Asymetric Triblock Copolymer Nanocarriers for Controlled Localization and pH-Sensitive Release of Proteins

Designing nanocarriers to release proteins under specific conditions is required to improve therapeutic approaches, especially in treating cancer and protein deficiency diseases. We present here supramolecular assemblies based on asymmetric poly(ethylene glycol)-b-poly(methylcaprolactone)-b-poly(2-(N,Ndiethylamino)ethyl methacrylate) (PEG-b-PMCL-b-PDMAEMA) copolymers for controlled localization and pH-sensitive release of proteins. Copolymers self-assembled in soft nanoparticles with a core domain formed by PMCL, and a hydrophilic domain based on PEG mainly embedded inside, and the branched PDMAEMA exposed at the particle surface. We selected as model proteins to be attached to the nanoparticles bovine serum albumin (BSA) and acid sphingomyelinase (ASM), the latter being an ideal candidate for protein replacement therapy. The hydrophilic/hydrophobic ratio, nanoparticle size, and the nature of biomolecules are key factors for modulating protein localization and attachment efficiency. The predominant outer shell of PDMAEMA allows efficient pH-triggered release of BSA and ASM, and in acidic conditions >70% of the bound proteins were released. Uptake of protein-attached nanoparticles by HELA cells, together with low toxicity and pH-responsive release, supports such protein-bound nanoparticles as efficient stimuli-responsive candidates for protein therapy

Live Follow-Up of Enzymatic Reactions Inside the Cavities of Synthetic Giant Unilamellar Vesicles Equipped with Membrane Proteins Mimicking Cell Architecture

Compartmentalization of functional biological units, cells, and organelles serves as an inspiration for the development of biomimetic materials with unprecedented properties and applications in biosensing and medicine. Because of the complexity of cells, the design of ideal functional materials remains a challenge. An elegant strategy to obtain cell-like compartments as novel materials with biofunctionality is the combination of synthetic micrometer-sized giant unilamellar vesicles (GUVs) with biomolecules because it enables studying the behavior of biomolecules and processes within confined cavities. Here we introduce a functional cell-mimetic compartment formed by insertion of the model biopore bacterial membrane protein OmpF in thick synthetic membranes of an artificial GUV compartment that enclosesâEuro"as a modelâEuro"the oxidative enzyme horseradish peroxidase. In this manner, a simple and robust cell mimic is designed: the biopore serves as a gate that allows substrates to enter cavities of the GUVs, where they are converted into products by the encapsulated enzyme and then released in the environments of GUVs. Our bioequipped GUVs facilitate the control of specific catalytic reactions in confined microscale spaces mimicking cell size and architecture and thus provide a straightforward approach serving to obtain deeper insights into biological processes inside cells in real time

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

Artificial Organelles: Reactions inside Protein-Polymer Supramolecular Assemblies

Reactions inside confined compartments at the nanoscale represent an essential step in the development of complex multifunctional systems to serve as molecular factories. In this respect, the biomimetic approach of combining biomolecules (proteins, enzymes, mimics) with synthetic membranes is an elegant way to create functional nanoreactors, or even simple artificial organelles, that function inside cells after uptake. Functionality is provided by the specificity of the biomolecule(s), whilst the synthetic compartment provides mechanical stability and robustness. The availability of a large variety of biomolecules and synthetic membranes allows the properties and functionality of these reaction spaces to be tailored and adjusted for building complex self-organized systems as the basis for molecular factories

Bioinspired Molecular Factories with Architecture and In Vivo Functionalities as Cell Mimics

Despite huge need in the medical domain and significant development efforts, artificial cells to date have limited composition and functionality. Whereas some artificial cells have proven successful for producing therapeutics or performing in vitro specific reactions, they have not been investigated in vivo to determine whether they preserve their architecture and functionality while avoiding toxicity. Here we overcome these limitations and achieve customizable cell mimic - molecular factories (MFs) - by supplementing giant plasma membrane vesicles derived from donor cells with nanometer-sized artificial organelles (AOs). MFs inherit the donor cell's natural cytoplasm and membrane, while the AOs house reactive components and provide cell-like architecture and functionality. We demonstrate that reactions inside AOs take place in a close-to-nature environment due to the unprecedented level of complexity in the composition of the MFs. We further demonstrate that in a zebrafish vertebrate animal model these cell mimics showed no apparent toxicity and retained their integrity and function. The unique advantages of highly varied composition, multi-compartmentalized architecture, and preserved functionality in vivo open new biological avenues ranging from the study of bio-relevant processes in robust cell-like environments to the production of specific bioactive compounds

DNA-directed arrangement of soft synthetic compartments and their behavior in vitro and in vivo

DNA has been widely used as a key tether to promote self-organization of super-assemblies with emergent properties. However, control of this process is still challenging for compartment assemblies and to date the resulting assemblies have unstable membranes precluding in vitro and in vivo testing. Here we present our approach to overcome these limitations, by manipulating molecular factors such as compartment membrane composition and DNA surface density, thereby controlling the size and stability of the resulting DNA-linked compartment clusters. The soft, flexible character of the polymer membrane and low number of ssDNA remaining exposed after cluster formation determine the interaction of these clusters with the cell surface. These clusters exhibit in vivo stability and lack of toxicity in a zebrafish model. To display the breadth of therapeutic applications attainable with our system, we encapsulated the medically established enzyme laccase within the inner compartment and demonstrated its activity within the clustered compartments. Most importantly, these clusters can interact selectively with different cell lines, opening a new strategy to modify and expand cellular functions by attaching such pre-organized soft DNA-mediated compartment clusters on cell surfaces for cell engineering or therapeutic applications

Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy

How Can Giant Plasma Membrane Vesicles Serve as a Cellular Model for Controlled Transfer of Nanoparticles?

Cellular model systems are essential platforms used across multiple research fields for exploring the fundaments of biology and biochemistry. Here, we present giant plasma membrane vesicles (GPMVs) as a platform of cell-like compartments that will facilitate the study of particles within a biorelevant environment and promote their further development. We studied how cellularly taken up nanoparticles (NPs) can be transferred into formed GPMVs and which are the molecular factors that play a role in successful transfer (size, concentration, and surface charge along with 3 different cell lines: HepG2, HeLa, and Caco-2). We observed that polystyrene (PS) carboxylated NPs with a size of 40 and 100 nm were successfully and efficiently transferred to GPMVs derived from all cell lines. We then investigated the distribution of NPs inside formed GPMVs and established the average number of NPs/GPMVs and the percentage of all GPMVs with NPs in their cavity. We pave the way for GPMV usage as superior cell-like mimics in medically relevant applications

Manufacturing of Liposomes: A Direct Comparison of Extrusion and Microfluidics Protocols

Liposomal formulations are frequently used for oral, topical, or parenteral drug administration. However, liposome manufacturing and industrial scale-up remains a challenge, in particular if it comes to the preparation of liposome populations with a homogenous size distribution. Therefore, extrusion through filter membranes with defined pore size is traditionally used during the preparation of small unilamellar liposomes. Microfluidics is considered to be an alternative manufacturing method. Lipids, solvents and excipients are thereby passively mixed using a microfluidics device. While the microfluidic approach is highly scalable, most of the traditional liposome preparation protocols rely on extrusion. It was therefore the aim of the present study to compare liposomal formulations with identical composition, which were prepared using either extrusion or microfluidics protocols. Liposomal formulations produced by both methods were analyzed using dynamic light scattering (DLS) to compare size, polydispersity, and ζ-potential. Our results indicate significant differences between liposomal preparations obtained using the two manufacturing methods. We conclude that the two preparation methods should not be used interchangeably

Manufacturing of Liposomes: A Direct Comparison of Extrusion and Microfluidics Protocols

Liposomal formulations are frequently used for oral, topical, or parenteral drug administration. However, liposome manufacturing and industrial scale-up remains a challenge, in particular if it comes to the preparation of liposome populations with a homogenous size distribution. Therefore, extrusion through filter membranes with defined pore size is traditionally used during the preparation of small unilamellar liposomes. Microfluidics is considered to be an alternative manufacturing method. Lipids, solvents and excipients are thereby passively mixed using a microfluidics device. While the microfluidic approach is highly scalable, most of the traditional liposome preparation protocols rely on extrusion. It was therefore the aim of the present study to compare liposomal formulations with identical composition, which were prepared using either extrusion or microfluidics protocols. Liposomal formulations produced by both methods were analyzed using dynamic light scattering (DLS) to compare size, polydispersity, and ζ-potential. Our results indicate significant differences between liposomal preparations obtained using the two manufacturing methods. We conclude that the two preparation methods should not be used interchangeably.<br /