Time-correlated single photon counting (TCSPC) is afluorescence technique used to determine two properties offluorescent particles: lifetime and correlation. After pulsing alase rapidly into a fluorescent sample and recording the lengthof time between the excitation and de-excitation of afluorophore within the laser focus, the resulting microtime canbe used to calculate the fluorescence lifetime. Fluorescencelifetime is extremely sensitive to the fluorophore’s localenvironment, and changes noticeably due to steric orelectromagnetic factors. The time difference from a particularde-excitation and the start of the experiment, known asmacrotime, can be autocorrelated. This autocorrelation datacan be fit to established autocorrelation curves from literaturebased on diffusion and particle assumptions to determine thesize of the particle via the diffusion time through the focalvolume and the average number of particles within the focalvolume of the laser.

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Use of Time-Correlated Single Photon Counting to Evaluate Nanocarriers for Drug Delivery1Nick Frazzette, 2James H. Adair, 1Peter J. Butler

Departments of 1Biomedical Engineering and 2Materials Science and Engineering, The Pennsylvania State University, University Park, PA

IntroductionCancer and genetic-based diseases are among the most difficultto treat today. Small-interfering RNA presents a reliable methodfor oncogene knockdown, but needs protection from filtrationand degradation. Nanocarriers, in particular the biomineral-based calcium-phosphate silicatee nanoparticles (CPSNPs),present an encouraging, effective, and nontoxic solution to thisproblem. However, nanocarriers’ size range on the order of 10-100 nm and their dopants are even smaller, making traditionalmicroscopy impossible for evaluation. Additionally, dosageconcentrations are critical to medical use, but particleconcentration is difficult to obtain as a function of synthesisprotocol. This work focuses on using fluorescence and a time-correlated single photon counting system to determine theencapsulation of calcium-phosphate around certain dopantsand to determine the concentration of particles in solution; inparticular, cyanine3 amidite and cyanine3-tagged dsDNA areencapsulated.

Experimental Methods

Results

Conclusions and Future Work References and AcknowledgementsThe currently used protocol produces CPNPs of regular and consistent size and very capable of encapsulating organic dye moleculessuch as Cy3. However, substitution of Cy3-tagged dsDNA into the same protocol does not result in encapsulation, as evidenced bythe similarity in fluorophore radius. However, it is also notable that the concentration of Cy3-doped CPNPs can be found easily viathis method. Future work primarily involves determined what changes to protocol are necessary to induce particle growth arounddsDNA or inducing dsDNA incorporation into particles. Moreover, this method of particle number counting can be validated againstparticle counting methods that do not rely on fluorescence; in this way, non-fluorescent nanocarrier-doped drugs can be reliablycounted to determine dosing concentrations.

• Gullapalli, R. et al. “ Integrated multimodal microscopy, time-resolved fluorescence, and optical trap rheometry: towards singlemolecule mechanobiology”. J Biomed Opt 12. 2007. p 014012

• Morgan, T. et al. “Encapsulation of Organic Molecules in CalciumPhosphate Nanocomposite Particles for Intracellular Imaging andDrug Delivery”. Nano Lett 8. 2008. p 4108-15

• Muddana, H. et al. “Photophysics of Cy3-Encapsulated CalciumPhosphate 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

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FCS Results on CPNP Encapsulating Cy3-dsDNAFCS Results on Free Cy3-dsDNA

Δt (ms)

G(Δt

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Δt (ms)

G(Δt

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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 · 1011 mL-1

Hydrodynamic Radius = 1.855 ± 0.220 nm

Fluorescence Lifetime = 0.7682 ± 0.0148 ns

Number of particles = 1.8 · 1012 mL-1

Hydrodynamic Radius = 1.754 ± 0.068 nm

Fluorescence Lifetime = 0.9513 ± 0.0092 ns

Number of particles = 1.5 · 1012 mL-1