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Published on: April 29, 2015
Liposome-based nanocarriers loaded with anthrax lethal factor and armed with anti-CD19 VHH for effectively inhibiting
S Reza Banihashemi1, Fatemeh Rahbarizadeh2, Ahmad Zavaran Hosseini3
1Department of Medical Immunology, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran; Department of Immunology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran.
Objective:
One of the vital signaling pathways in cancer development and metastasis is mitogen-activated protein kinases (MAPKs). Bacillus anthracis Lethal Toxin (LT) is a potent MAPK signaling inhibitor. This toxin is comprised of two distinct domains, Lethal Factor (LF), MAPK inhibitor, and Protective Antigen (PA). To enter various cell lines, LF must be associated with the protective antigen (PA), which facilitates LF delivery. In the current study, to block MAPK signaling, LF was loaded into anti-CD19 immunoliposomes nanoparticle to deliver the cargo to Raji B cells.
Methods:
The liposome nanoparticle was prepared using classical lipid film formation, then conjugated to anti-CD19 VHH. The binding efficiency was measured through flow cytometry. The targeted cytotoxicity of LF immunoliposome was confirmed by BrdU lymphoproliferation assay. This was followed by Real-Time PCR to assess the effect of formulation on pro-apoptotic genes. The inhibitory effect of LF on MAPK signaling was confirmed by western blot.
Results:
Liposome nano-formulation was optimized to reach the maximum LF encapsulation and targeted delivery. Next, phosphorylation of MAPK pathway mediators like MEK1/2, P38 and JNK were inhibited following the treatment of Raji cells with LF-immunoliposome. The treatment also upregulated caspase genes, clearly illustrating cell death induced by LF through pyroptosis and caspase-dependent apoptosis.
Conclusions:
In conclusion, anti-CD19 VHH immunoliposome was loaded with LF, a potent MAPK inhibitor targeting B cells, which curbs proliferation and ushers B cells toward apoptosis. Thus, immunoliposome presents as a versatile nanoparticle for delivery of LF to block aberrant MAPK activation. To use LF as a therapy, it would be necessary to materialize LF without PA. In the current study, PA was substituted with anti-CD19 immunoliposome to make it targeted to CD19+ while keeping the normal cells intact.
Insights
Bacillus anthracis Lethal Factor (LF) was encapsulated in anti-CD19 immunoliposomes to inhibit MAPK signaling in B cells. This targeted nanoparticle delivery effectively curbed proliferation and induced apoptosis in cancer cells.
Area of Science:
- Biochemistry
- Nanotechnology
- Immunology
Background:
- Mitogen-activated protein kinases (MAPKs) are crucial in cancer development and metastasis.
- Bacillus anthracis Lethal Toxin (LT) inhibits MAPK signaling, with Lethal Factor (LF) being the active component.
- LF requires Protective Antigen (PA) for cell entry, posing challenges for therapeutic applications.
Purpose of the Study:
- To develop a targeted nanoparticle delivery system for LF to inhibit MAPK signaling in B cells.
- To utilize anti-CD19 immunoliposomes for selective delivery of LF to CD19-expressing B cells.
- To evaluate the efficacy of LF-loaded immunoliposomes in curbing B cell proliferation and inducing apoptosis.
Main Methods:
- Liposome nanoparticles were prepared and conjugated with anti-CD19 VHH.
- Binding efficiency was assessed using flow cytometry.
- Cytotoxicity, gene expression (pro-apoptotic), and MAPK pathway inhibition were evaluated using BrdU lymphoproliferation assay, Real-Time PCR, and western blot, respectively.
Main Results:
- Optimized liposome nano-formulation achieved high LF encapsulation and targeted delivery.
- LF-immunoliposome treatment inhibited MAPK pathway mediators (MEK1/2, P38, JNK) in Raji cells.
- Upregulation of caspase genes indicated LF-induced pyroptosis and caspase-dependent apoptosis.
Conclusions:
- Anti-CD19 VHH immunoliposomes effectively delivered LF to target B cells, inhibiting proliferation and inducing apoptosis.
- Immunoliposomes serve as a versatile nanoparticle for LF delivery, blocking aberrant MAPK activation.
- Substituting PA with anti-CD19 immunoliposomes enables targeted therapy for CD19+ cells while sparing normal cells.
