Plasmonic Photothermal Cancer Therapy: Nanoparticle-embedded Tumor-tissue-mimicking Phantoms for Visualizing Photothermal Temperature Distribution

This article presents the protocol for preparing tumor-tissue phantoms that replicate optical properties for plasmonic photothermal therapy. It details phantom preparation, photothermal evaluations, and validation of the developed numerical model based on photothermal temperature measurements for assessing therapeutic parameters, offering an ethical, cost-effective alternative to in vivo studies for preliminary testing.

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) irradiation to generate localized heat that destroys cancer cells. Before administering PPTT, the therapeutic parameters -- NP concentration, irradiation intensity, and duration -- need to be estimated. For this, numerical simulations are performed. However, to ensure robust computation, these simulations must be validated through photothermal experiments on tumor-tissue-mimicking phantoms replicating the optical properties of tumor tissue. For PPTT, therapeutic parameters are governed by the scattering and absorption of incident radiation by the tissue and NPs. Therefore, validation experiments can be conducted on phantoms mimicking the reduced scattering coefficient (µ_s') and absorption coefficient (µ_a) of the target tumor/tissue.

Specifically, this protocol provides instructions for preparing phantoms mimicking µ_s' and µ_a of breast tumor injected with gold nanorods, surrounded by normal breast tissue. The protocol also details NIR irradiation, temperature monitoring, and validation of numerical results by comparing spatiotemporal temperatures with those measured using thermocouples. The protocols presented in this study facilitated the preparation of hydrogel-based cylindrical breast tumor-tissue phantoms with dimensions (ϕ40 x 12 mm) and a central tumor region (ϕ20 x 6 mm), comprising 1% agarose as the base matrix and intralipid as the scattering constituent and tumor region embedded with gold nanorods at 25 µg/mL concentration. Representative results from a case study illustrate the application of fabricated phantoms for validating numerical simulations for PPTT. The study concludes that the demonstrated protocols are valuable for conducting photothermal experiments aimed at optimizing and planning therapeutic parameters prior to in vivo experiments and validating numerical simulations for PPTT.

Plasmonic photothermal therapy (PPTT) is an emerging localized cancer treatment modality that involves delivering nanoparticles (NPs) to the tumor site, followed by irradiation with near-infrared (NIR) radiation. The NPs are typically administered via intratumoral (IT) or intravenous (IV) routes¹. Upon NIR irradiation, the plasmonic interaction of the incident radiation and NPs leads to the generation of localized heat at the surface of the NPs, which then dissipates into the surrounding tumor tissue^2,3. This localized heating elevates the temperature in the tumor region, leading to cancer cell death through thermal ablation^4,5. Effective cancer treatment can be achieved by maintaining specific temperatures, such as 46 ºC for 1 h⁶, 50-52 ºC for 4-6 min⁷, or 60 ºC for instantaneous damage⁸ via various biological processes.

Various photothermal agents have been explored and reported for photothermal therapy application, and their therapeutic efficacy has been evaluated through in vitro or in vivo studies. These agents include organic materials⁹ such as near-infrared dyes (e.g., Indocyanine Green, IR780, IR820), polymer-based photothermal agents (e.g., polydopamine), and inorganic materials¹⁰, including noble metal-based NPs or plasmonic NPs (e.g., gold NPs)¹¹, transition metal sulfur/oxides¹², and MXenes¹³. Among these, plasmonic NPs, specifically gold NPs, offer several advantages over traditional photothermal agents (e.g., dyes), such as better photothermal stability, higher photothermal conversion efficiency, and tunable plasmonic response through shape and size variations¹⁰. These attributes make gold NPs ideal candidates for photothermal therapy, with some currently undergoing clinical trials¹⁴.

To optimize therapeutic efficacy and ensure sufficient tumor thermal damage during PPTT, it is essential to estimate treatment parameters such as NP dosage (in terms of concentration) and NIR radiation parameters (including irradiation intensity and duration) before the preclinical/clinical application of PPTT. Numerical simulations are typically employed to establish these parameters. Various numerical methods have been developed to assess thermal damage within tumor tissue, with the lattice Boltzmann method being one such approach^15,16. However, for these simulations to be reliable, they must be validated using tissue analogs known as tissue-mimicking phantoms. These phantoms can be prepared to replicate the optical, thermal, biological, or mechanical properties of real tissues, serving as substitutes to conduct preliminary testing, treatment evaluation, and validation of newly developed devices, materials, or methods intended for biological applications^17,18. This can reduce unnecessary suffering of animals or human subjects and address ethical concerns associated with such experiments^19,20.

The design and fabrication of a phantom depend on the intended application²¹. For instance, during phototherapies like PPTT, the dose of incident radiation is influenced by the amount of light absorbed or scattered by the NPs and tissues^22,23. Therefore, optical phantoms that mimic the optical properties, specifically the reduced scattering coefficient (µ_s^') and absorption coefficient (µ_a) of biological tissues, are used for PPTT evaluations and subsequent validation of numerical simulations^24,25. Optical phantoms are typically composed of three main constituents: a base matrix, scattering agents, and absorption agents^17,26. The base matrix holds the scattering and absorption components in suitable concentrations to replicate the desired optical properties. These phantoms can be classified into solid, liquid, and semi-solid (hydrogel) phantoms, depending on the type of base matrix. For thermal therapeutic studies like PPTT, hydrogel phantoms, particularly agarose-based phantoms, are preferred due to their biocompatibility, negligible inherent scattering and absorption, simple fabrication process, and flexibility to be cast into desired shapes and sizes corresponding to tumor geometries^19,22. Most importantly, the prepared agarose-based phantoms can be used up to ~70-80 ºC bulk temperatures, as the melting temperature of agarose-type phantoms is ~80 ºC¹⁹. For PPTT, as a temperature range of ~50-80 ºC is sufficient, such agarose-based phantoms can be used for PPTT-based photothermal evaluations.

Various hydrogel-based tissue-mimicking phantoms have been developed and reported for various applications. Mustari et al. developed agarose-based tissue-mimicking phantoms and demonstrated their utility in validating a newly designed optical system¹⁸. In another study, tissue-mimicking thermochromic phantoms were prepared to measure the extent of thermal damage during high-intensity focused ultrasound (HIFU) therapy²⁷. Polyacrylamide-based tissue-mimicking phantoms have also been prepared to analyze the cavitation effect during HIFU-based cancer therapy²⁸. The objective of this study is to demonstrate a step-by-step method to fabricate tumor-tissue-mimicking phantoms along with the protocol for phantom-based photothermal experiments for PPTT evaluations. This proposed detailed protocol aims to promote the adoption and reproducibility of the phantom preparation and subsequent phantom-based photothermal experimentation methods for testing the photothermal performance of newly developed nanostructures, thereby validating the numerical simulations and helping pretreatment planning or optimization of therapeutic parameters of PPTT. This article describes a phantom preparation protocol specifically designed for sub-surface breast tumors; however, the same steps can be adapted for fabricating various tumor-tissue types (of various shapes and sizes) by altering the composition of optical absorption and scattering agents. As an example, the demonstrated tissue-mimicking phantom-based photothermal evaluations have been employed in previously reported studies to validate PPTT simulations for sub-surface forearm tumor²⁴, sub-surface IDC²⁵, and skin tumors²⁹.

This paper describes the preparation steps of an optical phantom that mimics the µ_s' of a sub-surface or subcutaneous breast tumor, specifically invasive ductal carcinoma (IDC), located 3 mm beneath the skin surface and surrounded by normal breast tissue. The phantom is of cylindrical geometry prepared using agarose as a base matrix and intralipid (IL) as the scattering agent added in suitable concentrations to mimic µ_s' of normal and cancerous breast tissue. Agarose, a transparent hydrogel with negligible scattering and absorption, is an ideal base matrix for optical phantoms^18,30. Further, IL, a 20% fat emulsion that mimics the bilayer structure of cell membranes, is widely used as a scattering agent^31,32 and was chosen for this study to replicate the µ_s' of normal and cancerous breast tissue. The phantom is designed to mimic breast tumor (IDC) injected with gold nanorods (AuNRs) as plasmonic NPs, surrounded by normal breast tissue without AuNRs. Among various gold NPs used in PPTT, AuNRs were selected for this study due to their strong plasmonic response in the NIR region and their widespread use in preclinical PPTT studies, including those involving canine and feline patients¹⁴. The protocol demonstrates the preparation of two types of phantoms: one with a tumor featuring AuNR distribution as seen with IV injection and the other with a tumor reflecting the AuNR distribution achieved via IT injection. Following the phantom preparation protocol, the experimental setup for NIR irradiation and the steps for conducting photothermal evaluations on the phantoms are described. Finally, a step-by-step guide is provided for interpreting the temperature distribution results obtained from these evaluations and for comparing experimental data with numerical simulation results. This comparison helps validate a developed numerical method, enabling the tuning for optimal treatment parameters tailored specifically to a tumor.

NOTE: The phantoms were prepared using agarose and intralipid based on literature-reported compositions to achieve the desired optical properties. No real biological tissue from patients or cadavers was used. Therefore, the preparation of these phantoms is free from ethical constraints and does not require informed consent.

1. Selection or fabrication of a suitable mold

1. .Selection of a suitable mold
   1. Choose a mold that matches the desired shape and dimensions for the phantoms. For cylindrical phantoms with a tumor region uniformly distributed with NPs and surrounded by normal tissue, use a glass Petri dish and a small beaker as the mold⁸ (Figure 1A).
      NOTE: These steps are for preparing tumor-tissue-mimicking phantoms in cylindrical geometry. For other shapes or sizes, select an appropriate mold. If a suitable mold is unavailable, fabricate one using three-dimensional (3D) printing, as detailed in step 1.2.
2. Fabrication of mold by 3D printing
   1. Design a 3D model using Computer-aided Design (CAD) software (e.g., SolidWorks, Autodesk Inventor, or CATIA) according to the desired shape and size. To follow this protocol, design one hollow cylinder (inner diameter 40 mm, thickness 2 mm, and height 12 mm; see Supplemental File 1) and two solid cylindrical masking molds (dimensions ϕ20 x 6 mm and ϕ14 x 3 mm), as shown in Figure 1B.
      1. For hollow cylinder design/drafting, in CAD software, create two circles with diameters of 40 mm and 44 mm. Then, extrude the geometry for 12 mm.
      2. For solid cylindrical masking molds, create circles with diameters 20 mm and 14 mm, then extrude for 6 mm and 3 mm, respectively (see Supplemental File 2 and Supplemental File 3). Draw a rectangle (sides 44 mm and 5 mm) on one side of the cylinder and extrude it for 2 mm.
   2. Convert the 3D models into Gcode format using 3D printer-compatible software (e.g., Cura) for printing.
   3. Use this Gcode to print the molds (e.g., herein, using polylactic acid [PLA] φ1.75 mm,  1 kg of eSun) using a 3D printer.
      NOTE: The rectangle is drawn to suspend the masking molds. Different molds can be designed and fabricated to prepare phantoms with other desired shapes.

2. Preparation of tumor-tissue-mimicking phantom solutions²⁵

NOTE: In this study, agarose-based optical phantoms of cylindrical geometry mimicking tumor tissue are prepared to resemble a sub-surface breast tumor injected with AuNRs, via IT or IV injection, as shown in Figure 2. The IT phantom has two regions: a central tumor region with AuNRs and a surrounding normal tissue region. The IV phantom has three regions: a tumor region with AuNRs at the tumor periphery, a central bare tumor region without AuNRs, and a surrounding normal tissue region. Since the optical properties (µ_a and µ_s') differ for tumor and normal tissue, separate phantom solutions are prepared for each region having different compositions and will be discussed separately.

1. Preparation of phantom solution mimicking normal breast tissue (Solution 1)
   NOTE: This solution will be used for both IT and IV phantoms. The preparation steps of Solution 1 are shown in Figure 3A.
   1. Calculate the theoretical volume of the solution based on the mold's dimensions.
      NOTE: Here, for the cylindrical mold of ϕ40 x 12 mm, the calculated volume is 15 cm³ or 15 mL per phantom. As two such phantoms need to be prepared, the total volume is 15 mL x 2, which is 30 mL. Therefore, prepare 35 mL of the solution to account for evaporation or spillage during the phantom preparation step.
   2. Calculate the amount (weight/concentration/volume) of all the phantom constituents-agarose (as base material) and IL (as scattering constituent)-to be added to the 35 mL solution.
      1. Add 0.35 g of agarose to prepare 35 mL of solution corresponding to 1% w/v concentration.
      2. Estimate IL concentration corresponding to desired µ_s' of normal breast tissue (i.e., 10.1 cm^{-1 33}) based on the IL concentration versus µ_s' data available in the literature. Next, calculate the volume of IL (20% emulsion stock) to be added to the phantom solution using equation (1):
         (1)
         Where C₁ and V₁ are the concentration of the reagent stock (here 20% IL stock) and the volume of the reagent stock solution to be added (here it is to be calculated), respectively. C₂ is the required concentration of the reagent (to be obtained from the literature) in the final working solution, and V₂ is the total volume of the final working solution (here, 35 mL).
         NOTE: Here, for µ_s' of 10.1 cm^-1, the IL concentration estimated from the reported literature is 1.04%^34,35.Using the above steps, the volume of IL (20% IL stock) to be added is 1.82 mL.
   3. Weigh 0.35 g of agarose and add it to 33.18 mL of deionized (DI) water in a beaker. Cover the beaker with aluminum foil to avoid water loss.
   4. Heat the beaker containing the solution on a hot plate at 120 °C while stirring until the solution becomes transparent.
   5. Lower the temperature of the hot plate to 60 ºC. After 15 min, add 1.82 mL of IL while stirring. Keep the resulting solution, Solution 1, under stirring at 60 ºC until use (ready for pouring).
      NOTE: The phantom solution at 60 ºC needs to be kept under stirring conditions. Otherwise, it leads to the solidification of the solution.
2. Preparation of AuNR-embedded tumor phantom solution (Solution 2)
   NOTE: This solution will be used for both IT and IV phantoms. The preparation steps of Solution 2 are shown in Figure 3B.
   1. Calculate the volume of the tumor region to be filled (ϕ20 x 6 mm).
      NOTE: The approximate volume for two such tumor phantoms is 3.8 mL. So, the solution volume to be prepared would be 4.5 mL.
   2. Calculate the amount of tumor phantom constituents to be added-agarose, IL, and AuNRs-using similar steps as mentioned in Section 2.1.
      1. Add 45 mg to prepare 4.5 mL of solution corresponding to 1% w/v concentration.
      2. IL:µ_s' of breast tumor is 4.6 cm^{-1 33} and to mimic the same, the corresponding IL concentration required is 0.472%^34,35. Therefore, add 106.2 µL of IL from 20% IL stock to 4.5 mL of tumor phantom solution.
      3. The desired concentration of AuNRs in the phantom is 25 µg/mL. To achieve the same, add 3.21 mL of AuNRs solution (stock concentration: 35 µg/mL) to the tumor phantom solution.
   3. Add 45 mg of agarose to 1.18 mL of DI water in a beaker and cover it with an aluminum foil.
   4. Place the beaker on a hot plate and stir at 120 ºC until the solution becomes transparent.
   5. Reduce the temperature of the hot plate to 60 ºC and leave the solution for 15 min.
   6. Add 106.2 µL of IL and 3.21 mL of AuNR suspension (35 µg/mL) under stirring conditions. Keep the resulting solution, Solution 2, under stirring at 60 ºC until pouring.
3. Preparation of bare tumor (without AuNRs) phantom solution (Solution 3)
   NOTE: This solution will be used for IV phantom only. The preparation steps of Solution 2 are shown in Figure 3C.
   1. Calculate the theoretical volume of the suspension to be added to create a bare tumor region (~ ϕ20 x 6 mm).
      NOTE: The approximate volume for tumor phantoms is 1.9 mL. So, the solution volume to be prepared would be 2.5 mL.
   2. Calculate the amount of tumor phantom constituents to be added-agarose and IL-using similar steps as mentioned in Section 2.1.
      1. Add 25 mg of agarose to prepare 2.5 mL of solution so as to achieve 1% w/v concentration.
      2. IL:µ_s' of breast tumor is 4.6 cm^{-1 33} and to mimic the same, the corresponding IL concentration required is 0.472%^34,35. Add 59 µL of 20% IL stock.
   3. Add 25 mg of agarose to 2.44 mL of DI water in a beaker and cover it with an aluminum foil.
   4. Place the beaker on a hot plate and stir at 120 ºC till the solution becomes transparent.
   5. Reduce the temperature of the hot plate to 60 ºC and leave the solution for 15 min.
   6. Add 59 µL of IL to the solution under stirring conditions. Keep the resulting solution, Solution 3, under stirring conditions at 60 ºC until pouring.

3. Preparation of tumor-tissue-mimicking phantom^24,25,36

1. Prepare the molds for the pouring step. For this, seal the bottom of the cylindrical molds with parafilm and place the masking mold (ϕ20 x 6 mm) in the center.
2. Pour Solution 1 into the cylindrical molds up to the top mark of the masking mold and allow it to solidify (Figure 4A).
3. After solidification, remove the masking mold to create a cavity for the tumor region (Figure 4B).
   NOTE: The protocol will be the same for both IT and IV phantoms up to step 3.3. The process will be discussed separately for IT and IV phantoms after step 3.3.
4. IT phantom^24,25,36
   1. Fill the cavity with Solution 2 and allow it to solidify (Figure 4C).
   2. Add Solution 1 to the top of the phantom and allow it to solidify completely (Figure 4D).
5. IV phantom^24,25
   1. Insert a smaller masking mold (ϕ14 x 3 mm) and fill the cavity around it with Solution 2 (Figure 4E).
   2. After solidifying, remove the smaller mold and fill the remaining cavity with Solution 3 (Figure 4F).
   3. Add Solution 1 to the top and allow complete solidification (Figure 4G).

4. Insertion of the thermocouples within the phantom^24,25,36

NOTE: To monitor the spatial temperature distribution, type K thermocouples are inserted within the phantom at various radial (r) and axial (z) locations, as illustrated in Figure 2. For thermocouple insertion at accurate locations, glass capillaries are used as guides to ensure precision. The thermocouple locations are denoted as (r, z), where the midpoint on the top surface of the tumor at depth z = 3 mm serves as the reference point for both IT and IV phantoms and is designated as (0, 3), as shown in Figure 2A,B. When selecting radial and axial locations to quantify thermal damage in the tumor region, the locations at the tumor periphery (both radial and axial) are critical. Achieving the required temperatures at these peripheral points during NIR irradiation ensures complete tumor ablation. Thus, the thermocouples are placed at radial extreme points (of tumor) at z = 3 and 9 mm, i.e., (10, 3) and (10, 9), and one thermocouple is placed at the tumor-tissue interface at z = 9 mm (peripheral axial location), i.e., (0, 9) as depicted in Figure 2A,B. Additionally, to assess axial temperature distribution, a thermocouple is inserted between locations (0, 3) and (0, 9), designated as (0, 6). Lastly, to assess the temperature rise in the surrounding healthy tissue region, one thermocouple is inserted at (15, 3).

1. Cut the glass capillaries to suitable lengths so that these reach the desired radial and axial locations within the phantom.
2. Insert thermocouples within these glass capillaries and puncture at specified radial and axial phantom locations one by one.
3. Once all thermocouples are in place, carefully place the phantom in a glass Petri dish for subsequent NIR irradiation, as shown in Figure 5A.

5. Exposure to NIR irradiation and measurement of resulting photothermal temperatures³⁶

1. Place the glass Petri dish containing the phantom (inserted with thermocouples) so that the central region of the phantom's top surface is perpendicular to the tip of the optical fiber of the NIR light source, as depicted in Figure 5A.
   NOTE: The beam diameter on the phantom surface can be adjusted by changing the distance between the surface and the optical fiber tip. Here, a 9 mm distance is kept to achieve a 20 mm beam diameter, covering the central tumor region.
2. Connect the Data Acquisition (DAQ) system to the computer and open the LabVIEW software.
3. Turn on the NIR light source (Figure 5B) and play button in the software simultaneously to record temperature data at the start of irradiation.
4. Irradiate the phantom for 20 min, then switch off the NIR light source and stop the recording.
5. Plot the recorded temperature versus time data.

6. Temperature comparison with simulation results^24,25

NOTE: Experiments are generally repeated, and temperatures are recorded at set time intervals at all thermocouple locations. For validation, the following steps are performed:

1. Compute the mean and standard deviation of the experimental temperatures at all thermocouple locations (r, z).
2. Compute temperatures at considered thermocouple locations numerically.
3. Plot mean temperature obtained from the experiments and temperature obtained by simulation at all thermocouple locations with respect to time, shown in Figure 6.
4. Calculate root mean square error (RMSE) and mean absolute error (MAE) for all thermocouple locations to quantify the difference in temperature to assess the validation, as shown in Table 1.
   NOTE: MAE and RMSE are calculated using Equations 2 and 3, respectively.
   (2)
   (3)
   Where TE, TS, and N are the temperature obtained experimentally, temperature computed numerically, and number of data points (here, temperatures are recorded each second for 20 min; hence, = 1,200), respectively. i represents time instants.

Figure 6 shows the temporal mean temperatures obtained during experiments with an AuNR-embedded tumor-tissue phantom at all thermocouple locations, as shown in Figure 2, compared with the temperatures obtained during simulations at corresponding thermocouple locations. Here, the experiments were performed 4x for each distribution, i.e., IT and IV distributions of AuNRs. During the experiments, the room temperature was 25
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This paper presents the protocol for the preparation of agarose-based tumor-tissue mimicking optical phantoms. The phantoms are designed in a way to mimic the optical properties of tumor and normal tissues for their use in studies for PPTT. In this study, the application of these phantoms for validation of numerical methods during PPTT is highlighted. The most critical step in this protocol is maintaining the temperature of the phantom solutions containing agarose and IL at 60 ºC under constant stirring. If the...

The authors have no competing interests to disclose.

This study was conducted without any financial support from any public, commercial, or non-profit funding bodies. The authors acknowledge CSIR-Central Scientific Instruments Organisation, Chandigarh, India, for infrastructure and support.