The effect of hyperthermic cycling on intratumoral liposome accumulation and triggered drug release

Nanomedicine offers a promising approach in cancer therapy. Systemic side effects of chemotherapy may be reduced, and therapeutic efficacy may be improved through controlled drug release in the tumor. Previous studies show that combining mild hyperthermia with thermosensitive liposomes (TSLs) significantly increases intratumoral accumulation of doxorubicin and improves therapeutic outcomes in animal tumor models. These findings have supported the initiation of clinical trials involving TSLs and local hyperthermia for the treatment of sarcoma. However, the application of localized heat can inadvertently damage surrounding healthy tissues and disrupt blood flow. Although longer durations of heating have been associated with improved drug accumulation, prolonged exposure may increase the risk of adverse effects. To mitigate these risks, one potential strategy is to shorten the heating duration. However, this may compromise efficacy. Alternatively, cycling between heating and cooling phases could reduce tissue damage while maintaining an overall effective exposure period. The aim of this study is to evaluate mild hyperthermic heat cycling (HC) as an alternative to continuous hyperthermia for triggered drug release from TSLs. Each heat cycle consists of a 5-min exposure to mild hyperthermia at 42 °C followed by a 10-min normothermic period, repeated for a total of five cycles. Normothermia is defined as the normal temperature of the mouse and when in a controlled setting the heating set at 37 °C. When we reach a temperature of 37 °C in a controlled setting (i.e., after a heating episode) this is indicated with: normothermic cooling at 37 °C. We compared HC versus continuous heating (25 min) to induce liposome extravasation in four tumor types using the dorsal skinfold window chamber model. We employed intravital confocal microscopy for real-time observation. We assessed heat cycle-triggered release of carboxyfluorescein (CF) and doxorubicin (DXR) from TSLs. We then determined tissue penetration depth, release kinetics, and interstitial drug concentrations through image analysis. Therapeutic efficacy of DXR-TSLs under HC (5 cycles of 42 °C for 5 min followed by cooling for 10 min) was compared to conventional continuous hyperthermia (42 °C for 25 min) in subcutaneous tumor models, based on tumor growth inhibition and intratumoral DXR accumulation. Our results show that consecutive heat cycles promote liposome extravasation through leaky tumor vasculature, with progressively deeper and more pronounced nanoparticle penetration after each cycle. Heat cycling effectively triggered content release from both CF-TSLs and DXR-TSLs. In mice bearing subcutaneous human BLM melanoma, HC combined with DXR-TSL delayed tumor growth for over 20 days. This was comparable to the effect achieved with a single continuous hyperthermia treatment. In conclusion, heat cycling represents a viable and effective alternative to continuous hyperthermia for inducing liposome extravasation and drug release from TSLs, with the added benefit of potentially minimizing thermal damage to surrounding tissues.