Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

Dixon, James E.; Osman, Gizem; Morris, George E.; Markides, Hareklea; Rotherham, Michael; Bayoussef, Zahia; Haj, Alicia J. El; Denning, Chris; Shakesheff, Kevin M.

doi:10.1073/pnas.1518634113

Cited by 94 publications

(133 citation statements)

References 34 publications

(42 reference statements)

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“…Non-transducing monomeric red fluorescent protein (mRFP1; mR), CPP (eight arginine, 8R) -tagged mRFP1 (mR-8R), heparin binding domain; HBD (P21) -tagged mRFP1 (P21-mR), or the complete Glycosaminoglycan-binding enhanced transduction (GET is CPP and HBD) -tagged mRFP1 or MyoD protein (P21-mR-8R and P21-mR-MyoD-8R, respectively) were expressed and purified from E.coli using GST affinity purification as previously described by Dixon et al [26]. Sink compartments were loaded with 500 µL growth medium.…”

Section: Diffusion Studies and Get Proteinsmentioning

confidence: 99%

“…In our previous studies [26], we characterized the dynamics of GET-mediated transduction and compared the efficiency of intracellular transduction to nontransducing controls (mRFP1 only), less efficient transducing proteins (tagged with a CPP; mRFP-8R), and proteins with limited transduction but efficient cell interaction (P21-mR). We aimed to use all these variants within the gradient system which would allow us to determine whether cell binding verses transduction had the most significant influence of gradient profile.…”

Section: Using Get-tagged Proteins To Assess the Effect Of Intracellumentioning

confidence: 99%

“…These data demonstrate that early gradients (up to 24 hours) are dissipated and an equilibrium state is achieved regardless of presence or absence of cells within 72 hours. It is important that mRFP1 protein diffusion is statistically unaffected by the presence of cells in our system as we have previously shown it cannot efficiently interact with or transduce into cells [26].…”

Section: Cells Do Not Prevent Equilibration Of Non-transducing Proteinsmentioning

confidence: 99%

“…The ultimate realization of signalling is the interpretation by the cell to activate key regulatory genes, which in turn, instruct cell behaviour. We and others have shown that by delivering specific transcriptional master regulators either genetically (via viruses [25]) or recombinantly (using purified proteins, [26]) powerful cell fate changes can be programmed, effectively by-passing this highly-complicated interaction of extracellular signalling.…”

Section: Introductionmentioning

confidence: 99%

“…Even though CPPs significantly increase cellular uptake, their activity requires them in vast extracellular excess to drive uptake, with the quantities required to elicit any cell behaviour change in the micromolar scale. To overcome this inefficiency in delivery, we recently described a new technology named glycosaminoglycan GAG enhanced transduction (GET) [26]. We demonstrated enhanced intracellular transduction upon coupling CPPs with a GAG-binding peptide (the composite peptide termed GET peptides).…”

Section: Introductionmentioning

confidence: 99%

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Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

et al. 2016

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Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally.We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells.Using a compartmentalized diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time.The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications.

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Section: Diffusion Studies and Get Proteinsmentioning

confidence: 99%

Section: Using Get-tagged Proteins To Assess the Effect Of Intracellumentioning

confidence: 99%

Section: Cells Do Not Prevent Equilibration Of Non-transducing Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

et al. 2016

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Enhanced Cellular Transduction of Nanoparticles Resistant to Rapidly Forming Plasma Protein Coronas

et al. 2020

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physical properties suitable for directly targeting therapeutic sites using magnetic fields. [2] Furthermore, the ability of MNPs (a subtype being superparamagnetic iron oxide NPs; SPIONs) to efficiently convert magnetic energy into thermal energy, makes them a focus of interest for use in hyperthermia-based cell ablation anticancer therapies (such as Magforce Nanotechnologies AG). [3-6] Other relevant applications include imaging (including a focus on regenerative medicine), tissue engineering, for mechanical stimulation for cell differentiation in vivo [7-17] and, most recently, their use as synthetic enzymes in biocatalytic processes. [18] Targeted delivery strategies for MNPs have been developed toward specific-or overexpressed receptors on diseased cells by functionalizing the NP surface with proteins, antibodies, or other biomolecules as targeting ligands. These strategies efficiently enhance NP delivery to the target cells in vitro; however, there is mounting evidence that targeting ability of functionalized NPs disappears when placed in an in vivo biological environment. [19-21] It is now well established that when any material surface encounters a biological system, interactions occur between the material and the system components (i.e. proteins, lipids, DNA) forming a layer termed the protein corona. The protein corona defines the biological identity of the particle or surface, and has proven to be an obstacle in the past for effective targeted delivery of ligands, since it affects the physicochemical 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.

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Effective and Selective Anti‐Cancer Protein Delivery via All‐Functions‐in‐One Nanocarriers Coupled with Visible Light‐Responsive, Reversible Protein Engineering

Chen

et al. 2018

Adv Funct Materials

100

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Efficient intracellular delivery of protein drugs and tumor-specific activation of protein functions are critical toward anti-cancer protein therapy. However, an omnipotent protein delivery system that can harmonize the complicated systemic barriers as well as spatiotemporally manipulate protein function is lacking. Herein, an "all-functions-in-one" nanocarrier doped with photosensitizer (PS) is developed and coupled with reactive oxygen species (ROS)-responsive, reversible protein engineering to realize cancer-targeted protein delivery, and spatiotemporal manipulation of protein activities using long-wavelength visible light (635 nm) at low power density (5 mW cm −2 ). Particularly, RNase A is caged with H 2 O 2 -cleavable phenylboronic acid to form 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonate (NBC)-modified RNase (RNBC), which is encapsulated in aciddegradable, ketal-crosslinked PEI (KPEI)-based nanocomplexes (NCs) coatedwith PS-modified hyaluronic acid (HA). Such NCs harmonize the critical processes for protein delivery, wherein HA coating renders NCs with long blood circulation and cancer cell targeting, and KPEI enables endosomal escape as well as acid-triggered intracellular RNBC release. Tumor-specific light irradiation generates H 2 O 2 to kill cancer cells and restore the protein activity, thus achieving synergistic anti-cancer efficacy. It is the first time to photomanipulate protein functions by coupling ROS-cleavable protein caging with PS-mediated ROS generation, and the "all-functions-in-one" nanocarrier represents a promising example for the programmed anti-cancer protein delivery.

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Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

Cited by 94 publications

References 34 publications

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

Enhanced Cellular Transduction of Nanoparticles Resistant to Rapidly Forming Plasma Protein Coronas

Effective and Selective Anti‐Cancer Protein Delivery via All‐Functions‐in‐One Nanocarriers Coupled with Visible Light‐Responsive, Reversible Protein Engineering

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