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.

Systematic modulation of the lipid composition enables the tuning of liposome cellular uptake.

As liposomes have been widely explored as drug delivery carriers over the past decades, they are one of the most promising platforms due to their biocompatibility and versatility for surface functionalization. However, to improve the specific design of liposomes for future biomedical applications such as nanovaccines, it is necessary to understand how these systems interact with cell membranes, as most of their potential applications require them to be internalized by cells. Even though several investigations on the cellular uptake of liposomes were conducted, the effect of the liposome membrane properties on internalization in different cell lines remains unclear. Here, we demonstrate how the cellular uptake behavior of liposomes can be driven towards preferential interaction with dendritic cells (DC2.4) as compared to macrophages (RAW264.7) by tuning the lipid composition with varied molar ratios of the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cellular internalization efficiency was analyzed by flow cytometry, as well as liposome-cell membrane co-localization by confocal laser scanning microscopy. The corresponding proteomic analysis of the protein corona was performed in order to unravel the possible effect on the internalization. The obtained results of this work reveal that it is possible to modulate the cellular uptake towards enhanced internalization by dendritic cells just by modifying the applied lipids and, thus, mainly the physico-chemical properties of the liposomes. STATEMENT OF SIGNIFICANCE: In the field of nanomedicine, it is of key importance to develop new specific and efficient drug carriers. In this sense, liposomes are one of the most widely known carrier types and used in clinics with good results. However, the exact interaction mechanisms of liposomes with cells remain unclear, which is of great importance for the design of new drug delivery platforms. Therefore, in this work we demonstrate that cellular uptake depends on the lipid composition. We are able to enhance the uptake in a specific cell type just by tuning the content of a lipid in the liposome membrane. This finding could be a step towards the selective design of liposomes to be internalized by specific cells with promising applications in biomedicine.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Stimuli-Responsive Microarray Films for Real-Time Sensing of Surrounding Media, Temperature, and Solution Properties via Diffraction Patterns.

Stimuli-responsive polymers have attracted increasing attention over the years due to their ability to alter physiochemical properties upon external stimuli. However, many stimuli-responsive polymer-based sensors require specialized and expensive equipment, which limits their applications. Here an inexpensive and portable sensing platform of novel microarray films made of stimuli-responsive polymers is introduced for the real-time sensing of various environmental changes. When illuminated by laser light, microarray films generate diffraction patterns that can reflect and magnify variations of the periodical microstructure induced by surrounding invisible parameters in real time. Stimuli-responsive polyelectrolyte complexes are structured into micropillar arrays to monitor the pH variation and the presence of calcium ions based on reversible swelling/shrinking behaviors of the polymers. A pH hysteretic effect of the selected polyelectrolyte pair is determined and explained. Furthermore, polycaprolactone microchamber arrays are fabricated and display a thermal-driven structural change, which is exploited for photonic threshold temperature detection. Experimentally observed diffraction patterns are additionally compared with rigorous coupled-wave analysis simulations that prove that induced diffraction pattern alterations are solely caused by geometrical microstructure changes. Microarray-based diffraction patterns are a novel sensing platform with versatile sensing capabilities that will likely pave the way for the use of microarray structures as photonic sensors.

Laser-triggered drug release from polymeric 3-D micro-structured films via optical fibers.

Photosensitive polymeric three-dimensional microstructured film (PTMF) is a new type of patterned polymeric films functionalized with an array of sealed hollow 3D containers. The microstructured system with enclosed chemicals provides a tool for the even distribution of biologically active substances on a given surface that can be deposited on medical implants or used as a cells substrate. In this work, we proposed a way for photothermally activating and releasing encapsulated substances at picogram amounts from the PTMF surface in different environments using laser radiation delivered with a multimode optical fiber. The photosensitive PTMFs were prepared by the layer-by-layer (LbL) assembly from alternatively charged polyelectrolytes followed by covering with a layer of hydrophobic polylactic acid (PLA) and a layer of gold nanoparticles (AuNPs). Moreover, the typical photothermal cargo release amounts were determined on the surface of the PTMF for a range of laser powers delivered to films placed in the air, deionized (DI) water, and 1% agarose gel. The agarose gel was used as a soft tissue model for developing a technique for the laser activation of PTMFs deep in tissues using optical waveguides. The number of PTMF chambers activated by a near-infrared (NIR) laser beam was evaluated as the function of optical parameters.

pH dependent degradation properties of lactide based 3D microchamber arrays for sustained cargo release.

Encapsulation of small water soluble molecules is important in a large variety of applications, ranging from medical substance releasing implants in the field of medicine over release of catalytically active substances in the field of chemical processing to anti-corrosion agents in industry. In this work polylactic acid (PLA) based hollow-structured microchamber (MC) arrays are fabricated via one-step dip coating of a silicone rubber stamp into PLA solution. These PLA MCs are able to retain small water soluble molecules (Rhodamine B) stably entrapped within aqueous environments. It is shown, that degradation of PLA MCs strongly depends on environmental conditions like surrounding pH and follows first order degradation kinetics. This pH dependent PLA MC degradation can be utilized to control the release kinetics of encapsulated cargo.

Alginate Microparticle Arrays as Self-Polishing Bio-Fouling Release Coatings.

Biofouling is a severe problem of any water borne structure. Since the ban of tin-organic compounds and expected bans of other poisonous chemical formulations in the near future, replacement of these compounds are sought. This study investigates antibiofouling properties of micro- and nanostructured alginate layer films. Alginate is a natural product from brown algae and was in this study electro-sprayed and crosslinked with divalent calcium and copper ions to produce homogeneous micro- and nanoparticles. These calcium- and copper-alginate particles were assembled into micro- and nanostructured layer films and the antifouling properties of these low elastic modulus, hydrophilic, biodegradable system were evaluated with the microalgae chlorella. Comparison with pristine glass slides, smooth copper- and calcium alginate bulk films was performed. The comparison shows less biofouling for smooth bulk films compared to micro-nanostructured alginate, while all alginate systems are less bio-fouled on long timescales compared to pristine glass.

Polymer microchamber arrays for geometry-controlled drug release: a functional study in human cells of neuronal phenotype.

Polyelectrolyte multilayer (PEM) microchambers can provide a versatile cargo delivery system enabling rapid, site-specific drug release on demand. However, experimental evidence for their potential benefits in live human cells is scarce. Equally, practical applications often require substance delivery that is geometrically constrained and highly localized. Here, we establish human-cell biocompatibility and on-demand cargo release properties of the PEM or polylactic acid (PLA)-based microchamber arrays fabricated on a patterned film base. We grow human N2A cells (a neuroblastoma cell line widely used for studies of neurotoxicity) on the surface of the patterned microchamber arrays loaded with either a fluorescent indicator or the ubiquitous excitatory neurotransmitter glutamate. The differentiating human N2A cells show no detrimental effects on viability when growing on either PEM@PLA or PLA-based arrays for up to ten days in vitro. Firstly, we use two-photon (2P) excitation with femtosecond laser pulses to open individual microchambers in a controlled way while monitoring release and diffusion of the fluorescent cargo (rhodamine or FITC fluorescent dye). Secondly, we document the increases in intracellular Ca2+ in local N2A cells in response to the laser-triggered glutamate release from individual microchambers. The functional cell response is site-specific and reproducible on demand and could be replicated by applying glutamate to the cells using a pressurised micropipette. Time-resolved fluorescence imaging confirms the physiological range of the glutamate-evoked intracellular Ca2+ dynamics in the differentiating N2A cells. Our data indicate that the nano-engineering design of the fabricated PEM or PLA-based patterned microchamber arrays could provide a biologically safe and efficient tool for targeted, geometrically constrained drug delivery.

Magnetically-guided hydrogel capsule motors produced via ultrasound assisted hydrodynamic electrospray ionization jetting.

Hydrogel capsules are a potential candidate for drug delivery and an interesting alternative to polyelectrolyte multilayer capsules which are under investigation since 20 years. Recently introduced polyelectrolyte complex capsules produced by spraying are non-biodegradable and not biocompatible, which limits their practical application, while biodegradable alginate capsules require complex coaxial electrospray ionization jetting. In this work, biodegradable alginate capsules cross-linked by calcium are successfully produced by hydrodynamic electrospray ionization jetting with the assistance of low frequency ultrasound. The size and shape of most capsules show significant differences with respect to different spraying distance, spraying mode, electrode shape and spraying concentration. Capsules in the shape of vase, mushrooms and spheres were successfully produced. Average capsule size can be adjusted from 10 µm to 2 mm. These capsules are used to encapsulate a model drug. Encapsulated paramagnetic particles enable defined directional motion under the propulsion of a rotating magnetic field, while model drugs can be released by ultrasound.

Polylactic acid sealed polyelectrolyte complex microcontainers for controlled encapsulation and NIR-Laser based release of cargo.

Surface mediated drug delivery is important for a large variety of applications, especially in medicine to control cell growth, prevent blood platelet activation on implants or for self-disinfecting devices (e.g. catheters). In industrial applications, controlled release of substances from surfaces is needed in a broad range of applications from anti-corrosion systems to anti-biofouling. Polyelectrolyte multilayers (PEM) based microcontainers (MCs) require several days production time, while MCs composed out of polylactic acid (PLA) are entirely hydrophobic, offering no functionality. We hereby present an approach to fabricate PLA coated synthetic as well as biopolymer based biodegradable polyelectrolyte complex MCs able to encapsulate small hydrophilic cargo within less than one hour. The chambers facilitate laser controlled release of cargo within submerged conditions.

Magnetically-propelled hydrogel particle motors produced by ultrasound assisted hydrodynamic electrospray ionization jetting.

One of the most promising future applications of hydrogels is drug delivery. The hydrogels act as a biomedical cargo model to reach the target and release drugs to cure diseases. This application requires no side effects of the hydrogel and the ability to pass through porous media (e.g. membranes, interstitial tissue etc.) with nanoscaled channels. At the same time, the hydrogel must be mass-producible in an economic way. In this work, we show that hydrodynamic electrospray ionization jetting combined with ultrasound can fulfill these high requirements. This method can produce mucoadhesive micro-/nano-particles, which are small enough to pass through the gastrointestinal epithelium. The average size of the produced particles is exactly predictable by controlling the spraying distance, spraying mode, alginate concentration, ultrasound bath frequency and counter electrode shape. These micro-/nano-particles are loaded with biocompatible magnetite nanoparticles, and propelled by a rotating magnetic field between 5 to 20 m T and a frequency from 1 Hz to 100 Hz. These rotating micro-/nano-particle motors perform directional motion in solution, offering a promising possibility for magnetically controlled drug delivery.

Effect of a Controlled Release of Epinephrine Hydrochloride from PLGA Microchamber Array: In Vivo Studies.

This paper presents the synthesis of highly biocompatible and biodegradable poly(lactide- co-glycolide) (PLGA) microchamber arrays sensitive to low-intensity therapeutic ultrasound (1 MHz, 1-2 W, 1 min). A reliable method was elaborated that allowed the microchambers to be uniformly filled with epinephrine hydrochloride (EH), with the possibility of varying the cargo amount. The maximum load of EH was 4.5 µg per array of 5 mm × 5 mm (about 24 pg of EH per single microchamber). A gradual, spontaneous drug release was observed to start on the first day, which is especially important in the treatment of acute patients. Ultrasound triggered a sudden substantial release of EH from the films. In vivo real-time studies using a laser speckle contrast imaging system demonstrated changes in the hemodynamic parameters as a consequence of EH release under ultrasound exposure. We recorded a decrease in blood flow as a vascular response to EH release from a PLGA microchamber array implanted subcutaneously in a mouse. This response was immediate and delayed (1 and 2 days after the implantation of the array). The PLGA microchamber array is a new, promising drug depot implantable system that is sensitive to external stimuli.

In-situ NIR-laser mediated bioactive substance delivery to single cell for EGFP expression based on biocompatible microchamber-arrays.

Controlled drug delivery and gene expression is required for a large variety of applications including cancer therapy, wound healing, cell migration, cell modification, cell-analysis, reproductive and regenerative medicine. Controlled delivery of precise amounts of drugs to a single cell is especially interesting for cell and tissue engineering as well as therapeutics and has until now required the application of micro-pipettes, precisely placed dispersed drug delivery vehicles, or injections close to or into the cell. Here we present surface bound micro-chamber arrays able to store small hydrophilic molecules for prolonged times in subaqueous conditions supporting spatiotemporal near infrared laser mediated release. The micro-chambers (MCs) are composed of biocompatible and biodegradable polylactic acid (PLA). Biocompatible gold nanoparticles are employed as light harvesting agents to facilitate photothermal MC opening. The degree of photothermal heating is determined by numerical simulations utilizing optical properties of the MC, and confirmed by Brownian motion measurements of laser-irradiated micro-particles exhibiting similar optical properties like the MCs. The amount of bioactive small molecular cargo (doxycycline) from local release is determined by fluorescence spectroscopy and gene expression in isolated C2C12 cells via enhanced green fluorescent protein (EGFP) biosynthesis.

Hydrodynamic electrospray ionization jetting of calcium alginate particles: effect of spray-mode, spraying distance and concentration.

Hydrodynamic electrospray ionization jetting was applied for generating and characterizing calcium cross-linked alginate microparticles. These microparticles show different diameters and aspect ratios for three electrospray modes (dripping, conejet and multijet modes), four spraying distances (5, 10, 15 and 20 cm), and six spraying concentrations. Comparing the three different electrospray modes, we found that the conejet mode results in the smallest particle diameters, lowest aspect ratio and smallest variations over the parameter space mentioned above. For all spraying modes, the resultant particle diameters become independent of the spraying distance at a sprayed solute concentration ≥ 2.5%. The aspect ratio of microparticles varies significantly for different spraying modes and distances. An increasing aspect ratio of all spray modes was determined for sodium alginate spraying concentrations ≤ 1.5% and spraying distances of 20 cm; this phenomenon can be explained with the chain ejection effect. This systematic investigation offers a basic database for industrial applications of hydrodynamic electrospray ionization.

Polylactic acid nano- and microchamber arrays for encapsulation of small hydrophilic molecules featuring drug release via high intensity focused ultrasound.

Long term encapsulation combined with spatiotemporal release for a precisely defined quantity of small hydrophilic molecules on demand remains a challenge in various fields ranging from medical drug delivery, controlled release of catalysts to industrial anti-corrosion systems. Free-standing individually sealed polylactic acid (PLA) nano- and microchamber arrays were produced by one-step dip-coating a PDMS stamp into PLA solution for 5 s followed by drying under ambient conditions. The wall thickness of these hydrophobic nano-microchambers is tunable from 150 nm to 7 µm by varying the PLA solution concentration. Furthermore, small hydrophilic molecules were successfully in situ precipitated within individual microchambers in the course of solvent evaporation after sonicating the PLA@PDMS stamp to remove air-bubbles and to load the active substance containing solvent. The cargo capacity of single chambers was determined to be in the range of several picograms, while it amounts to several micrograms per cm2. Two different methods for sealing chambers were compared: microcontact printing versus dip-coating whereby microcontact printing onto a flat PLA sheet allows for entrapment of micro-air-bubbles enabling microchambers with both ultrasound responsiveness and reduced permeability. Cargo release triggered by external high intensity focused ultrasound (HIFU) stimuli is demonstrated by experiment and compared with numerical simulations.

Polylactic Acid Sealed Polyelectrolyte Multilayer Microchambers for Entrapment of Salts and Small Hydrophilic Molecules Precipitates.

Efficient depot systems for entrapment and storage of small water-soluble molecules are of high demand for wide variety of applications ranging from implant based drug delivery in medicine and catalysis in chemical processes to anticorrosive systems in industry where surface-mediated active component delivery is required on a time and site specific manner. This work reports the fabrication of individually sealed hollow-structured polyelectrolyte multilayer (PEM) microchamber arrays based on layer-by-layer self-assembly as scaffolds and microcontact printing. These PEM chambers are composed out of biocompatible polyelectrolytes and sealed by a monolayer of hydrophobic biocompatible and biodegradable polylactic acid (PLA). Coating the chambers with hydrophobic PLA allows for entrapment of a microair-bubble in each chamber that seals and hence drastically reduces the PEM permeability. PLA@PEM microchambers are proven to enable prolonged subaqueous storage of small hydrophilic salts and molecules such as crystalline NaCl, doxicycline, and fluorescent dye rhodamine B. The presented microchambers are able to entrap air bubbles and demonstrate a novel strategy for entrapment, storage, and protection of micropackaged water-soluble substances in precipitated form. These chambers allow triggered release as demonstrated by ultrasound responsiveness of the chambers. Low-frequency ultrasound exposure is utilized for microchamber opening and payload release.

Forecastable and Guidable Bubble-Propelled Microplate Motors for Cell Transport.

Cell transport is important to renew body functions and organs with stem cells, or to attack cancer cells with immune cells. The main hindrances of this method are the lack of understanding of cell motion as well as proper transport systems. In this publication, bubble-propelled polyelectrolyte microplates are used for controlled transport and guidance of HeLa cells. Cells survive attachment on the microplates and up to 22 min in 5% hydrogen peroxide solution. They can be guided by a magnetic field whereby increased friction of cells attached to microplates decreases the speed by 90% compared to pristine microplates. The motion direction of the cell-motor system is easier to predict due to the cell being opposite to the bubbles.

Guidable Thermophoretic Janus Micromotors Containing Gold Nanocolorifiers for Infrared Laser Assisted Tissue Welding.

Current wound sealing systems such as nanoparticle-based gluing of tissues allow almost immediate wound sealing. The assistance of a laser beam allows the wound sealing with higher controllability due to the collagen fiber melting which is defined by loss of tertiary protein structure and restoration upon cooling. Usually one employs dyes to paint onto the wound, if water absorption bands are absent. In case of strong bleeding or internal wounds such applications are not feasible due to low welding depth in case of water absorption bands, dyes washing off, or the dyes becoming diluted within the wound. One possible solution of these drawbacks is to use autonomously movable particles composing of biocompatible gold and magnetite nanoparticles and biocompatible polyelectrolyte complexes. In this paper a proof of principle study is presented on the utilization of thermophoretic Janus particles and capsules employed as dyes for infrared laser-assisted tissue welding. This approach proves to be efficient in sealing the wound on the mouse in vivo. The temperature measurement of single particle level proves successful photothermal heating, while the mechanical characterizations of welded liver, skin, and meat confirm mechanical restoration of the welded biological samples.

Patterned Microstructure Fabrication: Polyelectrolyte Complexes vs Polyelectrolyte Multilayers.

Polyelectrolyte complexes (PEC) are formed by mixing the solutions of oppositely charged polyelectrolytes, which were hitherto deemed "impossible" to process, since they are infusible and brittle when dry. Here, we describe the process of fabricating free-standing micro-patterned PEC films containing array of hollow or filled microchambers by one-step casting with small applied pressure and a PDMS mould. These structures are compared with polyelectrolyte multilayers (PEM) thin films having array of hollow microchambers produced from a layer-by-layer self-assembly of the same polyelectrolytes on the same PDMS moulds. PEM microchambers "cap" and "wall" thickness depend on the number of PEM bilayers, while the "cap" and "wall" of the PEC microchambers can be tuned by varying the applied pressure and the type of patterned mould. The proposed PEC production process omits layering approaches currently employed for PEMs, reducing the production time from ~2 days down to 2 hours. The error-free structured PEC area was found to be significantly larger compared to the currently-employed microcontact printing for PEMs. The sensitivity of PEC chambers towards aqueous environments was found to be higher compared to those composed of PEM.