Publications by authors named "Jan C M van Hest"

207 Publications

Author Correction: Engineering transient dynamics of artificial cells by stochastic distribution of enzymes.

Nat Commun 2021 Dec 16;12(1):7351. Epub 2021 Dec 16.

Department of Bio-Organic Chemistry, Institute of Complex Molecular Systems (ICMS), Eindhoven University of Technology, 5600 MB, Eindhoven, The Netherlands.

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http://dx.doi.org/10.1038/s41467-021-27682-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8677841PMC
December 2021

Therapeutic Stomatocytes with Aggregation Induced Emission for Intracellular Delivery.

Pharmaceutics 2021 Nov 2;13(11). Epub 2021 Nov 2.

Bio-Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.

Bowl-shaped biodegradable polymersomes, or stomatocytes, have much potential as drug delivery systems, due to their intriguing properties, such as controllable size, programmable morphology, and versatile cargo encapsulation capability. In this contribution, we developed well-defined therapeutically active stomatocytes with aggregation-induced emission (AIE) features by self-assembly of biodegradable amphiphilic block copolymers, comprising poly(ethylene glycol) (PEG) and AIEgenic poly(trimethylene carbonate) (PTMC) moieties. The presence of the AIEgens endowed the as-prepared stomatocytes with intrinsic fluorescence, which was employed for imaging of cellular uptake of the particles. It simultaneously enabled the photo-mediated generation of reactive oxygen species (ROS) for photodynamic therapy. The potential of the therapeutic stomatocytes as cargo carriers was demonstrated by loading enzymes (catalase and glucose oxidase) in the nanocavity, followed by a cross-linking reaction to achieve stable encapsulation. This provided the particles with a robust motile function, which further strengthened their therapeutic effect. With these unique features, enzyme-loaded AIEgenic stomatocytes are an attractive platform to be exploited in the field of nanomedicine.
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http://dx.doi.org/10.3390/pharmaceutics13111833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8617661PMC
November 2021

Engineering transient dynamics of artificial cells by stochastic distribution of enzymes.

Nat Commun 2021 11 25;12(1):6897. Epub 2021 Nov 25.

Department of Bio-Organic Chemistry, Institute of Complex Molecular Systems (ICMS), Eindhoven University of Technology, 5600 MB, Eindhoven, The Netherlands.

Random fluctuations are inherent to all complex molecular systems. Although nature has evolved mechanisms to control stochastic events to achieve the desired biological output, reproducing this in synthetic systems represents a significant challenge. Here we present an artificial platform that enables us to exploit stochasticity to direct motile behavior. We found that enzymes, when confined to the fluidic polymer membrane of a core-shell coacervate, were distributed stochastically in time and space. This resulted in a transient, asymmetric configuration of propulsive units, which imparted motility to such coacervates in presence of substrate. This mechanism was confirmed by stochastic modelling and simulations in silico. Furthermore, we showed that a deeper understanding of the mechanism of stochasticity could be utilized to modulate the motion output. Conceptually, this work represents a leap in design philosophy in the construction of synthetic systems with life-like behaviors.
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http://dx.doi.org/10.1038/s41467-021-27229-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8617035PMC
November 2021

Cucurbit-Like Polymersomes with Aggregation-Induced Emission Properties Show Enzyme-Mediated Motility.

ACS Nano 2021 Oct 20. Epub 2021 Oct 20.

Bio-Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.

Polymersomes that incorporate aggregation-induced emission (AIE) moieties are attractive inherently fluorescent nanoparticles with biomedical application potential for cell/tissue imaging and tracking, as well as phototherapeutics. An intriguing feature that has not been explored yet is their ability to adopt a range of asymmetric morphologies. Structural asymmetry allows nanoparticles to be exploited as active (motile) systems. Here, we present the design and preparation of AIE fluorophore integrated (AIEgenic) cucurbit-shaped polymersome nanomotors with enzyme-powered motility. The cucurbit scaffold was constructed via morphology engineering of biodegradable fluorescent AIE-polymersomes, followed by functionalization with enzymatic machinery via a layer-by-layer (LBL) self-assembly process. Because of the enzyme-mediated decomposition of chemical fuel on the cucurbit-like nanomotor surface, enhanced directed motion was attained, when compared with the spherical counterparts. These cucurbit-shaped biodegradable AIE-nanomotors provide a promising platform for the development of active delivery systems with potential for biomedical applications.
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http://dx.doi.org/10.1021/acsnano.1c07343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8613902PMC
October 2021

Bone-Adhesive Hydrogels Based on Dual Crosslinked Poly(2-oxazoline)s.

Macromol Biosci 2021 Dec 5;21(12):e2100257. Epub 2021 Oct 5.

Department of Dentistry-Regenerative Biomaterials, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, 6525 EX, the Netherlands.

The development of bone glues based on bone-adhesive hydrogels to allow for facile bone fracture fixation remains a major challenge. Herein, dual crosslinked hydrogels that combine tunable stiffness, ductility, and self-healing capacity are successfully synthesized. The resulting double network hydrogel is formed by chemical crosslinking of N-hydroxysuccinimide-functionalized poly(2-oxazoline)s(POx-NHS)"?> with amine-functionalized poly(2-oxazoline)s, and physical crosslinking of alendronate-functionalized poly(2-oxazoline)s (POx-Ale) with calcium ions in solution. The use of an excess of alendronate-functionalized POx-Ale polymers also ensures affinity toward calcium cations in the mineral phase of bone, thereby rendering these hydrogels adhesive to bone. The mechanical and bone-adhesive properties of these novel hydrogels are superior to commercially available fibrin sealants. Moreover, hydrogels retain their bone-adhesive properties under wet conditions. Although the dual crosslinked hydrogels swell considerably, they are stable upon immersion in phosphate-buffered saline (up to 12 d) and even in ethylenediaminetetraacetic acid solution. The enhanced mechanical and bone-adhesive properties of these hydrogels, as well as their in vitro stability, indicate that they have much application potential as bone-adhesive glues.
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http://dx.doi.org/10.1002/mabi.202100257DOI Listing
December 2021

Bone-adhesive barrier membranes based on alendronate-functionalized poly(2-oxazoline)s.

J Mater Chem B 2021 07;9(29):5848-5860

Department of Dentistry-Regenerative Biomaterials, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 EX Nijmegen, The Netherlands.

To create a novel generation of barrier membranes with bone-adhesive properties, three-component membranes were successfully developed using a solvent-free approach by combining an occlusive polyester backing layer with a bone-adhesive fibrous gelatin carrier impregnated with calcium-binding alendronate-functionalized poly(2-oxazoline)s (POx-Ale). The mechanical properties of these novel membranes were similar to other commercially available barrier membranes. In contrast, the adhesion of our membranes towards bone was by far superior (i.e. 62-fold) compared to conventional commercially available Bio-Gide® membranes. Moreover, alendronate-functionalized membranes retained their bone-adhesive properties under wet conditions in phosphate-buffered saline (PBS) solutions with and without collagenase. Finally, the in vitro degradation of the membranes was studied by monitoring their weight loss upon immersion in PBS solutions with and without collagenase. The membranes degraded in a sustained manner, which was accelerated by the presence of collagenase due to enzymatic degradation of the carrier. In conclusion, our results show that surface functionalization of barrier membranes with alendronate moieties renders them adhesive to bone. As such, the biomaterials design strategy presented herein opens up new avenues of research on bone-adhesive membranes for guided bone regeneration.
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http://dx.doi.org/10.1039/d1tb00502bDOI Listing
July 2021

Dynamic spatial and structural organization in artificial cells regulates signal processing by protein scaffolding.

Chem Sci 2020 Nov 5;11(47):12829-12834. Epub 2020 Nov 5.

Department of Chemical Engineering and Chemistry, Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology PO Box 513 5600 MB Eindhoven The Netherlands

Structural and spatial organization are fundamental properties of biological systems that allow cells to regulate a wide range of biochemical processes. This organization is often transient and governed by external cues that initiate dynamic self-assembly processes. The construction of synthetic cell-like materials with similar properties requires the hierarchical and reversible organization of selected functional components on molecular scaffolds to dynamically regulate signaling pathways. The realization of such transient molecular programs in synthetic cells, however, remains underexplored due to the associated complexity of such hierarchical platforms. In this contribution, we effectuate dynamic spatial organization of effector protein subunits in a synthetic biomimetic compartment, a giant unilamellar vesicle (GUV), by associating in a reversible manner two fragments of a split luciferase to the membrane. This induces their structural dimerization, which consequently leads to the activation of enzymatic signaling. Importantly, such organization and activation are dynamic processes, and can be autonomously regulated - thus opening up avenues toward continuous spatiotemporal control over supramolecular organization and signaling in an artificial cell.
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http://dx.doi.org/10.1039/d0sc03933kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163283PMC
November 2020

Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles.

Polymers (Basel) 2021 May 1;13(9). Epub 2021 May 1.

University Bordeaux, CNRS, Bordeaux INP, LCPO, UMR 5629, 33600 Pessac, France.

Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[MV-]-[I-]. First, ELP-[MV-]-[I-] ( = , , ; = , ) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[MV-] and ELP-[I-]. By selectively oxidizing methionines of ELP-[MV-] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.
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http://dx.doi.org/10.3390/polym13091470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125372PMC
May 2021

Biodegradable Polymersomes with Structure Inherent Fluorescence and Targeting Capacity for Enhanced Photo-Dynamic Therapy.

Angew Chem Int Ed Engl 2021 08 30;60(32):17629-17637. Epub 2021 Jun 30.

Bio-Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513 (STO 3.41), 5600, MB, Eindhoven, The Netherlands.

Biodegradable nanostructures displaying aggregation-induced emission (AIE) are desirable from a biomedical point of view, due to the advantageous features of loading capacity, emission brightness, and fluorescence stability. Herein, biodegradable polymers comprising poly (ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-P(CLgTMC)), with tetraphenylethylene pyridinium-TMC (PAIE) side chains have been developed, which self-assembled into well-defined polymersomes. The resultant AIEgenic polymersomes are intrinsically fluorescent delivery vehicles. The presence of the pyridinium moiety endows the polymersomes with mitochondrial targeting ability, which improves the efficiency of co-encapsulated photosensitizers and improves therapeutic index against cancer cells both in vitro and in vivo. This contribution showcases the ability to engineer AIEgenic polymersomes with structure inherent fluorescence and targeting capacity for enhanced photodynamic therapy.
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http://dx.doi.org/10.1002/anie.202105103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361757PMC
August 2021

Cowpea Chlorotic Mottle Virus-Like Particles as Potential Platform for Antisense Oligonucleotide Delivery in Posterior Segment Ocular Diseases.

Macromol Biosci 2021 Aug 24;21(8):e2100095. Epub 2021 May 24.

Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, Eindhoven, 5600 MB, The Netherlands.

Due to its small size, easy accessibility and immune privileged environment, the eye represents an ideal target for therapeutic nucleic acids in the treatment of posterior segment ocular diseases, such as age-related macular degeneration (AMD). Among nanocarriers that can be used to achieve nucleic acid delivery, virus-like particles (VLPs) obtained from the Cowpea chlorotic mottle virus (CCMV) are an appealing platform, because of their loading capacity, ease of manufacture and amenability for functionalization. Herein, antisense oligonucleotide-loaded CCMV nanoparticles, intended for intravitreal injection, are evaluated for selective silencing of miR-23, an important target in AMD. CCMV nanoparticles loaded with anti-miR-23 locked nucleic acid and stabilized using the 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) cross-linker, are assembled in vitro with a loading efficiency up to 80%. VLPs are found to be stable at 37 °C in the vitreous humor up to 24 hours. Nanoparticle cytotoxicity, cellular uptake and transfection efficacy are evaluated in endothelial cells. Selective miRNA down-regulation is achieved by the loaded CCMV VLPs both in absence and presence of Lipofectamine, with efficacies of ≈40% and more than 80%, respectively. The authors' findings pave the way for the future development of CCMV nanoparticles as oligonucleotide delivery platform to treat posterior segment ocular diseases.
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http://dx.doi.org/10.1002/mabi.202100095DOI Listing
August 2021

Self-Assembly or Coassembly of Multiresponsive Histidine-Containing Elastin-Like Polypeptide Block Copolymers.

Macromol Biosci 2021 06 4;21(6):e2100081. Epub 2021 May 4.

Bio-Organic Chemistry, Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, Helix (STO 3.41), P. O. Box 513, Eindhoven, 5600 MB, The Netherlands.

In this study a histidine containing elastin-like polypeptide (ELP) diblock copolymer is described with multiresponsive assembly behavior. Self-assembly into micelles is examined by two methods. First, the self-assembly is triggered by the addition of divalent metal ions, with Zn being the most suitable one. Increasing the Zn concentration stabilizes the nanoparticles over a large temperature window (4-45 °C). This diblock exhibits furthermore pH-responsiveness, and particles disassemble under mildly acidic conditions. Second, the coassembly of this ELP with a diblock ELP is examined, which is not responsive to pH and metal ions. Coassembly is triggered by heating the ELPs quickly above the transition temperature of the less hydrophobic block, which results in stable nanoparticles without the need to add metal ions. This novel ELP system offers a versatile modular nanocarrier platform that can respond to different stimuli and can be tuned effectively.
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http://dx.doi.org/10.1002/mabi.202100081DOI Listing
June 2021

Dual Site-Selective Presentation of Functional Handles on Protein-Engineered Cowpea Chlorotic Mottle Virus-Like Particles.

Bioconjug Chem 2021 05 16;32(5):958-963. Epub 2021 Apr 16.

Eindhoven University of Technology, Institute for Complex Molecular Systems, PO Box 513 (STO 3.41), 5600 MB Eindhoven, The Netherlands.

Protein cages hold much promise as carrier systems in nanomedicine, due to their well-defined size, cargo-loading capacity, and inherent biodegradability. In order to make them suitable for drug delivery, they have to be stable under physiological conditions. In addition, often surface modifications are required, for example, to improve cell targeting or reduce the particle immunogenicity by PEGylation. For this purpose, we investigated the functionalization capacity of the capsid of cowpea chlorotic mottle virus (CCMV), modified at the interior with a stabilizing elastin-like polypeptide (ELP) tag, by employing a combination of protein engineering and bio-orthogonal chemistry. We first demonstrated the accessibility of the native cysteine residue in ELP-CCMV as a site-selective surface-exposed functional handle, which was not available in the native CCMV capsid. An additional bio-orthogonal functional handle was introduced by incorporation of the noncanonical amino acid, azido-phenylalanine (AzF), using the amber suppression mechanism. Dual site-selective presentation of both a cell-penetrating TAT peptide and a fluorophore to track the particles was demonstrated successfully in HeLa cell uptake studies.
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http://dx.doi.org/10.1021/acs.bioconjchem.1c00108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154214PMC
May 2021

Photoactivated nanomotors via aggregation induced emission for enhanced phototherapy.

Nat Commun 2021 04 6;12(1):2077. Epub 2021 Apr 6.

Bio-Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, MB, Eindhoven, The Netherlands.

Aggregation-induced emission (AIE) has, since its discovery, become a valuable tool in the field of nanoscience. AIEgenic molecules, which display highly stable fluorescence in an assembled state, have applications in various biomedical fields-including photodynamic therapy. Engineering structure-inherent, AIEgenic nanomaterials with motile properties is, however, still an unexplored frontier in the evolution of this potent technology. Here, we present phototactic/phototherapeutic nanomotors where biodegradable block copolymers decorated with AIE motifs can transduce radiant energy into motion and enhance thermophoretic motility driven by an asymmetric Au nanoshell. The hybrid nanomotors can harness two photon near-infrared radiation, triggering autonomous propulsion and simultaneous phototherapeutic generation of reactive oxygen species. The potential of these nanomotors to be applied in photodynamic therapy is demonstrated in vitro, where near-infrared light directed motion and reactive oxygen species induction synergistically enhance efficacy with a high level of spatial control.
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http://dx.doi.org/10.1038/s41467-021-22279-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024279PMC
April 2021

Intravitreal Polymeric Nanocarriers with Long Ocular Retention and Targeted Delivery to the Retina and Optic Nerve Head Region.

Pharmaceutics 2021 Mar 26;13(4). Epub 2021 Mar 26.

Drug Research Program, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, 00790 Helsinki, Finland.

Posterior eye tissues, such as retina, are affected in many serious eye diseases, but drug delivery to these targets is challenging due to various anatomical eye barriers. Intravitreal injections are widely used, but the intervals between invasive injections should be prolonged. We synthesized and characterized (H NMR, gel permeation chromatography) block copolymers of poly(ethylene glycol), poly(caprolactone), and trimethylene carbonate. These polymers self-assembled to polymersomes and polymeric micelles. The mean diameters of polymersomes and polymeric micelles, about 100 nm and 30-50 nm, respectively, were obtained with dynamic light scattering. Based on single particle tracking and asymmetric flow field-flow fractionation, the polymeric micelles and polymersomes were stable and diffusible in the vitreous. The materials did not show cellular toxicity in cultured human umbilical vein endothelial cells in the Alamar Blue Assay. Pharmacokinetics of the intravitreal nanocarriers in the rabbits were evaluated using in vivo fluorophotometry. The half-lives of the polymersomes (100 nm) and the micelles (30 nm) were 11.4-32.7 days and 4.3-9.5 days. The intravitreal clearance values were 1.7-8.7 µL/h and 3.6-5.4 µL/h for polymersomes and polymeric micelles, respectively. Apparent volumes of distribution of the particles in the rabbit vitreous were 0.6-1.3 mL for polymeric micelles and 1.9-3.4 mL for polymersomes. Polymersomes were found in the vitreous for at least 92 days post-dosing. Furthermore, fundus imaging revealed that the polymersomes accumulated near the optic nerve and retained there even at 111 days post-injection. Polymersomes represent a promising technology for controlled and site-specific drug delivery in the posterior eye segment.
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http://dx.doi.org/10.3390/pharmaceutics13040445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066548PMC
March 2021

Pathway-Dependent Co-Assembly of Elastin-Like Polypeptides.

Small 2021 04 10;17(13):e2007234. Epub 2021 Mar 10.

Department of Biomedical Engineering & Department of Chemical Engineering and Chemistry, Institute for Complex Molecular Systems Eindhoven University of Technology, PO Box 513, Eindhoven, 5600 MB, the Netherlands.

In natural systems, temperature-induced assembly of biomolecules can lead to the formation of distinct assembly states, created out of the same set of starting compounds, based on the heating trajectory followed. Until now it has been difficult to achieve similar behavior in synthetic polymer mixtures. Here, a novel pathway-dependent assembly based on stimulus-responsive polymers is shown. When a mixture of mono- and diblock copolymers, based on elastin-like polypeptides, is heated with a critical heating rate co-assembled particles are created that are monodisperse, stable, and have tunable hydrodynamic radii between 20 and 120 nm. Below this critical heating rate, the constituents separately form polymer assemblies. This process is kinetically driven and reversible in thermodynamically closed systems. Using the co-assembly pathway, fluorescent proteins and bioluminescent enzymes are encapsulated with high efficiency. Encapsulated cargo shows unperturbed function even after delivery into cells. The pathway-dependent co-assembly of elastin-like polypeptides is not only of fundamental interest from a materials science perspective, allowing the formation of multiple distinct assemblies from the same starting compounds, which can be interconverted by going back to the molecularly dissolved states. It also enables a versatile way for constructing highly effective vehicles for the cellular delivery of biomolecular cargo.
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http://dx.doi.org/10.1002/smll.202007234DOI Listing
April 2021

Single Enzyme Nanoparticles with Improved Biocatalytic Activity through Protein Entrapment in a Surfactant Shell.

Biomacromolecules 2021 03 25;22(3):1159-1166. Epub 2021 Feb 25.

Laboratory of Self-Organizing Soft Matter, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.

A polymeric corona consisting of an alkyl-glycolic acid ethoxylate (CEO) surfactant offers a promising approach toward endowing proteins with thermotropic phase behavior and hyperthermal activity. Typically, preparation of protein-surfactant biohybrids is performed chemical modification of acidic residues followed by electrostatic conjugation of an anionic surfactant to encapsulate single proteins. While this procedure has been applied to a broad range of proteins, modification of acidic residues may be detrimental to function for specific enzymes. Herein, we report on the one-pot preparation of biohybrids covalent conjugation of surfactants to accessible lysine residues. We entrap the model enzyme hen egg-white lysozyme (HEWL) in a shell of carboxyl-functionalized CEO or CEO surfactants. With fewer surfactants, our covalent biohybrids display similar thermotropic phase behavior to their electrostatically conjugated analogues. Through a combination of small-angle X-ray scattering and circular dichroism spectroscopy, we find that both classes of biohybrids consist of a folded single-protein core decorated by surfactants. Whilst traditional biohybrids retain densely packed surfactant coronas, our biohybrids display a less dense and heterogeneously distributed surfactant coverage located opposite to the catalytic cleft of HEWL. In solution, this surfactant coating permits 7- or 3.5-fold improvements in activity retention for biohybrids containing CEO or CEO, respectively. The reported alternative pathway for biohybrid preparation offers a new horizon to expand upon the library of proteins for which functional biohybrid materials can be prepared. We also expect that an improved understanding of the distribution of tethered surfactants in the corona will be crucial for future structure-function investigations.
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http://dx.doi.org/10.1021/acs.biomac.0c01663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944482PMC
March 2021

Engineering of Biocompatible Coacervate-Based Synthetic Cells.

ACS Appl Mater Interfaces 2021 Feb 15;13(7):7879-7889. Epub 2021 Feb 15.

Institute for Complex Molecular Systems, Eindhoven University of Technology, P. O. Box 513 (STO 3.41), 5600MB Eindhoven, The Netherlands.

Polymer-stabilized complex coacervate microdroplets have emerged as a robust platform for synthetic cell research. Their unique core-shell properties enable the sequestration of high concentrations of biologically relevant macromolecules and their subsequent release through the semipermeable membrane. These unique properties render the synthetic cell platform highly suitable for a range of biomedical applications, as long as its biocompatibility upon interaction with biological cells is ensured. The purpose of this study is to investigate how the structure and formulation of these coacervate-based synthetic cells impact the viability of several different cell lines. Through careful examination of the individual synthetic cell components, it became evident that the presence of free polycation and membrane-forming polymer had to be prevented to ensure cell viability. After closely examining the structure-toxicity relationship, a set of conditions could be found whereby no detrimental effects were observed, when the artificial cells were cocultured with RAW264.7 cells. This opens up a range of possibilities to use this modular system for biomedical applications and creates design rules for the next generation of coacervate-based, biomedically relevant particles.
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http://dx.doi.org/10.1021/acsami.0c19052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908014PMC
February 2021

Terpolymer-stabilized complex coacervates: A robust and versatile synthetic cell platform.

Methods Enzymol 2021 9;646:51-82. Epub 2020 Jul 9.

Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands; Department of Chemical Engineering and Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands. Electronic address:

The utilization of liquid-liquid phase separated systems has seen increased attention as synthetic cell platforms due to their innate ability to sequester interesting, functional, and biologically relevant materials. However, their applications are limited by the temporal stability of such condensed phases. While there are a number of strategies toward droplet stabilization, in our group we have developed a polymer-based approach to stabilize complex coacervate microdroplets. These protocells are remarkably robust and have been utilized to support a number of new protocellular applications. Here, we describe in detail the methodologies we have developed for the synthesis of the starting components, their formation into stable, cargo-loaded protocells, and how these protocells are treated post-formation to purify and analyze the resultant functional self-assembled systems.
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http://dx.doi.org/10.1016/bs.mie.2020.06.008DOI Listing
June 2021

Artificial Organelles: Towards Adding or Restoring Intracellular Activity.

Chembiochem 2021 06 4;22(12):2051-2078. Epub 2021 Mar 4.

Bio-Organic Chemistry Research Group, Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513 (STO3.41), 5600 MB, Eindhoven, The Netherlands.

Compartmentalization is one of the main characteristics that define living systems. Creating a physically separated microenvironment allows nature a better control over biological processes, as is clearly specified by the role of organelles in living cells. Inspired by this phenomenon, researchers have developed a range of different approaches to create artificial organelles: compartments with catalytic activity that add new function to living cells. In this review we will discuss three complementary lines of investigation. First, orthogonal chemistry approaches are discussed, which are based on the incorporation of catalytically active transition metal-containing nanoparticles in living cells. The second approach involves the use of premade hybrid nanoreactors, which show transient function when taken up by living cells. The third approach utilizes mostly genetic engineering methods to create bio-based structures that can be ultimately integrated with the cell's genome to make them constitutively active. The current state of the art and the scope and limitations of the field will be highlighted with selected examples from the three approaches.
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http://dx.doi.org/10.1002/cbic.202000850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252369PMC
June 2021

Engineered protein cages for selective heparin encapsulation.

J Mater Chem B 2021 02 11;9(5):1272-1276. Epub 2021 Jan 11.

Department of Bioproducts and Biosystems, Aalto University, P.O. Box 16100, Aalto FI-00076, Espoo, Finland. and HYBER Centre, Department of Applied Physics, Aalto University, Aalto FI-00076, Finland.

A heparin-specific binding peptide was conjugated to a cowpea chlorotic mottle virus (CCMV) capsid protein, which was subsequently allowed to encapsulate heparin and form capsid-like protein cages. The encapsulation is specific and the capsid-heparin assemblies display negligible hemolytic activity, indicating proper blood compatibility and promising possibilities for heparin antidote applications.
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http://dx.doi.org/10.1039/d0tb02541kDOI Listing
February 2021

Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells.

Nat Commun 2020 12 8;11(1):6282. Epub 2020 Dec 8.

Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB, Eindhoven, The Netherlands.

The cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.
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http://dx.doi.org/10.1038/s41467-020-20124-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722712PMC
December 2020

Tuning Size and Morphology of mPEG--p(HPMA-Bz) Copolymer Self-Assemblies Using Microfluidics.

Polymers (Basel) 2020 Nov 2;12(11). Epub 2020 Nov 2.

Department of Bio-Organic Chemistry, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.

The careful design of nanoparticles, in terms of size and morphology, is of great importance to developing effective drug delivery systems. The ability to precisely tailor nanoparticles in size and morphology during polymer self-assembly was therefore investigated. Four poly(ethylene glycol)--poly(-2-benzoyloxypropyl methacrylamide) mPEG--p(HPMA-Bz) block copolymers with a fixed hydrophilic block of mPEG 5 kDa and a varying molecular weight of the hydrophobic p(HPMA-Bz) block (A: 17.1, B: 10.0, C: 5.2 and D: 2.7 kDa) were self-assembled into nanoparticles by nanoprecipitation under well-defined flow conditions, using microfluidics, at different concentrations. The nanoparticles from polymer A, increased in size from 55 to 90 nm using lower polymer concentrations and slower flow rates and even polymer vesicles were formed along with micelles. Similarly, nanoparticles from polymer D increased in size from 35 to 70 nm at slower flow rates and also formed vesicles along with micelles, regardless of the used concentration. Differently, polymers B and C mainly self-assembled into micelles at the different applied flow rates with negligible size difference. In conclusion, this study demonstrates that the self-assembly of mPEG--p(HPMA-Bz) block copolymers can be easily tailored in size and morphology using microfluidics and is therefore an attractive option for further scaled-up production activities.
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http://dx.doi.org/10.3390/polym12112572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693845PMC
November 2020

Scale-Up of the Manufacturing Process To Produce Docetaxel-Loaded mPEG--p(HPMA-Bz) Block Copolymer Micelles for Pharmaceutical Applications.

Org Process Res Dev 2019 Dec 29;23(12):2707-2715. Epub 2019 Oct 29.

Department of Bio-Organic chemistry, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.

An efficient, scalable, and good manufacturing practice (GMP) compatible process was developed for the production of docetaxel-loaded poly(ethylene glycol)--poly(-2-benzoyloxypropyl methacrylamide) (mPEG--p(HPMA-Bz)) micelles. First, the synthesis of the mPEG-p(HPMA-Bz) block copolymer was optimized through step-by-step investigation of the batch synthesis procedures. This resulted in the production of 1 kg of mPEG-p(HPMA-Bz) block copolymer with a 5 kDa PEG block and an overall molecular weight of 22.5 kDa. Second, the reproducibility and scalability of micelle formation was investigated for both batch and continuous flow setups by assessing critical process parameters. This resulted in the development of a new and highly efficient continuous flow process, which led to the production of 100 mL of unloaded micelles with a size of 55 nm. Finally, the loading of the micelles with the anticancer drug docetaxel was successfully fine-tuned to obtain precise control on the loaded micelle characteristics. As a result, 100 mL of docetaxel-loaded micelles (20 mg/mL polymer and 5 mg/mL docetaxel in the feed) with a size of 55 nm, an encapsulation efficiency of 65%, a loading capacity of 14%, and stable for at least 2 months in water at room temperature were produced with the newly developed continuous flow process. In conclusion, this study paves the way for efficient and robust large-scale production of docetaxel-loaded micelles with high encapsulation efficiencies and stability, which is crucial for their applicability as a clinically relevant drug delivery platform.
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http://dx.doi.org/10.1021/acs.oprd.9b00387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493301PMC
December 2019

Surface-Charge-Switchable Nanoclusters for Magnetic Resonance Imaging-Guided and Glutathione Depletion-Enhanced Photodynamic Therapy.

ACS Nano 2020 09 18;14(9):11225-11237. Epub 2020 Aug 18.

Bio-Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.

Photodynamic therapy (PDT) is an effective noninvasive therapeutic method that employs photosensitizers (PSs) converting oxygen to highly cytotoxic singlet oxygen (O) under light irradiation. The conventional PDT efficacy is, however, compromised by the nonspecific delivery of PSs to tumor tissue, the hypoxic tumor microenvironment, and the reduction of generated O by the intracellular antioxidant glutathione (GSH). Herein, an intelligent multifunctional synergistic nanoplatform (CMGCC) for -weighted magnetic resonance (MR) imaging-guided enhanced PDT is presented, which consists of nanoparticles composed of catalase (CAT) and manganese dioxide (MnO) that are integrated within chlorin-e6-modified glycol chitosan (GC) polymeric micelles. In this system, (1) GC polymers with pH-sensitive surface charge switchability from neutral to positive could improve the PS accumulation within the tumor region, (2) CAT could effectively reoxygenate the hypoxic tumor catalyzing endogenous hydrogen peroxide to O, and (3) MnO could consume the intracellular GSH while simultaneously producing Mn as a contrast agent for -weighted MR imaging. The CMGCC particles possess uniform size distribution, well-defined structure, favorable enzyme activity, and superior O generation ability. Both and experiments demonstrate that the CMGCC exhibit significantly enhanced PDT efficacy toward HeLa cells and subcutaneous HeLa tumors. Our study thereby demonstrates this to be a promising synergistic theranostic nanoplatform with highly efficient PDT performance for cancer therapy.
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http://dx.doi.org/10.1021/acsnano.0c03080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513467PMC
September 2020

Acid-Activatable Transmorphic Peptide-Based Nanomaterials for Photodynamic Therapy.

Angew Chem Int Ed Engl 2020 11 2;59(46):20582-20588. Epub 2020 Sep 2.

Bio-Organic Chemistry, Institute of Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, P. O. Box 513, 5600 MB, Eindhoven, The Netherlands.

Inspired by the dynamic morphology control of molecular assemblies in biological systems, we have developed pH-responsive transformable peptide-based nanoparticles for photodynamic therapy (PDT) with prolonged tumor retention times. The self-assembled peptide-porphyrin nanoparticles transformed into nanofibers when exposed to the acidic tumor microenvironment, which was mainly driven by enhanced intermolecular hydrogen bond formation between the protonated molecules. The nanoparticle transformation into fibrils improved their singlet oxygen generation ability and enabled high accumulation and long-term retention at tumor sites. Strong fluorescent signals of these nanomaterials were detected in tumor tissue up to 7 days after administration. Moreover, the peptide assemblies exhibited excellent anti-tumor efficacy via PDT in vivo. This in situ fibrillar transformation strategy could be utilized to design effective stimuli-responsive biomaterials for long-term imaging and therapy.
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http://dx.doi.org/10.1002/anie.202008708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693186PMC
November 2020

Bone-Targeting Prodrug Mesoporous Silica-Based Nanoreactor with Reactive Oxygen Species Burst for Enhanced Chemotherapy.

ACS Appl Mater Interfaces 2020 Aug 21;12(31):34630-34642. Epub 2020 Jul 21.

School of Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of New Drug Screening, Southern Medical University, Guangzhou 510515, China.

Cancer remains a primary threat to human lives. Recently, amplification of tumor-associated reactive oxygen species (ROS) has been used as a boosting strategy to improve tumor therapy. Here, we report on a bone-targeting prodrug mesoporous silica-based nanoreactor for combined photodynamic therapy (PDT) and enhanced chemotherapy for osteosarcoma. Because of surface modification of a bone-targeting biphosphate moiety and the enhanced permeability and retention effect, the formed nanoreactor shows efficient accumulation in osteosarcoma and exhibits long-term retention in the tumor microenvironment. Upon laser irradiation, the loaded photosensitizer chlorin e6 (Ce6) produces in situ ROS, which not only works for PDT but also functions as a trigger for controlled release of doxorubicin (DOX) and doxycycline (DOXY) from the prodrugs based on a thioketal () linkage. The released DOXY further promotes ROS production, thus perpetuating subsequent DOX/DOXY release and ROS burst. The ROS amplification induces long-term high oxidative stress, which increases the sensitivity of the osteosarcoma to chemotherapy, therefore resulting in enhanced tumor cell inhibition and apoptosis. The as-developed nanoreactor with combined PDT and enhanced chemotherapy based on ROS amplification shows significant promise as a potential platform for cancer treatment.
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http://dx.doi.org/10.1021/acsami.0c08992DOI Listing
August 2020

GE11 peptide-installed chimaeric polymersomes tailor-made for high-efficiency EGFR-targeted protein therapy of orthotopic hepatocellular carcinoma.

Acta Biomater 2020 09 18;113:512-521. Epub 2020 Jun 18.

Biomedical Polymers Laboratory, College of Chemistry, Chemical Engineering and Materials Science, and State Key Laboratory of Radiation Medicine and Protection, Soochow University, Soochow University, Suzhou, 215123, PR China. Electronic address:

Hepatocellular carcinoma (HCC) remains a leading malignancy with a high mortality and little improvement in treatments. Protein drugs though known for their extraordinary potency and specificity have rarely been investigated for HCC therapy owing to lack of appropriate delivery systems. Here, we designed GE11 peptide-installed chimaeric polymersomes (GE11-CPs) for high-efficiency EGFR-targeted protein therapy of orthotopic SMMC-7721 HCC-bearing nude mice. GE11-CPs were assembled from poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-b-poly(aspartic acid) (PEG-P(TMC-DTC)-PAsp) and GE11-functionalized PEG-P(TMC-DTC), which allowed efficient loading and protection of proteins in the watery interior and fine-tuning of GE11 densities at the surface. CPs with short PAsp segments (degree of polymerization (DP) = 5, 10 and 15) exhibited a protein loading efficiency of 60%-72% and glutathione-responsive protein release. Saporin-loaded GE11-CPs had a size of 36 - 62 nm depending on GE11 densities and DP of PAsp. Notably, GE11-CPs with 10% GE11 revealed greatly enhanced uptake in SMMC-7721 cells, boosting the anticancer potency of saporin for over 3-folds compared with non-targeted control (half-maximal inhibitory concentration (IC) = 11.0 versus 36.3 nM). The biodistribution studies using Cy5-labeled cytochrome C as a model protein demonstrated about 3-fold higher accumulation of GE11-CPs formulation than CPs counterpart in both subcutaneous and orthotopic SMMC-7721 tumor models. Notably, saporin-loaded GE11-CPs revealed low toxicity, effective tumor inhibition and significant improvement of survival rate compared with PBS and non-targeted groups (median survival time: 99 versus 37 and 42 days). EGFR-targeted chimaeric polymersomes carrying proteins appear an interesting HCC treatment modality.
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http://dx.doi.org/10.1016/j.actbio.2020.06.020DOI Listing
September 2020

Hyperthermia-Triggered On-Demand Biomimetic Nanocarriers for Synergetic Photothermal and Chemotherapy.

Adv Sci (Weinh) 2020 Jun 20;7(11):1903642. Epub 2020 Apr 20.

School of Pharmaceutical Science Guangdong Provincial Key Laboratory of New Drug Screening Southern Medical University Guangzhou 510515 China.

Nanoparticle-based drug delivery systems with low side effects and enhanced efficacy hold great potential in the treatment of various malignancies, in particular cancer; however, they are still challenging to attain. Herein, an anticancer drug delivery system based on a cisplatin (CDDP) containing nanogel, functionalized with photothermal gold nanorods (GNRs) which are electrostatically decorated with doxorubicin (DOX) is reported. The nanoparticles are formed via the crosslinking reaction of hyaluronic acid with the ancillary anticarcinogen CDDP in the presence of DOX-decorated GNRs. The nanogel is furthermore cloaked with a cancer cell membrane, and the resulting biomimetic nanocarrier (4T1-HANG-GNR-DC) shows efficient accumulation by homologous tumor targeting and possesses long-time retention in the tumor microenvironment. Upon near-infrared (NIR) laser irradiation, in situ photothermal therapy is conducted which further induces hyperthermia-triggered on-demand drug release from the nanogel reservoir to achieve a synergistic photothermal/chemo-therapy. The as-developed biomimetic nanocarriers, with their dual-drug delivery features, homotypic tumor targeting and synergetic photothermal/chemo-therapy, show much promise as a potential platform for cancer treatment.
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http://dx.doi.org/10.1002/advs.201903642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284223PMC
June 2020

Photoactivated Polymersome Nanomotors: Traversing Biological Barriers.

Angew Chem Int Ed Engl 2020 09 27;59(39):16918-16925. Epub 2020 Jul 27.

Bio-Organic Chemistry, Institute of Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Helix (STO 3.41), P. O. Box 513, 5600 MB, Eindhoven, The Netherlands.

Synthetic nanomotors are appealing delivery vehicles for the dynamic transport of functional cargo. Their translation toward biological applications is limited owing to the use of non-degradable components. Furthermore, size has been an impediment owing to the importance of achieving nanoscale (ca. 100 nm) dimensions, as opposed to microscale examples that are prevalent. Herein, we present a hybrid nanomotor that can be activated by near-infrared (NIR)-irradiation for the triggered delivery of internal cargo and facilitated transport of external agents to the cell. Utilizing biodegradable poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PDLLA) block copolymers, with the two blocks connected via a pH sensitive imine bond, we generate nanoscopic polymersomes that are then modified with a hemispherical gold nanocoat. This Janus morphology allows such hybrid polymersomes to undergoing photothermal motility in response to thermal gradients generated by plasmonic absorbance of NIR irradiation, with velocities ranging up to 6.2±1.10 μm s . These polymersome nanomotors (PNMs) are capable of traversing cellular membranes allowing intracellular delivery of molecular and macromolecular cargo.
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http://dx.doi.org/10.1002/anie.202003748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540338PMC
September 2020

Nanoparticles based on natural, engineered or synthetic proteins and polypeptides for drug delivery applications.

Int J Pharm 2020 Aug 9;586:119537. Epub 2020 Jun 9.

Univ. Bordeaux, CNRS, Bordeaux INP, LCPO, UMR 5629, F-33600 Pessac, France. Electronic address:

Medicine formulations at the nanoscale, referred to as nanomedicines, have managed to overcome key challenges encountered during the development of new medical treatments and entered clinical practice, but considerable improvement in terms of local efficacy and reduced toxicity still need to be achieved. Currently, the fourth-generation of nanomedicines is being developed, employing biocompatible nanocarriers that are targeted, multifunctional, and stimuli-responsive. Proteins and polypeptides can fit the standards of an efficient nanovector because of their biodegradability, intrinsic bioactivity, chemical reactivity, stimuli-responsiveness, and ability to participate in complex supramolecular assemblies. These biomacromolecules can be obtained from natural resources, produced in heterologous hosts, or chemically synthesized, allowing for different designs to access suitable carriers for a variety of drugs. To enhance targeting or therapeutic functionality, additional chemical modifications can be applied. This review demonstrates the potential of polypeptide and protein materials for the design of drug delivery nanocarriers with a special focus on their preclinical evaluation in vitro and in vivo.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119537DOI Listing
August 2020
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