Simplified Method for Removing Direct Oral Anticoagulant Interference in Mechanical Coagulation Test Systems-A Proof of Concept.

BACKGROUND: Direct oral anticoagulants (DOACs) cause unwanted interference in various hemostasis assays, including lupus anticoagulant (LA) testing, where false positive and false negative identification may occur. DOAC Stop (DS) is an activated charcoal (AC) product used to specifically and effectively adsorb DOACs from test plasma. This process normally requires plasma treatment, centrifugation and plasma separation prior to tests, but inexperienced operators may also inadvertently transfer residual AC particles, thereby potentially adversely affecting clot detection. METHODS: We hypothesized that residual DS might not be problematic for mechanical clot detection. We therefore investigated the potential impact of DS and a new DS liquid (DS-L) product on clotting tests using a mechanical clot detection system. Varying concentrations of DS were added to normal and abnormal plasmas with and without DOAC presence. Clotting tests including PT, APTT and dRVVT were performed directly in the analyzer without plasma/DS centrifugation. RESULTS: DS up to double the recommended treatment level had only minor effects on all test results, despite completely obscuring visibility in the plasma/reagent mix. This confirms that the centrifugation step may be able to be omitted when using mechanical detection systems. CONCLUSIONS: Should DS carryover into treated plasmas occur, this should not cause issues with testing performed on mechanical clot-sensing devices. Moreover, we hypothesize that DS can be used directly in these systems, without the need for centrifugation, thereby simplifying its many potential applications.

Simple and customizable method for fabrication of high-aspect ratio microneedle molds using low-cost 3D printing.

We present a simple and customizable microneedle mold fabrication technique using a low-cost desktop SLA 3D printer. As opposed to conventional microneedle fabrication methods, this technique neither requires complex and expensive manufacturing facilities nor expertise in microfabrication. While most low-cost 3D-printed microneedles to date display low aspect ratios and poor tip sharpness, we show that by introducing a two-step "Print & Fill" mold fabrication method, it is possible to obtain high-aspect ratio sharp needles that are capable of penetrating tissue. Studying first the effect of varying design input parameters and print settings, it is shown that printed needles are always shorter than specified. With decreasing input height, needles also begin displaying an increasingly greater than specified needle base diameter. Both factors contribute to low aspect ratio needles when attempting to print sub-millimeter height needles. By setting input height tall enough, it is possible to print needles with high-aspect ratios and tip radii of 20-40 µm. This tip sharpness is smaller than the specified printer resolution. Consequently, high-aspect ratio sharp needle arrays are printed in basins which are backfilled and cured in a second step, leaving sub-millimeter microneedles exposed resulting microneedle arrays which can be used as male masters. Silicone female master molds are then formed from the fabricated microneedle arrays. Using the molds, both carboxymethyl cellulose loaded with rhodamine B as well as polylactic acid microneedle arrays are produced and their quality examined. A skin insertion study is performed to demonstrate the functional capabilities of arrays made from the fabricated molds. This method can be easily adopted by the microneedle research community for in-house master mold fabrication and parametric optimization of microneedle arrays.

Metallic microneedles with interconnected porosity: A scalable platform for biosensing and drug delivery.

Metallic-based microneedles (MNs) offer a robust platform for minimally invasive drug delivery and biosensing applications due to their mechanical strength and proven tissue and drug compatibility. However, current designs suffer from limited functional surface area or challenges in manufacturing scalability. Here, porous 316L stainless steel MN patches are proposed. Fabricated through a scalable manufacturing process, they are suitable for storage and delivery of drugs and rapid absorption of fluids for biosensing. Fabrication of these MNs involves hot embossing a patch of stainless steel-based feedstock, sintering at 1100 °C and subsequent electropolishing. Optimisation of this manufacturing process yields devices that maintain mechanical integrity yet possess high surface area and associated porosity (36%) to maximise loading capacity. Similarly, a small pore size has been targeted (average diameter 2.22 µm, with 90% between 1.56 µm and 2.93 µm) to maximise capillarity and loading efficiency. This porous network has a theoretical wicking rate of 4.7 µl/s and can wick-up 27 ±â€¯5 µl of fluid through capillary action which allows for absorption of pharmaceuticals for delivery. When inserted into a metabolite-loaded skin model, the MNs absorbed and recovered 17 ±â€¯3 µl of the metabolite solution. The drug delivery performance of the porous metallic MNs (22.4 ±â€¯4.9 µg/cm2) was found to be threefold higher than that of topical administration (7.1 ±â€¯4.3 µg/cm2). The porous metallic MN patches have been shown to insert into porcine skin under a 19 N load. These results indicate the potential of design-for-manufacturing porous stainless steel MNs in biosensing and drug delivery applications. STATEMENT OF SIGNIFICANCE: Microneedles are micro-scale sharp protrusions used to bypass the stratum corneum, the skin's outer protective layer, and painlessly access dermal layers suitable for drug delivery and biosensing. Despite a depth of research in the area we have not yet seen large-scale clinical adoption of microneedle devices. Here we describe a device designed to address the potential barriers to adoption seen by other microneedles devices. We have developed a scalable, cost effective process to produce medical grade stainless steel microneedle patches which passively absorb and store drugs or interstitial fluid though a porous network and capillary action. This device, with low manufacturing and regulatory burdens may help the large-scale adoption of microneedles.

Transcutaneous implantation of valproic acid-encapsulated dissolving microneedles induces hair regrowth.

The interest in alternative material systems and delivery methods for treatment of androgenetic alopecia has been increasing in the recent decades. Topical application of valproic acid (VPA), an FDA-approved anticonvulsant drug, has been shown to effectively stimulate hair follicle (HF) regrowth by upregulating Wnt/ß-catenin, a key pathway involved in initiation of HF development. Moreover, a majority of studies have suggested that cutaneous wound re-epithelialization is capable of inducing HF through Wnt/ß-catenin pathway. Here, we report fabrication and evaluation of a novel VPA-encapsulating dissolving microneedle (DMN-VPA) that creates minimally invasive dermal micro-wounds upon application, significantly improving the VPA delivery efficiency. DMN-VPA not only delivers encapsulated VPA with higher accuracy than topical application, it also stimulates wound re-epithelialization signals involved in HF regrowth. Through a series of in vivo studies, we show that micro-wounding-mediated implantation of DMN-VPA upregulates expression of Wnt/ß-catenin pathway, alkaline phosphatase, proliferating cell nuclear antigen, loricrin and HF stem cell markers, including keratin 15, and CD34 more effectively than topical application.

Effects of dissolving microneedle fabrication parameters on the activity of encapsulated lysozyme.

Dissolving microneedle (DMN) is referred to a microscale needle that encapsulates drug(s) within a biodegradable polymer matrix and delivers it into the skin in a minimally invasive manner. Although vast majority of studies have emphasized DMN as an efficient drug delivery system, the activity of DMN-encapsulated proteins or antigens can be significantly affected due to a series of thermal, physical and chemical stress factors during DMN fabrication process and storage period. The objective of this study is to evaluate the effects of DMN fabrication parameters including polymer type, polymer concentration, fabrication and storage temperature, and drying conditions on the activity of the encapsulated therapeutic proteins by employing lysozyme (LYS) as a model protein. Our results indicate that a combination of low temperature fabrication, mild drying condition, specific polymer concentration, and addition of protein stabilizer can maintain the activity of encapsulated LYS up to 99.8 ±â€¯3.8%. Overall, findings of this study highlight the importance of optimizing DMN fabrication parameters and paves way for the commercialization of an efficient delivery system for therapeutics.

Enhanced Transdermal Delivery by Combined Application of Dissolving Microneedle Patch on Serum-Treated Skin.

Dissolving microneedle (DMN), a transdermal drug delivery system in which drugs are encapsulated in a biodegradable polymeric microstructure, is designed to dissolve after skin penetration and release the encapsulated drugs into the body. However, because of limited loading capacity of drugs within microsized structures, only a small dosage can be delivered, which is often insufficient for patients. We propose a novel DMN application that combines topical and DMN application simultaneously to improve skin permeation efficiency. Drugs in pretreated topical formulation and encapsulated drugs in DMN patch are delivered into the skin through microchannels created by DMN application, thus greatly increasing the delivered dose. We used 4-n-butylresorcinol to treat human hyperpigmentation and found that sequential application of serum formulation and DMNs was successful. In skin distribution experiments using Alexa Fluor 488 and 568 dyes as model drugs, we confirmed that the pretreated serum formulation was delivered into the skin through microchannels created by the DMNs. In vitro skin permeation and retention experiments confirmed that this novel combined application delivered more 4-n-butylresorcinol into the skin than traditional DMN-only and serum-only applications. Moreover, this combined application showed a higher efficacy in reducing patients' melanin index and hyperpigmented regions compared with the serum-only application. As combined application of DMNs on serum-treated skin can overcome both dose limitations and safety concerns, this novel approach can advance developments in transdermal drug delivery.

Anti-obesity effect of a novel caffeine-loaded dissolving microneedle patch in high-fat diet-induced obese C57BL/6J mice.

Natural products such as caffeine have been found to be effective in reducing body weight through lipolysis. Here, we report the successful loading of caffeine onto dissolving microneedle following inhibition of its crystal growth by hyaluronic acid (HA), the matrix material of the dissolving microneedle (DMN). Further, the anti-obesity activity of caffeine was evaluated in high-fat diet-induced obese C57BL/6J mice. After 6weeks of caffeine loaded dissolving microneedle patch (CMP) administration, lipolysis improved significantly as shown by leptin and adiponectin activity, which resulted in considerable weight loss of about 12.8±0.75% in high-fat diet-induced obese mice. Comparison of the levels of triglyceride, total cholesterol, high-density lipoprotein (HDL)-cholesterol, and low-density lipoprotein (LDL)-cholesterol after CMP administration with the initial levels in obese mice indicated significant anti-obesity activity of CMP. These findings suggested that a novel CMP with an increased amount of caffeine loaded onto DMN has therapeutic activity against obesity.

Development of a quantitative method for active epidermal growth factor extracted from dissolving microneedle by solid phase extraction and liquid chromatography electrospray ionization mass spectrometry.

Dissolving microneedle (DMN), a transdermal drug delivery in which biological drugs are encapsulated in biodegradable and biocompatible polymers, was fabricated using epidermal growth factor (EGF) as a model drug and hyaluronic acid (HA) as a backbone polymeric matrix. After mixing calibration and DMN samples with insulin, an internal standard, solid phase extraction (SPE) was performed to separate EGF and insulin from HA, and then liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) was conducted for microgram-scale quantitation. The method showed good linearity (R2=0.997) within a specified range (1-4µg). Additionally, the decrease in EGF levels during DMN fabrication was compared using the SPE/LC-ESI-MS and enzyme-linked immunosorbent assay (ELISA), a traditional analytical method. The ELISA method detected an EGF loss of only 3.88±4.67%, whereas SPE/LC-ESI-MS detected a loss of 16.75±4.39%. Qualitative analysis by circular dichroism showed wavelength shift and splitting after DMN fabrication indicating that EGF was denatured during DMN fabrication and cell viability test showed SPE/LC-ESI-MS is more accurate and reliable for detecting the amount of active EGF loaded into the DMN than ELISA.

Innovative polymeric system (IPS) for solvent-free lipophilic drug transdermal delivery via dissolving microneedles.

Lipophilic drugs are potential drug candidates during drug development. However, due to the need for hazardous organic solvents for their solubilization, these drugs often fail to reach the pharmaceutical market, and in doing so highlight the importance of solvent free systems. Although transdermal drug delivery systems (TDDSs) are considered prospective safe drug delivery routes, a system involving lipophilic drugs in solvent free or powder form has not yet been described. Here, we report, for the first time, a novel approach for the delivery of every kind of lipophilic drug in powder form based on an innovative polymeric system (IPS). The phase transition of powder form of lipophilic drugs due to interior chemical bonds between drugs and biodegradable polymers and formation of nano-sized colloidal structures allowed the fabrication of dissolving microneedles (DMNs) to generate a powerful TDDS. We showed that IPS based DMN with powder capsaicin enhances the therapeutic effect for treatment of the rheumatic arthritis in a DBA/1 mouse model compared to a solvent-based system, indicating the promising potential of this new solvent-free platform for lipophilic drug delivery.

A patchless dissolving microneedle delivery system enabling rapid and efficient transdermal drug delivery.

Dissolving microneedles (DMNs) are polymeric, microscopic needles that deliver encapsulated drugs in a minimally invasive manner. Currently, DMN arrays are superimposed onto patches that facilitate their insertion into skin. However, due to wide variations in skin elasticity and the amount of hair on the skin, the arrays fabricated on the patch are often not completely inserted and large amount of loaded materials are not delivered. Here, we report "Microlancer", a novel micropillar based system by which patients can self-administer DMNs and which would also be capable of achieving 97 ± 2% delivery efficiency of the loaded drugs regardless of skin type or the amount of hair on the skin in less than a second.

A self-powered one-touch blood extraction system: a novel polymer-capped hollow microneedle integrated with a pre-vacuum actuator.

Blood is the gold standard sample medium that can provide a wide variety of useful biological information for the diagnosis of various diseases. For portable point-of-care diagnosis, blood extraction systems have attracted attention as easier, safer, and more rapid methods of collecting small blood volumes. In this paper, we introduce a novel self-powered one-touch blood extraction system created by assembling a smart polymer-capped hollow microneedle in a pre-vacuum polydimethylsiloxane actuator. The optimized hollow microneedle was precisely fabricated by drawing lithography for minimally invasive blood extraction, with a length of 1800 µm, an inner diameter of 60 µm, an outer diameter of 130 µm, and a bevel angle of 15°. The system utilizes only a single step for operation; a finger press activates the blood sampling process based on the negative pressure-driven force built into the pre-vacuum activated actuator. A sufficient volume of blood (31.3 ± 2.0 µl) was successfully extracted from a rabbit for evaluation using a micro total analysis system. The entire system was made of low-cost and disposable materials to achieve easy operation with a miniature structure and to meet the challenging requirements for single-use application in a point-of-care system without the use of any external power equipment.

A minimally invasive blood-extraction system: elastic self-recovery actuator integrated with an ultrahigh- aspect-ratio microneedle.

A minimally invasive blood-extraction system is fabricated by the integration of an elastic self-recovery actuator and an ultrahigh-aspect-ratio microneedle. The simple elastic self-recovery actuator converts finger force to elastic energy to provide power for blood extraction and transport without requiring an external source of power. This device has potential utility in the biomedical field within the framework of complete micro-electromechanical systems.