Panels of head and neck cancer cell lines were ordered from ATCC and mouse-derived squamous cell carcinoma, SCCVIISF, was captured spontaneously from a mouse tumor and grown in culture. All cell lines were cultured under the following conditions: 37°C, 5% CO2 incubator with Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. The medium was changed twice a week until the cells were confluent. Confluent cells were then passaged or harvested for use.
Synthesis of multilayer films
Poly(D,L-lactide-co-glycolide) (PLGA, 50:50, Mn 25,000), polyvinyl alcohol (PVA, MW: 31,000–50,000) and dichloromethane (DCM) were purchased from Sigma-Aldrich (Santa Barbara, USA). Phosphate-buffered saline (pH 7.4) was obtained from Gibco Life Technologies (AG, Switzerland). All chemicals used in this study were of analytical reagent grade and were used without further purification.
The multilayer films were prepared layer by layer, and the fabrication of each layer depended on various factors of the material, including its stiffness, toughness, adhesion, degradability, porosity, hydrophilicity, and load-carrying capacity, among others. One manufacturing technique involved a mechanical centrifuge followed by heat and solvent evaporation. First, 10 g of PVA powder was added to 100 ml of deionized water, then centrifuged for 1 h at 95°C to obtain a 10% (w/v) PVA solution. Then 0.4 g of calcium carbonate (CaCO3) the microparticles were dispersed in 10 mL of a PVA solution after sonicating the bath for 5 min and the mixture was vortexed for 2 min (Fig. 1). The homogeneous solution obtained was deposited on a plastic Petri dish and placed in a stirrer so that the solution could be evenly distributed. Then the Petri dish was transferred to an oven at 70°C for 2 h to ensure that the water was completely evaporated. Finally, the first layer (CaCO3/PVA film) for CT imaging was obtained. Further, 0.5 g of the PLGA powders were added in 10 ml of DCM and then centrifuged for 30 min to obtain a 5% (w/v) PLGA solution. Then, 20 mg of the TQ powders were dispersed in 2 ml of a PLGA solution after sonicating the bath for 5 min and the mixture was vortexed for 2 min. The resulting mixture was deposited on the first layer in the Petri dish, then evaporated in ambient air under appropriate ventilation for 30 min. The multilayer polymer film was imaged by scanning electron microscopy (Fig. 2). The tensile stress was measured for the polymer as shown in Figure 3. Finally, the multilayer films with the first layer (CaCO3/PVA film) for CT imaging and the second layer (TQ/PLGA film) for chemotherapy were obtained (Fig. 4).
Mouse surgery protocol
Male C3H/HeJ mice (The Jackson Laboratories, Bar Harbor, ME, USA) were used in the study (Animal Research Committee (ARC), protocol number 2008-147). The University of California, Los Angeles Chancellor’s Animal Research Committee and Animal Research: Reports of In Vivo Experiments (ARRIVE)26 guidelines and protocols were approved and followed. To test the polymer platform, 20 8-week-old C3H/HeJ mice were injected with 400,000 cells of a mouse-derived squamous cell carcinoma line, SCCVIISF, into the right hind flank. Tumor growth was assessed with calipers three times a week after polymer implantation for 18-31 days to assess the antitumor efficacy of the different treatments. Control mice required euthanasia, as determined by the University of California, Los Angeles Animal Research Committee and in accordance with American Veterinary Medical Association (AVMA) guidelines for euthanasia of animals (2020 )27 due to tumor burden. The lengths, widths and heights (in mm) of the tumors were measured and the volume of the tumor (cm3) was calculated using the formula: Tumor volume= π/6×length×lenght×the size. When the tumors reached an average size of 0.5 to 1 cm, all animals underwent surgery to shrink their tumors by 50%. This was done to approximate the surgical situation when a patient’s tumor is unresectable and some tumor is left before polymer therapy. The animals were then randomly assigned to the different treatment groups. Treatment groups included: (1) Inert polymer without drug; (2) TQ polymer; (3) Inert polymer + 3 × 4 Gy RT; (4) TQ Polymer + 3×4 Gy RT. No systemic TQ treatment was administered. Each tumor bed was covered with 2.25 cm2 cut polymer in the shape of the remaining tumor. The polymer was draped over the edges of the tumor and sutured in place.
Mice in the RT group were anesthetized with ketamine/xylazine and eye ointment applied on days 1, 2, and 3 after surgery for RT treatment. The mice were placed under ½ inch of lead shielding, leaving only the tumor exposed. An X-ray dose was delivered at 0.4299 Gy/min for 9.3 min until 4 Gy of the total dose was received. The mice received 3 × 4 Gy RT, which is the scale-comparable dose given to patients with head and neck cancer.
In vivo small animal imaging was performed at the Crump Institute’s Preclinical Imaging Technology Center. Mice were injected via the lateral tail vein with a radiotracer (70 μCi for 18F-FDG and 200–400 μCi for 11C-L-glutamine), followed by 60 min absorption under anesthesia. with 2% isoflurane, followed by microPET (G8 PET/CT, PerkinElmer) and microCT (CrumpCAT, Arion Hadjioannou laboratory). Quantification of 18F-FDG uptake was performed using AMIDE software by drawing a region of interest on the tumor and the whole body, as well as calculating each of the maximum uptake values ( SUVmax) as a percentage of the injected dose per gram (%ID/g).
After the animals were sacrificed, gross necropsy examination was performed on the surrounding tissues. No discernible difference in implantation site could be observed between the groups. Tumor samples were taken for sectioning and hemotoxin and eosin staining was performed through the UCLA Translational Pathology Laboratory. Histopathological examination was performed with the assistance of a senior pathologist at the UCLA Medical Center.
Estimated dose rate administration TQ
The incorporated polymers were weighed before implantation and during resection. The imaging and drug treatment layer was peeled off and weighed separately, as the imaging layer was formulated to last three months, while the drug treatment layer was formulated to last 1-1.5 months. By subtracting the difference in weight between before and after one month of implantation, we found that the CaCO3 the imaging layer lost 5% of its weight and the drug treatment layer lost 60% of its weight, equivalent to an estimated delivery of 0.77 mg per day or 4.68 µM/uM3.
Statistical significance was set atP= 0.05. The dose escalation experiment for the optimal dose of TQ required to induce LD50 across eight cell lines was generated via a fitted nonlinear regression curve and determined via a one-sample t-test. Tumor volume was compared between treatment groups with a one-way ANOVA model. Additionally, the statistical significance of the differences in tumor volume when comparing the control group with all other treatment groups was determined via an unpaired two-sample t-test. A linear quadratic survival curve representing TQ-treated versus control SCCVIISF was analyzed via GraphPad Prism.