Multistage Nanocarrier Based on an Oil Core-Graphene Oxide Shell.

Potent synthetic drugs, as well as biomolecules extracted from plants, have been investigated for their selectivity toward cancer cells. The main limitation in cancer treatment is the ability to bring such molecules within each single cancer cell, which requires accumulation in the peritumoral region followed by homogeneous spreading within the entire tissue. In the last decades, nanotechnology has emerged as a powerful tool due to its ability to protect the drug during blood circulation and allow enhanced accumulation around the leaky regions of the tumor vasculature. However, the ideal size for accumulation of around 100 nm is too large for effective penetration into the dense collagen matrix. Therefore, we propose a multistage system based on graphene oxide nanosheet-based quantum dots (GOQDs) with dimensions that are 12 nm, functionalized with hyaluronic acid (GOQDs-HA), and deposited using the layer-by-layer technique onto an oil-in-water nanoemulsion (O/W NE) template that is around 100 nm in size, previously stabilized by a biodegradable polymer, chitosan. The choice of a biodegradable core for the nanocarrier is to degrade once inside the tumor, thus promoting the release of smaller compounds, GOQDs-HA, carrying the adsorbed anticancer compound, which in this work is represented by curcumin as a model bioactive anticancer molecule. Additionally, modification with HA aims to promote active targeting of stromal and cancer cells. Cell uptake experiments and preliminary penetration experiments in three-dimensional microtissues were performed to assess the proposed multistage nanocarrier.

Exploring the evolution of bacterial cellulose precursors and their potential use as cellulose-based building blocks.

Natural polymers have found increased use in a wider range of applications due to their less harmful effects. Notably, bacterial cellulose has gained significant consideration due to its exceptional physical and chemical properties and its substantial biocompatibility, which makes it an attractive candidate for several biomedical applications. This study attempts to thoroughly unravel the microstructure of bacterial cellulose precursors, known as bioflocculants, which to date have been poorly characterised, by employing both electron and optical microscopy techniques. Here, starting from bioflocculants from Symbiotic Culture of Bacteria and Yeast (SCOBY), we proved that their microstructural features, such as porosity percentage, cellulose assembly degree, fibres' density and fraction, change in a spatio-temporal manner during their rising toward the liquid-air interface. Furthermore, our research identified a correlation between electron and optical microscopy parameters, enabling the assessment of bioflocculants' microstructure without necessitating offline sample preparation procedures. The ultimate goal was to determine their potential suitability as a novel cellulose-based building block material with tuneable structural properties. Our investigations substantiate the capability of SCOBY bioflocculants, characterized by distinct microstructures, to successfully assemble within a microfluidic device, thereby generating a cellulose sheet endowed with specific and purposefully designed structural features.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Fundamentals of Skin Bioimpedances.

The bioimpedances of tissues beyond the stratum corneum, which is the outermost layer of skin, contain crucial clinical information. Nevertheless, bioimpedance measurements of both the viable skin and the adipose tissue are not widely used, mainly because of the complex multilayered skin structure and the electrically insulating nature of the stratum corneum. Here, a theoretical framework is established for analyzing the impedances of multilayered tissues and, in particular, of skin. Then, strategies are determined for the system-level design of electrodes and electronics, which minimize 4-wire (or tetrapolar) measurement errors even in the presence of a top insulating tissue, thus enabling non-invasive characterizations of tissues beyond the stratum corneum. As an example, non-invasive measurements of bioimpedances of living tissues are demonstrated in the presence of parasitic impedances which are much (e.g., up to 350 times) higher than the bioimpedances of the living tissues beyond the stratum corneum, independently on extreme variations of the barrier (tape stripping) or of the skin-electrode contact impedances (sweat). The results can advance the development of bioimpedance systems for the characterization of viable skin and adipose tissues in several applications, including transdermal drug delivery and the assessment of skin cancer, obesity, dehydration, type 2 diabetes mellitus, cardiovascular risk, and multipotent adult stem cells.

Colorectal Cancer Bioengineered Microtissues as a Model to Replicate Tumor-ECM Crosstalk and Assess Drug Delivery Systems In Vitro.

Current 3D cancer models (in vitro) fail to reproduce complex cancer cell extracellular matrices (ECMs) and the interrelationships occurring (in vivo) in the tumor microenvironment (TME). Herein, we propose 3D in vitro colorectal cancer microtissues (3D CRC µTs), which reproduce the TME more faithfully in vitro. Normal human fibroblasts were seeded onto porous biodegradable gelatin microbeads (GPMs) and were continuously induced to synthesize and assemble their own ECMs (3D Stroma µTs) in a spinner flask bioreactor. Then, human colon cancer cells were dynamically seeded onto the 3D Stroma µTs to achieve the 3D CRC µTs. Morphological characterization of the 3D CRC µTs was performed to assess the presence of different complex macromolecular components that feature in vivo in the ECM. The results showed the 3D CRC µTs recapitulated the TME in terms of ECM remodeling, cell growth, and the activation of normal fibroblasts toward an activated phenotype. Then, the microtissues were assessed as a drug screening platform by evaluating the effect of 5-Fluorouracil (5-FU), curcumin-loaded nanoemulsions (CT-NE-Curc), and the combination of the two. When taken together, the results showed that our microtissues are promising in that they can help clarify complex cancer-ECM interactions and evaluate the efficacy of therapies. Moreover, they may be combined with tissue-on-chip technologies aimed at addressing further studies in cancer progression and drug discovery.

PLGA microparticle formulations for tunable delivery of a nano-engineered filamentous bacteriophage-based vaccine: in vitro and in silico-supported approach.

Bacteriophages have attracted great attention in the bioengineering field in diverse research areas from tissue engineering to therapeutic and clinical applications. Recombinant filamentous bacteriophage, carrying multiple copies of foreign peptides on protein capsid has been successfully used in the vaccine delivery setting, even if their plasma instability and degradation have limited their use on the pharmaceutical market. Encapsulation techniques in polymeric materials can be applied to preserve bacteriophage activity, extend its half-life, and finely regulate their release in the target environment. The main goal of this study was to provide tunable formulations of the bacteriophage encapsulated in polymeric microparticles (MPs). We used poly (lactic-co-glycolic-acid) as a biocompatible and biodegradable polymer with ammonium bicarbonate as a porogen to encapsulate bacteriophage expressing OVA (257-264) antigenic peptide. We demonstrate that nano-engineered fdOVA bacteriophages encapsulated in MPs preserve their structure and are immunologically active, inducing a strong immune response towards the delivered peptide. Moreover, MP encapsulation prolongs bacteriophage stability over time also at room temperature. Additionally, in this study, we show the ability of in silico-supported approach to predict and tune the release of bacteriophages. These results lay the framework for a versatile bacteriophage-based vaccine delivery system that could successfully generate robust immune responses in a sustained manner, to be used as a platform against cancer and new emerging diseases. Graphical abstract: Synopsis: administration of recombinant bacteriophage-loaded PLGA microparticles for antigen delivery. PLGA microparticles release the bacteriophages, inducing activation of dendritic cells and enhancing antigen presentation and specific T cell response. Bacteriophage-encapsulated microneedles potentially can be administered into human body and generate robust immune responses.

Immunoresponsive microbiota-gut-on-chip reproduces barrier dysfunction, stromal reshaping and probiotics translocation under inflammation.

Here, we propose an immune-responsive human Microbiota-Intestine axis on-chip as a platform able to reproduce the architecture and vertical topography of the microbiota with a complex extracellular microenvironment consisting of a responsive extra cellular matrix (ECM) and a plethora of immune-modulatory mediators released from different cell populations such as epithelial, stromal, blood and microbial species in homeostatic and inflamed conditions. Firstly, we developed a three-dimensional human intestine model (3D-hI), represented by an instructive and histologically competent ECM and a well-differentiated epithelium with mucus-covered microvilli. Then, we replicated the microenvironmental anaerobic condition of human intestinal lumen by fabricating a custom-made microbiota chamber (MC) on the apical side of the Microbiota-human Intestine on chip (MihI-oC), establishing the physiological oxygen gradient occurring along the thickness of human small intestine from the serosal to the luminal side. The complexity of the intestinal extracellular microenvironment was improved by integrating cells populations that are directly involved in the inflammatory response such as peripheral blood mononuclear cells (PBMCs) and two species of the intestinal commensal microbiota (Lactobacillus rhamnosus and Bifidobacterium longum). We found that lipopolysaccharide (LPS)-induced inflammation elicits microbiota's geographical change and induce Bifidobacterium longum iper-proliferation, highlighting a role of such probiotic in anti-inflammatory process. Moreover, we proved, for the first time, the indirect role of the microbiota on stromal reshaping in immune-responsive MihI-oC in terms of collagen fibers orientation and ECM remodeling, and demonstrated the role of microbiota in alleviating gastrointestinal, immunological and infectious diseases by analyzing the release of key immune-mediators after inflammatory stimulus (reactive oxygen species (ROS), pro- and anti-inflammatory cytokines).

Engineered Bacterial Cellulose Nanostructured Matrix for Incubation and Release of Drug-Loaded Oil in Water Nanoemulsion.

Bacterial cellulose (BC) is a highly pure form of cellulose produced by bacteria, which possesses numerous advantages such as good mechanical properties, high chemical flexibility, and the ability to assemble in nanostructures. Thanks to these features, it achieved a key role in the biomedical field and in drug delivery applications. BC showed its ability to modulate the release of several drugs and biomolecules to the skin, thus improving their clinical outcomes. This work displays the loading of a 3D BC nanonetwork with an innovative drug delivery nanoemulsion system. BC was optimized by static culture of SCOBY (symbiotic colony of bacteria and yeast) and characterized by morphological and ultrastructural analyses, which indicate a cellulose fiber diameter range of 30-50 nm. BC layers were then incubated at different time points with a nanocarrier based on a secondary nanoemulsion (SNE) previously loaded with a well-known antioxidant and anti-inflammatory agent, namely, coenzyme-Q10 (Co-Q10). Incubation of Co-Q10-SNE in the BC nanonetwork and its release were analyzed by fluorescence spectroscopy.

Biocompatible, photo-responsive layer-by-layer polymer nanocapsules with an oil core: in vitro and in vivo study.

In cancer therapy, stimulus-responsive drug delivery systems are of particular interest for reducing side effects in healthy tissues and improving drug selectivity in the tumoral ones. Here, a strategy for the preparation of a photo-responsive cross-linked trilayer deposited onto an oil-in-water nanoemulsion via a layer-by-layer technique is reported. The system is made of completely biocompatible materials such as soybean oil, egg lecithin and glycol chitosan, with heparin as the polymeric shell. The oil core is pre-loaded with curcumin as a model lipophilic active molecule with anti-tumoral properties. The trilayer cross-linkage is performed via a photoinitiator-free thiol-ene 'click' reaction. In particular, the system is implemented with an o-nitrobenzyl group functionalized with a thiol moiety which can perform both the thiol-ene 'click' reaction and the cleavage meant for controlled drug release at two different wavelengths, respectively. So the preparation and characterization of a photo-responsive natural nanocarrier (PNC) that is stable under physiological conditions owing to the thiol-ene cross-linkage are reported. PNC performance has been assessed in vitro on melanoma cells as well as in vivo on xenograft tumour-induced mice.

Twin-Wire Networks for Zero Interconnect, High-Density 4-Wire Electrical Characterizations of Materials.

Four-wire measurements have been introduced by Lord Kelvin in 1861 and have since become the standard technique for characterizing small resistances and impedances. However, high-density 4-wire measurements are generally complex, time-consuming, and inefficient because of constraints on interconnects, pads, external wires, and mechanical contacts, thus reducing reproducibility, statistical significance, and throughput. Here, we introduce, systematically design, analyze, and experimentally validate zero interconnect networks interfaced to external instrumentation by couples of twin wire. 3D-printed holders with magnets, interconnects, nonadhesive layers, and spacers can effortlessly establish excellent electrical connections with tunable or minimum contact forces and enable accurate measurements even for delicate devices, such as thin metals on soft polymers. As an example, we measured all the resistances of a twin-wire 29-resistor network made of silver-nanoparticle ink printed on polyimide, paper, or photo paper, including during sintering or temperature calibration, resulting in an unprecedentedly easy and accurate characterization of both resistivity and its temperature coefficient. The theoretical framework and experimental strategies reported here represent a breakthrough toward zero interconnect, simple, and efficient high-density 4-wire characterizations, can be generalized to other 4-wire measurements (impedances, sensors) and can open the way to more statistically meaningful and reproducible analyses of materials, high-throughput measurements, and minimally invasive characterizations of biomaterials.

Cell Membrane-Coated Oil in Water Nano-Emulsions as Biomimetic Nanocarriers for Lipophilic Compounds Conveyance.

Recently, we developed ultra-stable oil in water nano-emulsions (O/W NEs), able to carry both internal and external cargos (Somes), such as lipophilic compounds and hydrophilic coatings, respectively, that we call here NEsoSomes. O/W NEs are an excellent bioengineering tool for drug and molecules delivery, due to their ability to dissolve a large number of hydrophobic compounds and protect them from hydrolysis and degradation under biological conditions. At present, no report is available on the combination of cell membrane coatings with such nanocarriers, probably due to their typical instability feature. Since then, we have reported, for the first time, a new cell membrane (CM)-coated nanomaterial composed of membranes extracted from glioblastoma cancer cells (U87-MG) deposited on NEsoSomes, through a liquid-liquid interface method, to produce highly controllable membrane caked nano-capsules, namely CM-NEsoSomes. CM-NEsoSomes were physically characterized by dynamic light scattering (DLS) over time and their correct morphology was analyzed by confocal and transmission electron microscopy (TEM) microscopy. Moreover, CM-NEsoSomes biocompatibility was tested on the healthy model cell line, performing cell cytotoxicity and uptake assay, showing nanocarriers uptake by cells with no induced cytotoxicity.

Morphological and Rheological Guided Design for the Microencapsulation Process of Lactobacillus paracasei CBA L74 in Calcium Alginate Microspheres.

The intestinal microbiota is a real ecosystem composed of several bacterial species and a very huge amount of strains that through their metabolic activities play a crucial role in the development and performance of the immune system and other functions. Microbiota modulation by probiotics establishes a new era into the pharmaceutical and healthcare market. Probiotics play, in fact, an important role in helping and sustaining human health, but in order to produce benefits, their viability must be preserved throughout the production process up to consumption, and in addition, their bioactivity required to be safeguarded while passing through the gastrointestinal tract. In this frame, encouraging results come from encapsulation strategies that have proven to be very promising in protecting bacteria and their viability. However, specific effort has to be dedicated to the design optimization of the encapsulation process and, in particular, to the processing parameters that affect capsules microstructure. Herein, focusing on calcium alginate microspheres, after a preliminary selection of their processing conditions based on size distribution, we implemented a micro-rheological analysis, by using the multiple-particle tracking technique, to correlate the inner microstructure to the selected process conditions and to the viability of the Lactobacillus paracasei CBA L74. It was assessed that the explored levels of cross-linking, although changing the microorganism constriction, did not affect its viability. The obtained results confirm how this technology is a promising and a valid strategy to protect the microorganism viability and ensure its stability during the production process.

Easy Surface Functionalization and Bioconjugation of Peptides as Capture Agents of a Microfluidic Biosensing Platform for Multiplex Assay in Serum.

The development of assays for protein biomarkers in complex matrices is a demanding task that still needs implementation of new approaches. Antibodies as capture agents have been largely used in bioassays but their low stability, low-efficiency production, and cross-reactivity in multiplex approaches impairs their larger applications. Instead, synthetic peptides, even with higher stability and easily adapted amino acid sequences, still remain largely unexplored in this field. Here, we provide a proof-of-concept of a microfluidic device for direct detection of biomarker overexpression. The multichannel microfluidic polydimethylsiloxane (PDMS) device was first derivatized with PAA (poly(acrylic acid)) solution. CRP-1, VEGF-114, and ΦG6 peptides were preliminarily tested to respectively bind the biomarkers, C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α). Each PDMS microchannel was then respectively bioconjugated with a specific peptide (CRP-1, VEGF-114, or ΦG6) to specifically capture CRP, VEGF, and TNF-α. With such microdevices, a fluorescence bioassay has been set up with sensitivity in the nanomolar range, both in buffered solution and in human serum. The proposed multiplex assay worked with a low amount of sample (25 µL) and detected biomarker overexpression (above nM concentration), representing a noninvasive and inexpensive screening platform.

Prolonged activity of a recombinant manganese superoxide dismutase through a formulation of polymeric multi-layer nanoassemblies targeting cancer cells.

A new isoform of human manganese superoxide dismutase (SOD) has been recently isolated and obtained in a synthetic recombinant form and termed rMnSOD. As compared to other SODs, this isoform exhibits a dramatically improved cellular uptake and an intense antioxidant and antitumoral activity. Unfortunately, its use is severely hampered as this active pharmaceutical ingredient (API) in solution suffers from remarkable instability, which realizes as an interplay of unfolding and aggregation phenomena. This leads the API to be ineffective after three weeks only when stored at 4°C. A formulation strategy was undertaken to mitigate this instability. This was based on the incorporation of the API in hyaluronic acid and its layer-by-layer deposition over a chitosan-n-acetyl cysteine- monolayer nanoemulsion (NE) and its subsequent coverage with a further external interface of a chitosan-n-acetyl cysteine. The obtained constructs were tested over a selected panel of healthy and cancerous cell lines. The undertaken formulation strategy enhanced the API's effect in vitro already at time zero, maintaining the efficacy of this anticancer agent until up to 30 weeks when stored at 4°C.

Experimental and Theoretical Studies on Sustainable Synthesis of Gold Sol Displaying Dichroic Effect.

Gold nanoparticles depending on their shape and mixtures of multiple shapes can exhibit peculiar optical properties, including the dichroic effect typical of the Lycurgus cup, which has puzzled scientists for a long time. Such optical properties have been recently exploited in several fields such as paint technology, sensors, dichroic polarizers, display (LCD) devices, laser applications, solar cells and photothermal therapy among others. In this article, we have demonstrated a simple room temperature one-pot synthesis of gold sol displaying a dichroic effect using a slow reduction protocol involving only trisodium citrate as a reducing agent. We found that the dichroic gold sol can be easily formed at room temperature by reducing gold salt by trisodium citrate below a certain critical concentration. The sol displayed an orangish-brown color in scattered/reflected light and violet/blue/indigo/purple/red/pink in transmitted light, depending on the experimental conditions. With minor changes such as the introduction of a third molecule or replacing a small amount of water in the reaction mixture with ethanol, the color of the gold sol under transmitted light changed and a variety of shades of red, pink, cobalt blue, violet, magenta and purple were obtained. The main advantage of the proposed method lies in its simplicity, which involves the identification of the right ratio of the reactants, and simple mixing of reactants at room temperature with no other requirements. TEM micrographs displayed the formation of two main types of particles viz. single crystal gold nanoplates and polycrystalline faceted polyhedron nanoparticles. The mechanism of growth of the nanoplates and faceted polyhedron particles have been described by an enhanced diffusion limited aggregation numerical scheme, where it was assumed that both trisodium citrate and the gold ions in solution undergo a stochastic Brownian motion, and that the evolution of the entire system is regulated by a principle of energy minimization. The predictions of the model matched with the experiments with a good accuracy, indicating that the initial hypothesis is correct.

Stimuli-responsive transdermal microneedle patches.

Microneedle (MN) patches consisting of miniature needles have emerged as a promising tool to perforate the stratum corneum and translocate biomolecules into the dermis in a minimally invasive manner. Stimuli-responsive MN patches represent emerging drug delivery systems that release cargos on-demand as a response to internal or external triggers. In this review, a variety of stimuli-responsive MN patches for controlled drug release are introduced, covering the mechanisms of action toward different indications. Future opportunities and challenges with respect to clinical translation are also discussed.

A theoretical and experimental study on l-tyrosine and citrate mediated sustainable production of near infrared absorbing twisted gold nanorods.

Anisotropic gold nanoparticles displaying plasmon band in the near infrared region can play a crucial role in cancer therapy particularly with techniques such as photothermal therapy (PTT) and photodynamic therapy (PDT). Herein, we report an efficient, sustainable, one pot protocol for the fabrication of an unusual gold anisotropic shape, which we have named as twisted gold nanorods. These particles, though having dimensions in the nanoscale regime comparable to those of gold nanorods, display a continuous flat plasmon band like that of 2-D gold nanowire networks, extended up to the NIR-III (SWIR) range. The proposed strategy is simple and does not require any seed mediation, heating or potential toxic templates or organic solvents. Our process is based on the slow reduction of gold salt in presence of two mild reducing agents viz. l-tyrosine (an amino acid) and trisodium citrate. We observed that when both molecules are present together in particular concentrations, they direct the growth in form of twisted gold nanorods. The mechanism of growth has been described by a Diffusion Limited Aggregation numerical scheme, where it was assumed that both l-tyrosine and the gold ions in solution undergo a stochastic Brownian motion. The predictions of the model matched with the experiments with a good accuracy, indicating that the initial hypothesis is correct. The final structure has been thoroughly characterized in terms of morphology, while SERS and cytotoxic activity have also been demonstrated.

Design of biodegradable bi-compartmental microneedles for the stabilization and the controlled release of the labile molecule collagenase for skin healthcare.

Proteins are widely explored as therapeutic agents, but some issues remain alive in their delivery versus target tissues and organs. Especially in the case of water-labile proteins, they undergo rapid failure if not properly stored or once they have encountered the biological environment. In this framework, delivery systems can be very useful to protect such proteins both during storage and during their administration. In particular, polymer microneedles (MNs) represent an interesting tool for the in vivo administration of proteins, avoiding the aggressive gastrointestinal or blood environment. Here, polymer microneedles for the encapsulation and delivery of the labile protein collagenase are presented. Polyvinylpyrrolidone-hyaluronic acid (PVP-HA) microneedles with embedded poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were designed in order to achieve a sustained but relatively fast release of the enzyme to avoid its long exposure to water upon administration. PLGA-MPs with tunable porosity were produced by means of a modified double emulsion protocol and their morphological and kinetic properties were characterized by different analytic techniques. Diffusion studies and in vivo experiments were used to assess the release and indentation ability of the proposed MP-based microneedles. The obtained results recommend our bi-compartmental system as a promising biomedical technique paving the way for its efficient use in treating human diseases with labile therapeutic agents.

Double-Framed Thin Elastomer Devices.

Elastomers and, in particular, polydimethylsiloxane (PDMS) are widely adopted as biocompatible mechanically compliant substrates for soft and flexible micro-nanosystems in medicine, biology, and engineering. However, several applications require such low thicknesses (e.g., <100 µm) that make peeling-off critical because very thin elastomers become delicate and tend to exhibit strong adhesion with carriers. Moreover, microfabrication techniques such as photolithography use solvents which swell PDMS, introducing complexity and possible contamination, thus limiting industrial scalability and preventing many biomedical applications. Here, we combine low-adhesion and rectangular carrier substrates, adhesive Kapton frames, micromilling-defined shadow masks, and adhesive-neutralizing paper frames for enabling fast, easy, green, contaminant-free, and scalable manufacturing of thin elastomer devices, with both simplified peeling and handling. The accurate alignment between the frame and shadow masks can be further facilitated by micromilled marking lines on the back side of the low-adhesion carrier. As a proof of concept, we show epidermal sensors on a 50 µm-thick PDMS substrate for measuring strain, the skin bioimpedance and the heart rate. The proposed approach paves the way to a straightforward, green, and scalable fabrication of contaminant-free thin devices on elastomers for a wide variety of applications.