Microfluidic-assisted preparation of RGD-decorated nanoparticles: exploring integrin-facilitated uptake in cancer cell lines

Abstract

This study is about fine tuning the targeting capacity of peptide-decorated nanoparticles to discriminate between cells that express different integrin make-ups. Using microfluidic-assisted nanoprecipitation, we have prepared poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles with a PEGylated surface decorated with two different arginine-glycine-aspartic acid (RGD) peptides: one is cyclic (RGDFC) and has specific affinity towards α v β 3 integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally α v β 3 and α 5 β 1. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no α v β 3 , moderate in α 5 β 1) and glioma U87MG (very high in α v β 3 , moderate/high in α 5 β 1). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-α v β 3) but not by A2780 (same as PEGylated particles). The linear peptide, on the other hand, did not differentiate between the cell lines, and the uptake increase vs. control particles was never higher than 50%, indicating a possible low and unselective affinity for various integrins. The strong preference of U87MG for cyclic (vs. linear) peptide-decorated nanoparticles was shown in 2D culture and further demonstrated in spheroids. Our results demonstrate that targeting specific integrin make-ups is possible and may open the way to more precise treatment, but more efforts need to be devoted to a better understanding of the relation between RGD structure and their integrinbinding capacity. Integrins are targetable receptors. They are transmembrane glycoproteins, which connect cell bodies to pericellular structures and therefore are critical in cell-cell and cell-environment interactions 1. Structurally, they are heterodimeric proteins composed of non-covalently linked α and β subunits 2. To date 18α and 8β subunits, and 24 different heterodimers have been reported in humans, with each heterodimer displaying unique binding properties, tissue distribution, and biological functions 1 ,3. Although integrins are virtually ubiquitous, their heterodimeric identity and level of expression are a physio-pathological signature, which has a specific relevance in cancer 4 , e.g. in the formation of metastatic niches or in angiogenetic processes 5. Most studies have focused either on integrins in peritumoral environments, e.g. on endothelial cells (ECs), critical to (neo)angiogenesis, or on those directly present on tumor cells 6. It is now well known that α v-containing integrins are highly expressed in both scenarios, but poorly expressed on healthy cells 7. In particular, α v β 3 (e.g. in pro-tumoral ECs) is the most abundant integrin capable of regulating angiogenesis 8. Among other heterodimers, also α 5 β 1 has been

Smart MSN-Drug-Delivery System for Tumor Cell Targeting and Tumor Microenvironment Release

“…The invasive growth of tumors requires a large amount of nutrients, which promotes the formation of new blood vessels. Endogenous α v β 3 integrin transmembrane glycoprotein, considered as a marker of angiogenesis, is widely distributed on tumor neovascular. , Research findings indicate that the arginine-glycine-aspartate sequence (RGD) can combine with α v β 3 integrin to inhibit tumor angiogenesis and thus prevent tumor growth and metastasis. , The binding ability of the cyclic RGD peptide (cRGD) to α v β 3 integrin and the inhibition of tumor neovascularization have been shown to be stronger than those of linear RGD peptide. − Therefore, the functionalization of drug-delivery carriers with cRGD targeting ligand can effectively increase their retention effect and enrich on the tumor cell surface. − In addition, according to the literature, − folate receptor (FR) is highly expressed on the surface of most cancer cells to absorb folic acid (FA), which is required for the rapid growth of cancer cells. The introduction of FA into the carrier surface can increase the probability of the carrier being captured and internalized by cancer cells via the active targeting function.…”

Microfluidic Nanoparticles for Drug Delivery

“…The versatile HFF device, staggered herringbone micromixer, [105] two-phase gas-liquid microfluidic mixers (Figure 6b), [102,[106][107][108][109][110] automated microfluidic Asia 320 flow reactor, [111] the microfluidic coflow capillary device, [112] and impact-jet micromixers [113] have been developed to prepare the synthetic polymer NPs in recent years.…”

Recent Advances in the Surface Functionalization of PLGA-Based Nanomedicines

“…RGDSP demonstrated similar internalization in both cell lines and no more than 50% of the uptake shown by the cyclic peptide, which may be related to its possible low and non-specific affinity with different integrins. Thus, these findings show the possibility of targeting cells with specific integrins, using various RGD peptides [ 177 ].…”