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

A delayed-lysis attenuated strain of Salmonella χ11802 was used as the foundation. The strain has deletions in the asd and murA genes, which are critical for bacterial cell wall synthesis. These genes were reintroduced on a plasmid regulated by the arabinose operon. Building on χ11802, we generated the new χ11803 strain by knocking out the msbB gene and inserting the T7 RNA polymerase gene at the same locus using homologous recombination. This modification further reduces the strain's virulence while allowing it to express T7 RNA polymerase. PCR analysis confirmed the successful construction. As shown in Figure 1, the msbB band (~982 bp) is absent in the modified strain, while a distinct band corresponding to T7 RNA polymerase (~2749 bp) is present, confirming the gene replacement.

These results validate the successful creation of the ΔmsbB::T7 RNA Polymerase strain χ11803.

Figure 1. Colony PCR was used to confirm the knockout of msbB and its replacement with T7 RNA polymerase
① WT; ② △msbB; ③ ΔmsbB::T7 RNAP; ④ marker
The PCR product length of msbB is approximately 982 bp, while that of T7 RNA polymerase is approximately 2749 bp.

Assessment of LPS Activity in χ11803

The LPS activity has been implicated in the safety concerns associated with Salmonella. To assess LPS activity, we measured the ability of endotoxin to stimulate the production of pro-inflammatory cytokine TNF-α. LPS was isolated from Salmonella mutants and used to induce TNF-α production in the macrophage-like cell line RAW264.7. Stimulation of RAW264.7 cells with LPS from the △msbB χ11802 strain resulted in lower levels of TNF-α production compared to LPS from χ11802, across concentration ranging from 10-3 to 103 pmol/L. As expected, the ability to induce TNF-α was no significantly altered in the χ11803 (ΔmsbB::T7 RP) mutant compared to the parent strain △msbB χ11802.

Figure 2. Analysis of the TNF-α level induced by purified LPS derived from the Salmonella mutant strains in RAW264.7

Delayed Lysis System

To verify that the growth of the engineered bacteria can be regulated by arabinose, the transformed bacteria were cultured in LB medium, LB medium containing arabinose, and LB medium containing carbenicillin at 37°C for 24 hours. As expected, bacterial growth was observed only in the LB medium supplemented with arabinose, confirming the system’s dependence on arabinose for bacterial survival and replication.

Figure 4. Growth of bacteria in LB, LB with arabinose, and LB with carbenicillin media

Additionally, the transformed bacteria were plated on LB and arabinose-containing LB agar plates. Consistent with the broth culture results, bacterial colonies were only observed on the plates containing arabinose, further supporting the effective functionality of the delayed lysis system.

The results indicated that our engineered Salmonella strain dissolves and dies as the concentration of arabinose gradually decreases, demonstrating strong safety and targeting capabilities.

Figure 5. Growth of bacteria on LB plates and LB plates with arabinose

Anaerobic-Induced hlyA Expression

To validate the hypoxia-inducible expression system, we cultured Salmonella χ11803 harboring the plasmid pSilencer-CLDN6 under anaerobic conditions. The bacterial culture was transferred to a low-oxygen environment using Na₂SO₃ and pre-reduced LB medium, followed by incubation at 37°C for 24 hours. After incubation, bacterial lysates were collected, and Western blot analysis was performed to detect the expression of listeriolysin O, the product of the hlyA gene.

The results showed a marked increase in listeriolysin O expression in bacteria exposed to hypoxic conditions, while the normoxic control group exhibited minimal or no expression. This confirms the successful induction of hlyA expression in response to low-oxygen environments, validating the system’s functionality under hypoxic conditions similar to those in tumor tissues.

Figure 6. Listeriolysin O Expression under Hypoxic Conditions

RGD Peptide Expression

To confirm the surface expression of the RGD peptide on Salmonella χ11803, flow cytometry analysis was performed. Bacterial cultures were grown to an OD600 ≈ 0.8, and approximately 1.0 × 10⁹ CFU of bacterial cells were harvested by centrifugation. The bacterial cells were washed with PBS and fixed using 4% paraformaldehyde. For immunofluorescence staining, the cells were incubated overnight at 4 °C with a mouse anti-His tag monoclonal antibody (diluted in PBA containing 3% BSA), followed by incubation with a goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 for 1 hour at room temperature.

The fluorescence signal was detected using a flow cytometer in the FITC channel, with a detection threshold of forward scatter (FSC) set to 1000 and 100,000 events recorded. The control group (χ11803 without the RGD construct) exhibited low fluorescence intensity, while the experimental group (χ11803 carrying the RGD construct) showed a significant increase in fluorescence. This indicates successful surface expression of the RGD peptide on the bacterial membrane.

These results validate the functional design of the system, confirming that the RGD peptide was effectively displayed on the bacterial surface through the use of immunofluorescence staining and flow cytometry detection.

Figure 7. Validation of RGD expression using flow cytometry

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

To establish a CLDN6-overexpressing MCF-7 cell line, we transfected MCF-7 cells with the pCLDN6-GFP plasmid using electroporation. The GFP reporter gene on the plasmid allowed us to assess transfection efficiency via fluorescence microscopy.

Figure 8. plasmid pCLDN6-GFP

Successful transfection was confirmed by the observation of strong GFP fluorescence and gene expression in the transfected cells, indicating effective introduction and expression of the pCLDN6-GFP plasmid in the MCF-7 cells.

Figure 9. Fluorescence image of MCF-7 cells with plasmid pCLDN6-GFP

Before conducting the Salmonella-mediated RNA interference (RNAi) knockdown experiment, we assessed the expression of CLDN6 protein in three cell lines: MCF-7, CLDN6-overexpressing MCF-7 (MCF-7/CLDN6), and MCF-7/MDR through Western blot analysis. The results demonstrated that CLDN6 expression was significantly higher in the MCF-7/CLDN6 cells compared to the parental MCF-7 cells. Additionally, CLDN6 expression was also elevated in the MCF-7/MDR drug-resistant cells.

Figure 10. Western blot analysis of CLDN6 expression in MCF-7, MCF-7/CLDN6, and MCF-7/MDR cell lines

To assess the efficacy of Salmonella-mediated RNA interference (RNAi) in knocking down drug resistance-related genes, MCF-7/CLDN6 cells and MCF-7/MDR cells were co-cultured with the engineered χ11803/pSilencer-CLDN6 bacteria. The goal was to determine whether the bacteria could successfully deliver shRNA targeting CLDN6 into tumor cells, thereby reducing the expression of CLDN6.

Western blot analysis was performed to evaluate the expression of CLDN6 in the treated MCF-7/CLDN6 cells and MCF-7/MDR cells. The results, as shown in the figure, indicate that the group co-cultured with χ11803/pSilencer-CLDN6 showed a significant reduction in CLDN6 expression compared to the control group and other experimental conditions. Specifically, the χ11803/pSilencer-CLDN6 treatment group exhibited a marked decrease in CLDN6 levels, demonstrating the successful shRNA delivery into tumor cells by Salmonella and effective gene knockdown.

Figure 11. Western Blot Analysis of CLDN6 Knockdown in MCF-7/CLDN6

Figure 12. Western Blot Analysis of CLDN6 Knockdown in MCF-7/MDR

CCK-8 Assay to Assess MCF-7/MDR Cells Sensitivity to Chemotherapy Drugs

To evaluate whether knocking down the CLDN6 gene in drug-resistant tumor cells would enhance their sensitivity to chemotherapy drugs, a CCK-8 assay was performed. MCF-7/MDR cells were co-cultured with χ11803/pSilencer-CLDN6, followed by treatment with three chemotherapy drugs: ADM, 5-FU, and DDP. The results showed that cells treated with χ11803/pSilencer-CLDN6 exhibited significantly reduced IC50 values for all three drugs compared to the control group. This indicates that knocking down CLDN6 successfully enhanced the drug sensitivity of tumor cells.

Figure 13. CCK-8 Assay to Assess MCF-7/MDR Cells Sensitivity to Chemotherapy Drugs

Western Blot Analysis of Drug Resistance-Related Protein Expression

After co-culturing χ11803/pSilencer-CLDN6 with drug-resistant tumor cells, Western blot analysis was performed to assess the expression of drug resistance-related proteins: P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 1 (MRP-1). As shown in the figure, the expression of both P-gp and BCRP was significantly reduced in the group treated with χ11803/pSilencer-CLDN6 compared to the negative control (shRNA-NEG). No significant change in MRP-1 expression was observed. These results suggest that the knockdown of CLDN6 reduces the expression of key drug resistance proteins, thereby potentially enhancing the sensitivity of tumor cells to chemotherapy.

Figure 14. Western Blot Analysis of Drug Resistance-Related Protein Expression

Apoptosis Rate Detection by Annexin V/PI Staining

To examine the effects of CLDN6 knockdown on apoptosis, Annexin V/PI staining followed by flow cytometry analysis was conducted. Tumor cells treated with χ11803/pSilencer-CLDN6 and cisplatin (DDP) showed a higher apoptosis rate compared to other groups. These results suggest that the knockdown of CLDN6 enhances the ability of chemotherapy drugs to induce apoptosis in drug-resistant tumor cells.

Figure 15. Annexin V/PI Staining to Assess the Effect on Cell Apoptosis

Western Blot Analysis of Apoptosis-Related Protein Expression

The expression of cleaved-PARP and cleaved-caspase-9, markers of apoptosis, was significantly increased in the χ11803/pSilencer-CLDN6 treated group, especially when combined with ADM, further validating the effect of CLDN6 knockdown on promoting apoptosis in MCF-7/MDR cells.

Figure 16. Western Blot Analysis of Apoptosis-Related Protein Expression

Chemosensitivity Evaluation by CCK-8 Assay

The sensitivity of the tumor cells to chemotherapy drugs was assessed using CCK-8 assays. The χ11803/pSilencer-PGC1α treated group displayed significantly lower IC50 values for cisplatin (CDDP) in both SKOV3/CDDP and A2780/CDDP cells, indicating enhanced chemotherapy sensitivity.

Figure 17. CCK-8 Assay to Assess SKOV3/CDDP and A2780/CDDP Cells Sensitivity to Cisplatin

Apoptosis Rate Detection by Annexin V/PI Staining

Apoptosis was measured using Annexin V/PI staining and flow cytometry. Tumor cells treated with χ11803/pSilencer-PGC1α and cisplatin showed a higher apoptosis rate compared to other groups, demonstrating the knockdown's effect on promoting apoptosis.

Figure 18. Annexin V/PI Staining to Assess the Effect on Cell Apoptosis

Western Blot Analysis of Apoptosis-Related Protein Expression

Western blot analysis showed that the significant increase in caspase-9, cleaved-caspase-3, and BAX expression, along with the decrease in BCL-2 expression, was primarily observed when χ11803/pSilencer-PGC1α was combined with cisplatin. This further highlights the synergistic effect of PGC1α knockdown and cisplatin in inducing apoptosis in cisplatin-resistant ovarian cancer cells.

Figure 19. Western Blot Analysis of Apoptosis-Related Protein Expression

Synthesis of Hydroxyl-Terminated mPEG-PLGA

Through the ring-opening polymerization (ROP) reaction of D,L-lactide and glycolide catalyzed by mPEG and stannous octoate (Sn(Oct)2), we successfully synthesized hydroxyl-terminated mPEG-PLGA. This process was carried out in an oil bath at 130°C for 8 hours. The structural integrity of the product was confirmed by ^1H NMR characterization (Figure 1).

Figure 20. mPEG-PLGA in CDCl³

Synthesis of Boc-L-Phe-Terminated mPEG-PLGA

Using the hydroxyl-terminated mPEG-PLGA mentioned above, we successfully converted the hydroxyl end group into Boc-L-Phe by reacting it with Boc-L-Phe in anhydrous dichloromethane at 0°C for 48 hours, using DCC and DMAP as reagents. After the reaction, the product was purified by filtration and washing, and recovered by precipitation from the dichloromethane solution. This process resulted in Boc-L-Phe-terminated mPEG-PLGA (Figure 2).

Figure 21. Boc-L-Phe end-capped mPEG-PLGA in CDCl³

Removal of the Boc Protecting Group

To further functionalize the polymer, we treated Boc-L-Phe-terminated mPEG-PLGA with trifluoroacetic acid under nitrogen protection, removing the Boc protecting group and obtaining amine-terminated mPEG-PLGA. The product was concentrated and precipitated in methanol (Figure 3).

Figure 22. amino-terminated mPEG-PLGA in CDCl³

Synthesis of mPEG-PLGA-b-Poly(Nε-(Z)-L-lysine)

Using amine-terminated mPEG-PLGA as an initiator, Nε-(Z)-L-lysine N-carboxyanhydride (NCA) underwent ring-opening polymerization (ROP) in dry chloroform to synthesize the block copolymer mPEG-PLGA-b-poly(Nε-(Z)-L-lysine). The reaction was carried out at room temperature for 72 hours, and the product was separated by precipitation in cold ether, yielding the block copolymer (Figure 4).

Figure 23. mPEG-PLGA-b-poly(Nε-(Z)-L-lysine) in DMSO-d⁶

Removal of the Nε-(Carbobenzyloxy) Protecting Group

The aforementioned block copolymer was treated with hydrogen bromide/acetate to remove the Nε-(carbobenzyloxy) protecting group, yielding the mPEG-PLGA-b-PLL block copolymer. The reaction mixture was precipitated in cold ether, resulting in the final product (Figure 5).

Figure 24. mPEG-PLGA-b-PLL in DMSO-d⁶

Characterization of mPEG-PLGA-b-PLL Block Copolymer

The mPEG-PLGA-b-PLL block copolymer was thoroughly characterized by FTIR and GPC, confirming its chemical structure and molecular weight characteristics (Figures 24, 25).

Figure 25. The FTIR spectra of mPEG-PLGA-b-PLL

Figure 26. The GPC traces of mPEG-PLGA-b-PLL(Mn=41400 g/mol, PDI=2.26)

Preparation and Characterization of Chemotherapeutic Drug-Loaded mPEG-PLGA-PLL Nanoparticles

mPEG-PLGA-PLL nanoparticles were prepared using the ultrasonic emulsification method and successfully loaded with cisplatin. The resulting nanoparticles exhibited a uniform spherical shape with good dispersibility, an average particle size of 140.2 nanometers, a zeta potential of +12.1 millivolts, and a loading efficiency of 88.45%.