Validation of nanoparticle mediated photodynamic therapy for drug resistant patient derived Head and Neck Cancer xenograft models

Introduction:: Human Papillomavirus (HPV)-negative head and neck cancer (HNC) is an aggressive malignancy with a poor prognosis. Conventional chemotherapies such as cisplatin, have a narrow therapeutic window therapeutic index. To improve outcomes, we developed a novel liposomal system embedded with 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), a chlorin-based photosensitizer. HPPH photo-triggering with ~660 nm NIR beam generates reactive oxygen species (Figure 1.A). The objective of this study was to evaluate biodistribution and test efficacy of HPPH-liposomal therapy in a patient-derived xenograft (PDX) model of chemo-radioresistant HNC.


Materials and Methods:: Patient samples were obtained in accordance with Medical College of Wisconsin (MCW) Institutional Review Board (PRO00040992) with appropriate patient consent. Experimental procedures were on mice were approved by the MCW Institutional Animal Care and Use Committees (IACUC). Equal numbers of male and female NSG-SGM3 (The Jackson Laboratory; strain 013062) were subcutaneously implanted with tumor pieces (~10-20 mm³). The Liposome-HPPH encapsulation was carried out according to method described by Sine J. et al. Phospholipids (Avanti Polar Lipids, Inc. Alabaster, AL, USA) were desiccated overnight to remove chloroform solvent and obtain dried lipid films and were resuspended in HEPES buffered saline (HBS) (pH=7.4) and 2 mg HPPH was added. Freeze-thaw cycles were employed to ensure thorough dispersion of lipids throughout the mixture. Probe sonication was used to form the vesicles for 1 minute followed by 1 minute of rest 5–10 times. The lipid dispersion was cooled at 4°C during the process to avoid lipid degradation. To enable assessment of in vivo biodistribution, trace amounts of a near infrared lipid probe DiR (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide; Ex/Em785/830 nm) was added. Liposome size and charge was measured by Dynamic Light Scattering measurements on a Malvern^TM Zetasizer, with hydrodynamic size of ~160nm, and near-neutral surface charge. 4h after injection, mice were anesthetized with isoflurane the PDX tumor was treated for 5 minutes with a continuous wave 660nm diode laser (Thorlabs, HL6545MG) at 90 mW/cm². Tumor response was tracked with caliper measurements.


Results, Conclusions, and Discussions:: Results and Discussion. We evaluated the extravasation of DiR-tagged liposomes into the vascularized tumor microenvironment at 30 minutes, 2 hours, and 4 hours following liposome injection (Figure 1. B). 24 hours following phototherapy, individual organs were harvested and NIR imaging was performed (Figure 1. B). DiR-tagged liposomes were not detectable in the heart, lungs, and kidney (Fig 1.B, lower panel). However, at this time point, DiR-tagged liposomes were retained in the treated tumor and were also visible in the spleen and liver (Fig 1. A, lower panel). The mice were randomly assigned to one of three treatment groups: laser treatment control, HPPH-liposome control, or laser treatment with HPPH-liposomes. Four hours following a single intravenous injection of vehicle or HPPH-liposomes, phototherapy to the subcutaneous tumor was applied. One week following treatment, gross differences in tumor size between treatment groups were observed (Fig 1.C). P033 tumors in both control groups continued to grow at the same rate as prior to treatment; however, P033 tumors treated with a combination of HPPH-liposomes and laser therapy were reduced to their starting volume at implantation (Fig 1.C).

Conclusion. In this study, we demonstrate tumor-specific uptake of HPPH-liposomes in a PDX model of HNC. HPPH-liposomes are cleared from most end-organs by 24 hours after delivery and no systemic toxicity was observed. Tumor treatment with red laser light photoactivated liposomal HPPH within the tumor resulted in ROS generation with associated tumor regression and necrosis.

Figure Caption. A) The schematic showing the HPPH- loaded liposomes which are triggered by 660 nm NIR right and cause ROS in tumor site. B) The biodistribution of DiR- tagged liposomes in the tumor at 0.5, 2, and 4 hours after intravenous injection (up), and organ imaging after 24 hours after injection (down). C) PDX growth in control groups (Laser-treatment alone or HPPH-liposome alone) and experimental treatment group (Laser and HPPH-liposome). On day 48, tumors were randomly assigned to control or experimental treatment groups and administered the corresponding therapy. Representative gross images of tumors at 1-week post-treatment (middle). Tumor volume at endpoint for individual PDX tumors (right).


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