Chemical and Biomolecular Engineering - Research Publications

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    The mechanical regulation of RNA binding protein hnRNPC in the failing heart
    Martino, F ; Varadarajan, NM ; Perestrelo, AR ; Hejret, V ; Durikova, H ; Vukic, D ; Horvath, V ; Cavalieri, F ; Caruso, F ; Albihlal, WS ; Gerber, AP ; O'Connell, MA ; Vanacova, S ; Pagliari, S ; Forte, G (AMER ASSOC ADVANCEMENT SCIENCE, 2022-11-23)
    Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by reexpressing fetal contractile proteins via transcriptional and posttranscriptional processes, such as alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is up-regulated and relocates to the sarcomeric Z-disc upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates with the translation machinery. Alterations in hnRNPC expression, phosphorylation, and localization can be mechanically determined and affect the AS of mRNAs involved in mechanotransduction and cardiovascular diseases, including Hippo pathway effector Yes-associated protein 1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by affecting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNA homeostatic mechanoregulation in pathological conditions.
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    Engineering Poly(ethylene glycol) Nanoparticles for Accelerated Blood Clearance Inhibition and Targeted Drug Delivery
    Tian, Y ; Gao, Z ; Wang, N ; Hu, M ; Ju, Y ; Li, Q ; Caruso, F ; Hao, J ; Cui, J (AMER CHEMICAL SOC, 2022-10-12)
    Surface modification with poly(ethylene glycol) (PEGylation) is an effective strategy to improve the colloidal stability of nanoparticles (NPs) and is often used to minimize cellular uptake and clearance of NPs by the immune system. However, PEGylation can also trigger the accelerated blood clearance (ABC) phenomenon, which is known to reduce the circulation time of PEGylated NPs. Herein, we report the engineering of stealth PEG NPs that can avoid the ABC phenomenon and, when modified with hyaluronic acid (HA), show specific cancer cell targeting and drug delivery. PEG NPs cross-linked with disulfide bonds are prepared by using zeolitic imidazolate framework-8 NPs as templates. The reported templating strategy enables the simultaneous removal of the template and formation of PEG NPs under mild conditions (pH 5.5 buffer). Compared to PEGylated liposomes, PEG NPs avoid the secretion of anti-PEG antibodies and the presence of anti-PEG IgM and IgG did not significantly accelerate the blood clearance of PEG NPs, indicating the inhibition of the ABC effect for the PEG NPs. Functionalization of the PEG NPs with HA affords PEG NPs that retain their stealth properties against macrophages, target CD44-expressed cancer cells and, when loaded with the anticancer drug doxorubicin, effectively inhibit tumor growth. The innovation of this study lies in the engineering of PEG NPs that can circumvent the ABC phenomenon and that can be functionalized for the improved and targeted delivery of drugs.
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    Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation
    Cevaal, PM ; Roche, M ; Lewin, SR ; Caruso, F ; Faria, M (JOURNAL OF VISUALIZED EXPERIMENTS, 2022-09)
    A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a chemotherapeutic might be functionalized with an antibody to enhance binding to cancer cells ("targeting") or functionalized with polyethylene glycol to help evade immune cell recognition ("stealth"). Even at a cellular level, optimizing the binding and uptake of a drug carrier is a complex biological design problem. Thus, it is valuable to separate how strongly a new carrier interacts with a cell from the functional efficacy of a carrier's cargo once delivered to that cell. To continue the chemotherapeutic example, "how well it binds to a cancer cell" is a separate problem from "how well it kills a cancer cell". Quantitative in vitro assays for the latter are well established and usually rely on measuring viability. However, most published research on cell-carrier interactions is qualitative or semiquantitative. Generally, these measurements rely on fluorescent labeling of the carrier and, consequently, report interactions with cells in relative or arbitrary units. However, this work can be standardized and be made absolutely quantitative with a small number of characterization experiments. Such absolute quantification is valuable, as it facilitates rational, inter- and intra-class comparisons of various drug delivery systems-nanoparticles, microparticles, viruses, antibody-drug conjugates, engineered therapeutic cells, or extracellular vesicles. Furthermore, quantification is a prerequisite for subsequent meta-analyses or in silico modeling approaches. In this article, video guides, as well as a decision tree for how to achieve in vitro quantification for carrier drug delivery systems, are presented, which take into account differences in carrier size and labeling modality. Additionally, further considerations for the quantitative assessment of advanced drug delivery systems are discussed. This is intended to serve as a valuable resource to improve rational evaluation and design for the next generation of medicine.
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    mRNA Treatment Rescues Niemann-Pick Disease Type C1 in Patient Fibroblasts
    Furtado, D ; Cortez-Jugo, C ; Hung, YH ; Bush, AI ; Caruso, F (AMER CHEMICAL SOC, 2022-11-07)
    Messenger RNA (mRNA) holds great potential as a disease-modifying treatment for a wide array of monogenic disorders. Niemann-Pick disease type C1 (NP-C1) is an ultrarare monogenic disease that arises due to loss-of-function mutations in the NPC1 gene, resulting in the entrapment of unesterified cholesterol in the lysosomes of affected cells and a subsequent reduction in their capacity for cholesterol esterification. This causes severe damage to various organs including the brain, liver, and spleen. In this work, we describe the use of NPC1-encoded mRNA to rescue the protein insufficiency and pathogenic phenotype caused by biallelic NPC1 mutations in cultured fibroblasts derived from an NP-C1 patient. We first evaluated engineering strategies for the generation of potent mRNAs capable of eliciting high protein expression across multiple cell types. We observed that "GC3" codon optimization, coupled with N1-methylpseudouridine base modification, yielded an mRNA that was approximately 1000-fold more potent than wild-type, unmodified mRNA in a luciferase reporter assay and consistently superior to other mRNA variants. Our data suggest that the improved expression associated with this design strategy was due in large part to the increased secondary structure of the designed mRNAs. Both codon optimization and base modification appear to contribute to increased secondary structure. Applying these principles to the engineering of NPC1-encoded mRNA, we observed a normalization in NPC1 protein levels after mRNA treatment, as well as a rescue of the mutant phenotype. Specifically, mRNA treatment restored the cholesterol esterification capacity of patient cells to wild-type levels and induced a significant reduction in both unesterified cholesterol levels (>57% reduction compared to Lipofectamine-treated control in a cholesterol esterification assay) and lysosome size (157 μm2 reduction compared to Lipofectamine-treated control). These findings show that engineered mRNA can correct the deficit caused by NPC1 mutations. More broadly, they also serve to further validate the potential of this technology to correct diseases associated with loss-of-function mutations in genes coding for large, complex, intracellular proteins.
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    Polyphenol-Functionalized Cubosomes as Thrombolytic Drug Carriers
    Yu, H ; Palazzolo, JS ; Ju, Y ; Niego, B ; Pan, S ; Hagemeyer, CE ; Caruso, F (WILEY, 2022-11)
    The safe administration of thrombolytic agents is a challenge for the treatment of acute thrombosis. Lipid-based nanoparticle drug delivery technologies present opportunities to overcome the existing clinical limitations and deliver thrombolytic therapy with enhanced therapeutic outcomes and safety. Herein, lipid cubosomes are examined as nanocarriers for the encapsulation of thrombolytic drugs. The lipid cubosomes are loaded with the thrombolytic drug urokinase-type plasminogen activator (uPA) and coated with a low-fouling peptide that is incorporated within a metal-phenolic network (MPN). The peptide-containing MPN (pep-MPN) coating inhibits the direct contact of uPA with the surrounding environment, as assessed by an in vitro plasminogen activation assay and an ex vivo whole blood clot degradation assay. The pep-MPN-coated cubosomes prepared with 22 wt% peptide demonstrate a cell membrane-dependent thrombolytic activity, which is attributed to their fusogenic lipid behavior. Moreover, compared with the uncoated lipid cubosomes, the uPA-loaded pep-MPN-coated cubosomes demonstrate significantly reduced nonspecific cell association (<10% of the uncoated cubosomes) in the whole blood assay, a prolonged circulating half-life, and reduced splenic uPA accumulation in mice. These studies confirm the preserved bioactivity and cell membrane-dependent release of uPA within pep-MPN-coated lipid cubosomes, highlighting their potential as a delivery vehicle for thrombolytic drugs.
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    Influence of protein corona on the interaction of glycogen-siRNA constructs with ex vivo human blood immune cells
    Wojnilowicz, M ; Laznickova, P ; Ju, Y ; Ang, C-S ; Tidu, F ; Bendickova, K ; Forte, G ; Plebanski, M ; Caruso, F ; Cavalieri, F ; Fric, J (ELSEVIER, 2022-09)
    Glycogen-nucleic acid constructs i.e., glycoplexes are emerging promising platforms for the alteration of gene expression and transcription. Understanding the interaction of glycoplexes with human blood components, such as serum proteins and peripheral blood mononuclear cells (PBMCs), is important to overcome immune cell activation and control biodistribution upon administration of the glycoplexes in vivo. Herein, we investigated the interactions of polyethylene glycol (PEG)ylated and non-PEGylated glycoplexes carrying siRNA molecules with PBMCs isolated from the blood of healthy donors. We found that both types of glycoplexes were non-toxic and were primarily phagocytosed by monocytes without triggering a pro-inflammatory interleukin 6 cytokine production. Furthermore, we investigated the role of the protein corona on controlling the internalization efficiency in immune cells - we found that the adsorption of serum proteins, in particular haptoglobin, alpha-1-antitrypsin and apolipoprotein A-II, onto the non-PEGylated glycoplexes, significantly reduced the uptake of the glycoplexes by PBMCs. Moreover, the non-PEGylated glycoplexes were efficient in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) knockdown in monocytic THP-1 cell line. This study provides an insight into the rational design of glycogen-based nanocarriers for the safe delivery of siRNA without eliciting unwanted immune cell activation and efficient siRNA activity upon its delivery.
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    Engineering Programmable DNA Particles and Capsules Using Catechol-Functionalized DNA Block Copolymers
    Kim, C-J ; Ercole, F ; Goudeli, E ; Bhangu, SK ; Chen, J ; Faria, M ; Quinn, JF ; Caruso, F (AMER CHEMICAL SOC, 2022-08-02)
    DNA block copolymer (DBC) assemblies have attracted attention because of their tunable properties (e.g., programmability, high biocompatibility, efficient cellular uptake, and stability against enzymatic degradation); however, controlling the size of DNA block copolymer assemblies and preparing well-defined DNA-functionalized particle systems are challenging. Herein, we report the preparation of DBC-based particles and capsules with different sizes (i.e., from approximately 0.15 to 3.2 μm) and a narrow size distribution (i.e., polydispersity index <0.2) through the assembly of catechol-functionalized DBC, DNA-b-poly(methyl methacrylate-co-2-methacryloylethyl dihydrocaffeate, with metal ions (e.g., FeIII). This assembly process largely exploits the coordination bonding of the metal ions and phenolic (i.e., catechol) groups, forming metal-phenolic networks (MPNs). The DBC-FeIIIMPN capsules formed are stable under acidic, metal-chelating, and surfactant solutions because of the coexistence of metal coordination, hydrogen bonding, and hydrophobic interactions. The molecular recognition properties of the DNA strands enable tailorable interactions with small molecules and nanoparticles and are used to tune the permeability of the assembled capsules (>40% permeability decrease for 2000 kDa fluorescein isothiocyanate dextran compared with untreated capsules). The DBC-FeIIIMPN particles show efficient cellular uptake and endosomal escape capability, allowing the efficient delivery of small-interfering RNA for gene silencing (89% downregulation). The reported approach provides the rational design of a range of DNA-functionalized particles, which can potentially be applied in materials science and biomedical applications.
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    Next-generation enhanced-efficiency fertilizers for sustained food security
    Lam, SK ; Wille, U ; Hu, H-W ; Caruso, F ; Mumford, K ; Liang, X ; Pan, B ; Malcolm, B ; Roessner, U ; Suter, H ; Stevens, G ; Walker, C ; Tang, C ; He, J-Z ; Chen, D (NATURE PORTFOLIO, 2022-07-21)
    Nitrogen losses in agricultural systems can be reduced through enhanced-efficiency fertilizers (EEFs), which control the physicochemical release from fertilizers and biological nitrogen transformations in soils. The adoption of EEFs by farmers requires evidence of consistent performance across soils, crops and climates, paired with information on the economic advantages. Here we show that the benefits of EEFs due to avoided social costs of nitrogen pollution considerably outweigh their costs—and must be incorporated in fertilizer policies. We outline new approaches to the design of EEFs using enzyme inhibitors with modifiable chemical structures and engineered, biodegradable coatings that respond to plant rhizosphere signalling molecules.
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    Site-Selective Coordination Assembly of Dynamic Metal-Phenolic Networks
    Xu, W ; Pan, S ; Noble, BB ; Chen, J ; Lin, Z ; Han, Y ; Zhou, J ; Richardson, JJ ; Yarovsky, I ; Caruso, F (WILEY-V C H VERLAG GMBH, 2022-08-22)
    Coordination states of metal-organic materials are known to dictate their physicochemical properties and applications in various fields. However, understanding and controlling coordination sites in metal-organic systems is challenging. Herein, we report the synthesis of site-selective coordinated metal-phenolic networks (MPNs) using flavonoids as coordination modulators. The site-selective coordination was systematically investigated experimentally and computationally using ligands with one, two, and multiple different coordination sites. Tuning the multimodal Fe coordination with catechol, carbonyl, and hydroxyl groups within the MPNs enabled the facile engineering of diverse physicochemical properties including size, selective permeability (20-2000 kDa), and pH-dependent degradability. This study expands our understanding of metal-phenolic chemistry and provides new routes for the rational design of structurally tailorable coordination-based materials.
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    Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine
    Ju, Y ; Lee, WS ; Pilkington, EH ; Kelly, HG ; Li, S ; Selva, KJ ; Wragg, KM ; Subbarao, K ; Nguyen, THO ; Rowntree, LC ; Allen, LF ; Bond, K ; Williamson, DA ; Truong, NP ; Plebanski, M ; Kedzierska, K ; Mahanty, S ; Chung, AW ; Caruso, F ; Wheatley, AK ; Juno, JA ; Kent, SJ (AMER CHEMICAL SOC, 2022-08-23)
    Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG, but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle-immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0-70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68-16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9-377.1) and 2.64-fold (0.76-12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo. PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle-leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA vaccines should be monitored. It may be useful to identify suitable alternatives to PEG for developing next-generation LNP vaccines to overcome PEG immunogenicity in the future.