My Research

My doctoral research revolves around the following three ideas:

1. Unraveling the early-stage dynamics of Cytolysin A⇔lipid interactions and understanding the effect of membrane-component Sphingomyelin on its oligomerization kinetics

Cytolysin A monomer

Various pathogenic strains of microorganisms employ pore-forming toxins to disrupt host cells by inducing osmotic imbalance across the cell membranes. The widely studied bacterial α pore-forming toxin Cytolysin A is known to follow a typical modus operandi, i.e., its water-soluble ‘inactive’ monomer undergoes a conformational transition to an ‘active’ or ‘protomeric’ form upon introduction of detergent micelles or lipid-based structures (vesicles, bilayers, etc.). It’s the protomeric form that oligomerises further into higher structures and eventually into full-grown pores that induce lysis in the case of cells.

Fully formed dodecameric pore structure

We aim to understand this segment of the pore-forming mechanism, where the toxin monomer binds and assembles on the membrane surface, in greater depth to frame design principles for synthetic nanopores that have the potential to lyse, selectively and in a controlled manner, the cancer cells. Experiments conducted so far in the detergent-micellar environment led us to understand that the presence of raft-like lipid nano-domains on the membrane surface enhances the pore-forming activity of the protein. We also observed that if the protein is introduced in a ‘molten-globular’ form rather than a ‘protomeric’ form, it leads to faster lysis, both in the case of phase-segregated vesicles and erythrocytes.

2. Studying the effect of macromolecular crowding at the membrane surface on various biomolecular phenomena

Natural cell membranes are highly crowded environments due to embedded proteins and sugars. Molecular crowding is important in various biophysical and biochemical phenomena like binding, surface diffusion, macromolecular assembly, and even biomolecular reactions. This makes studying the impact of molecular crowding an interesting research arena. We model the cell membranes as supported lipid bilayers to study various physicochemical phenomena occurring at the cell surface. We usually incorporate polymeric molecules (like PEG) of different chain lengths at different densities to mimic different degrees of macromolecular crowding. 

Visualization of molecular crowding

We know from previously reported studies that the polymeric crowder PEG can assume different conformations (mushroom or brush) based on its surface concentration and length. Single particle tracking experiments with Satyaghosh Maurya generated data on the lateral diffusion coefficients of various molecular species (ssDNA conjugated to tocopherol, rhodamine-labelled lipid DOPE, and proteins). Real cellular membranes are seldom crowded with linear species alone, or all the diffusing species are not necessarily point-species or linear ones. The famous Saffman-Delbrück equation accounts for the embedded volume of the diffusing species and provides a relation featuring weak dependence of the particle diffusivity on the size of the embedded region of the particle. This theory assumes a dilute surface, i.e., a diffusing molecule enjoys an interaction-free environment. Various studies conducted in the recent past established the dependence of the diffusivity of particles with varying sizes of their extracellular regions in crowder-free environments. Although various macromolecules or virus particles interacting with highly crowded cell membranes have large ectodomains associated with them, there is a dearth of research that accounts for the size of the extracellular domains of these molecules in highly crowded environments.

3. Designing DNA-based nanostructures for the intracellular delivery of genetic material

Virus life cycles typically follow a five-step process: attachment, entry, genome replication and expression, assembly, and exit. Based on the mode of entry, many viruses use different kinds of protein-receptor interactions. These interactions help viruses enter the host cell.

A non-viral vector that utilizes DNA as structural entity

It is important to study how an evolved virus particle uses cellular receptors in multiple ways to gain access to the cellular replication machinery. The following diagram shows how different steps that occur in tandem help a viral particle get endocytosed. Some of these learnings can be used to design a synthetic particle that can potentially act as a vector for intracellular delivery of the gene of interest. The same vector can also be utilised for generating innate immune responses and thus act as a potential vaccine candidate. A non-viral vector that mimics the non-pathogenic functions of a viral particle helps a long way in lowering the cost of handling and mass-scale production of vectors and can also curb potential long-term side effects due to utilizing a viral vector.

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📜During my Masters, I worked on the following idea:

Synthesizing lipid-based nanoparticles for intrapulmonary delivery of insulin

I conducted comprehensive research on commercial inhaled insulin-delivery systems, analyzing their features and limitations compared to injectable insulin.

All hail the inhaled-insulin!

My work culminated in developing lipid encapsulated nanoparticles through aerosol synthesis for efficient intrapulmonary insulin delivery. I focused on achieving optimal bioavailability, biocompatibility, and patient compliance by synthesizing nanoparticles with aerodynamic sizes of 50 to 200 nm and encapsulating insulin in a crystalline lipid shell. I utilized air-jet atomization to form nanoparticles from a precursor solution.

Insulin release profile in PBS 

The wet-lab work involved carrying out in-depth characterization of the synthesized particles using TEM and FTIR for substructure and surface composition analysis, respectively. I also conducted in-vitro studies to evaluate insulin release in PBS, measuring insulin concentration with a fluorescence spectrophotometer. The project resulted in development of an innovative approach for intrapulmonary insulin delivery with potential clinical significance.