Nanoparticle (NP) delivery to solid tumors remains an actively studied field, where several recent studies have shed new insights into the underlying mechanisms and the still overall poor efficacy. In the present study, Au NPs of different sizes were used as model systems to address this topic, where delivery of the systemically administered NPs to the tumor as a whole or to tumor cells specifically was examined in view of a broad range of tumor-associated parameters. Using non-invasive imaging combined with histology, immunohistochemistry, single-cell spatial RNA expression and image-based single cell cytometry revealed a size-dependent complex interaction of multiple parameters that promoted tumor and tumor-cell specific NP delivery. Interestingly, the data show that most NPs are sequestered by tumor-associated macrophages and cancer-associated fibroblasts, while only few NPs reach the actual tumor cells. While perfusion is important, leaky blood vessels were found not to promote NP delivery, but rather that delivery efficacy correlated with the maturity level of tumor-associated blood vessels. In line with recent studies, we found that the presence of specialized endothelial cells, expressing high levels of CD276 and Plvap promoted both tumor delivery and tumor cell-specific delivery of NPs. This study identifies several parameters that can be used to determine the suitability of NP delivery to the tumor region or to tumor cells specifically, and enables personalized approaches for maximal delivery of nanoformulations to the targeted tumor. Graphical Abstract

Nanoparticle delivery to solid tumors is known to be an inefficient process and various studies have tried to increase efficacy, but mechanistic and comparative studies remain scarce. Here, we use pharmacological agents to study the effect of vessel normalization or vessel disintegration on nanoparticle delivery to solid tumors. Using a multiparametric approach, we find that vessel disintegration fails to improve nanoparticle delivery and instead seems to have a limiting effect. Vessel normalization, however, improves delivery efficacy for nanoparticles ranging from 20 to 60 nm diameter. The normalization of the tumor blood vessels results in reduced hypoxia, reduced necrosis and an increase in Plvap+ CD276+ endothelial cells, which have been linked with nanoparticle delivery. Interestingly, where vessel disintegration stimulated cancer cell intravasation and associated metastases, vessel normalization impeded these processes. Together, these data reveal that, vessel normalization may be a safer and more suited approach for improving nanoparticle delivery to solid tumors, but its efficacy is limited by nanoparticle diameter and tumor parameters.

The delivery of nanomaterials (NMs) to solid tumors remains a heavily researched topic, and while significant efforts have been aimed at optimizing NM design for active tumor targeting, it has become clear that the aberrant and heterogeneous nature of the tumor microenvironment (TME) serves as a physical barrier to NM uptake by tumor cells. In our previous work, we show that NM delivery efficacy is dependent on NM size and is linked to various TME parameters, with the levels of tumorassociated macrophages (TAMs) and hypoxia, and density of the extracellular matrix (ECM) having the largest impact. In this study, we examined the effect of modulating these three components on the delivery of 20 and 60 nm gold nanoparticles (Au NPs) to the tumor in view of various TME parameters, including tumor size, necrosis, hypoxia, ECM density, blood vessel area and maturity, as well as different cell populations within the TME. While TAM depletion led to significant improvements in tumor vasculature maturity and coverage and increased NP uptake by cancer cells, it had no significant effect on overall NP delivery efficacy, possibly due to the failure of completely removing Kupffer cells. Additionally, reduction of hypoxia using acriflavine, a potent inhibitor of HIF-1α, was expected to improve NP delivery, but the results were also not significant. However, ECM breakdown using both acriflavine and collagenase led to significant improvements in NP delivery, especially for the larger NPs, although collagenase treatment alone resulted in increased tumor cell intravasation and metastasis. This study highlights the complexity and heterogeneity of the TME and the difficulties in optimizing NP delivery to solid tumors.

The ability to improve nanoparticle delivery to solid tumors is an actively studied domain, where various mechanisms are looked into. In previous work, the authors have looked into nanoparticle size, tumor vessel normalization, and disintegration, and here it is aimed to continue this work by performing an in‐depth mechanistic study on the use of ciRGD peptide co‐administration. Using a multiparametric approach, it is observed that ciRGD can improve nanoparticle delivery to the tumor itself, but also to tumor cells specifically better than vessel normalization strategies. The effect depends on the level of tumor perfusion, hypoxia, neutrophil levels, and vessel permeability. This work shows that upon characterizing tumors for these parameters, conditions can be selected that can optimally benefit from ciRGD co‐administration as a means to improve NP delivery to solid tumors.