ABSTRACT: Upregulation of NADPH oxidases (NOXs) in
cancer cells leads to chronic increase in intracellular reactive
oxygen species (ROS) and adaptation to a high ROS level for
cell survival and, thereby, low sensitivity to radiotherapy. To
overcome resistance to radiotherapy, we have developed a
bioactive and CD44 targeted hyaluronic acid nanoparticle
encapsulated with an NOX inhibitor, GKT831 (HANP/
GKT831). We found that HANP/GKT831 had stronger
inhibitory effects on ROS generation and cell proliferation
than that of GKT831 alone in cancer cells. Systemic delivery of
HANP/GKT831 led to the targeted accumulation in breast
cancer patient derived xenograft (PDX) tumors in nude mice.
Importantly, the combination of systemic delivery of HANP/
GKT831 with a low dose of local radiotherapy significantly enhanced tumor growth inhibition in breast cancer PDX models.
Our results showed that HANP/GKT831 primed tumor cells to radiation-induced DNA damage and cell death by
downregulation of DNA repair function and oncogenic signal pathways.
METHODS/EXPERIMENTAL
Preparation of Hyaluronic Acid Nanoparticle Carrying
GKT831 (HANP/GKT831). HANP/GKT831 nanocomplexes were
produced according to our established protocol with minor
modifications42 (Figure 2A). Sodium hyaluronic acid (HA, 234
kDa) was purchased from Lifecore Biomedical Company. HA was
first converted to the tetrabutylammonium (TBA) salt of HA
according to previously reported methods. The HA-TBA was then
chemically modified with 5β-cholanic acid (CA) in the presence of 1-
ethyl-3(3-(dimethylamino) propyl)carbodiimide (EDC) and N
hydroxysuccinimide (NHS) at 60 °C in dimethyl sulfoxide
(DMSO) (Sigma-Aldrich) to produce HA-CA. After dialysis against
methanol/ultrapure water (1:1, v/v) and then ultrapure water for 4?
12 h, HA-CA was lyophilized and stored at 4 °C in the dark until
further use. Next, 80 mg of HA-CA conjugates were dissolved in 14
mL of distilled water and subjected to a high-pressure homogenizer
(D-3L, PhD Technology) for 5 min to form HA nanoparticles
P(HANPs). Hydrophobic GKT 831 was loaded to HANP via a high
pressure homogenizer at a ratio of 10 mg GKT831/40 mg of HANP.
Thirty milligrams of GKT831 (MedKoo Biosciences #522357)
dissolved in 1 mL of DMSO was added dropwise into the solution
containing 120 mg of HANP and then circulated for an additional 5
min in the homogenizer. HANP/GKT831 nanocomplex was then
dialyzed for 8 h in distilled water to remove free drugs and organic
solvents. Finally, HANP/GKT831 was lyophilized into white powder
and kept at 4 °C.
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