CN111939129A - Application of small-molecule-drug-carrying polymer vesicle in preparation of drugs for treating acute lymphatic leukemia - Google Patents

Abstract

The invention discloses the application of small molecule drug-carrying polymer vesicles in the preparation of drugs for treating acute lymphocytic leukemia; the small molecule drug-carrying polymer vesicles are prepared from small molecule drugs and amphiphilic block polymers; or Small molecule drug polymer vesicles are prepared from small molecule drugs, amphiphilic block polymers, functionalized amphiphilic block polymers, and targeting monoclonal antibody molecules. The vesicle system of the present invention has many unique advantages, including small size, simple and controllable preparation, excellent biocompatibility, high circulation stability in vivo, strong tumor cell specific selectivity, fast intracellular drug release, and tumor growth inhibition. The effect is remarkable, etc.

Description

Application of small molecule drug-loaded polymer vesicles in the preparation of drugs for the treatment of acute lymphocytic leukemia application

technical field

The invention belongs to the technical field of polymer nano-drugs, and in particular relates to a reversibly cross-linked degradable polymer vesicle loaded with vincristine sulfate and a preparation method thereof and application in tumor targeted therapy, especially in the preparation of anti-acute lymphatic system Applications in leukemia nanomedicine.

Background technique

In the prior art, ELISA was used to detect the expression of sB7-H3 in the cerebrospinal fluid of 176 leukemia patients, and acute leukemia patients were classified according to the internationally recognized FAB (French-Amer-ican-British) classification system; The expression difference between lymphocytic leukemia and acute myeloid leukemia is significant. In the subtype of acute lymphocytic leukemia, there is no significant difference in the expression of sB7-H3. In the subtype of acute myeloid leukemia, the expression of sB7-H3 is in the There were significant differences between M3 and M5 and M4 and M5. Vincristine sulfate (VCR) is a water-soluble, potent drug that acts primarily on tubulin to arrest mitosis in metaphase, but its severe neurotoxicity results in lower doses available. Although the liposomal vincristine sulfate (Marqibo®) nanomedicine approved for marketing in 2012 can prolong the circulation time of VCR and reduce toxic and side effects, the overall improvement is limited. Therefore, how to achieve efficient and stable encapsulation and tumor-targeted delivery of VCR is crucial. The prior art discloses a vincristine sulfate liposome and a preparation method thereof. The vincristine sulfate liposome is composed of vincristine sulfate and nanoliposomes prepared by using sphingomyelin, wherein the vincristine sulfate liposome is Alkali is encapsulated in the nano-liposomes, sphingomyelin is used to prepare nano-liposomes, and vincristine sulfate liposomes are prepared after encapsulating vincristine sulfate, wherein sphingomyelin contains more amide bonds, which can be better It can resist chemical and biological degradation, protect the stability of liposome structure, and improve the drug enrichment of tumor cells, thereby improving the anti-tumor effect. Preparation and characterization of vincristine sulfate apoferritin nanoparticles (VCR-APO-NPs) in the prior art, to investigate the ability of the drug-loaded ferritin nanoparticles to cross the blood-brain barrier in vitro and in vivo, and to study its effect on brain glioma. In vitro and in vivo targeting and anti-tumor effects, the vincristine sulfate ferritin nanoparticles were prepared by pH gradient method, and the encapsulation efficiency of vincristine sulfate ferritin nanoparticles was determined by high performance liquid phase, and the prepared vincristine sulfate ferritin nanoparticles The particle size of the nanoparticles can meet the design requirements, the shape is round, the encapsulation efficiency and drug loading are relatively good, and the stability is good, which is conducive to the release of drugs in an acidic environment. In the prior art, leukemia cell-specific transmembrane peptide-modified vincristine sulfate-loaded vesicle nanomedicine CPP44-PS-VCR was prepared for active targeted therapy of leukemia, and the VCR was actively loaded into vesicles by a pH gradient method. cavity with a particle size of 90-100 nm. The existing liposome-structured polymer vesicles have a hydrophilic inner cavity, which can be used to load hydrophilic small-molecule drugs. However, the loading efficiency of hydrophilic drugs such as VCR is low, and the stability of the collective internal circulation is still lacking. It has multi-functional characteristics such as tumor-specific targeting, rapid intracellular drug release and excellent biocompatibility.

SUMMARY OF THE INVENTION

The purpose of the present invention is to disclose amphiphilic block polymers, drug-loaded polymer vesicles and their preparation methods and their application in the preparation of anti-acute lymphoid leukemia nano-drugs, specifically a loaded vincristine sulfate (VCR) Reversible cross-linking degradable polymer vesicles and preparation method and application thereof.

In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

Application of small molecule drug-loaded polymer vesicles in the preparation of drugs for treating acute lymphocytic leukemia.

The application of the amphiphilic block polymer in the preparation of anti-acute lymphoid leukemia nano-drugs, wherein the active components of the nano-drugs are small molecule drugs.

The application of an amphiphilic block polymer, a functionalized amphiphilic block polymer and a targeting molecule in the preparation of anti-acute lymphoid leukemia nano-drugs, wherein the active components of the nano-drugs are small-molecule drugs.

The small-molecule drug-carrying polymer vesicles of the present invention are prepared from small-molecule drugs, amphiphilic block polymers; Preparation of monoclonal antibody;

The molecular structural formula of the amphiphilic block polymer is as follows:

However, z is 5-15.

In the present invention, in the amphiphilic block polymer, the molecular weight of PEG is 3000-8000 Da; the molecular weight of the hydrophobic segment is 2.5-6 times the molecular weight of PEG; the molecular weight of the PDTC segment is 8 times the molecular weight of the hydrophobic segment %～30%. The amphiphilic block polymer of the present invention has hydrophilic segment (n segment), hydrophobic segment (x+y segment), KD _z segment (z segment), hydrophobic segment, KD _z segment Linked by urethane bonds; the amphiphilic block polymers are denoted PEG-P(TMC-DTC) _-KDz , PEG-P(LA-DTC) _-KDz , PEG-P(CL-DTC)- KD _z .

In the present invention, the small molecule drug is vincristine sulfate, doxorubicin hydrochloride, epirubicin hydrochloride, verapamil hydrochloride, irinotecan hydrochloride, requimod, preferably vinblastine sulfate Neoalkaloid (VCR); the targeting molecule is a targeting monoclonal antibody, preferably the targeting monoclonal antibody is a CD38-targeting monoclonal antibody, such as daratumumab (Dar), isatuximab (Isa) or other Monoclonal antibody targeting CD38.

The preparation method of the above drug-loaded polymer vesicles is as follows: using small molecule drugs and the amphiphilic block polymer as raw materials, and preparing drug-loaded polymer vesicles by a solvent replacement method; Affinity block polymer, functionalized amphiphilic block polymer, and targeting monoclonal antibody are used as raw materials, and drug-loaded polymer vesicles are prepared by solvent replacement method. Preferably, the functionalized amphiphilic block polymer is assembled and cross-linked with the amphiphilic block polymer and loaded with a drug, and then reacted with a monoclonal antibody targeting CD38 to prepare a drug-loaded polymer vesicle.

The reversibly cross-linked and degradable polymer vesicles loaded with vincristine sulfate (VCR) of the present invention are obtained by assembling and cross-linking amphiphilic block polymers, and have an asymmetric membrane structure, and the outer shell is polyethylene glycol ( PEG), the membrane layer is reversibly cross-linked hydrophobic polycarbonate, and the inner shell is KD _z , which can realize the efficient loading of VCR. The drug-loaded vesicles of the present invention are targeting or non-targeting structures, and the targeting molecules of the present invention are monoclonal antibody molecules or monoclonal antibody fragments, and the like, such as daratumumab (Dar), isartor ciximab (Isa) or other mAbs targeting CD38.

In the present invention, the amphiphilic block polymer is first combined with the functionalized amphiphilic block polymer as a raw material to prepare drug-carrying vesicles, and then a CD38-targeting monoclonal antibody is connected to obtain CD38-targeting drug-carrying vesicles. The functional groups are derived from PEG initiators, and the resulting polymer PEG ends carry reactive functional groups, such as azide (N ₃ ), maleimide (Mal) or N-hydroxysuccinimide (NHS), to amphiphilic Taking the functional block polymer PEG-P(TMC-DTC) as an example, the functionalized amphiphilic block polymer can be N3 _- PEG-P(TMC-DTC), Mal-PEG-P(TMC-DTC), NHS-PEG-P (TMC-DTC).

The drug-loaded vesicle of the present invention is composed of a drug and a vesicle, and the vesicle is obtained by cross-linking a polymer, and the targeting molecule may or may not be modified; the amphiphilic block polymer PEG-P (TMC-DTC), Taking vincristine sulfate as an example, the preparation method of the drug-loaded vesicles of the present invention can be as follows:

(1) The terminal hydroxyl group of PEG-P(TMC-DTC) was activated by p-nitrophenyl chloroformate, and then reacted with _KDz to obtain PEG-P(TMC-DTC) _-KDz ;

(2) Functional groups such as N ₃ , Mal or NHS were introduced into the PEG end of PEG-P(TMC-DTC) to obtain functionalized PEG-P(TMC-DTC);

(3) Using vincristine sulfate and PEG-P(TMC-DTC)-KD _z as raw materials, the reversible cross-linking and degradable polymer vesicles loaded with VCR were prepared by solvent replacement method; or vincristine sulfate, PEG- Using P(TMC-DTC)-KD _z and functionalized PEG-P(TMC-DTC) as raw materials, the surface-containing reactive functional groups, VCR-loaded, reversibly cross-linked, and degradable polymer capsules were prepared by solvent replacement. vesicles, and then react with mAbs to prepare mAb-directed VCR-loaded multifunctional vesicles.

The invention discloses the above-mentioned VCR-loaded reversibly cross-linked degradable polymer vesicles and a preparation method thereof. A solution of a PEG-P(TMC-DTC)-KD _z polymer is injected into a standing VCR aqueous solution, and dialyzed after stirring. , that is, to obtain VCR-loaded reversibly cross-linked degradable polymer vesicles (Ps-VCR); specifically, VCR was dissolved in ultrapure water and mixed with HEPES buffer (pH 6.8, 10 mM), and then allowed to stand The DMSO solution of PEG-P(TMC-DTC)-KD _z polymer was injected into it, stirred for 3-5 minutes and then dialyzed with HEPES (pH 7.4, 10 mM) to obtain Ps-VCR.

The invention also discloses a monoclonal antibody-directed, VCR-loaded, reversibly cross-linked degradable polymer vesicle and a preparation method thereof: a DMSO solution of PEG-P(TMC _- DTC) _-KDz and a functionalized polymer such as N3 -After the DMSO solution of PEG-P(TMC-DTC) was mixed uniformly, it was injected into the HEPES solution containing VCR, and after stirring for 3-5 minutes, the reversible cross-linked polymer capsules loaded with VCR containing N _on the surface were obtained by dialysis vesicles; mAbs modified by dibenzocyclooctyne, such as daratumumab (Dar), isatuximab (Isa), or other CD38-targeting mAbs with azide-functionalized VCR-loaded vesicles The tension-triggered click chemistry reaction of vesicles (N ₃ -Ps-VCR) can prepare mAb-directed VCR-loaded vesicles (Ab-Ps-VCR) under mild conditions. Using the same method, the Ab can be easily prepared by the Michael addition reaction between the thiol-functionalized monoclonal antibody molecule and the VCR-loaded vesicles containing Mal on the surface, or the amidation reaction between the monoclonal antibody and the NHS-functionalized VCR-loaded vesicles. -Ps-VCR.

In the polymer of the present invention, KD has good biocompatibility, and combined with the PEG segment and the hydrophobic segment, it can form asymmetric membrane structure vesicles, and realize the efficient and stable encapsulation of small molecule drugs (such as VCR). The force encapsulates the VCR, and is separated from the outside world by the disulfide cross-linked vesicle membrane, which can avoid the loss and toxic side effects caused by leakage and cell adhesion during the delivery process, and can be efficiently delivered to the lesion and restored in the body. Under the action of glutathione (GSH), VCR is rapidly released to effectively kill tumor cells.

The polymer vesicles in the present invention are reduction-sensitive reversible cross-linking, intracellular reversible cross-linking and biodegradable polymer vesicles with negatively charged inner membrane; the polymer is PEG-P (TMC-DTC) -KD _z , in which the TMC (LA or CL) in the middle block is randomly arranged with DTC; the molecular weight of KD _z is 700-2000 Da , which is much smaller than the molecular weight of the PEG segment, and the inner membrane band is obtained after self-assembly and cross-linking Reversibly cross-linked polymer vesicles with negative charges, the inner shell of the vesicles is KD _z for compounding small molecule drugs. The vesicle membrane is a reversibly cross-linked biodegradable PTMC with good compatibility. The dithiopentane structure of the side chain is similar to the human body's natural antioxidant lipoic acid, which can spontaneously form a reduction-sensitive reversible cross-linking, which not only guarantees the drug Stable and long-term circulation in blood can also achieve rapid intracellular de-crosslinking and rapid release of drugs into target cells.

Compared with the prior art, the present invention has the following advantages:

1. The present invention designs a new small-molecule hydrophilic drug VCR drug-loaded vesicle and tumor-targeted delivery; Alkane can provide reduction-sensitive reversible cross-linking, which can not only ensure the long-term circulation of drugs in the blood, but also can quickly de-cross-link in cells and release drugs into target cells; the shell is PEG and has targeting molecules such as monoclonal antibodies. Can specifically bind to cancer cells; the small size of the vesicles and tumor-specific targeting allow the vesicles to efficiently deliver VCRs into tumor cells.

2. The drug-loaded vesicles disclosed in the present invention have significant anti-tumor effects in vitro and in vivo, the polymers have good biocompatibility, can form vesicles with asymmetric membrane structures, and have good drug-carrying effects.

3. The degradable polymer vesicle carrier of the present invention avoids the defects of the existing nanocarriers such as large particle size, poor circulation stability in vivo, low tumor cell selectivity, and slow release of intracellular VCR.

4. The vesicle system of the present invention has many unique advantages, including small size, simple and controllable preparation, excellent biocompatibility, high in vivo circulation stability, strong tumor cell specific selectivity, fast intracellular drug release, tumor The growth inhibitory effect is remarkable and so on. Therefore, this vesicle system is expected to be a simple and all-in-one nanoplatform for efficient and specific targeted delivery of VCR to acute lymphoblastic leukemia cells.

Description of drawings

Fig. 1 is the nuclear magnetic spectrum of N ₃ -PEG-P(TMC-DTC) in Example 1.

Fig. 2 is the nuclear magnetic spectrum of PEG-P(TMC-DTC)-NPC in Example 2.

Fig. 3 is the nuclear magnetic spectrum of PEG-P(TMC-DTC)-KD ₅ in Example 2.

4 is the macromolecular mass spectrum of Dar and Dar-DBCO in Example 5.

FIG. 5 is a graph showing the stability of Dar-Ps-VCR in Example 6 in the presence of high dilution and serum.

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

Figure 7 shows (A) the endocytosis of Dar-Ps-Cy5 with different targeting densities in 697 cells in Example 7 and (B) 697 cells incubated with Dar _4.4 -Ps-Cy5 and Ps-Cy5 for 4 hours CLSM image (ruler bar: 25 μm).

Figure 8 shows the endocytosis of Dar-Ps-Cy5 with different targeting densities in CCRF-CEM cells in Example 7.

Figure 9 shows the toxicity of Dar-Ps-VCR, Ps-VCR and free VCR with different targeting densities in 697 cells in Example 8.

Figure 10 shows the toxicity of Dar-Ps-VCR and Ps-VCR in (A) MV4-11 cells and (B) L929 cells in Example 8.

Figure 11 is a graph showing the body weight change and Kaplan-Meier survival curve of the Dutch in situ 697 B-line acute lymphoblastic leukemia mice in Example 10 after receiving different treatments.

Detailed ways

The reversibly cross-linked and degradable polymer vesicles loaded with VCR of the present invention are obtained by self-cross-linking while self-assembly of amphiphilic tri-block polymers; the molecular chains of the tri-block polymers include sequentially connected hydrophilic Segment, hydrophobic segment and KD molecule; the hydrophilic segment is polyethylene glycol (PEG) with a molecular weight of 3000-8000 Da; the hydrophobic segment is a polycarbonate segment, and the molecular weight is a hydrophilic segment 2.1-5.7 times the molecular weight; the molecular weight of KD polypeptide is 15%-50% of the hydrophilic segment of PEG.

The PEG-P(TMC-DTC) _-KDz polymer of the present invention is prepared by reacting with _KDz after activating the terminal hydroxyl group of PEG-P(TMC-DTC) by p-nitrophenyl chloroformate (p-NPC). The route is as follows:

Wherein, in step (i), the reaction conditions are anhydrous dichloromethane (DCM), pyridine, 25 ºC, 24 hours; in step (ii), the reaction conditions are anhydrous dimethyl sulfoxide (DMSO), KD _z , triethylamine, 30 ºC, 48 hours.

The specific synthesis steps are as follows:

(1) In an ice-water bath, pyridine was added to the anhydrous DCM solution of PEG-P(TMC-DTC), and after stirring for 10 minutes, the DCM solution of p-NPC was slowly added dropwise thereto. After the completion of the dropwise addition (about 30 minutes), the reaction was continued at room temperature for 24 hours, and then the pyridinium salt was removed by suction filtration, and the polymer solution was collected and concentrated to ~100 mg/mL by rotary evaporation. P(TMC-DTC)-NPC;

(2) Under nitrogen protection, weigh the KD _z polypeptide into a double-necked round-bottomed flask and add anhydrous DMSO to dissolve it completely, add triethylamine under stirring, and then add PEG-P(TMC) dropwise to it. The anhydrous DMSO solution of -DTC)-NPC was added dropwise in 30 minutes. After reacting at 30 ºC for 2 days, dialyze against DMSO containing 5% anhydrous methanol for 36 hours (replace the medium 4-5 times) to remove unreacted KD _z and p-nitrophenol produced by the reaction, and then dialyze against DCM for 6 hours. After 2 hours, the polymer solution was collected and concentrated to a polymer concentration of about 50 mg/mL by rotary evaporation. After precipitation in ice ether, vacuum drying was performed to obtain a white cotton-like polymer PEG-P(TMC-DTC)-KD _z . The TMC was routinely replaced with LA or CL to obtain PEG-P(LA-DTC)-KD _z and PEG-P(CL-DTC)-KD _z .

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 is further described below in conjunction with the examples and accompanying drawings:

Example 1 Synthesis of polymer N ₃ -PEG-P (TMC-DTC)

The polymer N ₃ -PEG-P (TMC-DTC) is obtained by using DPP as a catalyst and N ₃ -PEG-OH as a macromolecular initiator to initiate ring-opening copolymerization of TMC and DTC. First, _{N 3} -PEG-OH ( Mn = 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 under nitrogen in a glove box. In a closed reactor, add 5.0 mL of anhydrous DCM to dissolve, then add DPP (0.25 g, 1.2 mmol), seal the reactor, transfer it out of the glove box, and place it at 30ºC to react for four days. After the reaction, it was precipitated twice with glacial ether, and dried in vacuo to obtain a white flocculent polymer N ₃ -PEG-P(TMC-DTC), yield: 85.4%. Characteristic peaks for N3 _- PEG at δ 3.38 and 3.63 ppm, characteristic peaks for TMC at δ 2.03 and 4.18 ppm, and characteristic peaks for DTC at δ 2.99 and 4.22 ppm can be seen in Figure 1 . The molecular weight of N ₃ -PEG-P(TMC-DTC) polymer can be calculated by the ratio of the integral area of methylene hydrogen at δ 2.03 and δ 2.99 ppm to the integral area of PEG methylene hydrogen at δ 3.63 ppm, which is 7.9-( 15.0-2.0) kg/mol, and its molecular weight distribution measured by GPC was 1.1, which was used in the following examples.

N ₃ -PEG-OH was replaced with CH ₃ O-PEG-OH with a molecular weight of 5K, and the rest remained unchanged. 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 _z

The synthesis of the polymer PEG-P(TMC-DTC)-KD _z is divided into two steps, namely, using p-NPC to activate the terminal hydroxyl group of PEG-P(TMC-DTC) (5.0-(15.0-2.0) kg/mol) , obtained by reacting with KD _z polypeptide molecules. Taking the synthesis of PEG-P(TMC-DTC)-KD ₅ as an example, the specific operation is as follows. PEG-P(TMC-DTC) (1.0 g, 45.5 μmol) was dissolved in 10 mL of anhydrous DCM under nitrogen atmosphere, It was then transferred to an ice-water bath and pyridine (18.0 mg, 227.5 μmol) was added, and after stirring for 10 minutes, a solution of p-NPC (48.4 mg, 240.3 μmol) in DCM (1.0 mL) was added dropwise. After the completion of the dropwise addition in 30 minutes, the reaction was continued for 24 hours at room temperature, and then the pyridinium salt was removed by suction filtration, and the polymer solution was collected by rotary evaporation and concentrated to ~100 mg/mL, precipitated with glacial ether, and dried in vacuo to obtain the product PEG-P ( TMC-DTC)-NPC, yield: 90.0%. Subsequently, under nitrogen protection, KD ₅ (60.0 mg, 83.4 μmol) was weighed and dissolved in 4 mL of anhydrous DMSO and triethylamine (4.2 mg, 41.7 μmol) was added, and then PEG- Anhydrous DMSO solution (9.0 mL) of P(TMC-DTC)-NPC was added dropwise for 30 minutes. After reacting at 30 ºC for 2 days, dialyze against DMSO containing 5% anhydrous methanol for 36 hours (replace the medium 4~5 times) to remove unreacted KD ₅ and the p-nitrophenol produced by the reaction, and then dialyze against DCM for 6 hours , then the polymer solution was collected and concentrated to a polymer concentration of 50 mg/mL by rotary evaporation, precipitated in ice ether and dried in vacuo to obtain a white cotton-like polymer PEG-P(TMC-DTC)-KD ₅ . Rate: 91.0%. Figures 2 and 3 are hydrogen NMR spectra of PEG-P(TMC-DTC)-NPC and PEG-P(TMC-DTC) _-KD5 . The characteristic peaks of p-NPC (δ 7.41 and δ 8.30 ppm) and the characteristic peaks of PEG-P(TMC-DTC) (δ 2.03, 2.99, 3.38, 3.63, 4.18 and 4.22 ppm) can be seen from Fig. 2, According to the ratio of the integral area of p-NPC characteristic peak to the area of PEG methyl hydrogen peak at δ3.38 ppm, the grafting rate of NPC was about 100%. Figure 3 shows that the characteristic peaks of NPC at δ 7.41 and δ 8.30 ppm disappear, and a new signal peak appears at δ 4.54 ppm, which is the characteristic peak of methine in KD ₅ . The degree of substitution of KD ₅ was calculated to be ~100% by comparing the ratio of the peak area at δ 4.54 ppm to the TMC methylene hydrogen peak area at δ 1.95 ppm. In addition, the grafting ratio of KD ₅ was 100% measured by high performance liquid chromatography (HPLC), demonstrating the successful synthesis of PEG-P(TMC-DTC)-KD ₅ , which was used in the following examples.

Example 3 Preparation of VCR-loaded reversibly cross-linked biodegradable vesicles (Ps-VCR)

Ps-VCR was prepared by solvent displacement method, in which VCR was encapsulated by electrostatic interaction with KD _z . PEG-P(TMC-DTC)-KD _z was dissolved in DMSO (40 mg/mL), 100 µL was dispensed into standing 900 µL HEPES (pH 6.8, 10 mM) containing VCR, and stirred at 300 rpm After 3 minutes, Ps-VCR was obtained by dialysis against HEPES (pH 7.4, 10 mM) for 8 hours. The theoretical drug loading of VCR was set at 4.8-11.1 wt.%, and it was found that the particle size of the obtained Ps-VCR was between 26-40 nm and the particle size distribution was 0.05-0.20 (Table 1). The encapsulation efficiency of Ps-VCR was calculated as high as 97.2% by measuring its absorbance at 298 nm by UV-Vis spectroscopy. Based on the same method, under the theoretical drug loading of 4.8%, the encapsulation efficiency of Ps-VCR prepared by PEG-P(LA-DTC)-KD ₅ and PEG-P(CL-DTC)-KD ₅ was 88.3%, The particle size of the drug-loaded vesicles prepared by PEG-P (TMC-DTC) diblock polymer is about 75 nm, and the encapsulation efficiency of VCR is low, only 14.1%.

Example 4 Preparation of reversibly cross-linked biodegradable vesicles (Ps-drug) loaded with other drugs

Using a similar method in Example 3, the encapsulation of other drugs such as verapamil hydrochloride (VER), irinotecan hydrochloride (CPT), and requimod (R848) by reversibly cross-linked degradable vesicles was studied. The study found that after encapsulating different drugs, the particle size of the obtained Ps-drug was between 20 and 40 nm. The specific results are shown in Table 2.

Example 5 Preparation of VCR-loaded mAb-directed polymersomes (Ab-Ps-VCR)

Ab-Ps-VCR was obtained by post-modification of a dibenzocyclooctyne-functionalized monoclonal antibody (Ab-DBCO) on the surface of an azide-functionalized polymersome VCR nanomedicine (N3 _- Ps-VCR). N ₃ -Ps-VCR is obtained by co-assembly of N ₃ -PEG-P(TMC-DTC) and PEG-P(TMC-DTC)-KD _z while wrapping VCR, wherein N ₃ -PEG-P(TMC-DTC ) content is 1~10 wt.%. Specifically, taking the preparation of N ₃ -Ps-VCR containing 2% N ₃ -PEG-P(TMC-DTC) as an example, 8.0 mg N ₃ -PEG-P(TMC-DTC) and 392.0 mg PEG- P(TMC-DTC)-KD ₅ (molar ratio 2:98) was dissolved in DMSO (total polymer concentration of 40 mg/mL), and 4.0 mL of VCR in water (5 mg/mL) was added to 90 mL of HEPES ( pH 6.8, 10 mM) and mix well, pour 10 mL of the polymer solution into it under standing, stir for 5 minutes, and place it at 37 ºC for 4 hours. After the organic solvent was removed by dialysis (MWCO: 14 kDa) with HEPES (pH 7.4, 10 mM) for 8 hours, the free VCR was removed by a nanofiltration system to obtain N ₃ -Ps-VCR. The particle size of N ₃ -Ps-VCR measured by dynamic light scattering (DLS) was 36 nm, and the distribution was narrow (PDI: 0.11). When the theoretical drug loading of VCR was 4.8 wt.%, the encapsulation efficiency was as high as 97.2%, and the drug loading was 4.6 wt.%. In order to efficiently bond the mAb, the N ₃ -Ps-VCR was then 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 mAb. The particle size of N ₃ -Ps-VCR after concentration was 42 nm and the PDI was 0.07. During the 180-day storage at 4 ºC, the particle size remained around 40 nm, the PDI was less than 0.17, and the leakage of VCR was less than 0.6%, indicating that N ₃ -Ps-VCR has excellent long-term storage stability (Table 3).

Ab-DBCO was prepared by amidation reaction of small molecule NHS-OEG ₄ -DBCO with the amino group on the monoclonal antibody, wherein the functionalization degree of DBCO can be adjusted by changing the molar ratio of Ab to NHS-OEG ₄ -DBCO. Taking the preparation of DBCO-functionalized daratumumab (Dar-DBCO) as an example, a solution of Dar in PBS (21.7 mg/mL) was diluted to 10 mg/mL with PB (pH 8.5, 10 mM), and 200 μL of 3 or 5 times molar equivalent of NHS-OEG ₄ -DBCO in DMSO solution (5 mg/mL) was added under shaking, and the reaction was carried out overnight at 27 ºC and 120 rpm in a shaker. After the reaction, the unreacted NHS-OEG ₄ -DBCO was removed by ultrafiltration tube centrifugation (MWCO: 10 kDa, 3000 rpm), and washed with PBS (pH 7.4, 10 mM) for ultrafiltration twice to obtain Dar-DBCO. When the molar ratio of Dar to NHS-OEG ₄ -DBCO was 1:3 and 1:5, 1.5 and 2.8 DBCOs were modified on each Dar by time-of-flight mass spectrometry (MALDI-TOF-MS), respectively (Fig. 4), expressed as Dar-DBCO _1.5 and Dar-DBCO _2.8 . In order to maximize the targeting and biological activity of the mAb, Dar-DBCO _1.5 or other mAbs modified with 1.5-2 DBCOs were used for subsequent experiments.

Dar-Ps-VCR can be easily prepared by tension-triggered click chemistry between N ₃ on the surface of N ₃ -Ps-VCR and Dar-DBCO, and the surface density of Dar can be adjusted by changing the feed ratio. The molar ratios of Dar-DBCO to N3 _were set to be 0.25:1, 0.5:1 and 1:1, respectively, that is, 10.4, 20.9 and 41.8 μL were added to 107.5 μL of N3 _- Ps-VCR (18.6 mg/mL), respectively of Dar-DBCO solution (5.6 mg/mL), then reacted overnight in a shaker at 25 ºC, 100 rpm. Unbound Dar-DBCO was removed by ultracentrifugation (58 krpm, 4 ºC, 30 min) and washed twice with HEPES (pH 7.4, 10 mM), while Dar-Ps-VCR and supernatant were collected to determine Dar binding combined amount. The unbound Dar-DBCO in the supernatant was determined by HPLC, and the content of Dar per mg of the surface of the polymersomes was calculated to be 28.6, 56.4 and 112.2 μg, respectively, according to the multi-angle laser light scattering. The absolute molecular weight (1.15×10 ⁷ g/mol) and aggregation number (523) were calculated to show that 2.2, 4.4 and 8.7 Dar were bound to each Dar-Ps-VCR surface, 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 narrow (PDI: 0.14-0.21). Example N3 _- Ps-VCR is the same.

The preparation methods of other mAb-directed VCR-loaded polymersomes, such as Isa-Ps-VCR and Anti-CD38-Ps-VCR, are similar to Dar-Ps-VCR. The particle size is between 40-60 nm, the particle size distribution is narrow (PDI: 0.10-0.30), and the number of mAbs on the surface of each vesicle is 1-10.

After the saporin-loaded protein (SAP) non-targeted vesicles (KD ₅ ) disclosed in Table 7 of the prior art CN110229323A were subjected to ultrafiltration or ultracentrifugation (58 krpm, 4 ºC, 30 minutes), the DLE decreased from 68.3% to 23 %, a large amount of drugs leaked, indicating that they cannot receive targeted mAbs.

Example 6 The stability and in vitro drug release of Ab-Ps-VCR targeting polymer vesicle nanomedicine

Dar _4.4 -Ps-VCR containing 4.4 Dar on each vesicle surface 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 diluted 50 times with phosphate buffer solution or added with 10% fetal bovine serum, and the particle size changes were detected by dynamic light scattering. Figure 5 is a particle size distribution diagram of Dar-Ps-VCR stability. The results showed that the Dar-Ps-VCR-targeted vesicle nanomedicine kept intact particle size and particle size distribution after being diluted 50 times and adding 10% FBS for 24 hours, and had good stability.

The in vitro drug release behavior of Dar-Ps-VCR was studied by dialysis, in which there were two release media, HEPES (pH 7.4, 10 mM) and HEPES solution containing 10 mM GSH (nitrogen atmosphere). 0.5 mL of Dar-Ps-VCR (0.5 mg/mL) was first loaded into a release bag (MWCO: 14 kDa) and then placed in 20 mL of the corresponding release medium in a shaker at 37 ºC, 100 rpm. At the set time points (0, 1, 2, 4, 6, 8, 10, 12, 24 h), 5 mL of dialysate was withdrawn and supplemented with 5 mL of fresh medium. The content of VCR in the dialysate was determined by HPLC (mobile phase was methanol:water (15% triethylamine was added, pH was adjusted to 7.0 with phosphoric acid) = 70:30). Figure 6 is a graph showing the results of in vitro release of Dar-Ps-VCR targeting vesicle nanomedicine. The results showed that under the reducing condition of 10 mM GSH, Dar-Ps-VCR released more than 85% of VCR within 12 hours, while under non-reducing conditions, the cumulative release of VCR within 24 hours was only about 22%.

Example 7 Endocytosis behavior of Dar-Ps-VCR targeting polymer vesicle nanomedicine

Since VCR itself has no fluorescence, Cy5 is used to label the polymer vesicles. The preparation method of Dar-Ps-Cy5 refers to Example 5, and the preparation method of Ps-Cy5 refers to Example 3; flow cytometry and laser scanning confocal microscopy ( CLSM) to study the uptake of Dar-Ps-Cy5 with different Dar densities in 697 cells. In the flow experiment, the 697 cell suspension was firstly plated in a 6-well plate (5×10 ⁵ cells/well), placed in an incubator for 12 hours, and 200 μL of Dar-Ps-Cy5 and Ps-Cy5 were added to each well. (The concentration of Cy5 in the well is 2.0 μg/mL), and the PBS group was used as a control. After a further 4 hours of incubation, cells were collected by centrifugation (800 rpm, 5 min), washed twice with PBS, and finally dispersed with 500 μL of PBS and placed in a flow tube for assay. The test results showed that the endocytosis of Dar-Ps-Cy5 in 697 cells was significantly higher than that of Ps-Cy5, and the cells incubated with Dar _4.4 -Ps-Cy5 had the highest fluorescence intensity, which was the same as that of the Ps-Cy5 control group. 5.7 times (Fig. 7A), indicating that the introduction of Dar can significantly enhance the cellular uptake of Ps-Cy5, and the targeting is optimal when 4.4 Dar are bound to the surface of each vesicle.

The endocytic behavior of Dar _4.4 -Ps-Cy5 and Ps-Cy5 in 697 cells was further studied by CLSM. The specific experimental steps are as follows. The small discs pretreated with poly-lysine (300 μL, 0.1 mg/mL) were placed in a 24-well plate, and 697 cell suspension (3×10 ⁵ cells/well) was added. After 24 hours in the incubator, 100 μL of Dar _4.4 -Ps-Cy5 and Ps-Cy5 (40 μg/mL in Cy5 well) were added, respectively. After incubation for 4 hours, the medium was carefully removed, washed 3 times with PBS, then fixed with 4% paraformaldehyde solution for 15 min, washed 3 times with PBS, stained with DAPI for 3 min, washed 3 times with PBS, and finally Mounted with glycerol and observed and photographed with CLSM (Leica, TCS SP5). Figure 7B is a graph showing the uptake results of Dar _4.4 -Ps-Cy5 and Ps-Cy5 in 697 cells. The results showed that when 697 cells were incubated with Dar _4.4 -Ps-Cy5 for 4 hours, there was obvious red fluorescence around the nucleus, while the fluorescence in the cells incubated with Ps-Cy5 was weak, indicating that Dar-Ps-Cy5 has an excellent target tropism and efficient and fast endocytosis.

Meanwhile, the uptake of Dar-Ps-Cy5 with different Dar densities in CCRF-CEM cells was also investigated by flow cytometry and laser scanning confocal microscopy (CLSM). In the flow experiment, the CCRF-CEM cell suspension was firstly plated in a 6-well plate (5×10 ⁵ cells/well), placed in an incubator for 12 hours, and 200 μL of Dar-Ps-Cy5 and Ps were added to each well. -Cy5 (the concentration of Cy5 in the well is 2.0 μg/mL), the PBS group was used as a control. After a further 4 hours of incubation, cells were collected by centrifugation (800 rpm, 5 min), washed twice with PBS, and finally dispersed with 500 μL of PBS and placed in a flow tube for assay. The test results showed that the endocytosis of Dar-Ps-Cy5 in CCRF-CEM cells was significantly higher than that of Ps-Cy5, and the cells incubated with Dar _4.4 -Ps-Cy5 had the highest fluorescence intensity, and its fluorescence intensity was Ps-Cy5 4.1 times that of the control group (Fig. 8), indicating that the introduction of Dar can significantly enhance the cellular uptake of Ps-Cy5, and the targeting is the best when 4.4 Dar are bound to the surface of each vesicle.

The endocytic behavior of Dar _4.4 -Ps-Cy5 and Ps-Cy5 in CCRF-CEM cells was further studied by CLSM. The specific experimental steps are as follows. The small discs pretreated with polylysine (300 μL, 0.1 mg/mL) were placed in a 24-well plate, and CCRF-CEM cell suspension (5×10 ⁵ cells/well) was added. , after culturing in the incubator for 24 hours, add 100 μL of Dar _4.4 -Ps-Cy5 and Ps-Cy5 respectively (the concentration of Cy5 in the well is 40 μg/mL). After incubation for 4 hours, the medium was carefully removed, washed 3 times with PBS, then fixed with 4% paraformaldehyde solution for 15 min, washed 3 times with PBS, stained with DAPI for 3 min, washed 3 times with PBS, and finally Mounted with glycerol and observed and photographed with CLSM (Leica, TCS SP5).

Example 8 Cytotoxicity experiment of Dar-Ps-VCR targeting polymer vesicle nanomedicine

The in vitro antitumor activity of Dar-Ps-VCR on B-lineage acute lymphoblastic leukemia (B-ALL) 697 cells was determined by CCK-8 kit. First, 697 cells were plated in 96-well plates (18,000 cells/well), placed in a 37 ºC, 5% CO ₂ incubator for 12 hours, and then 20 μL of Dar-containing Dar-containing different Dar surface densities were added to each well. The final concentrations of VCR in the wells were 0.001, 0.01, 0.05, 0.1, 0.5, 1, 10 and 100 ng/mL for Ps-VCR, Ps-VCR and free VCR, respectively. After 48 hours of incubation at 37 ºC, 10 μL of CCK-8 solution was added to each well and incubated for 4 hours. Finally, the absorbance at 450 nm was measured with a microplate reader. Figure 9 is a graph showing the cytotoxicity results of Dar-Ps-VCR vesicle nanomedicines with different targeting densities (z is 5) on 697 cells. The results showed that when 4.4 Dar were bound to the surface of each vesicle (Dar _4.4 -Ps-VCR), the cytotoxicity was the strongest, and its half-lethal concentration (IC ₅₀ ) was as low as 0.05 ng/mL, compared with free VCR (IC ₅₀ ) : 0.79 ng/mL) and the non-targeting control group Ps-VCR (z = 5, IC ₅₀ : 0.23 ng/mL) were reduced by 16- and 5-fold, respectively.

The in vitro antitumor activity of Dar-Ps-VCR on T-lineage acute lymphoblastic leukemia (T-ALL) CCRF-CEM cells was determined using CCK-8 kit. First, CCRF-CEM cells were plated in 96-well plates (16,000 cells/well), placed in a 37 ºC, 5% CO ₂ incubator for 12 hours, and 20 μL of cells containing different Dar surface densities were added to each well. Dar-Ps-VCR, Ps-VCR and free VCR, the final concentrations of VCR in the wells were 0.001, 0.01, 0.05, 0.1, 0.5, 1, 10 and 100 ng/mL, respectively. After 48 hours of incubation at 37 ºC, 10 μL of CCK-8 solution was added to each well and incubated for 4 hours. Finally, the absorbance at 450 nm was measured with a microplate reader.

MV4-11 cells (12,000 cells/well) and L929 fibroblasts (3,000 cells/well) were plated in 96-well plates for 24 hours, and then 20 μL of Dar _4.4 -Ps-VCR (z = 5) was added to each well. ) and Ps-VCR (z is 5), the final concentration of VCR in the wells was 0.0001-100 ng/mL. After MV4-11 cells were incubated at 37 ºC for 48 hours, 10 μL of CCK-8 solution was added to each well for further incubation for 4 hours, and the absorbance at 492 nm was measured with a microplate reader. After L929 cells were incubated at 37 ºC for 48 hours, 10 μL of MTT in PBS (5 mg/mL) was added to each well for 4 hours, after which the medium was carefully removed and 150 μL of DMSO was added to dissolve the resulting formazan crystals. The absorbance at 570 nm was measured by a standard spectrometer; the results showed that in MV4-11 cells, the _IC50 was 27 times higher than that in 697 cells (Fig. 10A). More interestingly, for L929 normal cells, Dar _4.4 -Ps-VCR and Ps-VCR did not show obvious toxicity even when the VCR concentration was as high as 100 ng/mL, and the cell viability was close to 100% ( Figure 10B). These results together indicate that Dar-Ps-VCR can selectively target and efficiently kill acute lymphoblastic leukemia cells with less toxicity to normal cells.

In addition, the same method was used to test the toxicity of Dar-Ps empty vesicles and Ps empty vesicles and free Dar on 697 cells.

In the following examples, Dar-Ps-VCR refers to Dar _4.4 -Ps-VCR vesicle nanomedicine (z is 5), and Dar-Ps-Cy5 refers to Dar _4.4 -Ps-Cy5 (z is 5).

Example 9 Construction of He 697 in situ B-lineage acute lymphoblastic leukemia mouse model

Establishment of Orthotopic B-ALL Tumor Model: All animal experiments and operations were approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University. ZOD/SCID female mice aged 6-8 weeks with an average body weight of approximately 20 g were irradiated on day 0 with a dose of 1.5 Gy and injected intraperitoneally with 0.2 mg (1 mg/mL) of anti-CD122 antibody against Mice were myeloablated, and then 697 cells (1×10 ⁵ cells/mice) were injected into mice through the tail vein. On the 6th day, the mice were randomly divided into groups for treatment, and the body weight, posture changes and survival period of the mice were continuously monitored.

Example 10 Antitumor effect of Dar-Ps-VCR in 697-bearing orthotopic B-lineage acute lymphoblastic leukemia mice

In order to study the antitumor effect of Dar-Ps-VCR on 697-bearing orthotopic B-lineage acute lymphoblastic leukemia mice, the treatment experiment was started on the 6th day after inoculation. The dose of VCR was 0.25 mg/kg, one injection for 4 days, a total of 4 injections. The groups were Dar-Ps-VCR, Ps-VCR and free VCR, and the PBS group was used as a control (z = 6). The study found that the mice in the PBS group began to develop disease 21 days after inoculation, showing paralysis of both legs, weight loss and death. Figure 11 shows the body weight change and survival period of mice in each treatment group. The results showed that all the mice in the treatment groups had stable body weight and no abnormal posture during the administration period (6-18 days), indicating no obvious toxic and side effects (Fig. 11A). After the administration, the mice in the PBS, Ps-VCR and free VCR treatment groups rapidly developed hindlimb paralysis, weight loss, and death. However, the mice in the Dar-Ps-VCR treatment group continued to maintain a stable body weight after the administration, and the survival period was significantly prolonged compared with the above three groups (Fig. 11B). Taken together, these results suggest that Dar-Ps-VCR can efficiently target and deliver VCR to the tumor site, thereby effectively inhibiting the growth of B-lineage acute lymphoblastic leukemia in situ.

Claims (10)

1. The application of the polymer vesicle carrying the small molecular drug in the preparation of the drug for treating acute lymphatic leukemia; the polymer vesicle carrying the micromolecular drug is prepared from the micromolecular drug and an amphiphilic block polymer; or the polymer vesicle carrying the small molecular drug is prepared from the small molecular drug, the amphiphilic block polymer, the functionalized amphiphilic block polymer and the targeting molecule;

the molecular structural formula of the amphiphilic block polymer is as follows:

wherein z is 5-15.

2. The application of the small-molecule-drug-carrying polymer vesicle in the preparation of drugs for treating acute lymphatic leukemia according to claim 1, wherein 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.

3. The use of the small molecule drug-loaded polymersome according to claim 1, wherein the small molecule drug is vincristine sulfate, doxorubicin hydrochloride, epirubicin hydrochloride, verapamil hydrochloride, irinotecan hydrochloride, and resiquimod.

4. The use of the small molecule drug-loaded polymersome according to claim 1 in the preparation of a drug for treating acute lymphatic leukemia, wherein the targeting molecule is a targeting monoclonal antibody.

5. The use of the small molecule drug-loaded polymersome according to claim 1 in the preparation of a drug for treating acute lymphatic leukemia, wherein the targeting monoclonal antibody is a targeting CD38 monoclonal antibody.

6. The application of the small-molecule-drug-carrying polymer vesicle in the preparation of drugs for treating acute lymphatic leukemia according to claim 1, wherein the preparation method of the small-molecule-drug-carrying polymer vesicle comprises the following steps of preparing drug-carrying polymer vesicles by a solvent displacement method by 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.

7. The application of the small-molecule-loaded polymer vesicle in the preparation of the drug for treating acute lymphatic leukemia according to claim 6, wherein the functionalized amphiphilic block polymer and the amphiphilic block polymer are assembled and crosslinked to load the drug, and then the drug-loaded polymer vesicle is reacted with the targeted monoclonal antibody to prepare the drug-loaded polymer vesicle.

8. The use of the small molecule drug-loaded polymer vesicle of claim 6 in the preparation of drugs for treating acute lymphatic leukemia, wherein the functional group in the functionalized amphiphilic block polymer is N₃-, Mal-or NHS-.

9. The application of the amphiphilic block polymer in preparing the nano-drug for resisting acute lymphatic leukemia according to claim 1, wherein the active ingredient of the nano-drug is a small molecule drug.

10. The application of the amphiphilic block polymer, the functionalized amphiphilic block polymer and the targeting molecule in the preparation of the anti-acute lymphatic leukemia nano-drug as claimed in claim 1, wherein the active ingredient of the nano-drug is a small molecule drug.

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