br Fig A Microscopic photographs showing
Fig. 7. A) Microscopic photographs showing H&E and TUNEL staining of tumor slices from mice of each treatment group: (a) PBS (non-treated); (b) Dox (intravenous injection); (c) AuNRs BSA-Gel without 660-nm light and with 808-nm laser plus Dox; (d) Chlorella AuNRs BSA-Gel without 660-nm light and with 808-nm laser plus Dox; (e) Chlorella AuNRs BSA-Gel with 660-nm light and without 808-nm laser plus Dox; or (f) Chlorella AuNRs BSA-Gel with both 660-nm light and 808-nm laser plus Dox. B) Immunofluorescence imaging of tumor slices from mice treated with (a) PBS, (b) Chlorella AuNRs BSA-Gel with 808-nm light, (c) Chlorella AuNRs BSA-Gel with 660-nm light, or (d) Chlorella AuNRs BSA-Gel with both 660- and 808-nm light. Cell nuclei, blood vessels, and hypoxic regions were stained with DAPI (blue), anti-CD31 antibody (red), and antipimonidazole (green), respectively. Dox was not included for this set of experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
hypoxic tumors compared with the simple Chlorella BSA-Gel system. The tumors obtained from each treatment group were evaluated
histologically by two staining assays: hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) (Fig. 7A). The H&E-stained tumor tissue specimens from mice treated with Chlorella AuNRs BSA-Gel showed noticeably lower tumor cell density than specimens from other mouse groups. The TUNEL histologies revealed that cell death of 4 T1 tumor groups sup-plied with oxygen by chlorella photosynthesis (Fig. 7Ae, f) was pri-marily due to substantial apoptosis. Notably, tumors treated with Dox plus Chlorella AuNRs BSA-Gel illuminated by 660 nm and 808 nm dis-played the highest TUNEL fluorescence signals, and this appeared to be associated with the enhanced ROS-mediated apoptosis induced by Dox by virtue of the elevated oxygen level.
Separately, the enhanced tumor oxygenation caused by chlorella photosynthesis and m-HT was confirmed using the Hydroxyprobe™-1 (pimonidazole hydrochloride) immunofluorescence staining assay for tissue hypoxia. Pimonidazole is a 2-nitroimidazole that is activated specifically in hypoxic Fulvestrant and forms stable adducts [55,56]. As shown in Fig. 7B, only 4 T1 tumors treated with Chlorella AuNRs BSA-Gels with 660-nm illumination alone or both 660-nm and 808-nm illumi-nation were able to overcome the hypoxic state, whereas other tumors showed clear green fluorescence indicating hypoxia. Especially, 4 T1 tumors treated with Chlorella AuNRs BSA-Gel with both 660-nm and 808-nm illumination showed a noticeably diminished hypoxia state. Again, these overall data demonstrated that our Chlorella AuNRs BSA-Gel played a key role as an O2-generating depot capable of delivering oxygen to hypoxic tumor tissues.
3.5. Toxicological assessment of Chlorella AuNRs BSA-Gel
Despite the remarkable antitumor efficacy induced by Chlorella AuNRs BSA-Gel, there was a concern about clinical toxicity derived from the gel matrix including chlorella and AuNRs. We therefore in-vestigated the biodegradability of Chlorella AuNRs BSA-Gel formed in the flanks of mice. The apparent volume of Chlorella AuNRs BSA-Gel on day 7 seemed slightly larger than that on day 1 due to the hydrogel swelling process in subcutaneous tissue (Fig. 8A). However, the weight of Chlorella AuNRs BSA-Gel subsequently decreased gradually and reached almost zero over 21 days (data not shown). This showed that Chlorella AuNRs BSA-Gel was apparently biodegradable and thus probably biocompatible as an in situ injectable hydrogel. Also, the main building materials for this hydrogel are albumin and PEG, both of which are among the most biocompatible/biodegradable materials developed to date and have been approved by the Food and Drug Ad-ministration (FDA). The biodegradability of our Chlorella AuNRs BSA-Gel appeared to be due to the biomedical merits of albumin and PEG. Although there are no reports on the subcutaneous clinical toxicity or safety of chlorella, to our best knowledge, chlorella is considered to be potentially non-toxic via the oral route because it has shown no sig-nificant mutagenicity or acute and subacute toxicity in rats and has been used for a long time as a nutrient . Gold nanorods might in-duce potential systemic toxicity to animals and humans, and there is controversy over whether this toxicity derives from the gold core or cetyltrimethylammnonium bromide (CTAB). However, the toxicity of gold nanorods is reported to mainly originate from the surfactant, CTAB, and not the gold core. Many papers have shown that in-travenously administered AuNRs do not display significant systemic toxicity in various tissue organs when assessed histologically [35,58,59]. Nevertheless, we made an effort to use small-sized AuNRs (traverse size: < 5 nm) in our hydrogel system to help renal excretion and reduce potential toxicity because particles smaller than ~6 nm are more likely to be removed by kidney filtration due to the pore size