Laparoscopic fluorescence image-guided photothermal therapy enhances cancer diagnosis and treatment

Endoscopy is the gold standard investigation in the diagnosis of gastrointestinal cancers and the management of early and pre-malignant lesions either by resection or ablation. Recently gold nanoparticles have shown promise in cancer diagnosis and therapeutics (theranostics). The combination of multifunctional gold nanoparticles with near infrared fluorescence endoscopy for accurate mapping of early or pre-malignant lesions can potentially enhance diagnostic efficiency while precisely directing endoscopic near infrared photothermal therapy for established cancers. The integration of endoscopy with near infrared fluorescence imaging and photothermal therapy was aided by the accumulation of our multifunctionalized PEG-GNR-Cy5.5-anti-EGFR-antibody gold nanorods within gastrointestinal tumor xenografts in BALB/c mice. Control mice (with tumors) received either gold nanorods or photothermal therapy, while study mice received both treatment modalities. Local (tumor-centric) and systemic effects were examined for 30 days. Clear endoscopic near infrared fluorescence signals were observed emanating specifically from tumor sites and these corresponded precisely to the tumor margins. Endoscopic fluorescence-guided near infrared photothermal therapy successfully induced tumor ablations in all 20 mice studied, with complete histological clearance and minimal collateral damage. Multi-source analysis from histology, electron microscopy, mass spectrometry, blood, clinical evaluation, psychosocial and weight monitoring demonstrated the inherent safety of this technology. The combination of this innovative nanotechnology with gold standard clinical practice will be of value in enhancing the early optical detection of gastrointestinal cancers and a useful adjunct for its therapy.

After an hour, the solution was transferred into an Eppendorf tube and centrifuged at 8500 rpm for 15 minutes at 30C. Then the supernatant was decanted and the pellet was resuspended in 10 ml of phosphate buffered saline (PBS) (pH = 7.4). The GNR solution was stored at room temperature.

PEGylation of GNRs
Briefly, 10 ml of synthesized GNR solution was centrifuged twice at 8500 rpm for 15 minutes and then resuspended in 4 ml of water in order to reduce the CTAB concentration. Subsequently, 2 ml of K2CO3 buffer (2 mM) and 2 ml of HS-PEG-COOH solution (maintaining the stoichiometric ratio between GNRs and HS-PEG-COOH at 500,000:1) were added. The solution was sonicated for two hours to prevent aggregation and then left under continuous agitation overnight at room temperature in the dark. Next, the solution was centrifuged at 8500 rpm for 15 minutes at 30C. The supernatant was decanted and the pellet precipitate was redispersed in 10 ml of PBS buffer (pH = 7.4) in an Eppendorf tube covered with aluminum foil. Spectral and ζ-potential measurements were performed before and after ligand conjugation to confirm functionalization of GNRs with thiol carboxyl PEG (COOH-PEG-GNRs).

Functionalization of GNRs
In a classical experiment, 4 ml of GNR-PEG-COOH solution was placed into a glass vial. Then 0.5 ml 0.4 M 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 0.5 ml 0.1 M N-hydroxysulfosuccinimide (NHS) agents were added respectively. The mixture was put on a rotary shaker for 20 minutes and then centrifuged at 8500 rpm for 15 minutes. The supernatant was decanted and distilled DNAse/RNAse-free water was added to the precipitate to make up to a total volume of 4 ml before adding modified Cy5.5 anti-human EGFR antibody (GNR:Anti-EGFR = 1:1000; PerCP/Cy5.5 anti-human EGFR antibody, BioLegend, London, UK). The solution was left on a rotary shaker for four hours and then centrifuged. The supernatant was decanted and the pellet was redispersed in 2 ml of distilled DNAse/RNAse-free water in an Eppendorf tube covered with aluminum foil. Absorption spectra, ζ-potential measurements and fluorescence emission spectra were checked to verify GNR-PEG-Anti-EGFR-Cy5.5 conjugation (see Supplementary Fig. S5). The final solution was stored in the fridge at 4C.

Heat profile assessment
Background and baseline body temperatures were pre-determined prior to laser application using a thermal imaging camera (FLIR Systems ThemaCAM S65 Infrared Camera -76,000 pixel display, Wilsonville USA) mounted above the specimen. This was manually triggered at the beginning of the irradiation and set to acquire images at 1 Hz frame rate for the length of the irradiation. The CW laser was also manually triggered at the start of the thermal image acquisitions. The heating effect on both healthy and tumor tissues were observed. Temperature rises were calculated and plotted by measuring the heating occurring in a manually selected region of interest (ROI) within the irradiated tumor site and subsequently subtracting from the background temperature which was defined as an area remote to the mouse.

Psychosocial, weight and tumor site assessments
Animals were observed for four weeks during which serial photographs of progress were taken. The animals were weighed once weekly while tumor and/or irradiation sites and psychosocial behavior was reviewed daily. Monitoring was by at least thrice weekly clinical examinations and physical measurements of tumor size if there was still a tumor present post irradiation.

Histological assessment
Tissue from irradiated tumor sites was excised for microscopic assessment in all mice at the end of the study period. In selected cases histological examination was conducted on major organs. The specimen was fixed in 10% neutral buffered formal saline and embedded in paraffin prior to performing 5 m sections which were then stained with hematoxylin & eosin (H & E) and 0.1% Picrosirius red with Miller's elastic stain to demonstrate collagen. Histological slides were imaged with an Olympus BX 40 light microscope and with polarized light microscopy.

Transmission electron microscopy (TEM) and EDX analysis
Tissue from tumor sites was harvested during terminations in mice that received either IV or IT GNRs (without laser) and also from tumor sites that underwent PTT. This excision was done to visualize the position of GNRs within the tumor tissue from both routes of GNR administration, both before and after irradiation. The tissues were visualized by TEM (TEM JEOL JEM-2100FX, Tokyo, Japan, operated at 200 kV). Energy-dispersive X-ray spectroscopy (EDX/EDS, X-Max 80 mm 2 , Oxford Instruments, UK) was used to detect the presence of Au (gold) within tissues. A minimum of 20 cells per region of interest (ROI) were observed from each tissue (n=2), with a total of 40 cells viewed.
Tissues for TEM were processed as follows: Briefly, dissected tissue specimens were washed in 0.9% NaCl (saline) solution followed by another wash with sodium cacodylate buffer solution (pH = 7.4), before they were fixed with 2.5% glutaraldehyde solution at 4C for two hours. The tissue specimens were then washed three times with sodium cacodylate buffer solution, and stained with 1% osmium containing 2% of potassium ferrocyanide for two hours and washed with distilled deionized water (x3) and incubated overnight with 1% uranyl acetate in cacodylate buffer at 4°C before en bloc Walton's lead aspartate staining was performed. The samples were subsequently dehydrated with 25%, 50%, 70%, 90% and 100% anhydrous ethanol for 15 minutes (x3) each, before being placed in anhydrous ice-cold acetone and left at room temperature for 20 minutes (x3). Durcupan ACM was used as the resin. The samples were then placed into 25%, 50% and finally 75% Durcupan:acetone for two hours each. Tissues were placed in 100% Durcupan overnight then into fresh 100% Durcupan for two hours. The resin block was then precision sectioned with a diamond knife ultramicrotome. For TEM analysis, sections of fixed and embedded cells were cut with an ultramicrotome using a 35° wedge angle diamond knife and floated on distilled water. Sections were immediately collected on uncoated 300 mesh copper grids (Agar Scientific, Stansted, UK) and dried for 30 minutes at 37°C. Sections of 70 -100 nm thickness were used for imaging studies.

Blood profile assessment
Under terminal general anesthesia blood was drawn by intracardiac venesection for clinical pathology screen from mice receiving both IV and IT GNRs and PTT at the end of 30 days. Blood was examined for biochemical parameters which included urea, creatinine, liver function and a full blood count. These blood values were compared to published normal parameters for the type of mice and used to identify any systemic toxicity, hematological or biochemical abnormalities from either GNRs or PTT (results shown in Supplementary Tab. S2).

Inductively coupled plasma mass spectrometry (ICP-MS)
The concentration of gold ([Au]) within organs was determined at the end of the study using ICP-MS. This was performed on blood using intracardiac venesection during terminal anesthesia, and harvesting organs such as liver, spleen, kidneys, lungs, heart, brain and tumor sites. The [Au] at 30 days (or end of study) was also contrasted with [Au] at the tumor sites and in vital organs on Day 0 (defined as the day after IV injections of GNR or the day of IT injections) to see the initial [Au] concentration values at the time when PTT would be instituted and to observe its variation at the end of 30 days.
Tissue samples were placed in individual microwave digestion vessels with 7 ml concentrated (15.4 M) HNO3 and 3 ml 30% H2O2 (Suprapur 30% H2O2 from Merck) and left overnight at room temperature. Blood samples were weighed into the microwave digestion vessels. Sample solutions were produced using an Ethos EZ microwave digestion system, whereby the samples were increased up to a temperature of 210°C over one hour. Once clear sample solutions were obtained, they were evaporated to dryness before dissolving in 5% aqua regia. Samples were analyzed for the gold concentration using an Agilent 7700x

Supplementary tables
Supplementary