Chapter V: Overall Summary and Conclusions
2.2 Results and Discussion
2.2.5 In Vivo Investigation of PLGA Nanoparticles Containing
high avidity to TfRs, but once Tf falls off the nanoparticles they bind non-specifically to the Neuro2A cells just as the non-targeted nanoparticles. This indicates that a disulfide bond is present between the Tf and PLGA nanoparticle core and when cleaved causes loss of the targeting ligand from the nanoparticle and subsequent loss of binding avidity for the targeting ligand’s receptor.
2.2.5 In Vivo Investigation of PLGA Nanoparticles Containing Surface Tf
Fig 2.10 Fluorescent image of fresh-frozen brain section of mouse injected with non- targeted PLGA-PEG nanoparticles.
Fig 2.11 Fluorescent image of fresh-frozen brain section of mouse injected with low-Tf PLGA-PEG nanoparticles.
Fig 2.12 Fluorescent image of fresh-frozen brain section of mouse injected with high-Tf PLGA-PEG nanoparticles.
Fig 2.13 Fluorescent image of fresh-frozen brain section of mouse injected with high-Tf PLGA-PEG nanoparticles containing a disulfide link between Tf and the nanoparticle core.
Making conclusions using fresh-frozen sections, however, is limited due to the poorly preserved tissue architecture preventing definitive morphological identification of vessels, so the assay was repeated using formalin-fixed paraffin-embedded brain tissue sections. Figures 2.14-2.17 illustrate to what extent each nanoparticle formulation reaches the brain parenchyma using this method. Nanoparticles were positively identified as
distinct fluorescent signal above autofluorescence and clearly away from the blood vessels and in the parenchyma. Fluorescence associated with cell nuclei was seen in the negative controls and was therefore not considered to be specific to nanoparticles.
Untargeted PLGA-mPEG nanoparticles did not access the brain parenchyma and remained exclusively in the vasculature (Fig 2.14). Low-Tf PLGA-PEG nanoparticles were present in the parenchyma (Fig 2.15). High-Tf PLGA-PEG nanoparticles were not clearly seen in the brain parenchyma, with a similar fluorescent pattern to the PLGA-mPEG formulation (Fig 2.16). This is consistent with the necessity for the nanoparticles’ avidity to be tuned for successful release into the brain parenchyma. High-Tf with disulfide nanoparticles showed the greatest amount of fluorescence within the brain parenchyma (Fig 2.17).
Fig 2.14 Confocal images of PLGA-mPEG nanoparticle in formalin-fixed mouse brain sections. Panel A: 488nm excitation, panel B: DAPI signal, panel C: merged image of Panels A and B. Panel D shows an enlarged view of the merged image in Panel C. Solid white arrows indicate fluoresecence co-localized with cell nuclei. Since this phenomenon was seen with non-targeted particles, it was considered normal tissue background fluorescence. Dotted white arrows indicate blood vessels.
Fig 2.15 Confocal images of low-Tf PLGA-PEG nanoparticles in formalin-fixed mouse brain sections. Panel A shows fluorescence from 488nm excitation. Panel B shows the DAPI signal. Panel C shows a merged image of Panels A and B. Panel D shows an enlarged view of the merged image in Panel C. Solid white arrows indicate fluoresecence co- localized with cell nuclei. Dotted white arrows indicate blood vessels. Hollow white arrows indicate fluorescence in the parenchyma not associated with cell nuclei determined to be nanoparticle signal.
Fig 2.16 Confocal images of high-Tf PLGA-PEG nanoparticles in formalin-fixed mouse brain sections. Panel A shows fluorescence from 488nm excitation. Panel B shows the DAPI signal. Panel C shows a merged image of Panels A and B. Panel D shows an enlarged view of the merged image in Panel C. Solid white arrows indicate fluoresecence co- localized with cell nuclei. Dotted white arrows indicate blood vessels. Hollow white arrows indicate fluorescence in the parenchyma not associated with cell nuclei determined to be nanoparticle signal.
Fig 2.17 Confocal images of high-Tf plus disulfide PLGA-PEG nanoparticle in formalin- fixed mouse brain sections. Panel A shows fluorescence from 488nm excitation. Panel B shows the DAPI signal. Panel C shows a merged image of Panels A and B. Panel D shows an enlarged view of the merged image in Panel C. Solid white arrows indicate fluoresecence co-localized with cell nuclei. Dotted white arrows indicate blood vessels.
Hollow white arrows indicate fluorescence in the parenchyma not associated with cell nuclei determined to be nanoparticle signal.
Recent work by Wiley et al. demonstrated that nanoparticle avidity to transcytosing receptors need to be tuned to deliver the nanoparticles into the brain parenchyma. In this work, nanoparticles of intermediate avidity were able to attach to receptors on the blood-side of the BBB and detach from the receptors on the brain side of the BBB. These optimally tuned nanoparticles reached the brain parenchyma; however, they did so in very small numbers (much less than 1% of the injected dose reached the brain parenchyma). Nanoparticles of high avidity accumulated more in the bulk of the brain mainly by remaining stuck to receptors in or on the endothelial cells of the BBB.
Here we demonstrate that nanoparticles whose targeting molecules are attached to the nanoparticle core through a disulfide bond can access the brain in greater numbers than nanoparticles with fixed targeting molecules. Nanoparticles of high avidity can associate with receptors in larger numbers, and these nanoparticles, when released from their targeting ligand while en route through the BBB, can accumulate more in the brain parenchyma.
Disulfide bonds inserted into PEG linkers between the nanoparticle and the targeting molecules can be cleaved by reducing agents including BME, DTT, and GSH.
When nanoparticles of high avidity are injected, they do not appreciably enter the brain parenchyma. However, when nanoparticles of high avidity with the disulfide-based linker are injected, the nanoparticles access the brain parenchyma in much greater numbers.
Nanoparticles of high avidity with disulfide linkers also access the brain parenchyma to a greater extent than nanoparticles of tuned avidity. Therefore nanoparticles with targeting molecules that fall off may be better able to deliver drugs to the brain than nanoparticles of tuned avidity.