Dissertation Defense – Stefany Y. Holguin
Dr. Mark Prausnitz, Advisor, CHBE
Dr. Naresh Thadhani, Advisor, MSE
Dr. Valeria Milam, MSE
Dr. David Bucknall, MSE
Dr. Michelle LaPlaca, BME
Dr. Michael Gray, ME
“PHYSICAL MECHANISMS OF LASER-ACTIVATED NANOPARTICLES FOR INTRACELLULAR DRUG DELIVERY”
Novel intracellular drug delivery techniques are needed to overcome the barrier of the cell’s plasma membrane. Since the cell membrane is highly selective, it is difficult for potential therapeutics, like those for gene-based therapy and other active molecules to enter the cytosol by methods other than active transport. To bypass the membrane, physical drug delivery methods can be employed to create transient openings in the membrane that enable the passive transport of molecules. Optimizing physical methods is often challenging due to a need to balance molecular delivery and high cell viability. A novel, laser-mediated technique known as transient nanoparticle energy transduction (TNET) has been shown to successfully balance these two parameters. When carbon black (CB) nanoparticles in suspension with cells and small molecules are irradiated by nanosecond-pulsed near infrared (NIR) laser energy, efficacious delivery while maintaining high cell viability are achieved.
This novel drug delivery platform is driven by the laser-activation of CB nanoparticles and the subsequent energy transduction which induces bioeffects (i.e., uptake and viability loss). Upon NIR absorption, the CB nanoparticles rapidly heat up to hundreds of degrees Celsius and undergo thermal expansion. This rapid heating leads to vaporization of surrounding water, which creates vapor-bubbles that transfer heat and pressure to nearby cells. To gain mechanistic insight into TNET, we studied various aspects of this in vitro system, including cellular mechanics, cell-CB nanoparticle interaction, and the role of photoacoustics.
First, we studied the role of cellular mechanics in TNET by way of the cytoskeleton and plasma membrane fluidity. Destabilizing the cytoskeleton caused greater intracellular uptake and lower viability loss compared to cells with intact cytoskeletons. Additional studies showed that altering the fluidity of the membrane had no significant effect on bioeffects. From these studies, we concluded that cytoskeletal mechanics are integral to resulting bioeffects achieved with TNET, whereas the fluidity of the plasma membrane is not.
Next, we studied the effect of energy input into the system, which was increased by increasing laser fluence, CB nanoparticle concentration and number of laser pulses. We found that at low energy, intracellular uptake increased with increasing energy input. At higher energy, cell viability loss increased and viable cells with intracellular uptake decreased. At the highest energy inputs, cell fragmentation increased, while intracellular uptake and loss of viability decreased. Increasing cell concentration had the opposite effect (i.e., it reduced the intensity of bioeffects), which suggests that neighboring cells shielded each other from the effects of TNET. Increasing medium viscosity also decreased the intensity of bioeffects, suggesting a mechanical (i.e., not thermal) cause of bioeffects.
Finally, we studied the effects of three different carbon-based nanoparticles – CB, multi-walled carbon nanotubes (MWCNT) and single-walled (SWCNT) carbon nanotubes – on cellular bioeffects. As mentioned above, increasing energy input with CB nanoparticles progressed through increasing intracellular uptake, followed by increasing cell viability loss, which was followed by increasing cell fragmentation. For MWCNT, increasing energy input first increased intracellular uptake and then increased cell fragmentation, but did not cause cell viability loss. For SWCNT, no significant bioeffects were seen, except at extreme conditions where cell viability loss was seen. The intensity of photoacoustic output in the form of a single, mostly positive-pressure pulse of ~100 µs duration varied among the different types of nanoparticles, where SWCNT had the highest peak pressure, followed by CB and then by MWCNT. Lack of a universal correlation between peak pressure and cellular bioeffects, suggested that total energy input rather than pressure output was more mechanistically relevant to TNET.
Overall, this work provides functional characterization and mechanistic understanding the cellular bioeffects cause by TNET. These studies will contribute an understanding of TNET that will enable rational design of TNET systems for future applications and possible translation into the clinic.