Previously, we demonstrated that the IN route is an effective peptide delivery route to treat cerebellar disorders, like SCA1 (Vig et al. 2006b, 2011). We also reported that the peptide carrier, Synb1-ELP, can be delivered to the brain via i.p. injection (Hearst et al. 2011). However, our peptide's localization to the brain at a cellular level has never been assessed. Therefore, we tested the IN and i.p. brain delivery capabilities of our peptide carrier Synb1-ELP and examine the cerebellar uptake of our Synb1-ELP carrier at a cellular level. We treated 3-week-old WT mice with rhodamine labeled Synb1-ELP via IN and i.p. and used saline as the control. The animals were perfused 3 h post treatment and their brains removed for frozen sectioning and visualization under the fluorescent microscope. Both IN (Fig. 5a) and i.p. (Fig. 5b) routes effectively delivered the Synb1-ELP carrier to the cerebellum. We saw strong localization of Synb1-ELP in the molecular layer, the PC layer and slightly weaker localization in the granular cell layer. These data indicate that both IN and i.p. may be possible routes to deliver our therapeutic peptides to the SCA1 cerebellum. We also looked at localization of rhodamine labeled Synb1-ELP, via IN, throughout the brain and found localization in the olfactory bulb, the cerebral cortex, the hippocampus, and again the cerebellum (Fig. 5c). The olfactory bulb showed high localization of Synb1-ELP in the form of aggregates after IN administration (Fig. 5c, 40×). The olfactory bulb is the closest brain tissue to the nasal passage and alongside the trigeminal pathway provides a route for IN peptide delivery to other regions of the brain (Renner et al. 2012). I.P. injections of rhodamine labeled Synb1-ELP showed similar localization to other regions of the brain as compared to IN (data not shown). Our earlier report indicates that IN route has rapid absorption (Vig et al. 2006b), therefore we chose to analyzed brain uptake over time by scanning the brains starting at 30 min post treatment in these experiments. We treated 3 week old WT mice with Alexa 750 labeled Synb1-ELP via the IN route at 50 mg/kg and used the IVIS Live Imager to scan their brains 30 min, 1, 2, 3, 4, 8 and 18 hs post treatment. Interestingly, in the body scan, Synb1-ELP was highly localized to the nasal cavity after IN treatment (Fig. 6a). We also saw some uptake of Synb1-ELP to what appeared to be the stomach after IN treatment (Fig. 6a). It is not uncommon for small amounts of IN delivered agents to be swallowed into the stomach (Southam et al. 2002). The brain scans revealed Synb1-ELP uptake to the brain post IN treatment as compared to control (Fig. 6a). As seen earlier, we saw a high localization of Synb1-ELP to the olfactory bulbs (Fig. 6a). Next, we measured the kinetic uptake of Synb1-ELP carrier to the brain after IN treatment. Fig. 6b displays the total brain uptake of Synb1-ELP as μg of Synb1ELP per gram of brain tissue over time. Synb1-ELP peaks the brain 30 min after treatment and then drops at 2 h and then increases again at 4 h (Fig. 6b). The rise in brain levels seen at 4 h post treatment is most likely because of the redelivery of Synb1-ELP to the brain via the blood stream. Next, we quantified the uptake in different regions of the brain by taking an ROI of the olfactory bulb, cerebral cortex and the cerebellum over time as described previously (Hearst et al. 2011). We found that Synb1-ELP was highly localized to the olfactory bulb and peaked at 30 min (Fig. 6c). Synb1-ELP peaked the cerebellum and cerebral cortex at 4 h, which may be because of both the trigeminal pathway delivery and blood stream delivery of Synb1-ELP to these regions of the brain (Fig. 6c). To ensure that our peptide Synb1-ELP-PKI had similar IN and i.p. delivery capabilities as the Synb1-ELP carrier, we labeled the Synb1-ELP-PKI peptide with rhodamine and compared the brain distribution, CSF level, and plasma level 6 h after IN or i.p. administration of 100 mg/kg Synb1-ELP-PKI-Rho. We found that Synb1-ELP-PKI localized to the brain in both IN- and i.p.-treated animals (Fig. 7a). However, i.p.-treated mice showed higher brain uptake as compared to IN-treated mice (Fig. 7a and b). Also, CSF and plasma levels were higher in i.p.-treated mice as compared to IN-treated mice (Fig. 7c and d). Although, the i.p. route delivered more peptide to the brain, it had a lower CSF to plasma ratio as compared to the IN route (Fig. 7e). Our data suggest that IN may be the better delivery route to target the Synb1-ELP-PKI peptide to the brain while limiting its delivery to the blood stream and non-targeted organs and tissues.
Figure 5. 3-week-old WT mice were given Synb1-elastin-like polypeptide (ELP)-Rho via intranasal (IN) or i.p. route and saline as the Control. (a) Shown are cerebellar sections from IN-treated mice. Shown in red is the localization of Synb1-ELP in the cerebellum and DAPI nuclear stain in blue, left scale bar 10 μm, right scale bar 25 μm. (b) Shown are cerebellar sections from i.p.-treated mice. Shown in red is the localization of Synb1-ELP in the cerebellum and DAPI nuclear stain in blue, left scale bar 10 μm, right scale bar 25 μm. (c) Shown in red is the localization of Synb1-ELP in the olfactory bulb, the cerebral cortex, the hippocampus, and the cerebellum and DAPI nuclear stain in blue, left scale bar 25 μm (20×), right scale bar 10 μm (40×).
Download figure to PowerPoint
Figure 6. 3-week-old WT mice were given Alexa-750 labeled Synb1-elastin-like polypeptide (ELP) peptide via intranasal (50 mg/kg). 3 h post treatment their bodies and then their brains were scanned with the IVIS imager as previously described (Hearst et al. 2011). (a) The images show the localization of Synb1-ELP after intranasal administration. The Color Scale shows Epi-fluorescence in Counts from 0 to 4000 where green is a high level of Synb1-ELP localization, red is a low level, and blue is background fluorescence. Scale bars 4 mm. (b) Shown is the Mean brain uptake or Mean plasma level of Alexa-750 labeled Synb1-ELP peptide via intranasal (50 mg/kg) over time. (c) Shown is the Mean localization of Synb1-ELP in the olfactory bulb, cerebral cortex, and cerebellum over time.
Download figure to PowerPoint