The toxicity, biodistribution, pharmacokinetics and excretion pathways of PAA and PAA NPs in rats were investigated. The dose of nanoparticle administered to each rat was based on the specific (radioactive) activity and the need to achieve a minimum dose of 10 uCi per animal. This resulted in a 61% higher dose for the PAA cohort when compared to PAApeg, however, the compartmental analysis indicated that once the saturation of certain organs were accounted for, there was no major kinetic difference between the two formulations.
Although the acrylamide monomer used in the synthesis of PAA NPs is a neurotoxin (Dearfield et al. 1988; Friedman 2003), its polymerized form is non-toxic and does not degrade back into its monomeric chemical form (Caulfield et al. 2002). Previous experiments with PAA NPs up to a dose of 500 μg/g over 4 weeks showed no visible alterations in histopathology or clinical chemistry values indicating acute toxicity by the NPs. Because of PAA long-term stability, high biocompatibility (Lemperle et al. 2003), and flexibility for incorporating targeting moieties, it is a promising material for medical applications. Consistently, our acute toxicity study indicates that PAA nanoparticles do not induce detectable organ damages or other measurable signs of toxicity. The modest elevation in alkaline phosphate could suggest impairment of liver, intestine or bone dysfunction although histopathological studies do not indicate toxicity at these targets. In addition, other clinical chemistry markers in the study do not support toxicity of these systems and the modest elevation in alkaline phosphatase is just beyond the published reference range (200-300IU/L) for Fisher344 rats at this age. Future, chronic toxicity studies and multiple exposure studies will be important due to the slow degradative process of the polyacrylamide matrix.
The SDS-PAGE analysis of the proteins bound to either the PAA or PAApeg NPs revealed similar patterns, although proteins associated with naked PAA were more abundant. Apolipoproteins were detected, and it has been previously reported that this class of proteins is involved in the translocation of poly(butyl-cyanoacrylate) (PBCA) NPs across the blood brain barrier (Kreuter et al., 2002
). In this study from Kreuter et al, translocation was specifically triggered by apolipoprotein-E and B, which were not detected in our experiments. However, the presence of other detectable proteins and their concurrence with published studies suggests that failure to detect other key proteins may be a product of the limits of detection inherent in the technology employed.
The pharmacokinetic analysis shows an initial rapid transfer from blood to organs with half-lives of 0.6 (PAA) and 1.3h (PAApeg). The liver was the major deposition site, followed by the kidneys and the lungs. The concentration in the popliteal lymph nodes increased steadily and was the most concentrated tissue at 48h postdose, whereas inguinal and popliteal lymph nodes contained modest concentrations. The lowest concentrations were measured in the brain. Part of an explanation for the important liver confiscation might be that it receives approximately 30% of the cardiac output and contains the largest fraction of the total fixed macrophages (Kupffer cells) in the body (Wake et al., 1989
). The total recovery in organs accounted only for less than 22% of the total dose administered.
The initial transfer from blood to organs was followed by a slower distribution to the carcass representing more than 75% of the initial dose with a kinetic of appearance of radioactive material at this location corresponding to the kinetic of disappearance observed in the blood (half-life of approximately 40h). compares the maximum half-life in blood and the Area Under the Curve (AUC) obtained in the present study to results for five other polymeric nanoparticles reported in the literature (see table S5, supporting information
). The residence times observed in our study were typically longer than those retrieved in experiments with other polymeric NPs such as poly(lactic-coglycolic acid) (PLGA or PLGA-mPEG) nanoparticles (half-lives of 35 sec and 7.0 h, respectively)(Panagi et al., 2001
), PLA (Gaucher at al., 2009
; Liu, 2008
), PRINT-PEG (Gratton et al., 2007
), and Nanogel and Nanolatex (Yang et al., 2009
). The studied PAA and PAA-PEG particles are at the highest half-lives and AUC. These two parameters decrease by two to three orders of magnitude when nanoparticle size increases from the 20 to 30 nm range up to 250 nm. For similar sizes, pegylated nanoparticles are always more persistent in the blood than non pegylated ones, but this difference is much smaller in the 30 nm and relatively high dose range of the present experiment than in the range above 100 nm.
FIG. 9A and 9B
Maximum half-lives in blood (9A) and Area Under the Curve (9B) obtained in this study (Wenger et al., 2010) compared with results of five other experiments on pegylated (empty symbols) and non-pegylated (filled symbols) polymeric nanoparticles.
Neither PAA nanoparticle formulation resulted in significant amounts of recovery in urine and feces after 120h (less than 6% excreted from the body after 120h). This long residence time in the body is not surprising, based on studies reporting the low biodegradability of cross-linked polyacrylamide (Smith et al., 1996
; Smith et al., 1997
), and the fact that most of the NPs used where too large to undergo glomerular filtration. However, the synthesis of most NPs results in a suspension of NPs of slightly varying diameters that can be defined by a Gaussian distribution. As a result, NPs parts of a single suspension, but having different diameters, may not follow identical kinetics or tissue distribution profiles. Particles with a diameter smaller than 10nm are typically capable of being filtered by the renal system. Based on the mean diameter of the PAA NPs used here (mean diameter 30 nm), renal filtration of the particles may filter a small percentage of the particle suspension in the low end distribution (<10nm), consistently with what was observed.
On a therapeutical level, the rapid saturation observed in the organs of the RES suggests that a pre-treatment with a type of NPs that can be rapidly degraded, immediately followed by the injection of long-lasting PAA NPs would allow the latter to escape first pass capture by the macrophages, and thus extend their circulation time. Finally, the confiscation of PAA and PAApeg NPs by the organs of the RES, probably involving the saturation of their resident macrophages, raises the question of a potential increased susceptibility of treated subjects to superinfections, as macrophages are believed to be major players of body's defense against foreign pathogens.