ABSTRACT
Inflammatory bowel disease (IBD) has become a globally prevalent chronic disease with no causal therapeutic options. Targeted drug delivery systems with selectivity for inflamed areas in the gastrointestinal tract promise to reduce severe drug-related side effects. By creating three distinct nanostructures (vesicles, spherical, and wormlike micelles) from the same amphiphilic block copolymer poly(butyl acrylate)-block-poly(ethylene oxide) (PBA-b-PEO), the effect of nanoparticle shape on human mucosal penetration is systematically identified. An Ussing chamber technique is established to perform the ex vivo experiments on human colonic biopsies, demonstrating that the shape of polymeric nanostructures represents a rarely addressed key to tissue selectivity required for efficient IBD treatment. Wormlike micelles specifically enter inflamed mucosa from patients with IBD, but no significant uptake is observed in healthy tissue. Spheres (≈25 nm) and vesicles (≈120 nm) enter either both normal and inflamed tissue types or do not penetrate any tissue. According to quantitative image analysis, the wormlike nanoparticles localize mainly within immune cells, facilitating specific targeting, which is crucial for further increasing the efficacy of IBD treatment. These findings therefore demonstrate the untapped potential of wormlike nanoparticles not only to selectively target the inflamed human mucosa, but also to target key pro-inflammatory cells.
Subject(s)
Inflammatory Bowel Diseases , Micelles , Polymers , Humans , Polymers/chemistry , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Nanoparticles/chemistry , Inflammation/drug therapy , Inflammation/pathology , Drug Delivery SystemsABSTRACT
Within this study, an amphiphilic and potentially biodegradable polypeptide library based on poly[(4-aminobutyl)-l-glutamine-stat-hexyl-l-glutamine] [P(AB-l-Gln-stat-Hex-l-Gln)] was investigated for gene delivery. The influence of varying proportions of aliphatic and cationic side chains affecting the physicochemical properties of the polypeptides on transfection efficiency was investigated. A composition of 40 mol% Hex-l-Gln and 60 mol % AB-l-Gln (P3) was identified as best performer over polypeptides with higher proportions of protonatable monomers. Detailed studies of the transfection mechanism revealed the strongest interaction of P3 with cell membranes, promoting efficient endocytic cell uptake and high endosomal release. Spectrally, time-, and z-resolved fluorescence microscopy further revealed the crucial role of filopodia surfing in polyplex-cell interaction and particle internalization in lamellipodia regions, followed by rapid particle transport into cells. This study demonstrates the great potential of polypeptides for gene delivery. The amphiphilic character improves performance over cationic homopolypeptides, and the potential biodegradability is advantageous toward other synthetic polymeric delivery systems.
Subject(s)
Gene Transfer Techniques , Glutamine , Genetic Therapy , Transfection , Cations , PeptidesABSTRACT
Controlling the length of one-dimensional (1D) polymer nanostructures remains a key challenge on the way toward the applications of these structures. Here, we demonstrate that top-down processing facilitates a straightforward adjustment of the length of polyethylene oxide (PEO)-based supramolecular polymer bottlebrushes (SPBs) in aqueous solutions. These cylindrical structures self-assemble via directional hydrogen bonds formed by benzenetrisurea (BTU) or benzenetrispeptide (BTP) motifs located within the hydrophobic core of the fiber. A slow transition from different organic solvents to water leads first to the formation of µm-long fibers, which can subsequently be fragmented by ultrasonication or dual asymmetric centrifugation. The latter allows for a better adjustment of applied shear stresses, and thus enables access to differently sized fragments depending on time and rotation rate. Extended sonication and scission analysis further allowed an estimation of tensile strengths of around 16 MPa for both the BTU and BTP systems. In combination with the high kinetic stability of these SPBs, the applied top-down methods represent an easily implementable technique toward 1D polymer nanostructures with an adjustable length in the range of interest for perspective biomedical applications.
ABSTRACT
This review revisits essential staining protocols for electron microscopy focussing on the visualization of active sites, i.e. enzymes, metabolites or proteins, in cells and tissues, which have been developed 50 to 60 years ago, however, never were established as standard protocols being used in electron microscopy in a routine fashion. These approaches offer numerous possibilities to expand the knowledge of cellular function and specifically address the localization of active compounds of these systems. It is our conviction, that many of these techniques are still useful, in particular when applied in conjunction with correlative light and electron microscopy. Revisiting specialized classical electron microscopy staining protocols for use in correlative microscopy is particularly promising, as some of these protocols were originally developed as staining methods for light microscopy. To account for this history, rather than summarizing the most recent achievements in literature, we instead first provide an overview of techniques that have been used in the past. While some of these techniques have been successfully implemented into modern microscopy techniques during recent years already, more possibilities are yet to be re-discovered and provide exciting new perspectives for their future use.
Subject(s)
Microscopy, Electron , Staining and LabelingABSTRACT
Cationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymerization. The copolymer poly[(n-butyl acrylate)-b-(2-(dimethyl amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphology, interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addition of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.
Subject(s)
Gene Transfer Techniques , Genetic Therapy , Polymers , Acrylic Resins , Animals , Cations , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Mice , Micelles , Plasmids , Polymerization , TransfectionABSTRACT
Reactive polymersomes represent a versatile artificial cargo carrier system that can facilitate an immediate release in response to a specific stimulus. The herein presented oxidation-sensitive polymersomes feature a time-delayed release mechanism in an oxidative environment, which can be precisely adjusted by either tuning the membrane thickness or partial pre-oxidation. These polymeric vesicles are conveniently prepared by PISA allowing the straightforward and effective in situ encapsulation of cargo molecules, as shown for dyes and enzymes. Kinetic studies revealed a critical degree of oxidation causing the destabilization of the membrane, while no release of the cargo is observed beforehand. The encapsulation of glucose oxidase directly transforms these polymersomes into glucose-sensitive vesicles, as small molecules including sugars can passively penetrate their membrane. Considering the ease of preparation, these polymersomes represent a versatile platform for the confinement and burst release of cargo molecules after a precisely adjustable time span in the presence of specific triggers, such as H2 O2 or glucose.
ABSTRACT
Strong directional hydrogen bonds represent a suitable supramolecular force to drive the one-dimensional (1D) aqueous self-assembly of polymeric amphiphiles resulting in cylindrical polymer brushes. However, our understanding of the kinetics in these assembly processes is still limited. We here demonstrate that the obtained morphologies for our recently reported benzene tris-urea and tris-peptide conjugates are strongly pathway-dependent. A controlled transfer from solutions in organic solvents to aqueous environments enabled a rate-dependent formation of kinetically trapped but stable nanostructures ranging from small cylindrical or spherical objects (<50 nm) to remarkably large fibers (>2 µm). A detailed analysis of the underlying assembly mechanism revealed a cooperative nature despite the steric demands of the polymers. Nucleation is induced by hydrophobic interactions crossing a critical water content, followed by an elongation process due to the strong hydrogen bonds. These findings open an interesting new pathway to control the length of 1D polymer nanostructures.
Subject(s)
Nanofibers , Nanostructures , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Nanofibers/chemistry , Nanostructures/chemistry , Polymers/chemistry , Water/chemistryABSTRACT
The assembly of supramolecular polymer bottlebrushes in aqueous systems is, in most cases, associated with a lateral aggregation of the supramolecular building blocks in addition to their axial stacking. Here, it is demonstrated that this limitation can be overcome by attaching three polymer chains to a central supramolecular unit that possesses a sufficiently high number of hydrogen bonding units to compensate for the increased steric strain. Therefore, a 1,3,5-benzenetrisurea-polyethylene oxide conjugate is modified with different peptide units located next to the urea groups which should facilitate self-assembly in water. For a single amino acid per arm, spherical micelles are obtained for all three tested amino acids (alanine, leucine, and phenylalanine) featuring different hydrophobicities. Only a slight increase in size and solution stability of spherical micelles is observed with increasing hydrophobicity of amino acid unit. In contrast, introducing two amino acid units per arm and thus increasing the number of hydrogen bonds per unimer molecule results in the formation of cylindrical structures, that is, supramolecular polymer bottlebrushes, despite a suppressed lateral aggregation. Consequently, it can be concluded that the number of hydrogen bonds has a more profound impact on the resulting solution morphology than the hydrophobicity of the amino acid unit.
Subject(s)
Polymers , Water , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , MicellesABSTRACT
Roughening by anisotropic etching of N-face gallium nitride is the key aspect in today's production of blue and white light emitting diodes (LEDs). Both surface area and number of surface angles are increased, facilitating light outcoupling from the LED chip. The structure of a GaN layer stack grown by metal organic chemical vapour deposition (MOCVD) was varied in the unintentionally doped u-GaN bulk region. Different sequences of 2D and 3D grown layers led to a variation in dislocation density, which was monitored by photoluminescence microscopy (PLM) and X-ray diffraction (XRD). Thin-film processing including laser lift off (LLO) was applied. The influence of epitaxial changes on the N-face etch kinetics was determined in aqueous KOH solution at elevated temperature. Inductively-coupled plasma optical emission spectroscopy (ICP-OES) was used to measure the etch progress in small time increments with high precision. Thereby, the disadvantages of other techniques such as determination of weight loss or height difference were overcome, achieving high accuracy and reproducibility.