*Purpose: Hepatic ischemia/reperfusion injury (IRI) is a major inflammatory event in liver transplantation that is mediated by oxidative stress. Doxorubicin (DOX) can mitigate oxidative stress by inducing the expression of heme oxygenase-1; however its clinical use in IRI has been hampered by significant systemic toxicity. Nanodiamonds (ND) are carbon nanoparticles with potential to be high-affinity carriers for the selective delivery of anthracyclines, such as DOX, and thus mitigate the negative effects of systemic delivery. This study aims to characterize ND-adsorbed DOX (NDX) uptake and evaluate its efficacy in a mouse model of hepatic IRI.

*Methods: ND solution was mixed with aqueous DOX and filtered to produce NDX complexes. Particle diameters were determined using dynamic light scattering (DLS). UV spectrophotometry was usedto quantify DOX loading efficacy onto NDs. Uptake of DOX, ND-FITC, and NDX were tested on hepatic cells and visualized using fluorescence microscopy. DOX (1 mg/kg), ND, NDX (0.5 mg/kg equivalent of DOX) and PBS were administeredi.v. to Balb/c mice 48 hours prior to our surgical model. Warm hepatic ischemia was produced in the left and median lobes for 90 minutes followed by 24 hours of reperfusion.

*Results: NDX 5:1 ratio (w:w ND:DOX) exhibited an initial DOX loading efficacy of 67%; 30% NDX remained after filtration. DOX did not elute from NDs in PBS. Mean ND and NDX particle sizes were 51.45 ± 24.98nm and 249.07 ± 61.6nm, respectively. While DOX alone significantly reduced the viability of THLE-3 hepatocytes, cells treated with ND or NDX retained the same viability as untreated cells. Fluorescent microscopy confirmed ND/NDX uptake and localization to the cytoplasm of THLE-3 cells. In contrast, DOX uptake was localized to the nucleus. Next, we evaluated the efficacy of NDX in hepatic IRI. Plasma ALT levels were significantly lower in DOX and NDX treated mice after 24h of IRI, when compared to ND and PBS treated mice (P=0.002,P=0.006 and P=0.002, P<0.001). ALT levels of the DOX and NDX treated mice were comparable (P=0.981). Finally, NDX and DOX markedly reduced hepatocellular vacuolization, necrosis and hepatic inflammatory leukocyte infiltration present in livers of ND and PBS treated mice.

*Conclusions: We establish that NDs bind DOX efficiently to form NDX complexes that are readily taken up by hepatic cells with minimal toxicity. Moreover, NDX complexes enhance the physiological efficacy of DOX and protect mice from hepatic IRI with half the DOX dose. These results support the development of a more clinically effective strategy for DOX in liver transplantation.

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