shown in Figure 1 shown.

[0158] Synthesis Example 3: Synthesis of a polyethylene glycol macro chain transfer agent by reversible addition-fragmentation chain transfer (RAFT) polymerization

[0159]

[0160] Weigh 500 mg of 4-cyano-4-(((ethylthio)thiocarbonyl)thio)pentanoic acid (C2-CTA) and 6.33 g of polyethylene glycol 5000 capped with a methoxy group at one end into a reaction flask and dissolve them in dichloromethane. Separately, take 1.5-fold molar equivalents of dicyclohexylcarbodiimide and 0.2-fold molar equivalents of 4-dimethylaminopyridine with respect to polyethylene glycol 5000, dissolve them separately in dichloromethane, and add them to the reaction system successively under stirring in an ice bath. After reacting at room temperature for 12 h, cool the reaction mixture in an ice bath, concentrate the reaction solution, and filter to remove insoluble substances. Obtain a yellow solid product by precipitation with ice-cold diethyl ether and store it after vacuum drying.

[0161] Synthesis Example 4: Fluorescent Molecule Coupling of pH-Sensitive Polymers Containing Non-pH-Sensitive Monomers

[0162] In this example, a fluorescent molecule was chemically coupled to a pH-sensitive polymer containing a non-pH-sensitive monomer. The fluorescent molecule selected in this example was 5(6)-carboxytetramethylrhodamine, and its structure is shown in Formula C:

[0163]

[0164] Taking the three pH-sensitive polymers of Synthesis Example 2 as examples, the structures of the fluorescently labeled polymers obtained are shown in Formula D:

[0165]

[0166] Specific implementation method: Accurately weigh an appropriate amount of TMR fluorescent probe, 1.2 times the molar equivalent of the fluorescent probe of dicyclohexylcarbodiimide, and 1.2 times the molar equivalent of N-hydroxysuccinimide, dissolve them in an appropriate amount of ultra-dry N,N-dimethylformamide, and stir the reaction at room temperature for 6 h. Add a pH-sensitive polymer containing a non-pH-sensitive monomer obtained from Synthesis Example 2 equivalent to 1 / 4 of the molar equivalent of the fluorescent probe. Stir the reaction at room temperature for 12 h. After the reaction is completed, gel permeation chromatography is used for the separation and purification of the pH-sensitive polymer linked with the fluorescent molecule. During the above reaction process and after separation and purification, thin-layer chromatography can be used to monitor the coupling of the fluorescent molecule.

[0167] Example 1: Preparation of Micelles of pH-Sensitive Polymers Containing Non-pH-Sensitive Monomers and Verification of pH-Sensitive Properties

[0168] In this example, according to the method described in Synthesis Example 2, isopropyl methacrylate (iDPA) was polymerized with the hydrophobic non-pH-sensitive monomer n-butyl methacrylate (BMA) and the hydrophilic non-pH-sensitive monomer 2-hydroxyethyl methacrylate (HEMA) respectively to obtain a pH-sensitive polymer with the structural formula shown in Formula E. The ratios of the pH-sensitive monomers and non-pH-sensitive monomers of this series of pH-sensitive polymers are diverse. In this example, there are 6 different ratios: x = 100 and y = 0, x = 90 and y = 10, x = 80 and y = 20, x = 70 and y = 30, x = 60 and y = 40, x = 50 and y = 50, and finally 11 specific polymers were obtained.

[0169]

[0170] The proton nuclear magnetic resonance spectra of each polymer are as Figure 2 、 Figure 3 shown.

[0171] The pH-sensitive micelles containing non-pH-sensitive monomers were prepared by probe ultrasound-desolvation method. Specific implementation method: Accurately weigh 2.0 mg of the polymer and completely dissolve it in 150 μL of methanol. Place the ultrasonic probe below the liquid level of 3 mL of deionized water. While turning on the probe ultrasound, add the polymer solution dissolved in methanol to the deionized water and ultrasonicate for 30 s at a power of 45 W. Subsequently, transfer it to a 100 kD ultrafiltration tube and ultrafilter 4 times with deionized water to remove the organic solvent. Finally, quantitatively determine the micelle concentrate to 5 mg / mL (or other required concentration) with a balance.

[0172] Verification of the pH responsiveness of pH-sensitive micelles: Dilute the 5 mg / mL micelle solution prepared according to the above steps to PBS buffer solutions with pH 7.4 and 5.0 at a ratio of 1:20 respectively. Measure the particle sizes of the micelle solutions under physiological conditions of pH 7.4 and acidic conditions of pH 5.0 with a dynamic light scattering particle size analyzer. The particle size distribution diagram is as Figure 4 shown. The results show that the micelles can maintain a particle size in the range of 30 - 50 nm at pH 7.4, while they will dissociate into a form of 6 - 10 nm under acidic conditions of pH 5.0, indicating its pH-sensitive characteristics responsive to acidic conditions.

[0173] Example 2: Regulation ability of non-pH-sensitive monomers with different hydrophobicities on the pH transition point of pH-sensitive polymers

[0174] In this example, N-(n-propyl)acrylamide (DPA) was copolymerized with 7 non-pH-sensitive monomers respectively. The specific implementation method is the same as that described in Synthesis Example 2. Among them, DPA monomer and each specific non-pH-sensitive monomer were copolymerized at molar ratios of 90:10, 80:20, 70:30, 60:40, 50:50 respectively, to obtain a series of 35 polymers with linearly varying proportions of non-pH-sensitive monomers, and one polymer with 100% DPA. Finally, a total of 36 polymers were obtained. The polymer structural formulas and their corresponding proton nuclear magnetic resonance spectra are as Figures 5 - 11 shown.

[0175] After preparing the blank polymer, conjugate the fluorescent probe by the method described in Synthesis Example 4. Fluorescently label the polymer with TMR and prepare it into a 4 mg / mL micelle solution by the method described in Example 1. Dilute the micelle solution to 100 μg / mL with a series of pre-prepared phosphate buffer solutions (PBS buffer solutions) with different pH values, and perform fluorescence measurement with a fluorescence analyzer (excitation wavelength 545 nm, emission wavelength reception range 560 - 660 nm) to obtain the fluorescence spectra of each polymer micelle at different pH values, as Figures 12 - 18 shown. Normalize the fluorescence peaks of the micelle solution at different pH values and fit as Figure 19For the curve shown, the pH corresponding to a fluorescence signal intensity of 50% is the pH transition point of the pH-sensitive polymer of the micelle. Subsequently, with the proportion of non-pH-sensitive monomers as the abscissa and the pH transition point as the ordinate, the variation rule of the pH transition points of a series of polymers with different proportions of non-pH-sensitive monomers is obtained, as shown in Figure 20 shown. This rule indicates that by changing the proportion of non-pH-sensitive monomers in the polymer, the pH transition point of the polymer will change linearly, and the ability of non-pH-sensitive monomers to regulate the pH transition point is related to their hydrophilicity and hydrophobicity (characterized by log P).

[0176] Example 3: pH-sensitive polymers with different pH transition points are used for the conjugation and imaging of various fluorescent probes

[0177] In this example, a series of pH-sensitive polymers synthesized in Example 1 with pH transition points in the range of pH 5.2 - 7.0 were used for the conjugation of fluorescent probes. In this example, eight different fluorescent probes, namely ICG, BODIPY FL, BODIPY650, TMR, Cy3.5, Cy5, Cy5.5, and Pacific Blue, were used, and their respective structures are shown in Formula F. The specific conjugation method is the same as that in Synthesis Example 4.

[0178]

[0179] Subsequently, a series of pH-sensitive polymers conjugated with different fluorescent molecules were prepared into micelles, and the number of conjugated fluorescent molecules was characterized using a UV spectrophotometer, and their pH-sensitive properties were characterized using a fluorescence analyzer. The results are shown in Table 1. Among them, UPS 6.9 represents PEG 5k -P(iDPA 80 -HEMA 20 ), UPS 6.7 represents PEG 5k -P(iDPA 60 -HEMA 40 ), UPS 6.5 represents PEG 5k -iPDPA 100 ), UPS 6.3 represents PEG 5k -P(iDPA 90 -BMA 10 ), UPS 6.0 represents PEG 5k -P(iDPA 80 -BMA 20 ), UPS 5.7 represents PEG 5k -P(iDPA 70 -BMA30 ),UPS 5.5 represents PEG 5k -P(iDPA 60 -BMA 40 ),UPS 5.3 represents PEG 5k -P(iDPA 50 -BMA 50 ).

[0180]

[0181] Table 1

[0182] Subsequently, the pH-sensitive micelle solutions at different pH values were added into black 384-well plates, and the fluorescence signals of each well were collected separately in each fluorescence channel using a live imaging system to obtain multi-color fluorescence imaging as Figure 21 shown. The results showed that the 8 pH-sensitive micelles constructed in this example with different pH transition points and respectively connected with different fluorescent probes could all exhibit the phenomenon of stable fluorescence signal "on" or "off" in environments with different pH values. When the environmental pH was higher than its pH transition point, its fluorescence signal was "off", and when the environmental pH was lower than its pH transition point, its fluorescence signal was "on", and the difference in fluorescence signals between the "on" and "off" states could be clearly presented under conventional fluorescence imaging conditions.

[0183] Example 4 Multi-color fluorescence imaging of different pH-sensitive polymers for the maturation process of cell endosomes

[0184] In this example, five fluorescent probes, namely Pacific blue, Bodipy FL, mFluor610, TMR, and Cy5, were respectively conjugated with five pH-sensitive polymers with pH transition points at pH 6.9, 6.5, 5.7, 5.5, and 5.3 described in Example 3, and micelles labeled with the five fluorescent probes were prepared using the above polymers. The characterization of the five micelles is shown in Table 2.

[0185]

[0186] Table 2

[0187] In this example, human non-small cell lung cancer A549 cells were used for five-color fluorescence imaging of the endosome maturation process. Specifically, first, the mixed solution of the above five micelles (each micelle concentration was 50 μg / mL) was added to the A549 cell culture medium at 0 °C, and incubated for 10 min to allow the micelles to bind to the cell membrane. Then, the cell culture medium was replaced with fresh culture medium and the culture conditions were restored to 37 °C to allow the cells to uptake the micelles. As Figure 22As shown, five different fluorescent signals are turned on in sequence, indicating that the pH of the endosome is gradually decreasing, which indicates the entire process of endosome maturation. Among them, the blue signal lights up indicating that the pH is in the range of 6.5 - 6.9; the green signal lights up indicating that the pH is in the range of 5.7 - 6.5; the red signal lights up indicating that the pH is in the range of 5.5 - 5.7; the cyan signal lights up indicating that the pH is in the range of 5.3 - 5.5; the purple signal lights up indicating that the pH is below 5.3.

[0188] Example 5 Series of pH-Sensitive Polymers for In Vivo Imaging of Mouse Tumors

[0189] In this example, the Indocyanine Green (ICG) fluorescent probe was conjugated with a series of pH-sensitive polymers with different transition points in Example 3 and prepared into nanomicelles. The characterization of the pH-sensitive micelles conjugated with ICG is shown in Table 3.

[0190]

[0191] Table 3

[0192] In this example, a tumor model was constructed by orthotopically inoculating 4T1 mouse breast cancer cells into the mammary pads of female BALB / c mice. When the volume of the tumor carried by the mouse reached 150 mm 3 , 20 mg / kg of the ICG nanomicelle preparation was injected via the tail vein. The results of in vivo imaging of small animals showed that the pH-sensitive micelle preparation with a low pH transition point (such as UPS 5.3 ) showed stronger fluorescent signals at the tumor site, and the quantitative results of the contrast to noise ratio (CNR) also showed that the preparation with a low pH transition point showed stronger tumor-targeted imaging characteristics. The results of in vivo imaging are shown in Figure 23 , and the results of the contrast to noise ratio are shown in Figure 24 .

[0193] Example 6 Enzyme-Responsive Series of pH-Sensitive Polymers for Efficient Delivery of Chemotherapeutic Drug Docetaxel

[0194] In this example, the conjugation of the pH-sensitive polymer with the chemotherapeutic drug docetaxel was achieved through a cathepsin B-sensitive glycine-phenylalanine-leucine-glycine tetrapeptide. The structural formula of the polymer-drug conjugate is shown as Formula G:

[0195]

[0196] Among the polymers involved in this embodiment, the non-pH-sensitive monomer is selected from one or both of n-butyl methacrylate (BMA) and 2-hydroxyethyl methacrylate (HEMA). By changing the ratio of x and y, polymer-drug conjugates with different pH transition points are obtained. The proton nuclear magnetic resonance spectra of several polymer-drug conjugates described above are as shown in Figure 25 shown. According to the method described in Example 2, the polymer was prepared into a micelle preparation. The preparation was treated with papain, and the drug release amount of docetaxel was quantitatively detected by high performance liquid chromatography at a series of time points, and the enzyme-sensitive drug release curve as shown in Figure 26 was obtained. The results showed that each preparation group exhibited high drug release performance, and more than 80% of the drug was released within 24 - 48 h. After the drug preparation was intravenously injected into 4T1 breast cancer-bearing mice via the tail vein, the tumor growth curve of the mice was as shown in Figure 27 shown, and each preparation group exhibited significantly better anti-tumor characteristics than the control group.

[0197] Example 7 Enzyme-responsive guanidine group-containing pH-sensitive polymer conjugate immunostimulant for immunotherapy

[0198] In this embodiment, the pH-sensitive polymer and the immunostimulant IMDQ were conjugated through a cathepsin B-sensitive valine-alanine dipeptide unit. The structure of the polymer conjugate is as shown in formula H:

[0199]

[0200] For the pH-sensitive polymer used in this embodiment, the non-pH-sensitive monomer is a methacrylate monomer containing a phenylguanidine group structure, the number of x is 80, the number of y is 8, and the number of z is 5. The proton nuclear magnetic resonance spectrum of the polymer is as shown in Figure 28 shown. The polymer was prepared into a micelle preparation according to the method described in Example 2, and an enzyme-sensitive cleavage experiment was carried out with papain, and the release of the drug was quantitatively detected by high performance liquid chromatography. The results are as shown in Figure 29 shown, indicating that 50% of IMDQ can be released within 30 min and 90% of IMDQ can be released within 1 h under the conditions of pH 6.0 and 1 mM papain.

[0201] The present invention has been described as above. The present invention includes various changes within its scope, and these changes do not deviate from the scope of the present invention. In addition, all such situations that are obviously considered to be modifications of the present invention by those skilled in the art are included within the scope of the appended claims.

Claims

1. A pH-sensitive polymer, comprising two parts: a hydrophilic segment and a hydrophobic segment, wherein, The hydrophilic segment is one or more of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC), poly (oligoethylene glycol methacrylate) (POEG), polysaccharide, poly (betaine methacrylate) (PCB), The hydrophobic segment has the structure shown in Formula 1: Wherein, R’, R”, R”’, X1 and X2 may be the same or different, and are each independently selected from -H, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl, C1-C12 substituted cycloalkyl, R”” is an end group generated by polymerization reaction, and is selected from halogen or thiol or thioester; R1 and R2 may be the same or different, and are each independently selected from C1-C16 alkyl, or R1 and R2 may also combine together to form a C1-C16 alkylene group; M1 and M2 are each independently selected from one of -O- and -NH-; R3 is a non-pH-sensitive group, and is selected from: C1-C16 alkyl, C1-C16 cycloalkyl, and the above groups substituted with one or more of hydroxyl, mercapto, guanidyl, halogen, ether bond, ester bond, amide bond, aryl, alkyl, carbamate bond; a and b are each an integer from 1 to 10; x and y are each integers, and the sum of x and y is an integer from 10 to 200; z is an integer from 1 to 50, The three parts of x, y, and z are arranged in any order in the polymer hydrophobic segment; L is a linker arm, and each L is independent of each other and may be different from each other; F is a labeling molecule, and each F is independent of each other and may be different from each other.

2. The pH-sensitive polymer according to claim 1, wherein, The labeling molecule is selected from one or more of fluorescent molecules, photosensitizers, fluorescence quenching molecules, self-luminescent molecules, chemotherapeutic drugs, immunotherapeutic drugs, metal chelating molecules.

3. The pH-sensitive polymer according to claim 2, wherein, The fluorescent molecules are selected from: 5(6)-carboxytetramethylrhodamine (TMR), Cyanine 3, Cyanine 3.5, Cyanine 5, Cyanine 5.5, Cyanine 7.5, indocyanine green (ICG), Bodipy FL, Bodipy 650, Pacific blue, coumarin and one or more of the structural derivatives of the above molecules; The photosensitizers are selected from: porphine, porphyrin, bacteriochlorophyll, phthalocyanine, cationic photosensitizers, quinone photosensitizers, curcumin photosensitizers, Bodipy photosensitizers and one or more of the structural derivatives of the above molecules; The fluorescence quenching molecules are selected from one or more of DABCYL, DABSYL, QXL, QSY, BHQ, ATTO; The self-luminescent molecule is selected from luminol; The chemotherapeutic drugs are selected from one or more of paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, doxorubicin, epirubicin, daunorubicin, cisplatin; the immunotherapeutic drugs are selected from one or more of indoleamine-(2,3)-dioxygenase inhibitors IDOi, Toll-like receptor agonists; The metal chelating group is selected from: DOTA, NOTA, TETA, Diamsar, NETA, TACN-TM, and derivatives of the above structures.

4. The pH-sensitive polymer according to any one of claims 1-3, wherein R’, R”, R”’ may be the same or different and are each independently selected from -H, C1-C4 alkyl X1 and X2 are each -H; R”” is selected from halogens; R1 and R2 may be the same or different and are each independently selected from C1-C5 alkyl, or R1 and R2 may together form a C2-C8 alkylene group; M1 and M2 are each independently selected from -O-- or -NH-; R3 is a non-pH-sensitive group selected from: C1-C8 alkyl, and C1-C8 alkyl substituted with one or more of hydroxyl, ether bond structures; a and b are each an integer from 2 to 4; x and y are each integers, and the sum of x and y is an integer from 40 to 150; z is an integer from 1 to 20; Other symbols are as defined in claims 1-3.

5. The pH-sensitive polymer according to any one of claims 1-4, which has the structure shown in Formula 2 below: Among them, Y1 is selected from -H, -COOH, -NH2, -SH, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl, C1-C12 substituted cycloalkyl, or or a metal chelating group; n is an integer from 1 to 500; Y2 and Y3 are each independently selected from -H, C1-C12 alkyl, C1-C12 cycloalkyl, C1-C12 substituted alkyl or C1-C12 substituted cycloalkyl; Other symbols are as defined in claims 1-4.

6. The pH-sensitive polymer according to any one of claims 1-5, which has the structure shown in Formula 3 below: Among them, M1 and M2 are -O- or -NH-; R1’ is selected from the following structures: R2’ is selected from the following structures: wherein the sum of x and y is an integer from 40 to 150; z is an integer from 2 to 10; The three parts x, y, and z are arranged in any order in the hydrophobic segment of the polymer; Other symbols are as defined in claims 1-5.

7. The pH-sensitive polymer according to claim 6, wherein M1 and M2 are -O; R1’ is selected from the following structures: R2’ is selected from the following structures: wherein the sum of x and y is 80-120; z is 3-5; Other symbols are as defined in claim 6.

8. The pH-sensitive polymer according to any one of claims 1-7, wherein the sum of x and y is 100.

9. The pH-sensitive polymer according to any one of claims 1-8, wherein, F is a fluorescent molecule selected from one or more of 5(6)-carboxytetramethylrhodamine (TMR), Cyanine 3, Cyanine 3.5, Cyanine 5, Cyanine 5.5, Cyanine7.5, indocyanine green (ICG), Bodipy FL, Bodipy 650, Pacific blue, coumarin, and structural derivatives of the above molecules.

10. A pH-sensitive micelle formed from one or more of the pH-sensitive polymers according to any one of claims 1-9.

11. The pH-sensitive micelle according to claim 10, wherein, The particle size of the micelle is 10-200 nm.

12. The pH-sensitive micelle according to any one of claims 10-11, wherein, The pH value at which the pH-sensitive micelles exhibit a pH response is 4.0 - 8.0, and the pH range for the pH-sensitive micelles to complete the pH response is less than 1 pH unit.

13. The pH-sensitive micelle according to claim 10, wherein The fluorescence switching multiple of the pH-sensitive micelles conjugated with fluorescent molecules is greater than 10.

14. Use of the pH-sensitive micelles according to claim 10 in the preparation of a reagent for fluorescence imaging.

15. Use of the pH-sensitive micelles according to claim 10 in the preparation of a reagent for cell imaging, or animal tumor imaging, or multimodal imaging.

16. Use of the pH-sensitive micelles according to claim 10 in the preparation of a reagent for tumor chemotherapy drug delivery, or tumor immunotherapy, or targeted diagnosis and treatment.

17. Use of the pH-sensitive micelles according to claim 10 in the preparation of a drug for diagnosing and treating diseases selected from the following: malignant tumors, inflammatory diseases, autoimmune diseases, atherosclerosis, skin diseases, eye diseases, infectious diseases caused by pathogenic microorganisms.

18. A kit, which comprises the pH-sensitive micelles according to claim 10.

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