Single-wall carbon nanohorns with oxidative holes (SWNHox) were shown in 2008 to suppress the growth of transplanted human lung cancer tumors in mice after intratumoral injection even without any drug cargo, and to remain in the tumor tissue for at least 25 days, while the reason for this drug-free effect, the determinants of host adverse responses, and the long-term fate of the retained material were left unresolved. Later work has identified candidate cellular pathways (lysosomal accumulation with reactive oxygen species, mitochondrial dysfunction via the mitochondrial regulatory enzyme sirtuin 3, endoplasmic reticulum stress) for the intrinsic cytotoxicity of carbon nanohorns in vitro, and has mapped route-dependent biodistribution and excretion after intravenous and oral dosing, but no study has established which processes actually dominate in vivo at the intratumoral injection site, whether the effect is tumor-selective in vivo, or what happens to intratumorally retained nanohorns and their drug payloads beyond 25 days. The open problem is to determine the in vivo mechanism of the drug-free antitumor action of SWNHox and the long-term fate and systemic safety of intratumorally retained SWNHox and its drug-loaded formulations.

Background

Single-wall carbon nanohorns (SWNHs) are horn-shaped tubules made of a single rolled graphene sheet, roughly 2-5 nm in diameter and tens of nanometers long, produced by CO2 laser ablation of graphite without any metal catalyst; as synthesized they aggregate into spherical 'dahlia-like' clusters about 100 nm across. Heating the aggregates in oxygen opens small holes ('nanowindows') in the graphene walls and tips, giving oxidized nanohorns (SWNHox) whose internal nanoscale cavities can host small molecules, which has made SWNHox an attractive candidate carrier for anticancer drugs. Cisplatin (CDDP, cis-diamminedichloroplatinum(II), Pt(NH3)2Cl2Pt(NH_{3})_{2}Cl_{2}) is a platinum chemotherapy drug that kills cells by crosslinking DNA but suffers from dose-limiting systemic toxicity, so a carrier that keeps the drug inside tumor tissue while releasing it slowly is clinically attractive.

Building on earlier work that optimized hole-opening conditions for cisplatin incorporation in SWNHs (Ajima et al., J. Phys. Chem. B 2006), Ajima et al. in 2008 (ACS Nano, DOI 10.1021/nn800395t) incorporated CDDP into SWNHox by nanoprecipitation: CDDP and SWNHox were dispersed together in a solvent that was then evaporated. Switching the solvent from dimethylformamide (DMF) to water raised the incorporated CDDP fraction from about 15% to about 46%, restored the Pt:Cl≈1:2Pt:Cl\approx 1:2 stoichiometry of intact CDDP, and increased the total released fraction from roughly 60% to essentially 100% over about 150 hours in culture medium. The resulting CDDP@SWNHox was 4-6 times more potent than intact CDDP against human lung cancer NCI-H460 cells in vitro, and intratumoral injection of CDDP@SWNHox (0.5 mg/kg CDDP with 0.5 mg/kg SWNHox) suppressed transplanted NCI-H460 tumor growth in nude mice (a hairless, immunodeficient strain that accepts transplanted human tumors) more effectively than intact CDDP, consistent with the observation that CDDP@SWNHox aggregates adhered to cancer-cell surfaces in vitro and remained within tumor tissue in vivo.

The 2008 study also produced an unexplained finding: SWNHox alone, injected intratumorally at 0.5 mg/kg without any drug, appreciably suppressed tumor growth relative to saline, and histology showed vacuolar-like degeneration of tumor tissue in regions where SWNHox agglomerates were present. Prior data had shown little toxicity of SWNHs in normal animals and in vitro, so this intrinsic antitumor action of the bare carrier was unexpected; the authors could not infer why the SWNHox had this effect and called for further investigation. Related loose ends left by the same study were that one mouse treated with CDDP@SWNHox showed an anomalously large body-weight decrease whose cause was not determined, and that the observation window ended at day 25, leaving the long-term biodistribution, clearance, and safety of the intratumorally retained nanohorns (with or without cargo) unaddressed. Subsequent literature has since established that intravenously injected carbon nanohorns accumulate mainly in liver and spleen (with size- and functionalization-dependent kinetics and no obvious short-term toxicological lesions), that orally administered oxidized nanohorns are excreted mainly through the gut, and that macrophages can partially biodegrade nanohorns enzymatically, but these results concern administration routes other than intratumoral retention in tumor tissue.

Problem Statement

Determine how and why oxidized single-wall carbon nanohorns (SWNHox), injected intratumorally without any drug cargo, suppress tumor growth in vivo, and what happens to that material — and to drug-loaded formulations of it — in the host over the long term. Concretely: (1) identify the cellular and microenvironmental processes that dominate the drug-free antitumor effect at the intratumoral injection site in vivo, distinguishing among direct induction of tumor-cell death (for example by the reactive-oxygen-species, mitochondrial, or endoplasmic-reticulum-stress pathways observed for carbon nanohorns in vitro), local tissue-level physical or microenvironmental disruption, and immune or inflammatory modulation, and establish whether the effect is selective for tumor tissue rather than a generic response of any injected tissue; (2) establish the long-term in vivo fate of the nanohorns retained in tumor tissue beyond the approximately 25-day observation window of the original study — their persistence, redistribution to other organs, degradation, and clearance — together with the determinants and variability of host adverse responses (including the severe body-weight loss observed in one treated mouse whose cause was never determined), for both bare SWNHox and cisplatin-loaded CDDP@SWNHox. Both parts serve one objective: establishing whether the retained carbon nanocarrier is itself a biologically active and safe agent in vivo, which is required both to interpret carrier-based efficacy results and to assess the translational safety of SWNHox drug-delivery formulations.

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