Singhai, Gaurav

English

DNA and viruses are biological building blocks that in recent year have also been used to create new nanomaterials with applications in material science, nanomedicine and bio-nanotechnology. DNA nanostructures (DNs) have been used, for example, as promising drug carriers owing to their properties related to drug loading efficiency, controlled drug release mechanisms, biocompatibility and surface functionalisation. However, the literature suggests some significant challenges for the use of DNs in effective and targeted drug delivery such as their stability against nuclease digestion and increasing their circulation half-life in the cellular environments. In order to tackle the challenges mentioned above, virus capsids can be often used to coat the DN’s surface, facilitating their entry into cells. Such surface-modified DNs exhibit also improved stability and low non-specific interactions in the cellular milieu. Thus, creating multifunctional biohybrid nanocarriers from DN and virus proteins capable of targeting specific cells and ensure stimuli-responsive drug release is of great utility in therapeutic applications.The main objective of this research project was to create ‘smart’ nanocarriers by coating drug-loaded modular DNs with viral capsid proteins (CP), isolated from the cowpea chlorotic mottle virus (CCMV). Four modular DNs were designed and validated in-silico using designing and structure prediction softwares. DNs were then self-assembled using a bottom-up approach and characterised using electrophoresis and imaging techniques. Next, two purified DNs were coated with virus capsid proteins forming assemblies at neutral pH and tested as potential delivery vehicles. Imaging of encapsulated assemblies showed the presence of a “protein corona” around the DNs, confirming the formation of encapsulated DNs with CPs. One of the critical parameters in the encapsulation process is the DN-CP interaction, which was studied as a CP:DNA mass ratio and successful encapsulation of DNs was only reported at only increased ratios. The resulting nano-assemblies were found to be monodisperse with an average particle size between 40-50 nm.Following the formation of nanoassemblies, anthracycline daunorubicin, a chemotherapeutic drug was incorporated into two selected DNs and evaluated for their drug-loading efficiency. DNs were then, encapsulated with CPs to form a functional drug-loaded nanocarrier. The resulting nanocarriers were purified and characterised for monitoring the structural modification with analytical techniques. Next, the stability and drug-releasing properties of the nanocarriers were investigated, towards time-dependent controlled drug delivery at the target site. The formulated CP-coated nanocarriers were observed to outperform uncoated DNs and unfolded ds-DNA in stability studies against enzymatic degradation. The designed nanocarriers were also evaluated for their drug retention properties and were found to be capable of transporting a large amount of drug inside cells. Additionally, the triggered drug release within the cellular environment marked the utility of the created nanocarriers in delivery, indicating its potential in biomedical applications.Furthermore, to study the anticancer therapeutic efficacy, the intracellular fate of these nanocarriers was studied. Drug-loaded and CP-encapsulated DNs were tested in pancreatic cancer cell lines (PANC-1) for their cellular viability and drug uptake. Nanocarriers were found to prefer the clathrin-mediated endocytosis as the main uptake route, and their cellular internalisation kinetics appears to be size and cell-type dependent. Moreover, nanocarriers were identified to be endocytosed by cells after 2 hours of incubation and attain a controlled drug-release state with a protein coating on their surface, thereby facilitating drug enrichment inside cancer cells.Overall, we successfully demonstrated the potential of using Drug/DNs-virus hybrid nanoassemblies as smart drug nanocarriers. The designed nanocarriers were reported to mimic the morphology and functionality of virus-like particles with increased stability, enhanced cellular uptake, and efficiently targeting the cancer cells. DNs provide a robust platforms for delivering chemotherapeutic agents into cancer cells. However, extending the horizon of using these designed DNs-based nanocarriers in cancer therapeutics requires more in -vitro and in- vivo studies sweeping various cancer cell lines. Hence, the results presented in this thesis form a factual basis for further research using self-assembled biohybrid materials in cancer theranostics and drug delivery. There lie different opportunities to synchronise the properties of two biomolecules and investigate their use in a wide range of other biomedical applications. Thus, on a lighter note, we can say all viruses are not dreadful, but few eventually make us better instead

University of Twente Research Information

Virus Coated DNA Nanostructures:A Biological Way for Drug Delivery

https://ris.utwente.nl/ws/files/264089270/Thesis_Gaurav_Singhai.pdf

Virus Coated DNA Nanostructures:A Biological Way for Drug Delivery

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University of Twente Research Information