Engineered Protein Nano-Compartments for Targeted Enzyme Localization

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis

Development of bio-mimetic nano-compartments for solar energy capture

A growing range of artificial cell-mimicking compartments(e.g., liposomes) have been demonstrated as technological platforms for applications ranging from model systems in bottom-up cell biology to miniature chemical reactors. Here, I describe work on developing a liposomal compartment for capturing light-energy. The harvesting of light energy starts at a photoactive centre, where light-excited electrons are generated and then transferred to an electron acceptor. The efficiency of this electron transfer is often limited due to charge recombination (i.e., re-assembly of photo-separated electrons and electron holes) within the photoactive chromophore. Inspired by natural photosynthesis, this study envisions a strategy to limit charge recombination by rapid transfer of the lightexcited electrons away from the photoactive molecules (dye-sensitized TiO2 nanoparticles or carbon dots) and across the liposome membrane via conductive transmembrane protein complex MtrCAB from Shewanella oneidensis MR-1. Furthermore, such compartment enables localisation of the oxidation and reduction processes in separate environments. The assembly of the envisioned compartment begins with a study of the molecular interface between TiO2 nanoparticles, a commonly used material for photocatalysis studies, and the MtrC(AB) conduit. This interface is mapped using an approach called protein footprinting, which involves protein labelling and subsequent analysis of the modified peptides by mass spectrometry. Understanding the molecular interactions at this bio-inorganic interface is crucial for engineering electronic communication between these materials. Then, a proof of concept is demonstrated of a half-reaction: light energy capture, charge separation across the membrane and use of the energy to drive a chemical reaction. Transmembrane electron transfer is achieved chemically and photochemically using dye sensitized TiO2 nanoparticles or carbon dots located outside the liposomes. The electron transfer through MtrCAB conduit is confirmed optically by monitoring the destructive reduction of an encapsulated azo-dye Reactive Red 120. Finally, work on encapsulation of fuel evolving catalysts (i.e., hydrogen producing Pt nanoparticles and a hydrogenase HydA1) within the lipid-enclosed compartment (i.e., liposome lumen and porous silica support) is discussed alongside the challenges for combining different materials within ordered structures

DNA-Mediated Self-Organization of Polymeric Nanocompartments Leads to Interconnected Artificial Organelles

Self-organization of nanocomponents was mainly focused on solid nanoparticles, quantum dots, or liposomes to generate complex architectures with specific properties, but intrinsically limited or not developed enough, to mimic sophisticated structures with biological functions in cells. Here, we present a biomimetic strategy to self-organize synthetic nanocompartments (polymersomes) into clusters with controlled properties and topology by exploiting DNA hybridization to interconnect polymersomes. Molecular and external factors affecting the self-organization served to design clusters mimicking the connection of natural organelles: fine-tune of the distance between tethered polymersomes, different topologies, no fusion of clustered polymersomes, and no aggregation. Unexpected, extended DNA bridges that result from migration of the DNA strands inside the thick polymer membrane (about 12 nm) represent a key stability and control factor, not yet exploited for other synthetic nano-object networks. The replacement of the empty polymersomes with artificial organelles, already reported for single polymersome architecture, will provide an excellent platform for the development of artificial systems mimicking natural organelles or cells and represents a fundamental step in the engineering of molecular factories

Chemical Cascading Between Polymersomal Nanoreactor Populations

[EN] Harnessing interactions of functional nano-compartments to generate larger particle assemblies allows studying diverse biological behaviors based on their population states and can lead to the development of smart materials. Herein, thiol-functionalized polymersome nanoreactors are utilized as responsive organelle-like nano-compartments-with inherent capacity to associate into larger aggregates in response to change in the redox state of their environment-to study the kinetics of cascade reactions and explore functions of their collective under different population states. Two nanoreactor populations, glucose oxidase- and horseradish peroxidase-loaded polymersomes, are prepared, and the results of their cascading upon addition of glucose are investigated. The kinetics of resorufin production in associated polymersomes and non-associated polymersome populations are compared, observing a decreased rate upon association. For the associated populations, faster chemical cascading is found when the two types of nanoreactors are associated in a concerted step, as compared to sequential association. The addition of competing agents such as catalase impacts the communication between non-associated polymersomes, whereas such an effect is less pronounced for the associated ones. Altogether, the results showcase the impact of collective associations on enzymatic cascading between organelle-like nanoreactors.Y.A. and A.L.-L. contributed equally to this work. The authors would like to acknowledge the support from the Dutch Ministry of Education, Culture, and Science (Gravitation program 024.001.035 and Spinoza premium) and the ERC Advanced Grant (Artisym 694120).A.L.-L. acknowledges support from the MSCA Cofund project oLife, which has received funding from the European Union's Horizon 2020 research and innovation program under the Grant Agreement 847675; and the Maria Zambrano Program from the Spanish Government funded by NextGenerationEU from the European Union. Dr. Imke Pijpers is thanked for cryo-TEM imaging. Dr. Pascal Welzen is acknowledged for advice and useful discussion on polymer and polymersome preparation.Altay, Y.; Llopis-Lorente, A.; Abdelmohsen, LKEA.; Van Hest, JC. (2023). Chemical Cascading Between Polymersomal Nanoreactor Populations. Macromolecular Chemistry and Physics. 224(1):1-5. https://doi.org/10.1002/macp.20220026915224

Nanostructured Lipid Carriers for Delivery of Chemotherapeutics: A Review

The efficacy of current standard chemotherapy is suboptimal due to the poor solubility and short half-lives of chemotherapeutic agents, as well as their high toxicity and lack of specificity which may result in severe side effects, noncompliance and patient inconvenience. The application of nanotechnology has revolutionized the pharmaceutical industry and attracted increasing attention as a significant means for optimizing the delivery of chemotherapeutic agents and enhancing their efficiency and safety profiles. Nanostructured lipid carriers (NLCs) are lipid-based formulations that have been broadly studied as drug delivery systems. They have a solid matrix at room temperature and are considered superior to many other traditional lipid-based nanocarriers such as nanoemulsions, liposomes and solid lipid nanoparticles (SLNs) due to their enhanced physical stability, improved drug loading capacity, and biocompatibility. This review focuses on the latest advances in the use of NLCs as drug delivery systems and their preparation and characterization techniques with special emphasis on their applications as delivery systems for chemotherapeutic agents and different strategies for their use in tumor targeting.This research was funded by Aljalila foundation, grant number AJF201777

Solid Lipid Nanoparticles: A Potential Approach for Dermal Drug Delivery

Solid lipid nanoparticles (SLNs) have attracted increasing attention during recent years. Due to their unique size dependent properties, lipid nanoparticles offer possibilities to develop new therapeutics. The ability to incorporate drugs into nanoparticles offers a new prototype in drug delivery thus realizing the dual goal of both controlled release and site-specific drug delivery. Drug delivery to the skin is widely used for local and systemic delivery and has potential to be improved by application of nanoparticulate formulations. If investigated appropriately, solid lipid nanoparticles may open new opportunities in therapy of complex diseases which is difficult to treat

Reaction between Energy Particle Ion Beam with Carbon Nanotube

Carbon nanotubes (CNTs) have attracted considerable attention due to their high aspect ratio, whisker-like form for best possible geometrical field enhancement, high electrical conductivity, and extraordinary thermal stability. Ion beam technology is a potential technique for controlled construction of CNTs. During collision with energetic ions, carbon atom of CNTs can get an adequate amount of energy to escape from the graphite lattice and produce a large number of defects. These defects are advantageous for adding some new functional groups and nanoparticles to modify CNTs. Meanwhile, the structure and atoms in the region of the defects can be rearranged and changed into amorphous structure, onion structure, and so on. These defects also can be used to form the junctions of CNTs and realize welding of CNTs and network formation of amorphous carbon nanowires

Biomimetic Strategy To Reversibly Trigger Functionality of Catalytic Nanocompartments by the Insertion of pH-Responsive Biovalves

We describe an innovative strategy to generate catalytic compartments with triggered functionality at the nanoscale level by combining pH-reversible biovalves and enzyme-loaded synthetic compartments. The biovalve has been engineered by the attachment of stimuli-responsive peptides to a genetically modified channel porin, enabling a reversible change of the molecular flow through the pores of the porin in response to a pH change in the local environment. The biovalve functionality triggers the reaction inside the cavity of the enzyme-loaded compartments by switching the in situ activity of the enzymes on/off based on a reversible change of the permeability of the membrane, which blocks or allows the passage of substrates and products. The complex functionality of our catalytic compartments is based on the preservation of the integrity of the compartments to protect encapsulated enzymes. An increase of the in situ activity compared to that of the free enzyme and a reversible on/off switch of the activity upon the presence of a specific stimulus is achieved. This strategy provides straightforward solutions for the development of catalytic nanocompartments efficiently producing desired molecules in a controlled, stimuli-responsive manner with high potential in areas, such as medicine, analytical chemistry, and catalysis