*3.5. Light-Triggered <sup>1</sup>O2-Generation Ability of Ce6@FDC + O<sup>2</sup>*

Light-triggered <sup>1</sup>O2-generation potency of Ce6@FDC + O<sup>2</sup> nanoemulsion was quantitated using 1,3-diphenylisobenzofuran (DPBF) as probe [11]. For that, Ce6, Ce6 + O2, Ce6@FDC, or Ce6@FDC + O<sup>2</sup> (5 µg mL−<sup>1</sup> of Ce6) was mixed with deoxygenated dimethyl formamide (DMF) containing DPBF (100 µM) and irradiated with a 660 nm continuous wave diode laser beam (50 mW cm−<sup>2</sup> , Rayan Tech., Changchun, China) for 3 min. The generated <sup>1</sup>O<sup>2</sup> was detected with DPBF every 20 s by quantitating the reduction of its absorption at 410 nm via microplate spectrophotometer. DMF containing DPBF and deoxygenated PBS was used as a negative control.

#### *3.6. Bacterial Association of Ce6@FDC*

The interaction between Ce6@FDC nanoemulsion and bacteria was evaluated by flow cytometry. Briefly, *E. coli* or *A. baumannii* were cultured in Luria−Bertani (LB) broth medium at 37 ◦C with shaking overnight and harvested at the exponential growth phase. After being washed with PBS three times, bacteria were resuspended in PBS at <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>8</sup> CFU mL−<sup>1</sup> . Following that, bacteria suspension was treated with Ce6 or Ce6@FDC (10 µg mL−<sup>1</sup> , equivalent to Ce6) at 37 ◦C for 30 min and washed with cold PBS for semiquantitative analysis by NovoCyte 2060R flow cytometry and ACEA NovoExpress software (ACEA Biosciences inc., San Diego, CA, USA). Meanwhile, the zeta potential of bacteria treated as above was measured by Zetasizer.

#### *3.7. In Vitro Photodynamic Antibacterial Activity*

The photodynamic bactericidal activity of Ce6@FDC + O<sup>2</sup> nanoemulsion against two Gram-negative strains, *E. coli* and *A. baumannii*, was evaluated by colony counting method. In short, bacteria (10<sup>8</sup> CFU mL−<sup>1</sup> ) were incubated with Ce6, Ce6 + O2, Ce6@FDC, or Ce6@FDC + O<sup>2</sup> (concentration of Ce6 ranging from 0 to 60 µg mL−<sup>1</sup> ) at 37 ◦C for 30 min in dark and then exposed to 660 nm laser at an intensity of 100 mW cm−<sup>2</sup> for 20 min. Finally, 100 µL of bacteria suspension was diluted several times and spread onto LB agar plate and grown at 37 ◦C overnight. PBS was set as the negative control. All experiments were independently performed in triplicate.

The morphology of Ce6@FDC + O2-treated bacteria was observed by field emission scanning electron microscopy (FESEM). In brief, *A. baumannii* or *E. coli* suspension (10<sup>8</sup> CFU mL−<sup>1</sup> ) was incubated with Ce6@FDC + O<sup>2</sup> (20 µg mL−<sup>1</sup> of Ce6) and irradiated as indicated above. Then bacteria were fixed with 2.5% glutaric dialdehyde for 2 h. After washing with PBS, samples were dehydrated by graded ethanol (30%, 50%, 70%, 80%, 90%, and 100% for 10 min each). The fixed bacteria were coated with gold and imaged with FESEM (JEOL, Osaka, Japan).

#### *3.8. In Vitro Ablation Capacity of Bacterial Biofilm*

The performance of biofilm ablation was assessed via crystal violet (CV) staining assay and plate counting assay. For biofilm construction, 10 µL of *E. coli* or *A. baumannii* (10<sup>8</sup> CFU mL−<sup>1</sup> ) were added to culture dishes supplemented with 1 mL of fresh LB + 1% sucrose solid medium in 24-well plates and incubated at 37 ◦C under static conditions for 24 h to form fresh biofilms [28]. Then, the formed biofilms were gently washed with PBS and treated with Ce6, Ce6 + O2, Ce6@FDC, or Ce6@FDC + O<sup>2</sup> (concentration of Ce6 ranging from 0 to 60 µg mL−<sup>1</sup> ) at 37 ◦C for 30 min followed by irradiation (660 nm, 100 mW cm−<sup>2</sup> ) for 20 min. Biofilms subjected to PBS treatment as above were used as negative control. The viable bacteria in the biofilms were detached into sterile PBS via vortexing. Obtained bacteria suspension was diluted and spread onto LB agar plates and then incubated at 37 ◦C for 12 h. The CFU on different plates were imaged and counted.

Meanwhile, CV staining assay was carried out for quantitative analysis of the ablation effect of Ce6@FDC + O<sup>2</sup> nanoemulsion on *E. coli* or *A. baumannii* biofilm. Biofilms subjected to various treatments as above were washed with cold PBS and subsequently stained with CV at 37 ◦C for 30 min. The stained residual biofilms were photographed and resuspended with acetic acid (33%, *w*/*v*), and then the absorbance of supernatant was measured at 595 nm for quantitative analysis. All experiments were independently performed in triplicate.

#### *3.9. Statistics Analysis*

ANOVA was used to analyze all data with a Student−Newman−Keuls test for post hoc pairwise comparisons. All statistical analyses were performed using the GraphPad Prism software (version 8, Prism Software, Beijing, China). Differences with *p*-values < 0.05 were considered statistically significant.

#### **4. Conclusions**

In summary, we have successfully fabricated a photodynamic perfluorocarbon nanoemulsion, Ce6@FDC, which can simultaneously deliver photosensitizer and oxygen for the sensitization of antibiotic-resistant Gram-negative bacteria to APDT. Beyond the superior colloidal stability, Ce6@FDC also exhibits remarkable oxygen loading and releasing potency in an aqueous environment and excellent light-triggered singlet oxygen generation. Furthermore, we also demonstrated that Ce6@FDC could strongly bind with bacteria and alter their surface properties due to its positively charged surface, resulting in great bactericidal activity in combating antibiotic-resistant *A. baumannii* and *E. coli* compared with free Ce6. Ce6@FDC with oxygen loading also displayed a notable performance in the photodynamic eradication of Gram-negative bacteria biofilm. Therefore, Ce6@FDC, as a

novel bactericidal agent enabling efficient delivery of Ce6 and oxygen, and enhanced APDT provide a new strategy for killing antibiotic-resistant Gram-negative bacteria. In future, the formulation of such a nanoemulsion needs to be further optimized for maximizing the safety and pathogen targetability, and its in vivo therapeutical efficacy is also yet to be investigated.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ph15020156/s1, Figure S1. Field emission scanning electron microscopy images of bacteria after being treated with PBS or Ce6@FDC + O<sup>2</sup> plus light (660 nm, 100 mW, 20 min). Red arrows indicate lesions and holes on bacterial wall. Scale bar: 1 µm.

**Author Contributions:** Conceptualization, P.N., J.D. and W.M.; formal analysis, P.N., Z.W. and D.F.; investigation, P.N. and Y.W.; writing—original draft, P.N. and W.M.; writing—review and editing, Y.L. and W.M.; funding acquisition, W.M.; methodology, P.N. and Y.L.; supervision, W.M. All authors have read and agreed to the published version of this manuscript.

**Funding:** This research was supported by research grants from the National Natural Science Foundation of China (project number: 51603101) and the Postgraduate Research and Practice Innovation Program of Jiangsu Province for project SJCX21\_0545.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary material.

**Conflicts of Interest:** The authors declare no competing financial interests.

#### **References**

