Organelle-specific targeting of polymersomes into the cell nucleus.

Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.

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.

DNA-tethered Polymersome Clusters as Nanotheranostic Platform.

Nanotheranostics combine the use of nanomaterials and biologically active compounds to achieve diagnosis and treatment at the same time. To date, severe limitations compromise the use of nanotheranostic systems as potent nanomaterials are often incompatible with potent biomolecules. Herein we emphasize how a novel type of polymersome clusters loaded with active molecules can be optimized to obtain an efficient nanotheranostic platform. Polymersomes loaded with enzymes and specific dyes, respectively and exposing complementary DNA strands at their external surface formed clusters by means of DNA hybridization. We describe factors at the molecular level and other conditions that need to be optimized at each step of the cluster formation to favor theranostic efficiency.

Segregated Nanocompartments Containing Therapeutic Enzymes and Imaging Compounds within DNA-Zipped Polymersome Clusters for Advanced Nanotheranostic Platform.

Nanotheranostics is an emerging field that brings together nanoscale-engineered materials with biological systems providing a combination of therapeutic and diagnostic strategies. However, current theranostic nanoplatforms have serious limitations, mainly due to a mismatch between the physical properties of the selected nanomaterials and their functionalization ease, loading ability, or overall compatibility with bioactive molecules. Herein, a nanotheranostic system is proposed based on nanocompartment clusters composed of two different polymersomes linked together by DNA. Careful design and procedure optimization result in clusters segregating the therapeutic enzyme human Dopa decarboxylase (DDC) and fluorescent probes for the detection unit in distinct but colocalized nanocompartments. The diagnostic compartment provides a twofold function: trackability via dye loading as the imaging component and the ability to attach the cluster construct to the surface of cells. The therapeutic compartment, loaded with active DDC, triggers the cellular expression of a secreted reporter enzyme via production of dopamine and activation of dopaminergic receptors implicated in atherosclerosis. This two-compartment nanotheranostic platform is expected to provide the basis of a new treatment strategy for atherosclerosis, to expand versatility and diversify the types of utilizable active molecules, and thus by extension expand the breadth of attainable applications.

How Do the Properties of Amphiphilic Polymer Membranes Influence the Functional Insertion of Peptide Pores?

Pore-forming peptides are of high biological relevance particularly as cytotoxic agents, but their properties are also applicable for the permeabilization of lipid membranes for biotechnological applications, which can then be translated to the more stable and versatile polymeric membranes. However, their interactions with synthetic membranes leading to pore formation are still poorly understood, hampering the development of peptide-based nanotechnological applications, such as biosensors or catalytic compartments. To elucidate these interactions, we chose the model peptide melittin, the main component of bee venom. Here, we present our systematic investigation on how melittin interacts with and inserts into synthetic membranes, based on amphiphilic block copolymers, to induce pore formation in three different setups (planar membranes and micrometric and nanometric vesicles). By varying selected molecular properties of block copolymers and resulting membranes (e.g., hydrophilic to hydrophobic block ratio, membrane thickness, surface roughness, and membrane curvature) and the stage of melittin addition to the synthetic membranes, we gained a deeper understanding of melittin insertion requirements. In the case of solid-supported planar membranes, melittin interaction was favored by membrane roughness and thickness, but its insertion and pore formation were hindered when the membrane was excessively thick. The additional property provided by micrometric vesicles, curvature, increased the functional insertion of melittin, which was evidenced by the even more curved nanometric vesicles. Using nanometric vesicles allowed us to estimate the pore size and density, and by changing the stage of melittin addition, we overcame the limitations of peptide-polymer membrane interaction. Mirroring the functionality assay of planar membranes, we produced glucose-sensing vesicles. The design of synthetic membranes permeabilized with melittin opens a new path toward the development of biosensors and catalytic compartments based on pore-forming peptides functionally inserted in synthetic planar or three-dimensional membranes.

Biomolecule-polymer hybrid compartments: combining the best of both worlds.

Compartmentalization is a fundamental principle in biology that is needed for the temporal and spatial separation of chemically incompatible reactions and biomolecules. Nano- or micro-sized compartments made of synthetic polymers are used to mimick this principle. The self-assembly of these polymers into vesicular objects is highly compatible with the integration of biomolecules, either into the lumen, the membrane or onto the surface of the vesicles. Thus, a great variety of biohybrid nano- and microscaled compartments has been developed exploiting the specific function and properties of targeting peptides, antibodies, enzymes, nucleic acids or lipids. Such biohybrid compartments have moved from simple systems encapsulating e.g. a model protein into complex multicompartmentalized structures that are able to combine the activity of different biomolecular cargos getting closer to the realization of artifical organelles or cells. Encapsulation of medically relevant cargos combined with careful design of the polymeric scaffold and specific surface functionalization have led to a significant progress in therapeutical applications such as targeted drug delivery or enzyme replacement therapy.

Nanoscale Enzymatic Compartments in Tandem Support Cascade Reactions in Vitro.

Compartmentalization at the nanoscale is fundamental in nature, where the spatial segregation of biochemical reactions within cells ensures optimal conditions for regulating metabolic pathways. Here, we present a nature-inspired approach to engineer enzymatic cascade reactions taking place between separate vesicular nanocompartments (polymersomes), each containing one enzyme type. We propose, by the selected combination of enzymes, an efficient solution to detoxify the harmful effect of uric acid and prevent the accumulation of the derived H2O2, both being associated with various pathological conditions (e.g., gout and oxidative stress). Fungal uricase and horseradish peroxidase combined to act in tandem, and they were separately encapsulated within nanocompartments that were equipped with channel porins as gates to allow passage of substrates and products from each step of the reaction. We established the molecular factors affecting the efficiency of the overall reaction, and the protective role of the compartments. Interestingly, the cascade reaction between separate nanocompartments was as efficient as for free enzymes in complex media, such as human serum. The nanocompartments were nontoxic toward cells, and more importantly, addition of the tandem catalytic nanocompartments to cells exposed to uric acid provided simultaneous detoxification of uric acid and the H2O2. Such catalytic nanocompartments can be used as a platform for understanding fundamental factors affecting intracellular communication and can introduce non-native metabolic reactions into living systems for therapeutic applications.

Amphiphilic Peptide Self-Assembly: Expansion to Hybrid Materials.

The design of functional systems with sizes in the nanometer range is a key challenge in fields such as biomedicine, nanotechnology, and engineering. Some of the most promising materials nowadays consist of self-assembling peptides or peptide-polymer hybrid materials because of their versatility and the resulting properties that can be achieved with these structures. Self-assembly of pure amphiphilic peptides or in combination with block copolymers results in a large variety of nanostructures (micelles, nanoparticles (NPs), compartments, planar membranes) each with different characteristics and tunable properties. Here, we describe such novel peptide- or peptide-polymer-based supramolecular nanostructures and emphasize their functionality and various promising applications.

N-Arylacyl O-sulfonated aminoglycosides as novel inhibitors of human neutrophil elastase, cathepsin G and proteinase 3.

The balance between neutrophil serine proteases (NSPs) and protease inhibitors (PIs) in the lung is a critical determinant for a number of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis and acute lung injury. During activation at inflammatory sites, excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, PIs counteract these effects by inactivating NSPs; however, in chronic inflammatory lung diseases, there are insufficient amounts of PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP activity for the clinical management of chronic inflammatory lung diseases. In the study reported here, a panel of N-arylacyl O-sulfonated aminoglycosides was screened to identify inhibitors of the NSPs. Dose-dependent inhibitors for each individual serine protease were identified. Select compounds were found to inhibit multiple NSPs, including one lead structure that is shown to inhibit all three NSPs. Two lead compounds identified during the screen for each individual NSP were further characterized as partial mixed inhibitors of CatG. Concentration-dependent inhibition of protease-mediated detachment of lung epithelial cells is demonstrated.

Contrast-Enhanced Low-Field MRI Occasionally Alters the Surgical Approach for Canine Intervertebral Disc Extrusions.

OBJECTIVES: The aim of this study was to describe how the addition of contrast-enhanced low-field magnetic resonance imaging sequences can confirm or change the initially planned surgical approach for canine intervertebral disc extrusions. STUDY DESIGN: Magnetic resonance imagings of 20 dogs diagnosed with intervertebral disc extrusions were retrospectively reviewed by a board-certified neurologist for the location of extradural disc material, contrast enhancement, and whether enhancement reinforced or changed the initially planned surgical approach. RESULTS: Extradural compressive material contrast-enhanced in 17/20 dogs. In 14/20 dogs, enhancement was considered to increase the confidence level of the location for surgery including two cases where the surgical approach was altered. CONCLUSION: Gadolinium-based contrast agents in low-field magnetic resonance imaging can aid the surgical planning of intervertebral disc extrusions in dogs by improving the confidence level of location and extent of extradural material and occasionally altering the surgical approach.

Nanoparticle coatings for controlled release of quercetin from an angioplasty balloon.

Peripheral artery disease (PAD) is a systemic vascular disease of the legs that results in a blockage of blood flow from the heart to the lower extremities. Now one of the most common causes of mortality in the U.S., the first line of therapy for PAD is to mechanically open the blockages using balloon angioplasty. Coating the balloons with antiproliferative agents can potentially reduce vessel re-narrowing, or restenosis after surgical intervention, but current drug-coated balloons releasing chemotherapy agents like paclitaxel have in some cases shown increased mortality long-term. Our aim was to design a novel drug-coated balloon using a polymeric nanodelivery system for a sustained release of polyphenols that reduce restenosis but with reduced toxicity compared to chemotherapy agents. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles with entrapped quercetin, a dimethoxy quercetin (rhamnazin), as well as quercetin covalently attached to PLGA, were developed. Balloon catheters were coated with polymeric nanoparticles using an ultrasonic method, and nanoparticle characteristics, drug loading, coating uniformity and drug release were determined. The adhesion of nanoparticles to vascular smooth muscle cells and the antiproliferative effect of nano-delivered polyphenols were also assessed. Of the nanoparticle systems tested, those with covalently attached quercetin provided the most sustained release over a 6-day period. Although these particles adhered to cells to a smaller extent compared to other nanoparticle formulations, their attachment was resistant to washing. These particles also exhibited the greatest anti-proliferative effect. In addition, their attachment was not altered when the cells were grown in calcifying conditions, and in PAD tissue calcification is typically a condition that impedes drug delivery. Moreover, the ultrasonic coating method generated a uniform balloon coating. The polymeric nanoparticle system with covalently attached quercetin developed herein is thus proposed as a promising platform to reduce restenosis post-angioplasty.

Catalytic polymersomes to produce strong and long-lasting bioluminescence.

Here, we introduce an artificial bioluminescent nanocompartment based on the encapsulation of light-producing enzymes, luciferases, inside polymersomes. We exploit nanocompartmentalization to enhance luciferase stability in a cellular environment but also to positively modulate enzyme kinetics to achieve a long-lasting glow type signal. These features pave the way for expanding bioluminescence to nanotechnology-based applications.

Clustering of catalytic nanocompartments for enhancing an extracellular non-native cascade reaction.

Compartmentalization is fundamental in nature, where the spatial segregation of biochemical reactions within and between cells ensures optimal conditions for the regulation of cascade reactions. While the distance between compartments or their interaction are essential parameters supporting the efficiency of bio-reactions, so far they have not been exploited to regulate cascade reactions between bioinspired catalytic nanocompartments. Here, we generate individual catalytic nanocompartments (CNCs) by encapsulating within polymersomes or attaching to their surface enzymes involved in a cascade reaction and then, tether the polymersomes together into clusters. By conjugating complementary DNA strands to the polymersomes' surface, DNA hybridization drove the clusterization process of enzyme-loaded polymersomes and controlled the distance between the respective catalytic nanocompartments. Owing to the close proximity of CNCs within clusters and the overall stability of the cluster architecture, the cascade reaction between spatially segregated enzymes was significantly more efficient than when the catalytic nanocompartments were not linked together by DNA duplexes. Additionally, residual DNA single strands that were not engaged in clustering, allowed for an interaction of the clusters with the cell surface as evidenced by A549 cells, where clusters decorating the surface endowed the cells with a non-native enzymatic cascade. The self-organization into clusters of catalytic nanocompartments confining different enzymes of a cascade reaction allows for a distance control of the reaction spaces which opens new avenues for highly efficient applications in domains such as catalysis or nanomedicine.

Bioactive Catalytic Nanocompartments Integrated into Cell Physiology and Their Amplification of a Native Signaling Cascade.

Bioactive nanomaterials have the potential to overcome the limitations of classical pharmacological approaches by taking advantage of native pathways to influence cell behavior, interacting with them and eliciting responses. Herein, we propose a cascade system mediated by two catalytic nanocompartments (CNC) with biological activity. Activated by nitric oxide (NO) produced by inducible nitric oxidase synthase (iNOS), soluble guanylyl cyclase (sGC) produces cyclic guanosine monophosphate (cGMP), a second messenger that modulates a broad range of physiological functions. As alterations in cGMP signaling are implicated in a multitude of pathologies, its signaling cascade represents a viable target for therapeutic intervention. Following along this line, we encapsulated iNOS and sGC in two separate polymeric compartments that function in unison to produce NO and cGMP. Their action was tested in vitro by monitoring the derived changes in cytoplasmic calcium concentrations of HeLa and differentiated C2C12 myocytes, where the produced second messenger influenced the cellular homeostasis.

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.

Enzymatic reactions in polymeric compartments: nanotechnology meets nature.

One of the main features of living matter is compartmentalization, that is the temporal and spatial division of biological reactions and containment of the cellular components. Nanotechnology aims to replicate this, separating tiny environments from the exterior into nano-sized and micro-sized self-assembled compartments. Those synthetic compartments can perform reactions, be tracked and act in vivo. Here, an overview of the techniques to fabricate vesicular, polymer-based catalytic compartments and the parameters affecting their architecture is presented. How communication can be ensured across their membranes, recent developments in the enzymes that have been loaded into them and the latest advances in biological applications are discussed. This review highlights the characteristics that make polymers an enticing choice, the protection they offer, and their applications in compartmentalizing biologically relevant reactions.

Expanding the potential of MRI contrast agents through multifunctional polymeric nanocarriers.

MRI is a sought-after, noninvasive tool in medical diagnostics, yet the direct application of contrast agents to tissue suffers from several drawbacks. Hosting the contrast agents in polymeric nanocarriers can solve many of these issues while creating additional benefit through exploitation of the intrinsic characteristics of the polymeric carriers. In this report, the versatility is highlighted with recent examples of dendritic and hyperbranched polymers, polymer nanoparticles and micelles, and polymersomes as multifunctional bioresponsive nanocarriers for MRI contrast agents.