Controlled Membrane Transport in Polymeric Biomimetic Nanoreactors.

Polymersome-based biomimetic nanoreactors (PBNs) have generated great interest in nanomedicine and cell mimicry due to their robustness, tuneable chemistry, and broad applicability in biologically relevant fields. In this concept review, we mainly discuss the state of the art in functional polymersomes as biomimetic nanoreactors with membrane-controlled transport. PBNs that use environmental changes or external stimuli to adjust membrane permeability while maintaining structural integrity are highlighted. By encapsulating catalytic species, PBNs are able to convert inactive substrates into functional products in a controlled manner. In addition, special attention is paid to the use of PBNs as tailored artificial organelles with biomedical applications in vitro and in vivo, facilitating the fabrication of next-generation artificial organelles as therapeutic nanocompartments.

Polymer nano-systems for the encapsulation and delivery of active biomacromolecular therapeutic agents.

Biomacromolecular therapeutic agents, particularly proteins, antigens, enzymes, and nucleic acids are emerging as powerful candidates for the treatment of various diseases and the development of the recent vaccine based on mRNA highlights the enormous potential of this class of drugs for future medical applications. However, biomacromolecular therapeutic agents present an enormous delivery challenge compared to traditional small molecules due to both a high molecular weight and a sensitive structure. Hence, the translation of their inherent pharmaceutical capacity into functional therapies is often hindered by the limited performance of conventional delivery vehicles. Polymer drug delivery systems are a modular solution able to address those issues. In this review, we discuss recent developments in the design of polymer delivery systems specifically tailored to the delivery challenges of biomacromolecular therapeutic agents. In the future, only in combination with a multifaceted and highly tunable delivery system, biomacromolecular therapeutic agents will realize their promising potential for the treatment of diseases and for the future of human health.

Confining the Sol-Gel Reaction at the Water/Oil Interface: Creating Compartmentalized Enzymatic Nano-Organelles for Artificial Cells.

Living organisms compartmentalize their catalytic reactions in membranes for increased efficiency and selectivity. To mimic the organelles of eukaryotic cells, we develop a mild approach for in situ encapsulating enzymes in aqueous-core silica nanocapsules. In order to confine the sol-gel reaction at the water/oil interface of miniemulsion, we introduce an aminosilane to the silica precursors, which serves as both catalyst and an amphiphilic anchor that electrostatically assembles with negatively charged hydrolyzed alkoxysilanes at the interface. The semi-permeable shell protects enzymes from proteolytic attack, and allows the transport of reactants and products. The enzyme-carrying nanocapsules, as synthetic nano-organelles, are able to perform cascade reactions when enveloped in a polymer vesicle, mimicking the hierarchically compartmentalized reactions in eukaryotic cells. This in situ encapsulation approach provides a versatile platform for the delivery of biomacromolecules.

Liposomal Enzyme Nanoreactors Based on Nanoconfinement for Efficient Antitumor Therapy.

Enzymatic reactions can consume endogenous nutrients of tumors and produce cytotoxic species and are therefore promising tools for treating malignant tumors. Inspired by nature where enzymes are compartmentalized in membranes to achieve high reaction efficiency and separate biological processes with the environment, we develop liposomal nanoreactors that can perform enzymatic cascade reactions in the aqueous nanoconfinement of liposomes. The nanoreactors effectively inhibited tumor growth in vivo by consuming tumor nutrients (glucose and oxygen) and producing highly cytotoxic hydroxyl radicals (⋅OH). Co-compartmentalization of glucose oxidase (GOx) and horseradish peroxidase (HRP) in liposomes could increase local concentration of the intermediate product hydrogen peroxide (H2 O2 ) as well as the acidity due to the generation of gluconic acid by GOx. Both H2 O2 and acidity accelerate the second-step reaction by HRP, hence improving the overall efficiency of the cascade reaction. The biomimetic compartmentalization of enzymatic tandem reactions in biocompatible liposomes provides a promising direction for developing catalytic nanomedicines in antitumor therapy.

Poly(ethylene glycol)-Based Surfactant Reduces the Conformational Change of Adsorbed Proteins on Nanoparticles.

When in contact with a biological medium, the surfaces of nanoparticles are usually covered by proteins. In this regard, it was found that poly(ethylene glycol) (PEG) promotes the "stealth effect". This implies a reduction of unspecific protein adsorption and cellular uptake. Although information about the PEG-protein interaction was reported, more accurate and sophisticated structure and dynamics analyses are needed to understand the interaction processes in detail. This work studies the PEG-protein interaction using model nanoparticles stabilized either by the PEG-based surfactant Lutensol AT50 or sodium dodecyl sulfate. The interaction with human serum albumin was studied using neutron scattering techniques. The parameters obtained by small-angle neutron scattering yielded information about the adsorbed protein layer thickness. Protein structure changes were detected via differential scanning fluorimetry and elastic neutron scattering. This combination gives a better insight into the PEG-protein interaction, contributing to the design of nanomaterials for medical applications.

Squaric Ester-Based Nanogels Induce No Distinct Protein Corona but Entrap Plasma Proteins into their Porous Hydrogel Network.

After intravenous administration of nanocarriers, plasma proteins may rapidly adsorb onto their surfaces. This process hampers the prediction of the nanocarriers' pharmacokinetics as it determines their physiological identity in a complex biological environment. Toward clinical translation it is therefore an essential prerequisite to investigate the nanocarriers' interaction with plasma proteins. Here, this work evaluates a highly "PEGylated" squaric ester-based nanogel with inherent prolonged blood circulation properties. After incubation with human blood plasma, the nanogels are isolated by asymmetrical flow-field flow fractionation. Multiangle light scattering measurements confirm the absence of significant size increases as well as aggregation upon plasma incubation. However, proteomic analyses by gel electrophoresis find minor absolute amounts of proteins (3 wt%), whereas label-free liquid chromatography mass spectrometry identify 65 enriched proteins. Interestingly, the relative abundance of these proteins is almost similar to their proportion in pure native plasma. Due to the nanogels' hydrated and porous network morphology, it is concluded that the detected proteins rather result from passive diffusion into the nanogel network than from specific interactions at the plasma particle interface. Consequently, these results do not indicate a classical surface protein corona but rather reflect the highly outer and inner stealth-like behavior of the porous hydrogel network.

Brush Conformation of Polyethylene Glycol Determines the Stealth Effect of Nanocarriers in the Low Protein Adsorption Regime.

For nanocarriers with low protein affinity, we show that the interaction of nanocarriers with cells is mainly affected by the density, the molecular weight, and the conformation of polyethylene glycol (PEG) chains bound to the nanocarrier surface. We achieve a reduction of nonspecific uptake of ovalbumin nanocarriers by dendritic cells using densely packed PEG chains with a "brush" conformation instead of the collapsed "mushroom" conformation. We also control to a minor extent the dysopsonin adsorption by tailoring the conformation of attached PEG on the nanocarriers. The brush conformation of PEG leads to a stealth behavior of the nanocarriers with inhibited uptake by phagocytic cells, which is a prerequisite for successful in vivo translation of nanomedicine to achieve long blood circulation and targeted delivery. We can clearly correlate the brush conformation of PEG with inhibited phagocytic uptake of the nanocarriers. This study shows that, in addition to the surface's chemistry, the conformation of polymers controls cellular interactions of the nanocarriers.

Light-Activated Membrane Transport in Polymeric Cell-Mimics.

Giant polymersomes are versatile and stable biomimetic compartments that are ideal for building cell-like systems. However, the transport of hydrophilic molecules across the membrane, which controls the function of cell-like systems, is limited by the low permeability of polymeric bilayers. Therefore, mechanisms to control the permeability of polymersomes are necessary to create functional cell-like systems. Here, we describe the design of giant polymersomes equipped with spiropyran-based permeability modulators. Photo-isomerization of the modulators leads to perturbation of the polymer membrane, resulting in increased permeability. The photoactivated polymersomes were used to construct two cell-like systems controlled by light-activated transport of hydrophilic molecules. First, we designed an enzymatic micro-reactor activated by light irradiation. Second, we constructed a hybrid coacervate-in-polymersome system that mimics the adaptive formation of biological condensates in cells.

Synthetic Cells: From Simple Bio-Inspired Modules to Sophisticated Integrated Systems.

Bottom-up synthetic biology is the science of building systems that mimic the structure and function of living cells from scratch. To do this, researchers combine tools from chemistry, materials science, and biochemistry to develop functional and structural building blocks to construct synthetic cell-like systems. The many strategies and materials that have been developed in recent decades have enabled scientists to engineer synthetic cells and organelles that mimic the essential functions and behaviors of natural cells. Examples include synthetic cells that can synthesize their own ATP using light, maintain metabolic reactions through enzymatic networks, perform gene replication, and even grow and divide. In this Review, we discuss recent developments in the design and construction of synthetic cells and organelles using the bottom-up approach. Our goal is to present representative synthetic cells of increasing complexity as well as strategies for solving distinct challenges in bottom-up synthetic biology.

Synthetic Silica Nano-Organelles for Regulation of Cascade Reactions in Multi-Compartmentalized Systems.

In eukaryotic cells, enzymes are compartmentalized into specific organelles so that different reactions and processes can be performed efficiently and with a high degree of control. In this work, we show that these features can be artificially emulated in robust synthetic organelles constructed using an enzyme co-compartmentalization strategy. We describe an in situ encapsulation approach that allows enzymes to be loaded into silica nanoreactors in well-defined compositions. The nanoreactors can be combined into integrated systems to produce a desired reaction outcome. We used the selective enzyme co-compartmentalization and nanoreactor integration to regulate competitive cascade reactions and to modulate the kinetics of sequential reactions involving multiple nanoreactors. Furthermore, we show that the nanoreactors can be efficiently loaded into giant polymer vesicles, resulting in multi-compartmentalized microreactors.

Membrane Manipulation of Giant Unilamellar Polymer Vesicles with a Temperature-Responsive Polymer.

Understanding the complex behavior and dynamics of cellular membranes is integral to gain insight into cellular division and fusion processes. Bottom-up synthetic cells are as a platform for replicating and probing cellular behavior. Giant polymer vesicles are more robust than liposomal counterparts, as well as having a broad range of chemical functionalities. However, the stability of the membrane can prohibit dynamic processes such as membrane phase separation and division. Here, we present a method for manipulating the membrane of giant polymersomes using a temperature responsive polymer. Upon elevation of temperature deformation and phase separation of the membrane was observed. Upon cooling, the membrane relaxed and became homogeneous again, with infrequent division of the synthetic cells.

Bio-Orthogonal Nanogels for Multiresponsive Release.

Responsive nanogel systems are interesting for the drug delivery of bioactive molecules due to their high stability in aqueous media. The development of nanogels that are able to respond to biochemical cues and compatible with the encapsulation and the release of large and sensitive payloads remains challenging. Here, multistimuli-responsive nanogels were synthesized using a bio-orthogonal and reversible reaction and were designed for the selective release of encapsulated cargos in a spatiotemporally controlled manner. The nanogels were composed of a functionalized polysaccharide cross-linked with pH-responsive hydrazone linkages. The effect of the pH value of the environment on the nanogels was fully reversible, leading to a reversible control of the release of the payloads and a "stop-and-go" release profile. In addition to the pH-sensitive nature of the hydrazone network, the dextran backbone can be degraded through enzymatic cleavage. Furthermore, the cross-linkers were designed to be responsive to oxidoreductive cues. Disulfide groups, responsive to reducing environments, and thioketal groups, responsive to oxidative environments, were integrated into the nanogel network. The release of model payloads was investigated in response to changes in the pH value of the environment or to the presence of reducing or oxidizing agents.

Formation of giant polymer vesicles by simple double emulsification using block copolymers as the sole surfactant.

Polymer vesicles that mimic the function of cell membranes can be obtained through the self-assembly of amphiphilic block copolymers. The cell-like characteristics of polymer vesicles, such as the core-shell structure, semi-permeability and tunable surface chemistry make them excellent building blocks for artificial cells. However, the standard preparation methods for polymer vesicles can be time consuming, require special equipment, or have low encapsulation efficiency for large components, such as nanomaterials and proteins. Here, we introduce a new encapsulation strategy based on a simple double emulsification (SDE) approach which allows giant polymer vesicles to be formed in a short time and with basic laboratory equipment. The SDE method requires a single low molecular weight block copolymer that has the dual role of macromolecular surfactant and membrane building block. Giant polymer vesicles with diameters between 20-50 µm were produced, which allowed proteins and nanoparticles to be encapsulated. To demonstrate its practical application, we used the SDE method to assemble a simple artificial cell that mimics a two-step enzymatic cascade reaction. The SDE method described here introduces a new tool for simple and rapid fabrication of synthetic compartments.

Membrane Engineering: Phase Separation in Polymeric Giant Vesicles.

Cell membranes exhibit elaborate lipidic patterning to carry out a myriad of functions such as signaling and trafficking. Domain formation in giant unilamellar vesicles (GUVs) is thus of interest for understanding fundamental biological processes and to provide new prospects for biocompatible soft materials. Lipid rearrangements in lipidic GUVs and lipid/polymer GUVs are extensively studied whereas polymer/polymer hybrid GUVs remain evasive. Here, the focus is on the thermodynamically driven phase separation of amphiphilic polymers in GUVs. It is demonstrated that polymer phase separation is entropically dictated by hydrophobic block incompatibilities and that films topology can help to determine the outcome of polymeric phase separation in GUVs. Lastly, Janus-GUVs are obtained and GUVs exhibit a single large domain by using a compatibilizing hydrophobic block copolymer.

Polymeric Nanoparticles with Neglectable Protein Corona.

The current understanding of nanoparticle-protein interactions indicates that they rapidly adsorb proteins upon introduction into a living organism. The formed protein corona determines thereafter identity and fate of nanoparticles in the body. The present study evaluates the protein affinity of three core-crosslinked polymeric nanoparticles with long circulation times, differing in the hydrophilic polymer material forming the particle surface, namely poly(N-2-hydroxypropylmethacrylamide) (pHPMA), polysarcosine (pSar), and poly(ethylene glycol) (PEG). This includes the nanotherapeutic CPC634, which is currently in clinical phase II evaluation. To investigate possible protein corona formation, the nanoparticles are incubated in human blood plasma and separated by asymmetrical flow field-flow fractionation (AF4). Notably, light scattering shows no detectable differences in particle size or polydispersity upon incubation with plasma for all nanoparticles, while in gel electrophoresis, minor amounts of proteins can be detected in the particle fraction. Label-free quantitative proteomics is additionally applied to analyze and quantify the composition of the proteins. It proves that some proteins are enriched, but their concentration is significantly less than one protein per particle. Thus, most of the nanoparticles are not associated with any proteins. Therefore, this work underlines that polymeric nanoparticles can be synthesized, for which a protein corona formation does not take place.

Temperature Sensing in Cells Using Polymeric Upconversion Nanocapsules.

Monitoring local temperature inside cells is crucial when interpreting biological activities as enhanced cellular metabolism leads to higher heat production and is commonly correlated with the presence of diseases such as cancer. In this study, we report on polymeric upconversion nanocapsules for potential use as local nanothermometers in cells by exploiting the temperature dependence of the triplet-triplet annihilation upconversion phenomenon. Nanocapsules synthesized by the miniemulsion solvent evaporation technique are composed of a polymer shell and a liquid core of rice bran oil, hosting triplet-triplet annihilation upconversion active dyes as sensitizer and emitter molecules. The sensitivity of the triplet-triplet annihilation upconversion to the local oxygen concentration was overcome by the oxygen reduction ability of the rice bran oil core. The triplet-triplet annihilation upconversion process could thus successfully be applied at different levels of oxygen presence including at ambient conditions. Using this method, the local temperature within a range of 22 to 40 °C could be determined when the upconversion nanocapsules were taken up by HeLa cells with good cellular viability. Thus, the higher cell temperatures where the cells show enhanced metabolic activity led to a significant increase in the delayed fluorescence spectrum of the upconversion nanocapsules. These findings are promising for further development of novel treatment and diagnostic tools in medicine.

Polysaccharide-Based pH-Responsive Nanocapsules Prepared with Bio-Orthogonal Chemistry and Their Use as Responsive Delivery Systems.

Bio-orthogonal reactions have become an essential tool to prepare biomaterials; for example, in the synthesis of nanocarriers, bio-orthogonal chemistry allows circumventing common obstacles related to the encapsulation of delicate payloads or the occurrence of uncontrolled side reactions, which significantly limit the range of potential payloads to encapsulate. Here, we report a new approach to prepare pH-responsive nanocarriers using dynamic bio-orthogonal chemistry. The reaction between a poly(hydrazide) crosslinker and functionalized polysaccharides was used to form a pH-responsive hydrazone network. The network formation occurred at the interface of aqueous nanodroplets in miniemulsion and led to the production of nanocapsules that were able to encapsulate payloads of different molecular weights. The resulting nanocapsules displayed low cytotoxicity and were able to release the encapsulated payload, in a controlled manner, under mildly acidic conditions.

Bio-Based Lignin Nanocarriers Loaded with Fungicides as a Versatile Platform for Drug Delivery in Plants.

Lignin-based nano- and microcarriers are a promising biodegradable drug delivery platform inside of plants. Many wood-decaying fungi are capable of degrading the wood component lignin by segregated lignases. These fungi are responsible for severe financial damage in agriculture, and many of these plant diseases cannot be treated today. However, enzymatic degradation is also an attractive handle to achieve a controlled release of drugs from artificial lignin vehicles. Herein, chemically cross-linked lignin nanocarriers (NCs) were prepared by aza-Michael addition in miniemulsion, followed by solvent evaporation. The cross-linking of lignin was achieved with the bio-based amines (spermine and spermidine). Several fungicides-namely, azoxystrobin, pyraclostrobin, tebuconazole, and boscalid-were encapsulated in situ during the miniemulsion polymerization, demonstrating the versatility of the method. Lignin NCs with diameters of 200-300 nm (determined by dynamic light scattering) were obtained, with high encapsulation efficiencies (70-99%, depending on the drug solubility). Lignin NCs successfully inhibited the growth of Phaeomoniella chlamydospora and Phaeoacremonium minimum, which are lignase-producing fungi associated with the worldwide occurring fungal grapevine trunk disease Esca. In planta studies proved their efficiency for at least 4 years after a single injection into Vitis vinifera ("Portugieser") plants on a test vineyard in Germany. The lignin NCs are of high interest as biodegradable delivery vehicles to be applied by trunk injection against the devastating fungal disease Esca but might also be promising against other fungal plant diseases.

Amphiphilic Polyphenylene Dendron Conjugates for Surface Remodeling of Adenovirus†5.

Amphiphilic surface groups play an important role in many biological processes. The synthesis of amphiphilic polyphenylene dendrimer branches (dendrons), providing alternating hydrophilic and lipophilic surface groups and one reactive ethynyl group at the core is reported. The amphiphilic surface groups serve as biorecognition units that bind to the surface of adenovirus 5 (Ad5), which is a common vector in gene therapy. The Ad5/dendron complexes showed high gene transduction efficiencies in coxsackie-adenovirus receptor (CAR)-negative cells. Moreover, the dendrons offer incorporation of new functions at the dendron core by in situ post-modifications, even when bound to the Ad5 surface. Surfaces coated with these dendrons were analyzed for their blood-protein binding capacity, which is essential to predict their performance in the blood stream. A new platform for introducing bioactive groups to the Ad5 surface without chemically modifying the virus particles is provided.

Polymer-Based Module for NAD+ Regeneration with Visible Light.

The regeneration of enzymatic cofactors by cell-free synthetic modules is a key step towards producing a purely synthetic cell. Herein, we demonstrate the regeneration of the enzyme cofactor NAD+ by photo-oxidation of NADH under visible-light irradiation by using metal-free conjugated polymer nanoparticles. Encapsulation of the light-active nanoparticles in the lumen of polymeric vesicles produced a fully organic module able to regenerate NAD+ in an enzyme-free system. The polymer compartment conferred physical and chemical autonomy to the module, allowing the regeneration of NAD+ to occur efficiently, even in harsh chemical environments. Moreover, we show that regeneration of NAD+ by the photocatalyst nanoparticles can oxidize a model substrate, in conjunction with the enzyme glycerol dehydrogenase. To ensure the longevity of the enzyme, we immobilized it within a protective silica matrix; this yielded enzymatic silica nanoparticles with enhanced long-term performance and compatibility with the NAD+ -regeneration system.