A novel boron liposome to facilitate diagnosis and treatment

Abstract

The present disclosure provides a carborane-phospholipid conjugate which incorporates a carborane group at the end of one of the lipid arms of a phospholipid. The present disclosure also provides liposomal compositions comprising such carborane-phospholipid conjugates and their use for the delivery of at least one therapeutic agent and / or at least one diagnostic agent.

Description

Technical Field

[0001] This disclosure relates to the medical and diagnostic fields, specifically to a novel boron liposome that can aid in diagnosis and treatment. Background Technology

[0002] Li J, Tu Z, Liu Z, "Exploring the Niche Areas of Dawn: A Brief Overview of the Development of Boron Carriers," *Science in China: Chemistry*, 2020, Vol. 50, pp. 1296-1319, reviews boron carriers used in boron neutron capture therapy (BNCT). BNCT is a binary, cellular-scale, highly targeted radiotherapy with high energy density, exhibiting excellent killing effects against locally invasive malignancies such as melanoma, glioma, and recurrent head and neck cancer. The cytotoxicity of BNCT originates from non-radioactive isotopes. 10 After B captures a low-energy thermal neutron, a nuclear fission reaction occurs, producing alpha particles and recoil. 7 Li nuclei, with their high linear energy transfer and relatively high bioavailability, can cause lethal damage to cells; however, their limited penetration (5–9 μm, approximately the diameter of a cell) confines their destructive power to the single-cell scale. If a large number of... 10 Boolean atoms can selectively accumulate in tumor cells and absorb enough neutrons to maintain lethality. 10 B(n, α) 7 In the case of Li capture reaction, BNCT will have a significant killing effect on malignant tumors with almost no impact on nearby normal tissues. A key aspect of an effective therapy for malignant tumors is the delivery of sufficient boron to the tumor site via neutron irradiation.

[0003] Currently, 4-dihydroxyboron-L-phenylalanine (BPA) is a widely used boron delivery agent, but it suffers from insufficient boron content, limiting its efficacy. Using unusual boranes can sometimes lead to biotoxicity and immunogenicity. Furthermore, liposome encapsulation and delivery of drugs faces the problem of easily broken encapsulation structures. In actual treatment, doctors also need to make timely diagnoses, but current BNCT drugs have limited functions, often only meeting therapeutic needs, which restricts their clinical application. Against this backdrop, we hope to find new raw materials to synthesize a molecule with a novel structure to solve the above problems. Summary of the Invention

[0004] One aspect of this disclosure provides a carborane-phospholipid conjugate of formula (I),

[0005] in:

[0006] In R1 and R2, one is an -L-carboronic alkyl group, and the other is a straight-chain alkyl or alkenyl group with 10-24 carbon atoms; L is a divalent linker; X is -H, -CH2CH2NH3, or -CH2CH2N. + (CH3)3, -CH2CH(NH2)COOH, -CH2CH(OH)CH2OH, or cyclohexane-2,3,4,5,6-pentahydroxy-1-yl; Y is OH or O. - .

[0007] In one embodiment of this disclosure, the carboroalkyl group in the -L-carboalkyl group is 10 B-rich.

[0008] In one embodiment of this disclosure, X is -CH2CH2N + (CH3)3; Y is O - .

[0009] In a preferred embodiment of this disclosure, the two lipid arms comprising R1 or R2 respectively satisfy one or more of the following conditions: the length difference is within ±1 Å; the distance between the lipid arms is within 5 Å; and the dihedral angle is within 5 degrees. In a more preferred embodiment of this disclosure, the two lipid arms comprising R1 or R2 respectively simultaneously satisfy any two of these three conditions. In an even more preferred embodiment of this disclosure, the two lipid arms comprising R1 or R2 respectively simultaneously satisfy all three conditions.

[0010] In one embodiment of this disclosure, the carborane alkyl group is 1,2-C2B4H5-, 1,2-C2B8H9-, or 1,2-C2B 10 H 11 -,2,3-C2B4H7-,7,8-C2B9H 12 -or 5,6-C2B8H 11 - The preferred option is 1,2-C2B 10 H 11 -

[0011] In one embodiment of this disclosure, L is -(CH2). n+m -、-(CH2) n+m -S-、-(CH2) n+m -O-、-(CH2) n -CH = CH-(CH2) m -、-(CH2) n -CH = CH-(CH2) m -S-、-(CH2) n -CH = CH-(CH2) m -O-、-(CH2) n -S-(CH2)m -、-(CH2) n -S-(CH2) m+1 -S-、-(CH2) n -O-(CH2) m -、-(CH2)nO-(CH2) m+1 -O-、-(CH2) n -N(CH3)-(CH2) m -or-(CH2) n -N(CH3)-(CH2) m+1 -N(CH3)-, where n is an integer from 1 to 20 and m is an integer from 0 to 20.

[0012] For example, the carborane-phospholipid conjugate of formula (I) has the following structure

[0013]

[0014] Where Bo represents a carborane alkyl group, and n is an integer from 1 to 20. In one embodiment, Bo is 1,2-C2B. 10 H 11 - Preferably, it is a closed-dicarbododecanoyl group, more preferably a closed-1,2-dicarbododecanoyl group; n is 3, 7, 11 or 15, preferably 11.

[0015] Another aspect of this disclosure provides a liposome composition comprising the above-described carborane-phospholipid conjugate, wherein the conjugate is part of a lipid bilayer.

[0016] In one embodiment of this disclosure, the liposome composition further comprises cholesterol.

[0017] In one embodiment of this disclosure, the liposome composition further comprises phospholipids, preferably dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylglycerol (DMPG), distearyl phosphatidylglycerol (DSPG), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylserine (DMPS), distearyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylserine (DPPS), dioleoyl phosphatidyl ethanolamine (DOPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphatidyl ethanolamine (DMPE), distearyl phosphatidyl ethanolamine (DSPE), or ditransoleoyl phosphatidyl ethanolamine, more preferably DPPC.

[0018] In one embodiment of this disclosure, the liposome composition further comprises PEGylated phospholipids, such as DLPE-PEG, DPPE-PEG, DMPE-PEG or DSPE-PEG, preferably DSPE-PEG.

[0019] In one embodiment of this disclosure, the liposome composition further comprises at least one therapeutic agent and / or at least one diagnostic agent.

[0020] In a preferred embodiment, the liposome composition comprises at least one therapeutic agent, which is an anticancer drug. For example, the anticancer drug is selected from methylaurestatin E, methylaurestatin F, ibrutinib, acalabrutinib, zanubrutinib, doxorubicin, mitomycin-C, mitomycin-A, daunorubicin, aminopterin, actinomycin, bleomycin, 9-aminocamptothecin, N8-acetylspermethylene, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, yunnanycin, gemcitabine, cytarabine, dolalastatin, dacarbazine, 5-fluorouracil; paclitaxel, docetaxel, gemcitabine, cytarabine, 6-mercaptopurine, vincristine, cisplatin, oxaliplatin, and PARP inhibitors. In one embodiment, the anticancer drug is selected from PARP inhibitors, preferably olaparib, niraparib, rucaparib, fluzoparib, pamiparib, veriparib, tapazanib, and more preferably olaparib.

[0021] In a preferred embodiment of this disclosure, the liposome composition comprises at least one diagnostic agent, said diagnostic agent being 64 Cu-NOTA-PEG2000-DSPE.

[0022] Another aspect of this disclosure provides the use of the above-described liposome composition in a pharmaceutical.

[0023] Another aspect of this disclosure provides the use of the above-described liposome composition for delivering at least one therapeutic agent and / or at least one diagnostic agent. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit the invention.

[0025] Figure 1 Schematic diagram of the structure of novel boron liposomes and drug encapsulation strategies;

[0026] Figure 2Changes in tumor volume in mice under boron liposome encapsulation of different drugs (Dox: doxorubicin; PARPi: olaparib, a polyadenosine diphosphate ribose polymerase (PARP) inhibitor; N represents neutron irradiation).

[0027] Figure 3 Imaging of boron liposomes using transmission electron microscopy (TEM);

[0028] Figure 4 4T1 tumor-bearing mice were injected intravenously 64 Whole-body maximum density projection PET image 12 hours after Cu-NOTA-boron liposome administration. Tumors are circled in white dashed lines;

[0029] Figure 5 Cross-section of the BoP-1 membrane structure as simulated by molecular dynamics;

[0030] Figure 6 Cross-section of the BoP-2 membrane structure as simulated by molecular dynamics;

[0031] Figure 7 Cross-section of the BoP-3 membrane structure as simulated by molecular dynamics.

[0032] Figure 8 Cross-section of the BoP-4 membrane structure as simulated by molecular dynamics;

[0033] Figure 9 Cross-section of DPPC membrane structure from molecular dynamics simulation;

[0034] Figure 10 UV-vis absorption of BoPs and DPPC was measured under conditions of SRB encapsulation.

[0035] Figure 11 The percentage of SRB leakage was determined after culturing in 50% bovine serum albumin at 37°C for 24 hours.

[0036] Figure 12 Boron uptake in 4T1 cells incubated with different concentrations of boron liposomes for 24 hours;

[0037] Figure 13 Cell viability of 4T1 cells incubated with different concentrations of boron liposomes for 24 hours;

[0038] Figure 14 Mean tumor volume of mice in the PARP inhibitor-boron liposome and PARP inhibitor-boron liposome + N treatment groups (n=9) (paired t-test, **p<0.01);

[0039] Figure 15 Mean body weight of mice in each group treated with boron liposomes + N or PARP inhibitor-boron liposomes + N (n=9). Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the described embodiments of this disclosure without creative effort are within the scope of protection of this invention.

[0041] This invention may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this invention can be combined with technical features of any or more other embodiments to obtain further embodiments. This invention includes such combinations to obtain further embodiments.

[0042] Unless otherwise defined, the technical or scientific terms used in this disclosure shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0043] As used in this disclosure, the words “including,” “containing,” or “comprising” or similar terms mean that the element preceding the word covers the element listed after the word and its equivalents, without excluding other elements.

[0044] Unless otherwise indicated in the working embodiments or elsewhere, all figures set forth in the specification and claims expressing the amount of material, reaction conditions, duration, and quantitative properties of the material shall be understood to be modified by the term “about” in all cases. It should also be understood that any range of numbers listed in this application is intended to include all subranges within that range and any combination of the endpoints of that range or subranges; for example, integers 1-20 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and also include subranges 1-3, 1-4, 1-10, 2-4, 2-10, etc.

[0045] This disclosure should be interpreted as consistent with the laws and principles of chemical bonding. In some cases, it may be necessary to remove a hydrogen atom to accommodate a substituent at a given position.

[0046] In this disclosure, phospholipids refer to glycerophospholipids, which are derivatives of fatty acids (e.g., decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, erucic acid), glycerol, and phosphoric acid, wherein two hydroxyl groups in glycerol are replaced by fatty acids, and the other hydroxyl group is replaced by phosphoric acid and other groups. A hydrophilic phosphate group and two hydrophobic lipid arm tails are linked together by the glycerol molecule. The phospholipids mentioned in this disclosure can be natural or synthetic. Exemplary phospholipids include, but are not limited to, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol.

[0047] In this application, carborane-phospholipid conjugates refer to phospholipid derivatives in which a carborane alkyl group is introduced at the end of one lipid arm of a phospholipid.

[0048] One aspect of this disclosure provides a carborane-phospholipid conjugate of formula (I),

[0049] in:

[0050] In R1 and R2, one is an -L-carboronic alkyl group, and the other is a straight-chain alkyl or alkenyl group with 10-24 carbon atoms; L is a divalent linker; X is -H, -CH2CH2NH3, or -CH2CH2N. + (CH3)3, -CH2CH(NH2)COOH, -CH2CH(OH)CH2OH, or cyclohexane-2,3,4,5,6-pentahydroxy-1-yl; Y is OH or O. - .

[0051] The carborane-phospholipid conjugate disclosed herein introduces carborane into one lipid arm of a phospholipid, and its structure is similar to that of cellular phospholipids, thus exhibiting good compatibility with cells.

[0052] Carboranes are a class of polyhedral boron-carbon cluster compounds. In this application, carborane alkyl refers to the group formed by losing a hydrogen atom from the CH bond of a carborane.

[0053] In one embodiment of this disclosure, a carborane alkyl group is obtained by losing a hydrogen atom from the CH bond of the carborane as follows:

[0054] In other words, carborane alkyl groups are 1,2-C2B4H5-, 1,2-C2B8H9-, or 1,2-C2B 10 H 11 -,2,3-C2B4H7-,7,8-C2B9H 12 -or 5,6-C2B8H 11 -

[0055] In a preferred embodiment, the carborane is obtained by losing hydrogen atoms from the CH bond of a carborane containing 8-11 boron atoms. In a more preferred embodiment, the carborane is obtained from dicarba-closo-dodecaborane (C2B). 10 H 12 The CH bond of ) loses a hydrogen atom to obtain 1,2-C2B. 10 H 11 -

[0056] In this application, the boron atoms in carborane / carboranealkyl groups can be composed of naturally abundant boron, or they can be... 10 B-enriched. In one embodiment, one or more boron atoms in the carborane / carboranealkyl group are... 10 B-enriched, such as carborane / carborane-alkyl 10 B abundance is higher than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, or 97%, and even higher than 98% or 99%. 10 B-enriched carboranes can be used 10 Boric acid enriched with B can be prepared from raw materials or can be commercially available from Katchem spol.s ro, Czech Republic (http: / / www.katchem.cz / en).

[0057] In one embodiment, at least one boron atom in the carborane / carboranealkyl group is 10 B.

[0058] In one embodiment, the boron atom in the carborane-phospholipid conjugate is 10 B-enriched. For example, at least one boron atom in carborane-phospholipid conjugates is... 10 B.

[0059] In one implementation, 10 One or more boron atoms in the conjugate of general formula (I) enriched by B 10 The abundance of boron is greater than 30%, 40%, 50%, 60%, 70%, or 80%, preferably greater than 90%, 95%, 96%, or 97%, and even more preferably greater than 98% or 99%. It should be understood that the abundance of each boron atom... 10 The abundance of B can be compared with that of other boron atoms. 10 The abundance of B may be the same or different.

[0060] In one implementation, 10 Each boron atom in the conjugate of general formula (I) enriched by B 10 The abundance of B is higher than 90%.

[0061] Where X is -H, -CH2CH2NH3, or -CH2CH2N. + (CH3)3, -CH2CH(NH2)COOH, -CH2CH(OH)CH2OH, or cyclohexane-2,3,4,5,6-pentahydroxy-1-yl carborane-phospholipid conjugates are derivatives of phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, respectively.

[0062] In one implementation, X is -CH2CH2N + (CH3)3; Y is O - That is, carborane-phospholipid conjugates are phosphatidylcholine derivatives.

[0063] In this application, the length difference between lipid arms refers to the distance from the carboxyl C of the R1 head to the non-H atom at the very end of the R1 tail, minus the distance from the carboxyl C of the R2 head to the non-H atom at the very end of the R2 tail.

[0064] In this application, the distance between lipid arms is the distance between the first C of R1 near the glycerol group and the first C of R2 near the glycerol group in the compound of general formula (I).

[0065] In this application, the dihedral angle between lipid arms refers to the dihedral angle formed by the non-H atom at the very end of the R1 tail, the carboxyl C at the head of R1, the carboxyl C at the head of R2, and the non-H atom at the very end of the R2 tail.

[0066] In one embodiment of the carborane-phospholipid conjugate disclosed herein, the two lipid arms comprising R1 or R2 respectively satisfy one, two, or three of the following conditions:

[0067] The length difference is within ±3A;

[0068] The distance between lipid arms is within 15 Å;

[0069] The dihedral angle is within 15 degrees.

[0070] In one embodiment of the carborane-phospholipid conjugate disclosed herein, the two lipid arms comprising R1 or R2 respectively satisfy one, two, or three of the following conditions:

[0071] The length difference is within ±2A;

[0072] The distance between lipid arms is within 10 Å;

[0073] The dihedral angle is within 10 degrees.

[0074] In a preferred embodiment of the carborane-phospholipid conjugate disclosed herein, the two lipid arms comprising R1 or R2 respectively satisfy one, two, or three of the following conditions: the length difference is within ±1 Å; the distance between the lipid arms is within 5 Å; and the dihedral angle is within 5 degrees. In a more preferred embodiment of the carborane-phospholipid conjugate disclosed herein, all three conditions are preferably satisfied simultaneously.

[0075] One of R1 and R2 is a straight-chain alkyl or alkenyl group having 10-24 carbon atoms. In one embodiment, the straight-chain alkyl or alkenyl group comprises 11, 13, 15, 17, 19, 21, or 23 carbon atoms.

[0076] As used in this article, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon chain group consisting of only n carbon atoms and 2n+1 hydrogen atoms. For example, C 10 -C 24 Alkyl groups are saturated hydrocarbon chain groups with straight or branched chains having 10 to 24 carbon atoms. Numerical ranges such as "10 to 24" refer to every integer within the given range; for example, "10 to 24 carbon atoms" means that the alkyl group contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Straight-chain alkyl groups are unbranched alkyl groups, such as decyl (C10-C20). 10 H 21 -), Laureth (C 12 H 25 -), Myristyl (C 14 H 29 -), palmityl (C) 16 H 33 -), stearyl group (C 18 H 37 -), eicosyl (C 20 H 41 -), docosyl (C 22 H 45 -) or tetraalkyl (C 24 H 49 -).

[0077] In this application, alkenyl refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms and containing at least one double bond (e.g., one or two double bonds). Examples include cis-9-tetradecenyl, trans-9-tetradecenyl, cis-9-hexadecenyl, trans-9-hexadecenyl, cis-9-octadecenyl, trans-9-octadecenyl, cis-11-octadecenyl, cis,cis-9,12-octadecadienyl, trans,trans-9,12-octadecadienyl, cis-13-docodecenyl, etc. Straight-chain alkenyl refers to an alkenyl group without branches.

[0078] L is a divalent linker. In a preferred embodiment, L is a group having a flexible hydrophobic chain. In a more preferred embodiment, L has hydrophobicity and flexibility comparable to the lipid arm in a carborane-phospholipid conjugate that does not contain carborane. For example, L is -(CH2). n+m -、-(CH2) n+m -S-、-(CH2) n+m -O-、-(CH2) n -CH = CH-(CH2) m -、-(CH2) n -CH = CH-(CH2) m -S-、-(CH2) n -CH = CH-(CH2) m -O-、-(CH2) n -S-(CH2) m -、-(CH2) n -S-(CH2) m+1 -S-、-(CH2) n -O-(CH2) m -、-(CH2)nO-(CH2) m+1 -O-、-(CH2) n -N(CH3)-(CH2) m -or-(CH2) n -N(CH3)-(CH2) m+1 -N(CH3)-, where n is an integer from 1 to 20, and m is an integer from 0 to 20. In one specific implementation, L is -(CH2). n+m -S-, where n is an integer from 1 to 20, and m is an integer from 0 to 20. For example, L is -(CH2). 12 -S-、-(CH2) 13 -S-、-(CH2) 14 -S-、-(CH2) 15 -S-、-(CH2) 16 -S-、-(CH2) 17 -S-、-(CH2) 18 -S-、-(CH2) 19 -S-、-(CH2) 20 -S-、-(CH2) 21 -S-、-(CH2) 22 -S-、-(CH2) 23 -S- or -(CH2) 24 -S-.

[0079] In a preferred embodiment, L does not have branches.

[0080] In a preferred embodiment of this disclosure, the carborane-phospholipid conjugate has the following structure

[0081]

[0082] Where Bo represents a carborane alkyl group, and n is an integer from 1 to 20. In a more preferred embodiment of this disclosure, Bo is 1,2-C2B. 10 H 11 - Preferably, it is a closed-dicarbododecanoyl group, more preferably a closed-1,2-dicarbododecanoyl group; n is 3, 7, 11 or 15, preferably 11.

[0083] Synthesis of carborane-phospholipid conjugates

[0084] The carborane-phospholipid conjugates disclosed herein can be synthesized by reacting C-substituted carborane derivatives, such as carborane-L-COOH, with lysophospholipids in the presence of a catalyst, such as 4-dimethylaminopyridine (DMAP), with a dehydrating agent, such as dicyclohexylcarbodiimide (DCC).

[0085] Synthesis of C-substituted carborane derivatives

[0086] The strong electron-withdrawing property of carborane cages makes the H on the CH bond weakly acidic, allowing for the reaction with strong bases such as alkyllithium reagents to produce a series of C-substituted carborane derivatives. For example, carboranes can be reacted with oxetane, ethylene oxide, or formaldehyde in the presence of butyllithium to introduce -CH2CH2CH2OH, -CH2CH2OH, and -CH2OH groups onto the carborane, respectively.

[0087] Carboalkyl-L-COOH can be obtained by oxidation of carboalkyl-L-CH2OH with an oxidizing agent such as CrO3.

[0088] Carborane derivatives can be monosubstituted or disubstituted. In a preferred embodiment, the prepared carborane derivative is monosubstituted.

[0089] Carborane derivatives can be synthesized using tert-butyldimethylsilyl (TBDMS) or triphenylsilyl as the C-protecting group. For example, using triphenylsilyl as the C-protecting group can synthesize monosubstituted or disubstituted carborane derivatives.

[0090]

[0091] For the synthesis of C-substituted carborane derivatives, please refer to the review article "Research Progress in the Synthesis of Carborane Derivatives" by Lu Juyou et al. in Synthetic Chemistry, 2015, 23(9): 883, and A newseries of organoboranes III: Some reactions of 1,2-dicarbaclovododecaborane(12) and its derivatives by He YT, Ager J, Clark S et al., Inorganic Chemistry, 1963, 2: 1097-1105.

[0092] Synthesis of lysophospholipids

[0093] The synthetic method disclosed herein can utilize commercially available or homemade lysophosphatidylcholine. Lysophosphatidylcholine can be prepared by enzymatic hydrolysis of phospholipids, with phospholipase A1 hydrolyzing the 1-acyl group and phospholipase A2 hydrolyzing the 2-acyl group. Examples of lysophosphatidylcholine used include lysophosphatidylcholine, lysophosphatidyl acid, lysophosphatidylethanolamine, lysophosphatidylserine, and lysophosphatidylinositol. In one embodiment, the lysophosphatidylcholine used is lysophosphatidylcholine (Lyso-PPC).

[0094] For specific preparation methods of lysophospholipids, please refer to the review article by Li Zhao et al., China Oils and Fats, 2018, 43(6): 132-143. The phospholipids used for hydrolysis can be commercially available or synthesized as needed.

[0095] The following uses compound BoP 1-4 as an example to illustrate the synthesis of carborane-phospholipid conjugates.

[0096]

[0097] Chemical synthesis route of compound BoP

[0098] Liposomes

[0099] Another aspect of this disclosure provides a liposome composition comprising the above-described carborane-phospholipid conjugate, wherein the carborane-phospholipid conjugate is part of a lipid bilayer.

[0100] The liposomes disclosed herein can be monolayer liposomes, i.e., composed of a single lipid bilayer, and typically have a diameter in the range of approximately 20 nm to approximately 400 nm. The liposomes disclosed herein can also be multilayer liposomes, with a diameter typically in the range of 1 μm to 10 μm.

[0101] The liposomes disclosed herein, due to their cell membrane-like structure, exhibit a high affinity for cells and increase the permeability of active ingredients (therapeutic agents and / or diagnostic agents) to cells, thereby enhancing the concentration and efficacy of the active ingredients at the disease site. Because the active ingredients are encapsulated within the liposomes, their diffusion rate in tissues is reduced, and their release rate into the bloodstream is slowed, thus prolonging the duration of their effect. Protected by the liposome bilayer, the active ingredients are protected from oxidation, degradation, or destruction by acids or enzymes in the body, thereby ensuring or extending their stability.

[0102] In one embodiment of this disclosure, the liposome composition further comprises cholesterol. Cholesterol is believed to reinforce the lipid bilayer membrane, reduce the fluidity of the phospholipid bilayer membrane, decrease membrane permeability, and reduce leakage of active ingredients. It also helps maintain a certain degree of flexibility in the lipid membrane, enhancing the ability of liposome vesicles to resist changes in external conditions. Furthermore, it has a certain protective effect against phospholipid oxidation.

[0103] In one embodiment of this disclosure, the liposome composition further comprises phospholipids, preferably saturated phospholipids. Examples of phospholipids include dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylglycerol (DMPG), distearyl phosphatidylglycerol (DSPG), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), and dimyristoyl phosphatidylserine (DMPS). The phospholipids include, but are not limited to, distearylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), ditransoleoylphosphatidylethanolamine, and hydrogenated soybean phosphatidylcholine (HSPC), with DPPC being preferred. In a preferred embodiment, the phospholipids included in the liposome composition have a structure similar to that of the carborane-phospholipid conjugate of Formula I, i.e., having the same or similar R1 or R2 (e.g., carbon chain lengths differing by less than 1) and the same or similar X and Y. It is believed that the addition of phospholipids can improve the stability of the lipid bilayer molecules and reduce the amount of carborane-phospholipid conjugate used.

[0104] In one embodiment of this disclosure, the liposome composition further comprises PEGylated phospholipids. PEGylated phospholipids are formed by covalently bonding PEG molecules with phospholipid molecules to form PEG-derived phospholipids, which are believed to reduce the leakage rate of liposomes, decrease their aggregation and fusion, prolong their shelf life, and inhibit cell adhesion in systemic circulation, shielding RES from recognition and uptake of liposomes, thus extending the in vivo circulation time of liposomes. The molecular weight of PEG in the PEGylated phospholipids is typically from about 500 to about 5000 Daltons (Da; g / mol), for example, 750 Da, 1000 Da, 2000 Da, or 5000 Da. The PEG in the PEGylated phospholipids may have a linear or branched structure.

[0105] In one embodiment of this disclosure, the PEGylated phospholipid is preferably DLPE-PEG, DPPE-PEG, DMPE-PEG or DSPE-PEG, more preferably DSPE-PEG, such as DSPE-PEG2000 and DSPE-PEG5000.

[0106] In one embodiment of this disclosure, the liposome composition contains both cholesterol and phospholipids.

[0107] In one embodiment of this disclosure, the liposome composition simultaneously comprises cholesterol, phospholipids, and PEGylated phospholipids. In a preferred embodiment of this disclosure, the liposome composition simultaneously comprises cholesterol, phospholipids, and PEGylated phospholipids, wherein the lipid arms of the phospholipids and PEGylated phospholipids differ by 0, 1, or 2 carbon atoms.

[0108] The liposomes disclosed herein may also contain pharmaceutically acceptable formulation adjuvants, such as stabilizers, antioxidants, pH adjusters, flavoring agents, carriers, diluents, and excipients. Pharmaceutically acceptable means that the formulation adjuvants must be compatible with other components of the formulation and harmless to the subject receiving the formulation. In this disclosure, the subject refers to a mammal, which may be a human or a pet.

[0109] In one embodiment of this disclosure, the liposome composition further comprises at least one therapeutic agent and / or at least one diagnostic agent. The therapeutic agent or diagnostic agent may be encapsulated within the aqueous chambers of the liposome or may be embedded within a lipid bilayer. Figure 1 As shown, a water-soluble chemotherapy drug can be encapsulated in the water chamber of a liposome formed from boronized phospholipids.

[0110] Suitable molecules that can be used as therapeutic agents include, but are not limited to, polypeptides, oligopeptides, peptide mimics, amino acids, enzyme inhibitors, hormones, toxins, antibiotics, anti-inflammatory substances, anticancer drugs, immunosuppressants, bronchodilators, etc.

[0111] In a preferred embodiment, the liposome composition comprises at least one therapeutic agent, said therapeutic agent being an anticancer drug.

[0112] The therapeutic agents may be, for example, antimetabolites (e.g., antifolate (e.g., methotrexate), fluoropyrimidines (e.g., 5-fluorouracil), purine and adenosine analogs, cytarabine), intercalating antitumor antibiotics (e.g., anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin C, daunorubicin, and mithramycin)), platinum derivatives (e.g., cisplatin and carboplatin), alkylating agents (e.g., nitrogen mustard, melphalan, chlorambucil, busulfan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa), antimitotics (e.g., vinca alkaloids (e.g., vincristine)), and taxanes (e.g., ... (paclitaxel) (docetaxel) and newer microbtubule agents (e.g., epothilone analogs, discermolide analogs, and eleutherobin analogs), topoisomerase inhibitors (e.g., epipodophyllotoxins (e.g., etoposide, teniposide, saccharin, and topotecan)), cell cycle inhibitors (e.g., flavopyridol), biological response modifiers, and proteasome inhibitors (e.g., ... (bortezomib)

[0113] For example, the anticancer drugs are selected from methylaurestatin E, methylaurestatin F, ibrutinib, acalabrutinib, zanubrutinib, doxorubicin, mitomycin-C, mitomycin-A, daunorubicin, aminopterin, actinomycin, bleomycin, 9-aminocamptothecin, N8-acetylspermethylene, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, yunnanycin, gemcitabine, cytarabine, dolalastatin, dacarbazine, 5-fluorouracil; paclitaxel, docetaxel, gemcitabine, cytarabine, 6-mercaptopurine, vincristine, cisplatin, oxaliplatin, and PARP inhibitors.

[0114] In one embodiment, the anticancer drug is selected from PARP inhibitors, preferably olaparib, niraparib, rucaparib, fluzoparib, pamiparib, veriparib, tapazanib, and more preferably olaparib.

[0115] The liposomes disclosed herein may contain diagnostic agents. Diagnostic agents used in this disclosure may include any diagnostic agents known in the art, such as those provided in the following references: Armstrong et al., Diagnostic Imaging, 5th Edition, Blackwell Publishing (2004); Torchilin, VP ed., Targeted Delivery of Imaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET and SPECT, Springer (2009). Diagnostic agents can be detected in a variety of ways, including by providing and / or enhancing detectable signals, including but not limited to gamma emission, radioactivity, echo, optical, fluorescence, absorption, magnetic, or tomographic signals.

[0116] The techniques used for imaging diagnostic agents may include, but are not limited to, single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET), computed tomography (CT), X-ray imaging, gamma-ray imaging, etc. The diagnostic agent may be conjugated to therapeutic liposomes in various ways, including, for example, by embedding or encapsulating the liposomes.

[0117] In some embodiments, the diagnostic agent may include a chelating agent that binds to metal ions for use in a variety of diagnostic imaging techniques. Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), [4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl]benzoic acid (CPTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), citric acid, hydroxyethylethylenediaminetriacetic acid (HEDTA), iminodiacetic acid (IDA), triethylenetetraminehexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and their derivatives.

[0118] Radioactive isotopes may be incorporated into some of the diagnostic agents described herein, and may include radionuclides that emit gamma rays, positrons, beta and alpha particles, and X-rays. Suitable radionuclides include, but are not limited to, 225Ac, 72As, 211At, 11B, 128Ba, 212Bi, 75Br, 77Br, 14C, 109Cd, 62Cu, 64Cu, 67Cu, 18F, 67Ga, 68Ga, 3H, 123I, 125I, 130I, 131I, 111In, 177Lu, 13N, 15O, 32P, 33P, 212Pb, 103Pd, 186Re, 188Re, 47Sc, 153Sm, 89Sr, 99mTc, 88Y, and 90Y. In some embodiments, the radioactive reagent may include 111In-DTPA, 99mTc(CO)3-DTPA, 99mTc(CO)3-ENPy2, 62 / 64 / 67Cu-TETA, 99mTc(CO)3-IDA, and 99mTc(CO)3 triamine (cyclic or linear). In other embodiments, the reagent may include DOTA and various analogues having 111In, 177Lu, 153Sm, 88 / 90Y, 62 / 64 / 67Cu, or 6w / 68Ga. In some embodiments, the liposomes may be radiolabeled, for example, by incorporation of lipids linked to a chelating agent, such as DTPA-liposomes, as provided in the following references: Phillips et al., Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 1(1): 69-83 (2008); Torchilin, VP & Weissig, V. (eds.), Liposomes 2nd ed.: Oxford Univ. Press (2003); Elbayoumi, TA & Torchilin, VP, Eur. J. Nucl. Med. Mol. Imaging 33: 1196-1205 (2006); Mougin-Degraef, M. et al., Int'l J. Pharmaceutics 344: 110-117 (2007).

[0119] In other embodiments, the diagnostic agent may include optical agents such as fluorescent agents, phosphorescent agents, chemiluminescent agents, etc. Various agents (e.g., dyes, probes, labels, or indicators) are known in the art and can be used in this invention (see, for example, Invitrogen, The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, 10th Edition (2005)). Fluorescent agents may include various organic and / or inorganic small molecules or various fluorescent proteins and their derivatives. For example, fluorescent agents may include, but are not limited to, anthocyanins, phthalocyanines, porphyrins, indigo, rhodamine, phenoxazines, phenylxanthoxanes, phenothiazines, phenselenazines, fluorescein, benzoporphyrins, squaric acid cyanines, dipyrrolopyrimidinones, tetracenes, quinoline, pyrazines, croconiums, acridiniums, phenanthridines, rhodamine, acridinium, anthraquinones, chalcogenopyrylium analogs, dihydroporphyrins, naphthylphthalocyanines, methinedyes, indoleniumdyes, azo compounds, chamomile blue, azachamomile blue, triphenylmethane dyes, indole, benzoindole, indole carbocyanine, benzoindole carbocyanine, and BODIPY having the general structure of 4,4-difluoro-4-boron-3a,4a-diaza-s-dicyclopentadienbenzene. TM derivatives, and / or any conjugates and / or derivatives thereof.Other reagents that may be used include, but are not limited to, fluorescein, fluorescein-polyaspartic acid conjugate, fluorescein-polyglutamic acid conjugate, fluorescein-polyarginine conjugate, indigo green, indigo-dodecanoic acid conjugate, indigo-polyaspartic acid conjugate, isossurant blue, indole disulfonates, benzoindole disulfonates, di(ethylcarboxymethyl)indigo, di(pentylcarboxymethyl)indigo, polyhydroxyindole sulfonates, polyhydroxybenzoindole sulfonates, rigid heteroatom indole sulfonates, indigo dipropionic acid, indigo dihexanoic acid, 3,6-dicyano-2,5-[(N,N,',N'-tetra(carboxymethyl)amino]pyrazine, 3,6-[(N,N,',N'-tetra(2-hydroxy) [Ethyl)amino]pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-azatedino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid, 3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide, 2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide, indocarbocyaninetetrasulfonate, chloroindocarbocyanine, and 3,6-diaminopyrazine-2,5-dicarboxylic acid.

[0120] In a preferred embodiment of this disclosure, the liposome composition comprises at least one diagnostic agent, said diagnostic agent being 64 Cu-NOTA-PEG2000-DSPE.

[0121] The liposomes disclosed herein can be prepared by conventional methods in the art, such as injection (ethanol injection and ether injection), thin film dispersion, reverse phase evaporation, chemical gradient (pH gradient, ammonium sulfate gradient, calcium acetate gradient), double emulsion, freeze drying, etc. See Chinese Pharmaceutical Journal, 2011, Vol. 46, No. 14, pp. 1084-1088.

[0122] In one embodiment, blank liposomes containing ammonium sulfate solution are prepared by ethanol injection or thin-film dispersion. Then, after removing the ammonium sulfate from the liposome membrane, an ammonium sulfate gradient is formed. Liposomes loaded with therapeutic or diagnostic agents are formed by the ammonium sulfate gradient method.

[0123] The particle size of the liposomes disclosed herein can be determined by thermodynamic light scattering methods, such as a Zetasizer 3000SH laser particle size analyzer (Malvern Instruments Ltd), with diameters ranging primarily from about 20 to about 150 nm.

[0124] The encapsulation efficiency of the liposomes disclosed herein can be determined by centrifugation, dialysis, gel column chromatography, etc.

[0125] The liposomes according to this disclosure can be administered orally or non-orally (e.g., intravenously, subcutaneously, intraperitoneally, or locally) as needed. The injection volume varies depending on the patient's weight, age, sex, health status, diet, injection time, injection method, excretion rate, and severity of disease. For example, the liposomes of this disclosure can be administered intravenously, via the lungs (nebulized inhalation or dry powder inhalation), or via the skin as needed. The injection volume is approximately 0.1–10 mg / kg body weight, based on the therapeutic or diagnostic agent contained in the liposomes.

[0126] Example

[0127] The starting materials used in the examples were commercially available and / or could be prepared using a variety of methods well known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select the reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of the experiment, and post-treatment) from the synthetic methods described below. Those skilled in the art of organic synthesis will understand that the functional groups present on the various parts of the molecule should be compatible with the proposed reagents and reactions. NMR was recorded using a Bruker AVANCE 400MHz spectrometer. High-resolution mass spectrometry was performed using a Bruker Fourier Transform Ion Cyclotron resonance mass spectrometer. The liquid chromatography-mass spectrometry used was a Waters e2695 instrument equipped with a Waters 2995 PDA and a Waters Acquity QDA mass spectrometer.

[0128] Chemical synthesis of BoP series 1

[0129] The above synthetic route was used to synthesize a series of BoPs (BoP 1-4), in which the number of methylene groups in the carbon chain n = 3, 7, 11, 15, and the corresponding compound numbers were arranged in ascending order. The total yield was 11%-20%, and the purity was >97%.

[0130] The specific synthesis steps and characterization methods of BoP-3 are as follows:

[0131] (1) Chemical synthesis of carborane mercaptotriethylamine salt (1):

[0132] The substrate (o-carborane, 1.00 g, 6.93 mmol) was dissolved in THF (30 mL) and cooled to 0 °C under anhydrous and oxygen-free conditions. Then, n-butyllithium (4.4 mL, 6.93 mmol) was added dropwise to the solution for 15 min, followed by stirring for 30 min. The system was then allowed to return to room temperature for 30 min. Sublimed sulfur (225 mg, 6.93 mmol) was added, and the reaction was carried out in an ice-water bath for 30 min. The system was then allowed to return to room temperature for 30 min. The solution was evaporated to dryness to give a yellow liquid. Unreacted n-butyllithium was quenched with hydrochloric acid (1 M, 7 mL). The solution was extracted with n-hexane, washed successively with water and saturated brine, and evaporated to dryness. Triethylamine (1 mL, 7.18 mmol) was added dropwise to a hexane solution (6 mL) of the above product at room temperature and stirred for 15 min. A white precipitate formed, which was then filtered, washed three times with 6 mL of n-hexane, and dried in air.

[0133] (2) Preparation of carborane-modified fatty acids: in 1-HOOC(CH2) 11 Br (9.3 mmol) was added dropwise to an ethanol (50 mL) solution with 5 mL of concentrated sulfuric acid. The mixture was stirred at room temperature for 15 min and then refluxed for 12 h. The mixture was cooled and evaporated to dryness. Ethyl acetate (30 mL) and water (30 mL) were added for extraction. The extract was washed with pure water and saturated brine, dried over anhydrous Na₂SO₄, and evaporated to dryness. The crude product was subjected to silica gel column chromatography using petroleum ether and ethyl acetate as eluents. After evaporating the solvent under reduced pressure, 1-C₂H₅OC(O)(CH₂) was obtained. 11 Br. Add Br(CH2) to a 1 (1.86 mmol) ethanol solution (42 mL). 11 C(O)OC2H5 (1.86 mmol) was stirred at room temperature for 15 min and then heated for 16 h. The reaction mixture was cooled and evaporated to dryness. The residue was extracted with ethyl acetate (30 mL) and washed with pure water (30 mL). After separation of the organic phase, the mixture was washed with water and saturated brine, dried over anhydrous Na2SO4, and evaporated to dryness. The crude product was subjected to column chromatography on a silica gel column. After evaporating the solvent under reduced pressure, 1-C2H5OC(O)(CH2) was given. 11 S-1,2-C2B 10 H 11 It was dissolved in glacial acetic acid (29 mL) to form a colorless and transparent solution. After stirring at room temperature for 15 min, pure water (9.8 mL) and concentrated sulfuric acid (9.8 mL) were added dropwise for hydrolysis for 16 h to obtain 1-HOOCC(O)(CH2). 11 S-1,2-C2B 10 H 11 (2).

[0134] (3) Preparation of BoP-3: Dicyclohexylcarbodiimide (DCC, 2.6 equivalents) and 4-dimethylaminopyridine (DMAP, 4.0 equivalents) were added to chloroform in a 0.1 M solution of carboroalkyl fatty acids (12,2 carbon atoms). The solution was stirred at room temperature for 5 minutes. Lyso-PPC (1.3 equivalents) was then added, and the reaction was stirred for 24 hours. The reaction mixture was concentrated by rotary evaporation. BoP-3 was purified by silica gel column chromatography (65:25:4CH2Cl2:MeOH:H2O).

[0135] (4) Characterization of BoP-3: Molecular dynamics simulations were used to observe the conformations of membrane structures formed by BoPs with different carbon atom numbers (BoP-1, BoP-2, BoP-3, BoP-4). It was found that the conformation of BoP-3 was the most stable, with the least degree of folding of its long chains, and it most closely resembled the membrane structure formed by DPPC. Figures 5-9 Simultaneously, drug loading efficiency was tested. By encapsulating sulfonylrhodamine B (SRB), the absorption at 550 nm was measured, revealing that BoP-3 exhibited the highest absorption. Figure 10 This indicates that the membrane structure is the most intact. SRB leakage was also measured; after culturing in 50% bovine serum at 37°C for 24 hours, BoP-3 showed the lowest SRB leakage percentage. Figure 11 Simultaneously, the structure was characterized using 1H NMR spectroscopy, revealing the correct structure. To obtain more intuitive information about the membrane-forming structure of BoP-3, boron liposomes composed of BoP-3, DPPC, cholesterol, and DPPE-PEG2000 were prepared. Transmission electron microscopy (TEM) revealed that, after particle size control via extrusion and dialysis purification, the boron liposomes were obtained as spherical carriers with a diameter of 30-100 nm, exhibiting regular shapes and almost no breakage (see...). Figure 3 The polydispersity index and zeta potential of this spherical carrier were also measured. After encapsulating doxorubicin (Dox), the carrier was electrically neutral to -3.1 mV, demonstrating effective drug loading performance.

[0136] Preparation of BoP-1, BoP-2, and BoP-4:

[0137] The preparation of BoP-1, BoP-2, BoP-4, and BoP-3 is almost identical, except for the different carbon chain lengths of the bromocarboxylic acids chosen during the initial synthesis of carboronized fatty acids. Specifically, the substrate for BoP-1 is 1-HOOC(CH2)3Br, for BoP-2 it is 1-HOOC(CH2)7Br, and for BoP-4 it is 1-HOOC(CH2). 15 Br. The detailed process is as follows:

[0138] (1) BoP-1: 5 mL of concentrated sulfuric acid was added dropwise to a 50 mL solution of 1-HOOC(CH2)3Br (9.3 mmol) in ethanol. The mixture was stirred at room temperature for 15 min and then refluxed for 12 h. The mixture was cooled and evaporated to dryness. Ethyl acetate (30 mL) and water (30 mL) were added for extraction. The product was washed with pure water and saturated brine, dried over anhydrous Na2SO4, and evaporated to dryness. The crude product was subjected to column chromatography using silica gel, with petroleum ether and ethyl acetate as eluents. After evaporating the solvent under reduced pressure, 1-C2H5OC(O)(CH2)3Br was obtained. Br(CH2)3C(O)OC2H5 (1.86 mmol) was added to a 42 mL solution of 1-HOOC(CH2)3Br (1.86 mmol). The mixture was stirred at room temperature for 15 min and then heated for 16 h. The reaction mixture was cooled and evaporated to dryness. The residue was extracted with ethyl acetate (30 mL) and washed with pure water (30 mL). After organic phase separation, the product was washed with water and saturated brine, dried over anhydrous Na₂SO₄, and then evaporated to dryness. The crude product was subjected to silica gel column chromatography. After evaporation under reduced pressure, 1-C₂H₅OC(O)(CH₂)₃S⁻¹,²-C₂B⁻ was obtained. 10 H 11 It was dissolved in glacial acetic acid (29 mL) to form a colorless and transparent solution. After stirring at room temperature for 15 min, pure water (9.8 mL) and concentrated sulfuric acid (9.8 mL) were added dropwise for hydrolysis for 16 h to obtain 1-HOOCC(O)(CH2)3S-1,2-C2B 10 H 11 (3); Dicyclohexylcarbodiimide (DCC, 2.6 equivalents) and 4-dimethylaminopyridine (DMAP, 4.0 equivalents) were added to chloroform in a 0.1 M solution of carboroalkyl fatty acids (4,3 carbon atoms). The solution was stirred at room temperature for 5 minutes. Lyso-PPC (1.3 equivalents) was then added, and the reaction was stirred for 24 hours. The reaction mixture was concentrated using rotary evaporation. BoP-1 was purified by silica gel column chromatography (65:25:4CH2Cl2:MeOH:H2O).

[0139] (2) BoP-2: 5 mL of concentrated sulfuric acid was added dropwise to a solution of 1-HOOC(CH2)7Br (9.3 mmol) in ethanol (50 mL). The mixture was stirred at room temperature for 15 min and then refluxed for 12 h. The mixture was cooled and evaporated to dryness. Ethyl acetate (30 mL) and water (30 mL) were added for extraction. The product was washed with pure water and saturated brine, dried over anhydrous Na2SO4, and evaporated to dryness. The crude product was subjected to column chromatography using silica gel, with petroleum ether and ethyl acetate as eluents. After evaporating the solvent under reduced pressure, 1-C2H5OC(O)(CH2)7Br was obtained. Br(CH2)7C(O)OC2H5 (1.86 mmol) was added to a solution of 1-HOOC(CH2)7Br (42 mL). The mixture was stirred at room temperature for 15 min and then heated for 16 h. The reaction mixture was cooled and evaporated to dryness. The residue was extracted with ethyl acetate (30 mL) and washed with pure water (30 mL). After organic phase separation, the product was washed with water and saturated brine, dried over anhydrous Na₂SO₄, and then evaporated to dryness. The crude product was subjected to silica gel column chromatography. After evaporating the solvent under reduced pressure, 1-C₂H₂O was obtained. s OC(O)(CH2)7S-1,2-C2B 10 H 11 It was dissolved in glacial acetic acid (29 mL) to form a colorless and transparent solution. After stirring at room temperature for 15 min, pure water (9.8 mL) and concentrated sulfuric acid (9.8 mL) were added dropwise for hydrolysis for 16 h to obtain 1-HOOCC(O)(CH2)7s-1,2-C2B 10 H 11 (4); Dicyclohexylcarbodiimide (DCC, 2.6 equivalents) and 4-dimethylaminopyridine (DMAP, 4.0 equivalents) were added to chloroform in a 0.1 M solution of carboroalkyl fatty acids (8, 4 carbon atoms). The solution was stirred at room temperature for 5 minutes. Lyso-PPC (1.3 equivalents) was then added, and the reaction was stirred for 24 hours. The reaction mixture was concentrated using rotary evaporation. BoP-2 was purified by silica gel column chromatography (65:25:4CH2Cl2:MeOH:H2O).

[0140] (3) BoP-4: in 1-HOOC(CH2) 15 Br (9.3 mmol) was added dropwise to an ethanol (50 mL) solution with 5 mL of concentrated sulfuric acid. The mixture was stirred at room temperature for 15 min and then refluxed for 12 h. The mixture was cooled and evaporated to dryness. Ethyl acetate (30 mL) and water (30 mL) were added for extraction. The extract was washed with pure water and saturated brine, dried over anhydrous Na₂SO₄, and evaporated to dryness. The crude product was subjected to silica gel column chromatography using petroleum ether and ethyl acetate as eluents. After evaporating the solvent under reduced pressure, 1-C₂H₅OC(O)(CH₂) was obtained. 15 Br. Add Br(CH2) to a 1 (1.86 mmol) ethanol solution (42 mL).15 C(O)OC2H5 (1.86 mmol) was stirred at room temperature for 15 min and then heated for 16 h. The reaction mixture was cooled and evaporated to dryness. The residue was extracted with ethyl acetate (30 mL) and washed with pure water (30 mL). After separation of the organic phase, the mixture was washed with water and saturated brine, dried over anhydrous Na2SO4, and evaporated to dryness. The crude product was subjected to column chromatography on a silica gel column. After evaporating the solvent under reduced pressure, 1-C2H5OC(O)(CH2) was given. 15 S-1,2-C2B 10 H 11 It was dissolved in glacial acetic acid (29 mL) to form a colorless and transparent solution. After stirring at room temperature for 15 min, pure water (9.8 mL) and concentrated sulfuric acid (9.8 mL) were added dropwise for hydrolysis for 16 h to obtain 1-HOOCC(O)(CH2). 15 S-1,2-C2B 10 H 11 (4); Dicyclohexylcarbodiimide (DCC, 2.6 equivalents) and 4-dimethylaminopyridine (DMAP, 4.0 equivalents) were added to chloroform in a 0.1 M solution of carboroalkyl fatty acids (16, 5 carbon atoms). The solution was stirred at room temperature for 5 minutes. Lyso-PPC (1.3 equivalents) was then added, and the reaction was stirred for 24 hours. The reaction mixture was concentrated using rotary evaporation. BoP-4 was purified by silica gel column chromatography (65:25:4CH2Cl2:MeOH:H2O).

[0141] Composition and preparation of boron liposomes

[0142] A total of 20 mg of distearylphosphatidylcholine (DPPC), cholesterol (Chol), distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2k), and BoP were dissolved in 1 mL of ethanol at 60 °C in a ratio of (BoP:DPPC:Chol:DSPE-PEG2k = 50:10:40:1, mol%). Then, ammonium sulfate buffer (4 mL, 250 mM, pH = 5.5) was injected at 60 °C. The liposome solution was then passed through a liposome extruder with a pore size of 200 nm for 10 pushes at 60 °C. Afterward, a membrane with a pore size of 100 nm was used, and the extrusion was repeated 10 times. Free ammonium sulfate was removed by dialyzing in 800 mL of a solution composed of 10% sucrose and 10 mM histidine (pH = 6.5), with the dialysate changed at least twice within 12 hours.

[0143] Drug delivery via 3-boron liposomes

[0144] For boron liposomes loaded with sulfonylrhodamine (SRB), the lipids of the formulation were dissolved in ethanol, hydrated with 50 mM SRB, and sonicated at 45°C for 30 min. Dox was loaded using an ammonium sulfate gradient method. Dox at a drug-to-lipid molar ratio of 1:8 was added to the liposome solution, and the mixture was incubated at 60°C for 1 h. The SRB release rate (%SRB release rate = (A...)) was then calculated. final -A initial ) / (A Triton-X-100 -A initial The absorbance is calculated as ()×100% to evaluate its stability and encapsulation ability.

[0145] 4. Characterization of boron liposomes (dynamic light scattering, zeta potential analysis, morphological examination, 3D model construction)

[0146] Dynamic light scattering and zeta potential analysis were performed on a Malvern Zetasizer Nano ZS; morphological examination of boron liposomes was performed using a 200 kV TEM (JEM-2100, JEOL, Japan). A drop of boron liposome solution was added to a copper mesh coated with an ultrathin carbon film and dried at room temperature for 10 min. Excess solution was removed before negative staining with uranyl acetate solution (4% w / v), followed by washing with water; the three-dimensional structure of BoPs was predicted using Spartan 14 software after optimization on density functional theory / Becke, three-parameter, Lee-Yang-Parr, and 6-31G* basis sets (DFT / B3LYP / 6-31G*). I c , I b , Δl, ,d, And parameters such as θ, fat arm dihedral angle (°).

[0147]

[0148] Radiolabeling of 5-boron liposomes

[0149] Provided by Peking University Cancer Hospital 64 CuCl2 was dissolved in 0.01 M hydrochloric acid. NOTA-boron liposomes were prepared according to the specified composition (BoP-3:DPPC:cholesterol:DSPE-PEG2k-NOTA = 50:10:40:1, mol%). NaAc buffer (1 mL, pH = 5.5, 0.2 M) was added to the boron liposome solution (100 μL, 0.5 mg / mL) and... 64 CuCl2 (18.5 MBq) was co-incubated at 37°C for 2 hours, and then purified by PD-10 size exclusion chromatography using PBS as the eluent. 64Cu-NOTA-boron liposomes, with a radiochemical yield greater than 90%.

[0150] In selecting boron-containing molecules, we chose carboranes (C2B) which are highly hydrophobic and have low nucleophilicity. 10 H 12 As a boron-containing molecule that binds to liposome membranes, carboranes possess a considerable boron content, offering good therapeutic effects for BNCT. Therefore, we linked boronized acyl groups (carborane-containing fatty acids) to phospholipid molecules (Lyso-PPCs), utilizing the Thiol-halo reaction instead of click chemistry to maintain the flexibility of phospholipids. However, since carboranes are three-dimensional molecules, the impact of steric hindrance on the stability of lipid bilayer formation needs to be considered. Therefore, we screened carborane-containing fatty acids of different lengths (4, 8, 12, and 16 carbon atoms in the carbon chain, although practical applications are not limited to these numbers). First, we examined the three-dimensional structures of BoPs with different carbon numbers, using dipalmitoylphosphatidylcholine (DPPC) as a reference, comparing the lengths of the two lipid arms under different carbon number conditions for carborane-containing fatty acids. We found that BoP-3 had the smallest length difference, even smaller than DPPC, which is conducive to forming the most stable membrane structure. For other parameters, such as the distance between lipid arms and dihedral angle, we found that BoP-3 was closest to DPPC. Building upon this foundation, molecular dynamics simulations were used to model a bilayer system formed by 128 BoP-3 and DPPC molecules. The results showed that the membrane structure formed by BoP-3 was relatively regular and well-organized, with minimal chain folding deformation. Simultaneously, at the experimental level, the drug-loading capacity of BoP-3 was determined by encapsulating sulfonylrhodamine B (SRB) to assess drug loading efficiency. Liposomes were obtained by hydrating the lipids with a 50 mM solution followed by gel filtration. The highest absorption was observed at 550 nm in BoP-3, approaching that of the DPPC group. After 24 hours of incubation, SRB release was measured in 50% bovine serum at 37°C to assess stability. The conclusion is that the nanostructure formed by BoP-3 exhibits high retention efficiency for SRB. Finally, BoP-3 (with 12 carbon atoms in the carbon chain) was selected as the optimal structure for self-assembly.

[0151] DSPE-PEG2000-NOTA was used as a chelating agent for the positron-emitting nuclide Cu-64 in PET imaging. The aforementioned NOA-liposome was incorporated into boron liposomes and then incubated with an aqueous solution of Cu-64 ions. Purification was achieved by gel filtration. 64 Cu-NOTA-boron liposomes. Stability was verified for up to 24 hours by radiolabeled thin-layer chromatography. The radiolabeled boron liposomes (7.4 MBq, 150 μL) were then intravenously injected into 4T1 tumor-bearing mice for PET-CT imaging. Figure 4The study evaluated the biodistribution and tumor specificity of boron liposomes in mice, further confirming that the biodistribution (tumor / normal site ratio) of this novel boron liposome in vivo is acceptable. In clinical oncology treatment, this can be used to determine tumor location, assess treatment efficacy in real time, and develop further treatment plans.

[0152] Boron concentration in cells after incubation with boron liposomes was assessed using ICP-OES. Mouse triple-negative breast cancer 4T1 cells were co-incubated with boron liposomes at concentrations of 0.1-10 mg / mL for 24 h. The experiment showed that intracellular boron concentration gradually increased with increasing boron liposome incubation dose. Figure 12 At a dose of 5 mg / mL, the boron concentration in 4T1 cells can reach 182.5 ppm (n=4), which fully meets the requirements for clinical BNCT.

[0153] Next, the cytotoxicity of boron liposomes was assessed using the CCK-8 assay. Figure 13 As shown, the cell viability of 4T1 cells was measured after incubation with different concentrations of boron liposomes for 24 hours. The experiment found that when the incubation concentration reached 5 mg / mL, the cells showed good tolerance to boron liposomes.

[0154] Based on the above research, neutron irradiation experiments were conducted on 4T1 tumor-bearing mice using boron liposomes. 4T1 tumor-bearing mice were injected intravenously with PBS (400 μL), boron liposomes (500 mg / kg), or Dox-boron liposomes (500 mg / kg). Twelve hours later, the mice were anesthetized and fixed at the slow neutron beam exit point (@IHNI), with the mouse's head pointing towards the center. The tumor was exposed to full power irradiation within the thermal neutron irradiation field for 30 minutes; the liver, spleen, and intestines were located outside the irradiation field to avoid unnecessary damage.

[0155] The in vivo antitumor efficacy of PARPi-boron liposome-BNCT was further investigated. Experiments showed that the tumor volume in the PARPi@BLNP group (i.e., PARPi loaded with boron liposomes without neutron irradiation) significantly increased with increasing treatment duration; while the average tumor volume in the PARPi@BLNP+N group (i.e., PARPi loaded with boron liposomes and neutron irradiation) shrank to one-fifth of its original volume. Figure 14 The tumor suppression effect was better than that of the other control groups.

[0156] The body weight of mice in each group was measured, either by irradiation with boron liposomes and neutrons or by irradiation with boron liposomes loaded with PARP inhibitors. The resulting mouse body weight curves ( Figure 15 This indicates that the above treatment has no obvious toxicity.

[0157] Regarding integrated chemoradiotherapy, doxorubicin was initially chosen as the encapsulated drug, and it was found that some tumors could be eradicated, initially validating the efficacy of boron liposome-BNCT therapy as adjuvant chemotherapy. Next, we considered that selecting individual drugs that disrupt the DNA replication process, based on the principles of BNCT, could further enhance its efficacy. Therefore, we chose the poly(ADP-ribose) polymerase (PARP) inhibitor (olaparib). We verified its interference with the DNA repair system using γ-h2ax staining, and in vivo experiments in 4T1 tumor-bearing mice showed that PARPi-boron liposomes had better efficacy than doxorubicin-boron liposomes (see [link to relevant documentation]). Figure 2 ).

[0158] All references cited in this application (including bibliographic references, published patents, published patent applications, and concurrently pending patent applications) are expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly known to one of ordinary skill in the art.

[0159] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature having the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a series of equivalent or similar features.

[0160] Based on the above description, those skilled in the art can readily identify the essential features of the present invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the appended claims.

Claims

1. A carborane-phospholipid conjugate of formula (I), (I), where: Of R1 and R2, one is an -L-carboronic alkyl group, and the other is a straight-chain alkyl or alkenyl group with 10-24 carbon atoms, where L is -(CH2). n+m -S-, where m+n is 11; X represents -H, -CH2CH2NH3, or -CH2CH2N. + (CH3)3, -CH2CH(NH2)COOH, -CH2CH(OH)CH2OH, or cyclohexane-2,3,4,5,6-pentahydroxy-1-yl; Y is OH or O - .

2. The conjugate according to claim 1, wherein the carboroalkyl group in the -L-carboroalkyl group is 10 B-rich.

3. The conjugate according to claim 1, wherein X is -CH2CH2N + (CH3)3; Y is O - .

4. The conjugate according to claim 1, wherein the carborane alkyl group is 1,2-C2B4H5-, 1,2-C2B8H9-, or 1,2-C2B 10 H 11 -、2,3-C2B4H7-、7,8-C2B9H 12 -or 5,6-C2B8H 11 - 5. The conjugate according to claim 1, wherein the carborane alkyl group is 1,2-C2B. 10 H 11 - 6. A carborane-phospholipid conjugate having the following structure ， Where Bo represents a carborane alkyl group and n is 11.

7. The conjugate according to claim 6, wherein... Bo is 1,2-C2B 10 H 11 - 8. The conjugate according to claim 6, wherein Bo is a closed-dicarbododecyl group.

9. The conjugate according to claim 6, wherein Bo is a closed-form-1,2-dicarbododecyl group.

10. A liposome composition comprising: The conjugate according to any one of claims 1-9 is part of a lipid bilayer.

11. The liposome composition according to claim 10, further comprising: cholesterol.

12. The liposome composition according to claim 11, further comprising phospholipids.

13. The liposome composition according to claim 12, wherein the phospholipid is dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylglycerol (DMPG), distearyl phosphatidylglycerol (DSPG), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylserine (DMPS), distearyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylserine (DPPS), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidyl ethanolamine (DMPE), distearyl phosphatidyl ethanolamine (DSPE), or ditransoleoyl phosphatidyl ethanolamine.

14. The liposome composition according to claim 12, wherein the phospholipid is DPPC.

15. The liposome composition of claim 10, further comprising polyethylene glycolated phospholipids.

16. The liposome composition of claim 15, wherein the polyethylene glycol-modified phospholipid is selected from DLPE-PEG, DPPE-PEG, DMPE-PEG or DSPE-PEG.

17. The liposome composition of claim 15, wherein the polyethylene glycolated phospholipid is DSPE-PEG.

18. The liposome composition of claim 10, further comprising at least one therapeutic agent and / or at least one diagnostic agent.

19. The liposome composition of claim 18, comprising at least one therapeutic agent, said therapeutic agent being an anticancer drug.

20. The liposome composition according to claim 19, wherein the anticancer drug is selected from methylaurestatin E, methylaurestatin F, ibrutinib, acalabrutinib, zanubrutinib, doxorubicin, mitomycin-C, mitomycin-A, daunorubicin, aminopterin, actinomycin, bleomycin, 9-aminocamptothecin, N8-acetylspermethylene, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, yunnanycin, gemcitabine, cytarabine, dolalastatin, dacarbazine, 5-fluorouracil; paclitaxel, docetaxel, gemcitabine, cytarabine, 6-mercaptopurine, vincristine, cisplatin, oxaliplatin, and PARP inhibitors.

21. The liposome composition according to claim 20, wherein the anticancer drug is a PARP inhibitor.

22. The liposome composition of claim 21, wherein the PARP inhibitor is selected from olaparib, niraparib, rucaparib, fluzoparib, pamiparib, veliparib, or tapazopanib.

23. The liposome composition of claim 21, wherein the PARP inhibitor is olaparib.

24. The liposome composition of claim 18, comprising at least one diagnostic agent, said diagnostic agent being 64 Cu-NOTA-PEG2000-DSPE.

25. Use of the liposome composition according to any one of claims 10 to 24 in the preparation of formulations for delivering at least one therapeutic agent and / or at least one diagnostic agent.

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