Enhanced Intracellular Delivery of BCG Cell Wall Skeleton into Bladder Cancer Cells Using Liposomes Functionalized with Folic Acid and Pep-1 Peptide

Abstract

Although bacillus Calmette–Guérin cell wall skeleton (BCG-CWS) might function as a potential substitute for live BCG, its use in the treatment of bladder cancer remains limited owing to issues such as insolubility and micrometer-size following exposure to an aqueous environment. Thus, to develop a novel nanoparticulate system for efficient BCG-CWS delivery, liposomal encapsulation was carried out using a modified emulsification-solvent evaporation method (targets: Size, <200 nm; encapsulation efficiency, ~60%). Further, the liposomal surface was functionalized with specific ligands, folic acid (FA), and Pep-1 peptide (Pep1), as targeting and cell-penetrating moieties, respectively. Functionalized liposomes greatly increased the intracellular uptake of BCG-CWS in the bladder cancer cell lines, 5637 and MBT2. The immunoactivity was verified through elevated cytokine production and a THP-1 migration assay. In vivo antitumor efficacy revealed that the BCG-CWS-loaded liposomes effectively inhibited tumor growth in mice bearing MBT2 tumors. Dual ligand-functionalized liposome was also superior to single ligand-functionalized liposomes. Immunohistochemistry supported the enhanced antitumor effect of BCG-CWS, with IL-6 production and CD4 infiltration. Thus, we conclude that FA- and Pep1-modified liposomes encapsulating BCG-CWS might be a good candidate for bladder cancer treatment with high target selectivity.

Development, Characterization, and Ex Vivo Assessment of Elastic Liposomes for Enhancing the Buccal Delivery of Insulin

“…Several options have been explored to enhance the permeability, pharmacokinetics, and targeted selectivity across the buccal membrane. Accordingly, permeation enhancers, adhesive polymers, prodrugs, lipid-based nanocarriers, polymer-based nanocarriers, and hybrid nanocarriers have been designed [5][6][7][8][9][10]. Among these, lipid-based nanocarriers, particularly liposomes that are composed of natural or synthetic phospholipids, with or without cholesterol, have shown tremendous potential in increasing the permeability and bioavailability of peptide drugs [11][12][13].…”

Liposome-Encapsulated Bacillus Calmette–Guérin Cell Wall Skeleton Enhances Antitumor Efficiency for Bladder Cancer In Vitro and In Vivo via Induction of AMP-Activated Protein Kinase

“…Importantly, in this study, we developed an improved system for the intravesical delivery of BCG-CWS in an orthotopic tumor mouse model of bladder cancer. Previously, we performed flow cytometry and a confocal laser scanning microscopy assay to evaluate the cellular uptake of CWS-Nano-CL in 5637 and MBT2 cells [12]. We showed that the fluorescence peak shift (MFI) value was 34.95 according to flow cytometry, and a strong fluorescence was visualized by confocal laser scanning microscopy (CLSM) for 2 h. As for the results, the cellular uptake of CWS-Nano-CL in MBT2 cells was clearly demonstrated, indicating internalized fluorescence within the cytoplasm for 2 h. To observe the in vivo antitumor efficacies, MBT2-Luc cells were instilled into the bladders of C3H/HeN mice.…”

“…Although BCG-CWS is a potent candidate non-infectious immunotherapeutic drug that could serve as an alternative to live BCG [6,8,9], its aggregation in both aqueous and non-aqueous solvents limits its efficient use in anticancer therapy. To overcome this issue of insolubility and improve the internalization of BCG-CWS into bladder cancer cells, we encapsulated it within a liposomal nanoparticle using an emulsification-solvent evaporation method [12,35]. We showed that, in vitro, though both CWS and CWS-Nano-CL showed antitumor effects on bladder cancer cells, the latter displayed greater efficiency than the former.…”

“…Paik (Chungnam National University, Daejeon, Korea) and was prepared as previously described [6], with slight modifications [16]. To prepare the nanoparticles containing liposome-encapsulated BCG-CWS, CWS was encapsulated into the liposomes using the "liposome evaporated via emulsified lipid" method, with slight modifications [10,12]. For BCG-CWS encapsulation, plain liposomal vesicles composed of soy phosphatidylcholine and cholesterol (in a 9:1 molar ratio) were prepared using the thin film hydration method.…”

“…Therefore, it has been a challenge to develop a formulation that disperses uniformly without forming aggregates in an aqueous environment [11]. We recently reported an intracellular delivery system for BCG-CWS involving liposome-encapsulated BCG-CWS nanoparticles that circumvented the limitations of the intravesical instillation of BCG-CWS in bladder cancer [12].…”