Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood–brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines. Fluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells. In the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible to tune the intracellular drug release rate by choosing appropriate monomers from the PACA family or by using hybrid PACA nanoparticles containing different monomers. In addition, we showed that the uptake of PACA nanoparticles depends not only on the monomer material, but also on the cell type, and that different cell lines can use different internalization pathways.

Although nanotoxicology has become a large research field, assessment of cytotoxicity is often reduced to analysis of one cell line only. Cytotoxicity of nanoparticles is complex and should, preferentially, be evaluated in several cell lines with different methods and on multiple nanoparticle batches. Here we report the toxicity of poly(alkyl cyanoacrylate) nanoparticles in 12 different cell lines after synthesizing and analyzing 19 different nanoparticle batches and report that large variations were obtained when using different cell lines or various toxicity assays. Surprisingly, we found that nanoparticles with intermediate degradation rates were less toxic than particles that were degraded faster or more slowly in a cell-free system. The toxicity did not vary significantly with either the three different combinations of polyethylene glycol surfactants or with particle size (range 100–200 nm). No acute pro- or anti-inflammatory activity on cells in whole blood was observed.

Preclinical research has demonstrated that nanoparticles and macromolecules can accumulate in solid tumors due to the enhanced permeability and retention effect. However, drug loaded nanoparticles often fail to show increased efficacy in clinical trials. A better understanding of how tumor heterogeneity affects nanoparticle accumulation could help elucidate this discrepancy and help in patient selection for nanomedicine therapy. Here we studied five human tumor models with varying morphology and evaluated the accumulation of 100 nm polystyrene nanoparticles. Each tumor model was characterized in vivo using micro-computed tomography, contrast-enhanced ultrasound and diffusion-weighted and dynamic contrast-enhanced magnetic resonance imaging. Ex vivo, the tumors were sectioned for both fluorescence microscopy and histology. Nanoparticle uptake and distribution in the tumors were generally heterogeneous. Density of functional blood vessels measured by fluorescence microscopy correlated significantly (p = 0.0056) with nanoparticle accumulation and interestingly, inflow of microbubbles measured with ultrasound also showed a moderate but significant (p = 0.041) correlation with nanoparticle accumulation indicating that both amount of vessels and vessel morphology and perfusion predict nanoparticle accumulation. This indicates that blood vessel characterization using contrast-enhanced ultrasound imaging or other methods could be valuable for patient stratification for treatment with nanomedicines.

Treatment of glioblastoma and other diseases in the brain is especially challenging due to the blood-brain barrier, which effectively protects the brain parenchyma. In this study we show for the first time that cabazitaxel, a semi-synthetic derivative of docetaxel can cross the blood-brain barrier and give a significant therapeutic effect in a patient-derived orthotopic model of glioblastoma. We show that the drug crosses the blood-brain barrier more effectively in the tumor than in the healthy brain due to reduced expression of p-glycoprotein efflux pumps in the vasculature of the tumor. Surprisingly, neither ultrasound-mediated blood-brain barrier opening (sonopermeation) nor drug formulation in polymeric nanoparticles could increase either accumulation of the drug in the brain or therapeutic effect. This indicates that for hydrophobic drugs, sonopermeation of the blood brain barrier might not be sufficient to achieve improved drug delivery. Nonetheless, our study shows that cabazitaxel is a promising drug for the treatment of brain tumors.

Immunocompetent murine models are important tools for preclinical evaluation of immunotherapies. Here, six different immunocompetent tumor models based on four different cell lines were characterized, including metastatic lung cancer (CMT 167), triple‐negative breast cancer (4T1), pancreatic cancer (KPCY), and colon cancer (MC38). The tumors were implanted subcutaneously or orthotopically before the animals were treated with anti‐PD1 checkpoint inhibitor. A range of innate and adaptive immune cells were then quantified by flow cytometry of single‐cell suspensions from the tumors. Furthermore, confocal laser scanning microscopy was used to quantify the density and distribution of T‐cells in frozen sections. A model‐dependent cellular immune landscape was observed, with variable responsiveness toward anti‐PD1, ranging from the most responsive MC38 colon cancer model to the least responsive 4T1 breast cancer model. The study provides an overview of the immune landscape of these tumor models, and a foundation for further elucidation of pro‐tumor and anti‐tumor mechanisms behind heterogeneous responses towards immunotherapies.

Drug delivery to tumors is challenging due to biological barriers obstructing effective delivery. Sonopermeation with ultrasound and microbubbles has been shown to improve therapeutic effect of many classes of drugs, but the underlying mechanism is not fully understood. In this study, two subcutaneous xenograft tumor models, that differed substantially in blood vessel density and stiffness, is treated with poly(alkyl cyanoacrylate) nanoparticles and nanoparticle‐stabilized microbubbles combined with ultrasound. Improved nanoparticle accumulation and extracellular matrix (ECM) penetration is found. The stiffness and solid stress in the tumors are measured and it is discovered that sonopermeation can reduce the solid stress in both models, with the highest effect in the stiffest tumor model. This suggests that sonopermeation affects not only the blood vessel wall which has been described previously, but also the ECM to reduce solid stress and increase diffusion and transport of nanomedicines.