Bifidobacterium bifidum-Mediated Specific Delivery of Nanoparticles for Tumor Therapy

Abstract

Purpose: Hypoxia is considered to be obstructive to tumor treatment, but the reduced oxygen surroundings provide a suitable habitat for Bifidobacterium bifidum (BF) to colonize. The anaerobe BF selectively colonizes into tumors following systemic injection due to its preference for the hypoxia in the tumor cores. Therefore, BF may be a potential targeting agent which could be used effectively in tumor treatment. We aimed to determine whether a novel BF-mediated strategy, that was designed to deliver AP-PFH/PLGA NPs (aptamers CCFM641-5-functionalized Perfluorohexane (PFH) loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles) by aptamer-directed approach into solid tumor based on the tumor-targeting ability of BF, could improve efficiency of high intensity focused ultrasound (HIFU) treatment of breast cancer.

Methods: We synthesized AP-PFH/PLGA NPs using double emulsion method and carbodiimide method. Then, we evaluated targeting ability of AP-PFH/PLGA NPs to BF in vivo. Finally, we studied the efficacy of HIFU ablation based on BF plus AP-PFH/PLGA NPs (BF-mediated HIFU ablation) in tumor.

Results: The elaborately designed AP-PFH/PLGA NPs can target BF colonized in tumor to achieve high tumor accumulation, which can significantly enhance HIFU therapeutic efficiency. We also found that, compared with traditional chemotherapy, this therapy not only inhibits tumor growth, but also significantly prolongs the survival time of mice. More importantly, this treatment strategy has no obvious side effects.

Conclusion: We successfully established a novel therapy method, BF-mediated HIFU ablation, which provides an excellent platform for highly efficient and non-invasive therapy of tumor.

Keywords: Bifidobacterium bifidum; aptamers; high intensity focused ultrasound; nanoparticles; tumor treatment.

Scheme 1

Schematic illustration of targeting AP-PFH/PLGA NPs for BF-mediated HIFU ablation.

Figure 1

Tumor-Targeting Ability of BF. (A) Homogenates of tumor tissues and the five organs on different time points after the third injection of BF were cultured on solid LB agar at 37°C. (B) Quantification of BF per gram of tumor tissues and the five organs at different time points after the third injection of BF (n = 3).

Figure 2

Characterization of AP-PFH/PLGA NPs. (A) The schematic diagram for the synthesis of AP-PFH/PLGA NPs. (B) Flow cytometry analysis binding between AP and PFH/PLGA NPs. (C) Binding efficiency between AP and PFH/PLGA NPs (n = 3, ***P < 0.001). (D) SEM image and (E) TEM image of AP-PFH/PLGA NPs. (F) Size distribution of PFH/PLGA NPs and AP-PFH/PLGA NPs. (G) The size distribution of AP-PFH/PLGA NPs in PBS with prolonged time duration (n = 3); Inset: digital photos of the AP-PFH/PLGA NPs dispersed in PBS (15 mg/mL).

Figure 3

The binding between BF and AP-PFH/PLGA NPs in vitro. (A) CLSM images analysis binding between FITC-labeled BF and DiI-labeled NPs. (B) Flow cytometry analysis binding between BF and DiI-labeled NPs. (C) Binding efficiency between BF and DiI-labeled NPs (n = 3, ***P < 0.001).

Figure 4

Targeting ability of AP-PFH/PLGA NPs to BF in vivo. (A) Fluorescence imaging and (B) Quantitative fluorescence intensity of tumor tissue before injection and at 1 h, 8 h, 24 h, 48 h after injection NPs (n = 3, ***P < 0.001). (C) Fluorescence imaging and (D) Quantitative analysis of fluorescence intensity of tumor and major organs excised from mice at 48 h after injection of NPs (n = 3, ***P < 0.001). (E) Ultrathin section of tumor tissues at 1 h, 8 h, 24 h, 48 h after injection of NPs detected by CLSM. The scale bar is 50 μm.

Figure 5

Evaluation of synergistic effect of AP-PFH/PLGA NPs for BF-mediated HIFU ablation in vivo. (A) ultrasonic grayscale changes of tumor tissues (red circle) before and after HIFU ablation and (B) quantitative analysis of grayscale value (n = 10, ***P < 0.001). (C) Coagulation necrosis (red arrow) of tumors by TTC staining after HIFU ablation and (D) quantitative analysis of necrosis volume (n = 5, ***P < 0.001).

Figure 6

(A) H&E staining and (B) TUNEL assay in tumor tissue in different groups after HIFU ablation (H&E: ×200 magnification, TUNEL assay: ×400 magnification). All the scale bars are 50 μm.

Figure 7

In vivo anti-tumor effect evaluation. (A) Tumor growth inhibition, (B) survival, (C) body weights and (D) photographs after different treatment in mice bearing MDA-MB-231 xenografts (n = 5, ***P < 0.001).