Pancreatic ductal adenocarcinoma (PDAC) remains a challenging malignancy, mainly due to its resistance to chemotherapy and its complex tumour microenvironment characterised by stromal desmoplasia. There is a need for new strategies to improve the delivery of drugs and therapeutic response. Relevant preclinical tumour models are needed to test potential treatments. This paper compared orthotopic and subcutaneous PDAC tumour models and their suitability for drug delivery studies. A novel aspect was the broad range of tumour properties that were studied, including tumour growth, histopathology, functional vasculature, perfusion, immune cell infiltration, biomechanical characteristics, and especially the extensive analysis of the structure and the orientation of the collagen fibres in the two tumour models. The study unveiled new insights into how these factors impact the uptake of a fluorescent model drug, the macromolecule called 800CW. While the orthotopic model offered a more clinically relevant microenvironment, the subcutaneous model offered advantages for drug delivery studies, primarily due to its reproducibility, and it was characterised by a more efficient drug uptake facilitated by its collagen organisation and well-perfused vasculature. The tumour uptake seemed to be influenced mainly by the structural organisation and the alignment of the collagen fibres and perfusion. Recognising the diverse characteristics of these models and their multifaceted impacts on drug delivery is crucial for designing clinically relevant experiments and improving our understanding of pancreatic cancer biology.

Alginate hydrogels have been broadly investigated for use in medical applications due to their biocompatibility and the possibility to encapsulate cells, proteins, and drugs. In the treatment of peritoneal metastasis, rapid drug clearance from the peritoneal cavity is a major challenge. Aiming to delay drug absorption and reduce toxic side effects, cabazitaxel (CAB)-loaded poly(alkyl cyanoacrylate) (PACA) nanoparticles were encapsulated in alginate microspheres. The PACAlg alginate microspheres were synthesized by electrostatic droplet generation and the physicochemical properties, stability, drug release kinetics, and mesothelial cytotoxicity were analyzed before biodistribution and therapeutic efficacy were studied in mice. The 450 µm microspheres were stable at in vivo conditions for at least 21 days after intraperitoneal implantation in mice, and distributed evenly throughout the peritoneal cavity without aggregation or adhesion. The nanoparticles were stably retained in the alginate microspheres, and nanoparticle toxicity to mesothelial cells was reduced, while the therapeutic efficacy of free CAB was maintained or improved in vivo. Altogether, this work presents the alginate encapsulation of drug-loaded nanoparticles as a promising novel strategy for the treatment of peritoneal metastasis that can improve the therapeutic ratio between toxicity and therapeutic efficacy.

The delivery of nanoparticles to tumors has been shown preclinically to be improved by microbubble-mediated ultrasound. However, the mechanisms and biological effects are not fully understood. In this study, we explored the influence of the tumor microenvironment on nanoparticle uptake and microdistribution both with and without ultrasound and microbubble treatment. Three murine tumor models, KPC (pancreatic ductal adenocarcinoma), 4T1 (triple negative mammary carcinoma) and CT26 (colon carcinoma), were characterized with respect to extracellular matrix composition, tumor stiffness and perfusion. KPC and 4T1 tumors presented higher levels of collagen and hyaluronic acid and were stiffer compared to CT26, whereas all three tumors had similar levels of sulfated glycosaminoglycans. Furthermore, the 4T1 tumors appeared poorly vascularized with a lower cell density compared to KPC and CT26. All three tumors presented similar nanoparticle uptake, but extravasated nanoparticles traveled significantly shorter in KPC tumors compared to 4T1 and CT26. The effect of ultrasound and microbubble treatment on the tumor uptake and penetration of polymer nanoparticles into the extracellular matrix were evaluated using a treatment protocol previously shown to increase nanoparticle delivery to tumors. Interestingly, we found a significant increase in nanoparticle uptake in the soft CT26 tumor, but no effect of the ultrasound treatment in the stiff KPC and 4T1 tumors, suggesting that tumor stiffness is an important parameter for treatment with ultrasound and microbubbles. Ultrasound treatment resulted in a modest but not statistically significant improvement in nanoparticle penetration through the extracellular matrix. In tumors demonstrating increased uptake of nanoparticles following ultrasound treatment, the uptake correlated positively with blood volume. These findings emphasize the importance of taking the tumor microenvironment into consideration when optimizing ultrasound parameters for delivery of nanomedicine.

Pulsed focused ultrasound (FUS) in combination with microbubbles has been shown to improve delivery and penetration of nanoparticles in tumors. To understand the mechanisms behind this treatment, it is important to evaluate the contribution of FUS without microbubbles on increased nanoparticle penetration and transport in the tumor extracellular matrix (ECM). A composite agarose hydrogel was made to model the porous structure, the acoustic attenuation and the hydraulic conductivity of the tumor ECM. Single-particle tracking was used as a novel method to monitor nanoparticle dynamics in the hydrogel during FUS exposure. FUS exposure at 1 MHz and 1 MPa was performed to detect any increase in nanoparticle diffusion or particle streaming at acoustic parameters relevant for FUS in combination with microbubbles. Results were compared to a model of acoustic streaming. The nanoparticles displayed anomalous diffusion in the hydrogel, and FUS with a duty cycle of 20% increased the nanoparticle diffusion coefficient by 23%. No increase in diffusion was found for lower duty cycles. FUS displaced the hydrogel itself at duty cycles above 10%; however, acoustic streaming was found to be negligible. In conclusion, pulsed FUS alone cannot explain the enhanced penetration of nanoparticles seen when using FUS and microbubbles for nanoparticle delivery, but it could be used as a tool to enhance diffusion of particles in the tumor ECM.

Focused ultrasound has experimentally been found to enhance the diffusion of nanoparticles; our aim with this work is to study this effect closer using both experiments and non-equilibrium molecular dynamics. Measurements from single particle tracking of 40 nm polystyrene nanoparticles in an agarose hydrogel with and without focused ultrasound are presented and compared with a previous experimental study using 100 nm polystyrene nanoparticles. In both cases, we observed an increase in the mean square displacement during focused ultrasound treatment. We developed a coarse-grained non-equilibrium molecular dynamics model with an implicit solvent to investigate the increase in the mean square displacement and its frequency and amplitude dependencies. This model consists of polymer fibers and two sizes of nanoparticles, and the effect of the focused ultrasound was modeled as an external oscillating force field. A comparison between the simulation and experimental results shows similar mean square displacement trends, suggesting that the particle velocity is a significant contributor to the observed ultrasound-enhanced mean square displacement. The resulting diffusion coefficients from the model are compared to the diffusion equation for a two-time continuous time random walk. The model is found to have the same frequency dependency. At lower particle velocity amplitude values, the model has a quadratic relation with the particle velocity amplitude as described by the two-time continuous time random walk derived diffusion equation, but at higher amplitudes, the model deviates, and its diffusion coefficient reaches the non-hindered diffusion coefficient. This observation suggests that at higher ultrasound intensities in hydrogels, the non-hindered diffusion coefficient can be used.