Chemical and Biomolecular Engineering - Research Publications

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    Generation and maturation of human iPSC-derived 3D organotypic cardiac microtissues in long-term culture
    Ergir, E ; Oliver-De la Cruz, J ; Fernandes, S ; Cassani, M ; Niro, F ; Pereira-Sousa, D ; Vrbsky, J ; Vinarsky, V ; Perestrelo, AR ; Debellis, D ; Vadovicova, N ; Uldrijan, S ; Cavalieri, F ; Pagliari, S ; Redl, H ; Ertl, P ; Forte, G (NATURE PORTFOLIO, 2022-10-18)
    Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.
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    Chemoenzymatic surface decoration of Nisin-shelled nanoemulsions: Novel targeted drug-nanocarriers for cancer applications
    Hashad, RA ; Singla, R ; Bhangu, SK ; Jap, E ; Zhu, H ; Peleg, AY ; Blakeway, L ; Hagemeyer, CE ; Cavalieri, F ; Ashokkumar, M ; Alt, K (ELSEVIER, 2022-11)
    Nisin, a peptide used as a natural food preservative, is employed in this work for the development of a novel nanocarrier system. Stable and uniform nisin-shelled nanoemulsions (NSNE) with a diameter of 100 ± 20 nm were successfully prepared using 20 kHz flow-through ultrasonication technique. The NSNE showed limited toxicity, high bactericidal activity and high drug loading capacity (EE 65 % w/w). In addition, the nisin shell was exploited for the site-specific attachment of a recombinantly produced cancer targeting ligand (αHER2LPETG IgG). Employing a unique two phases (bio-click) approach which involved both Sortase A mediated Azide Bioconjugation (SMAB) and Strain Promoted Azide Alkyne Cycloaddition (SPAAC) reactions, targeted NSNE (NSNEDOX-αHER2 IgG) were successfully assembled and loaded with the chemotherapeutic drug Doxorubicin (DOX). Finally, NSNEDOX-αHER2 IgG showed cancer-specific binding and augmented cytotoxicity to HER2 expressing tumour cells.
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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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    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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    Sonosynthesis of nanobiotics with antimicrobial and antioxidant properties

    Zhu, H ; Wen, Q ; Bhangu, SK ; Ashokkumar, M ; Cavalieri, F (ELSEVIER, 2022-05)
    Transforming small-molecule antibiotics into carrier-free nanoantibiotics represents an opportunity for developing new multifunctional therapeutic agents. In this study, we demonstrate that acoustic cavitation produced by high-frequency ultrasound transforms the antibiotic doxycycline into carrier-free nanobiotics. Upon sonication for 1 h at 10-15 W cm-3, doxycycline molecules underwent hydroxylation and dimerization processes to ultimately self-assemble into nanoparticles of ∼100-200 nm in size. Micrometer sized particles can be also obtained by increasing the acoustic power to 20 W cm-3. The nanodrugs exhibited antioxidant properties, along with antimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial strains. Our results highlight the feasibility of the ultrasound-based approach for engineering drug molecules into a nanosized formulation with controlled and multiple bio-functionalities.
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    Lysozyme microspheres incorporated with anisotropic gold nanorods for ultrasound activated drug delivery
    Bhargawa, B ; Sharma, V ; Ganesh, M-R ; Cavalieri, F ; Ashokkumar, M ; Neppolian, B ; Sundaramurthy, A (ELSEVIER, 2022-05)
    We report on the fabrication of lysozyme microspheres (LyMs) incorporated with gold nanorods (NRs) as a distinctive approach for the encapsulation and release of an anticancer drug, 5-Fluorouracil (5-FU). LyMs with an average size of 4.0 ± 1.0 µm were prepared by a sonochemical method and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). The LyMs were examined using hydrophobic (nile red) as well as hydrophilic (trypan blue) dyes under confocal laser scanning microscopy (CLSM) to obtain information about the preferential distribution of fluorescent molecules. Notably, the fluorescent molecules were accumulated in the inner lining of LyMs as the core was occupied with air. The encapsulation efficiency of 5-FU for LyMs-NR was found to be ∼64%. The drug release from control LyMs as well as LyMs incorporated with NRs was investigated under the influence of ultrasound (US) at 200 kHz. The total release for control LyMs and LyMs incorporated with gold NRs was found to be ∼70 and 95% after 1 h, respectively. The density difference caused by NR incorporation on the shell played a key role in rupturing the LyMs-NR under US irradiation. Furthermore, 5-FU loaded LyMs-NR exhibited excellent anti-cancer activity against the THP-1 cell line (∼90% cell death) when irradiated with US of 200 kHz. The enhanced anti-cancer activity of LyMs-NR was caused by the transfer of released 5-FU molecules from bulk to the interior of the cell via temporary pores formed on the surface of cancer cells, i.e., sonoporation. Thus, LyMs-NR demonstrated here has a high potential for use as carriers in the field of drug delivery, bio-imaging and therapy.
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    Aggregation properties of a therapeutic peptide for rheumatoid arthritis: A spectroscopic and molecular dynamics study
    Cimino, R ; Savioli, M ; Carrante, NF ; Placidi, E ; Garay-Perez, H ; López-Abad, M ; Lasa, AM ; Domínguez-Horta, MDC ; Gatto, E ; Cavalieri, F ; Bocchinfuso, G ; Venanzi, M (Elsevier BV, 2022-01-01)
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    Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation
    Savioli, M ; Antonelli, L ; Bocchinfuso, G ; Cavalieri, F ; Cimino, R ; Gatto, E ; Placidi, E ; Fernandez Masso, JR ; Garay Perez, H ; Santana, H ; Guerra-Vallespi, M ; Venanzi, M (WILEY, 2022-01)
    Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH2 ), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.
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    Ultrasonic microencapsulation of oil-soluble vitamins by hen egg white and green tea for fortification of food
    Zhu, H ; Mettu, S ; Cavalieri, F ; Ashokkumar, M (ELSEVIER SCI LTD, 2021-08-15)
    We report the microencapsulation of oil soluble vitamins (A, D and E) using a one pot ultrasonic process and raw egg white proteins as a shell material. Green tea catechin/iron complex coating method was further developed to impart UV filtering property to the microcapsules in order to protect the encapsulated nutrients from photodegradation. The microcapsules showed antibacterial properties and long shelf-life. The encapsulated vitamins were protected from degradation upon heating, UV irradiation, simulated storage/transit and cooking processes. The in-vitro digestion study showed that functional vitamin D can be potentially released in the gastrointestinal tract improving vitamin D availability by more than 2-fold compared to the free vitamin. The vitamin D microcapsules were highly stable and maintained their microstructures once incorporated into staple food products. The low-cost egg white shell encapsulated vitamins can improve the nutritional value of staple food products to combat maternal and child malnutrition.