WO2020140600A1 - 跨屏障靶向病灶的递药系统、载体系统及宿主细胞系统 - Google Patents

跨屏障靶向病灶的递药系统、载体系统及宿主细胞系统 Download PDF

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WO2020140600A1
WO2020140600A1 PCT/CN2019/115128 CN2019115128W WO2020140600A1 WO 2020140600 A1 WO2020140600 A1 WO 2020140600A1 CN 2019115128 W CN2019115128 W CN 2019115128W WO 2020140600 A1 WO2020140600 A1 WO 2020140600A1
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virus
barrier
particles
drug delivery
delivery system
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French (fr)
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任明
毕胜利
张利平
曹健荣
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北京岱瑞汛生物科技发展有限公司
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  • the present disclosure relates to the field of pharmaceutical preparations, and in particular, to a drug delivery system, a carrier system, and a host cell system that target lesions across barriers.
  • Drugs usually need to be delivered to the lesion at a certain speed and concentration to maximize the efficacy and minimize the side effects. Such a treatment is considered effective.
  • endothelial cell barrier and mucus barrier Common natural barriers affecting drug delivery include endothelial cell barrier and mucus barrier, endothelial cells include blood brain barrier, blood eye barrier, blood emulsion barrier, blood testis barrier, blood ovarian barrier and blood placental barrier, mucus barrier includes gastrointestinal mucosal barrier, Respiratory mucosal barrier or cervical mucosal barrier.
  • the blood-brain barrier refers to the barrier between blood and brain tissue. It can selectively pass or hinder the substance, and is composed of the continuous capillary endothelium of the brain and the tight connection between the cells, the complete basement membrane, the pericytes, and the glial membrane surrounded by the astrocyte foot plate
  • the endothelium is the main structure of the blood-brain barrier.
  • the structure of the brain capillaries prevents macromolecules from entering the brain tissue from the blood, so that the environment in the brain is relatively stable and less affected and invaded by the outside world. Exogenous substances enter the brain tissue from the blood mainly through diffusion or carrier transport.
  • the protein carrier present on the membrane of brain capillary endothelial cells mainly transports glucose, amino acids and various ionic substances. Up to 95% of drugs will be blocked by the blood-brain barrier when given intravenously.
  • the current delivery vector system mainly selects viral vectors and non-viral vectors.
  • the transfection efficiency of viral vectors is high, but there are major safety problems in itself, which may produce mutations and immune reactions; while the currently studied non-viral vectors such as high molecular polymers, liposomes, inorganic nanoparticles, etc. exist
  • the transfection efficiency is low and the biocompatibility is poor, so it is necessary to find a more suitable carrier.
  • Objects of the present disclosure include, for example, to provide a drug delivery system that targets lesions across a barrier, the drug delivery system including virus-like particles across a barrier and virus-like particles targeting a lesion, which are self-assembled by the aforementioned two virus-like particles Heteropolymeric virus-like particles can be formed, and the dual functions of drug cross-barrier and targeted transport to lesions can be achieved at the same time.
  • the virus-like particles have the advantages of safety and high transportation efficiency.
  • the trans-barrier peptide of the drug delivery system is a TGN peptide
  • the lesion-targeting peptide is RGD, which can break through the blood-brain barrier and deliver drugs to gliomas efficiently, safely, and directionally;
  • both ends of the lesion targeting peptide are also inserted with a connecting peptide whose amino acid sequence is as shown in SEQ ID NO: 2, which can increase flexibility, help RGD form a natural conformation, and increase targeting efficiency;
  • the carboxy terminus of the monomer protein of the disease-targeting virus-like particle is further inserted with a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3, which improves the ability of the virus-like particle to entrap hydrophobic drugs ;
  • the heteropolymer virus-like particles are hepatitis B virus-like particles, typically virus-like particles of hepatitis B virus core protein, forming a symmetric icosahedral structure, and the spiked structure on the surface is conducive to display across the barrier Signal and focus target signal; in addition, it can be well expressed in eukaryotic, prokaryotic and plant expression systems;
  • a virus-like particle is a natural nano-delivery system directly expressed by genetic engineering, as compared to a carrier formed by attaching a targeting peptide to the surface of a nano-particle through a chemical reaction or other force, the present disclosure
  • the drug delivery system has better homogeneity and stronger biocompatibility and safety.
  • Objects of the present disclosure include, for example, to provide a vector system including a nucleic acid vector encoding the monomer protein of the aforementioned trans-barrier virus-like particles and focus-targeting virus-like particles.
  • Objects of the present disclosure include, for example, to provide a host cell system containing the aforementioned vector.
  • a drug delivery system for cross-barrier targeting lesions includes cross-barrier virus-like particles and focus-targeting virus-like particles, wherein the cross-barrier virus-like particles carry cross-barrier signals, and the focus-targeting virus-like particles carry focus targeting Signal, the cross-barrier virus-like particles and the disease-targeting virus-like particles can self-assemble to form heteropolymeric virus-like particles.
  • Virus-like particles are hollow particles of one or more structural proteins of a virus. They have similar biological properties as natural viruses, but do not contain the genome of the virus, so they are not infectious and infectious. Under conditions, it can depolymerize and self-assemble, and has good biocompatibility and target cell specificity.
  • the drug delivery system of the present disclosure includes virus-like particles carrying a cross-barrier signal and virus-like particles carrying a lesion-targeting signal. The system utilizes the depolymerization and self-assembly of virus-like particles to form a signal with a cross-barrier and The heterogeneous virus-like particles that target the signal of the lesion make the drug delivery system have the dual functions of crossing the barrier and targeting to the lesion. At the same time, the drug delivery system of the present disclosure also has the advantages of no infectivity and infectivity, good biocompatibility and specificity of target cells.
  • the cross-barrier virus-like particles self-assemble with the disease-targeting virus-like particles to form heteropolymeric virus-like particles.
  • the cross-barrier virus-like particles and the lesion-targeting virus-like particles do not self-assemble to form heteropolymeric virus-like particles.
  • the drug delivery system further includes therapeutic drugs.
  • the heteroviral-like particles encapsulate the therapeutic drugs; in one or more embodiments, the therapeutic drugs are selected from chemotherapy Agents and/or nucleic acid drugs, for example, paclitaxel and siRNA; in one or more embodiments, the chemotherapeutic agent is encapsulated by a hydrophobic effect, and the nucleic acid drug is encapsulated by an electrostatic effect.
  • the barrier includes: an endothelial cell barrier or a transmucosal barrier, such as a blood-brain barrier, a blood-eye barrier, a blood emulsion barrier, a blood testis barrier, a blood ovarian barrier, or a blood placental barrier, or a gastrointestinal mucosal barrier, a respiratory tract Mucosal barrier or cervical mucosal barrier.
  • an endothelial cell barrier or a transmucosal barrier such as a blood-brain barrier, a blood-eye barrier, a blood emulsion barrier, a blood testis barrier, a blood ovarian barrier, or a blood placental barrier, or a gastrointestinal mucosal barrier, a respiratory tract Mucosal barrier or cervical mucosal barrier.
  • the lesion is an inflammatory lesion or a tumor lesion; in one or more embodiments, the lesion is a brain tumor lesion, for example, a glioma lesion.
  • the cross-barrier signal is a cross-barrier peptide, and the cross-barrier peptide is inserted into the monomer protein of the cross-barrier virus-like particle.
  • the focus target signal is a focus target peptide, and the focus target signal is inserted into the monomer protein of the focus target virus-like particle.
  • the trans-barrier peptide and/or focus-targeting peptide are inserted by genetic engineering.
  • the virus-like particles are directly expressed by genetic engineering.
  • the drug delivery system of the present disclosure has better uniformity and biocompatibility With stronger security.
  • the trans-barrier peptide is a TGN peptide
  • the lesion targeting peptide is an RGD peptide.
  • TGN peptide (TGNYKALHPHNG, SEQ ID NO: 1) is a short peptide that can overcome the blood-brain barrier to reach the brain.
  • the RGD tripeptide can specifically recognize integrin receptors. Integrin is a type of cell adhesion receptor molecule widely present on the surface of cells, and a large number of integrin receptors are expressed on the surface of gliomas.
  • the drug delivery system of the present disclosure uses TGN peptides and RGD peptides as cross-barrier peptides and focus-targeting peptides, which can break through the blood-brain barrier and efficiently, safely, and directionally deliver drugs to gliomas.
  • both ends of the RGD peptide are also inserted with a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 2 (GTSGSSGSGSGGSGSGGGG).
  • Linking peptides can increase the flexibility of RGD peptides, help RGD to form a natural conformation, and increase targeting efficiency.
  • the carboxy terminus of the monomer protein of the disease-targeting virus-like particle is further inserted with a polypeptide whose amino acid sequence is as shown in SEQ ID NO: 3 (AGSWLRDIWDWICEVLSDFKTWLKAKAKLMPTM).
  • Peptides can increase the ability of virus-like particles to entrap hydrophobic drugs.
  • the heteropolymeric virus-like particle is a hepatitis B virus-like particle, in one or more embodiments is a virus-like particle of a hepatitis B virus core protein; in one or more embodiments, a trans-barrier peptide
  • the insertion site of the Hepatitis Targeting Peptide is the amino acids 78-81 of the hepatitis B virus core protein, the hydrophobic peptide is inserted at the carboxyl end of the hepatitis B virus core protein with truncated carboxyl ends, and the truncation occurs at the amino acids 140-150 , For example, amino acid 144.
  • HBcVLPs virus-like particles
  • HBcAg can form a symmetric icosahedral structure in eukaryotic, prokaryotic and plant expression systems, with 240 or 180 monomers forming particles with a diameter of about 34nm and 30nm.
  • the total length of HBcAg is composed of 183 amino acids, of which amino acids 1-144 are related to the assembly of the cage structure.
  • amino acids 150-177 are rich in arginine and embedded in the HBc particles, which has a strong packaging for nucleic acids Ability
  • amino acids 73-82 are the main immune region, and have a spike-like structure on the surface of HBc VLPs
  • amino acids 78-82 are located at the apex of the spike, which can be well exposed on the surface of HBc VLPs.
  • the system of the present disclosure inserts a cross-barrier peptide and/or a focus-targeting peptide in the amino acid region 78-82, which facilitates its display on the surface of virus-like particles.
  • the heteromeric virus-like particles are nanoparticles, and in one or more embodiments, the diameter is 25-40 nm, such as 30 nm or 34 nm.
  • the mass ratio of heteropolymer virus-like particles to nucleic acid drug is 150-250:1, for example 200:1.
  • the mass ratio of heteropolymer virus-like particles to chemotherapeutic agent is 2-3:1, for example, 2.5:1.
  • the present disclosure also relates to a vector system including a nucleic acid vector encoding the monomer protein of the aforementioned trans-barrier virus-like particles and focus-targeting virus-like particles.
  • the present disclosure also relates to a host cell system containing the aforementioned vector system.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cells are E. coli or Bacillus subtilis, such as E. coli BL21 (DE3), and the eukaryotic cells are yeast cells, insect cells, plant cells or mammalian cells.
  • the present disclosure also relates to a drug delivery method that targets a lesion across a barrier, including administering a drug delivery system or a carrier system or a host cell system of the present disclosure to a subject in need.
  • the present disclosure also relates to: the use of the drug delivery system or carrier system or host cell system of the present disclosure for drug delivery across a barrier targeted lesion.
  • the present disclosure also relates to the use of the drug delivery system or carrier system or host cell system of the present disclosure for the treatment of inflammatory diseases or tumors.
  • the tumor is a brain tumor, more preferably a glioma.
  • the subject's lesion has a barrier to drug delivery.
  • the barrier may include an endothelial cell barrier and a mucus barrier.
  • the endothelial cell barrier includes a blood-brain barrier, a blood-eye barrier, a blood milk barrier, a blood testis barrier, a blood ovarian barrier, and a blood placental barrier.
  • the mucus barrier includes the gastrointestinal mucosal barrier, the respiratory mucosal barrier, or the cervical mucosal barrier.
  • the subject can be a mammal, such as a human.
  • the beneficial effects of the present disclosure include, for example:
  • the drug delivery system includes cross-barrier virus-like particles and lesion-targeting virus-like particles, which form heteropolymeric virus-like particles through the self-assembly of the aforementioned two virus-like particles, and at the same time realize the double barrier-crossing of drugs and targeted transportation At the same time, virus-like particles have the advantages of safety and high transportation efficiency;
  • the trans-barrier peptide of the drug delivery system is a TGN peptide
  • the lesion targeting peptide is RGD, which can break through the blood-brain barrier and deliver drugs to gliomas efficiently, safely, and directionally;
  • Both ends of the lesion-targeting peptide are also inserted with a connecting peptide whose amino acid sequence is shown as SEQ ID NO: 2, which can increase flexibility, help RGD to form a natural conformation, and increase targeting efficiency;
  • Heteropolymeric virus-like particles are hepatitis B virus-like particles, in one or more embodiments are virus-like particles of hepatitis B virus core protein, forming a symmetric icosahedral structure, and the spiked structure on the surface is conducive to display the span Barrier signal and focus target signal; in addition, it can be well expressed in eukaryotic, prokaryotic and plant expression systems;
  • Figure 1 is the construction and expression of delivery vector
  • Figure 2 shows in vitro, using U87 glioma cells as a model, to detect the killing effect of the drug delivery system on glioma cells;
  • Figure 3 is an in situ glioma model of U87-bearing glioma cells.
  • the delivery vector is labeled with Cy5.5, and the distribution of the vector in the brain is observed;
  • Figure 4 shows the establishment of an in situ glioma cell model of U87-bearing glioma cells to evaluate the targeted therapy of the targeted drug delivery system.
  • TGN-HBc VLPs refer to: virus-like particles of the hepatitis B virus core protein with TGN peptide inserted, and the insertion position of the TGN peptide is at the amino acids 78-81 of the hepatitis B virus core protein;
  • RGD-HBc VLPs refer to the virus-like particles of the hepatitis B virus core protein with the RGD peptide inserted.
  • the RGD peptide is inserted at the amino acids 78-81 of the hepatitis B virus core protein, and the connecting peptide is also inserted on both sides of the RGD peptide.
  • the amino acid sequence is shown in SEQ ID NO: 2 (GTSGSSGSGSGGSGSGGGG);
  • RGD-HBc-NS5A ⁇ VLPs means: Compared with RGD-HBc VLPs, the carboxyl terminus of hepatitis B virus core protein is truncated at amino acid 144, and the truncated carboxy terminus is inserted as SEQ ID NO: 3 (AGSWLRDIWDWICEVLSDFKTWLKAKAKLMPTM).
  • TGN-RGD-HBc VLPs refer to: TGN-HBc VLPs and RGD-HBc VLPs heteropolymer virus-like particles.
  • HBc VLPs streak or lay the genetically modified E. coli in the solid medium, incubate at 37°C overnight and pick single colonies to expand to 2.5% LB medium until the OD600 reaches 0.6
  • the precipitate was resuspended with Tris buffer (10 mM Tris-HCl, 0.5% (v/v) Triton-X-100), and the resuspended solution was sonicated to the point that the bacterial solution was not drawn, and the ultrasonic power was 50%.
  • the crushed cells were centrifuged at 12,000 rpm for 30 minutes at 4°C, the supernatant was collected, and saturated (NH 4 ) 2 SO 4 solution was slowly added during the stirring process. The saturated (NH 4 ) 2 SO 4 solution accounted for 30% of the total volume -40%, continue to stir for 30min.
  • the precipitated protein solution was centrifuged at 12000 rpm for 30 min at 4°C.
  • the precipitate was collected, resuspended in Tris buffer, and the resuspended solution was dialyzed in 20 mM Tris buffer for 48 h using a dialysis bag (MWCO 8000-14000 Da). After dialysis, the solution was centrifuged at 4°C, 12000 rpm for 30 minutes, passed through a DEAE ion exchange chromatography column, and the product was collected to obtain a solution of HBc VLPs. The position of protein bands detected by SDS-PAGE electrophoresis is shown in Figure 1. Both TGN-HBc VLPs and RGD-HBc VLPs are obtained by this method.
  • TGN-RGD-HBc VLPs use the BCA protein detection kit to detect the concentration of VLPs, and dilute the concentration to 2 mg/mL, take equal volumes of TGN-HBc VLPs and RGD-HBc VLPs, mix well, add Incubate 2-3 times the volume of depolymerization buffer (pH 8, 50 mM Tris-HCl, 150 mM NaCl, 6 M urea, 0.1% glycine) for 30 minutes to completely depolymerize VLPs. In the depolymerized state, add siRNA or PTX solution, mix and let stand for 30min.
  • depolymerization buffer pH 8, 50 mM Tris-HCl, 150 mM NaCl, 6 M urea, 0.1% glycine
  • the resulting solution was added to a dialysis bag (MWCO 8000-14000Da), dialyzed in recombinant buffer I (pH 8, 50 mM Tris, 150 mM NaCl, 10% (V/V) glycerol, 1% glycine) for 12 hours, and then reconstituted Buffer II (pH 8, 50 mM Tris, 150 mM NaCl, 1% glycine) was dialyzed for 12 hours, and samples were collected to obtain targeted drug-loaded nanoparticles.
  • buffer I pH 8, 50 mM Tris, 150 mM NaCl, 10% (V/V) glycerol, 1% glycine
  • the nano drug-loaded particles are regular in shape, uniform in size, and have a particle size of about 30 nm.
  • U87 cells were seeded in 96-well plates at a concentration of 5000 cells per well. Add 100 ⁇ L of PTX carrier solution to each well, a total of three groups: PTX group, PTX@TGN-RGD-HBc VLPs group and PTX@TGN-RGD-HBc VLPs and siRNA@TGN-RGD-HBc VLPs group (PTX concentration is 0.001, 0.01, 0.1, 1, 10 ⁇ g/mL).
  • mice bearing U87-Luc glial cells were evenly divided into four groups, labeled as normal saline group (NS), free paclitaxel group (Taxol), and paclitaxel-targeted drug delivery group (PTX@TGN-RGD-HBc VLPs ), Paclitaxel and siRNA-loaded targeted co-administration group (PTX@TGN-RGD-HBc VLPs+YAP-siRNA@TGN-RGD-HBc VLPs), according to PTX 5mg/kg, once every two days, A total of three times (days 0, 2, and 4), on day 0, 5, 10, 15, and 20, the tumor tumor fluorescence intensity was monitored by in vivo imaging. It can be seen from the results in Figure 4 that the dual-targeting drug-administration group has a better tumor suppression effect than the free paclitaxel group.
  • the drug delivery system, carrier system and host cell system of the cross-barrier targeting lesions provided by the present disclosure can be mass produced in industry.
  • the drug delivery system provided by the present disclosure can simultaneously achieve the dual functions of drug cross-barrier and targeted transportation, and at the same time, virus-like particles have the advantages of safety and high transportation efficiency, and can break through the blood-brain barrier, efficiently, safely and directionally.
  • the drug is delivered to glioma, and it can be well expressed in eukaryotic, prokaryotic and plant expression systems, and it has good uniformity, strong biocompatibility and safety.

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Abstract

提供一种跨屏障靶向病灶的递药系统、载体细胞及宿主细胞系统，该递药系统包括跨屏障病毒样颗粒和病灶靶向病毒样颗粒，屏障病毒样颗粒携带跨屏障信号，病灶靶向病毒样颗粒携带病灶靶向信号，跨屏障病毒样颗粒与病灶靶向病毒样颗粒能够自组装形成杂聚病毒样颗粒。

Description

[根据细则37.2由ISA制定的发明名称]　跨屏障靶向病灶的递药系统、载体系统及宿主细胞系统
相关申请的交叉引用
本公开要求于2018年12月30日提交中国专利局的申请号为2018116491459、名称为“一种跨屏障靶向病灶的递药系统、载体系统及宿主细胞系统”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及药物制剂领域，具体而言，涉及一种跨屏障靶向病灶的递药系统、载体系统及宿主细胞系统。
背景技术
药物通常需要以一定的速度和浓度递送至病灶处，使疗效最大而副作用最小，这样的治疗才被认为是有效的。然而，在药物递送和靶向分布过程中存在许多天然屏障，使得原本有应用前景的药物无效或者失效。
影响药物递送的常见天然屏障包括内皮细胞屏障和粘液屏障，内皮细胞包括血脑屏障、血眼屏障、血乳屏障、血睾丸屏障、血卵巢屏障和血胎盘屏障，粘液屏障包括胃肠道粘膜屏障、呼吸道粘膜屏障或子宫颈粘膜屏障。
以血脑屏障为例，血脑屏障(blood-brain barrier,BBB)是指介于血液和脑组织之间的屏障。它对物质有选择性通过或阻碍作用，由脑的连续毛细血管内皮及其细胞间的紧密连接、完整的基膜、周细胞以及星形胶质细胞脚板围成的神经胶质膜构成，其中内皮是血脑屏障的主要结构.脑毛细血管阻止大分子物质由血液进入脑组织的结构，从而使脑内环境相对稳定，较少受外界影响和侵害。外源性物质由血液进入脑组织主要是通过扩散或载体转运，只有少量脂溶性的小分子量药物可以经扩散透过血脑屏障。存在于脑毛细血管内皮细胞膜上的蛋白质载体，主要转运葡萄糖、氨基酸和各种离子物质。高达95％的药物静脉给予时，将被血脑屏障阻挡住。
为突破包括血脑屏障在内的各种天然屏障，目前递药载体系统主要选择病毒载体和非病毒载体。病毒载体的转染效率高，但本身存在较大的安全问题，可能产生突变与免疫反应；而目前研究较多的非病毒载体如高分子聚合物、脂质体、无机纳米粒子等则存在着转染效率低、生物相容性差等缺点，因此需要寻找一种更为合适的载体。
发明内容
本公开的目的包括，例如，提供一种跨屏障靶向病灶的递药系统，该递药系统包括跨屏障病毒样颗粒和病灶靶向病毒样颗粒，其通过前述两种病毒样颗粒的自组装可形成杂聚病毒样颗粒，同时实现药物的跨屏障、靶向运输至病灶的双功能，同时，该病毒样颗粒具有安全、运输效率高的优点。
在一种或多种实施方式中，递药系统的跨屏障肽为TGN肽，病灶靶向肽为RGD，能够突破血脑屏障，高效、安全和定向地将药物递送至脑胶质瘤；
在一种或多种实施方式中，病灶靶向肽的两端还插入氨基酸序列如SEQ ID NO：2所示的连接肽，能够增加柔性，有利于RGD形成天然构象，增加靶向效率；
在一种或多种实施方式中，病灶靶向病毒样颗粒的单体蛋白的羧基端还插入氨基酸序列如SEQ ID NO：3所示的多肽，提高病毒样颗粒对疏水性药物的包载能力；
在一种或多种实施方式中，杂聚病毒样颗粒为乙肝病毒样颗粒，典型为乙肝病毒核心蛋白的病毒样颗粒，形成对称的二十面体结构，表面的刺突结构有利于展示跨屏障信号和病灶靶向信号；另外，其在真核、原核与植物表达系统中均能较好表达；
在一种或多种实施方式中，病毒样颗粒由基因工程直接表达的天然纳米递药系统，相比于通过化学反应或其他作用力将靶向肽接至纳米粒子表面形成的载体，本公开递药系统的均一性更好，生物相容性与安全性更强。
本公开的目的包括，例如，提供一种载体系统，该载体系统包括编码前述跨屏障病毒样颗粒和病灶靶向病毒样颗粒的单体蛋白的核酸载体。
本公开的目的包括，例如，提供一种宿主细胞系统，该宿主细胞系统含前述载体。
为了实现本公开的上述目的，特采用以下技术方案：
一种跨屏障靶向病灶的递药系统，该系统包括跨屏障病毒样颗粒和病灶靶向病毒样颗粒，其中，跨屏障病毒样颗粒携带跨屏障信号，病灶靶向病毒样颗粒携带病灶靶向信号，跨屏障病毒样颗粒与病灶靶向病毒样颗粒能够自组装形成杂聚病毒样颗粒。
病毒样颗粒(VLPs)是病毒的一个或多个结构蛋白的空心颗粒，它具有与天然病毒相似的生物学性质，但不含病毒的基因组，因此无病毒的传染性和感染性，且在一定条件下能解聚与自组装，具有较好的生物相容性与靶细胞特异性。本公开的递药系统，包括携带有跨屏障信号的病毒样颗粒和携带有病灶靶向信号的病毒样颗粒，该系统利用病毒样颗粒的解聚与自组装作用，形成同时具有跨屏障信号和病灶靶向信号的杂聚病毒样颗粒，使得递药系统同时具备跨屏障和靶向定位至病灶的双重作用。同时，本公开的递药系统还具有无传染性和感染性、生物相容性与靶细胞特异性好的优点。
在一些具体实施方式中，跨屏障病毒样颗粒与病灶靶向病毒样颗粒自组装形成杂聚病 毒样颗粒。
在一些具体实施方式中，跨屏障病毒样颗粒与病灶靶向病毒样颗粒未自组装形成杂聚病毒样颗粒。
在一些具体实施方式中，递药系统还包括治疗药物，在一种或多种实施方式中，杂聚病毒样颗粒包载治疗药物；在一种或多种实施方式中，治疗药物选自化疗剂和/或核酸药物，例如，紫杉醇和siRNA；在一种或多种实施方式中，化疗剂通过疏水作用包载，核酸药物通过静电作用包载。
在一些具体实施方式中，屏障包括：内皮细胞屏障或跨粘液屏障，例如血脑屏障、血眼屏障、血乳屏障、血睾丸屏障、血卵巢屏障或血胎盘屏障，或胃肠道粘膜屏障、呼吸道粘膜屏障或子宫颈粘膜屏障。
在一些具体实施方式中，病灶为炎性病灶或肿瘤病灶；在一种或多种实施方式中，病灶为脑部肿瘤病灶，例如，脑胶质瘤病灶。
在一些具体实施方式中，跨屏障信号为跨屏障肽，跨屏障肽插入至跨屏障病毒样颗粒的单体蛋白。
在一些具体实施方式中，病灶靶向信号为病灶靶向肽，病灶靶向信号插入至病灶靶向病毒样颗粒的单体蛋白。
在一些具体实施方式中，跨屏障肽和/或病灶靶向肽通过基因工程的方式插入。所述病毒样颗粒由基因工程直接表达，相比于通过化学反应或其他作用力将靶向肽接至纳米粒子表面形成的载体，本公开的递药系统的均一性更好，生物相容性与安全性更强。
在一些具体实施方式中，跨屏障肽为TGN肽，病灶靶向肽为RGD肽。TGN肽(TGNYKALHPHNG，SEQ ID NO：1)是一段可以克服血脑屏障到达脑部的短肽。RGD三肽能特异性识别整合素受体，整合素是一类广泛存在于细胞表面的细胞粘附受体分子，在脑胶质瘤表面有大量的整合素受体表达。本公开的递药系统以TGN肽、RGD肽为跨屏障肽和病灶靶向肽，能够突破血脑屏障，高效、安全和定向地将药物递送至脑胶质瘤。
在一些具体实施方式中，RGD肽的两端还插入连接肽，该连接肽的氨基酸序列如SEQ ID NO：2(GTSGSSGSGSGGSGSGGGG)所示。连接肽可以增加RGD肽的柔性，有利于RGD形成天然构象，增加靶向效率。
在一些具体实施方式中，病灶靶向病毒样颗粒的单体蛋白的羧基端还插入氨基酸序列如SEQ ID NO：3(AGSWLRDIWDWICEVLSDFKTWLKAKAKLMPTM)所示的多肽。多肽能够提高病毒样颗粒对疏水性药物的包载能力。
在一些具体实施方式中，杂聚病毒样颗粒为乙肝病毒样颗粒，在一种或多种实施方式中为乙肝病毒核心蛋白的病毒样颗粒；在一种或多种实施方式中，跨屏障肽和病灶靶向肽 的插入位点为乙肝病毒核心蛋白第78-81位氨基酸，疏水性多肽插入在羧基端被截短的乙肝病毒核心蛋白的羧基端，截短发生在第140-150位氨基酸，例如，第144位氨基酸。
本公开的系统用的是乙肝病毒核心蛋白的病毒样颗粒(HBc VLPs)。HBcAg能够在真核、原核以及植物表达系统中形成对称的二十面体结构，由240个或180个单体形成直径约34nm和30nm的颗粒。HBcAg全长由183个氨基酸组成，其中第1-144位氨基酸与笼状结构的组装有关，第150-177位氨基酸富含精氨酸并包埋于HBc颗粒内部，对核酸有较强的包装能力，第73-82位氨基酸是主要免疫区域，在HBc VLPs表面呈刺突状结构，而其中第78-82位氨基酸又位于刺突的顶点，能够很好的暴露于HBc VLPs表面。本公开的系统在第78-82位氨基酸区域插入跨屏障肽和/或病灶靶向肽，有利于其在病毒样颗粒表面的展示。
在一些具体的实施方式中，杂聚病毒样颗粒为纳米颗粒，在一种或多种实施方式中，直径为25-40nm，例如30nm或34nm。
在一些具体实施方式中，杂聚病毒样颗粒与核酸药物的质量比为150-250:1，例如为200:1。
在一些具体的实施方式中，杂聚病毒样颗粒与化疗剂的质量比为2-3:1，例如为2.5:1。
本公开还涉及：一种载体系统，该载体系统包括编码前述跨屏障病毒样颗粒和病灶靶向病毒样颗粒的单体蛋白的核酸载体。
本公开还涉及：一种宿主细胞系统，该宿主细胞系统含有前述载体系统，在一种或多种实施方式中，宿主细胞为原核细胞或真核细胞，在一种或多种实施方式中，原核细胞为大肠杆菌或枯草芽孢杆菌，例如大肠杆菌BL21(DE3)，真核细胞为酵母细胞、昆虫细胞、植物细胞或哺乳动物细胞。
本公开还涉及：一种跨屏障靶向病灶的递药方法，包括向有需要的受试者给药本公开的递药系统或载体系统或宿主细胞系统。
本公开还涉及：本公开的递药系统或载体系统或宿主细胞系统，用于跨屏障靶向病灶递药的用途。
本公开还涉及：本公开的递药系统或载体系统或宿主细胞系统，用于治疗炎性疾病或肿瘤中的用途，优选地，该肿瘤为脑部肿瘤，更优选地为脑胶质瘤。
在一种或多种实施方式中，受试者的病灶对于药物递送存在屏障。在一种或多种实施方式中，屏障可以包括内皮细胞屏障和粘液屏障。在一种或多种实施方式中，内皮细胞屏障包括血脑屏障、血眼屏障、血乳屏障、血睾丸屏障、血卵巢屏障和血胎盘屏障。在一种或多种实施方式中，粘液屏障包括胃肠道粘膜屏障、呼吸道粘膜屏障或子宫颈粘膜屏障。
在一种或多种实施方式中，受试者可以是哺乳动物，例如人类。
与现有技术相比，本公开的有益效果包括，例如：
(1)递药系统包括跨屏障病毒样颗粒和病灶靶向病毒样颗粒，其通过前述两种病毒样颗粒的自组装形成杂聚病毒样颗粒，同时实现药物的跨屏障、靶向运输的双功能，同时，病毒样颗粒具有安全、运输效率高的优点；
(2)递药系统的跨屏障肽为TGN肽，病灶靶向肽为RGD，能够突破血脑屏障，高效、安全和定向地将药物递送至脑胶质瘤；
(3)病灶靶向肽的两端还插入氨基酸序列如SEQ ID NO：2所示的连接肽，能够增加柔性，有利于RGD形成天然构象，增加靶向效率；
(4)病灶靶向病毒样颗粒的单体蛋白的羧基端还插入氨基酸序列如SEQ ID NO：3所示的多肽，提高病毒样颗粒对疏水性药物的包载能力；
(5)杂聚病毒样颗粒为乙肝病毒样颗粒，在一种或多种实施方式中为乙肝病毒核心蛋白的病毒样颗粒，形成对称的二十面体结构，表面的刺突结构有利于展示跨屏障信号和病灶靶向信号；另外，其在真核、原核与植物表达系统中均能较好表达；
(6)病毒样颗粒由基因工程直接表达的天然纳米递药系统，相比于通过化学反应或其他作用力将靶向肽接至纳米粒子表面形成的载体，本公开的递药系统的均一性更好，生物相容性与安全性更强。
附图说明
为了更清楚地说明本公开的具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为递送载体的构建及表达；
图2为在体外，以U87胶质瘤细胞为模型，检测递药系统对胶质瘤细胞的杀伤效果；
图3为建立荷U87胶质瘤细胞原位胶质瘤动物模型，以Cy5.5标记递送载体，观察载体在脑内的分布；
图4为建立荷U87胶质瘤细胞原位胶质瘤动物模型，评价靶向递药系统的靶向治疗情况。共分为4组：生理盐水组，PTX组，PTX@TGN-RGD-HBc VLPs组和PTX@TGN-RGD-HBc VLPs协同YAP-siRNA@TGN-RGD-HBc VLPs组。
具体实施方式
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解， 下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购买获得的常规产品。
TGN-HBc VLPs是指：插入有TGN肽的乙肝病毒核心蛋白的病毒样颗粒，TGN肽的插入位置为乙肝病毒核心蛋白第78-81位氨基酸处；
RGD-HBc VLPs是指：插入有RGD肽的乙肝病毒核心蛋白的病毒样颗粒，RGD肽的插入位置为乙肝病毒核心蛋白第78-81位氨基酸处，RGD肽两侧还插入连接肽，连接肽的氨基酸序列如SEQ ID NO：2(GTSGSSGSGSGGSGSGGGG)所示；
RGD-HBc-NS5A·VLPs是指：与RGD-HBc VLPs相比，乙肝病毒核心蛋白的羧基端在第144位氨基酸处被截短，并在截短后的羧基端插入如SEQ ID NO：3(AGSWLRDIWDWICEVLSDFKTWLKAKAKLMPTM)所示的多肽。
TGN-RGD-HBc VLPs是指：TGN-HBc VLPs和RGD-HBc VLPs的杂聚病毒样颗粒。
实施例1
HBc VLPs的表达与纯化：将经过目的基因改造的大肠杆菌划线或平铺在固体培养基中，37℃倒置过夜培养后挑取单菌落至2.5％LB培养基中扩大培养，直至OD600达到0.6-0.8时，加入0.1mM IPTG(体积比IPTG:LB培养基＝1:10000)于18℃诱导培养18h。诱导结束后，收集菌液，4℃，4000rpm离心12min，弃上清，收集沉淀。沉淀用Tris缓冲液(10mM Tris-HCl,0.5％(v/v)Triton-X-100)重悬，将重悬后的溶液超声破碎至菌液不拉丝，超声功率50％。破碎后的菌体在4℃，12000rpm条件下离心30min，收集上清，在搅拌过程中缓慢加入饱和(NH 4) 2SO 4溶液，饱和(NH 4) 2SO 4溶液占总体积的30％-40％，持续搅拌30min。将沉淀后的蛋白溶液4℃，12000rpm离心30min，收集沉淀，用Tris缓冲液重悬，重悬液用透析袋(MWCO 8000-14000Da)在20mM的Tris缓冲液中透析48h。透析后溶液在4℃，12000rpm，离心30min，过DEAE离子交换色谱柱，收集产物，即得到HBc VLPs溶液。用SDS-PAGE电泳检测蛋白条带位置如图1。TGN-HBc VLPs与RGD-HBc VLPs皆由此方法获得。
靶向递药载体TGN-RGD-HBc VLPs的制备：用BCA蛋白检测试剂盒检测VLPs浓度，并将浓度稀释成2mg/mL，取等体积TGN-HBc VLPs和RGD-HBc VLPs，混匀，加入2-3倍体积的解聚缓冲液(pH为8，50mM Tris-HCl,150mM NaCl，6M尿素，0.1％甘氨酸)共孵育30min，使VLPs完全解聚。在解聚的状态下，加入siRNA或PTX溶液，混匀后静置30min。将所得溶液加入透析袋(MWCO 8000-14000Da)中，在重组缓冲液I(pH为8，50mM Tris，150mM NaCl，10％(V/V)甘油，1％甘氨酸)中透析12h，再在重组缓冲液II(pH为8，50mM Tris，150mM NaCl，1％甘氨酸)中透析12h，收集样品即可得到靶向 载药纳米粒子。
通过电镜观察粒子形态，如图1，纳米载药粒子形状规整，大小均一，粒径约为30nm。
实施例2
将U87细胞以每孔5000个细胞的浓度接种于96孔板中。每孔加入100μL PTX载体溶液，共三组：PTX组，PTX@TGN-RGD-HBc VLPs组和PTX@TGN-RGD-HBc VLPs协同siRNA@TGN-RGD-HBc VLPs组(PTX的浓度为0.001、0.01、0.1、1、10μg/mL)。孵育24h后，弃掉上清，加入100μL MTT(5mg/mL)溶液孵育4h后，弃上清，加入150μL DMSO，37℃摇床摇30min，在490nm处测OD值。结果如图2显示，在PTX低浓度下，载PTX靶向给药组(PTX@TGN-RGD-HBc VLPs)对细胞的杀伤效果与游离药物组(PTX)区别不大，而协同给药(PTX@TGN-RGD-HBc VLPs+YAP-siRNA@TGN-RGD-HBc VLPs)表现出明显的杀伤效果，在高浓度下，载PTX靶向给药组与协同给药组均表现出明显的杀伤效果。
实施例3
建立荷U87细胞原位脑胶质瘤模型，用Cy5.5标记TGN-RGD-HBc VLPs与无靶向VLPs(HBc-183VLPs)，通过尾静脉注射给药，在不同时间点观察小鼠活体荧光分布情况。结果如图3所示，对同一时间点图片，其中左侧小鼠为生理盐水组，中间小鼠为无靶向VLPs组，右侧小鼠为双靶向VLPs组。根据图3所示结果可知，双靶向的VLPs在脑部的分布明显优于无靶向的VLPs。
实施例4
将12只荷U87-Luc胶质细胞的小鼠平均分成四组，标记为生理盐水组(NS)，游离紫杉醇组(Taxol)，载紫杉醇靶向给药组(PTX@TGN-RGD-HBc VLPs)，载紫杉醇与载siRNA靶向协同给药组(PTX@TGN-RGD-HBc VLPs+YAP-siRNA@TGN-RGD-HBc VLPs)，按PTX 5mg/kg给药，每两天给药一次，共三次(第0、2、4天)，于第0、5、10、15、20天用活体成像监测小鼠肿瘤荧光强度。从图4结果可以看出，双靶向给药组对肿瘤的抑制效果明显优于游离紫杉醇组。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供的一种跨屏障靶向病灶的递药系统、载体系统及宿主细胞系统，均能够在工业上批量生产。此外，本公开提供的递药系统能同时实现药物的跨屏障、靶向运输的双功能，同时，病毒样颗粒具有安全、运输效率高的优点，能够突破血脑屏障，高效、安全和定向地将药物递送至脑胶质瘤，且其在真核、原核与植物表达系统中均能较好表达，而且其均一性好，生物相容性与安全性强。

Claims (20)

  1. 一种跨屏障靶向病灶的递药系统，其包括跨屏障病毒样颗粒和病灶靶向病毒样颗粒，其中，所述跨屏障病毒样颗粒携带跨屏障信号，所述病灶靶向病毒样颗粒携带病灶靶向信号，所述跨屏障病毒样颗粒与所述病灶靶向病毒样颗粒能够自组装形成杂聚病毒样颗粒。
  2. 根据权利要求1所述的递药系统，其中，所述跨屏障病毒样颗粒与所述病灶靶向病毒样颗粒自组装形成杂聚病毒样颗粒，或者，所述跨屏障病毒样颗粒与所述病灶靶向病毒样颗粒未自组装形成杂聚病毒样颗粒。
  3. 根据权利要求1或2所述的递药系统，所述递药系统还包括治疗药物，可选地，所述杂聚病毒样颗粒包载所述治疗药物；优选地，所述治疗药物选自化疗剂和/或核酸药物，例如，紫杉醇和siRNA；更优选地，所述化疗剂通过疏水作用包载，所述核酸药物通过静电作用包载。
  4. 根据权利要求1至3中任一项所述的递药系统，其中，所述屏障包括：内皮细胞屏障或粘液屏障，例如血脑屏障、血眼屏障、血乳屏障、血睾丸屏障、血卵巢屏障、血胎盘屏障、胃肠道粘膜屏障、呼吸道粘膜屏障或子宫颈粘膜屏障。
  5. 根据权利要求1至4中任一项所述的递药系统，其中，所述病灶为炎性病灶或肿瘤病灶；优选地，所述病灶为脑部肿瘤病灶，例如，脑胶质瘤病灶。
  6. 根据权利要求1至5中任一项所述的递药系统，其中，所述跨屏障信号为跨屏障肽，所述跨屏障肽插入至所述跨屏障病毒样颗粒的单体蛋白，所述病灶靶向信号为病灶靶向肽，所述病灶靶向信号插入至所述病灶靶向病毒样颗粒的单体蛋白，可选地，所述跨屏障肽为TGN肽，所述病灶靶向肽为RGD肽；优选地，所述跨屏障肽和/或所述病灶靶向肽通过基因工程的方式插入；
    优选地，所述RGD肽的两端还插入连接肽，所述连接肽的氨基酸序列如SEQ ID NO：2所示。
    优选地，所述病灶靶向病毒样颗粒的单体蛋白的羧基端还插入氨基酸序列如SEQ ID NO：3所示的多肽。
  7. 根据权利要求6所述的递药系统，其中，所述杂聚病毒样颗粒为乙肝病毒样颗粒，优选为乙肝病毒核心蛋白的病毒样颗粒；优选地，所述跨屏障肽和病灶靶向肽的插入位点为乙肝病毒核心蛋白第78-81位氨基酸，SEQ ID NO：3所示的多肽插入在羧基端被截短的乙肝病毒核心蛋白的羧基端，所述截短发生在第140-150位氨基酸，例如，第144位氨基酸。
  8. 根据权利要求7所述的递药系统，其中，所述乙肝病毒核心蛋白的病毒样颗粒形成对称的二十面体结构。
  9. 根据权利要求7或8所述的递药系统，其中，所述乙肝病毒核心蛋白的病毒样颗粒的表面呈刺突结构。
  10. 根据权利要求7至9中任一项所述的递药系统，其中，所述乙肝病毒核心蛋白的病毒样颗粒由基因工程直接表达。
  11. 根据权利要求7至10中任一项所述的递药系统，其中，所述乙肝病毒核心蛋白的病毒样颗粒能够在真核、原核以及植物表达系统中形成对称的二十面体结构。
  12. 根据权利要求7至11中任一项所述的递药系统，其中，所述乙肝病毒核心蛋白的病毒样颗粒由240个或180个单体形成。
  13. 根据权利要求7至12中任一项所述的递药系统，其中，所述乙肝病毒核心蛋白的病毒样颗粒全长由183个氨基酸组成，其中第1-144位氨基酸与笼状结构的组装有关，第150-177位氨基酸富含精氨酸并包埋于HBc颗粒内部，第73-82位氨基酸是主要免疫区域。
  14. 根据权利要求13所述的递药系统，其中，第78-82位氨基酸位于所述刺突状结构的顶点。
  15. 根据权利要求1至14中任一项所述的递药系统，其中，所述杂聚病毒样颗粒为纳米颗粒，优选地，纳米颗粒的直径为25-40nm，例如30nm或34nm；
    优选地，所述杂聚病毒样颗粒与核酸药物的质量比为150-250:1，例如为200:1；
    优选地，所述杂聚病毒样颗粒与化疗剂的质量比为2-3:1，例如为2.5:1。
  16. 一种载体系统，其包括编码权利要求1-15任一项所述跨屏障病毒样颗粒和病灶靶向病毒样颗粒的单体蛋白的核酸载体。
  17. 一种宿主细胞系统，其含有如权利要求16所述的载体系统，可选地，所述宿主细胞为原核细胞或真核细胞，优选地，所述原核细胞为大肠杆菌或枯草芽孢杆菌，例如大肠杆菌BL21(DE3)，所述真核细胞为酵母细胞、昆虫细胞、植物细胞或哺乳动物细胞。
  18. 一种跨屏障靶向病灶的递药方法，包括向有需要的受试者给药根据权利要求1至15任一项所述的递药系统或根据权利要求16所述的载体系统或根据权利要求17所述的宿主细胞系统。
  19. 根据权利要求1至15任一项所述的递药系统或根据权利要求16所述的载体系统或根据权利要求17所述的宿主细胞系统，用于跨屏障靶向病灶递药的用途。
  20. 根据权利要求1至15任一项所述的递药系统或根据权利要求16所述的载体系 统或根据权利要求17所述的宿主细胞系统，用于治疗炎性疾病或肿瘤中的用途，优选地，所述肿瘤为脑部肿瘤，更优选地为脑胶质瘤。
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