SMALL MOLECULAR DRUG-LOADED POLYMER VESICLE, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

The present disclosed a preparation method preparation method for, and use of, a small molecular drug-loaded polymer vesicle. The small molecular drug-loaded polymer vesicle is prepared by assembling an amphiphilic block polymer and a small molecular drug; or is obtained by assembling and cross-linking the amphiphilic block polymer and a functionalized amphiphilic block polymer, loading the small molecular drug, and then reacting with a targeting monoclonal antibody. The vesicle system has many unique advantages, including small size, simple and controllable preparation, excellent biocompatibility, high stability of circulation in vivo, strong specific selectivity of tumor cells, high intracellular drug release rate, remarkable effect of tumor growth inhibition, etc. Therefore, the vesicle system is expected to become a simple and multi-functional nano-platform for efficient and specific targeted delivery of vincristine sulfate to multiple myeloma cells.

Claims

1. A drug-loaded polymer vesicle, wherein the drug-loaded polymer vesicle is prepared from a small molecule drug and an amphiphilic block polymer; or from the small molecule drug, the amphiphilic block polymer, a functionalized PEG-P (TMC-DTC), and a targeting molecule; the chemical structural of the amphiphilic block polymer is one of the following formulas: ##STR00003## wherein, z is 5-15.

2. The drug-loaded polymer vesicle according to claim 1, wherein in the amphiphilic block polymer, a molecular weight of a PEG is 3000-8000 Da; a molecular weight of a hydrophobic chain segment is 2.5-6 times that of the PEG; and a molecular weight of a PDTC chain segment is 8%-30% of that of the hydrophobic chain segment.

3. The drug-loaded polymer vesicle according to claim 1, wherein the small molecule drug is vincristine sulfate, adriamycin hydrochloride, epothilone hydrochloride, verapamil hydrochloride, irinotecan hydrochloride, or resiquimod.

4. The drug-loaded polymer vesicle according to claim 1, wherein a targeting molecule is a targeting monoclonal antibody.

5. The drug-loaded polymer vesicle according to claim 4, wherein the targeting monoclonal antibody is a CD38-targeting monoclonal antibody.

6. Use of the drug-loaded polymer vesicle according to claim 1 in preparing anti-myeloma drugs.

7. A method for preparing the drug-loaded polymer vesicle according to claim 1, wherein the method comprises the following steps: preparing the drug-loaded polymer vesicle by a solvent displacement method using a small molecule drug and an amphiphilic block polymer as starting materials; or preparing the drug-loaded polymer vesicle by a solvent displacement method using the small molecule drug, the amphiphilic block polymer, a functionalized amphiphilic block polymer, and a targeting monoclonal antibody as starting materials.

8. The method for drug-loaded polymer vesicle according to claim 7, wherein the drug-loaded polymer vesicle is prepared by assembling and cross-linking a functionalized amphiphilic block polymer with the amphiphilic block polymer and loading the small molecular drug, and then reacting with the monoclonal antibody targeting; the functional group in the functionalized amphiphilic block polymer is N.sub.3, Mal- or NHS.

9. Use of the drug-loaded polymer vesicle according to claim 1 in preparing an anti-myeloma nanomedicine, wherein an active ingredient of the nanomedicine is a small molecule drug.

10. The amphiphilic block polymer, the functionalized amphiphilic block polymer, and the targeting molecule according to claim 1; use of the drug-loaded polymer vesicle in preparing an anti-myeloma nanomedicine, wherein an active ingredient of the nanomedicine is a small molecule drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is nuclear magnetic map of N.sub.3-PEG-P (TMC-DTC) in Example 1.

[0027] FIG. 2 is nuclear magnetic map of PEG-P(TMC-DTC)-NPC in Example 2.

[0028] FIG. 3 is nuclear magnetic map of PEG-P(TMC-DTC)-KD.sub.5 in Example 2.

[0029] FIG. 4 shows the macromolecular mass spectra of Dar and Dar-DBCO in Example 5.

[0030] FIG. 5 shows a graph of the stability of Dar-Ps-VCR in Example 6 at high dilutions and in the presence of serum.

[0031] FIG. 6 shows the VCR release behavior of Dar-Ps-VCR in Example 6 under non-reducing conditions and 10 mM GSH.

[0032] FIG. 7 shows the endocytosis of (A) Dar-Ps-Cy5 at different targeting densities in LP-1 cells in Example 7 and (B) CLSM images of LP-1 cells after incubation with Dar.sub.4.4-Ps-Cy5 and Ps-Cy5 for 4 hr (scale bar: 25 ?m).

[0033] FIG. 8 shows the toxicity of Dar-Ps-VCR, Ps-VCR and free VCR in LP-1 cells at different targeting densities in Example 8.

[0034] FIG. 9 shows 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 8.

[0035] FIG. 10 shows flow cytometry determination of apoptosis induced by Dar-Ps-VCR, Ps-VCR and free VCR in LP-1 cells in Example 9.

[0036] FIG. 11 is ex-vivo bioluminescence imaging in Example 10 to observe the tumor distribution of LP-1-Luc tumors in various organs, skull, and hind leg bones of mice.

[0037] FIG. 12 shows in vivo fluorescence imaging of loaded in situ LP-1-Luc multiple myeloma mice in Example 11 after tail vein injection of Dar-Ps-Cy5 and Ps-Cy5 at different time points.

[0038] FIG. 13 shows a workflow for the construction and treatment of an in situ LP-1-Luc multiple myeloma mouse transplantation model and a graph of the therapeutic effect of in vivo imaging evaluating Dar-Ps-VCR on an in situ LP-1-Luc multiple myeloma mouse model in Example 12.

[0039] FIG. 14 shows graphs of Luc fluorescence signaling changes; body weight changes; and Kaplan-Meier survival curves for mice in different treatment groups in Example 12.

[0040] FIG. 15 shows micro-CT maps of the femur (Femur) and tibia (Tibia) of mice in different treatment groups in Example 12.

[0041] FIG. 16 shows the analysis of various metrics of (A) femur and (B) tibia of mice in different treatment groups in Example 12. (BMD: bone mineral density; BV/TV: bone volume fraction; Tb.N: number of trabeculae; Tb.Sp: trabecular separation; BS/TV: bone surface area; Tb.Th: trabecular thickness.

EXAMPLES OF THE INVENTION

[0042] The VCR-loaded reversibly cross-linked degradable polymer vesicles of the present invention are obtained by self-assembly of an amphiphilic triblock polymer undergoing simultaneous self-crosslinking; the molecular chain of triblock polymer comprises sequentially connected hydrophilic chain segments, hydrophobic chain segments, and a KD molecule; hydrophilic chain segment is a polyethylene glycol (PEG) with a molecular weight of 3000-8000 Da; hydrophobic chain segment is a polycarbonate chain segment with a molecular weight of 2.1-5.7 times the molecular weight of the hydrophilic chain segment; and the molecular weight of the KD polypeptide is 15%-50% of the molecular weight of the PEG hydrophilic chain segment.

[0043] The PEG-P(TMC-DTC)-KD.sub.z polymer of the present invention was produced by the reaction of KD.sub.z after activation of the terminal hydroxyl group of PEG-P(TMC-DTC) by p-nitrophenyl chloroformate (p-NPC) by the following synthetic route.

##STR00002##

[0044] Wherein, in step (i), the reaction conditions are anhydrous dichloromethane (DCM), pyridine, 25? C., 24 hrs; in step (ii), the reaction conditions are anhydrous dimethylsulfoxide (DMSO), KD.sub.z, triethylamine, 30? C., 48 hrs.

[0045] The specific synthesis steps are as follows. [0046] (1) Pyridine was added to an anhydrous DCM solution of PEG-P(TMC-DTC) in an ice water bath, stirred for 10 minutes and then a DCM solution of p-NPC was added to it slowly dropwise. After the dropwise addition was completed (?30 min) the reaction was continued at room temperature for 24 h. Then the pyridine salt was removed by filtration and the polymer solution was collected and concentrated by spinning to ?100 mg/mL, which was precipitated by ice ether and dried in vacuo to give the product PEG-P(TMC-DTC)-NPC. [0047] (2) Under nitrogen protection, the KD.sub.z peptide was weighed and placed in a two-necked round-bottomed flask and anhydrous DMSO was added to completely dissolve the peptide, triethylamine was added under stirring, and then anhydrous DMSO solution of PEG-P(TMC-DTC)-NPC was added to it drop by drop, and the dropwise addition was completed in 30 minutes. After 2 days of reaction at 30? C., the polymer solution was first dialyzed with DMSO containing 5% anhydrous methanol for 36 h (with 4-5 media changes) to remove unreacted KD.sub.z and reacted p-nitrophenol, and then dialyzed with DCM for 6 h. Then the polymer solution was collected and concentrated by spinning to a polymer concentration of about 50 mg/mL, and then precipitated in iced ethyl ether and then dried in vacuum, which resulted in the white cotton-wool-like polymer PEG-P(TMC-DTC)-KD.sub.z. TMC was routinely replaced with LA or CL to obtain PEG-P(LA-DTC)-KD.sub.z, PEG-P(CL-DTC)-KD.sub.z.

[0048] The raw materials involved in the present invention are existing commercially available raw materials, and the specific preparation methods and testing methods are conventional techniques in the art; the present invention will be further described below with reference to the examples and accompanying drawings.

Example 1 Synthesis of Polymer N.SUB.3.-PEG-P (TMC-DTC)

[0049] The polymer N.sub.3-PEG-P (TMC-DTC) was obtained by initiating the ring-opening co-polymerization of TMC and DTC using DPP as a catalyst and N.sub.3-PEG-OH as a macroinitiator. First, N.sub.3-PEG-OH (M.sub.n=7.9 kg/mol, 0.79 g, 0.1 mmol), TMC (1.50 g, 14.8 mmol) and DTC (0.20 g, 1.0 mmol) were weighed in a closed reactor under nitrogen environment in a glove box, and 5.0 mL of anhydrous DCM was added to dissolve them, followed by the addition of DPP (0.25 g, 1.2 mmol), and the reactor was sealed and transferred out of the glove box and placed at 30? C. for four days. At the end of the reaction, it was precipitated twice with ice ether and dried under vacuum to obtain the white flocculent polymer N.sub.3-PEG-P (TMC-DTC), yield: 85.4%. The characteristic peaks of N.sub.3-PEG at ? 3.38 and 3.63 ppm, TMC at ? 2.03 and 4.18 ppm, and DTC at ? 2.99 and 4.22 ppm can be seen in the attached FIG. 1. The molecular weight of the N.sub.3-PEG-P(TMC-DTC) polymer can be calculated from the ratio of the methylene hydrogen integral area at ? 2.03 and ? 2.99 ppm to the PEG methylene hydrogen integral area at ? 3.63 ppm to obtain a molecular weight of 7.9-(15.0-2.0) kg/mol, with a molecular weight distribution of 1.1 as measured by the GPC, and is used in the following examples.

[0050] Replacing N.sub.3-PEG-OH with CH.sub.3 O-PEG-OH having a molecular weight of 5 k, and leaving the rest unchanged, and referring to the above preparation method, PEG-P(TMC-DTC) (5.0-(15.0-2.0) kg/mol) was obtained.

Example 2 Synthesis of Polymer PEG-P(TMC-DTC)-KD.SUB.z

[0051] The polymer PEG-P(TMC-DTC)-KD.sub.z was synthesized in two steps, i.e., it was obtained by reacting with the KD.sub.z peptide molecule after activating the terminal hydroxyl group of PEG-P(TMC-DTC) (5.0-(15.0-2.0) kg/mol) using p-NPC. As an example, the synthesis of PEG-P(TMC-DTC)-KD.sub.5 was carried out as follows: PEG-P(TMC-DTC) (1.0 g, 45.5 ?mol) was dissolved in 10 mL of anhydrous DCM under nitrogen atmosphere, and then transferred to an ice-water bath with the addition of pyridine (18.0 mg, 227.5 ?mol) and stirred for 10 min, to which was added dropwise p-NPC (48.4 mg, 240.3 ?mol) to which was added dropwise DCM solution (1.0 mL). 3 0 min after completion of the dropwise addition, the reaction was continued at room temperature for 24 h. Then the pyridine salt was removed by filtration and the polymer solution was collected and concentrated by spinning to ?100 mg/mL, which was precipitated by iced ethyl ether and dried under vacuum to obtain the product PEG-P(TMC-DTC)-NPC in a yield of: 90.0%. Subsequently, KD.sub.5 (60.0 mg, 83.4 ?mol) was weighed and dissolved in 4 mL of anhydrous DMSO under nitrogen protection and triethylamine (4.2 mg, 41.7 ?mol) was added to it dropwise under stirring, and then anhydrous DMSO solution (9.0 mL) of PEG-P(TMC-DTC)-NPC was added to it dropwise, and the dropwise addition was completed in 30 min. After 2 days of reaction at 30? C., the polymer solution was dialyzed with DMSO containing 5% anhydrous methanol for 36 h (4-5 media changes) to remove unreacted KD.sub.5 and reacted p-nitrophenol, and then dialyzed with DCM for 6 h. Then the polymer solution was collected and concentrated by spinning to a polymer concentration of 50 mg/mL, and the polymer was precipitated in iced ether and dried in vacuum to give a white cotton-wool-like polymer PEG-P(TMC-DTC)-KD.sub.5, yield: 91.0%. The NMR hydrogen spectra of PEG-P(TMC-DTC)-NPC and PEG-P(TMC-DTC)-KD.sub.5 are shown in the attached FIGS. 2 and 3. The characteristic peaks of p-NPC (? 7.41 and ? 8.30 ppm) as well as the characteristic peaks of PEG-P(TMC-DTC) (? 2.03, 2.99, 3.38, 3.63, 4.18, and 4.22 ppm) can be seen in the accompanying FIG. 2, which were calculated based on the ratio of the integrated area of the characteristic peaks of p-NPC to the area of the peak of the methyl hydrogen of the PEG at ? 3.38 ppm the grafting rate of NPC was about 100%. Attachment 3 shows that the characteristic peaks of NPC at ? 7.41 and ? 8.30 ppm disappeared, and a new signal peak appeared at ? 4.54 ppm, which is the characteristic peak of hypomethyl group in KD.sub.5. The degree of substitution of KD.sub.5 was calculated to be ?100% by comparing the ratio of the peak area at ? 4.54 ppm to that of the TMC hypomethylidene hydrogen peak area at ? 1.95 ppm. In addition, the grafting ratio of KD.sub.5 was 100% as measured by high performance liquid chromatography (HPLC), demonstrating the successful synthesis of PEG-P(TMC-DTC)-KD.sub.5 for the following Examples.

Example 3 Preparation of Reversibly Cross-Linked Biodegradable Vesicles Loaded with VCR (Ps-VCR)

[0052] Ps-VCR was prepared by solvent displacement wherein the VCR was encapsulated by electrostatic interactions with KD.sub.z. Ps-VCR was obtained by dissolving PEG-P(TMC-DTC)-KD.sub.z in DMSO (40 mg/mL), taking 100 ?L and beating it into 900 ?L of HEPES (pH 6.8, 10 mM) containing the VCR at rest, stirring at 300 rpm for 3 min, and then dialyzing it with HEPES (pH 7.4, 10 mM) for 8 hr. where the VCR The theoretical drug loading was set at 4.8-11.1 wt. %, and it was found that the particle size of the resulting Ps-VCR ranged from 26-40 nm with a particle size distribution of 0.05-0.20 (Table 1). The encapsulation rate of Ps-VCR was calculated to be 97.2% by measuring its absorbance value at 298 nm by UV-visible spectroscopy. Based on the same method, the encapsulation rates of Ps-VCR prepared by PEG-P(LA-DTC)-KD.sub.5 and PEG-P(CL-DTC)-KD.sub.5 at a theoretical loading capacity of 4.8% were 88.3% and 83.9%, respectively; whereas, the particle size of the drug-loaded vesicles prepared by PEG-P(TMC-DTC) two-block copolymers was around 75 nm, and the encapsulation rate of the VCRs was lower which was only 14.1%.

TABLE-US-00001 TABLE 1 Index of Ps-VCR particle size DLC (wt. %).sup.b DLE.sup.b polymer (nm).sup.a PDI.sup.a theory test .sup.(%) PEG-P(TMC-DTC) 75 0.12 4.8 0.7 14.1 PEG-P(TMC-DIC)-KD.sub.5 36 0.11 4.8 4.6 97.2 38 0.12 7.0 4.3 60.2 PEG-P(TMC-DTC)-KD.sub.10 31 0.06 4.8 4.2 88.0 33 0.12 7.0 4.1 57.3 PEG-P(TMC-DTC)-KD.sub.15 26 0.08 4.8 4.1 86.2 28 0.08 7.0 4.8 66.7 .sup.atested by DLS .sup.btested by UV-vis

Example 4 Preparation of Reversibly Cross-Linked Biodegradable Vesicles Loaded with Other Drugs (Ps-Drug)

[0053] The encapsulation of other drugs such as verapamil hydrochloride (VER), irinotecan hydrochloride (CPT), and recoquimod (R848) by reversibly cross-linked degradable vesicles was investigated using a similar methodology as in Example 3. It was found that after encapsulation of different drugs, the particle size of the resulting Ps-drugs ranged from 20-40 nm, as shown in Table 2.

TABLE-US-00002 TABLE 2 Index of Ps-drug particle size DLC (wt. %).sup.a DLE.sup.b polymer Drug (nm).sup.a PDI.sup.a theory test (%) PEG-P(TMC-DTC)-KD.sub.10 VER 27 0.17 4.7 0.3 5.0 30 0.13 9.0 0.4 4.0 33 0.18 13.0 0.7 4.5 PEG-P(TMC-DTC)-KD.sub.5 CPT 30 0.17 5.0 0.48 9.5 PEG-P(TMC-DTC)-KD.sub.10 29 0.15 5.0 0.15 0.3 PEG-P(TMC-DTC)-KD.sub.10 31 0.16 10.0 0.06 0.6 PEG-P(TMC-DTC)-KD.sub.10 32 0.26 20.0 0.80 4.0 PEG-P(TMC-DTC)-KD.sub.15 39 0.23 20.0 0.82 4.1 PEG-P(TMC-DTC)-KD.sub.10 R848 23 0.11 12.0 0.7 6.1 24 0.12 40.0 1.6 4.0 .sup.atested by DLS .sup.btested by UV-vis

Example 5 Preparation of Monoclonal Antibody-Directed Polymeric Vesicles Loaded with VCR (Ab-Ps-VCR)

[0054] Ab-Ps-VCR was obtained by post-modifying a dibenzocyclooctyne-functionalized monoclonal antibody (Ab-DBCO) on the surface of an azide-functionalized polymeric vesicular VCR nanomedicine (N.sub.3-Ps-VCR). N.sub.3-Ps-VCR was obtained by coassembling N.sub.3-PEG-P (TMC-DTC) and PEG-P (TMC-DTC)-KD.sub.z while encapsulating a VCR obtained, wherein the content of N.sub.3-PEG-P(TMC-DTC) is 1 to 10 wt. %. Specifically, as an example of the preparation of N.sub.3-Ps-VCR containing 2% N.sub.3-PEG-P(TMC-DTC), 8.0 mg of N.sub.3-PEG-P(TMC-DTC) and 392.0 mg of PEG-P(TMC-DTC)-KD.sub.5 (molar ratio of 2:98) were weighed and dissolved in DMSO (the total polymer concentration of 40 mg/mL), and at the same time, 4.0 mL of An aqueous solution of VCR (5 mg/mL) was added to 90 mL of HEPES (pH 6.8, 10 mM) and mixed well, into which 10 mL of the polymer solution was injected under standstill, stirred for 5 min, and then placed at 37? C. for 4 hours. After removing the organic solvent by dialysis (MWCO: 14 kDa) with HEPES (pH 7.4, 10 mM) for 8 h, a nanofiltration system was used to remove the free VCR, yielding N.sub.3-Ps-VCR. The particle size of N.sub.3-Ps-VCR was measured by dynamic light scattering (DLS) to be 36 nm with a narrow distribution (PDJ: 0.11). The encapsulation rate was as high as 97.2% when the theoretical drug loading of VCR was 4.8 wt. % with 4.6 wt. %. In order to efficiently bond the monoclonal antibody, the N.sub.3-Ps-VCR was subsequently concentrated from 4 mg/mL to 18.6 mg/mL using a tangential flow device to facilitate storage and improve the bonding efficiency of the monoclonal antibody. The particle size of N.sub.3-Ps-VCR after concentration was 42 nm and the PDI was 0.07. Its particle size remained around 40 nm, the PDI was less than 0.17, and the leakage of VCR was less than 0.6% during 180 days of storage at 4? C., indicating that the N.sub.3-Ps-VCR has excellent long-term storage stability (Table 3).

TABLE-US-00003 TABLE 3 long-term storage stability at 4? C. of N3-Ps-VCR Particle size Leakage of Time (Day) (nm).sup.a PDI.sup.a VCR(%).sup.b 0 42 0.07 15 42 0.08 0.39 45 36 0.15 0.39 90 38 0.17 0.58 132 37 0.14 0.28 180 37 0.15 0.17 .sup.atested by DLS .sup.btested by UV-vis

[0055] Ab-DBCO was prepared by amidation of the small molecule NHS-OEG.sub.4-DBCO with the amino group on the monoclonal antibody, wherein the degree of DBCO functionalization can be adjusted by changing the molar ratio of Ab to NHIS-OEG.sub.4-DBCO. Taking the preparation of DBCO-functionalized daratumumab (Dar-DBCO) as an example, a PBS solution (21.7 mg/mL) of Dar was diluted to 10 mg/mL with PB (pH 8.5, 10 mM), and 200 ?L of it was taken and to it was added 3 or 5-fold molar equivalents of a DMSO solution (5 mg/mL) of NHS-OEG.sub.4-DBCO under shaking, and placed at 27? C., 120 rpm shaker for overnight reaction. At the end of the reaction, unreacted NHS-OEG.sub.4-DBCO was removed by centrifugation (MWCO: 10 kDa, 3000 rpm) in an ultrafiltration tube and the ultrafiltration was washed twice with PBS (pH 7.4, 10 mM) to obtain Dar-DBCO. when the molar ratios of Dar to NHS-OEG.sub.4-DBCO were 1:3 and 1:5, the reaction was analyzed by time-of-flight mass spectrometry (MALDI-TOF-MS), 1.5 and 2.8 DBCO were modified on each Dar, respectively (Supplementary FIG. 4), denoted as Dar-DBCO.sub.1.5 and Dar-DBCO.sub.2.8. In order to maximize the targeting and biological activity of the monoclonal antibodies, subsequent experiments were conducted with Dar-DBCO.sub.1.5 or other monoclonal antibodies modified with 1.5-2 DBCO.

[0056] Dar-Ps-VCR can be simply prepared by a click chemistry reaction of tensile touching between N.sub.3 and Dar-DBCO on the surface of N.sub.3-Ps-VCR, and the surface density of Dar can be adjusted by changing the feeding ratio. The molar ratios of Dar-DBCO to N.sub.3 were set to be 0.25:1, 0.5:1, and 1:1, respectively, i.e., 10.4, 20.9, and 41.8 ?L of Dar-DBCO solution (5.6 mg/mL) were added to 107.5 ?L of N.sub.3-Ps-VCR (18.6 mg/mL), respectively, and then the reaction was carried out in a shaker at 25? C., 100 rpm overnight. Unbonded Dar-DBCO was removed using ultracentrifugation (58 krpm, 4? C., 30 min) and washed twice with HEPES (pH 7.4, 10 mM), while Dar-Ps-VCR and supernatant were collected to determine the amount of Dar bonded. The unbonded Dar-DBCO in the supernatant was determined by HPLC, which led to the calculation of 28.6, 56.4, and 112.2 g of Dar per mg of polymer vesicle surface, respectively, and the calculation of the absolute molecular weight of the polymer vesicles (1.15?10.sup.7 g/mol) and the number of aggregates (523), measured by multiangle laser light scattering, showed that each Dar-Ps-VCR surface was bonded with 2.2, 4.4 and 8.7 Dar, respectively (Table 4). With the increase of Dar density, the particle size of Dar-Ps-VCR increased slightly (43-49 nm) and the particle size distribution was narrower (PDI: 0.14-0.21), and the encapsulation results were the same as the present Example of N.sub.3-Ps-VCR after the monoclonal antibody was received.

TABLE-US-00004 TABLE 4 Indexes of different density of Dar-Ps-VCR molar ratio of Bonding Dar particle Targeting drug Feeding efficiency ?g/mg number size vesicle Dar-DBCO:N.sub.3 (%) Ps.sup.a Per Ps (nm).sup.b PDI.sup.b Dar.sub.2.2-Ps-VCR 0.25:1 96.6 28.6 2.2 43 0.14 Dar.sub.4.4-Ps-VCR 0.5:1 95.3 56.4 4.4 45 0.15 Dar.sub.8.7-Ps-VCR 1:1 94.8 112.2 8.7 49 0.21 .sup.atested by HPLC; .sup.btested by DLS

[0057] Other monoclonal antibody-directed loaded VCR polymer vesicles such as Isa-Ps-VCR and Anti-CD38-Ps-VCR are prepared similarly to Dar-Ps-VCR. Their particle sizes ranged from 40-60 nm with a narrow particle size distribution (PDI: 0.10-0.30) and the number of monoclonal antibodies on the surface of each vesicle was 1-10.

[0058] The prior art CN110229323A Table 7 discloses that saponin-carrying protein (SAP) non-targeting vesicles (KD.sub.5) undergo ultracentrifugation (58 krpm, 4? C., 30 min) show a decrease in DLE from 68.3% to 23%, with a large amount of drug leakage, suggesting that they are unable to pick up a targeted monoclonal antibody.

Example 6 Stabilization and In Vitro Drug Release of Ab-Ps-VCR Targeted Polymeric Vesicle Nanomedicines

[0059] Dar.sub.4.4-Ps-VCR containing 4.4 Dar on the surface of each vesicle was used as a representative to study the stability and in vitro drug release behavior of Ab-Ps-VCR targeted vesicle nanomedicine. The stability of Dar-Ps-VCR was determined by 50-fold dilution of phosphate buffer solution or addition of 10% fetal bovine serum, respectively, and the particle size changes were detected by dynamic light scattering. The particle size distribution of Dar-Ps-VCR stability is shown in the accompanying FIG. 5. The results showed that Dar-Ps-VCR targeted vesicle nanomedicine maintained intact particle size and particle size distribution with good stability after 50-fold dilution and addition of 10% FBS for 24 hours.

[0060] The in vitro drug release behavior of Dar-Ps-VCR was studied using a dialysis method with 2 release media, HEPES (pH 7.4, 10 mM) and HEPES solution containing 10 mM GSH (nitrogen environment). Firstly, 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 at 37? C., 100 rpm in a shaker. At set time points (0, 1, 2, 4, 6, 8, 10, 12, 24 h) 5 mL of dialysate was removed and 5 mL of fresh medium was replenished. The amount of VCR in the dialysate was determined by HPLC (mobile phase methanol:water (15% triethylamine added and pH adjusted to 7.0 with phosphoric acid)=70:30). The results of in vitro release of Dar-Ps-VCR-targeted vesicular nanomedicine are plotted in the accompanying FIG. 6. The results showed that Dar-Ps-VCR released more than 85% of VCR in 12 h under the reducing condition of 10 mM GSH, while the cumulative release of VCR was only about 22% in 24 h under the non-reducing condition.

Example 7 Endocytosis Behavior of Dar-Ps-VCR Targeted Polymeric Vesicle Nanomedicines

[0061] Since the VCR itself is non-fluorescent, Cy5-labeled polymer vesicles were used, and Dar-Ps-Cy5 was prepared with reference to Example 5, and Ps-Cy5 was prepared with reference to Example 3; the uptake of Dar-Ps-Cy5 with different Dar densities in LP-1 cells was studied by flow cytometry and laser scanning confocal microscopy (CLSM). In flow experiments, LP-1 cell suspensions were first spread in 6-well plates (2?10.sup.5 cells/well) and placed in the incubator for 24 h. After incubation, 200 ?L of Dar-Ps-Cy5 and Ps-Cy5 were added to each well (the concentration of Cy5 in the wells was 2.0 g/mL), and the PBS group was used as a control. After continuing the incubation for 4 h, cells were collected by centrifugation (800 rpm, 5 min) and washed twice with PBS, and finally dispersed with 500 ?L PBS and placed in a flow-through tube for assay. The test results showed that the endocytosis of Dar-Ps-Cy5 in LP-1 cells was significantly higher than that of Ps-Cy5, where the cells incubated with Dar.sub.4.4-Ps-Cy5 had the highest fluorescence intensity, which was 6.4-fold higher than that of the Ps-Cy5 control (Supplementary FIG. 7A), indicating that the introduction of Dar significantly enhanced the cellular uptake of Ps-Cy5, and that when bonded to the surface of each vesicle 4.4 Dar was best targeted when the surface of each vesicle was bonded.

[0062] The endocytosis behavior of Dar.sub.4.4-Ps-Cy5 and Ps-Cy5 in LP-1 cells was then further investigated using CLSM. The specific experimental steps were as follows: polylysine (300 ?L, 0.1 mg/mL) pretreated pellets were placed in a 24-well plate with LP-1 cell suspension (3?10.sup.5 cells/well) and incubated in an incubator for 24 h. After 24 h, 200 ?L of Dar.sub.4.4-Ps-Cy5 and Ps-Cy5 were added respectively (the concentration of Cy5 in the wells was 40 g/mL). After continuing incubation for 4 h, the medium was carefully removed and washed 3 times with PBS, followed by fixation with 4% paraformaldehyde solution for 15 min, washed 3 times with PBS, then the nuclei of the cells were stained with DAPI for 3 min, and washed 3 times with PBS, and finally the slices were blocked with glycerol and observed and photographed with CLSM (Leica, TCS SP5). Attachment 7B shows the graph of the uptake results of Dar.sub.4.4-Ps-Cy5 and Ps-Cy5 in LP-1 cells. The results showed that when LP-1 cells were incubated with Dar.sub.4.4-Ps-Cy5 for 4 hours, obvious red fluorescence was presented around the nucleus, while the fluorescence was weaker in the cells incubated with Ps-Cy5, indicating that Dar-Ps-Cy5 possesses excellent targeting and efficient and rapid cellular endocytosis.

Example 8 Cytotoxicity Test of Dar-Ps-VCR-Targeted Polymeric Vesicle Nanomedicine

[0063] The in vitro anti-tumor activity of Dar-Ps-VCR against LP-1 multiple myeloma cells was determined using a CCK-8 kit, with MV4-11 cells as a control. LP-1 cells were first spread in 96-well plates (15,000/well) and placed in an incubator at 37? C. with 5% CO.sub.2 for 24 h. After that, 20 ?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. After incubation at 37? C. for 48 h, 10 ?L of CCK-8 solution was added to each well to continue the incubation for 4 h. Finally, its absorbance value at 492 nm was tested by an enzyme marker. Cell viability was calculated by the ratio of the absorbance value of the experimental group to the absorbance value of the cells incubated with the addition of PBS, and the experiment was performed in parallel for four groups (mean?SD, z=4). The cytotoxicity results of Dar-Ps-VCR vesicle nanomedicine with different targeting densities (z=5) on LP-1 cells are plotted in the accompanying FIG. 8. The results showed that the cytotoxicity was strongest when 4.4 Dar were bonded on the surface of each vesicle (Dar.sub.4.4-Ps-VCR), with the LC50 (IC.sub.50) as low as 0.07 ng/mL, which was 20- and 12-fold lower compared to the free VCR (IC.sub.50: 1.38 ng/mL) and the nontargeted control Ps-VCR (z of 5, IC.sub.50. 0.85 ng/mL), suggesting that the introduction of Dar significantly increased the targeted delivery and rapid intracellular release of VCR.

[0064] MV4-11 cells (12,000/well) and L929 fibroblasts (3,000/well) were spread in 96-well plates and incubated for 24 hours, then 20 ?L of Dar.sub.4.4-Ps-VCR (z of 5) and Ps-VCR (z of 5) were added to each well, with the final concentration of VCR in the wells ranging from 0.0001-100 ng/mL. MV4-11 cells were incubated at 37? C. for 48 h. After incubation at 37? C., 10 ?L of CCK-8 solution was added to each well to continue incubation for 4 h. The cells were tested for absorbance values at 492 nm using an enzyme marker. L929 cells were incubated at 37? C. for 48 h. After incubation at 37? C., 10 ?L of PBS solution (5 mg/mL) with MTT was added to each well and incubated for 4 h. Afterwards, the medium was carefully removed and 150 ?L of DMSO to dissolve the resulting metazan crystals, and their absorbance at 570 nm was tested with an enzyme marker; the results showed that the IC.sub.50 was 20-fold higher in MV4-11 cells than in LP-1 cells (Supplementary FIG. 9A). More interestingly, for L929 normal cells, Dar.sub.4.4-Ps-VCR and Ps-VCR did not show significant toxicity even at VCR concentrations as high as 100 ng/mL, and the cell survival rates were both close to 100% (Supplementary FIG. 9B). These results collectively indicated that Dar-Ps-VCR could selectively target and efficiently kill multiple myeloma cells with less toxicity to normal cells.

[0065] In addition, the same method was used to test the toxicity of Dar-Ps and Ps empty vesicles as well as free Dar on LP-1 cells, and the results showed that cell survival was close to 100% without significant cytotoxicity even at Ps concentrations up to 30 g/mL (Attachment 9C), and Dar concentrations of 9 ?g/mL (Attachment 9D).

[0066] Dar-Ps-VCR in the following Examples all refer to Dar.sub.4.4-Ps-VCR vesicular nanomedicine (z of 5) and Dar-Ps-Cy5 all refer to Dar.sub.4.4-Ps-Cy5 (z of 5).

Example 9 Dar-Ps-VCR-Targeted Polymeric Vesicle Nanomedicine Induced Apoptosis

[0067] Apoptosis assay of Dar-Ps-VCR was treated by double staining with fluorescent dye AnnexinV-FITC/PI and then tested by flow cytometry. LP-1 cells were first spread in 6-well plates at a density of 2?10.sup.5 cells/well and incubated in the incubator for 24 h. After 24 h, 200 ?L of Dar-Ps-VCR, Ps-VCR, and free VCR were added (the in-well concentration of VCR was 0.5 ng/mL), and the cells with only PBS were used as a control. After incubation in the incubator for 48 h, LP-1 cells were collected by centrifugation (800 rpm, 5 min) and washed twice with ice PBS, and finally 200 ?L of Binding buffer was added to each sample to resuspend the cells (the cell density was approximately 10.sup.6 cells/mL). After blowing uniformly, 100 ?L was taken into the flow tube and 5 ?L of AnnexinV-FITC and 10 ?L of PI solution were added sequentially, and the cells were stained at room temperature and protected from light for 15 min, then 400 ?L of PBS was added and mixed uniformly, and measured by flow cytometry within 1 h. The cells were then stained with PBS for 15 min at room temperature and protected from light for 15 min. Among them, the samples in the PBS group that were treated in a 50? C. water bath for 5 min and fixed with 4% paraformaldehyde for 5 min were used as the early apoptosis group and the late apoptosis group, respectively, and stained for 15 min by adding 5 ?L of AnnexinV-FITC solution and 10 ?L of PI solution, respectively. The results of apoptosis induced by Dar-Ps-VCR in LP-1 cells are shown in Appendix FIG. 10. The results showed that Dar-Ps-VCR could effectively induce apoptosis, and when the concentration of VCR was 0.5 ng/mL, it could cause 60.8% apoptosis, and the rate of apoptosis was significantly higher than that of the non-targeting control Ps-VCR group (43.4%) and the free VCR group (31.4%), and the number of late apoptotic cells in all the groups was significantly more than that of early apoptosis.

Example 10 Construction of Dutch LP-1-Luc In Situ Multiple Myeloma Mouse Model

[0068] All animal experiments and manipulations were approved by the Laboratory Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University. Establishment of in situ MM tumor model: 6-week-old ZOD/SCID female mice were used, firstly, the mice were cleared of marrow by intraperitoneal injection of 10 mg/mL of cyclophosphamide solution on two consecutive days, 2 mg per mouse per injection, and then on the third day, LP-1-Luc cells (8?10.sup.6 cells/each) were injected into the mice through the tail vein, and the in vivo imaging and treatment were started on the 10th day after inoculation. Mice were simultaneously weighed. To study tumor distribution in the LP-1-Luc in situ multiple myeloma mouse transplantation model, mice were injected intraperitoneally with fluorescein potassium salt through the mice on day 35 post-inoculation, and 8 minutes later, mice were dissected and collected for fluorescence imaging of the heart, liver, spleen, lungs, kidneys, intestines, skull, and hind leg bones. The attached FIG. 11 shows the results of ex vivo bioluminescence imaging of various organs, skull as well as hind leg bones of Hol LP-1-Luc in orthotopic multiple myeloma mice. From the images, it can be seen that 35 days after inoculation, LP-1-Luc tumors were mainly concentrated in the hind legs and skulls of the mice, and there was no obvious distribution of tumor Luc signals in the heart, liver, spleen, lungs, kidneys, and other organs.

Example 11 Dar-Ps-Cy5 In Vivo Imaging Experiments in Hol LP-1-Luc in Orthotopic Multiple Myeloma Mice

[0069] The distribution of Dar-Ps-Cy5 in LP-1-Luc in orthotopic multiple myeloma mice was obtained by mouse live imaging analysis. On day 37 post-inoculation (when the mice were about to develop the disease), 200 ?L of Dar-Ps-Cy5 and Ps-Cy5 solution (250 ?g Cy5 equiv./kg) were injected into the mice through the tail vein, respectively, and mice were anesthetized with isoflurane for in vivo fluorescence imaging at 1, 2, 4, 6, 8, 10, 12, and 24 hours after the injections, using the Lumia II software to image the biological distribution in mice with myeloma mice (Supplementary FIG. 12). The results showed that Dar-Ps-Cy5 was efficiently targeted and enriched to the tumor site, and its fluorescence signal in the legs and head of mice was significantly higher than that of the non-targeted Ps-Cy5 group.

Example 12 Anti-Tumor Effects of Dar-Ps-VCR in Hol LP-1-Luc in Orthotopic Multiple Myeloma Mice

[0070] To investigate the anti-tumor effect of Dar-Ps-VCR in Holo-Positive LP-1-Luc multiple myeloma mice, treatment experiments were initiated when the bioluminescence intensity reached 1.2?10.sup.6 p/sec/cm.sup.2/sr on day 10 post-inoculation. There were two dosing regimens that maintained the same total VCR administration: one with a VCR dose of 0.25 mg/kg, one injection given on 4 days for a total of 4 injections, denoted as Dar-Ps-VCR (0.25 mg VCR equiv./kg, Q4d); and the other with a VCR dose of 0.50 mg/kg, one injection given on 8 days for a total of 2 injections, denoted as Dar-Ps-VCR (0.50 mg VCR equiv./kg, Q8d). Based on the first dosing regimen, equal VCR doses of Ps-VCR and free VCR, equal equivalents of Dar-Ps, and PBS were used as controls. Each treatment group consisted of 10 hormonal mice, of which 4 were used for bioluminescence imaging and 6 were used to monitor body weight and observe survival. It was found that the mice in the PBS group continued to have rapid growth of LP-1-Luc cells, which started to develop when the bioluminescence intensity reached 1.0?109 p/sec/cm.sup.2/sr on days 37-45 post inoculation, and manifested as paralysis of both legs, weight loss and death occurred (Supplementary FIG. 13). During the administration treatment period (10-22 days), there was no significant increase or even a slight decrease in Luc signal in mice in the Dar-Ps-VCR two administration groups, Ps-VCR and free VCR groups, indicating that they could effectively inhibit the spreading and proliferation of LP-1-Luc cells in mice. At the end of drug administration, the tumors of mice in the non-targeted Ps-VCR and free VCR groups began to recur, and Luc bioluminescence signals increased rapidly, i.e., 157- and 53-fold by day 39, respectively. However, both groups of mice injected with Dar-Ps-VCR in the tail vein continued to inhibit the growth of LP-1-Luc tumors, and no significant Luc signal was detected even at day 39 post-inoculation, and their fluorescence values were comparable to the background signals of healthy mice, which were 235- and 114-fold lower compared to the Ps-VCR and free VCR groups, and about 5000-fold lower compared to the untreated PBS group. around 5000-fold. The tumor growth trend of mice in the empty vector Dar-Ps group was similar to that of the PBS group (Supplementary FIG. 14A). These results indicated that Dar-Ps-VCR could efficiently target deliver VCR to the tumor site and completely eliminate the tumor in the short term. Notably, mice in all treatment groups showed no significant changes in body weight during administration until mice would show weight loss at the onset of disease, indicating that they had relatively low toxicity (Supplementary FIG. 14B). In addition, the survival of mice in the Dar-Ps-VCR treatment group was significantly prolonged (Supplementary FIG. 14C), in which the median survival of mice in the 0.25 mg VCR equiv./kg, Q4d and 0.50 mg VCR equiv./kg, Q8d administration modes was 156 and 154 days, respectively, compared with that in the PBS group (43 days), Dar-Ps group (49.5 days), Ps-VCR (51 days) and free VCR group (52 days) were prolonged by 3.0-3.6-fold. Together, these results suggest that the introduction of Dar significantly increased the selective targeting of Ps-VCR, which efficiently inhibited the growth of in situ multiple myeloma.

[0071] Osteolytic lesions are one of the common clinical manifestations of MM patients, so the relevant indexes of femur and tibia in mice of each treatment group were evaluated using micro-CT. The results revealed that the hind leg bones of the mice in the PBS and Dar-Ps groups had severe osteolysis and a large number of bone trabeculae were missing, while the osteolytic lesions of the mice were significantly improved after treatment with Dar-Ps-VCR, which was similar to that of the healthy mice (Supplementary FIG. 15). Subsequently, by further quantitative analysis, it was found that the bone mineral density, bone volume fraction, number of trabeculae, trabecular separation, and bone surface area of the femur and tibia in the mice in the Dar-Ps-VCR-targeted treatment groups under both administration modalities were similar, and there were significant differences between them and the untreated PBS group, whereas there were no significant differences between the indices and those of the healthy mice (Supplementary FIG. 16). In addition, some indexes such as bone volume fraction were also significantly different from those of the non-targeted Ps-VCR control group, further indicating that the introduction of Dar significantly increased the efficacy of Ps-VCR in multiple myeloma in situ and effectively inhibited osteolytic lesions.