Self-Assembled DNA-Collagen Bioactive Scaffolds Promote Cellular Uptake and Neuronal Differentiation

Academic Background

In molecular biology research, the interaction between DNA and proteins has always been a crucial topic for understanding cellular processes. With the deepening understanding of DNA-protein interactions, this knowledge has been widely applied in fields such as tissue engineering, drug development, and gene editing. Among these, DNA/collagen complexes have garnered significant attention due to their applications in gene delivery studies. However, there has been limited research on the potential of these complexes as bioactive scaffolds, particularly regarding the properties of complexes formed by the interaction of self-assembled DNA macrostructures with collagen. This study aims to explore bioactive scaffolds formed by the interaction of self-assembled DNA macrostructures with Type I collagen and evaluate their potential applications in cell culture, drug delivery, and tissue engineering.

Source of the Paper

This paper was co-authored by Nihal Singh, Ankur Singh, and Dhiraj Bhatia from the Department of Biological Sciences and Engineering at the Indian Institute of Technology Gandhinagar. The paper was published on December 4, 2024, in the journal ACS Biomaterials Science & Engineering, titled “Self-Assembled DNA−Collagen Bioactive Scaffolds Promote Cellular Uptake and Neuronal Differentiation”.

Research Process

1. Synthesis of Self-Assembled DNA Macrostructures

The study first synthesized self-assembled DNA macrostructures (X-DNA Macrostructure, XDM). XDM was formed by the self-assembly of four single-stranded DNA (ssDNA) sequences into a branched four-way junction structure. The formation of XDM was confirmed through electrophoretic mobility shift assay (EMSA).

2. Synthesis of DNA/Collagen Scaffolds

The research team mixed XDM with Type I collagen (Collagen Type I, Coll I) at different mass fractions (20%, 50%, 90%) to prepare DNA/collagen composite scaffolds. The mixed solutions were vacuum-dried onto glass coverslips to form the scaffolds. Optical microscopy revealed that 20% and 50% XDM/Coll I scaffolds formed fibrous network structures, while the 90% XDM/Coll I scaffold did not exhibit fibrous structures.

3. Characterization of Scaffolds

The morphology of the scaffolds was characterized in detail using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The results showed that 20% and 50% XDM/Coll I scaffolds formed intertwined fibrous networks, with the fibers in the 50% scaffold being thicker. AFM further revealed the three-dimensional structure and height distribution of the fibers.

4. In Vitro Cell Culture

The research team seeded the SUM159 triple-negative breast cancer cell line onto the XDM/Coll I scaffolds to observe cell growth and proliferation. The results showed that the 50% XDM/Coll I scaffold promoted directional cell growth and acted as a soft matrix to support cell growth, reducing the organization of F-actin.

5. Cellular Uptake Experiment

To evaluate the effect of the scaffolds on cellular endocytosis, the research team conducted a cellular uptake experiment using FITC-labeled transferrin. The results showed that the 50% XDM/Coll I scaffold significantly enhanced the cellular uptake of transferrin, indicating that the scaffold, as a soft matrix, promoted cellular endocytosis.

6. Neuronal Differentiation Experiment

The research team seeded SH-SY5Y neuroblastoma cells onto the XDM/Coll I scaffolds to observe their differentiation into mature neurons. The results showed that the 20% and 50% XDM/Coll I scaffolds significantly promoted the differentiation of SH-SY5Y cells, resulting in extensive neurite outgrowth, with higher differentiation efficiency compared to the control groups.

Key Findings

1. Fibrous Structure of Scaffolds: The 20% and 50% XDM/Coll I scaffolds formed intertwined fibrous networks, with the fibers in the 50% scaffold being thicker. AFM and SEM further revealed the three-dimensional structure and height distribution of the fibers.
2. Cell Growth and Proliferation: The 50% XDM/Coll I scaffold promoted directional cell growth and acted as a soft matrix to support cell growth, reducing the organization of F-actin.
3. Cellular Uptake Capacity: The 50% XDM/Coll I scaffold significantly enhanced the cellular uptake of transferrin, indicating that the scaffold, as a soft matrix, promoted cellular endocytosis.
4. Neuronal Differentiation: The 20% and 50% XDM/Coll I scaffolds significantly promoted the differentiation of SH-SY5Y cells, resulting in extensive neurite outgrowth, with higher differentiation efficiency compared to the control groups.

Conclusion

This study is the first to report bioactive scaffolds formed by the interaction of self-assembled DNA macrostructures with Type I collagen. These scaffolds exhibit unique fibrous structures that support cell growth, promote cellular endocytosis, and induce neuronal differentiation. They hold broad application potential in neuroscience, drug delivery, tissue engineering, and in vitro cell culture.

Research Highlights

1. Novel Scaffold Material: This study is the first to combine self-assembled DNA macrostructures with collagen to form bioactive scaffolds with unique fibrous structures.
2. Multifunctional Applications: These scaffolds not only support cell growth but also promote cellular endocytosis and neuronal differentiation, offering wide-ranging application prospects.
3. Soft Matrix Effect: The scaffolds, acting as soft matrices, significantly influence cell growth patterns and cellular endocytosis, providing new insights for tissue engineering and drug delivery.

Additional Valuable Information

This study also provides detailed experimental methods and data analysis, serving as a reference for subsequent research. Additionally, the research team explored the potential applications of the scaffolds in different cell types, laying the groundwork for future studies.