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CN-121059561-B - Micro-nano robot based on probiotics and framework nucleic acid and preparation method thereof

CN121059561BCN 121059561 BCN121059561 BCN 121059561BCN-121059561-B

Abstract

The invention relates to the technical field of drug delivery systems, in particular to a double-targeting micro-nano robot based on probiotics and framework nucleic acid and a preparation method thereof. In the invention, the medicine is introduced into ECN engineering bacteria (ECN-pl) with tumor hypoxia micro-environmental cracking sensitivity AS a delivery strategy, so that activity resistance brought by surface modification to a strain can be avoided, DOX@FDN-A2.2@ECN-pl goes deep into a tumor core after entering the body, and tumor cells hypoxia is sensed to generate cracking protein, so that ECN structure is destroyed, DOX@FDN-A2.2 is successfully released to the tumor core, wherein AS1411 and S2.2 aptamers on the surface of FDN-A2.2 have the capability of targeting tumor cells, so that the uptake rate of the tumor cells is improved by the double-targeting aptamers on the surface of FDN-A2.2, the problem of tumor drug resistance is solved, and the growth of the tumor cells is inhibited.

Inventors

  • HUANG JIAOFANG
  • QIAN JIANGCHAO
  • JIANG NING
  • WANG YUJIE
  • ZHAO KUI
  • DING WANQING
  • HU JINYI

Assignees

  • 华东理工大学

Dates

Publication Date
20260512
Application Date
20250908

Claims (7)

  1. 1. The preparation method of the micro-nano robot based on the probiotics and the framework nucleic acid is characterized by comprising the following steps of: preparing DOX solution; mixing DOX solution with FDN-A2.2, and stirring for reaction to obtain DOX@FDN-A2.2; the DOX@FDN-A2.2 is converted into competent ECN to obtain DOX@FDN-A2.2@ECN, namely the micro-nano robot based on probiotics and framework nucleic acid; The FDN-A2.2 is FDN T -A2.2 or FDN R -A2.2,FDN T -A2.2, the nucleotide sequence of FDN R -A2.2 is shown as SEQ ID No.1, the nucleotide sequence of FDN R -A2.2 is shown as SEQ ID No.2, the competent ECN is ECN engineering bacteria capable of feeling the anoxic micro-environmental cracking of tumors, and DOX represents anthracycline.
  2. 2. The method of claim 1, wherein the concentration of the DOX solution is 4mg/mL to 10mg/mL.
  3. 3. The method of claim 2, wherein the concentration of the DOX solution is 5mg/mL.
  4. 4. The method of claim 1, wherein the DOX solution is an aqueous solution of doxorubicin.
  5. 5. The method of claim 1, further comprising the step of transforming the gene loop pGEX-pVHB-Lysis into competent ECN.
  6. 6. The method of claim 5, wherein the preparation method of the gene loop pGEX-pVHB-Lysis comprises the steps of cutting pGEX plasmid with EcoRI and BamHI, recovering linearized pGEX plasmid skeleton after electrophoresis, amplifying Lysis fragment by PCR, recovering Lysis fragment after electrophoresis, seamlessly cloning and connecting the Lysis fragment with pGEX skeleton, transferring the connection product into DH5 alpha competent cells, plating and culturing, picking single colony shake bacteria, PCR to obtain pGEX-Tac-GST-Lysis split plasmid, amplifying pGEX-Tac-GST-Lysis split plasmid skeleton by PCR, recovering pGEX-Lysis vector, seamlessly cloning and connecting pVHB promoter with pGEX-Lysis carried out, transferring the connection product into DH5 alpha strain for amplification, and producing gene loop pGEX-pVHB-Lysis by the obtained recombinant engineering bacteria.
  7. 7. A micro-nano robot based on probiotics and framework nucleic acid, characterized in that the micro-nano robot is obtained by the preparation method of the micro-nano robot based on probiotics and framework nucleic acid according to any one of claims 1-6.

Description

Micro-nano robot based on probiotics and framework nucleic acid and preparation method thereof Technical Field The invention relates to the technical field of drug delivery systems, in particular to a micro-nano robot based on probiotics and framework nucleic acid and a preparation method thereof. Background The dense extracellular matrix of solid tumors generates high osmotic pressure, so that drugs are difficult to enter the core of tumor cells, and traditional DOX cannot effectively clear the tumor cells. The nano-drug can accumulate at the tumor part through enhanced permeability and retention effect (Enhanced permeability and retention effect, EPR), so that the drug utilization rate is improved to a certain extent, but the nano-drug is still difficult to penetrate into the tumor core, and the tumor immunosuppression microenvironment can limit the immunocyte activity in the solid tumor, so that the tumor treatment difficulty is increased. Coli Nissle 1917 (ESCHERICHIA COLINISSLE 1917, ecn) is a facultative anaerobic probiotic that can penetrate the dense extracellular matrix of tumor tissue and specifically colonize the tumor core, and researchers attach drugs to its surface to achieve drug delivery to the tumor core. However, surface modification affects ECN activity, and drug loading rate is not ideal, and simultaneously tumor cells can enhance drug resistance by up-regulating P-glycoprotein, ABC transporter and the like, so that drugs delivered to tumor cores are difficult to be effectively ingested, and DOX therapeutic effects are further limited. Disclosure of Invention Based on this, the present invention provides a micro-nano robot based on probiotics and framework nucleic acid (FDN) and a preparation method thereof, which at least solves one problem in the prior art. In a first aspect, the present invention provides a method for preparing a micro-nano robot based on probiotics and framework nucleic acid, comprising the steps of: preparing DOX solution; mixing DOX solution with FDN-A2.2, and stirring for reaction to obtain DOX@FDN-A2.2; And (3) converting DOX@FDN-A2.2 into competent ECN to obtain DOX@FDN-A2.2@ECN, namely the micro-nano robot based on probiotics and framework nucleic acid. In a second aspect, the invention provides a micro-nano robot based on probiotics and framework nucleic acid, which is obtained by the micro-nano robot based on probiotics and framework nucleic acid and a preparation method thereof. Compared with a surface modified drug delivery strategy, the embodiment of the invention has the advantages that the drug is introduced into ECN engineering bacteria (ECN-pl) which are subjected to anoxic microenvironment cracking of tumors AS a delivery strategy, so that the activity resistance brought by surface modification to the strain can be avoided, DOX@FDN-A2.2@ECN-pl enters into the tumor core, and the tumor cells are subjected to hypoxia to generate cracking proteins to destroy the ECN structure, so that DOX@FDN-A2.2 is successfully released to the tumor core, wherein AS1411 and S2.2 aptamer on the surface of FDN-A2.2 have the capacity of targeting tumor cells, so that the double-targeting aptamer on the surface of FDN-A2.2 improves the uptake rate of tumor cells, overcomes the problem of tumor drug resistance, and inhibits the growth of tumor cells. Drawings FIG. 1 is an AFM picture of FDN T -A2.2 and FDN R -A2.2 of an embodiment of the invention, where A is an AFM picture of FDN T -A2.2 and B is an AFM picture of FDN R -A2.2. FIG. 2 is a statistical plot of loading rates of FDN T -A2.2 and FDN R -A2.2 loaded DOX in an embodiment of the invention. FIG. 3 is a TEM image of ECN and ECN-pl of an embodiment of the present invention, wherein A is a TEM image of ECN and B is a TEM image of ECN-pl. FIG. 4 is a graph showing the co-location of DOX and ECN-pl in DOX@FDN T -A2.2@ECN-pl using CLSM in an embodiment of the present invention. FIG. 5 is a graph showing the growth of ECN and DOX@FDN T -A2.2@ECN-pl under normoxic and anoxic conditions in an example of the present invention. FIG. 6 is a graph showing the effect of different framework nucleic acids on enhancing tumor cell uptake in the examples of the present invention. FIG. 7 is a graph showing the effect of flow cytometry in quantifying different framework nucleic acids in MCF7 cells according to an embodiment of the present invention. FIG. 8 is a graph showing the results of inducing polarization of RAW264.7 macrophages to M1 type by different drugs in the examples of the present invention. FIG. 9 is a graph showing the efficiency of inducing polarization of RAW264.7 macrophages to M1 type by different drugs in an embodiment of the present invention. FIG. 10 is a statistical chart of MCF-7 cytotoxicity test results after various drug treatments according to the present invention. FIG. 11 is a graph showing the results of the clonality test of MCF-7 cells after treatment with different drugs in the examples of the present