(575g) Sugar-Guided Organ and Cellular Targeting of PAMAM Dendrimers

Hydroxyl-terminated polyamidoamide (PAMAM) dendrimers
have been proven to be effective, versatile nanocarriers for delivering
therapeutics and imaging agents across the blood-brain barrier, where they are
selectively taken up by injured microglia and macrophages. This passive
targeting has been explored in great detail for the delivery of an
anti-inflammatory compound, N-acetyl cysteine, to activated glia in many small
and large animal models of central nervous system (CNS) disorders [1-3]. The
generation 4 dendrimer scaffold (D4-OH), which is a 4nm diameter spheroid with
64 hydroxyl end groups, is known to be non-toxic and non-immunogenic [4]. The
N-acetyl cysteine conjugate (D-NAC) has been shown to maintain that
biocompatibility and is currently undergoing clinical translation for a
pediatric neurological disorder.

Despite the advances that have been made with D4-OH,
there remains immense untapped potential in applications from diagnostics to
treatment due to the dendrimer’s propensity for residence exclusively in
injured glial cells and activated macrophages. Building on the unique
biophysics of hydroxyl PAMAM dendrimers, we seek to further tailor the
dendrimer’s pharmacokinetics and transport properties by utilizing various
sugars as targeting ligands to create a new catalog of dendrimer conjugates
with the ability to precisely target different cells and organs throughout the body.
Here we will discuss the synthesis of new sugar-dendrimer conjugates and their
performance in vitro and in vivo compared to D4-OH. The
conjugates of interest in this study are: dendrimer-galactose (D-Gal),
dendrimer-glucose (D-Glu), and dendrimer-mannose (D-Man) (Fig 1).

We have observed that conjugation of any of the three
sugars to the dendrimer surface results in stark deviations in dendrimer
pharmacokinetics as analyzed through tissue homogenization and fluorescence
spectroscopy. D4-OH has a diameter of about 4nm, which lends itself to rapid
renal clearance; however, in the cases of both D-Gal and D-Man the attachment
of just 12-14 sugar molecules to the dendrimer surface (<15 wt%, diameter~ 5
nm) allows the dendrimer to localize in the liver, which has not typically been
observed to metabolize the nanoparticle. In a neonatal rabbit model of cerebral
palsy, over 9% of the injected D-Man dose was present in the liver at 24 hours
post-injection compared with D4-OH, of which only less than 1% of the injected
dose had localized to the liver (Fig 2). Preliminary results indicate
that compared to D4-OH in healthy mice, D-Gal levels are ~3-fold higher in the
lungs and brain, ~2-fold higher in the kidneys, and over 10-fold higher in the
liver at 24 hours post-injection.

These modifications to overall dendrimer
biodistribution indicate that the conjugation of sugar to the dendrimer surface
alters dendrimer-cell interactions and dendrimer transport, which would also
effect the cellular localization of the dendrimer. We explored this phenomenon through
in vitro competitive uptake and binding assays as well as confocal
imaging and flow cytometry of ex vivo tissues. The conjugation of
mannose to the dendrimer surface gave the dendrimer a partiality for cellular
uptake through mannose receptor-mediated endocytosis, whereas D4-OH has been
observed to utilize a variety of fluid-phase endocytic pathways over mannose
receptor-mediated endocytosis [5]. This was confirmed in confocal imaging which
found D-Man localized in mannose receptor-expressing cells, such as the
meninges of the cortex and sinusoidal endothelial cells of the liver.
Hepatocytes comprise almost 80% of the liver by mass, yet have remained an
elusive target for nanoparticles; however, we have observed that D-Gal is
highly effective at penetrating and remaining in hepatocytes, with strong
dendrimer signal seen up to one week post injection in hepatocytes of healthy
animals (Fig 3) most likely due to the high expression of galactose
receptors, such as asialoglycoprotein receptor 1. D-Gal, D-Glu, and D-Man are
also under investigation in a mouse model of glioblastoma to see if sugar
conjugation allows the dendrimer to take advantage of the Warburg effect and
localize in tumor cells as well as tumor-associated macrophages.

Additional changes to cellular localization in other
organs and disease models are still being explored, but the results as such
indicate that the conjugation of sugar to the dendrimer surface modifies its
intrinsic transport properties, allowing the dendrimer to bind to different
surface receptors, which has dramatic effects on the pharmacokinetics of the
dendrimer. This new catalog of dendrimers gives new hope for treatment of
hepatic diseases, CNS disorders, and diseases of the back of the eye,
increasing the already expansive utility of this dendrimer nanoparticle.

This work was funded by grants from the National
Institute of Biomedical Imaging and Bioengineering (5R01EB018306-02) and the
National Institute of Child Health and Human Development (5R01HD076901-02).

[1] Kannan, et al. Dendrimer-based postnatal therapy
for neuroinflammation and cerebral palsy in a rabbit model. Sci. Trans. Med.
2012. 4(130):130-46.

[2] Grimm, et al. Nanotechnology Approaches to Targeting
Inflammation and Excitotoxicity in a Canine Model of Hypothermic Circulatory
Arrest-Induced Brain Injury. Ann Thorac. Surgery. 2016. 102(3):743-750.

[3] Nance, et al. Dendrimer-mediated delivery of
N-acetyl cysteine to microglia in a mouse model of Rett syndrome. J
Nueroinflammation. 2017. 14(1):252.

[4] Dobrovolskaia, MA. Dendrimers Effects on the
Immune System: Insights into Toxicity and Therapeutic Utility. Curr Pharm
Des. 2017. 23(21):3134-3141.

[5] Alnasser, et al. Preferential and increased uptake
of hydroxyl terminated PAMAM dendrimers by activated microglia in rabbit brain
mixed glial cultures. Molecules. 2018. In press.