Therapeutic intracellular delivery using magnetic fields, nanoparticle technology and enhanced cell penetrating peptides

The aim of this project was to develop a platform technology for the delivery of Magnetic Nanoparticles (MNPs). The delivery system is based on the incorporation of newly formulated multi domain delivery peptides termed GET (Glycosaminoglycan Enhanced Transduction) to biocompatible dextran coated iron oxide nanoparticles. GET synergistically combines a cell penetrating domain with a heparan sulphate binding unit and it has been previously demonstrated to efficiently deliver a wide range of cargoes without the disadvantages of a cell penetrating peptide such as cytotoxicity or low functionality of the delivered cargo. This technology was initially optimised for the delivery of MNPs as a theranostic complex with application to in vivo relevant environments and then tailored for magnetically mediated gene transfer for its application on modified cell therapies. Significant advancement has been made in the last couple of years in the development of MNP based therapies. Their applications rely on their physicochemical and magnetic properties and range from drug delivery systems for targeting therapeutics, to contrast agents for Magnetic Resonance Imaging (MRI), including their use in hyperthermia, cell and protein sorting or direct iron delivery. The most frequent application of MNPs in the clinic is in MRI with several MNP formulations already approved by the Food and Drug Administration (FDA). MNPs as gene delivery vectors allow for targeted gene transfer to a specific area by means of an external magnetic field (magnetofection). Although the potential of MNPs as drug/gene targeting agents has been consistently reported in vitro, their performance in preclinical studies has not been as successful. The main challenges that have prevented the incorporation of MNPs as targeted delivery systems include on the one hand, the alteration of the physicochemical properties of MNPs when they enter in contact with biological environments which makes it difficult to predict their behaviour in vivo. On the other hand, the lack of systems capable to generate a precise magnetic field capable to concentrate particles on a specific area against blood flow has restricted successful uptake in the targeted area in vivo. Since magnetic force is a function of the distance between the particle and the source of the magnetic field, this task becomes more complex the deeper the organ is inside the body. Asides from improving biomedical magnetic field settings, current efforts are focused on the formulation of stable (resistant to modification by interaction with biological matrixes) and long circulating MNPs that favour the fast cellular uptake at the target site, in order to reduce the need of long retention times at the target site. GET was able to safely mediate sustained intracellular transduction of MNPs even in the presence of plasma proteins. In order to exploit the ability of GET to promote the intracellular transduction of MNPs for gene delivery purposes, a modified version, able to efficiently condense DNA was conjugated with MNPs to develop a magnetic gene delivery vector. GET-MNPs mediated magnetofection significantly improved gene transfer speed achieving transfection efficiencies compared to commercially standard reagents in 1 hour. Additionally, external manipulation of the MNPs after delivery by the application of an external magnetic field, further enhanced transfection efficiency. Overall the two formulations of GET-MNPs were able to efficiently and safely deliver their cargo in vitro. With further development GET-MNPs could provide a flexible and tuneable platform technology for MNPs therapeutic delivery in vivo

Rapidly Transducing and Spatially Localized Magnetofection Using Peptide-Mediated Non-Viral Gene Delivery Based on Iron Oxide Nanoparticles

Non-viral delivery systems are generally of low efficiency, which limits their use in gene therapy and editing applications. We previously developed a technology termed glycosaminoglycan (GAG)-binding enhanced transduction (GET) to efficiently deliver a variety of cargos intracellularly; our system employs GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs), which enhance endocytotic cell internalization. Herein, we describe a further modification by combining gene delivery and magnetic targeting with the GET technology. We associated GET peptides, plasmid (p)DNA, and iron oxide superparamagnetic nanoparticles (MNPs), allowing rapid and targeted GET-mediated uptake by application of static magnetic fields in NIH3T3 cells. This produced effective transfection levels (significantly higher than the control) with seconds to minutes of exposure and localized gene delivery two orders of magnitude higher in targeted over non-targeted cell monolayers using magnetic fields (in 15 min exposure delivering GFP reporter pDNA). More importantly, high cell membrane targeting by GET-DNA and MNP co-complexes and magnetic fields allowed further enhancement to endocytotic uptake, meaning that the nucleic acid cargo was rapidly internalized beyond that of GET complexes alone (GET-DNA). Magnetofection by MNPs combined with GET-mediated delivery allows magnetic field-guided local transfection in vitro and could facilitate focused gene delivery for future regenerative and disease-targeted therapies in vivo

Highly versatile cell-penetrating peptide loaded scaffold for efficient and localised gene delivery to multiple cell types: From development to application in tissue engineering

Gene therapy has recently come of age with seven viral vector-based therapies gaining regulatory approval in recent years. In tissue engineering, non-viral vectors are preferred over viral vectors, however, lower transfection efficiencies and difficulties with delivery remain major limitations hampering clinical translation. This study describes the development of a novel multi-domain cell-penetrating peptide, GET, designed to enhance cell interaction and intracellular translocation of nucleic acids; combined with a series of porous collagen-based scaffolds with proven regenerative potential for different indications. GET was capable of transfecting cell types from all three germ layers, including stem cells, with an efficiency comparable to Lipofectamine® 3000, without inducing cytotoxicity. When implanted in vivo, GET gene-activated scaffolds allowed for host cell infiltration, transfection localized to the implantation site and sustained, but transient, changes in gene expression – demonstrating both the efficacy and safety of the approach. Finally, GET carrying osteogenic (pBMP-2) and angiogenic (pVEGF) genes were incorporated into collagen-hydroxyapatite scaffolds and with a single 2μg dose of therapeutic pDNA, induced complete repair of critical-sized bone defects within 4 weeks. GET represents an exciting development in gene therapy and by combining it with a scaffold-based delivery system offers tissue engineering solutions for a myriad of regenerative indications

Discovery of peptide ligands targeting a specific ubiquitin-like domain– binding site in the deubiquitinase USP11

© 2019 Spiliotopoulos et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (K D of 10 M) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11–peptide complex revealed a previously unknown binding site in USP11’s noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket– deficient double mutant disclosed that this binding site modulates USP11’s function in homologous recombination–mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets

Therapeutic intracellular delivery using magnetic fields, nanoparticle technology and enhanced cell penetrating peptides

The aim of this project was to develop a platform technology for the delivery of Magnetic Nanoparticles (MNPs). The delivery system is based on the incorporation of newly formulated multi domain delivery peptides termed GET (Glycosaminoglycan Enhanced Transduction) to biocompatible dextran coated iron oxide nanoparticles. GET synergistically combines a cell penetrating domain with a heparan sulphate binding unit and it has been previously demonstrated to efficiently deliver a wide range of cargoes without the disadvantages of a cell penetrating peptide such as cytotoxicity or low functionality of the delivered cargo. This technology was initially optimised for the delivery of MNPs as a theranostic complex with application to in vivo relevant environments and then tailored for magnetically mediated gene transfer for its application on modified cell therapies. Significant advancement has been made in the last couple of years in the development of MNP based therapies. Their applications rely on their physicochemical and magnetic properties and range from drug delivery systems for targeting therapeutics, to contrast agents for Magnetic Resonance Imaging (MRI), including their use in hyperthermia, cell and protein sorting or direct iron delivery. The most frequent application of MNPs in the clinic is in MRI with several MNP formulations already approved by the Food and Drug Administration (FDA). MNPs as gene delivery vectors allow for targeted gene transfer to a specific area by means of an external magnetic field (magnetofection). Although the potential of MNPs as drug/gene targeting agents has been consistently reported in vitro, their performance in preclinical studies has not been as successful. The main challenges that have prevented the incorporation of MNPs as targeted delivery systems include on the one hand, the alteration of the physicochemical properties of MNPs when they enter in contact with biological environments which makes it difficult to predict their behaviour in vivo. On the other hand, the lack of systems capable to generate a precise magnetic field capable to concentrate particles on a specific area against blood flow has restricted successful uptake in the targeted area in vivo. Since magnetic force is a function of the distance between the particle and the source of the magnetic field, this task becomes more complex the deeper the organ is inside the body. Asides from improving biomedical magnetic field settings, current efforts are focused on the formulation of stable (resistant to modification by interaction with biological matrixes) and long circulating MNPs that favour the fast cellular uptake at the target site, in order to reduce the need of long retention times at the target site. GET was able to safely mediate sustained intracellular transduction of MNPs even in the presence of plasma proteins. In order to exploit the ability of GET to promote the intracellular transduction of MNPs for gene delivery purposes, a modified version, able to efficiently condense DNA was conjugated with MNPs to develop a magnetic gene delivery vector. GET-MNPs mediated magnetofection significantly improved gene transfer speed achieving transfection efficiencies compared to commercially standard reagents in 1 hour. Additionally, external manipulation of the MNPs after delivery by the application of an external magnetic field, further enhanced transfection efficiency. Overall the two formulations of GET-MNPs were able to efficiently and safely deliver their cargo in vitro. With further development GET-MNPs could provide a flexible and tuneable platform technology for MNPs therapeutic delivery in vivo

Direct contact-mediated non-viral gene therapy using thermo-sensitive hydrogel-coated dressings

Nanotechnologies are being increasingly applied as systems for peptide and nucleic acid macromolecule drug delivery. However systemic targeting of these, or efficient topical and localized delivery remains an issue. A controlled release system that can be patterned and locally administered such as topically to accessible tissue (skin, eye, intestine) would therefore be transformative in realizing the potential of such strategies. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding peptides to mediate cell targeting, and cell penetrating peptides (CPPs) to promote uptake. Herein we demonstrate that the GET transfection system can be used with the moisturizing thermo-reversible hydrogel Pluronic-F127 (PF127) and methyl cellulose (MC) to mediate site specific and effective intracellular transduction and gene delivery through GET nanoparticles (NPs). We investigated hydrogel formulation and the temperature dependence of delivery, optimizing the delivery system. GET-NPs retain their activity to enhance gene transfer within our formulations, with uptake transferred to cells in direct contact with the therapy-laden hydrogel. By using Azowipe™ material in a bandage approach, we were able to show for the first-time localized gene transfer in vitro on cell monolayers. The ability to simply control localization of gene delivery on millimetre scales using contact-mediated transfer from moisture-providing thermo-reversible hydrogels will facilitate new drug delivery methods. Importantly our technology to site-specifically deliver the activity of novel nanotechnologies and gene therapeutics could be transformative for future regenerative medicine

Direct contact-mediated non-viral gene therapy using thermo-sensitive hydrogel-coated dressings

Nanotechnologies are being increasingly applied as systems for peptide and nucleic acid macromolecule drug delivery. However systemic targeting of these, or efficient topical and localized delivery remains an issue. A controlled release system that can be patterned and locally administered such as topically to accessible tissue (skin, eye, intestine) would therefore be transformative in realizing the potential of such strategies. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding peptides to mediate cell targeting, and cell penetrating peptides (CPPs) to promote uptake. Herein we demonstrate that the GET transfection system can be used with the moisturizing thermo-reversible hydrogel Pluronic-F127 (PF127) and methyl cellulose (MC) to mediate site specific and effective intracellular transduction and gene delivery through GET nanoparticles (NPs). We investigated hydrogel formulation and the temperature dependence of delivery, optimizing the delivery system. GET-NPs retain their activity to enhance gene transfer within our formulations, with uptake transferred to cells in direct contact with the therapy-laden hydrogel. By using Azowipe™ material in a bandage approach, we were able to show for the first-time localized gene transfer in vitro on cell monolayers. The ability to simply control localization of gene delivery on millimetre scales using contact-mediated transfer from moisture-providing thermo-reversible hydrogels will facilitate new drug delivery methods. Importantly our technology to site-specifically deliver the activity of novel nanotechnologies and gene therapeutics could be transformative for future regenerative medicine.</p

An innovative miR-activated scaffold for the delivery of a miR-221 inhibitor to enhance cartilage defect repair

The development of treatments to restore damaged cartilage that can provide functional recovery with minimal risk of revision surgery remains an unmet clinical need. Gene therapy shows increased promise as an innovative solution for enhanced tissue repair. Within this study a novel microRNA (miR)-activated scaffold is developed for enhanced mesenchymal stem/stromal cells (MSC) chondrogenesis and cartilage repair through the delivery of an inhibitor to microRNA-221 (miR-221), which is known to have a negative effect of chondrogenesis. To fabricate the miR-activated scaffolds, composite type II collagen-containing scaffolds designed specifically for cartilage repair are first manufactured by lyophilization and then functionalized with glycosaminoglycan-binding enhanced transduction (GET) system nanoparticles (NPs) encapsulating the miR-221 inhibitor. Subsequently, scaffolds are cultured with human-derived MSCs in vitro under chondrogenic conditions for 28 days. The miR-activated scaffolds successfully transfect human MSCs with the miR-221 cargo in a sustained and controlled manner up to 28 days. The silencing of miR-221 in human MSCs using the miR-activated scaffold promotes an improved and more robust cell-mediated chondrogenic response with repressed early-stage events related to MSC hypertrophy. Taken together, this innovative miR-activated scaffold for the delivery of a miR-221 inhibitor demonstrates capability to improve chondrogenesis with promise to enhance cartilage defect repair. </p

Systematic comparison of biomaterials-based strategies for osteochondral and chondral repair in large animal models

Joint repair remains a major challenge in orthopaedics. Recent progress in biomaterial design has led to the fabrication of a plethora of promising devices. Pre-clinical testing of any joint repair strategy typically requires the use of large animal models (e.g., sheep, goat, pig or horse). Despite the key role of such models in clinical translation, there is still a lack of consensus regarding optimal experimental design, making it difficult to draw conclusions on their efficacy. In this context, the authors performed a systematic literature review and a risk of bias assessment on large animal models published between 2010 and 2020, to identify key experimental parameters that significantly affect the biomaterial therapeutic outcome and clinical translation potential (including defect localization, animal age/maturity, selection of controls, cell-free versus cell-laden). They determined that mechanically strong biomaterials perform better at the femoral condyles; while highlighted the importance of including native tissue controls to better evaluate the quality of the newly formed tissue. Finally, in cell-laded biomaterials, the pre-culture conditions played a more important role in defect repair than the cell type. In summary, here they present a systematic evaluation on how the experimental design of preclinical models influences biomaterial-based therapeutic outcomes in joint repair

Enhanced cellular transduction of nanoparticles resistant to rapidly forming plasma protein coronas

© 2020 The Authors. Published by Wiley-VCH GmbH Nanoparticles (NPs) are increasingly being developed as biomedical platforms for drug/nucleic acid delivery and imaging. However, in biological fluids, NPs interact with a wide range of proteins that form a coating known as protein corona. Coronae can critically influence self-interaction and binding of other molecules, which can affect toxicity, promote cell activation, and inhibit general or specific cellular uptake. Glycosaminoglycan (GAG)-binding enhanced transduction (GET) is developed to efficiently deliver a variety of cargoes intracellularly; employing GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs) which enhance endocytotic cell internalization. Herein, it is demonstrated that GET peptide coatings can mediate sustained intracellular transduction of magnetic NPs (MNPs), even in the presence of serum or plasma. NP colloidal stability, physicochemical properties, toxicity and cellular uptake are investigated. Using label-free snapshot proteomics, time-resolved profiles of human plasma coronas formed on functionalized GET-MNPs demonstrate that coronae quickly form (<1 min), with their composition relatively stable but evolving. Importantly GET-MNPs present a subtly different corona composition to MNPs alone, consistent with GAG-binding activities. Understanding how NPs interact with biological systems and can retain enhanced intracellular transduction will facilitate novel drug delivery approaches for cell-type specific targeting of new nanomaterials