3D Printing of Biodegradable Polymeric Microneedles for Transdermal Drug Delivery Applications.

Microneedle (MN) technology is an optimal choice for the delivery of drugs via the transdermal route, with a minimally invasive procedure. MN applications are varied from drug delivery, cosmetics, tissue engineering, vaccine delivery, and disease diagnostics. The MN is a biomedical device that offers many advantages including but not limited to a painless experience, being time-effective, and real-time sensing. This research implements additive manufacturing (AM) technology to fabricate MN arrays for advanced therapeutic applications. Stereolithography (SLA) was used to fabricate six MN designs with three aspect ratios. The MN array included conical-shaped 100 needles (10 × 10 needle) in each array. The microneedles were characterized using optical and scanning electron microscopy to evaluate the dimensional accuracy. Further, mechanical and insertion tests were performed to analyze the mechanical strength and skin penetration capabilities of the polymeric MN. MNs with higher aspect ratios had higher deformation characteristics suitable for penetration to deeper levels beyond the stratum corneum. MNs with both 0.3 mm and 0.4 mm base diameters displayed consistent force-displacement behavior during a skin-equivalent penetration test. This research establishes guidelines for fabricating polymeric MN for high-accuracy and low-cost 3D printing.

Protection against Severe Illness versus Immunity-Redefining Vaccine Effectiveness in the Aftermath of COVID-19.

Anti-SARS-CoV-2 vaccines have played a pivotal role in reducing the risk of developing severe illness from COVID-19, thus helping end the COVID-19 global public health emergency after more than three years. Intriguingly, as SARS-CoV-2 variants emerged, individuals who were fully vaccinated did get infected in high numbers, and viral loads in vaccinated individuals were as high as those in the unvaccinated. However, even with high viral loads, vaccinated individuals were significantly less likely to develop severe illness; this begs the question as to whether the main effect of anti-SARS-CoV-2 vaccines is to confer protection against severe illness or immunity against infection. The answer to this question is consequential, not only to the understanding of how anti-SARS-CoV-2 vaccines work, but also to public health efforts against existing and novel pathogens. In this review, we argue that immune system sensitization-desensitization rather than sterilizing immunity may explain vaccine-mediated protection against severe COVID-19 illness even when the SARS-CoV-2 viral load is high. Through the lessons learned from COVID-19, we make the case that in the disease's aftermath, public health agencies must revisit healthcare policies, including redefining the term "vaccine effectiveness."

A novel, low-cost microfluidic device with an integrated filter for rapid, ultrasensitive, and high-throughput bioburden detection.

Rapid and accurate bioburden detection has become increasingly necessary for food, health, pharmaceutical and environmental applications. To detect bioburden accurately, and in a highly sensitive manner, we have fabricated a novel microfluidic device with an integrated filter to trap the cells. Bioburden is detected on the filter paper in situ using the redox reaction of fluorescent label resorufin and a portable multichannel fluorometer is used for fluorescence measurement. The microfluidic device was fabricated in a facile, low-cost, and rapid way with microwave-induced thermally assisted bonding. To characterize the bonding quality of the microfluidic cassettes, different tests were performed, and the filter paper material and size were optimized. Primary Bacillus subtilis culture bacterial samples were filtered through the device to validate and investigate the performance parameters. Our results show that a limit of detection (LOD) of 0.037 CFU/mL can be achieved through this microfluidic device whereas the LOD in a normal microfluidic cassette in the fluorometer and the golden standard spectrophotometer are 0.378 and 0.128 CFU/mL respectively. The results depict that three to ten times LOD improvement is possible through this microfluidic cassette and more sensitive detection is possible depending on the volume filtered within a rapid 3 min. This novel microfluidic device along with the fluorometer can be used as a rapid portable tool for highly sensitive, accurate and high-throughput bacterial detection for different applications.

Microwave induced thermally assisted solvent-based bonding of biodegradable thermoplastics: an eco-friendly rapid approach for fabrication of microfluidic devices and analyte detection.

There is an increasing interest in low-cost, facile and versatile thermoplastic bonding for microfluidic applications that can be easily transitioned from laboratory prototyping to industrial manufacturing. In addition, owing to the surge in the usage of thermoplastic microfluidics and its adverse effect on the environment, it is prudent to source alternative materials that are biodegradable, providing a sustainable, green approach. To address the problems, here we introduce an environment friendly, low-cost and safe welding technology used in the fabrication of microcassettes from biodegradable cellulose acetate (CA) thermoplastics. The thermally assisted solvent based bonding of the thermoplastics was accomplished in a domestic microwave oven with the aid of a polyether ether ketone (PEEK) vise. To characterize the quality of the bonding, our in-house technique was compared with a conventional thermally assisted solvent bonding configuration using a heat press machine and tested under different conditions. Our microwave induced bonding of CA presents three times reduced bonding time with higher bonding strength, good reliability and does not necessitate the use of cumbersome instrumentation. Finally, we demonstrate an electrophoresis application and vitamin C detection accomplished using this biodegradable microcassette presenting comparable results with traditional techniques, illustrating the potential of this fabrication technique in different microfluidic applications.

Rapid Ultrasensitive and High-Throughput Bioburden Detection: Microfluidics and Instrumentation.

Contamination detection often requires lengthy culturing steps to detect low-level bioburden. To increase the rate of detection and decrease the limit of detection (LOD), a system featuring microfluidics and a multichannel fluorometer has been developed. The eight-channel fluorometer enables parallel testing of multiple samples with the LOD as low as <1 cfu/mL. This low-cost system utilizes the slope of fluorescence intensity that serves as the criterion for bioburden detection. The redox indicator dye resazurin is used to monitor the presence of viable cells in this study and is reduced to resorufin with a high quantum yield at 585 nm. The sample under investigation is spiked with resazurin and loaded in a special-design microfluidic cassette, and the rate of change is observed via the fluorometer. The method was validated using primary Escherichia coli culture in comparison with a spectrophotometer which served as the gold standard. An optimized assay based on Luria-Bertani medium was developed. The impact on the assay sensitivity based on incubation and filtration steps was also explored. The assay is shown to pick up inadvertent contamination from test tubes and pipette tips showing its applicability in real-world settings. The data analysis demonstrated a comparable performance of the multichannel fluorometer vis-a-vis the conventional plate reader. The multichannel system is shown to detect bioburden presence in as low as 20 s for bacterial concentrations ≥5 cfu/mL after 6 h of incubation. Considering its portability, low cost, simplicity of operation, and relevant assay sensitivity, the system is well positioned to detect low-level bioburden in the laboratory, pharmaceutical, and field settings.

Surface Plasmon-Coupled Dual Emission Platform for Ultrafast Oxygen Monitoring after SARS-CoV-2 Infection.

The outbreak of the COVID-19 pandemic has had a major impact on the health and well-being of people with its long-term effect on lung function and oxygen uptake. In this work, we present a unique approach to augment the phosphorescence signal from phosphorescent gold(III) complexes based on a surface plasmon-coupled emission platform and use it for designing a ratiometric sensor with high sensitivity and ultrafast response time for monitoring oxygen uptake in SARS-CoV-2-recovered patients. Two monocyclometalated Au(III) complexes, one having exclusively phosphorescence emission (λPL = 578 nm) and the other having dual emission, fluorescence (λPL = 417 nm) and phosphorescence (λPL = 579 nm), were studied using the surface plasmon-coupled dual emission (SPCDE) platform for the first time, which showed 27-fold and 17-fold enhancements, respectively. The latter complex having the dual emission was then used for the fabrication of a ratiometric sensor for studying the oxygen quenching of phosphorescence emission with the fluorescence emission acting as an internal standard. Low-cost poly (methyl methacrylate) (PMMA) and biodegradable wood were used to fabricate the microfluidic chips for oxygen monitoring. The sensor showed a high sensitivity with a limit of detection ∼ 0.1%. Furthermore, real-time oxygen sensing was carried out and the response time of the sensor was calculated to be ∼0.2 s. The sensor chip was used for monitoring the oxygen uptake in SARS-CoV-2-recovered study participants, to assess their lung function post the viral infection.

A Comprehensive Review of Microneedles: Types, Materials, Processes, Characterizations and Applications.

Drug delivery through the skin offers many advantages such as avoidance of hepatic first-pass metabolism, maintenance of steady plasma concentration, safety, and compliance over oral or parenteral pathways. However, the biggest challenge for transdermal delivery is that only a limited number of potent drugs with ideal physicochemical properties can passively diffuse and intercellularly permeate through skin barriers and achieve therapeutic concentration by this route. Significant efforts have been made toward the development of approaches to enhance transdermal permeation of the drugs. Among them, microneedles represent one of the microscale physical enhancement methods that greatly expand the spectrum of drugs for transdermal and intradermal delivery. Microneedles typically measure 0.1-1 mm in length. In this review, microneedle materials, fabrication routes, characterization techniques, and applications for transdermal delivery are discussed. A variety of materials such as silicon, stainless steel, and polymers have been used to fabricate solid, coated, hollow, or dissolvable microneedles. Their implications for transdermal drug delivery have been discussed extensively. However, there remain challenges with sustained delivery, efficacy, cost-effective fabrication, and large-scale manufacturing. This review discusses different modes of characterization and the gaps in manufacturing technologies associated with microneedles. This review also discusses their potential impact on drug delivery, vaccine delivery, disease diagnostic, and cosmetics applications.

Manufacturing biological medicines on demand: Safety and efficacy of granulocyte colony-stimulating factor in a mouse model of total body irradiation.

Protein therapeutics, also known as biologics, are currently manufactured at centralized facilities according to rigorous protocols. The manufacturing process takes months and the delivery of the biological products needs a cold chain. This makes it less responsive to rapid changes in demand. Here, we report on technology application for on-demand biologics manufacturing (Bio-MOD) that can produce safe and effective biologics from cell-free systems at the point of care without the current challenges of long-term storage and cold-chain delivery. The objective of the current study is to establish proof-of-concept safety and efficacy of Bio-MOD-manufactured granulocyte colony-stimulating factor (G-CSF) in a mouse model of total body irradiation at a dose estimated to induce 30% lethality within the first 30 days postexposure. To illustrate on-demand Bio-MOD production feasibility, histidine-tagged G-CSF was manufactured daily under good manufacturing practice-like conditions prior to administration over a 16-day period. Bio-MOD-manufactured G-CSF improved 30-day survival when compared with saline alone (p = .073). In addition to accelerating recovery from neutropenia, the platelet and hemoglobin nadirs were significantly higher in G-CSF-treated animals compared with saline-treated animals (p < .05). The results of this study demonstrate the feasibility of consistently manufacturing safe and effective on-demand biologics suitable for real-time release.

Wood Microfluidics.

As nonbiodegradable plastics continue to pollute our land and oceans, countries are starting to ban the use of single-use plastics. In this paper, we demonstrated the fabrication of wood-based microfluidic devices and their adaptability for single-use, point-of-care (POC) applications. These devices are made from easily sourced renewable materials for fabrication while exhibiting all the advantages of plastic devices without the problem of nonbiodegradable waste and cost. To build these wood devices, we utilized laser engraving and traditional mechanical methods and have adapted specific surface coatings to counter the wicking effect of wood. To demonstrate their versatility, wood microfluidic devices were adapted for (i) surface plasmon coupled enhancement (SPCE) of fluorescence for detection of proteins, (ii) T-/Y-geometry microfluidic channel mixers, and (iii) devices for rapid detection of microbial contamination. These provide proof of concept for the use of wooden platforms for POC applications. In this study, we measured the fluorescence intensities of recombinant green fluorescent protein (GFP) standards (ranging from 1.5-25 ng/µL) and 6XHis-G-CSF (ranging from 0.1-100 ng/µL) expressed in cell-free translation systems. All tested devices perform as well as or better than their plastic counterparts.

Low-cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins.

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the µCols compared with conventional columns for similar protein yields.

Rapid recombinant protein expression in cell-free extracts from human blood.

Several groups have recently reported on the utility of cell-free expression systems to make therapeutic proteins, most of them employing CHO or E. coli cell-free extracts. Here, we propose an alternative that uses human blood derived leukocyte cell extracts for the expression of recombinant proteins. We demonstrate expression of nano luciferase (Nluc), Granulocyte-colony stimulating factor (G-CSF) and Erythropoietin (EPO) in cell-free leukocyte extracts within two hours. Human blood is readily available from donors and blood banks and leukocyte rich fractions are easy to obtain. The method described here demonstrates the ability to rapidly express recombinant proteins from human cell extracts that could provide the research community with a facile technology to make their target protein. Eventually, we envision that any recombinant protein can be produced from patient-supplied leukocytes, which can then be injected back into the patient. This approach could lead to an alternative model for personalized medicines and vaccines.

Point-of-care production of therapeutic proteins of good-manufacturing-practice quality.

Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins. We used the device to reproducibly manufacture His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technology may enable the rapid manufacturing of biologics at the point of care.

Measuring transdermal glucose levels in neonates by passive diffusion: an in vitro porcine skin model.

Current glucose monitoring techniques for neonates rely heavily on blood glucose monitors which require intermittent blood collection through skin-penetrating pricks on the heel or fingers. This procedure is painful and often not clinically conducive, which presents a need for a noninvasive method for monitoring glucose in neonates. Our motivation for this study was to develop an in vitro method for measuring passive diffusion of glucose in premature neonatal skin using a porcine skin model. Such a model will allow us to initially test new devices for noninvasive glucose monitoring without having to do in vivo testing of newborns. The in vitro model is demonstrated by comparing uncompromised and tape-stripped skin in an in-line flow-through diffusion apparatus with glucose concentrations that mimic the hypo-, normo-, and hyper-glycemic conditions in the neonate (2.0, 5.0, and 20 mM, respectively). Transepidermal water loss (TEWL) of the tape-stripped skin was approximately 20 g m-2 h-1, which closely mimics TEWL for neonatal skin at about 190 days post-conceptional age. The tape-stripped skin showed a >15-fold increase in glucose diffusion compared to the uncompromised skin. The very small concentrations of collected glucose were measured with a highly selective and highly sensitive fluorescent glucose biosensor based on the glucose binding protein (GBP). The demonstrated method of glucose determination is noninvasive and painless, which makes it especially desirable for glucose testing in neonates and children. This study is an important step towards an in vitro model for noninvasive real-time glucose monitoring that may be easily transferred to the clinic for glucose monitoring in neonates. Graphical Abstract Glucose diffusion through model skin was measured using an in-line flow-through diffusion apparatus with glucose solutions mimicking hypo-, normo- and hyperglycemia in the neonate. Phosphate buffered saline was added to the top chamber and the glucose that diffused through the model skin into the buffer was measured using a fluorescent glucose binding protein biosensor.

Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable Gas Vesicle Nanoparticles.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant protein organelles produced by the extremophilic microbe Halobacterium sp. NRC-1 and are being developed as bioengineerable and biocompatible antigen and drug-delivery systems (DDS). Dynamic light scattering measurements of purified GVNP suspensions showed a mean diameter of 245 nm. In vitro diffusion studies using Yucatan miniature pig skin showed GVNP permeation to be enhanced after MN-treatment compared to untreated skin. GVNPs were found to be nontoxic to mammalian cells (human kidney and rat mycocardial myoblasts). These findings support the use of GVNPs as DDS for intradermal/transdermal permeation of protein- and peptide-based drugs.

Intradermal delivery of Shigella IpaB and IpaD type III secretion proteins: kinetics of cell recruitment and antigen uptake, mucosal and systemic immunity, and protection across serotypes.

Shigella is one of the leading pathogens contributing to the vast pediatric diarrheal disease burden in low-income countries. No licensed vaccine is available, and the existing candidates are only partially effective and serotype specific. Shigella type III secretion system proteins IpaB and IpaD, which are conserved across Shigella spp., are candidates for a broadly protective, subunit-based vaccine. In this study, we investigated the immunogenicity and protective efficacy of IpaB and IpaD administered intradermally (i.d.) with a double-mutant of the Escherichia coli heat-labile enterotoxin (dmLT) adjuvant using microneedles. Different dosage levels of IpaB and IpaD, with or without dmLT, were tested in mice. Vaccine delivery into the dermis, recruitment of neutrophils, macrophages, dendritic cells, and Langerhans cells, and colocalization of vaccine Ag within skin-activated APC were demonstrated through histology and immunofluorescence microscopy. Ag-loaded neutrophils, macrophages, dendritic cells, and Langerhans cells remained in the tissue at least 1 wk. IpaB, IpaD, and dmLT-specific serum IgG- and IgG-secreting cells were produced following i.d. immunization. The protective efficacy was 70% against Shigella flexneri and 50% against Shigella sonnei. Similar results were obtained when the vaccine was administered intranasally, with the i.d. route requiring 25-40 times lower doses. Distinctively, IgG was detected in mucosal secretions; secretory IgA, as well as mucosal and systemic IgA Ab-secreting cells, were seemingly absent. Vaccine-induced T cells produced IFN-γ, IL-2, TNF-α, IL-17, IL-4, IL-5, and IL-10. These results demonstrate the potential of i.d. vaccination with IpaB and IpaD to prevent Shigella infection and support further studies in humans.

Microfluidic preparation of liposomes to determine particle size influence on cellular uptake mechanisms.

PURPOSE: This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40.6 nm to 276.6 nm. Liposomes were prepared by microfluidic hydrodynamic flow focusing, producing nearly-monodisperse populations and enabling size-dependent uptake to be effectively evaluated. METHODS: Populations of PEG-conjugated liposomes of various distinct sizes were prepared in a disposable microfluidic device using a simple continuous-flow microfluidic technique. Liposome cellular uptake was investigated using flow cytometry and confocal microscopy. RESULTS: Liposome uptake by Caco-2 cells was observed to be strongly size-dependent for liposomes with mean diameters ranging from 40.6 nm to 276.6 nm. When testing these liposomes against endocytosis inhibitors, cellular uptake of the largest (97.8 nm and 162.1 nm in diameter) liposomes were predominantly subjected to clathrin-dependent uptake mechanisms, the medium-sized (72.3 nm in diameter) liposomes seemed to be influenced by all investigated pathways and the smallest liposomes (40.6 nm in diameter) primarily followed a dynamin-dependent pathway. In addition, the 40.6 nm, 72.3 nm, and 162.1 nm diameter liposomes showed slightly decreased accumulation within endosomes after 1 h compared to liposomes which were 97.8 nm in diameter. Conversely, liposome co-localization with lysosomes was consistent for liposomes ranging from 40.6 nm to 97.8 nm in diameter. CONCLUSIONS: The continuous-flow synthesis of nearly-monodisperse populations of liposomes of distinct size via a microfluidic hydrodynamic flow focusing technique enabled unique in vitro studies in which specific effects of particle size on cellular uptake were elucidated. The results of this study highlight the significant influence of liposome size on cellular uptake mechanisms and may be further exploited for increasing specificity, improving efficacy, and reducing toxicity of liposomal drug delivery systems.

Genomic expression of mesenchymal stem cells to altered nanoscale topographies.

The understanding of cellular response to the shape of their environment would be of benefit in the development of artificial extracellular environments for potential use in the production of biomimetic surfaces. Specifically, the understanding of how cues from the extracellular environment can be used to understand stem cell differentiation would be of special interest in regenerative medicine. In this paper, the genetic profile of mesenchymal stem cells cultured on two osteogenic nanoscale topographies (pitted surface versus raised islands) are compared with cells treated with dexamethasone, a corticosteroid routinely used to stimulate bone formation in culture from mesenchymal stem cells, using 19k gene microarrays as well as 101 gene arrays specific for osteoblast and endothelial biology. The current studies show that by altering the shape of the matrix a cell response (genomic profile) similar to that achieved with chemical stimulation can be elicited. Here, we show that bone formation can be achieved with efficiency similar to that of dexamethasone with the added benefit that endothelial cell development is not inhibited. We further show that the mechanism of action of the topographies and dexamethasone differs. This could have an implication for tissue engineering in which a simultaneous, targeted, development of a tissue, such as bone, without the suppression of angiogenesis to supply nutrients to the new tissue is required. The results further demonstrate that perhaps the shape of the extracellular matrix is critical to tissue development.

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

Fluorescent aromatic platforms for cell patterning.

This paper describes a simple experimental method of patterning fluorescent organic dyes, fluorescein, and rhodamine on gold substrates by microcontact printing techniques. The development of this step-by-step protocol has allowed us to prepare striped and squared micropatterns with poly(ethylene glycol) (PEG) linkers terminated by these fluorophores using a fast, easy, and inexpensive technique. Although the rest of the surface was covered with aliphatic molecules (methyl terminated), human fibroblasts demonstrated an unexpected response, aligning themselves according to the aromatic patterns, despite the presence of PEG, which is a cell resistant molecule, in the fluorescent regions.