Time-correlated single photon counting (TCSPC) is a fluorescence technique used to determine two properties of fluorescent particles: lifetime and correlation. After pulsing a lase rapidly into a fluorescent sample and recording the length of time between the excitation and de-excitation of a fluorophore within the laser focus, the resulting microtime can be used to calculate the fluorescence lifetime. Fluorescence lifetime is extremely sensitive to the fluorophore’s local environment, and changes noticeably due to steric or electromagnetic factors. The time difference from a particular de-excitation and the start of the experiment, known as macrotime, can be autocorrelated. This autocorrelation data can be fit to established autocorrelation curves from literature based on diffusion and particle assumptions to determine the size of the particle via the diffusion time through the focal volume and the average number of particles within the focal volume of the laser. GE Global Research – Student Research Summit 2014 Use of Time-Correlated Single Photon Counting to Evaluate Nanocarriers for Drug Delivery 1 Nick Frazzette, 2 James H. Adair, 1 Peter J. Butler Departments of 1 Biomedical Engineering and 2 Materials Science and Engineering, The Pennsylvania State University, University Park, PA Introduction Cancer and genetic-based diseases are among the most difficult to treat today. Small-interfering RNA presents a reliable method for oncogene knockdown, but needs protection from filtration and degradation. Nanocarriers, in particular the biomineral- based calcium-phosphate silicatee nanoparticles (CPSNPs), present an encouraging, effective, and nontoxic solution to this problem. However, nanocarriers’ size range on the order of 10- 100 nm and their dopants are even smaller, making traditional microscopy impossible for evaluation. Additionally, dosage concentrations are critical to medical use, but particle concentration is difficult to obtain as a function of synthesis protocol. This work focuses on using fluorescence and a time- correlated single photon counting system to determine the encapsulation of calcium-phosphate around certain dopants and to determine the concentration of particles in solution; in particular, cyanine3 amidite and cyanine3-tagged dsDNA are encapsulated. Experimental Methods Results Conclusions and Future Work References and Acknowledgements The currently used protocol produces CPNPs of regular and consistent size and very capable of encapsulating organic dye molecules such as Cy3. However, substitution of Cy3-tagged dsDNA into the same protocol does not result in encapsulation, as evidenced by the similarity in fluorophore radius. However, it is also notable that the concentration of Cy3-doped CPNPs can be found easily via this method. Future work primarily involves determined what changes to protocol are necessary to induce particle growth around dsDNA or inducing dsDNA incorporation into particles. Moreover, this method of particle number counting can be validated against particle counting methods that do not rely on fluorescence; in this way, non-fluorescent nanocarrier-doped drugs can be reliably counted to determine dosing concentrations. • Gullapalli, R. et al. “ Integrated multimodal microscopy, time- resolved fluorescence, and optical trap rheometry: towards single molecule mechanobiology”. J Biomed Opt 12. 2007. p 014012 • Morgan, T. et al. “Encapsulation of Organic Molecules in Calcium Phosphate Nanocomposite Particles for Intracellular Imaging and Drug Delivery”. Nano Lett 8. 2008. p 4108-15 • Muddana, H. et al. “Photophysics of Cy3-Encapsulated Calcium Phosphate Nanoparticles”. Nano Lett 9. 2009. p 1559-66 This work was supported in part by NIH NCI 5 R01 CA167535-02 FCS Results on CPNP Encapsulating Cy3 Amidite Δt (ms) G(Δt) FCS Results on CPNP Encapsulating Cy3-dsDNA FCS Results on Free Cy3-dsDNA Δt (ms) G(Δt) Δt (ms) G(Δt) Lifetime Results on CPNP Encapsulating Cy3 Amidite Lifetime Results on Free Cy3-dsDNA Lifetime Results on CPNP Encapsulating Cy3-dsDNA Hydrodynamic Radius = 44.65 ± 9.35 nm Fluorescence Lifetime = 1.448 ± 0.034 ns Number of particles = 7.6 · 10 11 mL -1 Hydrodynamic Radius = 1.855 ± 0.220 nm Fluorescence Lifetime = 0.7682 ± 0.0148 ns Number of particles = 1.8 · 10 12 mL -1 Hydrodynamic Radius = 1.754 ± 0.068 nm Fluorescence Lifetime = 0.9513 ± 0.0092 ns Number of particles = 1.5 · 10 12 mL -1