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Published on: July 31, 2019
Delivery of Antisense DNA into Pathogenic Parasite Trypanosoma cruzi Using Virus-Like Protein-Based Nanoparticles
Rosa E Cárdenas-Guerra1, David S Moreno-Gutierrez2, Oscar de J Vargas-Dorantes2
1Laboratorio de Estudios sobre Tripanosomiasis, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.
Abstract:
Trypanosoma cruzi, which causes Chagas disease, is one of the most lacerating parasites in terms of health and social impacts. New approaches for its study and treatment are urgently needed since in more than 50 years only two drugs have been approved. Genetic approaches based on antisense oligonucleotides (AONs) are promising; however, to harness their full potential the development of effective carriers is paramount. Here, we report the use of an engineered virus-like protein C-BK12 to transfect AONs into T. cruzi. Using gel electrophoresis, Dynamic Light Scattering, and atomic force microscopy, we found that C-BK12 binds AONs and forms 10-25 nm nanoparticles (NPs), which are very stable when incubated in biological media, only releasing up to 25% of AON. Fluorescence microscopy and qPCR revealed that the NPs successfully delivered AONs into epimastigotes and reduced the expression of a target gene down to 68%. Importantly, the protein did not show cytotoxicity. The combination of high stability and capability to transfect and knock down gene expression without causing cell damage and death makes the protein C-BK12 a promising starting point for the further development of safe and effective carriers to deliver AONs into T. cruzi for biological studies.
Insights
An engineered protein, C-BK12, effectively delivers antisense oligonucleotides (AONs) into Trypanosoma cruzi, reducing gene expression without toxicity. This offers a promising new tool for studying Chagas disease.
Area of Science:
- Parasitology
- Molecular Biology
- Biotechnology
Background:
- Chagas disease, caused by *Trypanosoma cruzi*, has severe health and social impacts.
- Limited therapeutic options necessitate novel treatment and study approaches.
- Antisense oligonucleotides (AONs) show promise for genetic manipulation, but require effective delivery systems.
Purpose of the Study:
- To evaluate the engineered virus-like protein C-BK12 as a carrier for delivering AONs into *T. cruzi*.
- To assess the stability, transfection efficiency, and gene knockdown capability of C-BK12-AON nanoparticles.
- To determine the cytotoxicity of the C-BK12 protein in *T. cruzi*.
Main Methods:
- Characterization of C-BK12-AON complexes using gel electrophoresis, Dynamic Light Scattering, and atomic force microscopy.
- Assessment of nanoparticle stability in biological media.
- Evaluation of AON delivery and gene expression knockdown in *T. cruzi* epimastigotes via fluorescence microscopy and qPCR.
- Cytotoxicity assays to determine the safety of the C-BK12 protein.
Main Results:
- C-BK12 binds AONs, forming stable 10-25 nm nanoparticles.
- Nanoparticles demonstrated high stability in biological media with minimal AON release (≤25%).
- Successful delivery of AONs into epimastigotes and significant reduction (up to 68%) in target gene expression.
- The C-BK12 protein exhibited no observable cytotoxicity.
Conclusions:
- Engineered protein C-BK12 effectively forms stable nanoparticles with AONs for transfection into *T. cruzi*.
- The C-BK12-AON system demonstrates efficient gene knockdown without inducing cytotoxicity.
- C-BK12 represents a promising platform for developing safe and effective AON delivery strategies for *T. cruzi* research and potential therapeutics.
