Biodistribution of Fluorescently Labeled PAMAM Dendrimers in Neonatal Rabbits: Effect of Neuroinflammation

Abstract

Dendrimers are being explored in many preclinical studies as drug, gene, and imaging agent delivery systems. Understanding their detailed organ, tissue, cellular uptake, and retention can provide valuable insights into their effectiveness as delivery vehicles and the associated toxicity. This work explores a fluorescence-quantification based assay that enables simultaneous quantitative biodistribution and imaging of dendrimers with a single agent. We have labeled an ethylenediamine-core generation-4 hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimer using the fluorescent photostable, near-IR cyanine dye (Cy5) and performed quantitative and qualitative biodistribution of the dendrimer-Cy5 conjugates (D-Cy5) in healthy neonatal rabbits and neonatal rabbits with cerebral palsy (CP). The biodistribution of D-Cy5 and free Cy5 dye was evaluated in newborn rabbits, based on the developed quantification methods using fluorescence spectroscopy, high-performance liquid chromatography (HPLC), and size exclusion chromatography (SEC) and supported by microscopic imaging. The uptake was assessed in the brain, heart, liver, lungs, kidneys, blood serum, and urine. Results obtained based on these three independent methods are in good agreement and indicate the fast renal clearance of D-Cy5 and free Cy5 with relatively higher organs accumulation of the D-Cy5 conjugate. Following systemic administration, the D-Cy5 mainly accumulated in kidneys and bladder at 24 h. The quantitative biodistribution is in good agreement with previous studies based on radiolabeling. These methods for dendrimers quantification are easier and more practical, provide excellent sensitivity (reaching 0.1 ng per gram of tissue), and allow for quantification of dendrimers in different organs over longer time periods without concerns for radioactive decay, while also enabling tissue and cellular imaging in the same animal. In kits with fetal-neuroinflammation induced CP, there was a significantly higher uptake of D-Cy5 in the brain, while biodistribution in other organs was similar to that of healthy kits.

Uptake of dendrimer-drug by different cell types in the hippocampus after hypoxic–ischemic insult in neonatal mice: Effects of injury, microglial activation and hypothermia

“…Our analysis of cellular uptake of Cy5-labeled D-NAC in a mouse model of HIE examined the effects of therapeutic hypothermia and time of administration on uptake, and we used a correlation analysis to examine the effects of brain injury and microglial activation on D-NAC uptake in this model. These results expand upon previous findings that support the use of dendrimer therapy for brain injury in other neonatal 25,26,30 and large animal models. 28 …”

“…29,30 To prepare the novel conjugate, Cy5-D-NAC, for this dendrimer-drug uptake study in our neonatal hypoxic-ischemic mouse model, a modified synthetic scheme was followed. Our starting material consisted of a bifunctional G4-OH dendrimer with 17–18 amine groups on the surface of the hydroxyl dendrimer, so that the free amine groups would further react with NAC.…”

Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions

“…6d). More specifically, we employed the following diffusion tracers that are known to exhibit minimal adsorption to general proteins: D-Cy5 (4.3 nm in diameter) 42 , CdSeS/ZnS alloyed quantum dot (Q-dot) (6 nm in diameter), and fluorescent beads (20 nm in diameter). While the 4.3-nm tracer did penetrate into the hydrogel almost freely, the 6-nm and 20-nm tracers were clearly excluded from the hydrogel (Fig.…”

“…Fluorescently labeled ethylenediamine-core generation-4 hydroxyl-terminated poly(amidoamine) dendrimer, D-Cy5 42 , was kindly provided by Rangaramurajan M. Kannan’s group. CdSeS/ZnS alloyed quantum dots with 665 nm emission wavelength were purchased from Sigma Aldrich.…”

Nanoparticles for Brain-Specific Drug and Genetic Material delivery, Imaging and Diagnosis

“…The systemic administration of PAMAM dendrimers has shown minimal brain uptake in healthy animals. PAMAM dendrimers below generation 6 are rapidly cleared from systemic circulation by kidney filtration or by the RES, whereas PEGylated dendrimers or PAMAM dendrimers above generation 6 tend to circulate longer and be distributed to peripheral organs [52,53]. Only when BBB permeability is increased, as happens during neuroinflammation or meningitis, is the access of these dendrimers to the CNS favored.…”