Ultrasmall Glutathione-Protected Gold Nanoclusters as Next Generation Radiotherapy Sensitizers with High Tumor Uptake and High Renal Clearance

Radiotherapy is often the most straightforward first line cancer treatment for solid tumors. While it is highly effective against tumors, there is also collateral damage to healthy proximal tissues especially with high doses. The use of radiosensitizers is an effective way to boost the killing efficacy of radiotherapy against the tumor while drastically limiting the received dose and reducing the possible damage to normal tissues. Here, we report the design and application of a good radiosensitizer by using ultrasmall Au29–43(SG)27–37 nanoclusters (<2 nm) with a naturally-occurring peptide (e.g., glutathione or GSH) as the protecting shell. The GSH-coated Au29–43(SG)27–37 nanoclusters can escape the RES absorption, leading to a good tumor uptake (~8.1% ID/g at 24 h post injection). As a result, the as-designed Au nanoclusters led to a strong enhancement for radiotherapy, as well as a negligible damage to normal tissues. After the treatment, the ultrasmall Au29–43(SG)27–37 nanoclusters can be efficiently cleared by the kidney, thereby avoiding potential long-term side-effects caused by the accumulation of gold atoms in the body. Our data suggest that the ultrasmall peptide-protected Au nanoclusters are a promising radiosensitizer for cancer radiotherapy.

Cancer remains one of the world's most devastating diseases with more than 10 million new cases each year, and radiotherapy is a leading cancer treatment approach that addresses the needs of more than 50% cancer patients 1 . Though high-energy radiation can fatally damage tumor cells, it can also harm normal tissues. In fact, the mitotically active tumor cells are only slightly more susceptible to radiation damage than those in the essential normal tissues 2 . Hence, it is very important to strike the right balance between eradicating tumor and saving normal tissues by controlling the target and the dose of radiation administered to the patient. Many improvements have been made in radiotherapy to target tumors better, which could cause less damage to normal tissues. For example, megavolt (6-25 MV) X-rays are now used to avoid skin damage; tomotherapy and intensity-modulated radiation therapy (IMRT) are applied to better concentrate the radiation within the tumor volume; and optimal dose fractionation schedules are also developed to allow better cumulative damages to the tumor and adequate repairing of normal tissues [2][3][4][5] . Despite such advances, it is still challenging to use radiotherapy alone to eradicate tumor cells. A magic bullet to current challenges in radiotherapy is radiosensitizer, which can locally increase the efficacy of radiotherapy by enhancing the radiation damages to the cell.
In general, the radiosensitizing agents can be classified into two major categories according to their mechanisms of action: (type-1) chemotherapeutics that modulate the cell response to enhance the radiation damage, and (type-2) materials that interact directly with the radiation and generate additional damages to the cell 2,3,6,7 . The development of type-1 radiosensitizers started with Heidelberger's preclinical studies 8 in 1958, and this radiosensitizing approach is often referred to as combined chemotherapy and radiotherapy or chemoradiation 3,9 . Most organic radiosensitizers are type-1, which enhance radiotherapy by modulating cell responses, such as reducing the radioresistance of tumor cells, preventing the formation of blood vessels (or disrupting the existing vessels, anti-angiogenic), inducing apoptosis, and suppressing mitosis 3,6,8 . Although many preclinical and clinical studies have affirmed the efficacy of type-1 radiosensitizers, a major drawback of these chemotherapeutics is their inherent cytotoxicity and side effects. For example, gemcitabine is known to cause myelosuppression, anemia, vomiting, and diarrhea 10,11 . Similarly, cisplatin is known to have myelotoxicity, neurotoxicity, and nephrotoxicity, and it can also cause hemolytic anemia, hearing loss, and vomiting [12][13][14] .
Type-2 radiosensitizers are mostly metal-based materials that can strongly absorb, scatter, and reemit radiation energy, resulting in a local radiation dose increase when they are accumulated in tumors 15,16 . Intense research on nanoscale metallic materials in the past two decades has provided many novel materials for biomedical applications 17 . Among these emerging radiosensitizers, gold nanoparticles (Au NPs) are particularly attractive because of their strong interaction with the radiation (Au has a high atomic number of 79), excellent chemical stability and inertness, and good biocompatibility (low toxicity) [18][19][20][21] .
The enhancement of radiation dose received by the tumor tissue loaded with Au relative to the dose received by normal tissues without Au can be 200% or higher 22,23 . Such enhancement comes from the direct interaction between Au and radiation. When the incident radiation (gamma rays, X-rays) impinges on a Au NPs, the NPs becomes a new source of radiation and emits high energy through scattered photons (X-rays), photoelectrons, Compton electrons, Auger electrons, electron-positron pairs, and fluorescence photons, thereby causing radiochemical (free radicals and ionization) damages to the surrounding tumor tissue 22,24,25 . However, most of the Au NPs that have been demonstrated so far have large particle sizes (typically above 50 nm) and could be trapped by the reticulo-endothelial system (RES) absorption, which could result in low tumor uptake and unavoidable accumulation in liver and spleen [26][27][28][29][30][31] . Decreasing the particle size could benefit the escape of particles from the RES absorption. For example, one recent study showed that Au NPs with particle sizes below 20 nm could efficiently escape the RES absorption and showed good tumor uptake 32 . However, the sizes of these particles were still above the renal clearance barrier, that is ~5.5 nm, and could therefore induce the accumulation of NPs in RES, thus resulting in potential toxicity over the long term [33][34][35][36] . Besides the core size of NPs, the protecting ligands on the NPs surface can also affect the in vivo biodistribution. For example, the naked Au NPs of particle sizes of 1.9 and 4.8 nm, while small, have low colloidal stability due to the protein corona acquired in blood. These Au NPs eventually formed large aggregates of ~20-100 nm, which could not be rapidly metabolized and certainly unable to escape the RES 18,37 . Au NPs with different surface ligands can induce different NPs-protein corona in blood that could determine the RES absorption and cellular uptake efficiency 38 .
Taken together of the two key attributes (size and surface) for NPs-based radiosensitizers, we hypothesized that: 1) small naturally-occurring peptides, such as glutathione or GSH, could be a good surface ligand for Au NPs by helping them escape the RES absorption and improving their deposition in tumors; and 2) ultrasmall Au NPs with core sizes below 2 nm (hereafter referred to as nanoclusters, NCs) in combination with the GSH ligands can ensure a small hydrodynamic diameter (HD), which could provide good interface with the biological system, improve their in vivo pharmacokinetics, and enhance their deposition in tumors 39 . Here we demonstrate such concept by using sub-2-nm GSH-protected Au NCs with a well-defined molecular formula of Au 29-43 (SG) 27-37 40 . We show in this study that the Au 29-43 (SG) [27][28][29][30][31][32][33][34][35][36][37] NCs have attractive features of high tumor uptake, strong sensitizing enhancement for radiation, and low toxicity, and they could be a good candidate for next generation radiosensitizers for clinical use. This study has therefore enriched the family of Au NPs and NCs that could show good performance for cancer radiotherapy 33,37 .
In vivo Toxicity. The treated mice were weighed and assessed for behavioral changes. All mice were sacrificed at 28 days p.i., and their blood and organs were collected for hematology, biochemistry and toxicological investigation. The blood was drawn for hematology analysis (potassium EDTA collection tube) and serum biochemistry analysis (lithium heparin collection tube) using a standard saphenous vein blood collection technique. During necropsy, liver, kidney, spleen, heart, lung, brain, genitals, tumor, and thyroid were collected and weighed. Major organs from these mice were then fixed in 4% neutral buffered formalin, processed into paraffin, and stained with hematoxylin and eosin (H&E). Pathology was examined using a digital light microscope.       . Pathological data from the heart, liver, spleen, lung, and kidney of mice treated with Au 29-43 (SG) [27][28][29][30][31][32][33][34][35][36][37] NCs at the concentration of 5.9 mg-Au/kg-body. Scale bars, 100 µm.