Integrated simulator for heat dissipation and light propagation
We combined two separate MC-based simulations (heat dissipation39 and propagation/absorption of light40 (photons) in tissue (skin) and tumor) and created an integrated simulation platform. Here, we used C language-based MFC library to provide a user-friendly software interface. We conducted all simulations on a computer (Intel Core i7-7th gen, 8 GB RAM). The user-application guide of the developed simulation is found in Supplementary Fig. 1.
Modification to DLC model
We utilized, custom-trained, and modified the DLC Python package (Ver. 2.2b7). Specifically, we used the custom-trained DLC model to estimate the locations of the body parts such as snouts and tails of the mice within an image (i.e., video frame). Note that the original DLC python package does not support a real-time processing feature, instead it only runs on video files. Hence, we directly modified the Python package in such a way that it can infer the locations of the body parts of the mice and estimate the optimal coil antenna through the functional modules, illustrated in Fig. 3a, in a real-time manner. We conducted all experiments including training the DLC on a GPU workstation (Lambda workstation with Intel Core i9-9960X, 128 GB RAM, and two GEFORCE RTX 2080 Ti graphics cards).
Quantitative performance assessment of the AI-enabled motion tracking algorithm
The proposed AI algorithm yields the following information for each frame: 1) the position of the snout and tail of each mouse, 2) the direction in which each mouse is heading toward, and 3) the angle between a vector along the length of each mouse and the y-axis. Based on this information, the algorithm selects an antenna coil that leads to the best wireless coverage in a cage. The following are three antenna settings considered in this study: 1) Two pairs of X-shaped coil antenna, X-shaped coil antenna in 2) the x-axis direction and 3) the y-axis direction. For the quantitative performance assessment of the algorithm, we used three video recordings, each of which is 10 minutes running time. We randomly extracted and evaluated 20 frames from a total of 15,000 frames in each video recording, and repeated the procedures twenty times. For each set of 20 frames, we compared the decision made by the proposed algorithm for each frame in the given set with the one made by a human expert in each antenna setup. Supplementary Fig. 6 shows a representative example of an image (video frame) processed by the algorithm. To check the performance of each antenna setup and implanted devices, we focused on the following two statistics (in terms of the number of frames): how long a selected antenna remains activated (Fig. 3c) and how many frames (i.e., how long of a time interval) it takes between activation of an antenna and its reactivation after the first deactivation (Fig. 3d). Here, a human expert extracted and analyzed the data, which had been processed by the proposed algorithm, in every 20 frames. For Fig. 3c, we chose a mouse (implanted device) from the group and measured how long a selected antenna remains activated or aligned with a vector determined by the mouse as a function of frames. Similarly, for Fig. 3d, we measured a time interval as a function of the number of frames between deactivation of an antenna and subsequent reactivation of it. These were averaged for 20 trials, leading to the statistics shown in Figs. 3b-d.
Device fabrication
The pattern fabrication process began to mount a flexible copper/polyimide (Cu/PI) bilayer film (thickness; 12 µm/18 µm, AC181200RY, DupontTM Pyralux®) onto a glass slide (dimensions, 5.08 cm by 7.62 cm). In the cleanroom facility, we deposited the photoresistor onto the Cu layer for 2 µm thick (AZ 1518, AZ®, recipe; spin-coated at 4,000 r.p.m. for 20 sec), and illuminated UV lights to lithograph patterns for pads and interconnections (EVG610, EV Group, recipe; UV intensity for 100 mJ cm-2). To engrave the photoresistor layer, the Cu/PI film was immersed in developer solution (AZ Developer 1:1, AZ®) for 30 sec and washed with distilled water for 10 sec. Next, immersion in copper etchant (LOT: Z03E099, Alfa AesarTM) for 10 min and rinses with solvents: acetone, methanol, and isopropanol in order, and distilled water for 1 min defined Cu patterns such as interconnections and pads on the flexible PI layer. In the standard laboratory facility, chip components including SMD (surface-mount-device) LEDs, passive components, and IC components were mounted onto the pattern using a soldering machine. For encapsulations, we applied Polydimethylsiloxane (PDMS) (SylgardTM 184 silicone elastomer kit, Dow®; 10:1 mix ratio) with a dip-coating process (500 µm thick) to the sample, and then it was cured in the oven at 80 ºC for 1 h. These procedures yield a proposed multi-wavelength optoelectronic implant.
Antenna-coil fabrication and wireless, power-control system
We worked with 8-ga bare Cu wire for the sub-antenna coil and Cu tapes (0.635 mm thick by 2.54 cm wide) for the source-antenna coil. The sub-coils were placed under a cage and around all sides of the cage while the source-coil was situated in the center of the crossed-long side of the cage vertically. Impedance matching using Network Analyzer (ENA Series E5063A, Keysight) with a discrete capacitor component yielded source and sub-coils, each of which resonates at 13.56 MHz (the source coil) and 15 MHz (the sub-coils), respectively. Wireless power control systems consisted of a RF power generator (ID ISC.LRM2500-A, FEIG Electronics), and an auto-tunable matching board (ID ISC.DAT-A, FEIG Electronics). For the multi-cage system, it requires a TX controller including an RF multiplexer (ID ISC.ANT.MUX.M8, FEIG Electronics), a control board (nRF52832 Development Kit, Nordic semiconductor), and a customized decoupling multiplexer.
Finite element-methods analysis
For numerical electromagnetic simulations of the proposed antennas structure, we used a finite element-methods analysis tool (Ansys Electromagnetics Suite 17-HFSS, Ansys®) to compute distributions of the electromagnetic field in a home cage. Antenna coils made of copper stripes or wires were modeled to materials with finite conductivity, 58 MS s-1. We figured out the residual dependency of transmitted power on angles and orientations between an implanted device and the TX coil antenna in the experimental box. All simulations were conducted with a TX level of 4 W, which is far below guidelines suggested by IEEE or ICNIRP41,42 (Supplementary Fig. 12).
Optical and thermal characteristics of the implant device
The residue light intensity, which keeps in response to the capacitance after the cutoff of the power source, was measured using a photodiode and oscilloscope. This was conducted repeatedly in three colored LEDs that have different turn-on voltages. For thermal assessments of wireless devices, we measured heat dissipation of the light sources in a device using an infrared camera (VarioCAM HDx head 600, InfraTech) in two different conditions: a device installed under a sealed bag of saline solution (10 % PBS) instead of a mouse, and a device itself in the cage. The power supply was a function of time at duty cycles of 25 % with a 10 ms pulse train, which is the same as experimental conditions by the wireless TX system.
Materials for PDT in colorectal cancer models
The human colorectal adenocarcinoma cell line, HT29, was obtained from the European Collection of Authenticated Cell Cultures (Salisbury, UK) and cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium plus GlutaMAX™ (Gibco® by Life Technologies™, Paisley, UK) supplemented with 10 % (v/v) Foetal Bovine Serum (FBS) (Sigma-Aldrich, Gillingham, UK). Hypericin and Foscan were obtained from Sigma Aldrich and biolitec Pharma Ltd. (Jena, Germany) respectively and stock solutions of the photosensitizers were prepared in ethanol. Thiazolyl Blue Tetrazolium Bromide (MTT) was obtained from Sigma Aldrich.
Monitoring implantable device operating temperatures
Implantable LED devices were switched on at room temperature and allowed to continuously run for 48 hours. The surface operating temperature of the miniature LEDs was measured over 48 hours using an RS PRO medical infrared thermometer (RS Components Ltd., Corby, UK).
In vitro PDT cytotoxicity
HT29 cells were seeded into 24-well tissue culture plates (Corning Inc., New York, USA) at 2 x 105 cells per well and incubated at 37 °C/5 % CO2/95 % for 24 hours. Cells were then treated with 200 nM Hypericin or 100 nM Foscan in the dark for 16 hours. Cell cultures were then washed with Dulbecco’s Phosphate-Buffered Saline (DPBS, Gibco® by Life Technologies™) and Phenol red-free RPMI 1640 medium with L-glutamine (Gibco® by Life Technologies™) supplemented with 10 % (v/v) FBS was added to cultures. LED devices were positioned and affixed in the centre and underneath the wells and switched on. For single-channel LED devices, light treatment lasted for 1 hour at 10 µW cm-2, equating to 36 mJ cm-2 of total light dose. For dual-channel LED devices, light treatment lasted for 1 hour at 0.5 µW cm-2, equating to 1.8 mJ cm-2 of total light dose. Depending on the experimental conditions, cultures were either irradiated with light or kept in the dark at room temperature. After 24 hours, the MTT cell viability assay was performed by dissolving Thiazolyl Blue Tetrazolium Bromide into Phenol red-free RPMI 1640 medium (MTT solution). Cell media was discarded from cultures and replaced with the MTT solution and cultures were incubated in the dark for 3 hours. The MTT solution was then discarded and formazan crystals were dissolved using propan-1-ol. Optical absorbance values were measured using a Mithras LB 940 Microplate Reader (Ex: 570 nm) (Berthold Technologies Ltd., Harpenden, UK).
In vivo metronomic PDT
The in vivo experiment was conducted in accordance with the Animals (Scientific Procedures) Act 1986. Female BALB/c nude mice (6-8 weeks old) were purchased from Charles River UK, Ltd (Margate, UK). One million (1 x 106) HT29 cells were suspended in 100μL of FBS-free RPMI and injected subcutaneously into the right dorsal flank area of mice. Inoculated HT29 cells were grown for 8 days to generate heterotopic HT29 colorectal cancer tumor xenografts. Following the growth of tumor xenografts, the miniature implantable LED devices were sterilized in 70 % ethanol, and surgically implanted into mice and the LED-containing probes were positioned adjacent to tumor xenografts. 3M™ Vetbond™ Tissue Adhesive surgical glue (3M™ United Kingdom PLC., Bracknell, UK) was used to close up the incisions, through where the devices were inserted (Supplementary Fig. 13).
Immediately following the device implantation (Day 0), mice were intraperitoneally injected with 0.5 mg kg-1 Hypericin prepared as working solutions in DPBS, and LEDs were switched on. 0.5 mg kg-1 Hypericin injections were administered daily. Tumor xenograft volumes and the weights of mice were measured and recorded on Days 0, 2, 4, 6, and 7. HYP(-)LED(+) group received miniature LED light treatment only. HYP(+)LED(-) group received Hypericin treatment only. HYP(+)LED(+) group received both light and Hypericin treatments. Over 7 days, mice received total doses of 3.5 mg kg-1 Hypericin (0.5 mg kg-1 Hypericin per day) and 12.1J cm-2 of light (light fluency rate = 20 µW cm-2). Following the completion of in vivo experiment, the mice were euthanized in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986 and the tumor xenografts and mice livers were harvested.
Histological Analysis of Tissue
Harvested tumor xenografts and livers were fixed in 4 % (w/v) paraformaldehyde (PFA) for 24 hours and stored in 70 % (v/v) ethanol at 4 °C. Fixed tissue was then embedded into paraffin, sectioned onto glass slides, and subjected to Haematoxylin and Eosin (H&E) staining. Stained slides were imaged using the Nikon Eclipse E1000 Microscope (Nikon UK Ltd, Kingston upon Thames, UK).
Statistical Analysis
Unpaired two-tailed student’s t-test was used to perform statistical analysis using GraphPad Prism 9 (GraphPad Software, Inc., California, USA). p<0.05 was considered to be statistically significant. Data are presented as the mean ± standard deviation.