Analysis of In Vitro Cytotoxicity of Carbohydrate-Based Materials Used for Dissolvable Microneedle Arrays.

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.

Ultra-miniature ultra-compliant neural probes with dissolvable delivery needles: design, fabrication and characterization.

Stable chronic functionality of intracortical probes is of utmost importance toward realizing clinical application of brain-machine interfaces. Sustained immune response from the brain tissue to the neural probes is one of the major challenges that hinder stable chronic functionality. There is a growing body of evidence in the literature that highly compliant neural probes with sub-cellular dimensions may significantly reduce the foreign-body response, thereby enhancing long term stability of intracortical recordings. Since the prevailing commercial probes are considerably larger than neurons and of high stiffness, new approaches are needed for developing miniature probes with high compliance. In this paper, we present design, fabrication, and in vitro evaluation of ultra-miniature (2.7 µm x 10 µm cross section), ultra-compliant (1.4 × 10-2 µN/µm in the axial direction, and 2.6 × 10-5 µN/µm and 1.8 × 10-6 µN/µm in the lateral directions) neural probes and associated probe-encasing biodissolvable delivery needles toward addressing the aforementioned challenges. The high compliance of the probes is obtained by micron-scale cross-section and meandered shape of the parylene-C insulated platinum wiring. Finite-element analysis is performed to compare the strains within the tissue during micromotion when using the ultra-compliant meandered probes with that when using stiff silicon probes. The standard batch microfabrication techniques are used for creating the probes. A dissolvable delivery needle that encases the probe facilitates failure-free insertion and precise placement of the ultra-compliant probes. Upon completion of implantation, the needle gradually dissolves, leaving behind the ultra-compliant neural probe. A spin-casting based micromolding approach is used for the fabrication of the needle. To demonstrate the versatility of the process, needles from different biodissolvable materials, as well as two-dimensional needle arrays with different geometries and dimensions, are fabricated. Further, needles incorporating anti-inflammatory drugs are created to show the co-delivery potential of the needles. An automated insertion device is developed for repeatable and precise implantation of needle-encased probes into brain tissue. Insertion of the needles without mechanical failure, and their subsequent dissolution are demonstrated. It is concluded that ultra-miniature, ultra-compliant probes and associated biodissolvable delivery needles can be successfully fabricated, and the use of the ultra-compliant meandered probes results in drastic reduction in strains imposed in the tissue as compared to stiff probes, thereby showing promise toward chronic applications.

Processing fragile matter: effect of polymer graft modification on the mechanical properties and processibility of (nano-) particulate solids.

The effect of polymer modification on the deformation characteristics and processibility of particle assembly structures is analyzed as a function of particle size and degree of polymerization of surface-tethered chains. A pronounced increase of the fracture toughness (by approximately one order of magnitude) is observed as the degree of polymerization exceeds a threshold value that increases with particle size. The threshold value is interpreted as being related to the transition of tethered chains from stretched-to-relaxed conformation (and the associated entanglement of tethered chains) and agrees with predictions from scaling theory. The increase in toughness is reduced with increasing particle size - this effect is rationalized as a consequence of the decrease of entanglement density with increasing dimension of interstitial (void) space in particle array structures. The increased fracture toughness of particle brush materials (with sufficient degree of polymerization of tethered chains) enables the fabrication of ordered colloidal films and even complex 3D shapes by scalable polymer processing techniques, such as spin coating and micromolding. The results, therefore, suggest new opportunities for the processing of colloidal material systems that could find application in the economical fabrication of functional components or systems compromised of colloidal materials.

Dissolvable microneedle arrays for intradermal delivery of biologics: fabrication and application.

PURPOSE: Design and evaluate a new micro-machining based approach for fabricating dissolvable microneedle arrays (MNAs) with diverse geometries and from different materials for dry delivery to skin microenvironments. The aims are to describe the new fabrication method, to evaluate geometric and material capability as well as reproducibility of the method, and to demonstrate the effectiveness of fabricated MNAs in delivering bioactive molecules. METHODS: Precise master molds were created using micromilling. Micromolding was used to create elastomer production molds from master molds. The dissolvable MNAs were then fabricated using the spin-casting method. Fabricated MNAs with different geometries were evaluated for reproducibility. MNAs from different materials were fabricated to show material capability. MNAs with embedded bioactive components were tested for functionality on human and mice skin. RESULTS: MNAs with different geometries and from carboxymethyl cellulose, polyvinyl pyrrolidone and maltodextrin were created reproducibly using our method. MNAs successfully pierce the skin, precisely deliver their bioactive cargo to skin and induce specific immunity in mice. CONCLUSIONS: We demonstrated that the new fabrication approach enables creating dissolvable MNAs with diverse geometries and from different materials reproducibly. We also demonstrated the application of MNAs for precise and specific delivery of biomolecules to skin microenvironments in vitro and in vivo.

3D Assembly of MXene Networks using a Ceramic Backbone with Controlled Porosity.

Transition metal carbides (MXenes) are novel 2D nanomaterials with exceptional properties, promising significant impact in applications such as energy storage, catalysis, and energy conversion. A major barrier preventing the widespread use of MXenes is the lack of methods for assembling MXene in 3D space without significant restacking, which degrades their performance. Here, this challenge is successfully overcome by introducing a novel material system: a 3D network of MXene formed on a porous ceramic backbone. The backbone dictates the network's 3D architecture while providing mechanical strength, gas/liquid permeability, and other beneficial properties. Freeze casting is used to fabricate a silica backbone with open pores and controlled porosity. Next, capilary flow is used to infiltrate MXene into the backbone from a dispersion. The system is then dried to conformally coat the pore walls with MXene, creating an interconnected 3D-MXene network. The fabrication approach is reproducible, and the MXene-infiltrated porous silica (MX-PS) system is highly conductive (e.g., 340 S m-1 ). The electrical conductivity of MX-PS is controlled by the porosity distribution, MXene concentration, and the number of infiltration cycles. Sandwich-type supercapacitors with MX-PS electrodes are shown to produce excellent areal capacitance (7.24 F cm-2 ) and energy density (0.32 mWh cm-2 ) with only 6% added MXene mass. This approach of creating 3D architectures of 2D nanomaterials will significantly impact many engineering applications.

Freeform 3D Ice Printing (3D-ICE) at the Micro Scale.

Water is one of the most important elements for life on earth. Water's rapid phase-change ability along with its environmental and biological compatibility also makes it a unique structural material for 3D printing of ice structures reproducibly and accurately. This work introduces the freeform 3D ice printing (3D-ICE) process for high-speed and reproducible fabrication of ice structures with micro-scale resolution. Drop-on-demand deposition of water onto a -35 °C platform rapidly transforms water into ice. The dimension and geometry of the structures are critically controlled by droplet ejection frequency modulation and stage motions. The freeform approach obviates layer-by-layer construction and support structures, even for overhang geometries. Complex and overhang geometries, branched hierarchical structures with smooth transitions, circular cross-sections, smooth surfaces, and micro-scale features (as small as 50 µm) are demonstrated. As a sample application, the ice templates are used as sacrificial geometries to produce resin parts with well-defined internal features. This approach could bring exciting opportunities for microfluidics, biomedical devices, soft electronics, and art.

Skin-targeted delivery of extracellular vesicle-encapsulated curcumin using dissolvable microneedle arrays.

Therapeutic benefits of curcumin for inflammatory diseases have been demonstrated. However, curcumin's potential as a clinical therapeutic has been hindered due to its low solubility and stability in vivo. We hypothesized that a hybrid curcumin carrier that incorporates albumin-binding and extracellular vesicle (EV) encapsulation could effectively address the current challenges of curcumin delivery. We further postulated that using dissolvable microneedle arrays (dMNAs) for local delivery of curcumin-albumin-EVs (CA-EVs) could effectively control skin inflammation in vivo. Mild sonication was used to encapsulate curcumin and albumin into EVs, and the resulting CA-EVs were integrated into tip-loaded dMNAs. In vitro and in vivo studies were performed to assess the stability, cellular uptake, and anti-inflammatory bioactivity of dMNA-delivered CA-EVs. Curcumin in CA-EVs exhibited at least five-fold higher stability in vitro than naïve curcumin or curcumin-EVs without albumin. Incorporating CA-EVs into dMNAs did not alter their cellular uptake or anti-inflammatory bioactivity. The dMNA embedded CA-EVs retained their bioactivity when stored at room temperature for at least 12 months. In rat and mice models, dMNA delivered CA-EVs suppressed and significantly reduced lipopolysaccharide and Imiquimod-triggered inflammation. We conclude that dMNA delivery of CA-EVs has the potential to become an effective local-delivery strategy for inflammatory skin diseases. STATEMENT OF SIGNIFICANCE: We introduce and evaluate a skin-targeted delivery system for curcumin that synergistically combines albumin association, extracellular-vesicle encapsulation, and dissolvable microneedle arrays (dMNAs) . In vitro, curcumin-albumin encapsulated extracellular vesicles (CA-EVs) inhibit and reverse the LPS-triggered expression of inflammatory transcription factor NF-κB. The integration of CA-EVs into dMNAs does not affect them physically or functionally. Importantly, dMNAs extend EV storage stability for at least 12 months at room temperature with minimal loss in their bioactivity. We demonstrate that dMNA delivered CA-EVs effectively block and reverse skin inflammation in vivo in mouse and rat models.

Biomimetic scaffolds with three-dimensional undulated microtopographies.

Many human and animal tissues naturally possess three-dimensional (3D) micro-scale geometries enabling certain physiological functions. Absence of these microgeometries in engineered tissues may undermine the effectiveness of corresponding tissue repair and regeneration. This paper introduces a novel approach to create tissue scaffolds with biomimetic 3D undulated microtopographies. The mechanical micromilling technology is used for precise and reproducible fabrication of poly(methyl methacrylate) (PMMA) master molds with 3D undulated microtopographies. Poly(dimethylsiloxane) (PDMS) production molds are then created using the master molds through elastomer molding. Next, gelatin-chondroitin-6-sulfate-hyaluronic acid (Gel-C6S-HA) is filled into the PDMS molds, lyophilized to obtain solid porous scaffolds, and covalently cross-linked to control biodegradability. The utility of the final porous scaffolds with undulated microtopographies mimicking dermal papillae of skin is demonstrated in vitro by culturing neonatal human fibroblasts (NHFs) on the scaffold surfaces for up to 7 days. The assessment of the mold and scaffold geometries demonstrates high accuracy and reproducibility of the PMMA mold fabrication, as well as well-controlled undulated microtopographies and porous microstructures of the final scaffolds. The analysis of cell responses to the undulated microtopographies shows the biocompatibility and effectiveness of the final scaffolds, as well as unique cellular response to these biomimetic topographies at the macroscopic level.

Tip-Loaded Dissolvable Microneedle Arrays Effectively Deliver Polymer-Conjugated Antibody Inhibitors of Tumor-Necrosis-Factor-Alpha Into Human Skin.

Autoinflammatory skin diseases are characterized by a disequilibrium of cytokines in the local skin microenvironment, suggesting that local delivery of therapeutics, including anticytokine antibodies, may provide benefit without the unwanted off-target effects of systemically delivered therapies. Rapid diffusion of therapeutics away from the target site has been a challenge to the development of local therapies. Conjugation of high molecular weight hydrophilic polymers to cytokine neutralizing mAbs has been shown to be an effective strategy for local control of inflammation in healing burn wounds. However, the burn application is unique because the skin barrier is already breached. For the treatment of autoinflammatory skin diseases, the major challenge for local delivery lies in penetrating the stratum corneum. Here, we investigate a new therapeutic approach combining the use of tip-loaded dissolvable microneedle arrays (TL-dMNAs) for local application of polymer-conjugated antibody inhibitors of tumor-necrosis-factor-alpha (TNF-α). Specifically, intradermal delivery and pharmacokinetics of (anti-TNF-α-Ab)-(high molecular weight hyaluronic acid [HA]) conjugates from tip-loaded, obelisk-shaped dissolvable microneedle arrays were investigated in living human skin. The results indicate (1) TL-dMNAs can be successfully fabricated to integrate (anti-TNF-α-Ab)-HA at the tip portion of the microneedles while preserving the biological activity necessary for antibody ligand binding; (2) (anti-TNF-α-Ab)-HA can be effectively delivered into human skin using obelisk-shaped TL-dMNAs; and (3) polymer conjugation effectively inhibits antibody diffusion from the delivery site. Taken together, these results support the evaluation of microneedle array-based delivery of varying polymer-antibody conjugates for the treatment of inflammatory skin diseases.

High-density soft-matter electronics with micron-scale line width.

A method to produce soft and stretchable microelectronics composed of a liquid-phase Gallium-Indium alloy with micron-scale circuit features is introduced. Microchannels are molded onto the surface of a poly(dimethylsiloxane) (PDMS) elastomer and filled with EGaIn using a micro-transfer deposition step that exploits the unique wetting properties of EGaIn in air. The liquid-filled channels function as stretchable circuit wires or capacitor electrodes with a 2 µm linewidth and 1 µm spacing.

Chronic tissue response to carboxymethyl cellulose based dissolvable insertion needle for ultra-small neural probes.

Implantable neural electrodes must drastically improve chronic recording stability before they can be translated into long-term human clinical prosthetics. Previous studies suggest that sub-cellular sized and mechanically compliant probes may result in improved tissue integration and recording longevity. However, currently these design features are restricted by the opposing mechanical requirements needed for minimally damaging insertions. We designed a non-cytotoxic, carboxymethylcellulose (CMC) based dissolvable delivery vehicle (shuttle) to provide the mechanical support for insertion of ultra-small, ultra-compliant microfabricated neural probes. Stiff CMC-based shuttles rapidly soften immediately after being placed ∼1 mm above an open craniotomy as they absorb vapors from the brain. To address this, we developed a sophisticated targeting, high speed insertion (∼80 mm/s), and release system to implant these shuttles. After implantation, the goal is for the shuttle to dissolve away leaving only the electrodes behind. Here we show the histology of chronically implanted shuttles of large (300 µm × 125 µm) and small (100 µm × 125 µm) size at discrete time points over 12 weeks. Early time points show the CMC shuttle expanded after insertion as it absorbed moisture from the brain and slowly dissolved. At later time points neuronal cell bodies populate regions within the original shuttle tract. The large CMC shuttles show that the CMC expansion can cause extended secondary damage. On the other hand, the smaller CMC shuttles show limited secondary damage, wound closure by 4 weeks, absence of activated microglia at 12 weeks, as well as evidence suggesting neural regeneration at the implant site. This shuttle, therefore, shows great promise facilitating the implantation of nontraditional ultra-small, and ultra-compliant probes.

Ultra-compliant neural probes are subject to fluid forces during dissolution of polymer delivery vehicles.

Ultra-compliant neural probes implanted into tissue using a molded, biodissolvable sodium carboxymethyl cellulose (Na-CMC)-saccharide composite needle delivery vehicle are subjected to fluid-structure interactions that can displace the recording site of the probe with respect to its designed implant location. We applied particle velocimetry to analyze the behavior of ultra-compliant structures under different implantation conditions for a range of CMC-based materials and identified a fluid management protocol that resulted in the successful targeted depth placement of the recording sites.

An experimental investigation on thermal exposure during bone drilling.

This study presents an experimental investigation of the effects of spindle speed, feed rate, and depth of drilling on the temperature distribution during drilling of the cortical section of the bovine femur. In an effort to reduce measurement uncertainties, a new approach for temperature measurements during bone drilling is presented in this study. The new approach is based on a setup for precise positioning of multiple thermocouples, automated data logging system, and a computer numerically controlled (CNC) machining system. A battery of experiments that has been performed to assess the uncertainty and repeatability of the new approach displayed adequate results. Subsequently, a parametric study was conducted to determine the effects of spindle speed, feed rate, hole depth, and thermocouple location on the measured bone temperature. This study suggests that the exposure time during bone drilling far exceeds the commonly accepted threshold for thermal injury, which may prevail at significant distances from the drilled hole. Results of this study suggest that the correlation of the thermal exposure threshold for bone injury and viability should be further explored.

Modeling and experimentation of bone drilling forces.

Prediction and control of bone drilling forces are critical to the success of many orthopaedic operations. Uncontrolled and large forces can cause drill-bit breakage, drill breakthrough, excessive heat generation, and mechanical damage to the bone. This paper presents a mechanistic model for prediction of thrust forces and torques experienced during bone drilling. The model incorporates the radially varying drill-bit geometry and cutting conditions analytically, while capturing the material and friction properties empirically through a specific energy formulation. The forces from the chisel edge are modeled by considering the indentation process that occurs in the vicinity of the drill-bit axis. A procedure is outlined to calibrate the specific energies, where only a small number of calibration experiments are required for a wide range of drilling conditions and drill-bit geometry. The calibration parameters for the cortical portions of bovine tibia are identified through drilling tests. Subsequently, a series of validation tests are conducted under different feed rates and spindle speeds. The thrust forces and torques were observed to vary considerably between bones from different animals. The forces from the model were seen to match well with those from the experimentation within the inherent variations from the bone characteristics. The model can be used to select favorable drilling conditions, to assist in robotic surgeries, and to design optimal orthopaedic drill bits.

A new thermal model for bone drilling with applications to orthopaedic surgery.

This paper presents a new thermal model for bone drilling with applications to orthopaedic surgery. The new model combines a unique heat-balance equation for the system of the drill bit and the chip stream, an ordinary heat diffusion equation for the bone, and heat generation at the drill tip, arising from the cutting process and friction. Modeling of the drill bit-chip stream system assumes an axial temperature distribution and a lumped heat capacity effect in the transverse cross-section. The new model is solved numerically using a tailor-made finite-difference scheme for the drill bit-chip stream system, coupled with a classic finite-difference method for the bone. The theoretical investigation addresses the significance of heat transfer between the drill bit and the bone, heat convection from the drill bit to the surroundings, and the effect of the initial temperature of the drill bit on the developing thermal field. Using the new model, a parametric study on the effects of machining conditions and drill-bit geometries on the resulting temperature field in the bone and the drill bit is presented. Results of this study indicate that: (1) the maximum temperature in the bone decreases with increased chip flow; (2) the transient temperature distribution is strongly influenced by the initial temperature; (3) the continued cooling (irrigation) of the drill bit reduces the maximum temperature even when the tip is distant from the cooled portion of the drill bit; and (4) the maximum temperature increases with increasing spindle speed, increasing feed rate, decreasing drill-bit diameter, increasing point angle, and decreasing helix angle. The model is expected to be useful in determination of optimum drilling conditions and drill-bit geometries.

Fabrication of circular microfluidic channels by combining mechanical micromilling and soft lithography.

The fabrication of microfluidic channels with complex three-dimensional (3D) geometries presents a major challenge to the field of microfluidics, because conventional lithography methods are mainly limited to rectangular cross-sections. In this paper, we demonstrate the use of mechanical micromachining to fabricate microfluidic channels with complex cross-sectional geometries. Micro-scale milling tools are first used to fabricate semi-circular patterns on planar metallic surfaces to create a master mold. The micromilled pattern is then transferred to polydimethylsiloxane (PDMS) through a two-step reverse molding process. Using these semi-circular PDMS channels, circular cross-sectioned microchannels are created by aligning and adhering two channels face-to-face. Straight and serpentine-shaped microchannels were fabricated, and the channel geometry and precision of the metallic master and PDMS molds were assessed through scanning electron microscopy and non-contact profilometry. Channel functionality was tested by perfusion of liquid through the channels. This work demonstrates that micromachining enabled soft lithography is capable of fabricating non-rectangular cross-section channels for microfluidic applications. We believe that this approach will be important for many fields from biomimetics and vascular engineering to microfabrication and microreactor technologies.

A Rotating-Tip-Based Mechanical Nano-Manufacturing Process: Nanomilling.

We present a rotating-tip-based mechanical nanomanufacturing technique, referred to here as nanomilling. An atomic force microscopy (AFM) probe tip that is rotated at high speeds by out-of-phase motions of the axes of a three-axis piezoelectric actuator is used as the nanotool. By circumventing the high-compliance AFM beam and directly attaching the tip onto the piezoelectric actuator, a high-stiffness arrangement is realized. The feeding motions and depth prescription are provided by a nano-positioning stage. It is shown that nanomilling is capable of removing the material in the form of long curled chips, indicating shearing as the dominant material removal mechanism. Feature-size and shape control capabilities of the method are demonstrated.

Note: Aligned deposition and modal characterization of micron and submicron poly(methyl methacyrlate) fiber cantilevers.

Polymeric micro-/nanofibers are finding increasing use as sensors for novel applications. Here, we demonstrate the ability to deposit an array of poly(methyl methacyrlate) fibers with micron and submicron diameters in aligned configurations on customized piezoelectric shakers. Using lateral motion of an atomic force microscope tip, fibers are broken to obtain fiber cantilevers of high aspect ratio (length/diameter > 20). The resonant frequencies of fabricated microfiber cantilevers are experimentally measured using a laser Doppler vibrometer. An average Young's modulus of 3.5 GPa and quality factor of 20 were estimated from the experimentally obtained frequency responses.