Degradable cationic polymers with bone targeting function, and methods of making and using the same

Abstract

The present application relates to a kind of degradable cationic polymer with bone targeting function and its preparation method and application, specifically, the degradable cationic polymer with bone targeting function provided in the present application can effectively load nucleic acid (such as mRNA, miRNA, DNA), and the encapsulation efficiency is more than 90%, and stable nanoparticles are formed by electrostatic interaction, protect nucleic acid from degradation, while having the characteristics of slowing down particle positive electricity.This carrier shows better stability compared with LNP, supports long-term storage at low temperature.Its bone targeting is clear, promotes the accurate delivery of nucleic acid to bone tissue, enhances the efficacy and reduces side effects, while showing biocompatibility for osteoblasts without toxicity.The application range is wide, covering osteoporosis, osteoarthritis, bone tumor treatment and gene and stem cell therapy.Experiments have verified that the nanoparticles formed by the carrier and eGFP mRNA have high transfection efficiency, and the combination with miRNA effectively inhibits breast cancer bone metastasis, highlighting its therapeutic potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polymer material science and biomedical science, and particularly relates to a degradable cationic polymer with bone targeting function, a preparation method and application thereof. BACKGROUND

[0002] Gene therapy, as a cutting-edge treatment approach, provides a new way of thinking for the treatment of various diseases by specifically regulating gene expression in specific cells. The implementation of this method relies on the effective delivery of nucleic acids such as DNA, mRNA, saRNA, siRNA, miRNA, etc., which are the key mediators of gene regulation. However, due to the large size and hydrophilicity of nucleic acid molecules, they are difficult to cross the cell membrane alone, especially when administered systemically, they need to overcome multiple physiological barriers to reach the action site. Currently, although traditional delivery platforms such as viral vectors, lipid nanocomposites and polymers have been widely studied, they each have limitations such as cytotoxicity, instability and lack of targeting, etc.

[0003] Polymeric materials have shown significant advantages in nucleic acid delivery due to their multifunctionality, expandability and precise controllability. By adjusting the structure, molecular weight of the polymer and making specific modifications, polymer nucleic acid delivery systems that can target different organs have been successfully developed. In particular, cationic polymers form nanocomposites with negatively charged nucleic acids through electrostatic interactions, providing protection for nucleic acids to cross biological barriers and achieving precise organ targeting by regulating the physical properties of nanocomposites.

[0004] In the treatment of bone-related diseases, the unique extracellular matrix and complex bone marrow microenvironment of bone tissue make it difficult for drugs to effectively penetrate and accumulate at the lesion site. This non-specific biodistribution not only affects the treatment effect, but also can cause unnecessary side effects to patients. Therefore, it is particularly important to develop stable, efficient and degradable bone-targeting nucleic acid delivery carriers, which can ensure that nucleic acid drugs accurately and safely reach the target site, thereby exerting the best therapeutic effect. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a degradable cationic polymer with bone targeting function, a preparation method and application thereof. The polymer provided by the present application has bone targeting function, and the nanoparticles prepared by using the polymer as a carrier have good bone targeting performance, and have great application potential in the prevention, improvement or treatment of bone-related diseases such as osteoporosis, osteoarthritis and bone tumors, gene therapy and stem cell therapy, etc. medical treatment.

[0006] Specifically, the present application provides a polymer or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer or polymorph thereof, characterized in that the polymer has a structural formula as shown in formula (I):

[0007]

[0008] In formula (I):

[0009] R' is selected from L 1 , optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl, optionally substituted 3-8 membered cycloalkyl, optionally substituted 3-8 membered cycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 5-10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkynyl group can also be interrupted at random by one or more heteroatoms;

[0010] R'' is selected from , optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl, optionally substituted 3-8 membered cycloalkyl, optionally substituted 3-8 membered cycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 5-10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkynyl group can also be interrupted at random by one or more heteroatoms;

[0011] M is absent or -S-S-;

[0012] R 1 , R 2 are each independently hydrogen, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl;

[0013] R''' is selected from -L 5 -R 3 ;

[0014] R 3 is -OR 11 , -COOR 11 , -SO2OR 11 , (OCH2CH2) m OH, or NR 11 R 12 ;

[0015] R 11 and R 12each independently is hydrogen, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted 3-8 membered heterocycloalkyl, optionally substituted C 6-12 aryl or optionally substituted 5-10 membered heteroaryl, wherein adjacent carbon atoms in alkyl, alkenyl or alkynyl can also be randomly interrupted by one or more heteroatoms;

[0016] m is an integer between 1 and 10;

[0017] R’’’ is selected from L 4 , optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 8-membered cycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 5- to 10-membered heteroaryl, wherein adjacent carbon atoms in alkyl, alkenyl or alkynyl can also be randomly interrupted by one or more heteroatoms;

[0018] wherein L 1 to L 5 each independently is optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted 3- to 8-membered heterocycloalkyl, optionally substituted 3- to 8-membered heterocycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 5- to 10-membered heteroaryl, wherein adjacent carbon atoms in alkyl, alkenyl or alkynyl can also be randomly interrupted by one or more heteroatoms;

[0019] y / x + y is in the range of 0-0.5.

[0020] In some specific embodiments of the application,

[0021] R’ is selected from L 1 ;

[0022] R’’ is selected from ;

[0023] M is absent or -S-S-;

[0024] R 1 , R 2 each independently is hydrogen, optionally substituted C 1-12alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl;

[0025] R’’’ is selected from -L 5 -R 3 ;

[0026] R 3 is -OR 11 , -COOR 11 , -SO2OR 11 , (OCH2CH2) m OH, or NR 11 R 12 ;

[0027] R 11 and R 12 are each independently hydrogen, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted 3- to 8-membered heterocycloalkyl, optionally substituted 3- to 8-membered heterocycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 5- to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkylene or alkynylene group can also be randomly interrupted by one or more heteroatoms;

[0028] m is an integer between 1 and 10;

[0029] R’’’’ is selected from L 4 ;

[0030] wherein L 1 to L 5 are each independently optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted 3- to 8-membered heterocycloalkyl, optionally substituted 3- to 8-membered heterocycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 5- to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkynyl group can also be randomly interrupted by one or more heteroatoms;

[0031] y / x + y is in the range of 0-0.5.

[0032] In some specific embodiments of the application, said L 1 to L4 each independently is optionally substituted C 1-6 alkyl, optionally substituted C 1-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted 3- to 8-membered heterocycloalkyl.

[0033] In some embodiments of the application, the L 5 is optionally substituted C 2-8 alkyl, optionally substituted C 2-8 alkenyl, optionally substituted C 2-6 alkynyl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkynyl group can also be interrupted by one or more heteroatoms.

[0034] In some embodiments of the application, R 1 , R 2 are independently selected from hydrogen.

[0035] In some embodiments of the application, R 3 is -OH, -COOH, -SO3H, -NH2, -N(CH3)2, -N(CH2CH3)2.

[0036] In some embodiments of the application, the structure of formula (I) is selected from the following structural formulae:

[0037]

[0038] (Ia)

[0039]

[0040] (Ib)

[0041]

[0042] (Ic)

[0043]

[0044] (Id)

[0045]

[0046] (Ie)

[0047]

[0048] (If)

[0049]

[0050] (Ig)

[0051]

[0052] (Ih)

[0053]

[0054] (Ii)

[0055] wherein y / x+y is in the range of 0-0.5.

[0056] In some embodiments of the present application, the average molecular weight of the polymer of formula (I) is greater than 5 kg / mol and less than 150 kg / mol.

[0057] In some embodiments of the present application, the average molecular weight of the polymer of formula (Ih) is at least 5.5 kg / mol, at least 6 kg / mol, at least 7 kg / mol, at least 8 kg / mol, at least 9 kg / mol, at least 10 kg / mol, at least 12 kg / mol, at least 14 kg / mol, at least 16 kg / mol, at least 18 kg / mol, at least 20 kg / mol, at least 22 kg / mol, or at least 24 kg / mol.

[0058] The present application also provides a degradable cationic polymer having bone targeting function or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer or polymorph thereof, characterized in that the polymer has a structural formula as shown in formula (I):

[0059]

[0060] In formula (I):

[0061] R' is selected from L 1 , optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl, optionally substituted 3-8 membered cycloalkyl, optionally substituted 3-8 membered cycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 5-10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkynyl group can also be interrupted by one or more heteroatoms at random;

[0062] R" is selected from , optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl, optionally substituted 3-8 membered cycloalkyl, optionally substituted 3-8 membered cycloalkenyl, optionally substituted C 6-12Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms;

[0063] M does not exist or is -SS-;

[0064] R 1 R 2 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group;

[0065] R''' is selected from -L 5 -R 3 ;

[0066] R 3 Yes - OR 11 -COOR 11 -SO2OR 11 (OCH2CH2) m OH, or NR 11 R 12 ;

[0067] R 11 and R 12 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 Cycloalkyl, optionally substituted 3-8 membered heterocycloalkyl, optionally substituted C 6-12 Aryl or optionally substituted 5-10-membered heteroaryl groups, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl groups may be randomly interrupted by one or more heteroatoms;

[0068] m is an integer between 1 and 10;

[0069] R is selected from L 4 Optional substitution of C 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 Alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 8-membered cycloalkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms;

[0070] Among them, L 1 To L 5 Each is an optional substitution of C.1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms;

[0071] The range of y / x+y is 0-0.5.

[0072] In some specific embodiments of the present invention

[0073] R' is selected from L 1 ;

[0074] R'' is selected from ;

[0075] M does not exist or is -SS-;

[0076] R 1 R 2 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group;

[0077] R''' is selected from -L 5 -R 3 ;

[0078] R 3 Yes - OR 11 -COOR 11 -SO2OR 11 (OCH2CH2) m OH, or NR 11 R 12 ;

[0079] R 11 and R 12 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkylene or alkyleneyne may be randomly interrupted by one or more heteroatoms;

[0080] m is an integer between 1 and 10;

[0081] R is selected from L 4 ;

[0082] Among them, L 1 To L 5 Each is an optional substitution of C. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms;

[0083] The range of y / x+y is 0-0.5.

[0084] In some specific embodiments of the present invention, the L 1 To L 4 Each is an optional substitution of C. 1-6 Alkyl, optionally substituted C 1-6 alkenyl, optionally substituted C 2-6 Alkyne group, or optionally substituted 3 to 8-membered heterocyclic alkyl group.

[0085] In some specific embodiments of the present invention, the L 5 C is an optional substitute 2-8 Alkyl, optionally substituted C 2-8 alkenyl or optionally substituted C 2-6 Alkynyl groups, wherein adjacent carbon atoms in alkyl, alkenyl, or alkynyl groups may be randomly interrupted by one or more heteroatoms.

[0086] In some specific embodiments of the present invention, R 1 R 2 It is independently selected from hydrogen.

[0087] In some specific embodiments of the present invention, R 3 It is -OH, -COOH, -SO3H, -NH2, -N(CH3)2, -N(CH2CH3)2.

[0088] In some specific embodiments of the present invention, the structure of formula (Ⅰ) is selected from the following structural formulas:

[0089] (Ιa)

[0090] (Ib)

[0091] (IC)

[0092] (ID)

[0093] (Ie)

[0094] (If)

[0095]

[0096] (Ιg)

[0097]

[0098] (Ιh)

[0099]

[0100] (Ιi)

[0101] The range of y / x+y is 0-0.5.

[0102] In some specific embodiments of the present invention, the polymer represented by formula (Ⅰ) has an average molecular weight greater than 5 kg / mol and less than 150 kg / mol.

[0103] In some specific embodiments of the present invention, the polymer represented by formula (Ⅰh) has an average molecular weight of at least 5.5 kg / mol, at least 6 kg / mol, at least 7 kg / mol, at least 8 kg / mol, at least 9 kg / mol, at least 10 kg / mol, at least 12 kg / mol, at least 14 kg / mol, at least 16 kg / mol, at least 18 kg / mol, at least 20 kg / mol, at least 22 kg / mol, or at least 24 kg / mol.

[0104] This invention also provides a method for preparing the salt of the polymer described in formula (Ij) or formula (Ik), comprising the following preparation steps: adding N,N'-(propane-1,3-diyl)acrylamide or N,N'-bis(acryloyl)cysteine, the compound of formula (II), alendronic acid, and triethylamine to a flask equipped with a stir bar, and adding a mixed solvent of methanol and water to the flask; then reacting in a dark environment at 40-60°C under a nitrogen atmosphere; reacting for 5 to 14 days to produce a high-viscosity solution, taking out aliquots at predetermined time intervals for testing to monitor the conversion rate and molar mass; once the target molar mass is reached, stopping the reaction by dilution with MeOH; then acidifying the diluted reaction mixture to an acidic environment with HCl, then purifying by dialysis with acidic water, then lyophilizing the purified product, and collecting the polymer in hydrochloride form as a solid after lyophilization;

[0105]

[0106]

[0107] The structural formula of compound (II) is as follows:

[0108]

[0109] (II)

[0110] Where y / x+y ranges from 0 to 0.5;

[0111] L 5 C is an optional substitute 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group are randomly interrupted by one or more heteroatoms;

[0112] R 3 Yes - OR 11 -COOR 11 -SO2OR 11 (OCH2CH2) m OH, or NR 11 R 12 ;

[0113] R 11 and R 12 Each is independently hydrogen, and each C is optionally substituted.1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 Cycloalkyl, optionally substituted 3-8 membered heterocycloalkyl, optionally substituted C 6-12 aryl or optionally substituted 5-10-membered heteroaryl groups, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl groups are optionally interrupted by one or more heteroatoms;

[0114] m is an integer between 1 and 10.

[0115] In some specific embodiments of the present invention, formula (Ij) is prepared using N,N'-(propane-1,3-diyl)acrylamide, a compound of formula (II), alendronic acid, and triethylamine as raw materials.

[0116] In some specific embodiments of the present invention, formula (Ik) is prepared by reacting N,N'-bis(acryloyl)cysteine ​​with a compound of formula (II), alendronic acid and triethylamine.

[0117] In some specific embodiments of the present invention, the molar ratio of bisacrylamide, the compound shown in formula (III), alendronic acid and triethylamine is 10:9:1:1.

[0118] In some specific embodiments of the present invention, the volume ratio of methanol to water is 4:1.

[0119] In some specific embodiments of the present invention, the reaction temperature is 50°C.

[0120] In some specific embodiments of the present invention, the method of taking out equal portions of the sample for detection at predetermined time intervals is to take out equal portions of the sample for 1H NMR spectroscopy at predetermined time intervals. 1 Detection was performed using 1H NMR and gel permeation chromatography (SEC).

[0121] In some specific embodiments of the present invention, the diluent is made of 1.0M HCl.

[0122] In some specific embodiments of the present invention, the acidic environment has a pH of 4. In some specific embodiments of the present invention, purification is achieved by dialysis of acidic water (4.0 L, pH=5, replaced 6 times in 3 days).

[0123] The present invention also provides a bone-targeting nucleic acid delivery vector, characterized in that it comprises the above-mentioned polymer or a pharmaceutically acceptable complex thereof, composition, salt, solvate, tautomer or polymorph.

[0124] The present invention also provides a nanocomposite comprising the above-described polymer or a pharmaceutically acceptable complex thereof, a composition, a salt, a solvate, a tautomer or polymorph, and a nucleic acid.

[0125] In some specific embodiments of the present invention, the nucleic acid is selected from at least one of DNA, RNA, plasmid, or DNA / RNA hybrid sequences.

[0126] In some specific embodiments of the present invention, the RNA is selected from at least one of spontaneous RNA, microRNA, short interfering RNA, short hairpin RNA, self-amplifying RNA, interfering RNA, and small RNA.

[0127] In some specific embodiments of the present invention, the RNA is microRNA.

[0128] In some specific embodiments of the present invention, the weight ratio of the polymer to nucleic acid is between 5:1 and 100:1.

[0129] The present invention also provides nanoparticles comprising the above-described polymer or a pharmaceutically acceptable complex thereof, composition, salt, solvate, tautomer or polymorph, or the above-described nanocomposite.

[0130] In some specific embodiments of the present invention, the nanoparticles have a hydrodynamically average diameter D of less than 1000 nanometers. h ;

[0131] Preferably, the nanoparticles have a hydrodynamic average diameter D of less than 200 nanometers. h .

[0132] The present invention also provides a pharmaceutical composition, characterized in that it comprises the above-mentioned polymer or a pharmaceutically acceptable complex thereof, a composition, a salt, a solvate, a tautomer or polymorph, a polymer prepared by the preparation method of the above-mentioned polymer, the above-mentioned bone-targeting nucleic acid delivery carrier, the above-mentioned nanocomposite, and the above-mentioned nanoparticles.

[0133] In some specific embodiments of the present invention, the above-described pharmaceutical composition further includes the drug to be delivered and pharmaceutically acceptable excipients.

[0134] The use of the above-mentioned polymers or their pharmaceutically acceptable complexes, compositions, salts, solvates, tautomers or polymorphs, polymers prepared by the above-mentioned polymer preparation methods, the above-mentioned bone-targeting nucleic acid delivery carriers, the nanocomposites described in any one of the above-mentioned items, and the nanoparticles described in any one of the above-mentioned items in the preparation of drugs that stimulate and enhance protective immune responses, gene therapy drugs, or drugs for the prevention, improvement or treatment of bone-related diseases.

[0135] In some specific embodiments of the present invention, the bone-related disease is at least one of osteoporosis, osteoarthritis, or bone tumor.

[0136] The polymer provided by this invention has a series of significant and beneficial technical effects, which are mainly reflected in the following aspects:

[0137] (1) High nucleic acid loading rate and protection: The cationic polymer provided by the present invention can be compounded with various RNAs such as messenger RNA (mRNA), microRNA (miRNA) and nucleic acids such as DNA, and achieve a nucleic acid loading rate of more than 90%, which ensures the effective delivery of nucleic acid drugs and high therapeutic efficiency; in addition, the cationic polymer can combine with negatively charged nucleic acids through electrostatic interaction to form nanoparticles, which can protect nucleic acids from degradation and reduce the positive charge on the particle surface.

[0138] (2) Excellent stability: Compared with traditional nucleic acid delivery carriers LNP, the nanoparticles of the present invention are more stable and can be stored for a long time under refrigeration at 4°C, which provides convenience for the long-term storage and transportation of nucleic acid drugs.

[0139] (3) Clear bone-targeting properties: By introducing alendronate groups with bone-targeting function and bone repair pharmacological effects into the side chain, this invention endows the cationic polymer nucleic acid delivery carrier with clear bone-targeting properties. This enables nucleic acid drugs to reach bone tissue more precisely, thereby improving therapeutic effects and reducing side effects.

[0140] (4) Good biocompatibility: Both the bone-targeting degradable cationic polymer provided by the present invention and the nanoparticles based on the polymer provided by the present invention are non-toxic to normal osteoblasts and show good biocompatibility.

[0141] (5) Broad application prospects: The bone-targeting degradable cationic polymer and polymer-based bone-targeting nucleic acid delivery carrier provided by this invention can be widely used in the medical field, including the prevention, improvement or treatment of osteoporosis, osteoarthritis and bone tumors and other bone-related diseases; gene therapy; stem cell therapy, etc.

[0142] (6) High therapeutic effect: Experiments have shown that the nanoparticles formed by the bone-targeting polymer and eGFP mRNA have high transfection efficiency; when combined with miRNA, they can achieve the effect of treating bone metastases of breast cancer, showing its great potential in actual treatment.

[0143] In summary, the biodegradable cationic polymer with bone-targeting function proposed in this invention, as well as the nanoparticles, nanocomposites, and nucleic acid delivery carriers based thereon, have advantages such as high efficiency, stability, strong targeting, good biocompatibility, and significant therapeutic effects. They provide a new solution for the delivery of nucleic acid drugs and have broad application prospects and important medical value. Attached Figure Description

[0144] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0145] Figure 1 This is a comparison of the in vitro bone-targeting capabilities of polymers with different alendronate contents.

[0146] Figure 2 This is a transmission electron microscope image of the nanocomposite formed by the composite.

[0147] Figure 3 The DLS particle size test curve of the nanocomposite obtained by titration is shown.

[0148] Figure 4 The stability curve of the nanocomposite obtained by titration at 4°C is shown.

[0149] Figure 5 The degradation curves of the bone-targeting polymer / miRNA nanocomposite in the presence of glutathione are shown.

[0150] Figure 6 The fluorescence expression of MDA-MB-231 human breast cancer cells after transfection with nanocomposites formed by polymers with different alendronate contents and FAM-labeled miRNAs.

[0151] Figure 7 Data on cytotoxicity of miRNAs loaded with different delivery systems on MDA-MB-231 human breast cancer cells.

[0152] Figure 8 Data on the cytotoxicity of miRNAs loaded onto MC3T3-E1 osteoblasts using different delivery systems.

[0153] Figure 9 The expression of fluorescent proteins after transfection of HEK-293T human embryonic kidney cells with different polymer / mRNA nanocomposites.

[0154] Figure 10The effects of bone-targeting polymer / miRNA nanocomposite on MDA-MB-231 human breast cancer cells.

[0155] Figure 11 To investigate the effect of bone-targeting polymer / miRNA nanocomplex on the proliferation capacity of MC3T3-E1 osteoblasts.

[0156] Figure 12 This section compares the in vivo bone-targeting capabilities of different polymer / miRNA nanocomposites. Detailed Implementation

[0157] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0158] Example 1: Synthesis of polymers with different alendrolic acid grafting ratios

[0159] N,N'-bis(acryloyl)cysteine ​​(1 mmol), 4-aminobutanol, alendronate (1 mmol total), and triethylamine (0.1 mmol) were added to a flask equipped with a stir bar, along with a mixed solvent of methanol / water (4 / 1, v / v). The polymerization reaction was carried out in the dark at 50°C under a nitrogen atmosphere. The mixture was allowed to react for 5 to 14 days (depending on the target molecular weight) to produce a highly viscous solution. Aliquots were taken at predetermined time intervals for 1H NMR spectroscopy. 1 HNMR and gel permeation chromatography (SEC) were used to monitor conversion and molar mass. Once the target molar mass was reached, the reaction was stopped by dilution with MeOH. The diluted reaction mixture was then acidified to pH ~4 with 1.0 M HCl, and purified by dialysis against acidic water (4.0 L, pH ~5, changed 6 times over 3 days). The polymer was collected as a white solid in its hydrochloride form after lyophilization. The structural formula of the polymer is shown below (Ia1):

[0160] The values ​​of y / x+y are shown in Table 1 below.

[0161]

[0162] (Ia1)

[0163] Table 1. Polymers with different alendronate grafting ratios

[0164]

[0165] Example 2: Synthesis of polymers with different grafted monomers

[0166] N,N'-bis(acryloyl)cysteine ​​or N,N'-(propane-1,3-diyl)acrylamide (1 mmol), the compound shown in formula (II) (0.9 mmol), alendronic acid (0.1 mmol), and triethylamine (0.1 mmol) were added to a flask equipped with a stir bar, along with a mixed solvent of methanol / water (4 / 1, v / v). The polymerization reaction was carried out in the dark at 50°C under a nitrogen atmosphere. The mixture was allowed to react for 5 to 14 days (depending on the target molecular weight) to produce a highly viscous solution. Aliquots were taken at predetermined time intervals for 1H NMR spectroscopy. 1 ¹H NMR and gel permeation chromatography (SEC) were used to monitor conversion and molar mass. Once the target molar mass was reached, the reaction was stopped by dilution with MeOH. The diluted reaction mixture was then acidified to pH 4 with 1.0 M HCl, followed by purification by dialysis against acidic water (4.0 L, pH 5, changed 6 times over 3 days). The polymers were collected as white solids in hydrochloride form after lyophilization, with structural formulas (Ib1), (Id1), and (Ih1), respectively.

[0167]

[0168] (II)

[0169] In formula (II), L5 is -(CH2)3- and R3 is -COOH; or L5 is -(CH2)3- and R3 is -OH; or L5 is -(CH2)4- and R3 is -OH.

[0170] The structural formula of the polymer is shown below:

[0171]

[0172] (Ib1)

[0173]

[0174] (Id1)

[0175]

[0176] (Ih1)

[0177] The molecular weights of the polymers are shown in Table 2 below.

[0178] Table 2 Polymers with different grafted monomers

[0179]

[0180] Example 3: In vitro bone-targeting ability of polymers with different alendronate contents

[0181] Polymers with different alendronate contents (25 mg) prepared in Example 1 were dissolved in deionized water (5 mL), and fluorescein isothiocyanate (FITC) dissolved in 100 μL DMF (1 mg) was added to the polymer solutions. The mixtures were allowed to react in the dark at 25°C for 24 hours. The reaction mixture was then dialyzed against acidic water (pH ~5, refreshed 6 times over 3 days) in the dark (molecular weight cutoff 3.5 kDa). After freeze-drying, the fluorescently labeled polymers were collected as yellow solids. The FITC-labeled bone-targeting polymers and FITC-labeled non-targeting polymers were dissolved in deionized water to a concentration of 0.2 mg / mL, and the initial fluorescence intensity (IL) was measured using a multi-mode microplate reader (excitation wavelength = 485 ± 20 nm, emission wavelength = 535 ± 20 nm). t0 Then, hydroxyapatite powder (10.0 mg) was added to 1.50 mL of FITC-labeled polymer solution, and the mixture was stirred continuously at 37 °C for 6.0 h. After centrifugation, the fluorescence intensity (Ig) of the supernatant was measured. t The binding rate of the polymer to hydroxyapatite (HAP) is calculated using the following formula: HAP binding rate = (I t0 -I t ) / I t0 ×100%. The binding rate of pABOL to hydroxyapatite was 69.04%, the binding rate of the polymer with 5% alendronate to hydroxyapatite was 84.34%, the binding rate of the polymer with 10% alendronate to hydroxyapatite was 86.1%, and the binding rate of the polymer with 20% alendronate to hydroxyapatite was 87.18%. A comparison of the in vitro bone-targeting capabilities of polymers with different alendronate contents is shown in the attached graph. Figure 1 As shown.

[0182] Example 4: Preparation of nanocomposites by combining bone-targeting polymers with different nucleic acids

[0183] Before the experiment, prepare a HEPES buffer containing 20 mM HEPES, 5.0 wt% glucose, and pH=7 using RNase-free ultrapure water, and sterilize it by filtration through a 0.22 μm PES filter. Prepare polymer stock solutions of 1.0 μg / μL RNA and 10 μg / μL using RNase-free ultrapure water, and dilute to the appropriate concentrations with different volumes of HEPES buffer as needed during titration. Taking the preparation of a nanocomplex with a polymer / miRNA mass ratio of 20 / 1 (μg / μg) and a final miRNA concentration of 100 μg / mL as an example: Add 10 μL of miRNA stock solution to a 2.0 mL sterile centrifuge tube, add 50 μL of pH=7 HEPES buffer to dilute, and mix thoroughly to obtain the miRNA buffer. Add 20 μL of polymer stock solution to a 2.0 mL sterile centrifuge tube, add 20 μL of pH=7 HEPES buffer, and mix thoroughly to obtain the polymer buffer. Then, miRNA buffer was added to polymer buffer at a drip rate of 160 μL / min using a syringe pump, with magnetic stirring maintained at 1200 r / min during the addition. After the addition was complete, stirring was continued for 30 min until a stable polymer / miRNA nanocomposite was formed. The nanocomposite, with pH=7 and a ratio of 10% ALN-pABOL / miRNA=15 / 1 (w / w), was dropped onto one side of a carbon film on a copper mesh and dried at room temperature. It was then stained with 1% phosphotungstic acid solution, air-dried, and observed under a transmission electron microscope. The results are shown in the attached figure. Figure 2 As shown, the morphology of the nanocomposite is spherical particles with a particle size of approximately 200 nm. The DLS characterization results of the nanocomposite are shown in Table 3 below. The DLS particle size distribution curves for the nanocomposite at pH=7 and 10% ALN-pABOL / miRNA=15 / 1 (w / w) are attached. Figure 3 As shown, the hydrodynamic diameter (D) h The wavelength is 212.2 nm, and the polydispersity (PD) is 0.006.

[0184] Table 3. Parameter settings and DLS characterization results during the preparation of nanocomposites

[0185]

[0186] Example 5: In vitro stability and GSH responsiveness of bone-targeting polymer / miRNA complex

[0187] The nanocomposite, with pH=7 and 10% ALN-pABOL / miRNA=20 / 1 (w / w), was stored in HEPES buffer (20 mM HEPES, 5 wt% glucose, pH 7.0) at 4 °C. The hydrodynamic diameter (D) was determined using dynamic light scattering (DLS).h ) and polydispersity (PD), every other day. Results are attached. Figure 4 As shown, after the nanocomposite was stored at 4°C for two weeks, its hydrodynamic diameter (D) decreased. h The nanometer diameter (nm) remains below 300 nm, and the polydispersity (PD) remains below 0.2, indicating that it has good in vitro stability.

[0188] A 10% ALN-pABOL / miRNA = 20 / 1 (w / w) nanocomplex with a miRNA concentration of 10 μg / mL was prepared in HEPES buffer containing 20 mM HEPES, 5.0 wt% glucose, and pH=7. 20 μL of 100 mM reduced glutathione aqueous solution was added to 180 μL of the nanocomplex solution, and the hydrodynamic diameter (D) of the 10% ALN-pABOL / miRNA nanocomplex was measured over 5 hours at 25 °C using dynamic light scattering (DLS). h The variation of polydispersity (PD) over time was studied. Results are attached. Figure 5 As shown, the hydrodynamic diameter (D) of the nanocomposite decreased within 5 hours after the addition of a reduced glutathione aqueous solution. h The nanometer diameter (N) rises above 1000 nm, and the polydispersity (PD) rises above 0.3, indicating that it has good GSH responsiveness.

[0189] Example 6: Cellular uptake of bone-targeting polymer / miRNA complex

[0190] Nanocomplexes with a miRNA concentration of 1000 nM were prepared in HEPES buffer (20 mM HEPES, 5 wt% glucose aqueous solution, pH=7) using PEI, pABOL, 10% ALN-pABOL, 20% ALN-pABOL, and 5-carboxyfluorescein (5-FAM, excitation / emission wavelength 492 / 520 nm) labeled FAM-miRNA at a mass ratio of 20:1. Before transfection, MDA-MB-231 human breast cancer cells were cultured at 6 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in 100 μL of complete medium for 24 hours. The medium was then changed, and each well was washed three times with 100 μL of PBS. 10 μL of polymer / miRNA complex buffer was mixed thoroughly with 90 μL of serum-free and antibiotic-free medium and added to the 96-well plates. Cells were co-cultured for 3 hours. After the appropriate transfection time, the medium was removed, and each well was washed three times with 100 μL of PBS. The cells were then analyzed using a fluorescence microplate reader. The results are attached. Figure 6As shown in the results, the uptake efficiency of MDA-MB-231 human breast cancer cells after co-culturing with different nanocomplexes for 3 hours was higher than that of commercially available nanocomplexes formed by PEI and miRNA, demonstrating high uptake efficiency.

[0191] Example 7: Cytotoxicity of bone-targeting polymer / miRNA complex

[0192] Cytotoxicity of bone-targeting polymer / miRNA complexes was tested: PEI / miRNA, Lipo / miRNA, pABOL / miRNA, and 10% ALN-pABOL / miRNA nanocomplex solutions were prepared in HEPES buffer at optimal ratios using a 50 / 50 dilution method. MDA-MB-231 human breast cancer cells or MC3T3-E1 osteoblasts were inoculated at 8.0 × 10⁻⁶ cells / mL. 3 Cells were seeded per well in 96-well plates and cultured in 100 μL of DMEM or α-MEM complete medium for 24 hours. Afterward, the medium was changed, and 10 μL of different concentrations of different nanocomposite solutions and 90 μL of serum-free and antibiotic-free medium were added to each well. Three replicates were set for each concentration of each material, and the cells were co-cultured for 8 hours, after which the medium was changed again. 10 μL of CCK-8 chromogenic solution and 100 μL of fresh complete medium were added to each well, and the cells were cultured for another 2-4 hours. The absorbance at 450 nm was measured using a microplate reader. Cells not co-cultured with the nanocomposite solution served as a control group. The cytotoxicity of the materials was characterized by the relative cell viability of each group of cells. Relative cell viability can be calculated using the following formula, where A... s A represents the absorbance of the experimental well. c A represents the absorbance of the control well. b The absorbance of the culture medium in wells containing CCK-8 represents the absorbance of uninoculated cells. The results showed that the 10% ALN-pABOL / miRNA=20 / 1 (w / w) nanocomposite did not exhibit cytotoxicity against MDA-MB-231 human breast cancer cells or MC3T3-E1 osteoblasts (test data are shown below). Figure 7 and Figure 8 (As shown).

[0193] Relative cell viability = ×100%

[0194] Example 8: In vitro transfection of bone-targeting polymer / mRNA complex

[0195] The in vitro transfection efficiency of the bone-targeting polymer / mRNA complex was tested: A nanocomplex solution with an mRNA concentration of 1000 nM was prepared in HEPES buffer (20 mM HEPES, 5 wt% glucose aqueous solution, pH 7.4) using PEI, pABOL, 10% ALN-pABOL, and eGFP mRNA (eGFP, excitation / emission wavelength 488 / 507 nm) in the optimal ratio. One day before transfection, HEK-293T human embryonic kidney cells were transfected at 8.0 × 10⁻⁶ mM. 3 Cells / well were seeded in 96-well plates and cultured in 100 μL of DMEM complete medium for 24 hours. Afterward, the medium was changed, and 10 μL of nanocomposite solution and 90 μL of serum-free and antibiotic-free medium were added to each well. Three replicates were set up for each material. After a total of 4 hours of culture, the medium was replaced with 100 μL of fresh complete medium, and cultured for another 24 hours. Transfection was then observed under a fluorescence microscope. Fluorescence imaging is attached. Figure 9 As shown, from left to right, the transfection efficiencies of PEI / mRNA, pABOL / mRNA, and 10% ALN-pABOL / mRNA nanocomplexes are as follows. The results indicate that both pABOL / mRNA and 10% ALN-pABOL / mRNA nanocomplexes exhibit similar high in vitro transfection efficiencies to the commercially available transfection reagent PEI.

[0196] Example 9: Effects of bone-targeting polymer / miRNA complex on cell proliferation

[0197] A nanocomposite with a miRNA concentration of 1000 nM was prepared in HEPES buffer (20 mM HEPES, 5 wt% glucose aqueous solution, pH 7.4) using a polymer containing 10% alendronate at a mass ratio of 20:1 to miRNA. MDA-MB-231 human breast cancer cells and MC3T3-E1 osteoblasts were then cultured at 8.0 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of [number] cells / well in 96-well plates (at least 3 replicates per group), and cultured in 100 μL DMEM medium for 24 hours, followed by medium replacement. Each well was washed three times with 50 μL PBS. 10 μL of bone-targeting polymer / miRNA complex buffer or PBS was mixed thoroughly with 90 μL of serum-free and antibiotic-free medium and added to the 96-well plate. After co-culturing for 8 hours, the medium was changed, and each well was washed three times with 50 μL PBS. 100 μL of complete DMEM medium was added. CCK-8 was added at 24, 48, 72, and 96 hours of culture. The absorbance of cells at 450 nm was monitored over time using a microplate reader. All CCK-8 assays were performed triplicate to assess cell viability. Results are attached. Figure 10 and 11As shown, the results indicated that the 10% ALN-pABOL / miRNA complex significantly inhibited the proliferation of MDA-MB-231 human breast cancer cells, but had no significant effect on the proliferation of MC3T3-E1 osteoblasts.

[0198] Example 10: In vivo bone-targeting capability of the bone-targeting polymer / miRNA complex

[0199] A nanocomplex with a miRNA concentration of 100 μg / ml was prepared in HEPES buffer (20 mM HEPES, 5 wt% glucose aqueous solution, pH 7.4) using a 10% molar percentage of alendronate polymer and Cy5-labeled miRNA at a mass ratio of 20:1. Two 5-week-old female BALB / c nude mice of similar weight were intravenously injected with 100 μL of the pABOL / miRNA complex and 10% ALN-pABOL / miRNA complex, respectively. In vivo imaging (IVIS) was used to monitor the mice at 1 h, 4 h, 24 h, and 48 h post-administration. At 48 h, tissues from major organs (heart, liver, spleen, lung, kidney, tibia, femur, and spine) of both mice were collected for IVIS imaging. The test results are attached. Figure 12 As shown, the results indicate that the 10% ALN-pABOL / miRNA complex exhibits significantly better bone-targeting performance compared to the pABOL / miRNA complex.

[0200] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

[0201] Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and all such modifications and variations are within the protection scope of the present invention.

Claims

1. A polymer or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, The polymer has the structural formula shown in formula (I): ； In the formula: R' is selected from L 1 Optional substitution of C 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 Alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 8-membered cycloalkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms; R'' is selected from Optional substitution of C 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 Alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 8-membered cycloalkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms; M does not exist or is -SS-; R 1 R 2 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group; R''' is selected from -L 5 -R 3 ; R 3 Yes - OR 11 -COOR 11 -SO2OR 11 (OCH2CH2) m OH, or NR 11 R 12 ; R 11 and R 12 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 Cycloalkyl, optionally substituted 3-8 membered heterocycloalkyl, optionally substituted C 6-12 Aryl or optionally substituted 5-10-membered heteroaryl groups, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl groups may be randomly interrupted by one or more heteroatoms; m is an integer between 1 and 10; R is selected from L 4 Optional substitution of C 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 Alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 8-membered cycloalkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms; Among them, L 1 To L 5 Each is an optional substitution of C. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms; The range of y / x+y is greater than 0 and less than or equal to 0.

5.

2. The polymer as claimed in claim 1, or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, R' is selected from L 1 ; R'' is selected from ; M does not exist or is -SS-; R 1 R 2 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group; R''' is selected from -L 5 -R 3 ; R 3 Yes - OR 11 -COOR 11 -SO2OR 11 (OCH2CH2) m OH, or NR 11 R 12 ; R 11 and R 12 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkylene or alkyleneyne may be randomly interrupted by one or more heteroatoms; m is an integer between 1 and 10; R is selected from L 4 ; Among them, L 1 To L 5 Each is an optional substitution of C. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group may be randomly interrupted by one or more heteroatoms; The range of y / x+y is greater than 0 and less than or equal to 0.

5.

3. The polymer as claimed in claim 2, or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, The L 1 To L 4 Each is an optional substitution of C. 1-6 Alkyl, optionally substituted C 1-6 alkenyl, optionally substituted C 2-6 Alkyne group, optionally substituted 3 to 8-membered heterocyclic alkyl group.

4. The polymer as claimed in claim 2, or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, The L 5 C is an optional substitute 2-8 Alkyl, optionally substituted C 2-8 alkenyl or optionally substituted C 2-6 Alkynyl groups, wherein adjacent carbon atoms in alkyl, alkenyl, or alkynyl groups may be randomly interrupted by one or more heteroatoms.

5. The polymer as claimed in claim 1, or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, R 1 R 2 It is independently selected from hydrogen.

6. The polymer as claimed in claim 1, or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, R 3 It is -OH, -COOH, -SO3H, -NH2, -N(CH3)2, -N(CH2CH3)2.

7. The polymer as claimed in claim 2, or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, The structure of formula (Ⅰ) is selected from the following structural formula: (Ιa) (Ib) (IC) (ID) (Ie) (1f) (Ig) (Ιh) (Ιi) The range of y / x+y is greater than 0 and less than or equal to 0.

5.

8. The polymer or pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph as described in any one of claims 1-7, characterized in that, The polymer represented by formula (Ⅰ) has an average molecular weight greater than 5 kg / mol and less than 150 kg / mol.

9. The polymer of claim 7 or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer, or polymorph thereof, characterized in that, The polymer represented by formula (Ⅰh) has an average molecular weight of at least 5.5 kg / mol.

10. A method for preparing a salt of the polymer as described in formula (Ij) or formula (Ik), characterized in that, The preparation steps include: adding N,N'-(propane-1,3-diyl)acrylamide or N,N'-bis(acryloyl)cysteine, compound of formula (II), alendronic acid, and triethylamine to a flask equipped with a stir bar, and adding a mixed solvent of methanol and water to the flask; then reacting in a dark environment at 40-60°C under a nitrogen atmosphere; reacting for 5 to 14 days to produce a high-viscosity solution, taking aliquots at predetermined time intervals for testing to monitor conversion and molar mass; once the target molar mass is reached, the reaction is stopped by dilution with MeOH; then acidifying the diluted reaction mixture to an acidic environment with HCl, then purifying by dialysis with acidic water, then lyophilizing the purified product, and collecting its hydrochloride form as a solid after lyophilization; Where y / x+y is greater than 0 and less than or equal to 0.5; The structural formula of compound (II) is as follows: ； Formula (II) L 5 C is an optional substitute 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted 3- to 8-membered heterocyclic alkyl, optionally substituted 3- to 8-membered heterocyclic alkenyl, optionally substituted C 6-12 Aryl, optionally substituted 5 to 10-membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or ynyl group are randomly interrupted by one or more heteroatoms; R 3 Yes - OR 11 -COOR 11 -SO2OR 11 (OCH2CH2) m OH, or NR 11 R 12 ; R 11 and R 12 Each is independently hydrogen, and each C is optionally substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-6 Cycloalkyl, optionally substituted 3-8 membered heterocycloalkyl, optionally substituted C 6-12 aryl or optionally substituted 5-10-membered heteroaryl groups, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl groups are optionally interrupted by one or more heteroatoms; m is an integer between 1 and 10.

11. A bone-targeting nucleic acid delivery vector, characterized in that, The polymer, composition, salt, solvate, tautomer or polymorph thereof, comprising any one of claims 1-9.

12. A nanocomposite, characterized in that, It comprises the polymer or pharmaceutically acceptable complex of any one of claims 1-9, a composition, salt, solvate, tautomer or polymorph, and nucleic acid.

13. The nanocomposite according to claim 12, characterized in that, The nucleic acid is selected from at least one of DNA, RNA, and plasmid DNA / RNA hybrid sequences.

14. The nanocomposite according to claim 13, characterized in that, The RNA is selected from at least one of spontaneous RNA, microRNA, short interfering RNA, short hairpin RNA, and self-amplifying RNA.

15. The nanocomposite according to claim 13, characterized in that, The RNA in question is microRNA.

16. The nanocomposite according to any one of claims 12-15, characterized in that, The weight ratio of the polymer to nucleic acid is between 5:1 and 100:

1.

17. A nanoparticle, characterized in that, It comprises the polymer, pharmaceutically acceptable complex, composition, salt, solvate, tautomer or polymorph, or nanocomposite of any one of claims 1-9.

18. The nanoparticles as described in claim 17, characterized in that, The nanoparticles have a hydrodynamic average diameter D of less than 1000 nanometers. h ; 19. The nanoparticles as described in claim 18, characterized in that, The nanoparticles have a hydrodynamic average diameter D of less than 200 nanometers. h .

20. A pharmaceutical composition, characterized in that, Includes polymers or pharmaceutically acceptable complexes thereof as described in any one of claims 1-9, compositions, salts, solvates, tautomers or polymorphs, salts of polymers prepared by the preparation method of claim 10, bone-targeting nucleic acid delivery carriers as described in claim 11, nanocomposites as described in any one of claims 12-16, and nanoparticles as described in any one of claims 17-19.

21. The pharmaceutical composition of claim 20, characterized in that, It also includes the drug to be delivered and pharmaceutically acceptable excipients.

22. The use of the polymer of any one of claims 1-9 or a pharmaceutically acceptable complex, composition, salt, solvate, tautomer or polymorph thereof, salt of the polymer prepared by the method of claim 10, bone-targeting nucleic acid delivery carrier of claim 11, nanocomplex of any one of claims 12-16, and nanoparticle of any one of claims 17-19 in the preparation of medicaments for stimulating and enhancing protective immune responses, gene therapy medicaments, or medicaments for preventing, improving or treating bone-related diseases.

23. The use of the polymer or pharmaceutically acceptable complex, composition, salt, solvate, tautomer or polymorph of any one of claims 1-9, a salt of the polymer prepared by the method of claim 10, the bone-targeting nucleic acid delivery carrier of claim 11, the nanocomposite of any one of claims 12-16, or the nanoparticle of any one of claims 17-19 in the preparation of medicaments for stimulating and enhancing protective immune responses, gene therapy medicaments, or medicaments for preventing, improving, or treating bone-related diseases, characterized in that, The bone-related disease is at least one of osteoporosis, osteoarthritis, or bone tumor.

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