Injectable Hydrogel Containing Cowpea Mosaic Virus Nanoparticles Prevents Colon Cancer Growth

Abstract

Despite advances in laparoscopic surgery combined with neoadjuvant and adjuvant therapy, colon cancer management remains challenging in oncology. Recurrence of cancerous tissue locally or in distant organs (metastasis) is the major problem in colon cancer management. Vaccines and immunotherapies hold promise in preventing cancer recurrence through stimulation of the immune system. We and others have shown that nanoparticles from plant viruses, such as cowpea mosaic virus (CPMV) nanoparticles, are potent immune adjuvants for cancer vaccines and serve as immunostimulatory agents in the treatment or prevention of tumors. While being noninfectious toward mammals, CPMV activates the innate immune system through recognition by pattern recognition receptors (PRRs). While the particulate structure of CPMV is essential for prominent immune activation, the proteinaceous architecture makes CPMV subject to degradation in vivo; thus, CPMV immunotherapy requires repeated injections for optimal outcome. Frequent intraperitoneal (IP) injections however are not optimal from a clinical point of view and can worsen the patient's quality of life due to the hospitalization required for IP administration. To overcome the need for repeated IP injections, we loaded CPMV nanoparticles in injectable chitosan/glycerophosphate (GP) hydrogel formulations, characterized their slow-release potential, and assessed the antitumor preventative efficacy of CPMV-in-hydrogel single dose versus soluble CPMV (single and prime-boost administration). Using fluorescently labeled CPMV-in-hydrogel formulations, in vivo release data indicated that single IP injection of the hydrogel formulation yielded a gel depot that supplied intact CPMV over the study period of 3 weeks, while soluble CPMV lasted only for one week. IP administration of the CPMV-in-hydrogel formulation boosted with soluble CPMV for combined immediate and sustained immune activation significantly inhibited colon cancer growth after CT26 IP challenge in BALB/c mice. The observed antitumor efficacy suggests that CPMV can be formulated in a chitosan/GP hydrogel to achieve prolonged immunostimulatory effects as single-dose immunotherapy against colon cancer recurrence. The present findings illustrate the potential of injectable hydrogel technology to accommodate plant virus nanoparticles to boost the translational development of effective antitumor immunotherapies.

Keywords: antitumor immunotherapy; chitosan; colon cancer recurrence; cowpea mosaic virus; in situ forming depot; plant virus; thermosensitive hydrogels.

Figure 1.

Schematic presentation of the CPMV formulation in chitosan/GP hydrogels. Images of inverted Eppendorf tubes illustrate the no-flow behavior and increased turbidity occurring when gel forms upon heating.

Figure 2.

(A) Fluorescence intensity longitudinally determined using ROI analysis (n = 5), tracking the persistence of fluorescence signals from CPMV-Cy5 over 21 days following IP single injection of 450 μg of Cy5-CPMV in PBS (Cy5-CPMV group) or 450 μg of Cy5-CPMV formulated in hydrogels (F1, F2, and F3); the control group was untreated mice. (B) Fluorescence intensity measured by ROI analysis of organs (n = 5) collected at the study endpoint (day 21). Asterisks indicate statistical differences versus the control (*p < 0.05; **p < 0.01; ****p < 0.0001); hashtags show significant differences between a given hydrogel formulation and soluble Cy5-CPMV (^#p < 0.05; ^##p < 0.01; ^####p < 0.0001), ns denotes not statistically significant.

Figure 3.

CPMV-in-chitosan/GP hydrogel inhibits colon cancer growth in the intraperitoneal (IP) space (n = 5 mice per group). (A) Study design: BALB/c mice were inoculated IP with 1 million luciferase-labeled CT26 cells and then treated with single administration of the hydrogel F3 (containing 200 μg of CPMV) only or hydrogel F3 + 100 μg of soluble CPMV in PBS or single administration of 200 μg of soluble CPMV in PBS or prime and boost administration (weekly apart) of 100 μg of soluble CPMV in PBS as a prime-boost regimen. Cancer cell growth was longitudinally assessed by bioluminescence imaging (B) and intensity measurements (C) in the intraperitoneal space 5 min following I.P. injection of luciferin 15 mg mL⁻¹/150 μL. The luminescence was calculated using ROI analysis from Living Image 3.0 software. Asterisks indicate statistical differences versus the control (*p < 0.05; **p < 0.01); hashtags show significant differences between a given hydrogel treatment and soluble prime-boost CPMV particle (^#p < 0.05; ^##p < 0.01). No asterisk/hashtag means that the difference is not statistically significant. Tumor burden and ascites development were monitored by measuring abdominal circumferences (D). The p values indicate the difference with the control group (blank F3) on day 28.