High aspect-ratio nanomaterials have recently emerged as promising drug delivery vehicles due to evidence of strong cellular association and prolonged in vivo circulation times. Cyclic peptide - ...polymer conjugate nanotubes are excellent candidates due to their elongated morphology, their supramolecular composition and high degree of pliability due to the versatility in manipulating amino acid sequence and polymer type. In this work, we explore the use of a nanotube structure on which a potent anti-cancer drug, camptothecin, is attached alongside hydrophilic or amphiphilic RAFT polymers, which shield the cargo. We show that subtle modifications to the cleavable linker type and polymer architecture have a dramatic influence over the rate of drug release in biological conditions. In vitro studies revealed that multiple cancer cell lines in 2D and 3D models responded effectively to the nanotube treatment, and analogous fluorescently labelled materials revealed key mechanistic information regarding the degree of cellular uptake and intracellular fate. Importantly, the ability to instruct specific drug release profiles indicates a potential for these nanomaterials as vectors which can provide sustained drug concentrations for a maximal therapeutic effect.
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Here, we aimed to chemically modify PAMAM dendrimers using lysine as a site-selective anchor for successfully delivering mRNA while maintaining a low toxicity profile. PAMAM dendrimers were ...multi-functionalised by amidation reactions in a regioselective, quantitative and stepwise manner with carefully selected property-modifying surface groups. Alternatively, novel lysine-based dendrimers were prepared in the same manner with the aim to unlock their potential in gene delivery. The modified dendrimers were then formulated with Cy5-EGFP mRNA by bulk mixing via liquid handling robotics across different nitrogen to phosphate ratios. The resulting dendriplexes were characterised by size, charge, mRNA encapsulation, and mRNA binding affinity. Finally, their in-vitro delivery activity was systematically investigated across key cellular trafficking stages to relate chemical design to cellular effect. We demonstrate our findings in different cell lines and benchmarked relative to a commercially available transfection agent, jetPEI®. We demonstrate that specific surface modifications are required to generate small, reliable and well-encapsulated positively charged dendriplex complexes. Furthermore, we show that introduction of fusogenic groups is essential for driving endosomal escape and achieving cellular delivery and translation of mRNA in these cell lines.
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•PAMAM & lysine dendrimers were modified with functional groups for mRNA delivery•Automated formulation established optimal mRNA cargo loading conditions•In-vitro testing identified dendrimers with improved endosomal escape, particularly by p-toluylsulfonyl arginine groups•Dendrimers outperformed a commercial standard, jetPEI®•Effective dendrimers for mRNA delivery were well tolerated in vitro.
As activity in Earth orbit continues to grow, it is important to characterize the environment of near‐Earth space. One means of remotely sensing lower thermospheric neutrals is by measurement of O ...and N2 density through the observation of far‐ultraviolet (FUV) airglow of atomic oxygen at 135.6 nm and the N2 Lyman‐Birge‐Hopfield (LBH) bands (~130–180 nm), as has been done on the Ionospheric Connection Explorer (ICON), Global‐scale Observations of the Limb and Disk (GOLD), and Thermosphere Ionosphere Mesosphere Energetics and Dynamics (TIMED) missions. This technique is not without limitations, however, as the FUV measurements suffer from contamination by ionospheric emissions at low latitudes and auroral emissions excited by precipitating energetic electrons and protons at high latitudes. Previous work has shown the potential for making measurements of O and N2 density in the lower‐middle thermosphere using observations of extreme‐ultraviolet (EUV) airglow. This measurement approach has a potential advantage in that it does not have an inherent ionospheric emission that must be accounted for. Additionally, these emissions are primarily excited directly by solar UV rather than electron impact and thus have the potential to enable expansion of neutral density observations into the auroral zone and polar cap where the FUV measurement cannot be applied. This article demonstrates a new approach and algorithm designed to retrieve thermospheric O and N2 density from 150 to 400 km using measurements from the ICON EUV instrument. The retrieval results throughout 2020 are summarized and compared to measurements from ICON FUV, GOLD, and SWARM.
Plain Language Summary
The region of space where most satellites orbit is almost empty ‐ but not completely empty. The small amount of nitrogen and oxygen gas present can cause satellites to gradually fall out of orbit through drag. A failure to understand and evaluate changes in the density of the upper atmosphere led to the loss of 38 Starlink satellites in February 2022, for example, This is one reason that it is important to understand how much gas is in near‐Earth space and how the amount changes over time, but orbit decay due to drag makes it difficult to make direct measurements. One common solution is to measure the amount of gas from afar, taking advantage of the fact that these gases emit small amounts of light in the far‐UV spectrum. There are some limitations to this approach, one of which being that charged particles in the same region can create an extra and unrelated signal, which requires correction. This paper presents a novel approach which uses more energetic extreme‐UV light as an alternative to the far‐UV. We show the results of this new algorithm and demonstrate that the measurements line up well with those from other instruments and spacecraft.
Key Points
We present an algorithm concept for remote sensing thermospheric O and N2, novel in its use of EUV airglow measurements
Comparison to related measurements during conjunctions with ICON FUV, GOLD, and Swarm are favorable
Retrieval results from 2020 indicate cooler atmospheres than those predicted by MSIS
Polymeric micelles have been extensively used as nanocarriers for the delivery of chemotherapeutic agents, aiming to improve their efficacy in cancer treatment. However, the poor loading capacity, ...premature drug release, non-uniformity, and reproducibility still remain the major challenges. To create a stable polymeric micelle with high drug loading, a telodendrimer micelle was developed as a nanocarrier for fulvestrant, as an example of a drug that has extremely poor water solubility (sub-nanomolar range). Telodendrimers were prepared by the synthesis of hydrophilic linear poly(sarcosine) and growing a lysine dendron from the chain terminal amine by divergent synthesis. At the periphery of the dendritic block, either 4, 8, or 16 fulvestrant molecules were conjugated to the lysine dendron creating a hydrophobic block. Having drug molecules as a part of the carrier not only reduces the usage of the inert carrier materials but also prevents the drugs from leakage and premature release by diffusion. The self-assembled telodendrimer micelles demonstrated good colloidal stability (cmc < 2 μM) in buffer and were uniform in size. In addition, these telodendrimer micelles could solubilize additional fulvestrant yielding an excellent overall drug loading capacity of up to 77 wt % total drug load (summation of conjugated and encapsulated). Importantly, the size of the micelles could be tuned between 25 and 150 nm by controlling (i) the ratio between hydrophilic and hydrophobic blocks and (ii) the amount of encapsulated fulvestrant. The versatility of these telodendrimer-based micelle systems to both conjugated and encapsulated drugs with high efficiency and stability, in addition to possessing other tuneable properties, makes it a promising drug delivery system for a range of active pharmaceutical ingredients and therapeutic targets.
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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.
This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymerization of ...the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were observed, representing ideal properties for biophysical targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic molecule SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body weight loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chemical moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufacturing perspective and report characterization of the polymers with a variety of techniques.
Ion mobility mass spectrometry (IM-MS) measures the mass, size, and shape of ions in the same experiment, and structural information is provided via collision cross-section (CCS) values. The majority ...of commercially available IM-MS instrumentation relies on the use of CCS calibrants, and here, we present data from a family of poly(l-lysine) dendrimers and explore their suitability for this purpose. In order to test these compounds, we employed three different IM-MS platforms (Agilent 6560 IM-QToF, Waters Synapt G2, and a home-built variable temperature drift tube IM-MS) and used them to investigate six different generations of dendrimers in two buffer gases (helium and nitrogen). Each molecule gives a highly discrete CCS distribution suggestive of single conformers for each m/z value. The DTCCSN2 values of this series of molecules (molecular weight: 330–16,214 Da) range from 182 to 2941 Å2, which spans the CCS range that would be found by many synthetic molecules including supramolecular compounds and many biopolymers. The CCS values for each charge state were highly reproducible in day-to-day analysis on each instrument, although we found small variations in the absolute CCS values between instruments. The rigidity of each dendrimer was probed using collisionally activated and high-temperature IM-MS experiments, where no evidence for a significant CCS change ensued. Taken together, this data indicates that these polymers are candidates for CCS calibration and could also help to reconcile differences found in CCS measurements on different instrument geometries.
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.
Imaging mass cytometry (IMC) offers the opportunity to image metal- and heavy halogen-containing xenobiotics in a highly multiplexed experiment with other immunochemistry-based reagents to ...distinguish uptake into different tissue structures or cell types. However, in practice, many xenobiotics are not amenable to this analysis, as any compound which is not bound to the tissue matrix will delocalize during aqueous sample-processing steps required for IMC analysis. Here, we present a strategy to perform IMC experiments on a water-soluble polysarcosine-modified dendrimer drug-delivery system (S-Dends). This strategy involves two consecutive imaging acquisitions on the same tissue section using the same instrumental platform, an initial laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MSI) experiment followed by tissue staining and a standard IMC experiment. We demonstrated that settings can be found for the initial ablation step that leave sufficient residual tissue for subsequent antibody staining and visualization. This workflow results in lateral resolution for the S-Dends of 2 μm followed by imaging of metal-tagged antibodies at 1 μm.
Since the earliest space‐based observations of Earth's atmosphere, ultraviolet (UV) airglow has proven a useful resource for remote sensing of the ionosphere and thermosphere. The NASA Ionospheric ...Connection Explorer (ICON) spacecraft, whose mission is to explore the connections between ionosphere and thermosphere utilizes UV airglow in the typical way: an extreme‐UV (EUV) spectrometer uses dayglow between 54 and 88 nm to measure the density of O+, and a far‐UV spectrograph uses the O 135.6 nm doublet and N2 Lyman‐Birge‐Hopfield band dayglow to measure the column ratio of O to N2 in the upper thermosphere. Two EUV emission features, O+ 61.6 and 83.4 nm, are used for the O+ retrieval; however, many other features are captured along the EUV instrument's spectral dimension. In this study, we examine the other dayglow features observed by ICON EUV and demonstrate that it measures a nitrogen feature around 87.8 nm which can be used to observe the neutral thermosphere.
Plain Language Summary
The ionosphere is a region of near‐Earth space made up of plasma. NASA's Ionospheric Connection Explorer (ICON) mission seeks explore the factors influencing formation of the ionosphere and how it interacts with Earth and its atmosphere. One of the ways ICON does this is by measuring airglow: light released by the air in the upper atmosphere. This occurs with visible light, with the same shades seen in the aurora; it also occurs in the ultraviolet range, invisible to the human eye but visible to ICON instruments. An imager is included on ICON to measure extreme‐ultraviolet light, almost as energetic as X‐rays. Certain atoms and molecules in the atmosphere are known to glow at specific wavelengths. By measuring the brightness of airglow at certain wavelengths, ICON is able see the structure of ionospheric oxygen. The instrument also measures dimmer emissions at other wavelengths, some of which are known to come from certain atmospheric species and others which are unknown or uncertain. Here we look at the other wavelengths and attempt to find their origins. We find that most are likely coming from oxygen. Interestingly, we find one that we think comes from nitrogen. This could be useful for measuring the abundance of molecular nitrogen in the upper atmosphere, a task currently performed by another instrument on the ICON spacecraft. We make a case for the practicality of this approach.
Key Points
A comprehensive introduction to the Ionospheric Connection Explorer Extreme ultraviolet dayglow observations is presented
Some dim emission features are identified as originating from O+ from similarity to known features
Emission near 87.8 nm follows N2 and, combined with 61.6 nm data, contains information about ΣO/N2
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