Optics miniaturization strategy for demanding Raman spectroscopy applications.

Raman spectroscopy provides non-destructive, label-free quantitative studies of chemical compositions at the microscale as used on NASA's Perseverance rover on Mars. Such capabilities come at the cost of high requirements for instrumentation. Here we present a centimeter-scale miniaturization of a Raman spectrometer using cheap non-stabilized laser diodes, densely packed optics, and non-cooled small sensors. The performance is comparable with expensive bulky research-grade Raman systems. It has excellent sensitivity, low power consumption, perfect wavenumber, intensity calibration, and 7 cm-1 resolution within the 400-4000 cm-1 range using a built-in reference. High performance and versatility are demonstrated in use cases including quantification of methanol in beverages, in-vivo Raman measurements of human skin, fermentation monitoring, chemical Raman mapping at sub-micrometer resolution, quantitative SERS mapping of the anti-cancer drug methotrexate and in-vitro bacteria identification. We foresee that the miniaturization will allow realization of super-compact Raman spectrometers for integration in smartphones and medical devices, democratizing Raman technology.

Flexible Coatings Facilitate pH-Targeted Drug Release via Self-Unfolding Foils: Applications for Oral Drug Delivery.

Ingestible self-configurable proximity-enabling devices have been developed as a non-invasive platform to improve the bioavailability of drug compounds via swellable or self-unfolding devices. Self-unfolding foils support unidirectional drug release in close proximity to the intestinal epithelium, the main drug absorption site following oral administration. The foils are loaded with a solid-state formulation containing the active pharmaceutical ingredient and then coated and rolled into enteric capsules. The coated lid must remain intact to ensure drug protection in the rolled state until targeted release in the small intestine after capsule disintegration. Despite promising results in previous studies, the deposition of an enteric top coating that remains intact after rolling is still challenging. In this study, we compare different mixtures of enteric polymers and a plasticizer, PEG 6000, as potential coating materials. We evaluate mechanical properties as well as drug protection and targeted release in gastric and intestinal media, respectively. Commercially available Eudragit® FL30D-55 appears to be the most suitable material due to its high strain at failure and integrity after capsule fitting. In vitro studies of coated foils in gastric and intestinal media confirm successful pH-triggered drug release. This indicates the potential advantage of the selected material in the development of self-unfolding foils for oral drug delivery.

Magnetic and/or Radiopaque Functionalization of Self-Unfolding Foils for Improved Applicability within Oral Drug Delivery.

Various types of microfabricated devices have been proposed for overcoming the gastrointestinal (GI) challenges associated with oral administration of pharmaceutical compounds. However, unidirectional drug release in very close forced proximity to the intestinal wall still appears to be an unresolved issue for many of these microdevices, which typically show low drug absorption and thereby low bioavailabilities. This work explores how recently developed and promising self-unfolding foils (SUFs) can be magnetically and/or radiopaquely (M/R-) functionalized, by the addition of BaSO4 or Fe3O4 nanoparticles, for improving their applicability within oral drug delivery. Through surface characterization, mechanical testing, and X-ray imaging, the (M/R-)SUFs are generally inspected and their overall properties compared. Furthermore, R-SUFs are being used in an in vivo rat X-ray imaging study, whereas in situ rat testing of MR-SUFs are attempted together with an investigation of their general magnetic properties. Unfolding of the R-SUF, and its very close forced proximity to the small intestine, is very easily observed 2 h post-administration by applying both computed tomography scanning and planar X-ray imaging. In addition, MR-SUFs show a great magnetic response in water, which suggests the possibility for controlled motion and retention in the GI tract. However, the magnetic response does not seem strong enough for in situ rat testing, but most likely a strong magnetization of the MR-SUFs using for example an impulse magnetizer can be made for increasing the magnetic response. All of the results presented herein are highly relevant and applicable for future usage of (M/R-)SUFs, as well as similar devices, in pre-clinical studies and potential clinical trials.

Label-free SERS assay combined with multivariate spectral data analysis for lamotrigine quantification in human serum.

Considering the need for a more time and cost-effective method for lamotrigine (LTG) detection in clinics we developed a fast and robust label-free assay based on surface-enhanced Raman scattering (SERS) for LTG quantification from human serum. The optimization and application of the developed assay is presented  showing the: (i) exploration of different methods for LTG separation from human serum; (ii) implementation of a molecular adsorption step on an ordered Au nanopillar SERS substrate; (iii) adaptation of a fast scanning of the SERS substrate, performed with a custom-built compact Raman spectrometer; and (iv) development of LTG quantification methods with univariate and multivariate spectral data analysis. Our results showed, for the first time, the SERS-based characterization of LTG and its label-free identification in human serum. We found that combining a miniaturized solid phase extraction, as sample pre-treatment with the SERS assay, and using a multivariate model is an optimal strategy for LTG quantification in human serum in a linear range from 9.5 to 75 µM, with LoD and LoQ of 3.2 µM and 9.5 µM, respectively, covering the suggested clinical therapeutic window. We also showed that the developed assay allowed for quantifying LTG from human serum in the presence of other drugs, thereby demonstrating the robustness of label-free SERS. The sensing approach and instrumentation can be further automated and integrated in devices that can advance the drug monitoring in real clinical settings.

Delivery of E. coli Nissle to the mouse gut by mucoadhesive microcontainers does not improve its competitive ability against strains linked to ulcerative colitis.

For patients with ulcerative colitis (UC), administration of the probiotic E. coli Nissle (EcN) holds promise for alleviation of disease symptoms. The mechanisms are unclear, but it has been hypothesised that a capacity of the probiotic to outcompete potentially detrimental UC-associated E. coli strains plays an important role. However, this could previously not be confirmed in a mouse model of competition between EcN and two UC-associated strains, as reported by Petersen et al. 2011. In the present study, we re-evaluated the idea, hypothesising that delivery of EcN by a micro device dosing system (microcontainers), designed for delivery into the intestinal mucus, could support colonisation and confer a competition advantage compared to classical oral dosing. Six groups of mice were pre-colonised with one of two UC-associated E. coli strains followed by oral delivery of EcN, either in capsules containing microcontainers with freeze-dried EcN powder, capsules containing freeze-dried EcN powder, or as a fresh sucrose suspension. Co-colonisation between the probiotic and the disease-associated strains was observed regardless of dosing method, and no competition advantages linked to microcontainer delivery were identified within this setup. Other approaches are thus needed if the competitive capacity of EcN in the gut should be improved.

Nitroaromatic explosives' detection and quantification using an attention-based transformer on surface-enhanced Raman spectroscopy maps.

Rapidly and accurately detecting and quantifying the concentrations of nitroaromatic explosives is critical for public health and security. Among existing approaches, explosives' detection with Surface-Enhanced Raman Spectroscopy (SERS) has received considerable attention due to its high sensitivity. Typically, a preprocessed single spectrum that is the average of the entire or a selected subset of a SERS map is used to train various machine learning models for detection and quantification. Designing an appropriate averaging and preprocessing procedure for SERS maps across different concentrations is time-consuming and computationally costly, and the averaging of spectra may lead to the loss of crucial spectral information. We propose an attention-based vision transformer neural network for nitroaromatic explosives' detection and quantification that takes raw SERS maps as the input without any preprocessing. We produce two novel SERS datasets, 2,4-dinitrophenols (DNP) and picric acid (PA), and one benchmark SERS dataset, 4-nitrobenzenethiol (4-NBT), which have repeated measurements down to concentrations of 1 nM to illustrate the detection limit. We experimentally show that our approach outperforms or is on par with the existing methods in terms of detection and concentration prediction accuracy. With the produced attention maps, we can further identify the regions with a higher signal-to-noise ratio in the SERS maps. Based on our findings, the molecule of interest detection and concentration prediction using raw SERS maps is a promising alternative to existing approaches.

APELLA: Open-Source, miniaturized All-in-One powered Lab-on-a-Disc platform.

We present an unconventional approach to a common Lab-on-a-Disc (LoD) that combines a quadcopter propulsion system, a miniaturized 2.4 GHz Wi-Fi spy camera, 9.74 Watt Qi wireless power, and an Arduino into an open-source, miniaturized All-in-one powered lab-on-disc platform (APELLA). The quadcopter propulsion generates thrust to rotate (from 0.1 to 24.5 Hz) or shake the LoD device, while the spy camera enables a real-time (30 frames per second) and high definition (1280 × 720 pixels) visualization of microfluidic channels without requiring a bulky and heavy stroboscopic imaging setup. A mobile device can communicate with an Arduino microcontroller inside the APELLA through a Bluetooth interface for closed loop and sequential frequency control. In a proof-of-concept study, the APELLA achieved comparable mixing efficiency to a traditional spin stand and can capture rapid microfluidic events at low rotational frequencies (<5Hz). The APELLA is low-cost (c.a. 100 Euro), compact (15.6 × 15.6 × 10 cm3), lightweight (0.59 kg), portable (powered by a 5 V USB power bank), and energy efficient (uses < 6% power of the conventional system), making it ideal for field deployment, education, resource-limited labs.

A self-unfolding proximity enabling device for oral delivery of macromolecules.

Oral delivery of macromolecules remains highly challenging due to their rapid degradation in the gastrointestinal tract and poor absorption across the tight junctions of the epithelium. In the last decade, researchers have investigated several medical devices to overcome these challenges using various approaches, some of which involve piercing through the intestine using micro and macro needles. We have developed a new generation of medical devices called self-unfolding proximity enabling devices, which makes it possible to orally deliver macromolecules without perforating the intestine. These devices protect macromolecules from the harsh conditions in the stomach and release their active pharmaceutical ingredients in the vicinity of the intestinal epithelium. One device version is a self-unfolding foil that we have used to deliver insulin and nisin to rats and pigs respectively. In our study, this device has shown a great potential for delivering peptides, with a significant increase in the absorption of solid dosage of insulin by ∼12 times and nisin by ∼4 times in rats and pigs, respectively. With the ability to load solid dosage forms, our devices can facilitate enhanced absorption of minimally invasive oral macromolecule formulations.

Near-Infrared Light-Driven Mesoporous SiO2 /Au Nanomotors for Eradication of Pseudomonas aeruginosa Biofilm.

Bacterial biofilms are linked to several diseases and cause resistant and chronic infections in immune-compromised patients. Nanomotors comprise a new field of research showing a great promise within biomedicine but pose challenges in terms of biocompatibility. Nanomotors propelled by thermophoresis could overcome this challenge, as they leave no waste product during propulsion. In this study, mesoporous-silica nanoparticles are coated with a thin layer of gold to make nanomotors, which can be driven by near-infrared (NIR) light irradiation. The prepared mesoporous SiO2 -Au nanomotors exhibit efficient self-propulsion when exposed to NIR irradiation, they penetrate deep through a biofilm matrix, and disperse the biofilm in situ due to the photothermal effect on the Au part of the nanomotors. The velocities of such nanomotors are investigated at different wavelengths and laser powers. Furthermore, the study examines the ability of these nanomotors to eradicate Pseudomonas aeruginosa (P. aeruginosa) biofilm under NIR light irradiation. The conducted study shows that the nanomotor's velocity increases with increasing laser power. The mesoporous SiO2 /Au nanomotors show excellent capabilities to eradicate P. aeruginosa biofilms even under short (30 s-3 min) irradiation time. This study shows great promise for overcoming the challenges related to bacterial biofilm eradication.

Moving perfusion culture and live-cell imaging from lab to disc: proof of concept toxicity assay with AI-based image analysis.

In vitro, cell-based assays are essential in diagnostics and drug development. There are ongoing efforts to establish new technologies that enable real-time detection of cell-drug interaction during culture under flow conditions. Our compact (10 × 10 × 8.5 cm) cell culture and microscope on disc (CMoD) platform aims to decrease the application barriers of existing lab-on-a-chip (LoC) approaches. For the first time in a centrifugal device, (i) cells were cultured for up to six days while a spindle motor facilitated culture medium perfusion, and (ii) an onboard microscope enabled live bright-field imaging of cells while the data wirelessly transmitted to a computer. The quantification of cells from the acquired images was done using artificial intelligence (AI) software. After optimization, the obtained cell viability data from the AI-based image analysis proved to correlate well with data collected from commonly used image analysis software. The CMoD was also suitable for conducting a proof-of-concept toxicity assay with HeLa cells under continuous flow. The half-maximal inhibitory time (IT50) for various concentrations of doxorubicin (DOX) in the case of HeLa cells in flow, was shown to be lower than the IT50 obtained from a static cytotoxicity assay, indicating a faster onset of cell death in flow. The CMoD proved to be easy to handle, enabled cell culture and monitoring without assistance, and is a promising tool for examining the dynamic processes of cells in real-time assays.

Microscopic Cascading Devices for Boosting Mucus Penetration in Oral Drug Delivery-Micromotors Nesting Inside Microcontainers.

In the case of macromolecules and poorly permeable drugs, oral drug delivery features low bioavailability and low absorption across the intestinal wall. Intestinal absorption can be improved if the drug formulation could be transported close to the epithelium. To achieve this, a cascade delivery device comprising Magnesium-based Janus micromotors (MMs) nesting inside a microscale containers (MCs) has been conceptualized. The device aims at facilitating targeted drug delivery mediated by MMs that can lodge inside the intestinal mucosa. Loading MMs into MCs can potentially enhance drug absorption through increased proximity and unidirectional release. The MMs will be provided with optimal conditions for ejection into any residual mucus layer that the MCs have not penetrated. MMS confined inside MCs propel faster in the mucus environment as compared to non-confined MMs. Upon contact with a suitable fuel, the MM-loaded MC itself can also move. An in vitro study shows fast release profiles and linear motion properties in porcine intestinal mucus compared to more complex motion in aqueous media. The concept of dual-acting cascade devices holds great potential in applications where proximity to epithelium and deep mucus penetration are needed.

3D-Printed Radiopaque Microdevices with Enhanced Mucoadhesive Geometry for Oral Drug Delivery.

During the past decades, microdevices have been evaluated as a means to overcome challenges within oral drug delivery, thus improving bioavailability. Fabrication of microdevices is often limited to planar or simple 3D designs. Therefore, this work explores how microscale stereolithography 3D printing can be used to fabricate radiopaque microcontainers with enhanced mucoadhesive geometries, which can enhance bioavailability by increasing gastrointestinal retention. Ex vivo force measurements suggest increased mucoadhesion of microcontainers with adhering features, such as pillars and arrows, compared to a neutral design. In vivo studies, utilizing planar X-ray imaging, show the time-dependent gastrointestinal location of microcontainers, whereas computed tomography scanning and cryogenic scanning electron microscopy reveal information about their spatial dynamics and mucosal interactions, respectively. For the first time, the effect of 3D microdevice modifications on gastrointestinal retention is traced in vivo, and the applied methods provide a much-needed approach for investigating the impact of device design on gastrointestinal retention.

Oral vaccination using microdevices to deliver α-GalCer adjuvanted vaccine afford a mucosal immune response.

Oral vaccination has in the recent years gained a lot of attraction, mainly due to optimized patient compliance and logistics. However, the development of oral vaccines, especially oral subunit vaccines is challenging. Micro technology can be utilized to overcome some of these challenges, by facilitating protection and effective delivery of the vaccine components in the gastrointestinal tract (GI tract). One such technology is Microcontainers (MCs), which can be realized to be mucoadhesive and to target specific regions of the GI tract via oral delivery. Here, we test MCs, for oral delivery of the C. trachomatis vaccine candidate CTH522, in combination with effective mucosal adjuvants. The adjuvants alpha- galactosylceramide (α-GalCer), C-di-GMP and cholera toxin B were compared in vivo, to identify the most prominent adjuvant for formulation with CTH522. Formulations were administered both purely oral and as boosters following a subcutaneous (s.c.) prime with CTH522 in combination with the CAF®01 adjuvant. CTH522 formulated with α-GalCer showed to be the most efficient combination for the oral vaccine, based on the immunological analysis. Lyophilized formulation of CTH522 and α-GalCer was loaded into MCs and these were subsequently coated with Eudragit L100-55 and evaluated in vivo in mice for the ability of MCs to mediate intestinal vaccine delivery and increase immunogenicity of the vaccine. Mice receiving oral prime and boosters did show a significantly enhanced mucosal immune responses compared to naive mice. This indicates the MCs are indeed capable of delivering the vaccine formulation intact and able to stimulate the immune cells. Mice orally boosted with MCs following a s.c. prime with CAF01, demonstrated improved systemic and local Th17 responses, along with increased local IFN-γ and IgA levels compared to both the s.c. prime alone and the homologous oral prime-boost immunization. However, due to the relatively weak observed effect of the MC delivery on the immune responses, it was hypothesized that the MCs are proportionally too large for the GI tract of mice, and thus cleared before an effective immune response can be induced. To investigate this, MCs were loaded with BaSO4, and orally administered to mice. Analysis with X-ray and CT showed a transit time of approximately 1-1.5 h from the stomach to the cecum, corresponding to the standard transit time in mice, and an extremely narrow absorption window. This indicates that mice is not a suitable animal model for evaluation of MCs. These data should be taken into consideration in future in vivo trials with this and similar technologies, where larger animals might be a necessity for proof-of-concept studies.

Delivery of streptomycin to the rat colon by use of electrospun nanofibers.

Drug-loaded electrospun nanofibers are potential drug carrier systems that may optimize disease treatment while reducing the impact on commensal microbes. The feasibility of streptomycin-loaded pullulan nanofibers fabricated from a green electrospinning procedure using water as the solvent was assessed. We conducted a rat study including a group treated with streptomycin-loaded nanofibers (STR-F, n = 5), a group treated with similar concentrations of streptomycin in the drinking water (STR-W, n = 5), and a non-treated control group (CTR, n = 5). Streptomycin was successfully loaded into nanofibers and delivered by this vehicle, which minimized the quantity of the drug released in the ileal compartment of the gut. Ingested streptomycin-resistant E. coli colonized of up to 106 CFU/g feces, revealing a selective effect of streptomycin even when given in the low amounts allowed by the nanofiber-based delivery. 16S amplicon sequencing of the indigenous microbiota revealed differential effects in the three groups. An increase of Peptostreptococcaceae in the cecum of STR-F animals may indicate that the fermentation of nanofibers directly or indirectly promoted growth of bacteria within this family. Our results elucidate relevant properties of electrospun nanofibers as a novel vehicle for delivery of antimicrobials to the large intestine.

Bioadhesive Tannic-Acid-Functionalized Zein Coating Achieves Engineered Colonic Delivery of IBD Therapeutics via Reservoir Microdevices.

The biggest challenge in oral delivery of anti-inflammatory drugs such as 5-aminosalicylic acid (5-ASA) is to (i) prevent rapid absorption in the small intestine and (ii) achieve localized release at the site of inflammation in the lower gut, i.e., the colon. Here, we present an advanced biopolymeric coating comprising of tannic-acid-functionalized zein protein to provide a sustained, colon-targeted release profile for 5-ASA and enhance the mucoadhesion of the dosage form via a mussel-inspired mechanism. To enable localized delivery and provide high local concentration, 5-ASA is loaded into the microfabricated drug carriers (microcontainers) and sealed with the developed coating. The functionality and drug release profile of the coating are characterized and optimized in vitro, showing great tunability, scalability, and stability toward proteases. Further, ex vivo experiments demonstrate that the tannic acid functionalization can significantly enhance the mucoadhesion of the coating, which is followed up by in vivo investigations on the intestinal retention, and pharmacokinetic evaluation of the 5-ASA delivery system. Results indicate that the developed coating can provide prolonged colonic delivery of 5-ASA. Therefore, the here-developed biodegradable coating can be an eco-friendly substitute to the state-of-the-art commercial counterparts for targeted delivery of 5-ASA and other small molecule drugs.

Methotrexate Detection in Serum at Clinically Relevant Levels with Electrochemically Assisted SERS on a Benchtop, Custom Built Raman Spectrometer.

Therapeutic drug monitoring (TDM) is an essential clinical practice for optimizing drug dosing, thereby preventing adverse effects of drugs with a narrow therapeutic window, slow clearance, or high interperson pharmacokinetic variability. Monitoring methotrexate (MTX) during high-dose MTX (HD-MTX) therapy is necessary to avoid potentially fatal side effects caused by delayed elimination. Despite the efficacy of HD-MTX treatment, its clinical application in resource-limited settings is constrained due to the relatively high cost and time of analysis with conventional analysis methods. In this work, we developed (i) an electrochemically assisted surface-enhanced Raman spectroscopy (SERS) method for detecting MTX in human serum at a clinically relevant concentration range and (ii) a benchtop, Raman detection system with an integrated potentiostat, software, and data analysis unit that enables mapping of small areas of SERS substrates and quantitative SERS-based analysis. In the assay, by promoting electrostatic attraction between gold-coated nanopillar SERS substrates and MTX molecules in aqueous samples, a detection limit of 0.13 µM with a linear range of 0.43-2 µM was achieved in PBS. The implemented sample cleanup through gel filtration proved to be highly effective, resulting in a similar detection limit (0.55 µM) and linear range (1.81-5 µM) for both PBS and serum. The developed and optimized assay could also be used on the in-house built, Raman device. We showed that MTX detection can be carried out in less than 30 min with the Raman device, paving the way toward the TDM of MTX at the point-of-need and in resource-limited environments.

Local Delivery of Streptomycin in Microcontainers Facilitates Colonization of Streptomycin-Resistant Escherichia coli in the Rat Colon.

Oral antibiotic treatment is often applied in animal studies in order to allow establishment of an introduced antibiotic-resistant bacterium in the gut. Here, we compared the application of streptomycin dosed orally in microcontainers to dosage through drinking water. The selective effect on a resistant bacterial strain, as well as the effects on fecal, luminal, and mucosal microbiota composition, were investigated. Three groups of rats (n = 10 per group) were orally dosed with microcontainers daily for 3 days. One of these groups (STR-M) received streptomycin-loaded microcontainers designed for release in the distal ileum, while the other two groups (controls [CTR] and STR-W) received empty microcontainers. The STR-W group was additionally dosed with streptomycin through the drinking water. A streptomycin-resistant Escherichia coli strain was orally inoculated into all animals. Three days after inoculation, the resistant E. coli was found only in the cecum and colon of animals receiving streptomycin in microcontainers but in all intestinal compartments of animals receiving streptomycin in the drinking water. 16S rRNA amplicon sequencing revealed significant changes in the fecal microbiota of both groups of streptomycin-treated animals. Investigation of the inner colonic mucus layer by confocal laser scanning microscopy and laser capture microdissection revealed no significant effect of streptomycin treatment on the mucus-inhabiting microbiota or on E. coli encroachment into the inner mucus. Streptomycin-loaded microcontainers thus enhanced proliferation of an introduced streptomycin-resistant E. coli in the cecum and colon without affecting the small intestine environment. While improvements of the drug delivery system are needed to facilitate optimal local concentration and release of streptomycin, the application of microcontainers provides new prospects for antibiotic treatment. IMPORTANCE Delivery of antibiotics in microcontainer devices designed for release at specific sites of the gut represents a novel approach which might reduce the amount of antibiotic needed to obtain a local selective effect. We propose that the application of microcontainers may have the potential to open novel opportunities for antibiotic treatment of humans and animals with fewer side effects on nontarget bacterial populations. In the current study, we therefore elucidated the effects of streptomycin, delivered in microcontainers coated with pH-sensitive lids, on the selective effect on a resistant bacterium, as well as on the surrounding intestinal microbiota in rats.

Open-source force analyzer with broad sensing range based on an optical pickup unit.

In the pharmaceutical field, oral drug delivery devices continue to shrink down to the micrometer scale, driving a trending demand to investigate ex vivo mucoadhesive force down to the micro-Newton scale. However, owing to the limitation of measuring sensitivity, conventional methods (e.g., a texture analyzer) lack reliability while measuring forces in this range. Herein, we report on an open-source force analyzer that utilizes an optical-pickup-unit (from a DVD player) to detect cantilever-based force transducers and thereby, achieves a wide force-sensing range from 1.1 N to 0.99 nN. The cantilever force transducers can easily be adjusted to fit different force ranges by adjusting the steel shim, magnets, and 3D printed components. To validate the analyzer, we conducted a preliminary study to investigate the effect of time and humidity of mucoadhesion of porcine intestinal tissues. Besides, we measured the mucoadhesive force of a single oral drug delivery microdevice with an average force of 93.7 µN on the top sides of the device. This analyzer offers the possibility of measuring e.g. mucoadhesion of individual microdevices in the micro-Newton range. Hence, the analyzer can assist in the development of miniaturized oral drug delivery devices but has a much wider field of potential force sensing applications.