CN-118987254-B - Vein delivery system based on bimetallic nano-dot-oncolytic virus and preparation method and application thereof

Abstract

The invention belongs to the technical field of drug delivery carriers, and particularly relates to a bimetallic nano-dot-oncolytic virus intravenous delivery system, a preparation method thereof and application thereof in tumor immunotherapy. The invention uses oncolytic adenovirus, takes gadolinium-iron bimetallic nano-dots as raw materials, utilizes the covalent acting force of 4-maleimidobutyric acid-N-succinimidyl ester to construct a bimetallic nano-dot-adenovirus chimeric body, and evaluates the preparation performance, cytotoxicity, action mechanism, in-vivo anti-tumor effect, anti-tumor immunity, anti-tumor recurrence and metastasis effect and the like of the chimeric body. The preparation process is simple, the prepared bimetal nano dot-adenovirus chimeric has small and uniform particle size, is favorable for enriching in tumor tissues through permeation and retention Enhancement (EPR) effect, has high drug loading capacity and good safety, and can generate high-strength MRI signals at tumor positions. The bimetal nanodot-adenovirus chimeric body designed by the invention realizes the integration of diagnosis and treatment of tumors.

Inventors

• SUN JIN
• SUN MENGCHI
• WANG XU

Assignees

• 沈阳药科大学

Dates

Publication Date

20260512

Application Date

20240815

Claims (8)

1. 1. A vein delivery system based on bimetal nanodots-oncolytic viruses is characterized in that the system uses bimetal nanodots as carriers to coat oncolytic adenoviruses, the mass ratio of total proteins of the oncolytic adenoviruses to the bimetal nanodots is 1:2-1:20, the mass ratio of iron metal ions to albumin added in the forming process of the bimetal nanodots is 50:1-1:1, the mass ratio of gadolinium metal ions to total proteins is 50:1-1:1, the oncolytic viruses are oncolytic adenoviruses, the bimetal nanodots are nanodots formed by combining bovine serum albumin and metal ions in the interior of the bimetal nanodots, the metal ions are selected from gadolinium nitrate and ferric chloride, and the bimetal nanodots are covalently connected with the oncolytic viruses through 4-maleimidobutyric acid-N-succinimidyl ester.
2. 2. The method for preparing the bimetallic nano-dot-oncolytic adenovirus intravenous delivery system according to claim 1, comprising the steps of: (1) Construction and confirmation of the bimetal nano-dots: ① Gadolinium nitrate and ferric chloride solution are added into bovine serum albumin aqueous solution and mixed under intense stirring; ② The pH of the mixture was adjusted to 12 using 2.0M sodium hydroxide solution, then stirring the mixture was continued for 12 hours at 37 ℃ to form a crude product comprising gadolinium and iron bimetallic nanodots (Gd/Fe-ND); ③ Centrifuging the crude product by using an ultrafiltration concentration tube of 100kDa at a rotating speed of 3000: 3000rpm and centrifuging 5: 5min to obtain a product; ④ Dialyzing the product for 24 hours by distilled water to finally obtain a final product Gd/Fe-ND; ⑤ Verifying the particle size, potential and morphology of the final product Gd/Fe-ND by adopting dynamic light scattering, transmission electron microscopy, elemental analysis and circular dichroism; (2) Construction and confirmation of a bimetallic nanodot-oncolytic virus chimeric: ① Dissolving a proper amount of 4-maleimidobutyric acid-N-succinimidyl ester in dimethyl sulfoxide (DMSO); ② Diluting the solution prepared in the step ① with a proper amount of phosphate buffer solution, adding excessive Gd/Fe-ND, and incubating for 30 minutes; ③ Centrifuging the mixture to an original volume by using a 30 kDa ultrafiltration concentration tube; ④ Adding the concentrated solution into an oncolytic adenovirus solution which is diluted in a proper amount of phosphate buffer solution in advance, and incubating for 30 minutes again; ⑤ And verifying the particle size, potential and morphology of the chimera by adopting a dynamic light scattering and transmission electron microscopy method.
3. 3. The method according to claim 2, wherein in the step (1) ①, gadolinium nitrate having a concentration of 2.0 mL and 20.0 mM ferric chloride having a concentration of 2.0 mM and 2.0 mL are added to an aqueous solution of bovine serum albumin having a concentration of 10.0 mL and 50.0 mg/mL and mixed with vigorous stirring.
4. 4. The method of claim 2, wherein in the step (2) ②, the solution is diluted with an appropriate amount of phosphate buffer solution pH 8, and an excess of Gd/Fe-ND is added for 30 minutes of incubation.
5. 5. The method of claim 2, wherein in the step (2) ④, the concentrated solution is added to an oncolytic adenovirus solution previously diluted in an appropriate amount of phosphate buffer solution, pH 6.5, and the incubation is performed for 30 minutes again.
6. 6. Use of the bimetallic nanodot-oncolytic virus-based intravenous delivery system of claim 1 in the manufacture of a drug delivery system.
7. 7. Use of the bimetallic nanodot-oncolytic virus based intravenous delivery system of claim 1 in the manufacture of an anti-tumor drug.
8. 8. Use of the bimetallic nanodot-oncolytic virus intravenous delivery system of claim 1 for the preparation of a drug delivery system for injection, oral administration or topical administration.

Description

Vein delivery system based on bimetallic nano-dot-oncolytic virus and preparation method and application thereof Technical Field The invention belongs to the technical field of drug delivery carriers, and particularly relates to a bimetallic nano-dot-oncolytic virus intravenous delivery system, a preparation method thereof and application thereof in tumor immunotherapy. Background The incidence of cancer has increased over the years, becoming one of the leading causes of death worldwide. The traditional clinic mainly adopts the means of surgical excision, radiotherapy and chemotherapy to treat tumors. In recent years, with the progress and development of medicine, various emerging therapeutic means such as photothermal, photodynamic and oncolytic virus therapies and the like are emerging. Oncolytic virus therapy is an emerging cancer treatment of great interest, with unique advantages in the ability to selectively infect and kill tumor cells, and with the ability to specifically replicate and trigger an anti-tumor immune response in the body. As a new effort in the field of immunotherapy, oncolytic viral therapy has progressed significantly over decades. However, there is still a certain limitation in clinical application of oncolytic viruses, such as administration modes of intratumoral injection, and limited curative effects of pure oncolytic viruses, and lack of visual evaluation systems for oncolytic virus therapy. Therefore, how to enhance the curative effect of oncolytic viruses and establish an evaluation system become a major scientific problem to be solved in the field of oncolytic virus therapy. Disclosure of Invention In view of the above-mentioned drawbacks of the prior art, the present invention devised and constructed a novel intravenous delivery system based on bimetallic nanodot-oncolytic viruses. The bimetal nano-dot is a process that albumin can trigger metal ions to form a metal ion compound under mild conditions by simulating the process of chelating metal ions of organisms in nature, and the metal ion compound exists in a cavity of an albumin skeleton to finally form the nano-dot. Based on the method, the iron and gadolinium bimetallic nanodots are constructed, and the oncolytic adenovirus is connected covalently, so that the oncolytic virus intravenous injection can enhance the oncolytic virus tumor killing capability and nuclear magnetic resonance imaging can be realized. The bimetal nanodots are covalently connected with the oncolytic adenovirus, so that the bimetal nanodots are coated with the oncolytic adenovirus to shield antigens on the surface of the oncolytic adenovirus, avoid being cleared by the inherent immunity of the organism, and achieve the effect of long circulation in vivo. Meanwhile, the bimetallic nano-dots can enhance the tumor killing capability of oncolytic viruses and can be used as a contrast agent to realize nuclear magnetic resonance imaging. Oncolytic viruses activate an autophagy effect in cancer cells, the initiation of which provides support for the intimal structure required for viral replication, thereby increasing viral yield. Autophagy may also enhance the iron-dependent cell death mode-iron death. During excessive autophagy, nuclear receptor coactivator 4 (NCOA 4) is overexpressed, thereby promoting iron death. Notably, iron death can trigger and amplify autophagy by generating Reactive Oxygen Species (ROS), resulting in autophagic cell death. The invention provides a new solution strategy for the synergy of oncolytic adenovirus treatment, provides scientific basis for expanding the design of a novel high-efficiency low-toxicity microorganism delivery system, and has important research value. The invention realizes the aim through the following technical scheme: In a first aspect, the bimetallic nanodot-oncolytic viral intravenous delivery system (i.e., bimetallic nanodot-adenovirus chimera) of the present invention is achieved primarily using chemical bond cross-linking protein technology. The oncolytic virus is oncolytic adenovirus, the bimetallic nano-dots are iron nano-dots and gadolinium nano-dots generated in bovine serum albumin, and the metal ions are selected from gadolinium nitrate and ferric chloride. Alternatively, the bimetallic nanodot is covalently linked to the oncolytic virus via 4-maleimidobutyric acid-N-succinimidyl ester. The mass ratio of the oncolytic adenovirus to the total protein of the bimetallic nanodots is 1:2-1:20, in a preferred embodiment 1:20, the mass ratio of the iron metal ions added in the biomimetic mineralization process to the total protein is 50:1-1:1, in a preferred embodiment 46:1, and the mass ratio of the gadolinium metal ions added in the biomimetic mineralization process to the total protein is 50:1-1:1, in a preferred embodiment 36:1. In a second aspect, the bimetallic nanodot-oncolytic adenovirus intravenous delivery system of the invention (i.e., a bimetallic nanodot-adenovirus chimera) is prepared by t