Stage 1 (1.8~1.14)

Construction of the ΔmsbB::T7 RNA polymerase Strain χ11803 in Salmonella χ11802

1. We used PCR to amplify the T7 RNA polymerase gene and purify the PCR product for ligation and transformation into Escherichia coli. We verified the success of vector construction by PCR and sequencing.

2. We knocked out the msbB gene in attenuated Salmonella strain χ11802 and inserted T7 RNA polymerase at the same site. We named the modified strain as χ11803.

3. We identified χ11803 colonies PCR products through agarose gel electrophoresis. By designing primer, preparing individual PCR systems including pre-denaturation, denaturation, annealing, extension and agarose gel electrophoresis.

Stage 2 (1.14~1.27)

Electroporation of Designed Plasmids into χ11803

1. We transformed the plasmid pSilencer-CLDN6 into χ11803 through electroporation. The steps included cell preparation, preparation of DNA solution, preparation and disinfection of electrodes, pre-treatment of culture medium and electroporation experiment.

2. We transfected pCLDN6-EGFP into MCF-7 through electroporation to establish CLDN6-overexpressing cell lines. The steps included cell preparation, preparation of DNA solution, preparation and disinfection of electrodes, pre-treatment of culture medium and electroporation experiment.

Stage 3 (1.28~2.11)

Functional Validation of Plasmids in χ11803

1. Validation of the Delayed Lysis System: We used LB media, ara media with and without carb to evaluate the growth of attenuated Salmonella strain χ11802. We did this both in liquid medium and in solid medium.

2. Validation of Anaerobic-Induced hlyA Expression by Western Blot Anaerobic Treatment: We detected the expression levels of hlyA protein in χ11803 under hypoxic and non-hypoxic conditions through Western Blot. The steps included cell lysis, protein quantitation, SDS-PAGE electrophoresis, membrane transfer, blocking, primary antibody incubation, membrane wash, secondary antibody incubation, wash the membrane again and visualization and detection.

3. Validation of RGD Peptide Expression on Salmonella by Flow Cytometry: We analyzed the expression of RGD peptide on the cell wall of χ11803 using flow cytometry. The steps included preparation of solutions and reagents, cell fixation, staining, obtaining and maintaining a single-cell suspension, antibody incubation and analyzing stained bacteria with a flow cytometer and using FlowJo software to analyze the data.

Stage 4 (3.1~3.27)

Validation of Drug Resistance-caused Gene Knock-down by Salmonella-mediated RNAi

We co-cultured χ11803/pSilencer-CLDN6 with MCF-7/MDR and MCF-7/CLDN6 (MCF-7 Cell Transfection with pCLDN6-GFP), and then used Western Blot to detect the expression of CLDN6 in both cell lines. The Western Blot steps included cell lysis, protein quantitation, SDS-PAGE electrophoresis, membrane transfer, blocking, primary antibody incubation, membrane wash, secondary antibody incubation, wash the membrane again and visualization and detection.

Stage 5 (7.31~9.10)

Efficacy validation of modified Salmonella

IC50

We selected the multiple drug-resistant human breast cancer cell line MCF-7/MDR to verify that Bio-TARGET can reduce the resistance of drug-resistant tumor cells. Stable cell clones were screened by transfection of empty plasmids (MCF-7/MDR-sh-k) as control group. We used CCK8 to detect the sensitivity of MCF-7/MDR cells to ADM, 5-FU, and DDP before and after the addition of χ11803/pSilencer-CLDN6.

Expression levels of drug resistance-related proteins

We used Western Blot to detect the expression of P-gp, BCRP, and MRP-1, three drug resistance-related proteins, in MCF-7/MDR before and after the addition of χ11803/pSilencer-CLDN6.

Chemotherapy-induced apoptosis in tumor cells

1. We measured the apoptosis rate of MCF-7/MDR cells before and after the addition of χ11803/pSilencer-CLDN6 using Annexin V/PI double staining. First, set up control tubes, including blank tubes, single-staining tubes, experimental tubes, and negative tubes, then perform cell processing and staining.

2. We detected the expression of apoptotic proteins in MCF-7/MDR before and after the addition of χ11803/pSilencer-CLDN6 using Western Blot. The steps included cell lysis, protein quantitation, SDS-PAGE electrophoresis, membrane transfer, blocking, primary antibody incubation, membrane wash, secondary antibody incubation, wash the membrane again and visualization and detection.

We used the same efficacy validation methods to test the sensitivity of ovarian cancer cell lines SKOV3/CDDP and A2780/CDDP to DDP before and after the addition of χ11803/pSilencer-PGC1α.

Stage 6 (8.15~current)

Construction of Drug-loaded Nanoparticles

1. We added propyl acetate/ethyl acetate, PEG, and catalyst to a heat-resistant glass tube, flushed with nitrogen, heated to dissolve and vacuumed for 2 hours, and then sealed the tube to prepare mPEG-PLGA.

2. We dissolved mPEG-PLGA in dry organic solvent, added BOC-L-phenylalanine and N,N-dicyclohexylcarbodiimide, slowly added 4-dimethylaminopyridine, protected by nitrogen, stirred at room temperature for 1-3 days, filtered, washed with alkaline water, concentrated, precipitated, filtered, and vacuum dried to prepare mPEG-PLGA-Boc(Z).

3. We dissolved mPEG-PLGA-Boc(Z) in dry organic solvent, protected by nitrogen, slowly added dry trifluoroacetic acid at 0℃, continued the reaction, removed the solvent and unreacted trifluoroacetic acid by rotary evaporation, dissolved the residue in organic solvent, precipitated, filtered, and vacuum dried to prepare PEG-PLGA-NH2.

4. We dissolved PEG-PLGA-NH2 in dry organic solvent, added N-carboxylic anhydride, protected by nitrogen, reacted at room temperature, concentrated, precipitated, filtered, and vacuum dried to prepare mPEG-PLGA-PZLL.

5. We dissolved mPEG-PLGA-PZLL in a quantified amount of trifluoroacetic acid at 0°C, reacted, precipitated, filtered, and vacuum dried to prepare mPEG-PLGA-PLL.