Reversible addition-fragmentation chain-transfer (RAFT) agents 2-[(butylsulfanyl)carbonothioyl]sulfanyl propanoic acid (C4-RAFT) and methoxy-polyethylene glycol modified 2-[(butylsulfanyl)carbonothioyl]sulfanyl propanoic acid were kindly provided by Dr Algi Serelis (DuluxGroup). Fe(II) chloride tetrahydrate (99%), Fe(III) chloride hexahydrate (98%), Fe(III) nitrate nonahydrate (99%), acrylamide (> 98%), rhodamine B isothiocyanate (> 70%), DOX (> 98%) and nitric acid (HNO3, 65%, Suprapur®, Millipore) were purchased from Sigma-Aldrich (St. Louis, USA). Ammonium hydroxide (28% NH3 in water) and sodium hydroxide (NaOH) (> 98%) were obtained from Ajax Finechem (Australia). 1,4-Dioxane (Fluka-Sigma-Aldrich, St Louis, USA), monoacryloxyethyl phosphate (MAEP, > 98%, PolySciences, Warrington, USA) and 4,4-azobis(4-cyanovaleric acid) (Wako, USA) were used as received.
Maghemite nanoparticles (γ-Fe2O3, average 25 nm core diameter, unless stated otherwise) were synthesized by the coprecipitation method (Massart et al. 1995) and sterically stabilized with a mixture of 90% poly(ethylene glycol) methyl ether (MPEG)-end and 10% NH2-end macro-RAFT copolymers, prepared using RAFT polymerization (Pham et al. 2018). The polymers each contain a block of 10 units of MAEP to anchor MPEG or polyacrylamide to the SPIONs’ surface, and either a block of 40 units of acrylamide and 17 units of poly(ethylene glycol) (PEG) (in MPEG-end-copolymer) or a block of 60 units of acrylamide (in NH2-end copolymer) for steric stabilization. We refer to these copolymer-coated nanoparticles as “10% NH2-SPIONs”. Transmission electron microscopy images were obtained using a JEOL 1400 microscope. Hydrodynamic diameter was measured by dynamic light scattering (DLS) using Malvern’s Zetasizer nano-ZS. The polymer content was 13% (w/w) of the dried solid as analyzed by thermogravimetric analysis (Additional file 1: Figure S1d). Iron concentration of the SPIONs was determined by flame atomic absorption spectroscopy (Varian AA800 spectrometer). For in vivo studies, 10% NH2-SPIONs were dispersed in PBS to the final concentration of 5 mg/mL as Fe and then filtered through a 0.22 µm sterile syringe filter.
Rho-SPIONs were made by labeling 10% NH2-SPIONs with rhodamine B isothiocyanate in PBS at room temperature in the dark for 4 h. Free rhodamine was removed using Amicon Ultra MWCO 3000 centrifugation filter units. Fluorescence intensity of Rho-SPIONs was measured with a RF-5301 PC spectrofluorophotometer (Shimadzu, Kyoto, Japan).
Amelanotic tyrosinase-knockout B16-F10 melanoma cells were kindly provided by Dr Shweta Tikoo from the Centenary Institute, Sydney, Australia, and maintained in Advanced Dulbecco’s modified Eagle's medium and 5% fetal bovine serum (Thermo Fisher Scientific, Waltham, USA). They were stably transfected with CEFLP-tdTomato-H2B eukaryotic expression plasmids using GenePORTER®3000 (Genlantis, San Diego, CA, USA). Transfected cells were selected with puromycin (10 µg/mL) and tdTomato-expressing cells were purified by flow cytometry on a BD Influx Cell Sorter.
A colony of hairless albino C57BL/6 mice was established from a breeding pair obtained from the Jackson Laboratory (strain 017840) and maintained by breeding homozygous males (Hr −/−) with heterozygous (Hr ±) females. Mice were housed with dust-free bedding (Able Scientific, Australia), regular chow, water, environmental enrichment and 12/12 h light/dark cycles.
Transdermal delivery of SPIONs and chemotherapeutic drugs
Mice were anesthetized by intraperitoneal injection of ketamine (75 mg/kg) and medetomidine (1 mg/kg), placed on a warming pad and the back skin of the mice was cleaned with a 70% isopropyl alcohol swab (Livingstone International, Australia). Rho-SPIONs (20 µl of 2 mg/mL in PBS) were applied to the skin and left for 1 h, after which excess liquid was wiped off with a wet cotton bud. PBS only (20 µL) was used for the control mouse. For chemotherapeutic drug delivery, a 30 µL mixture in PBS containing either both 10% NH2-SPIONs (1.3 mg/mL as Fe) and DOX (0.17 mM), 10% NH2-SPIONs only (1.3 mg/mL as Fe), or DOX only (0.17 mM), was applied to the mouse skin and then left for 1–2 h, at which time unabsorbed material was removed by wiping. Anesthesia was reversed with atipamezole (0.2 mg/kg).
The mice were imaged under anesthesia on a Carestream In Vivo FX PRO (Carestream Health, Woodbridge, USA) using 550 nm excitation and 600 nm emission wavelength filters before application and 1, 2, 24 and 48 h after application. They were then euthanized, and treated skins were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C for fluorescent imaging.
In vivo melanoma model
Eight-week-old hairless albino C57BL/6 mice were injected subcutaneously with 1 × 104 B16F10-tdTomato amelanotic melanoma cells. Three days post-injection, the mice were randomized into different treatment groups. Each group was topically treated at the tumor site 3 times/week with 60 µL of either vehicle (0.9% saline), 5-FU (1 mM) alone, 10% NH2-SPIONs (1 mg/mL) alone, or a mixture of 5-FU (1 mM) and 10% NH2-SPIONs (1 mg/mL). Tumor growth was measured using calipers and micrographs taken with a Dino-Lite AM4515ZT Edge Handheld Microscope. Tumor volume was calculated by the modified ellipsoidal formula (Tomayko and Reynolds 1989). Mice were euthanized when tumors reached 1 cm3, or skin ulceration appeared to be imminent.
Histology and imaging
PFA-fixed skin samples from Rho-SPION-treated mice were embedded in Tissue-Tek® OCT (Sakura, Torrance, CA), frozen and cut into 7-µm-thick sections. Sections were counterstained with 1 µg/mL for 5 min 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St Louis, MO, USA), washed 3 times with PBS, mounted in Prolong Gold® anti-fade mounting media (Life Technologies, Carlsbad, CA) and cover-slipped. Skin sections were stained with F4/80 primary antibody (1:200, SC-52664, BM8 clone, Santa Cruz Biotechnology, Dallas, USA) and goat anti-rat IgG (H + L) Alexa Fluor 488-for secondary Antibody (1:200, A-11006, ThermoFisher Scientific, Australia) to detect macrophage distribution in the skin. Images were acquired using an Olympus FV1000 confocal microscope (Olympus, Japan) and Leica SP5 and SP8 confocal microscopes (Leica Biosystem, Wetzlar, Germany).
B16-F10 melanoma tumors were fixed in 4% PFA overnight, embedded in paraffin, and cut into 4-μm-thick sections for hematoxylin and eosin (H&E) staining. The sections were deparaffinized, rehydrated and then incubated in an antigen retrieval solution (RD913M, pH 6.0, Pacheco, USA) for 20 min over a boiling water bath to retrieve antigen. Endogenous peroxidase activity was blocked using 0.3% (w/v) hydrogen peroxide. Sections were incubated for 1 h at room temperature with antibodies: 1:25 anti-F4/80 monoclonal rat antibody (SC-52664, Santa Cruz Biotechnology, Dallas, USA) or the rat IgG2a isotype control (559073, R35-95 clone, BD Biosciences, North Ryde, Australia) and 1:75 anti-CD31 monoclonal rabbit antibody (77699T, D8V9E clone, Cell Signaling Technology, Danvers, USA) or the rabbit IgG isotype control (ab172730, EPR25A clone, Abcam, Melbourne, Australia). Detection was with Rat-on-Mouse HRP-Polymer, Biocare Medical (Pacheco, USA) or anti-rabbit EnVision + system HRP-polymer, and diaminobenzidine as substrate (Dako-Agilent, Santa Clara, USA). Slides were counterstained with Mayer’s haematoxylin.
Cell morphology of H&E stained sections were characterized by the pathologist (LFK) to assess tumor borders and areas of tumor tissue necrosis. The pathologist was blinded to the treatments the specimens had received. Three mice per group were used for analysis and 4–6 regions were selected randomly in each tumor. Necrosis and whole tumor areas were measured using ImageJ software, and the percentage of the necrotic region was calculated by the total tumor area. Tumor-infiltrating lymphocytes were identified by a small cytoplasm:nucleus ratio, an oval, dark stained nucleus and a cell diameter of 7–10 µm and were counted manually using ImageJ.
The vascular area was quantified from CD31-stained images in which the diaminobenzidine signal had been segmented by spectral deconvolution (Ruifrok and Johnston 2001) and thresholded using Fiji image analysis software. The image representing CD31 staining was then binarized and holes (vascular lumens) were filled. The CD31-positive stained area and total tumor area were measured in each tumor section, from which the percentage of CD31-stained area was calculated.
Statistical analyses were done using GraphPad Prism v. 8.4.