Gold nanoparticle delivery to solid tumors: a multiparametric study on particle size and the tumor microenvironment

Abstract

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

Gold Nanoparticle Delivery to Solid Tumors by Macrophage Depletion, Hypoxia Inhibition, and Collagen Degradation

“…Given that HIF-1α induces collagen gene expression, 36,37 ECM density was expected to be greatly reduced as a result of acriflavine treatment, and although there is some reduction and the overall distributions for ECM density are lower for this treatment group compared to the control, this difference was not significant. This can be due to the fact that ECM density in Renca tumors is not very high to begin with, and while perfusion in these tumors is relatively good, as already established in our previous studies, 40,41 hypoxia levels may be relatively low, thereby limiting the benefits of acriflavine on secondary outcomes. Blood vessel maturity was significantly Figure 3.…”

“…To measure hypoxia levels, animals were injected IV with Hypoxi-sense680 (2 nmol/100 uL per mouse, PerkinElmer, Life Sciences, Zaventem, Belgium) 24 h after the last pharmacological treatment (together with the Au NPs), and fluorescence signals were measured using the IVIS after 24 h. Levels of (1) tumor necrosis, (2) ECM density, and (3) blood vessel maturity and coverage were obtained by staining tissue slices with (1) hematoxylin and eosin, (2) Picrosirius Red and Fastgreen, and (3) antibodies against CD31 (endothelial cells), laminin, and NG2 (pericytes), respectively, as performed in our previous studies. 40,41 Analysis of Immune Cell Composition in the Tumor. Singlecell suspensions were obtained from isolated tumors using the GentleMACS tissue dissociator and accompanying kit (Miltenyi Biotec, Gladbach, Germany) as per company protocol.…”

“…To measure the amount of Au NPs present in the total tumor, ICP-MS was performed on each of the tumor samples as described in our previous studies. 40,41 Statistics. Statistical analyses were performed using GraphPad Prism 9.0, and comparisons between control and treated groups were obtained using one-way ANOVA.…”

“…Previous work had shown that NP delivery efficacy to solid tumors is linked to various parameters but that the level of TAMs, degree of hypoxia, and extent of ECM in the tumor play important roles. 40,41 In the present study, we study the effect of pharmacological interference with any of those parameters on NP delivery efficacy and uptake by cancer cells. For this, aCSFR1 treatment was chosen as a means to temporarily remove macrophages, as it is known that the majority of NPs are cleared from the blood by Kupffer cells 42,43 and the ones that do reach the tumor mainly end up in TAMs.…”

“…60 The lack of an effect here is most likely due to the fact that, while vessel normalization was observed, the extent of it was less outspoken as reported in previous works. 41 Treatment with acriflavine in combination with collagenase resulted in similar effects, leading to reductions in hypoxia and increased total vessel area and maturity levels, although this was less outspoken than for acriflavine alone. This may be due to the fact that collagenase-mediated ECM remodeling may activate CAFs and result in TGFβ increases, which are linked with reduced vessel maturation and perfusion.…”