Liposomal Phenylephrine Nanoparticles Enhance the Antitumor Activity of Intratumoral Chemotherapy in a Preclinical Model of Melanoma

Abstract

Intratumoral injection of anticancer agents has limited efficacy and is not routinely used for most cancers. In this study, we aimed to improve the efficacy of intratumoral chemotherapy using a novel approach comprising peri-tumoral injection of sustained-release liposomal nanoparticles containing phenylephrine, which is a potent vasoconstrictor. Using a preclinical model of melanoma, we have previously shown that systemically administered (intravenous) phenylephrine could transiently shunt blood flow to the tumor at the time of drug delivery, which in turn improved antitumor responses. This approach was called dynamic control of tumor-associated vessels. Herein, we used liposomal phenylephrine nanoparticles as a "local" dynamic control strategy for the B16 melanoma. Local dynamic control was shown to increase the retention and exposure time of tumors to intratumorally injected chemotherapy (melphalan). C57BL/6 mice bearing B16 tumors were treated with intratumoral melphalan and peri-tumoral injection of sustained-release liposomal phenylephrine nanoparticles (i.e., the local dynamic control protocol). These mice had statistically significantly improved antitumor responses compared to melphalan alone (p = 0.0011), whereby 58.3% obtained long-term complete clinical response. Our novel approach of local dynamic control demonstrated significantly enhanced antitumor efficacy and is the subject of future clinical trials being designed by our group.

Keywords: cancer therapies; drug delivery; tumor vessels.

Figure 1.

Physicochemical characterizations of phenylephrine loaded liposomal formulations. (A) Hydrodynamic diameters of blank (empty) liposomal nanoparticles (LNP) and liposomal phenylephrine nanoparticles (LNP-Phen). (B) Transmission electronic image (TEM) of liposomal phenylephrine nanoparticles. (C) The entrapment efficiency of phenylephrine was quantified by using UV-visible spectroscopy at 275 nm, which showed approximately 30% liposomal encapsulation of phenylephrine within the liposomal nanoparticles. (D) Stability of liposomal phenylephrine nanoparticles at 4°C over different time points. (E) In vitro phenylephrine release profile at different pH levels, ranging from 4.4 to 7.4. The more acidic pH levels were tested to replicate the tumor microenvironment, where pH levels have been shown to be more acidic than normal tissues.

Figure 2.

(A) C57BL/6 mice bearing B16 melanoma tumors within the dorsal skin fold were injected with intratumoral fluorescein (50 μl of a 1:50 dilution) and peri-tumoral injection of either (1) no liposomal nanoparticles, (2) blank/empty liposomal nanoparticles, or (3) liposomal phenylephrine-containing nanoparticles (10 μg per 100 μl per injection). Peri-tumoral injections (4–6 in total) were given along all 6 borders of the palpable tumors (when tumors reached approximately 10 mm³), creating wheals of injected nanoparticles. Fluorescence was measured at 2, 24, 48, and 72 hours after treatment. The highest radiant efficiency at each time point was recorded for tumors treated with liposomal phenylephrine-containing nanoparticles, demonstrating that these sustained release nanoparticles functioned as a “local” dynamic control mechanism to increase prolonged retention of intratumoral fluorescein. (B) Similar findings to (A) were detected in a 4T1 triple negative breast cancer model using BALB/c mice. Breast tumors growing orthotopically within the mammary fat pad had the highest retained fluorescence at all time points when treated with liposomal phenylephrine nanoparticles. (C) Examples of BALB/c mice treated with liposomal phenylephrine nanoparticles or blank liposomes, demonstrating radiant efficiency intensity over the 4 time points of the experiment.

Figure 3.

(A) Mass spectrometry analysis of intratumoral melphalan among B16 tumors treated with peri-tumoral injection of either (1) no liposomal nanoparticles, (2) “blank” liposomal nanoparticles, or (3) liposomal phenylephrine nanoparticles (10 μg per 100 μl per injection). Melphalan was injected into tumors at 20 μg in 100 μl. Tumors were resected at the same predefined endpoints (2, 24, 48, and 72 hours). Measurement of melphalan concentrations (standardized as ng per g of tumor tissue) was performed, which showed that the highest intratumoral melphalan concentrations were achieved with peri-tumoral injection of liposomal phenylephrine nanoparticles. (B) Mass spectrometry analysis was also performed on systemic tissues and peripheral blood at the time of necropsy. Very minimal levels of melphalan were detected systemically at only the 2 hour time point. Melphalan was not detected within these tissues at any other time point, and no levels were detected within the peripheral blood at any time point. These data suggest that there was minimal systemic spread or “leaking” of melphalan from the intratumoral injections.

Figure 4.

Peri-tumoral injection with phenylephrine dissolved in aqueous solution (standard formulation) did not have any effect when combined with intratumoral melphalan on anti-tumor responses, including tumor growth (A) or survival (B), when compared to intratumoral melphalan alone. In this experiment, C57BL/6 mice bearing orthotopic B16 tumors were treated with peri-tumoral aqueous phenylephrine. Controls included phenylephrine and melphalan only injections. While melphalan with or without peri-tumoral phenylephrine generated statistically significant improvements in tumor growth as expected, standard phenylephrine did not significantly enhance anti-tumor effects when compared to melphalan alone. (C) Example of a B16 tumor ulceration as confirmed on IHC on a C57BL/6 mouse, which developed during treatment. The combination treatment with intratumoral melphalan and peri-tumoral phenylephrine increased local toxicity in the form of tumor ulceration. Ulceration was a common adverse event, with 75 to 87.5% of mice demonstrating tumor ulceration that necessitated local wound care.

Figure 5.

Peri-tumoral injection with liposomal phenylephrine nanoparticles enhanced the effects of intratumoral injection of melphalan, as demonstrated by decreased tumor growth (A) and improved survival (B) compared to intratumoral melphalan alone. Mice treated with the combination of liposomal phenylephrine nanoparticles and intratumoral melphalan had a 58.3% (7/12 mice) complete response rate when compared to controls (including melphalan only controls). Tumor ulceration still developed for mice treated with liposomal phenylephrine, though to a lesser extent with the standard formulation (33.3 to 50% of mice per group that received liposomal phenylephrine nanoparticles).