Acoustically Driven Hybrid Nanocrystals for In Vivo Pancreatic Cancer Treatment

Abstract

New treatment strategies are urgently needed for pancreatic ductal adenocarcinoma (PDAC), which is one of the deadliest tumors nowadays. PDAC is marked by hypoxia, intrinsic chemoresistance, a "cold" tumor microenvironment, and dense desmoplastic stroma, which hinders drug penetration. This study investigates the combined effect of iron-doped, lipid-coated zinc oxide nanoparticles enhanced with a fluorescent sonosensitizer and local ultrasound stimulation in treating PDAC. Nanoparticles were synthesized and coated by lipids, and their physiochemical properties were characterized by assessing reproducibility, stability, and efficient inclusion of the sonosensitizer. In vitro, sonosensitizer-enhanced nanoconstructs were tested on a KPC murine PDAC cell line in combination with ultrasound to evaluate their cytotoxicity and assess their efficacy. In vivo, NPs were further coupled with AlexaFluor 700 to allow their localization over time, and the nanoconstructs were intratumorally administered to a subcutaneous murine PDAC model to enhance local bioavailability and tumor visualization and minimize off-target effects of systemic delivery. Biodistribution, efficacy, flow cytometry, and survival studies were carried out on different cohorts of mice. The sonosensitizer-enhanced nanoconstructs, combined with ultrasound, triggered significant reactive oxygen species (ROS) production, reducing the KPC cell viability. In vivo, the antitumor efficacy was particularly pronounced with ultrasound stimulation, demonstrating a synergistic interaction between the nanoparticles and ultrasound. Moreover, increased immune cell infiltration, enhanced cancer cell apoptosis, and prolonged survival of the treated animals were achieved. These findings highlight the potential of a synergistic therapeutic approach combining lipid-coated sonosensitizer-loaded nanoparticles and ultrasound stimulation as an effective therapy for PDAC and in situ monitoring.

Keywords: IR780 sonosensitizer; immune cells; in vivo models; pancreatic cancer; sonodynamic therapy; ultrasound; zinc oxide NPs.

Figure 1

(a) SEM picture, (b) CryoEM image, (c) EDX, and (d) XRD of the naked NPs. (e) Schematic representation of naked and lipid-coated NPs. (f) CryoEM image of lipid-coated NPs. (g) ζ-Potential and (h) NTA of naked (orange), lipid-coated (green), and lipid-coated-IR780 (red) NPs. (i) DLS of the naked (orange), lipid-coated (green), and lipid-coated-IR780 (red) NPs. (j) EPR measurements of ROS production of the various nanoconstructs in water upon ultrasound stimulation. (k) Representative image of the spin-adduct of the DMPO-OH complex obtained after US stimulation at 1.5 W/cm². Data are expressed as mean ± standard deviation. Significance was analyzed by two-way ANOVA. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. Tukey’s correction was applied for multiple comparison.

Figure 2

(a) Cytotoxicity of naked NPs, lipid-coated NPs, lipid-coated IR780 NPs, and free IR780 on KPC cells after 24 h exposure. (b) Schematic protocol of in vitro tests. (c) Cell viability of KPC cells after NPs administration, with and without US stimulation, measured 24 h after US. (d) Fluorescence confocal microscopy of KPC cells internalizing lipid-coated-IR780 NPs after US stimulation at 0.8 W/cm² for 1 min. The NPs, labeled with AlexaFluor 647, are shown in the pink channel, IR780 is imaged in the red channel, nuclei are stained with DAPI (blue channel), membranes with WGA 550 (orange channel), and the green channel shows DCF signal due to the generation of ROS. (e) One-dimensional histograms of the fluorescence intensity associated with NPs internalization (NPs-R660) and DCF production (DCF-B510) for all treatment groups. (f) Histograms reporting the percentage of DCF-producing cells, (g) dead cells, (h) cells internalizing NPs, and (i) cells internalizing IR780 (US treatment 0.8 W/cm², 1 min, 100% DC). Data are expressed as mean ± standard deviation. Significance was analyzed by two-way ANOVA. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. Tukey’s correction was applied for multiple comparison.

Figure 3

(a) Scheme of the treatment plan. (b) Tumor weights on day 14. (c) In vivo IVIS imaging over time of naked and lipid-coated NPs dyed with AlexaFluor 700 (excitation 675 nm, emission 720 nm). (d) IVIS pictures of organs explanted at various time points (excitation 675 nm, emission 720 nm). Data are expressed as mean ± standard deviation. Significance was analyzed by either one-way or two-way ANOVA. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. Tukey’s correction was applied for multiple comparison.

Figure 4

(a) Scheme of the treatment plan. (b) Tumor weights on day 14. (c) Digital photos of tumors explanted on day 14. (d) Tumor volume progression in vivo over time. (e) Total radiant efficiency (excitation 675 nm, emission 720 nm) of the explanted tumors. (f) In vivo total radiant efficiency progression within tumor regions of interest (ROIs) of the groups treated with NPs only and (g) those treated with NPs and ultrasound (excitation 675 nm, emission 720 nm). Data are expressed as mean ± standard deviation. Significance was analyzed by either one-way or two-way ANOVA. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. Tukey’s correction was applied for multiple comparison.

Figure 5

(a) Representative tumor sections, stained with the apoptosis assay, showing increasingly bigger apoptotic regions (brown) with respect to the total tumor slice area in groups receiving the combined treatment (scalebar 1 mm) and corresponding magnifications (scale bar 50 μm). (b) Percentage of apoptotic areas with respect to total tumor area for all treatment groups (n = 5/group). Data are expressed as mean ± standard deviation. Significance was analyzed by one-way ANOVA. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. (c) Representative Masson’s Trichrome staining of tumor slides where collagen fibers, stained in blue, decrease in the sample receiving the combined treatment (scalebar 50 μm).

Figure 6

(a) Scheme of the treatment plan. (b) Tumor volume progression in vivo. (c) Tumor weights of the explanted tumors. (d) Digital photos of the explanted tumors. (e) Percentage of CD4⁺Ki67⁺ cells (top) and effector Tregs (CD4⁺CD25⁺Foxp3⁺CTLA-4⁺, bottom) in TdLN. (f) Percentage of CD3⁺, CD4⁺ and CD8⁺ki67⁺ cells (top) and M1, M2, and M1/M2 ratio (bottom) in tumors. Data are expressed as mean ± standard deviation. Significance was analyzed by either one-way or two-way ANOVA. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. Tukey’s correction was applied for multiple comparison.

Figure 7

(a) Scheme of the treatment plan. (b) Tumor volume progression in vivo. Data are expressed as mean ± standard deviation. Significance was analyzed by two-way ANOVA. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001. Tukey’s correction was applied for multiple comparison. (c) Kaplan–Meier survival curves. Significance was analyzed by log-rank test; n = 8/group; **p < 0.001; ***p < 0.0005. (d) Tumor growth curve in vivo, each plot referred to a single experimental group.