(638d) Raltitrexed/Hyaluronic Acid Coated Nanoparticles for Chemoradiation of Colorectal Cancer

Purpose: Treatments for
various forms of cancer often involve chemotherapy and/or radiation. The
response of tumors to these treatments is not fully understood. More data, with
an improved understanding of cancer growth response, both in vitro and in
vivo, can lead to better treatment outcomes, and ultimately greater
clinical translation of the treatment regimen. In this work, we employ a
layer-by-layer strategy to load raltitrexed and hyaluronic acid, a
CD44-targeting ligand¹,
onto the surface of a polystyrene substrate. Raltitrexed, a potent thymidylate
synthase inhibitor²,
is a DNA-damaging agent, that has been used in combination with radiation
therapy clinically³.
We investigate the individual effects of ionizing radiation (IR) and the raltitrexed-loaded
nanoparticles, as well as their combined effects against the CT26 murine colorectal
cancer cell line.

Methods: Carboxylate-modified
polystyrene latex fluorescent nanoparticles were mixed with poly-L-arginine
(PLA) to deposit the positively-charged electrolyte on the surface, followed by
deposition of raltitrexed and hyaluronic acid. Between each deposition, the nanoparticles
were purified via tangential flow filtration (TFF), a means of quickly and
efficiently removing the excess polyelectrolytes from the mixture. Nanoparticle
uptake, cell viability, and DNA-damage quantifying assays were performed in
vitro to determine the effects of the nanotherapeutic against the CT26
cells, as well as its targeting ability. Tumor growth delay and survival
benefits were assessed in vivo with BALB/c mice inoculated with flank
tumors of the CT26 cells. (Experiments follow protocols approved by OHSU
IACUC.)

Results: The
nanotherapeutic (THANP), once constructed and purified, has a diameter of 115.2
± 5.0 nm, with a zeta potential of -22.8 ± 0.3 mV (Figure 1A/B). The
concentration of the drug present in the nanoparticle solution (via HPLC
quantification) was 22.2 µg/mL, with an encapsulation efficiency of 11.1 %. The
targeted nanotherapeutic showed greatly enhanced uptake (roughly 5-fold
increase in fluorescence for targeted treatment) compared to the untargeted substrate
when used with the CT26 cells, by both fluorescence microscopy and flow
cytometry. THANPs showed high toxicity against the same cells (IC₅₀
= 0.64 µg/mL THANPs), with little to no toxicity observed in cells treated with
the substrate. The nanoparticles imparted DNA damage (measured by fluorescence
from γH2AX assay), and the DNA damage increased when IR was also applied
with the therapeutic. The ability of the cells to form colonies post-IR
treatment (with and without THANPs) was assessed via the clonogenic assay.
Colony formation was inhibited further with the combination treatment relative
to the IR treatment alone (Figure 1C). Finally, mice inoculated with
CT26 flank tumors showed inhibited tumor growth and longer median survival for
all treatment regimens (3 x 2 Gy IR for 3 days and/or 6 x 100 µL IV injections
of THANPs for 6 days). IR and IR+THANPs showed the greatest tumor growth delay
(P < 0.01, n=7) compared to saline controls alone.

Figure 1: THANPs produced by layer-by-layer process inhibit
colony formation in vitro. (A) Mean diameter of the nanoparticles at
various stages of the layer-by-layer process. (B) Transmission electron
micrograph of THANP, with carbon dense polystyrene core shown as the darker
inner core of the structures, with a lighter, less carbon dense polymer layer
on the periphery. Scale bars: Large – 200 nm, Small – 20 nm. (C) Survival
fraction curve results from clonogenic assay show higher percent inhibition of
colony formation with THANP treatment + IR over IR alone.

Conclusions: To
better understand the outcomes of nanotherapeutics used with IR, we formulated
a targeted nanoparticle to use with radiation treatment. The formulation,
THANPs, proved efficacious in vitro, and showed tumor growth inhibition in
vivo. In addition, the THANPs alone showed median survival benefit in
vivo, and further benefit when combined with IR, when used for treatment of
the aggressive CT26 cell line. Future work is aimed at improving facets of the
formulation (drug concentration, monodispersity, etc.), as well as
investigating growth inhibition when used with lower total accumulated IR
doses.

References:

(1)          Seok, H. Y.; Sanoj Rejinold, N.; Lekshmi,
K. M.; Cherukula, K.; Park, I. K.; Kim, Y. C. CD44 Targeting Biocompatible and
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(2)          Zalcberg, J.
R.; Cunningham, D.; Van Cutsem, E.; Francois, E.; Schornagel, J.; Adenis, A.;
Green, M.; Iveson, A.; Azab, M.; Seymour, I. ZD1694: A Novel Thymidylate
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(3)          Botwood, N.;
James, R.; Vernon, C.; Price, P. Raltitrexed ('Tomudex’) and Radiotherapy Can
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