Nucleic Acid-Loaded Lipid Nanoparticle Interactions with Model Endosomal Membranes

[Image: see text] Lipid nanoparticles (LNPs) are important delivery systems for RNA-based therapeutics, yet the mechanism of their interaction with endosomal membranes remains unclear. Here, the interactions of nucleic acid-loaded LNPs that contain an ionizable lipid with models of the early and late endosomal membranes are studied, for the first time, using different reflectometry techniques. Novel insight is provided with respect to the subphase pH, the stage of the endosome, and the nature of the nucleic acid cargo. It is found that the insertion of lipids from the LNPs into the model membrane is greatest at pH 6.5 and 5.5, whereas at higher pH, lipid insertion is suppressed with evidence instead for the binding of intact LNPs, demonstrating the importance of the pH in the fusion of LNPs undergoing the endosomal pathway. Furthermore, and independently of the pH, the effect of the early- versus late-stage endosomal models is minimal, suggesting that the increased fluidity and anionic nature of the late endosome has little effect on the extent of LNP interaction. Last, there is greater nucleic acid delivery from LNPs containing mRNA than Poly(A), indicating that the extent of interaction can be tuned according to the nature of the nucleic acid cargo. Such new information on the relative impact of factors influencing nucleic acid delivery by LNP interactions with endosomal membranes is important in the design and tuning of vehicles with improved nucleic acid delivery capacities

Binding and Internalization in Receptor‐Targeted Carriers: The Complex Role of CD44 in the Uptake of Hyaluronic Acid‐Based Nanoparticles (siRNA Delivery)

This paper is about the actual role of CD44 in the perspective of a hyaluronic acid (HA)-based, targeted therapy. CD44 is the main HA receptor: it is present both in healthy and cancerous cells, but is overexpressed in many carcinomas, with important roles in their initiation and malignancy. This, and its endocytic capacities, have encouraged the use of HA to design CD44-targeting carriers. Here, we have used HA-decorated nanoparticles to deliver a siRNA payload to a panel of human cells comprising both tumoral (AsPC-1, PANC-1, HT-29, HCT-116) and non-tumoral (fibroblasts, differently polarized THP-1 macrophages, HUVEC) lines; we have evaluated in a comparative and quantitative fashion the initial binding of the nanoparticles, their internalization rate and the eventual silencing efficiency (cyclophilin B (PPIB) gene).A first result of our study is that, in general, tumoral cells internalized faster and experienced higher silencing than non-tumoral cells. This result is promising as it suggests that, when in a tumor environment, HA nanocarriers may have limited off-target effects.A more far-reaching result comes from the quantitative analysis of the inter-relation between the four parameters of our study (i.e. total CD44 expression, extent of HA cell surface binding, internalization rate and silencing efficiency). Our experiments showed no correlation between extent of binding (an early event) and any of the other parameters. On the contrary, silencing correlated well both with the speed of the internalization process and also with CD44 expression. This study, therefore, confirms on one hand that HA-based carriers can perform a targeted therapeutic action, but on the other it suggests that this may not be due to the selective binding of a cell surface marker, but possibly to a later recognition event leading to selective internalization

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

From Springer Nature via Jisc Publications RouterHistory: received 2020-02-23, accepted 2020-08-06, registration 2020-08-17, pub-electronic 2020-09-02, online 2020-09-02, collection 2020-12Publication status: PublishedFunder: AstraZeneca; doi: http://dx.doi.org/10.13039/100004325Funder: University of Manchester; doi: http://dx.doi.org/10.13039/501100000770Abstract: 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 αvβ3 integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally αvβ3 and α5β1. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no αvβ3, moderate in α5β1) and glioma U87MG (very high in αvβ3, moderate/high in α5β1). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-αvβ3) 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 integrin-binding capacity

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

Funder: AstraZeneca; doi: http://dx.doi.org/10.13039/100004325Funder: University of Manchester; doi: http://dx.doi.org/10.13039/501100000770This 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 αvβ3 integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally αvβ3 and α5β1. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no αvβ3, moderate in α5β1) and glioma U87MG (very high in αvβ3, moderate/high in α5β1). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-αvβ3) 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 integrin-binding capacity

The effectiveness of measles-mumps-rubella (MMR) vaccination in the prevention of pediatric hospitalizations for targeted and untargeted infections: a retrospective cohort study

Objectives: To evaluate the effectiveness of the measles-mumps-rubella (MMR) vaccine in reducing hospitalizations for infectious disease, targeted and not targeted, as well as from respiratory diseases in children in Rome. Methods: The cohort was recomposed through record linkage of 2 archives (vaccination register and hospital discharge records. Results: The analysis included 11,004 children. 20.9% did not receive the MMR vaccination, 49% and 30.1% received one and 2 doses. There were no hospitalizations for rubella, 2 for mumps, and 12 for measles. The vaccine was highly protective against measles and mumps hospitalizations (HR D 0.10; 95% CI: 0.03.0.34). Regarding all infectious diseases there were 414 hospitalizations, and the vaccine was protective (HR D 0.29; 95% CI: 0.25 to 0.34). Concerning respiratory diseases, there were 809 admissions (7.4%), and the vaccine was highly protective (HR: 0.18; 95% CI: 0.07 to 0.48). Conclusions: MMR vaccination is effective for the primary prevention of target and not targeted infectious diseases and may also limit hospitalizations for respiratory diseases

Evaluating the Efficiency of Hyaluronic Acid for Tumor Targeting via CD44

The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called “uptake”) into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44s^high cells bound and internalized more HA compared to CD44s^low cells. Moreover, CD44s^high HDFs were less efficient in uptaking HA compared to CD44s^high cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile

Review of life science applications using submicron O-PTIR and simultaneous Raman microscopy: a new paradigm in vibrational spectroscopy

The recent advent of Optical Photothermal IR (O-PTIR), has enabled for the first time, submicron infrared microscopy in far-field reflection mode with the combination of Raman for simultaneous, correlative IR+Raman microscopy. These unique and exciting synergistic capabilities are now spawning interest in life science application. A broad range of life science applications, otherwise impossible with traditional FTIR/QCL microscopy, will be presented, ranging from live cell imaging in water, to ultra-high resolution images of breast tissue calcifications, amyloid aggregates in neurons (neurites and dendritic spines), individual collagen fibrils with polarized IR and individual isotopically labelled bacterial cells and more