Uptake, efflux, and mass transfer coefficient of fluorescent PAMAM dendrimers into pancreatic cancer cells.

Targeted delivery of imaging agents to cells can be optimized with the understanding of uptake and efflux rates. Cellular uptake of macromolecules is studied frequently with fluorescent probes. We hypothesized that the internalization and efflux of fluorescently labeled macromolecules into and out of mammalian cells could be quantified by confocal microscopy to determine the rate of uptake and efflux, from which the mass transfer coefficient is calculated. The cellular influx and efflux of a third generation poly(amido amine) (PAMAM) dendrimer labeled with an Alexa Fluor 555 dye was measured in Capan-1 pancreatic cancer cells using confocal fluorescence microscopy. The Capan-1 cells were also labeled with 5-chloromethylfluorescein diacetate (CMFDA) green cell tracker dye to delineate cellular boundaries. A dilution curve of the fluorescently labeled PAMAM dendrimer enabled quantification of the concentration of dendrimer in the cell. A simple mass transfer model described the uptake and efflux behavior of the PAMAM dendrimer. The effective mass transfer coefficient was found to be 0.054±0.043µm/min, which corresponds to a rate constant of 0.035±0.023min(-1) for uptake of the PAMAM dendrimer into the Capan-1 cells. The effective mass transfer coefficient was shown to predict the efflux behavior of the PAMAM dendrimer from the cell if the fraction of labeled dendrimer undergoing non-specific binding is accounted for. This work introduces a novel method to quantify the mass transfer behavior of fluorescently labeled macromolecules into mammalian cells.

Physiologically based pharmacokinetics of molecular imaging nanoparticles for mRNA detection determined in tumor-bearing mice.

Disease detection and management might benefit from external imaging of disease gene mRNAs. Previously we designed molecular imaging nanoparticles (MINs) based on peptide nucleic acids complementary to cancer gene mRNAs. The MINs included contrast agents and analogs of insulin-like growth factor 1 (IGF-1). Analysis of MIN tumor uptake data showed stronger binding in tumors than in surrounding tissues. We hypothesized that MINs with an IGF-1 analog stay in circulation by binding to IGF-binding proteins. To test that hypothesis, we fit the tissue distribution results of several MINs in xenograft-bearing mice to a physiological pharmacokinetics model. Fitting experimental tissue distribution data to model-predicted mass transfer of MINs from blood into organs and tumors converged only when the parameter for MINs bound to circulating IGF-binding proteins was set to 10%-20% of the injected MIN dose. This result suggests that previous mouse imaging trials used more MINs than necessary. This prediction can be tested by a ramp of decreasing doses.

Design of (Gd-DO3A)n-polydiamidopropanoyl-peptide nucleic acid-D(Cys-Ser-Lys-Cys) magnetic resonance contrast agents.

We hypothesized that chelating Gd(III) to 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) on peptide nucleic acid (PNA) hybridization probes would provide a magnetic resonance genetic imaging agent capable of hybridization to a specific mRNA. Because of the low sensitivity of Gd(III) as an magnetic resonance imaging (MRI) contrast agent, a single Gd-DO3A complex per PNA hybridization agent could not provide enough contrast for detection of cancer gene mRNAs, even at thousands of mRNA copies per cell. To increase the Gd(III) shift intensity of MRI genetic imaging agents, we extended a novel DO3An-polydiamidopropanoyl (PDAPm) dendrimer, up to n = 16, from the N-terminus of KRAS PNA hybridization agents by solid phase synthesis. A C-terminal D(Cys-Ser-Lys-Cys) cyclized peptide analog of insulin-like growth factor 1 (IGF1) was included to enable receptor-mediated cellular uptake. Molecular dynamic simulation of the (Gd-DO3A-AEEA)16-PDAP4-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) genetic imaging nanoparticles in explicit water yielded a pair correlation function similar to that of PAMAM dendrimers, and a predicted structure in which the PDAP dendron did not sequester the PNA. Thermal melting measurements indicated that the size of the PDAP dendron included in the (DO3A-AEEA)n-PDAPm-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) probes (up to 16 Gd(III) cations per PNA) did not depress the melting temperatures (Tm) of the complementary PNA/RNA hybrid duplexes. The Gd(III) dendrimer PNA genetic imaging agents in phantom solutions displayed significantly greater T1 relaxivity per probe (r1 = 30.64 +/- 2.68 mM(-1) s(-1) for n = 2, r1 = 153.84 +/- 11.28 mM(-1) s(-1) for n = 8) than Gd-DTPA (r1 = 10.35 +/- 0.37 mM(-1) s(-1)), but less than that of (Gd-DO3A)32-PAMAM dendrimer (r1 = 771.84 +/- 20.48 mM(-1) s(-1)) (P < 0.05). Higher generations of PDAP dendrimers with 32 or more Gd-DO3A residues attached to PNA-D(Cys-Ser-Lys-Cys) genetic imaging agents might provide greater contrast for more sensitive detection.