Enhanced tumor uptake and penetration of virotherapy using polymer stealthing and focused ultrasound

Abstract

Background: Oncolytic viruses are among the most powerful and selective cancer therapeutics under development and are showing robust activity in clinical trials, particularly when administered directly into tumor nodules. However, their intravenous administration to treat metastatic disease has been stymied by unfavorable pharmacokinetics and inefficient accumulation in and penetration through tumors.

Methods: Adenovirus (Ad) was "stealthed" with a new N-(2-hydroxypropyl)methacrylamide polymer, and circulation kinetics were characterized in Balb/C SCID mice (n = 8 per group) bearing human ZR-75-1 xenograft tumors. Then, to noninvasively increase extravasation of the circulating polymer-coated Ad into the tumor, it was coinjected with gas microbubbles and the tumor was exposed to 0.5 MHz focused ultrasound at peak rarefactional pressure of 1.2 MPa. These ultrasound exposure conditions were designed to trigger inertial cavitation, an acoustic phenomenon that produces shock waves and can be remotely monitored in real-time. Groups were compared with Student t test or one-way analysis of variance with Tukey correction where groups were greater than two. All statistical tests were two-sided.

Results: Polymer-coating of Ad reduced hepatic sequestration, infection (>8000-fold; P < .001), and toxicity and improved circulation half-life (>50-fold; P = .001). Combination of polymer-coated Ad, gas bubbles, and focused ultrasound enhanced tumor infection >30-fold; (4 × 10(6) photons/sec/cm(2); standard deviation = 3 × 10(6) with ultrasound vs 1.3 × 10(5); standard deviation = 1 × 10(5) without ultrasound; P = .03) and penetration, enabling kill of cells more than 100 microns from the nearest blood vessel. This led to substantial and statistically significant retardation of tumor growth and increased survival.

Conclusions: Combining drug stealthing and ultrasound-induced cavitation may ultimately enhance the efficacy of a range of powerful therapeutics, thereby improving the treatment of metastatic cancer.

Figure 1.

Testing efficiency and reversibility of Adenovirus (Ad) polymer coating. A) After stealthing with nondegradable (ND PC-Ad) or low pH labile (PC-Ad) polymer, Ad shows reduced binding to enzyme-linked immunosorbent plates coated with antiadenovirus antibody, indicating effective protection of the Ad by stealthing with polymer. B) In vitro characterization of low pH triggered uncoating of PC-Ad by infection of a ZR-75-1 monolayer after pre-incubation of Ad, ND PC-Ad, or PC-Ad at pH 7.4 or 5.8. PC-Ad stealths Ad to effectively inhibit infection at pH 7.4 but stealthing is reversed and infection restored as pH is lowered. C) In vivo assessment of triggered uncoating after direct injection of 20 µL/5×10⁸ particles of ND PC-Ad (right flank) or PC-Ad (left flank) subcutaneously (SC) or into ZR-75-1 xenograft tumors. Measurement of luciferase transgene expression 24 hours later by IVIS. The image shows a representative mouse. The graph shows data from all three mice calculated as a ratio of intratumoral (IT) expression level divided by expression level after SC injection. Higher expression in tumors injected with PC-Ad indicates utility of pH triggered destealthing and infection reactivation in vivo. In the graph in (C), n = 3 mice; standard deviation (SD) is shown by bars. In other parts, n = 4; SD is shown by bars. ***P < .001, analysis using t test (C) or analysis of variance (A and B). Results typical of 3 independent experiments. All statistical tests were two-sided.

Figure 2.

Influence of polymer coating on interaction with bloodstream components. A) The effect of polymer coating on complement activation as assayed by C3a release after incubation with fresh human plasma for 30 minutes at 37°C. Reduced complement activation will lower immunogenicity and complement mediated clearance. B) The influence of polymer coating on sequestration by human blood cells by assessment of the association with cell and plasma fractions after 30 minutes at 37°C, using quantitative polymerase chain reaction to detect adenovirus (Ad) genomes. Reduced blood cell binding will extend the circulation of polymer coated Ad (PC-Ad) in vivo. C) Characterization of the effect of polymer coating on leukocyte infection using Ad_GFP and flow cytometry for detection of cells expressing the green fluorescent protein (GFP) transgene. Lower leukocyte infection will lower immunogenicity and enhance safety. All panels, n =3. Standard deviation is shown by bars. ***P < .001, analysis using t test (A) or analysis of variance with Tukey post test (B). Results typical of 3 experiments. All statistical tests were two-sided. White bar in (C) represents 10 microns.

Figure 3.

Impact of SonoVue microbubbles (SV) and focused ultrasound (US) on polymer coated adenovirus (PC-Ad) pharmacokinetics and tumor accumulation. A) The effect of focused US on the pharmacokinetics of intravenously injected 1×10¹⁰ copies of Ad or PC-Ad in Balbc/SCID mice bearing a ZR-75-1 xenograft tumor. Injection and US exposure regime as described in Methods. Blood was sampled 5, 15, and 30 minutes after injection, and Ad genome content was quantified by quantitative ploymerase chain reaction. The enhanced circulation of PC-Ad indicates the potential for improved passive uptake into tumors. B) Luciferase expression in the livers of mice injected with 1×10¹⁰ copies of Ad or PC-Ad as analyzed 20 hours after injection by IVIS imaging. Lower expression is indicative of lower capture by the liver and lowered potential toxicity. C) Assay of liver damage by quantification of ALT liver enzyme release 24 hours after injection with Ad or PC-Ad. Reduced ALT release shows PC-Ad to have reduced hepatic toxicity. D and E) The influence of focused US on tumor accumulation after intravenous delivery of Ad or PC-Ad and SV to Balbc/SCID mice bearing a ZR-75-1 xenograft on each hind leg, one of which was exposed to ultrasound, the other of which was not. Twenty hours after injection, mice were killed, and Ad genome content in tumors and liver was determined by quantitative polymerase chain reaction. Standard deviation is shown by bars. In (A) n = 8 mice, in (B–E) n = 4 mice. Analysis as done using analysis of variance with Tukey to compare all groups after test. Results are typical of two independent experiments. All statistical tests were two-sided. PBS = phosphate-buffered saline.

Figure 4.

Transgene expression and influence on tumor growth inhibition of polymer coated adenovirus (PC-Ad) with focused ultrasound (US) in the presence of SonoVue microbubbles (SV). A) Luciferase transgene expression in tumors of mice injected intravenously with PC-Ad and treated simultaneously with US or not, as quantified using IVIS for the first 20 days after injection. Improved luciferase expression in US-treated tumors indicates increased replication and spread. n = 8. Standard deviation is shown by bars. Two-sided t test performed at each time-point. At 1, 2, 3, and 7 days, P =.007, .004, .03, and .004, respectively. B) Staining of tumor sections with anti-CD31 (red) or anti-hexon (green) to determine the influence of US on intratumoral penetration of PC-Ad. Scale bar = 20 µm. Staining of Ad away from endothelial cells suggests improved penetration into tumors. C) Tumor growth in mice treated with phosphate-buffered saline (PBS) (black line), focused ultrasound and SV but no PC-Ad (gray line), PC-Ad with SV without focused ultrasound (blue line), or PC-Ad with SV with ultrasound (green line), as assessed by daily measurement with callipers. n = 8 mice. Mean values ± 95% confidence intervals are shown. Analysis of variance with Tukey correction comparing all groups post-test was used. D) Kaplan–Meier survival curve of mice treated in (C), in accordance with animal welfare legislation, mice were killed when tumor size reached a predefined limit of 700mm³. Log-rank test was performed. Results are typical of two independent experiments. All statistical tests were two-sided.