CN111973556A - Polymer vesicle carrying small molecular drug, preparation method and application thereof - Google Patents

Polymer vesicle carrying small molecular drug, preparation method and application thereof Download PDF

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CN111973556A
CN111973556A CN202010845920.9A CN202010845920A CN111973556A CN 111973556 A CN111973556 A CN 111973556A CN 202010845920 A CN202010845920 A CN 202010845920A CN 111973556 A CN111973556 A CN 111973556A
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drug
vcr
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amphiphilic block
vesicle
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CN111973556B (en
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孙欢利
余娜
张翼帆
钟志远
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Suzhou University
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Abstract

The invention discloses a polymer vesicle carrying small molecular drugs, a preparation method and application thereof, wherein the polymer vesicle carrying small molecular drugs is prepared by assembling amphiphilic block polymers and small molecular drugs; or the amphiphilic block polymer and the functionalized amphiphilic block polymer are assembled, crosslinked and loaded with small molecular drugs, and then the small molecular drugs react with the targeting monoclonal antibody to obtain the target monoclonal antibody. The vesicle system provided by the invention has a plurality of unique advantages, including small size, simple and controllable preparation, excellent biocompatibility, high in-vivo circulation stability, strong specific selectivity of tumor cells, high release speed of intracellular drugs, remarkable tumor growth inhibition effect and the like. Therefore, the vesicle system is expected to become a simple and multifunctional nano platform and is used for efficiently and specifically targeting vincristine sulfate to multiple myeloma cells.

Description

Polymer vesicle carrying small molecular drug, preparation method and application thereof
Technical Field
The invention belongs to the technical field of polymer nano-drugs, and particularly relates to a vincristine sulfate-loaded reversible cross-linked degradable polymer vesicle, a preparation method thereof and application thereof in tumor targeted therapy.
Background
Vincristine sulfate (VCR) is a water-soluble potent drug that acts mainly on tubulin, arresting mitosis in the metaphase, but at a lower dose due to its severe neurotoxicity. The liposome vincristine sulfate (Marqibo) nano-drugs approved to be marketed in 2012 can prolong the circulation time of the VCR and reduce the toxic and side effects, but the overall improvement is limited. Therefore, how to achieve efficient stable encapsulation and tumor-targeted delivery of VCRs is crucial. The vincristine sulfate liposome is composed of vincristine sulfate and a nano liposome prepared from sphingomyelin, wherein the vincristine sulfate is wrapped in the nano liposome, the nano liposome is prepared from the sphingomyelin, and the vincristine sulfate liposome is prepared after the vincristine sulfate is wrapped, wherein the sphingomyelin contains more amido bonds and can better resist chemical and biological degradation, the stability of the liposome structure is protected, and the drug enrichment amount of tumor cells is improved, so that the anti-tumor effect is improved. The prior art also discloses a preparation method of the difunctional nanoparticle preparation encapsulating vincristine sulfate, which encapsulates the vincristine sulfate in a PLGA-PEG polymer carrier modified by folic acid/cell penetrating peptide by a multiple emulsion method to prepare the difunctional nanoparticle preparation; the prepared folic acid/cell penetrating peptide modified PLGA-PEG difunctional nanoparticle has the particle size of 287.2 +/-0.8 nm, higher drug loading rate and encapsulation rate and good stability. The existing polymer vesicle with a liposome-like structure has a hydrophilic inner cavity and can be used for loading hydrophilic small-molecule drugs, but the loading efficiency of hydrophilic drugs such as VCR and the like is low, and the characteristics of integration of multiple functions such as internal circulation stability of a set, specific targeting of tumors, rapid release of drugs in cells, excellent biocompatibility and the like are lacked.
Disclosure of Invention
The invention aims to disclose an amphiphilic block polymer, a drug-loaded polymer vesicle and a preparation method and application thereof, and particularly relates to a vincristine sulfate (VCR) -loaded reversible cross-linked degradable polymer vesicle and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the drug-loaded polymer vesicle is prepared from a small molecular drug and an amphiphilic block polymer; or prepared from micromolecular drugs, amphiphilic block polymers, functionalized PEG-P (TMC-DTC) and targeted monoclonal antibodies;
the molecular structural formula of the amphiphilic block polymer is as follows:
Figure 426968DEST_PATH_IMAGE001
wherein z is 5-15.
In the amphiphilic block polymer, the molecular weight of PEG is 3000-8000 Da; the molecular weight of the hydrophobic chain segment is 2.5-6 times of the molecular weight of PEG; the molecular weight of the PDTC chain segment is 8-30% of that of the hydrophobic chain segment. The amphiphilic block polymer provided by the invention has a hydrophilic chain segment (n chain segment), a hydrophobic chain segment (x + y chain segment) and KDzSegment (z segment), hydrophobic segment, KDzThe chain segments being linked by urethane linkagesConnecting; the amphiphilic block polymer is expressed as PEG-P (TMC-DTC) -KDz、PEG-P(LA-DTC)-KDz、PEG-P(CL-DTC)-KDz。
In the invention, the small molecule drug is vincristine sulfate, adriamycin hydrochloride, epirubicin hydrochloride, verapamil hydrochloride, irinotecan hydrochloride and resiquimod, and is preferably vincristine sulfate (VCR); the targeting monoclonal antibody is a CD38 targeting monoclonal antibody, such as darunavir (Dar), isauximab (Isa) or other CD38 targeting monoclonal antibodies.
The preparation method of the drug-carrying polymer vesicle comprises the steps of preparing the drug-carrying polymer vesicle by using a solvent displacement method by using a small molecular drug and the amphiphilic block polymer as raw materials; or the drug-loaded polymer vesicle is prepared by a solvent displacement method by taking a small molecular drug, the amphiphilic block polymer, the functionalized amphiphilic block polymer and the targeted monoclonal antibody as raw materials. Preferably, the functionalized amphiphilic block polymer and the amphiphilic block polymer are assembled, crosslinked and loaded with a drug, and then the drug-loaded polymer vesicle is prepared by reacting with a monoclonal antibody targeting CD 38.
The invention discloses an application of the amphiphilic block polymer or the drug-loaded polymer vesicle in the preparation of nano drugs; the nanometer medicinal materials are antitumor drugs; the tumor is preferably orthotopic multiple myeloma.
The reversible cross-linked degradable polymer vesicle loaded with vincristine sulfate (VCR) is obtained by assembling and cross-linking amphiphilic block polymers, and has an asymmetric membrane structure, wherein the outer shell is polyethylene glycol (PEG), the membrane layer is hydrophobic polycarbonate with reversible cross-linking, and the inner shell is KDzEfficient loading of the VCR can be achieved. The drug-loaded vesicle is a targeting or non-targeting structure, the targeting molecule of the invention is a monoclonal antibody molecule or a monoclonal antibody fragment, and the monoclonal antibody molecule is darunavailamab (Dar), isauximab (Isa) or other monoclonal antibodies targeting CD 38.
The invention adopts amphiphilic block polymer and functional group amphiphilic block polymer as raw materials to prepare medicine-carrying vesicles, and then monoclonal antibody targeting CD38 is connected to obtain CD38 targeting drug-loaded vesicles. The functional group comes from PEG initiator, and the obtained polymer PEG end carries reactive functional group, such as azide (N)3) Maleimide (Mal) or N-hydroxysuccinimide (NHS), as exemplified by the amphiphilic block polymer PEG-P (TMC-DTC), the functionalized amphiphilic block polymer may be N3-PEG-P(TMC-DTC)、Mal-PEG-P(TMC-DTC)、NHS-PEG-P(TMC-DTC)。
The drug-loaded vesicle consists of drugs and vesicles, wherein the vesicles are obtained by polymer crosslinking and can be modified or not modified with targeting molecules; taking amphiphilic block polymer PEG-P (TMC-DTC) and vincristine sulfate as examples, the preparation method of the drug-loaded vesicle can be as follows:
(1) activating the end hydroxyl group of PEG-P (TMC-DTC) by chloroformic acid P-nitrophenyl ester, and reacting with KDzThe PEG-P (TMC-DTC) -KD is prepared by reactionz；
(2) Introducing N into PEG end of PEG-P (TMC-DTC)3Functional groups such as Mal or NHS and the like to obtain functionalized PEG-P (TMC-DTC);
(3) with vincristine sulfate and PEG-P (TMC-DTC) -KDzPreparing a reversible cross-linked degradable polymer vesicle loaded with a VCR by a solvent displacement method; or vincristine sulfate, PEG-P (TMC-DTC) -KDzAnd functionalized PEG-P (TMC-DTC) are used as raw materials, the VCR-loaded reversible crosslinking degradable polymer vesicle with the surface containing the reactive functional group is prepared by a solvent displacement method, and then the VCR-loaded reversible crosslinking degradable polymer vesicle is prepared by reacting with the monoclonal antibody.
The invention discloses the VCR-loaded reversible cross-linking degradable polymer vesicle and a preparation method thereof, and PEG-P (TMC-DTC) -KD is prepared byzInjecting the solution of the polymer into a standing VCR aqueous solution, stirring and dialyzing to obtain a VCR-loaded reversible cross-linked degradable polymer vesicle (Ps-VCR); specifically, VCR was dissolved in ultrapure water and mixed with HEPES buffer (pH 6.8, 10 mM) uniformly, and PEG-P (TMC-DTC) -KD was injected thereto under static conditionszThe polymer in DMSO solution was stirred for 3-5 minutes and dialyzed against HEPES (pH 7.4, 10 mM) to obtain Ps-VCR.
The invention also discloses a monoclonal antibody-oriented VCR-loaded reversible cross-linked degradable polymer vesicle and a preparation method thereof: PEG-P (TMC-DTC) -KDzWith a functionalized polymer such as N3Mixing the DMSO solution of-PEG-P (TMC-DTC), adding into HEPES solution containing VCR, stirring for 3-5 min, and dialyzing to obtain DMSO solution containing N on surface3The VCR-loaded reversibly cross-linked polymersomes of (a); a monoclonal antibody modified by dibenzocyclooctyne, such as darunavimab (Dar), isauximab (Isa), or other CD 38-targeting monoclonal antibodies, is conjugated with azide-functionalized VCR-loaded vesicles (N)3Ps-VCR) to generate a tension-triggered click chemistry reaction, and the monoclonal antibody-directed VCR-loaded vesicle (Ab-Ps-VCR) can be prepared under mild conditions. Ab-Ps-VCR can be simply prepared by adopting the same method through the Michael addition reaction of a sulfydryl functionalized monoclonal antibody molecule and a VCR-loaded vesicle with Mal on the surface or the amidation reaction of the monoclonal antibody and an NHS functionalized VCR-loaded vesicle.
In the polymer, KD has good biocompatibility, and by combining the PEG chain segment and the hydrophobic chain segment, vesicles with asymmetric membrane structures can be formed, so that high-efficiency and stable entrapment of small-molecule drugs (such as VCRs) is realized; the VCR is entrapped by electrostatic acting force, and meanwhile, the vesicle membrane crosslinked by the disulfide is separated from the outside, so that the loss and toxic and side effects caused by leakage and cell adhesion in the conveying process can be avoided, the VCR can be efficiently conveyed to a focus part, and the VCR is quickly released under the action of a reducing agent Glutathione (GSH) in vivo, so that tumor cells are effectively killed.
The polymersome in the invention is a reduction-sensitive reversible cross-linking polymersome with an inner membrane with negative charges, can be uncrosslinked in cells and is biodegradable; the polymer is PEG-P (TMC-DTC) -KDzWherein TMC (LA or CL) of the mid-block is randomly arranged with DTC; KDzThe molecular weight of the polymer is 700-2000 Da which is far less than that of the PEG section, the reversible cross-linked polymer vesicle with the inner membrane with negative charges is obtained after self-assembly and cross-linking, and the inner shell of the vesicle is KDzIs used for compounding small molecule medicines. The vesicle membrane is reversibly crosslinked, biodegradable and well-compatible PTMC, dithiopentane of a side chainThe structure of the lipoic acid is similar to that of the natural antioxidant lipoic acid of a human body, and the lipoic acid can spontaneously form reversible cross-linking sensitive to reduction, thereby not only ensuring the stable long circulation of the drug in blood, but also realizing the rapid de-crosslinking in cells and rapidly releasing the drug into target cells.
The invention discloses application of the VCR-loaded tumor-targeted reversibly-crosslinked degradable polymer vesicle in preparation of an anti-tumor targeted nano-drug. Preferably, the tumor is multiple myeloma.
Compared with the prior art, the invention has the following advantages:
1. the invention designs a new micromolecule hydrophilic drug VCR drug-carrying vesicle and tumor targeted delivery; the vesicle membrane is reversibly crosslinked, biodegradable and good-biocompatibility PTMC, the dithiolane on the side chain can provide reversible crosslinking sensitive to reduction, so that long circulation of the drug in blood can be ensured, and the drug can be rapidly crosslinked in cells to be released into target cells; the shell is PEG and has targeting molecules such as monoclonal antibody and the like, and can be specifically combined with cancer cells; the small size of the vesicles, as well as the tumor-specific targeting, allows the vesicles to efficiently deliver VCRs into tumor cells.
2. The medicine-carrying vesicle disclosed by the invention has obvious anti-tumor effect inside and outside the body, has good biocompatibility of the polymer, can form the vesicle with an asymmetric membrane structure, and has good medicine-carrying effect.
3. The degradable polymer vesicle carrier avoids the defects of large particle size, poor in vivo circulation stability, low tumor cell selectivity, slow release of a VCR in cells and the like of the conventional nano carrier.
4. The vesicle system provided by the invention has a plurality of unique advantages, including small size, simple and controllable preparation, excellent biocompatibility, high in-vivo circulation stability, strong specific selectivity of tumor cells, high release speed of intracellular drugs, remarkable tumor growth inhibition effect and the like. Therefore, the vesicle system is expected to become a simple and multifunctional nano platform for efficient and specific targeted delivery of VCRs to multiple myeloma cells.
Drawings
FIG. 1 shows example one in which N is3Nuclear magnetic spectrum of PEG-P (TMC-DTC).
FIG. 2 shows the nuclear magnetic spectrum of PEG-P (TMC-DTC) -NPC in example two.
FIG. 3 shows PEG-P (TMC-DTC) -KD in example two5Nuclear magnetic spectrum of (1).
FIG. 4 is the mass spectrum of macromolecules of Dar and Dar-DBCO in example five.
FIG. 5 is a graph of the stability of Dar-Ps-VCR in the presence of serum at high dilution in example six.
FIG. 6 is the VCR release behavior of Dar-Ps-VCR under non-reducing conditions and 10 mM GSH in example six.
FIG. 7 shows the endocytosis of Dar-Ps-Cy5 in LP-1 cells at different targeting densities and (B) the LP-1 cells and Dar in example seven4.4CLSM pictures (scale: 25 μm) after 4 hours of incubation with Ps-Cy5 and Ps-Cy 5.
FIG. 8 is a graph of the toxicity of Dar-Ps-VCR, Ps-VCR and free VCR in LP-1 cells at different targeting densities in example eight.
FIG. 9 is the toxicity of Dar-Ps-VCR and Ps-VCR in (A) MV4-11 cells and (B) L929 cells and (C) Dar-Ps and (D) Dar in LP-1 cells in example eight.
FIG. 10 is a flow cytometer as described in example nine to determine the apoptosis of LP-1 cells induced by Dar-Ps-VCR, Ps-VCR and free VCR.
FIG. 11 is the experimental example of ten in vitro bioluminescence imaging to observe the tumor distribution of LP-1-Luc tumor in each organ, skull and hind leg bone of mouse.
FIG. 12 is a graph of in vivo fluorescence images of the mice with mid-eleventh mid-maximal LP-1-Luc multiple myeloma obtained from the example, after tail vein injections of Dar-Ps-Cy5 and Ps-Cy5 at different time points.
FIG. 13 is a graph of the construction and treatment workflow of the mouse model for orthotopic LP-1-Luc multiple myeloma and in vivo imaging evaluation of the therapeutic effect of Dar-Ps-VCR on the mouse model for orthotopic LP-1-Luc multiple myeloma in the twelfth example.
FIG. 14 shows changes in Luc fluorescence signals of mice of different treatment groups in twelve examples; body weight change and Kaplan-Meier survival plots.
FIG. 15 is a micro-CT image of Femur (Femur) and Tibia (Tibia) of mice of different treatment groups in twelve examples.
FIG. 16 is an analysis of the indices of the femur (A) and tibia (B) of mice of different treatment groups in the twelve examples. (BMD: bone mineral density; BV/TV: bone volume fraction; Tb.N: number of trabeculae; Tb.Sp: trabecular resolution; BS/TV: bone surface area; Tb.Th: trabecular thickness).
Detailed Description
The VCR-loaded reversible cross-linked degradable polymer vesicle is obtained by self-assembling amphiphilic triblock polymers and simultaneously self-crosslinking; the molecular chain of the triblock polymer comprises a hydrophilic chain segment, a hydrophobic chain segment and KD molecules which are sequentially connected; the hydrophilic chain segment is polyethylene glycol (PEG) with the molecular weight of 3000-8000 Da; the hydrophobic chain segment is a polycarbonate chain segment, and the molecular weight of the hydrophobic chain segment is 2.1-5.7 times of the molecular weight of the hydrophilic chain segment; the molecular weight of the KD polypeptide is 15% -50% of that of the PEG hydrophilic segment.
PEG-P (TMC-DTC) -KD of the inventionzThe polymer is reacted with KD after the hydroxyl at the end of PEG-P (TMC-DTC) is activated by chloroformic acid P-nitrophenyl ester (P-NPC)zThe synthetic method is characterized by comprising the following steps:
Figure 16212DEST_PATH_IMAGE002
wherein in the step (i), the reaction conditions are anhydrous Dichloromethane (DCM) and pyridine at 25 ℃ for 24 hours; in step (ii), the reaction conditions are anhydrous Dimethylsulfoxide (DMSO), KDzTriethylamine, 30 ℃ and 48 hours.
The specific synthesis steps are as follows:
(1) in an ice-water bath, pyridine was added to a solution of PEG-P (TMC-DTC) in anhydrous DCM, and after stirring for 10 minutes, a solution of P-NPC in DCM was slowly added dropwise thereto. After dropwise adding, continuously reacting for 24 hours at room temperature (about 30 minutes), then carrying out suction filtration to remove pyridine salt, collecting polymer solution, carrying out rotary evaporation and concentration to 100 mg/mL, precipitating by using ethyl acetate, and carrying out vacuum drying to obtain a product PEG-P (TMC-DTC) -NPC;
(2) weighing KD under nitrogen protectionzThe polypeptide was placed in a two-necked round-bottomed flask and dissolved completely by adding anhydrous DMSO, triethylamine was added under stirring, and then a solution of PEG-P (TMC-DTC) -NPC in anhydrous DMSO was added dropwise thereto over 30 minutes. After reacting for 2 days at 30 ℃, dialyzing with DMSO containing 5% anhydrous methanol for 36 hours (replacing the medium for 4-5 times) to remove unreacted KDzAnd P-nitrophenol generated by the reaction is dialyzed for 6 hours by DCM, then polymer solution is collected and concentrated by rotary evaporation to the concentration of about 50 mg/mL, and the polymer solution is precipitated in glacial ethyl ether and dried in vacuum to obtain white flocculent polymer PEG-P (TMC-DTC) -KDz. Conventional replacement of TMC with LA or CL gives PEG-P (LA-DTC) -KDz、PEG-P(CL-DTC)-KDz。
The raw materials related to the invention are the existing commercial raw materials, and the specific preparation method and the test method are the conventional technologies in the field; the invention is further described below with reference to examples and figures:
EXAMPLE A Synthesis of Polymer N3-PEG-P(TMC-DTC)
Polymer N3PEG-P (TMC-DTC) is catalyzed by DPP, N3the-PEG-OH is a macroinitiator and is obtained by initiating TMC and DTC ring-opening copolymerization. Firstly, weighing N in a glove box under a nitrogen environment3-PEG-OH（M n Putting 0.79 g/mol, 0.1 mmol, TMC (1.50 g, 14.8 mmol) and DTC (0.20 g, 1.0 mmol) in a closed reactor, adding 5.0 mL of anhydrous DCM for dissolution, then adding DPP (0.25 g, 1.2 mmol), sealing the reactor, transferring out of a glove box, and placing at 30 ℃ for reaction for four days. After the reaction is finished, the mixture is precipitated twice by using glacial ethyl ether and dried in vacuum to obtain white flocculent polymer N3PEG-P (TMC-DTC), yield: 85.4 percent. In FIG. 1, N at 3.38 and 3.63 ppm can be seen3Characteristic peaks for PEG, for TMC at 2.03 and 4.18 ppm, and for DTC at 2.99 and 4.22 ppm. N can be calculated by the ratio of the integrated area of methylene hydrogen at 2.03 and 2.99 ppm to the integrated area of PEG methylene hydrogen at 3.63 ppm3-PEG-P(TMC-DTC) polymer having a molecular weight of 7.9- (15.0-2.0) kg/mol and a molecular weight distribution of 1.1 by GPC was used in the following examples.
Will N3-PEG-OH by CH with molecular weight of 5k3PEG-P (TMC-DTC) (5.0- (15.0-2.0) kg/mol) was obtained by the above preparation method without changing the amount of O-PEG-OH.
EXAMPLE two Synthesis of the Polymer PEG-P (TMC-DTC) -KDz
Polymer PEG-P (TMC-DTC) -KDzThe synthesis of (D) is divided into two steps, i.e. after the terminal hydroxyl of PEG-P (TMC-DTC) (5.0- (15.0-2.0) kg/mol) is activated by P-NPC, the hydroxyl and KD are reactedzPolypeptide molecule reaction. With PEG-P (TMC-DTC) -KD5The synthesis of (1.0 g, 45.5. mu. mol) of PEG-P (TMC-DTC) was dissolved in 10 mL of anhydrous DCM under nitrogen atmosphere, and then transferred to an ice-water bath and pyridine (18.0 mg, 227.5. mu. mol) was added, and after stirring for 10 minutes, a solution of P-NPC (48.4 mg, 240.3. mu. mol) in DCM (1.0 mL) was added dropwise. And (3) continuing to react for 24 hours at room temperature after dropwise adding for 30 minutes, then carrying out suction filtration to remove pyridine salt, collecting the polymer solution, carrying out rotary evaporation and concentration to 100 mg/mL, precipitating with glacial ethyl ether, and carrying out vacuum drying to obtain a product PEG-P (TMC-DTC) -NPC, wherein the yield is as follows: 90.0 percent. Subsequently, KD is weighed under nitrogen protection5(60.0 mg, 83.4. mu. mol) was dissolved in 4 mL of anhydrous DMSO and triethylamine (4.2 mg, 41.7. mu. mol) was added, and then a solution of PEG-P (TMC-DTC) -NPC in anhydrous DMSO (9.0 mL) was added dropwise thereto under stirring for 30 minutes, and the dropwise addition was completed. After 2 days at 30 ℃, dialyzing with DMSO containing 5% anhydrous methanol for 36 hours (exchange of 4-5 media) to remove unreacted KD5And P-nitrophenol generated by the reaction is dialyzed for 6 hours by DCM, then polymer solution is collected and concentrated by rotary evaporation until the concentration of the polymer is 50 mg/mL, and the polymer is precipitated in glacial ethyl ether and dried in vacuum, thus obtaining white flocculent polymer PEG-P (TMC-DTC) -KD5Yield, yield: 91.0 percent. FIGS. 2 and 3 are PEG-P (TMC-DTC) -NPC and PEG-P (TMC-DTC) -KD5Nuclear magnetic hydrogen spectrum diagram of (1). From FIG. 2, characteristic peaks of P-NPC (7.41 and 8.30 ppm) and of PEG-P (TMC-DTC) (2.03, 2.99, 3.38, 3.63, 4) can be seen18 and 4.22 ppm) and the grafting ratio of NPC was about 100% as calculated from the ratio of the integrated area of the characteristic peak of p-NPC to the area of the PEG methyl hydrogen peak at 3.38 ppm. FIG. 3 shows that the characteristic peaks of NPC at 7.41 ppm and 8.30 ppm disappear, and a new signal peak appears at 4.54 ppm, namely KD5Characteristic peak of the medium methine group. KD is calculated by comparing the ratio of the peak area at 4.54 ppm to the hydrogen peak area of TMC methylene at 1.95 ppm5The degree of substitution is-100%. In addition, KD was determined by High Performance Liquid Chromatography (HPLC)5The grafting ratio of (A) is 100%, and PEG-P (TMC-DTC) -KD is proved5The successful synthesis of (1) was used in the following examples.
Example preparation of reversibly Cross-Linked biodegradable vesicles (Ps-VCR) with triple Loading of VCR
The Ps-VCR is prepared by a solvent displacement method, wherein the VCR is reacted with KDzThe electrostatic interaction between them. PEG-P (TMC-DTC) -KDzDissolved in DMSO (40 mg/mL), 100. mu.L of the mixture was added to 900. mu.L of HEPES (pH 6.8, 10 mM) containing VCR which was allowed to stand, stirred at 300 rpm for 3 minutes, and dialyzed against HEPES (pH 7.4, 10 mM) for 8 hours to obtain Ps-VCR. Where the theoretical drug loading of the VCR was set at 4.8-11.1 wt.%, the resulting Ps-VCR was found to have a particle size between 26-40 nm and a particle size distribution between 0.05-0.20 (table 1). The encapsulation rate of the Ps-VCR is up to 97.2 percent by measuring the absorbance value of the Ps-VCR under the wavelength of 298 nm through an ultraviolet visible spectrum. Based on the same method, the PEG-P (LA-DTC) -KD is lower than the theoretical drug loading of 4.8 percent5、PEG-P(CL-DTC)-KD5The encapsulation efficiency of the prepared Ps-VCR is 88.3 percent and 83.9 percent respectively; the drug-carrying vesicle prepared by PEG-P (TMC-DTC) two-block polymer has a particle size of about 75 nm, and VCR has a low encapsulation rate of only 14.1%.
Figure 999212DEST_PATH_IMAGE003
EXAMPLE four preparation of reversibly Cross-Linked biodegradable vesicles (Ps-drug) loaded with other drugs
Using a similar method as in example three, the entrapment of reversibly cross-linked degradable vesicles on other drugs such as verapamil hydrochloride (VER), irinotecan hydrochloride (CPT), resiquimod (R848) was investigated. Research shows that after different medicines are loaded, the particle size of the obtained Ps-drug is between 20 and 40 nm, and specific results are shown in Table 2.
Figure 66525DEST_PATH_IMAGE004
Example five preparation of VCR-Supported monoclonal antibody-directed polymersome (Ab-Ps-VCR)
Ab-Ps-VCR through azide functionalized polymersome VCR nano-drug (N)3Ps-VCR) surface post-modified dibenzocyclooctyne functionalized monoclonal antibody (Ab-DBCO). N is a radical of3-Ps-VCR from N3PEG-P (TMC-DTC) and PEG-P (TMC-DTC) -KDzCo-assembling while wrapping a VCR, wherein N3The content of-PEG-P (TMC-DTC) is 1-10 wt.%. Specifically, so as to contain 2% of N3N of PEG-P (TMC-DTC)3Preparation of-Ps-VCR As an example, 8.0 mg N was weighed3PEG-P (TMC-DTC) and 392.0 mg PEG-P (TMC-DTC) -KD5(the molar ratio is 2: 98) is dissolved in DMSO (the total concentration of the polymer is 40 mg/mL), meanwhile, 4.0 mL of VCR aqueous solution (5 mg/mL) is added into 90 mL of HEPES (pH 6.8, 10 mM) to be uniformly mixed, 10 mL of polymer solution is injected into the mixture under static condition, and after stirring for 5 minutes, the mixture is placed at 37 ℃ and stands for 4 hours. Dialyzing (MWCO: 14 kDa) against HEPES (pH 7.4, 10 mM) for 8 hours to remove the organic solvent, and removing the free VCR by using a nanofiltration system to obtain N3-Ps-VCR. Dynamic Light Scattering (DLS) measurement of N3The particle size of the Ps-VCR was 36 nm and the distribution was narrow (PDI: 0.11). The encapsulation rate was as high as 97.2% with a VCR theoretical drug load of 4.8 wt.% and a drug load of 4.6 wt.%. To efficiently bind the mAbs, N is then introduced using a tangential flow device3the-Ps-VCR was concentrated from 4 mg/mL to 18.6 mg/mL for convenient storage and improved binding efficiency of the mAb. After concentration N3The particle size of the Ps-VCR was 42 nm, and the PDI was 0.07. The particle size of the composite material is kept about 40 nm during 180 days of storage at 4 ℃, PDI is less than 0.17, and the leakage rate of a VCR is less than 0.6 percent, which shows that N is3-Ps-VCR has advantagesExceptional long-term storage stability (table 3).
Figure 756263DEST_PATH_IMAGE005
Ab-DBCO through small-molecule NHS-OEG4the-DBCO is prepared by amidation reaction with amino on the monoclonal antibody, wherein the functionalization degree of DBCO can be changed by changing Ab and NHS-OEG4-the molar ratio of DBCO is adjusted. Taking the preparation of DBCO-functionalized darunavir (Dar-DBCO) as an example, a PBS solution of Dar (21.7 mg/mL) was diluted to 10 mg/mL with PB (pH 8.5, 10 mM), 200. mu.L of which was added with 3-or 5-fold molar equivalent of NHS-OEG under shaking4A DMSO solution of DBCO (5 mg/mL) in a shaker at 27 ℃ and 120 rpm for overnight reaction. After completion of the reaction, unreacted NHS-OEG was removed by centrifugation with an ultrafiltration tube (MWCO: 10 kDa, 3000 rpm)4DBCO and washed twice with PBS (pH 7.4, 10 mM) for ultrafiltration to give Dar-DBCO. When Dar and NHS-OEG4The molar ratio of DBCO was 1: 3 and 1: 5, the modification of each Dar by 1.5 and 2.8 DBCO respectively (FIG. 4), indicated as Dar-DBCO, was determined by time of flight mass spectrometry (MALDI-TOF-MS) and is expressed as Dar-DBCO1.5And Dar-DBCO2.8. In order to maintain the targeting property and biological activity of the monoclonal antibody to the maximum extent, Dar-DBCO is adopted in the follow-up process1.5Or other monoclonal antibodies modified with 1.5-2 DBCO.
By N3N of the surface of the Ps-VCR3The click chemistry reaction triggered by the tension between the Dar-DBCO can be simply prepared to obtain the Dar-Ps-VCR, and the surface density of the Dar can be adjusted by changing the feeding ratio. Setting Dar-DBCO and N3In a molar ratio of 0.25: 1, 0.5: 1 and 1: 1, respectively, i.e. at 107.5. mu. L N310.4, 20.9 and 41.8. mu.L of Dar-DBCO solution (5.6 mg/mL) were added to a Ps-VCR (18.6 mg/mL), respectively, and then reacted overnight in a shaker at 25 ℃ and 100 rpm. Unbound Dar-DBCO was removed by ultracentrifugation (58 krpm, 4 ℃ C., 30 minutes) and washed twice with HEPES (pH 7.4, 10 mM), while Dar-Ps-VCR and supernatant were collected to determine the amount of Dar bound. Unbound Dar-DBCO in the supernatant was determined by HPLCThe absolute molecular weights (1.15X 10) of the polymersome were determined by calculating the amount of Dar on the surface of the polymersome as 28.6, 56.4 and 112.2. mu.g per mg, respectively, and measured by multi-angle laser light scattering7g/mol) and aggregation number (523) calculation revealed 2.2, 4.4 and 8.7 dars bound to each Dar-Ps-VCR surface, respectively (Table 4). As the Dar density increases, the particle size of the Dar-Ps-VCR slightly increases (43-49 nm) and the particle size distribution is narrower (PDI: 0.14-0.21), and after the monoclonal antibody is connected, the encapsulation result is the same as that of the N in the example3-as in the Ps-VCR.
Figure 47567DEST_PATH_IMAGE006
Other monoclonal antibody-directed VCR-loaded polymersomes, such as Isa-Ps-VCR and Anti-CD38-Ps-VCR, were prepared in a manner similar to that of Dar-Ps-VCR. The particle size is between 40 nm and 60 nm, the particle size distribution is narrow (PDI: 0.10-0.30), and the number of monoclonal antibodies on the surface of each vesicle is 1-10.
Prior Art CN110229323A non-targeting vesicles (KD) carrying saporin protein (SAP) as disclosed in Table 75) After ultracentrifugation (58 krpm, 4 ℃ and 30 minutes), DLE is reduced from 68.3% to 23%, and a large amount of medicine leaks, indicating that the DLE cannot be connected with the targeting monoclonal antibody.
Example stability and in vitro drug Release of HeAb-Ps-VCR Targeted Polymer vesicle Nanomedicine
The Dar containing 4.4 dars per vesicle surface is adopted4.4And (4) taking the-Ps-VCR as a representative, and researching the stability and in-vitro drug release behavior of the Ab-Ps-VCR targeting vesicle nano-drug. The stability of the Dar-Ps-VCR is respectively diluted by 50 times by adopting a phosphoric acid buffer solution or 10 percent of fetal calf serum is added, and the particle size change of the Dar-Ps-VCR is detected by dynamic light scattering. FIG. 5 is a graph showing the particle size distribution of the Dar-Ps-VCR stability. The result shows that the Dar-Ps-VCR targeted vesicle nano-drug has good stability and maintains intact particle size and particle size distribution after being diluted by 50 times and added with 10% FBS for 24 hours.
The in vitro drug release behavior of Dar-Ps-VCR was studied by dialysis, in which there were 2 release media, HEPES (pH 7.4, 10 mM) and HEPES solution containing 10 mM GSH (nitrogen atmosphere). First 0.5 mL of Dar-Ps-VCR (0.5 mg/mL) was loaded into a release bag (MWCO: 14 kDa) and then placed in 20 mL of the corresponding release medium in a shaker at 37 ℃ and 100 rpm. At set time points (0, 1, 2, 4, 6, 8, 10, 12, 24 h) 5 mL of dialysate was removed and supplemented with 5 mL of fresh medium. VCR content in the dialysate was determined by HPLC (mobile phase methanol: water (15% triethylamine added, pH adjusted to 7.0 with phosphoric acid) = 70: 30). FIG. 6 is a graph showing the in vitro release results of Dar-Ps-VCR targeted vesicle nano-drugs. The results show that the release of the VCR in 12 hours by the Dar-Ps-VCR under the reducing condition of 10 mM GSH reaches more than 85%, while the cumulative release of the VCR in 24 hours under the non-reducing condition is only about 22%.
Example endocytosis of seven Dar-Ps-VCR targeting Polymer vesicle Nanoparticulate drugs
The VCR is not fluorescent, the polymer vesicle is marked by Cy5, the preparation method of Dar-Ps-Cy5 refers to example five, and the preparation method of Ps-Cy5 refers to example three; the uptake of Dar-Ps-Cy5 at different Dar densities in LP-1 cells was studied by flow cytometry and laser scanning confocal microscopy (CLSM). In flow experiments, LP-1 cell suspension was first plated in 6-well plates (2X 10)5One/well), after incubation in an incubator for 24 hours, 200. mu.L of Dar-Ps-Cy5 and Ps-Cy5 (2.0. mu.g/mL in Cy5 wells) was added to each well, using PBS group as a control. After a further 4 hours of incubation, the cells were harvested by centrifugation (800 rpm, 5 minutes) and washed twice with PBS, and finally dispersed with 500. mu.L PBS and placed in a flow tube for assay. The test result shows that the endocytosis amount of Dar-Ps-Cy5 in LP-1 cells is obviously higher than that of Ps-Cy5, wherein Dar and Cy are combined4.4The cells incubated with-Ps-Cy 5 had the highest fluorescence intensity, which was 6.4 times higher than that of the Ps-Cy5 control group (fig. 7A), indicating that the introduction of Dar significantly enhanced the cellular uptake of Ps-Cy5 and the targeting was optimal when 4.4 dars were bound to the surface of each vesicle.
Subsequent further study of Dar with CLSM4.4-endocytosis of Ps-Cy5 and Ps-Cy5 in LP-1 cells. The specific experimental steps are as followsSmall discs pretreated with polylysine (300. mu.L, 0.1 mg/mL) were placed in 24-well plates and LP-1 cell suspension (3X 10)5One/well), after 24 hours of incubation in an incubator, 200. mu.L of Dar was added thereto, respectively4.4Ps-Cy5 and Ps-Cy5 (Cy 5 in well at a concentration of 40. mu.g/mL). After a further incubation time of 4 hours the medium was carefully removed, washed 3 times with PBS, then fixed for 15 minutes with 4% paraformaldehyde solution, washed 3 times with PBS, stained nuclei with DAPI for 3 minutes, washed 3 times with PBS, finally mounted with glycerol and observed and photographed with CLSM (Leica, TCS SP 5). FIG. 7B shows Dar4.4Graphs of the results of uptake of Ps-Cy5 and Ps-Cy5 in LP-1 cells. The results show that when LP-1 cells were associated with Dar4.4After the-Ps-Cy 5 is incubated for 4 hours, the periphery of the cell nucleus presents obvious red fluorescence, and the fluorescence in the cell incubated with the Ps-Cy5 is weak, which indicates that the Dar-Ps-Cy5 has excellent targeting property and high-efficiency and rapid endocytosis.
Example cytotoxicity test of eight Dar-Ps-VCR Targeted polymersome Nanoparticles
In vitro anti-tumor activity of Dar-Ps-VCR on LP-1 multiple myeloma cells was determined using the CCK-8 kit with MV4-11 cells as a control. The LP-1 cells were first plated in 96-well plates (15000 cells/well) at 37 ℃ with 5% CO2After 24 hours of incubation in the incubator of (1), 20. mu.L of Dar-Ps-VCR, Ps-VCR and free VCR containing different Dar surface densities were added to each well, and the final concentrations of VCR in the wells were 0.001, 0.01, 0.05, 0.1, 0.5, 1 and 10 ng/mL, respectively. After 48 hours of incubation at 37 ℃, 10 μ L of CCK-8 solution was added to each well for further incubation for 4 hours, and finally the absorbance at 492 nm was measured with a microplate reader. Cell viability was calculated by the ratio of absorbance values of the experimental groups to those of cells cultured with PBS added, and the experiments were performed in parallel in four groups (mean ± SD, z = 4). FIG. 8 is a graph showing the cytotoxicity results of Dar-Ps-VCR vesicle nano-drugs (z is 5) with different targeting densities on LP-1 cells. The results show that when 4.4 dars are bonded to the surface of each vesicle (Dar)4.4Ps-VCR) was most cytotoxic, with a semi-lethal concentration (IC)50) As low as 0.07 ng/mL, compared to free VCR (IC)50: 1.38 ng/mL) and non-targetingControl group Ps-VCR (z 5, IC)50: 0.85 ng/mL) decreased by 20 and 12 fold, respectively, suggesting that the introduction of Dar significantly increased the targeted delivery and intracellular rapid release of VCRs.
MV4-11 cells (12000 cells/well) and L929 fibroblasts (3000 cells/well) were plated in 96-well plates and cultured for 24 hours, and then 20. mu.L of Dar was added to each well4.4Ps-VCR (z 5) and Ps-VCR (z 5), the final concentration of VCR in the wells was 0.0001-100 ng/mL. MV4-11 cells were incubated at 37 ℃ for 48 hours, 10. mu.L of CCK-8 solution was added per well for further incubation for 4 hours, and their absorbance at 492 nm was measured with a microplate reader. After 48 hours of incubation of L929 cells at 37 ℃, 10 μ L of MTT in PBS solution (5 mg/mL) was added to each well for 4 hours of incubation, after which the medium was carefully removed and 150 μ L of DMSO was added to dissolve formazan crystals produced, which were tested for absorbance at 570 nm with a microplate reader; the results showed that IC was in MV4-11 cells50Was 20 times higher in LP-1 cells (FIG. 9A). More interestingly, for L929 normal cells, Dar was observed even at VCR concentrations as high as 100 ng/mL4.4Neither the-Ps-VCR nor the Ps-VCR showed significant toxicity, and the cell viability was close to 100% (FIG. 9B). The results taken together indicate that Dar-Ps-VCR can selectively target and kill multiple myeloma cells with high efficiency and is less toxic to normal cells.
In addition, the toxicity of Dar-Ps and Ps vacuoles and free Dar to LP-1 cells was tested in the same manner, and the results showed that the cell viability was close to 100% and no cytotoxicity was evident even at a concentration of Ps as high as 30. mu.g/mL (FIG. 9C) and a concentration of Dar of 9. mu.g/mL (FIG. 9D).
In the following examples, the Dar-Ps-VCR means Dar4.4the-Ps-VCR vesicle nano-drug (z is 5) and the Dar-Ps-Cy5 are all Dar4.4-Ps-Cy5 (z is 5).
Example conditions of apoptosis induction by nine-Dar-Ps-VCR targeting polymersome Nanomedicines
Apoptosis experiments with Dar-Ps-VCR were performed by double staining with the fluorescent dye AnnexinV-FITC/PI, followed by testing using a flow cytometer. LP-1 cells were first plated at 2X 105Is/areThe wells were plated in 6-well plates and after 24 hours of incubation in an incubator, 200. mu.L of Dar-Ps-VCR, Ps-VCR and free VCR (VCR concentration in well: 0.5 ng/mL) were added, respectively, and PBS-only cells were used as controls. After 48 hours incubation in an incubator, LP-1 cells were harvested by centrifugation (800 rpm, 5 minutes) and washed twice with ice PBS, and finally 200. mu.L of Binding buffer (Binding buffer) was added to each sample to resuspend the cells (cell density approximately 10)6one/mL). After the mixture is blown to be uniform, 100 mu L of the mixture is taken out and put into a flow tube, 5 mu L of annexin V-FITC and 10 mu L of PI solution are sequentially added, after the mixture is dyed for 15 minutes in a dark place at room temperature, 400 mu L of PBS is added and mixed uniformly, and the flow cytometry is used for measuring within 1 hour. Wherein PBS group samples treated in a 50 ℃ water bath for 5 minutes and fixed with 4% paraformaldehyde for 5 minutes are respectively used as an early-withering group and a late-withering group, and 5 mul of AnnexinV-FITC solution and 10 mul of PI solution are respectively added for dyeing for 15 minutes. FIG. 10 is a graph showing the results of induction of apoptosis of LP-1 cells by Dar-Ps-VCR. The results show that Dar-Ps-VCR can effectively induce apoptosis, 60.8% of apoptosis can be caused when the concentration of VCR is 0.5 ng/mL, the apoptosis rate is obviously higher than that of a non-targeted control Ps-VCR group (43.4%) and a free VCR group (31.4%), and the number of late-stage apoptotic cells in each group is obviously more than that of early-stage apoptosis.
Example construction of Ten-Hold LP-1-Luc in situ multiple myeloma mouse model
All animal experiments and procedures were approved by the experimental animal center at suzhou university and the animal care and use committee at suzhou university. Establishing an in-situ MM tumor model: using ZOD/SCID female mice of 6 weeks of age, mice were first myeloablated by intraperitoneal injection of 10 mg/mL cyclophosphamide solution for two consecutive days, each mouse was injected with 2 mg each time, and LP-1-Luc cells (8X 10) were injected on the third day6One/one) was injected into the mice via tail vein, and live imaging and treatment started on day 10 after inoculation while the mice were weighed. To study tumor distribution in the LP-1-Luc orthotopic multiple myeloma mouse transplantation model, on day 35 post-inoculation, mice were injected intraperitoneally with fluorescein potassium salt, and 8 minutes later, mice were dissected and harvested for fluorescence imaging of heart, liver, spleen, lung, kidney, intestine, skull, and hind leg bones. Attached withFIG. 11 is a graph of in vitro bioluminescence imaging results for LP-1-Luc in situ multiple myeloma mouse organs, skull and hind leg bone. From the pictures, it can be seen that the LP-1-Luc tumor is mainly concentrated on the hind legs and the skull of the mice 35 days after inoculation, and no obvious tumor Luc signal distribution condition exists in organs such as heart, liver, spleen, lung, kidney and the like.
EXAMPLE in vivo imaging experiments of eleven Dar-Ps-Cy5 in LP-1-Luc in situ multiple myeloma mice
Distribution of Dar-Ps-Cy5 in LP-1-Luc in situ multiple myeloma mice was analyzed by in vivo imaging of mice. On day 37 after inoculation (when mice are about to develop disease), 200 μ L of Dar-Ps-Cy5 and Ps-Cy5 solutions (250 μ g Cy5 equiv./kg) were injected into the mice separately through the tail vein, and after injection live fluorography was performed on isoflurane anesthetized mice at 1, 2, 4, 6, 8, 10, 12, and 24 hours, respectively, and biodistribution maps in mouse imaging myeloma mice were performed using Lumia II software (fig. 12). The results show that Dar-Ps-Cy5 can be efficiently targeted and enriched to tumor sites, and the fluorescence signals of the Dar-Ps-Cy5 at the leg and the skull of the mouse are significantly higher than those of the non-targeted Ps-Cy5 group.
Example antitumor Effect of twelve Dar-Ps-VCR in LP-1-Luc in situ multiple myeloma mice
To investigate the antitumor effect of Dar-Ps-VCR on LP-1-Luc multiple myeloma mice in situ, the bioluminescence intensity reached 1.2X 10 at day 10 after inoculation6 p/sec/cm2Treatment experiments were started at/sr. There are two dosing regimens, keeping the total VCR dosed the same: one is a VCR dose of 0.25 mg/kg, 4 needles given for 4 days, for a total of 4 needles, denoted Dar-Ps-VCR (0.25 mg VCR equiv./kg, Q4 d); another is a VCR dose of 0.50 mg/kg, with one injection given for 8 days, for a total of 2 injections, denoted Dar-Ps-VCR (0.50 mg VCR equiv./kg, Q8 d). According to the first dosing regimen, equal VCR doses of Ps-VCR and free VCR, equal equivalents of Dar-Ps and PBS were used as controls. There were 10 tumor-bearing mice per treatment group, 4 of which were used for bioluminescence imaging and 6 were used to monitor body weight and to observe survival. The research finds that the LP-1-Luc cells of the mice in the PBS group continuously and rapidly grow and are inoculatedThe bioluminescence intensity reaches 1.0 multiplied by 10 after 37 to 45 days9 p/sec/cm2Onset of disease at/sr was manifested by paralysis of the legs, weight loss and death (FIG. 13). During the administration treatment period (10-22 days), the Luc signals of mice of two administration modes of Dar-Ps-VCR, Ps-VCR and free VCR groups have no obvious increase or even slightly decrease, which shows that the mice can effectively inhibit the diffusion and proliferation of LP-1-Luc cells in the mice. After the dosing was completed, tumors in the non-targeted Ps-VCR and free VCR groups of mice began to recur and the Luc bioluminescence signal increased rapidly, 157 and 53-fold at day 39, respectively. However, two groups of mice injected with Dar-Ps-VCR in tail vein still can continuously inhibit the growth of LP-1-Luc tumor, no obvious Luc signal is detected even at the 39 th day after inoculation, the fluorescence value of the Luc signal is equivalent to the background signal of healthy mice, and the fluorescence value is reduced by 235 and 114 times compared with the Ps-VCR and free VCR groups respectively and is reduced by about 5000 times compared with untreated PBS group. The tumor growth trend in mice in the empty vector Dar-Ps group was similar to that in the PBS group (fig. 14A). These results indicate that Dar-Ps-VCR can efficiently target VCR to the tumor site, completely eradicating the tumor in a short period of time. Notably, all treatment groups of mice showed no significant weight change during the dosing period until the mice developed weight loss, indicating their relatively low toxic side effects (fig. 14B). In addition, survival was significantly increased in the Dar-Ps-VCR treated group mice (fig. 14C), where median survival was 156 and 154 days for the mice with 0.25 mg VCR equiv/kg, Q4d and 0.50 mg VCR equiv/kg, Q8d, respectively, which was 3.0-3.6 fold longer than in the PBS group (43 days), Dar-Ps group (49.5 days), Ps-VCR (51 days) and free VCR group (52 days). Taken together, these results indicate that the introduction of Dar significantly increases the selective targeting of Ps-VCR, thereby efficiently inhibiting the growth of in situ multiple myeloma.
Osteolytic lesions are one of common clinical manifestations of MM patients, so the micro-CT is adopted to evaluate relevant indexes of femurs and tibias of mice in each treatment group. As a result, it was found that severe osteoclasts were present in the hind leg bones of the mice in PBS group and Dar-Ps group, and the trabecular bone was largely lost, and the osteolytic disease of the mice was significantly improved after the treatment with Dar-Ps-VCR, similarly to the case of healthy mice (FIG. 15). Then, through further quantitative analysis, the indexes of bone mineral density, bone volume fraction, number of trabeculae, separation degree of trabeculae and bone surface area of the thighbone and the tibia of mice in the Dar-Ps-VCR target treatment groups under the two administration modes are similar, and the indexes are remarkably different from those of an untreated PBS group and are not remarkably different from those of healthy mice (figure 16). In addition, some indexes such as bone volume fraction and the like also have significant difference with a non-target Ps-VCR control group, and further shows that the introduction of Dar obviously increases the curative effect of the Ps-VCR on in-situ multiple myeloma and effectively inhibits osteolytic lesion.

Claims (10)

1. The drug-loaded polymer vesicle is characterized by being prepared from a small molecular drug and an amphiphilic block polymer; or prepared from micromolecular drugs, amphiphilic block polymers, functionalized amphiphilic block polymers and targeting molecules;
the molecular structural formula of the amphiphilic block polymer is as follows:
Figure DEST_PATH_426968DEST_PATH_IMAGE001
wherein z is 5-15.
2. The drug-loaded polymer vesicle according to claim 1, wherein in the amphiphilic block polymer, the molecular weight of the PEG chain segment is 3000-8000 Da; the molecular weight of the hydrophobic chain segment is 2.5-6 times of the molecular weight of PEG; the molecular weight of the PDTC chain segment is 8-30% of that of the hydrophobic chain segment.
3. The drug-loaded polymersome of claim 1, wherein the small molecule drug is vincristine sulfate, doxorubicin hydrochloride, epirubicin hydrochloride, verapamil hydrochloride, irinotecan hydrochloride, and resiquimod.
4. The drug-loaded polymersome of claim 1, wherein the targeting molecule is a targeting mab.
5. The drug-loaded polymersome of claim 4, wherein the targeting monoclonal antibody is a targeting CD38 monoclonal antibody.
6. Use of the drug-loaded polymer vesicle of claim 1 for the preparation of an anti-myeloma drug.
7. The preparation method of the drug-carrying polymer vesicle of claim 1, comprising the steps of preparing the drug-carrying polymer vesicle by a solvent displacement method using a small molecule drug and the amphiphilic block polymer as raw materials; or the drug-loaded polymer vesicle is prepared by a solvent displacement method by taking a small molecular drug, the amphiphilic block polymer, the functionalized amphiphilic block polymer and the targeted monoclonal antibody as raw materials.
8. The preparation method of the drug-loaded polymer vesicle of claim 7, wherein a functionalized amphiphilic block polymer and the amphiphilic block polymer are assembled and cross-linked to carry a drug, and then reacted with a targeting monoclonal antibody to prepare the drug-loaded polymer vesicle; the functional group in the functionalized amphiphilic block polymer is N3-, Mal-or NHS-.
9. The use of the amphiphilic block polymer of claim 1 for the preparation of an antimyeloma nanomedicine, wherein the active ingredient of the nanomedicine is a small molecule drug.
10. The amphiphilic block polymer, functionalized amphiphilic block polymer, and targeting molecule of claim 1; the application of the nano-drug in preparation of the anti-myeloma nano-drug is characterized in that the active ingredient of the nano-drug is a small molecule drug.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112076159A (en) * 2020-09-14 2020-12-15 苏州大学 Drug-loaded polymer vesicle with asymmetric membrane structure, preparation method and application in preparation of drug for treating acute myeloid leukemia
CN113679670A (en) * 2021-08-01 2021-11-23 苏州大学 Vesicle nano-drug loaded with chloroquine compound and preparation method and application thereof
WO2022041017A1 (en) * 2020-08-26 2022-03-03 苏州大学 Small molecular drug-loaded polymer vesicle, preparation method therefor and use thereof
CN115887380A (en) * 2022-11-11 2023-04-04 北京大学深圳医院 Organ targeting polymer nano vesicle system and preparation method and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108451907A (en) * 2018-02-09 2018-08-28 苏州大学 Multifunctional polymer vesica is preparing the application in treating Huppert's disease drug
CN109053892A (en) * 2018-09-19 2018-12-21 苏州思坦维生物技术股份有限公司 The monoclonal antibody and the preparation method and application thereof of specific bond people and monkey CD38 antigen
CN110229323A (en) * 2019-05-31 2019-09-13 苏州大学 The polymer vesicle with asymmetric membrane structure for restoring sensitive reversible crosslink and its application in preparation treatment liver-cancer medicine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108451907A (en) * 2018-02-09 2018-08-28 苏州大学 Multifunctional polymer vesica is preparing the application in treating Huppert's disease drug
CN109053892A (en) * 2018-09-19 2018-12-21 苏州思坦维生物技术股份有限公司 The monoclonal antibody and the preparation method and application thereof of specific bond people and monkey CD38 antigen
CN110229323A (en) * 2019-05-31 2019-09-13 苏州大学 The polymer vesicle with asymmetric membrane structure for restoring sensitive reversible crosslink and its application in preparation treatment liver-cancer medicine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WENHAO ZHONG等: "Advancements in nanotechnology for the diagnosis and treatment of multiple myeloma", 《BIOMATERIALS》 *
YINAN ZHONG等: "CD44-targeted vesicles encapsulating granzyme B as artificial killer cells for potent inhibition of human multiple myeloma in mice", 《JOURNAL OF CONTROLLED RELEASE》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022041017A1 (en) * 2020-08-26 2022-03-03 苏州大学 Small molecular drug-loaded polymer vesicle, preparation method therefor and use thereof
CN112076159A (en) * 2020-09-14 2020-12-15 苏州大学 Drug-loaded polymer vesicle with asymmetric membrane structure, preparation method and application in preparation of drug for treating acute myeloid leukemia
CN113679670A (en) * 2021-08-01 2021-11-23 苏州大学 Vesicle nano-drug loaded with chloroquine compound and preparation method and application thereof
CN113679670B (en) * 2021-08-01 2023-05-02 苏州大学 Vesicle nano-drug carrying chloroquine compound, and preparation method and application thereof
CN115887380A (en) * 2022-11-11 2023-04-04 北京大学深圳医院 Organ targeting polymer nano vesicle system and preparation method and application thereof
CN115887380B (en) * 2022-11-11 2024-04-26 北京大学深圳医院 Organ targeting polymer nano vesicle system and preparation method and application thereof

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