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
β
integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally α
β
and α
β
. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no α
β
, moderate in α
β
) and glioma U87MG (very high in α
β
, moderate/high in α
β
). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-α
β
) 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.
Abstract
Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of ...phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.
Small interfering ribonucleic acids (siRNAs) form potentially the most important class of next generation therapeutics. However, achieving their efficient delivery in the correct dose, time and ...location in the body remains a significant challenge. Rapid developments in the chemistries of siRNA formulations are enabling new strategies to overcome the core obstacles to delivery which include poor ribonuclease (RNase) resistance, short biological half-life, lack of tissue targeting, inefficient cellular uptake and undesirable toxicity. In this review we describe these principal challenges and evaluate recent approaches proposed to overcome the chemical, biochemical and physiological barriers. The role of the specific chemical structure of siRNA is considered and an overview of selected literature-reported siRNA formulations is provided. These include chemically-modified siRNAs and analogues, aptamer-siRNA chimeras, self-assembled nanoparticles, lipid and polymer complexes, bioconjugates and fusion protein complexes. We conclude the review with an outlook for the clinical use of this highly promising, but pharmaceutically challenging biotherapeutic.
Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad ...application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.
Successfully translating anti-cancer nanomedicines from pre-clinical proof of concept to demonstration of therapeutic value in the clinic is challenging. Having made significant advances with drug ...delivery technologies, we must learn from other areas of oncology drug development, where patient stratification and target-driven design have improved patient outcomes. We should evolve our nanomedicine development strategies to build the patient and disease into the line of sight from the outset. The success of small molecule targeted therapies has been significantly improved by employing a specific decision-making framework, such as AstraZeneca's 5R principle: right target/efficacy, right tissue/exposure, right safety, right patient, and right commercial potential. With appropriate investment and collaboration to generate a platform of evidence supporting the end clinical application, a similar framework can be established for enhancing nanomedicine translation and performance. Building informative data packages to answer these questions requires the following: (I) an improved understanding of the heterogeneity of clinical cancers and of the biological factors influencing the behaviour of nanomedicines in patient tumours; (II) a transition from formulation-driven research to disease-driven development; (III) the implementation of more relevant animal models and testing protocols; and (IV) the pre-selection of the patients most likely to respond to nanomedicine therapies. These challenges must be overcome to improve (the cost-effectiveness of) nanomedicine development and translation, and they are key to establishing superior therapies for patients.
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Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver an RNA/DNA cargo to cells overexpressing HA receptors such as CD44. For these systems, unequivocal links have not been established ...yet between chitosan macromolecular (molecular weight; degree of deacetylation, i.e., charge density) and nanoparticle variables (complexation strength, i.e., stability; nucleic acid protection; internalization rate) on one hand, and transfection efficiency on the other hand. Here, we have focused on the role of avidity on transfection efficiency in the CD44-expressing HCT-116 as a cellular model; we have employed two differently sized payloads (a large luciferase-encoding mRNA and a much smaller anti-Luc siRNA), and a small library of chitosans (variable molecular weight and degree of deactylation). The RNA avidity for chitosan showedas expectedan inverse relationship: higher avidity–higher polyplex stability–lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity–higher polyplex stability but also higher transfection efficiency. Surprisingly, the best transfecting particles were those with the lowest propensity for RNA release, although this might be a misleading relationship: for example, the same macromolecular parameters that increase avidity can also boost chitosan’s endosomolytic activity, with a strong enhancement in transfection. The performance of these nonviral vectors appears therefore difficult to predict simply on the basis of carrier- or payload-related variables, and a more holistic consideration of the journey of the nanoparticle, from cell uptake to cytosolic bioavailability of payload, is needed. It is also noteworthy that the nanoparticles used in this study showed optimal performance under slightly acidic conditions (pH 6.4), which is promising for applications in a tumoral extracellular environment. It is also worth pointing out that under these conditions we have for the first time successfully delivered mRNA with chitosan/HA nanoparticles.
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Nanotechnology research over the past several decades has been aimed primarily at improving the physicochemical properties of small molecules to produce druggable candidates as well ...as for tumor targeting of cytotoxic molecules. The recent focus on genomic medicine and the success of lipid nanoparticles for mRNA vaccines have provided additional impetus for the development of nanoparticle drug carriers for nucleic acid delivery, including siRNA, mRNA, DNA, and oligonucleotides, to create therapeutics that can modulate protein deregulation. Bioassays and characterizations, including trafficking assays, stability, and endosomal escape, are key to understanding the properties of these novel nanomedicine formats. We review historical nanomedicine platforms, characterization methodologies, challenges to their clinical translation, and key quality attributes for commercial translation with a view to their developability into a genomic medicine. New nanoparticle systems for immune targeting, as well as in vivo gene editing and in situ CAR therapy, are also highlighted as emerging areas.
Messenger RNA (mRNA) is a biomolecule with a wide range of promising clinical applications. However, the unstable nature of mRNA and its susceptibility to degradation by ribonucleases (RNases) ...necessitate the use of specialized formulations for delivery. Polycations are an emerging class of synthetic carriers capable of packaging nucleic acids, and may serve as suitable RNase-resistant formulations for mRNA administration. Here, we explore the application of VIPER and sunflower polycations, two polycations previously synthesized by our group, for the delivery of mRNA in comparison to branched poly(ethylenimine); all three polycations have been shown to efficiently deliver plasmid DNA (pDNA) to cultured cells. Despite successful mRNA condensation and packaging, transfection studies reveal that these three polycations can only efficiently deliver mRNA under serum-free conditions, while pDNA delivery is achieved even in the presence of serum. RNase resistance studies confirm that nuclease degradation of mRNA cargo remains a significant barrier to mRNA delivery using these polycations. These results emphasize the need for additional strategies for nuclease protection of mRNA cargo beyond electrostatic complexation with polycation.
Sizing up the Next Generation of Nanomedicines Clogston, Jeffrey D.; Hackley, Vincent A.; Prina-Mello, Adriele ...
Pharmaceutical research,
01/2020, Letnik:
37, Številka:
1
Journal Article
Recenzirano
Odprti dostop
During the past two decades the nanomedicine field has experienced significant progress. To date, over sixty nanoparticle (NP) formulations have been approved in the US and EU while many others are ...in clinical or preclinical development, indicating a concerted effort to translate promising bench research to commercially viable pharmaceutical products. The use of NPs as novel drug delivery systems, for example, can improve drug safety and efficacy profiles and enable access to intracellular domains of diseased cells, thus paving the way to previously intractable biological targets. However, the measurement of their physicochemical properties presents substantial challenges relative to conventional injectable formulations. In this perspective, we focus exclusively on particle size, a core property and critical quality attribute of nanomedicines. We present an overview of relevant state-of-the-art technologies for particle sizing, highlighting the main parameters that can influence the selection of techniques suitable for a specific size range or material. We consider the increasing need, and associated challenge, to measure size in physiologically relevant media. We detail the importance of standards, key to validate any measurement, and the need for suitable reference materials for processes used to characterize novel and complex NPs. This perspective highlights issues critical to achieve compliance with regulatory guidelines and to support research and manufacturing quality control.
Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between ...in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.
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Lipid nanoparticles are promising vectors for delivery of macromolecular therapeutics, such as mRNA. Pre-clinical testing of vectors relies on cell models of disease, such as cancer. Here, using high-content endocytic profiling in different cell types, we have identified key endocytic features that are critical to effective cytosolic delivery of mRNA.
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