ABSTRACT
Selective detection and precise quantification of biomolecules in intracellular settings play a pivotal role in the diagnostics and therapeutics of diseases, including various cancers and infectious epidemics. Because of this clinical relevance, nanoprobes with high sensitivity, wide tunability, and excellent biological stability have become of high demand. In particular, nanoflares based on gold nanoparticles have emerged as an attractive candidate for intracellular detection due to their efficient cellular uptake, enhanced binding affinity with complementary targets, and improved biological compatibility. However, nanoprobes, including these nanoflares, are known to be susceptible to the adsorption of proteins present in the biological environment, which leads to the formation of a so-called protein corona layer on their surface, leading to an altered targeting efficiency and cellular uptake. In this work, we leverage the nanoflares platform to demonstrate the effect of protein corona on biomolecular detection, quantification, as well as biological stability against enzymatic degradation. Nanoflares incubated in a biologically relevant concentration of serum albumin proteins (0.50 wt %) were shown to result in more than 20% signal reduction in target detection, with a decrease varying proportionally with the protein concentrations. In addition, similar signal reduction was observed for different serum proteins, and PEG backfilling was found to be ineffective in mitigating the negative impact induced by the corona formation. Furthermore, nuclease resistance in nanoflares was also severely compromised by the presence of the corona shell (â¼2-fold increase in hydrolysis activity). This work demonstrates the consequences of an in situ formed protein corona layer on molecular detection/quantification and biological stability of nanoflares in the presence of nuclease enzymes, highlighting the importance of calibrating similar nanoprobes in proper biological media to improve the accuracy of molecular detection and quantification.
Subject(s)
Biosensing Techniques/methods , Nanoparticles/chemistry , Protein Corona/chemistry , Deoxyribonucleases/metabolism , Gold/chemistry , Metal Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Protein Corona/metabolismABSTRACT
Cellular reprogramming and stem cell-based therapies have shown tremendous potential in the field of regenerative medicine. To that end, developing tools to control stem cell fate is an attractive area of research for replacing damaged and diseased cells and reestablishing functional connections for tissue repair. Transcription factor (TFs) proteins are well known to regulate gene expression and direct stem cell fate. Inspired by natural TFs, NanoScript, a nanoparticle (NP)-based platform, mimics TFs to afford control over gene expression and stem cell fate for regenerative medicine. Here, we describe the construction of the NanoScript platform, which is designed with tunable properties to replicate the structure and function of TFs to bind to specific portions of the genome and regulate gene expression in a way that does not involve viral delivery.
Subject(s)
Gene Expression Regulation , Nanoparticles , Transcription Factors , Cell-Penetrating Peptides/chemistry , Gene Transfer Techniques , Gold/chemistry , Metal Nanoparticles , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurons/cytology , Neurons/metabolism , Nylons/chemistry , Protein Interaction Domains and Motifs , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
A novel cell-based biosensing platform is developed using a combination of sequential laser interference lithography and electrochemical deposition methods. This enables the sensitive discrimination of dopaminergic cells from other types of neural cells in a completely nondestructive manner. This platform and detection strategy may become an effective noninvasive in situ monitoring tool that can be used to determine stem cell fate for various regenerative applications.
Subject(s)
Cell Differentiation , Dopaminergic Neurons/metabolism , Electrochemical Techniques , Nanostructures/chemistry , Neural Stem Cells/metabolism , Animals , Biosensing Techniques , Dopamine/metabolism , Dopaminergic Neurons/cytology , Electrodes , Gold/chemistry , Humans , Levodopa/metabolism , Neural Stem Cells/cytology , PC12 Cells , Rats , Tin Compounds/chemistryABSTRACT
Even though gene repression is a powerful approach to exogenously regulate cellular behavior, developing a platform to effectively repress targeted genes, especially for stem-cell applications, remains elusive. Herein, we introduce a nanomaterial-based platform that is capable of mimicking the function of transcription repressor proteins to downregulate gene expression at the transcriptional level for enhancing stem-cell differentiation. We developed the "NanoScript" platform by integrating multiple gene repression molecules with a nanoparticle. First, we show a proof-of-concept demonstration using a GFP-specific NanoScript to knockdown GFP expression in neural stem cells (NSCs-GFP). Then, we show that a Sox9-specific NanoScript can repress Sox9 expression to initiate enhanced differentiation of NSCs into functional neurons. Overall, the tunable properties and gene-knockdown capabilities of NanoScript enables its utilization for gene-repression applications in stem cell biology.