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Biomedical subjects

Mark R Prausnitz

Publications and source records attributed to Mark R Prausnitz.

At least 19 recordsLinked to original sources
PubMed2007

Polymer particle-based micromolding to fabricate novel microstructures.

Conventional micromolding provides rapid and low-cost methods to fabricate polymer microstructures, but has limitations when producing sophisticated designs. To provide more versatile micromolding techniques, we developed methods based on filling micromolds with polymer microparticles, as opposed to polymer melts, to produce microstructures composed of multiple materials, having complex geometries, and made using mild processing conditions. Polymer microparticles of 1 to 30 microm in size were made from PLA, PGA and PLGA using established spray drying and emulsion techniques either with or without encapsulating model drug compounds. These polymer microparticles were filled into PDMS micromolds at room temperature and melted or bonded together to form microstructures according to different protocols. Porous microstructures were fabricated by ultrasonically welding microparticles together in the mold while maintaining the voids inherent in their packing structure. Multi-layered microstructures were fabricated to have different compositions of polymers and encapsulated compounds located in different regions of the microstructures. More complex arrowhead microstructures were fabricated in a two-step process using a single mold. To assess possible applications, microstructures were designed as microneedles for minimally invasive drug delivery. Multi-layer microneedles were shown to insert into cadaver tissue and, according to design, detach from their base substrate and remain embedded in the tissue for controlled release drug delivery over time. We conclude that polymer particle-based micromolding can encapsulate compounds within microstructures composed of multiple materials, having complex geometries, and made using mild processing conditions.

Biocompatible Materials↗
PubMed2006

Microinfusion using hollow microneedles.

PURPOSE: The aim of the study is to determine the effect of experimental parameters on microinfusion through hollow microneedles into skin to optimize drug delivery protocols and identify rate-limiting barriers to flow. METHODS: Glass microneedles were inserted to a depth of 720-1080 microm into human cadaver skin to microinfuse sulforhodamine solution at constant pressure. Flow rate was determined as a function of experimental parameters, such as microneedle insertion and retraction distance, infusion pressure, microneedle tip geometry, presence of hyaluronidase, and time. RESULTS: Single microneedles inserted into skin without retraction were able to infuse sulforhodamine solution into the skin at flow rates of 15-96 microl/h. Partial retraction of microneedles increased flow rate up to 11.6-fold. Infusion flow rate was also increased by greater insertion depth, larger infusion pressure, use of a beveled microneedle tip, and the presence of hyaluronidase such that flow rates ranging from 21 to 1130 microl/h were achieved. These effects can be explained by removing or overcoming the large flow resistance imposed by dense dermal tissue, compressed during microneedle insertion, which blocks flow from the needle tip. CONCLUSIONS: By partially retracting microneedles after insertion and other methods to overcome flow resistance of dense dermal tissue, protocols can be designed for hollow microneedles to microinfuse fluid at therapeutically relevant rates.

Drug Delivery Systems↗
PubMed2006

Coated microneedles for transdermal delivery.

Coated microneedles have been shown to deliver proteins and DNA into the skin in a minimally invasive manner. However, detailed studies examining coating methods and their breadth of applicability are lacking. This study's goal was to develop a simple, versatile and controlled microneedle coating process to make uniform coatings on microneedles and establish the breadth of molecules and particles that can be coated onto microneedles. First, microneedles were fabricated from stainless steel sheets as single microneedles or arrays of microneedles. Next, a novel micron-scale dip-coating process and a GRAS coating formulation were designed to reliably produce uniform coatings on both individual and arrays of microneedles. This process was used to coat compounds including calcein, vitamin B, bovine serum albumin and plasmid DNA. Modified vaccinia virus and microparticles of 1 to 20 micro m diameter were also coated. Coatings could be localized just to the needle shafts and formulated to dissolve within 20 s in porcine cadaver skin. Histological examination validated that microneedle coatings were delivered into the skin and did not wipe off during insertion. In conclusion, this study presents a simple, versatile, and controllable method to coat microneedles with proteins, DNA, viruses and microparticles for rapid delivery into the skin.

Administration, Cutaneous↗
PubMed2006

Polymer microneedles for controlled-release drug delivery.

PURPOSE: As an alternative to hypodermic injection or implantation of controlled-release systems, this study designed and evaluated biodegradable polymer microneedles that encapsulate drug for controlled release in skin and are suitable for self-administration by patients. METHODS: Arrays of microneedles were fabricated out of poly-lactide-co-glycolide using a mold-based technique to encapsulate model drugs--calcein and bovine serum albumin (BSA)--either as a single encapsulation within the needle matrix or as a double encapsulation, by first encapsulating the drug within carboxymethylcellulose or poly-L: -lactide microparticles and then encapsulating drug-loaded microparticles within needles. RESULTS: By measuring failure force over a range of conditions, poly-lactide-co-glycolide microneedles were shown to exhibit sufficient mechanical strength to insert into human skin. Microneedles were also shown to encapsulate drug at mass fractions up to 10% and to release encapsulated compounds within human cadaver skin. In vitro release of calcein and BSA from three different encapsulation formulations was measured over time and was shown to be controlled by the encapsulation method to achieve release kinetics ranging from hours to months. Release was modeled using the Higuchi equation with good agreement (r2 > or = 0.90). After microneedle fabrication at elevated temperature, up to 90% of encapsulated BSA remained in its native state, as determined by measuring effects on primary, secondary, and tertiary protein structure. CONCLUSIONS: Biodegradable polymer microneedles can encapsulate drug to provide controlled-release delivery in skin for hours to months.

Biocompatible Materials↗
PubMed2006

Mechanism of fluid infusion during microneedle insertion and retraction.

Previous work has shown that infusion flow rates can be increased by an order of magnitude by partially retracting microneedles after insertion into the skin. This study sought to determine the mechanism by which retraction increases fluid infusion by piercing human cadaver skin with single microneedles, fixing the skin after retracting microneedles to different distances, and examining skin microstructure by histology. We found that microneedle insertion to 1080 microm from the skin surface resulted primarily in skin indentation and only 100-300 microm penetration into the skin. This caused significant compaction of the skin, which probably pressed out most water and thereby dramatically lowered the flow conductivity of skin beneath the needle tip. Retraction of the microneedle allowed the skin to recoil back toward its original position, which relieved the skin compaction and increased local flow conductivity. Altogether, these results suggest that microneedle insertion to penetrate into the skin followed by microneedle retraction to relieve skin compaction is an effective approach to infuse fluid into the skin in a minimally invasive manner.

Humans↗
PubMed2006

Plasmid DNA and siRNA transfection of intestinal epithelial monolayers by electroporation.

This study was conducted to evaluate the ability of electroporation to efficiently transfect differentiated intestinal epithelial monolayers with plasmid DNA and to determine whether electroporation can transfect these monolayers with short-interfering RNA (siRNA) to cause gene silencing. Confluent T84 monolayers were transfected with reporter plasmids expressing luciferase or green-fluorescent protein or with siRNA directed against the nuclear envelope proteins lamin A/C using electroporation. Optimized electroporation conditions resulted in luciferase and GFP expression. Both intracellular uptake of fluorescently labeled plasmid and expression of the reporter genes increased with increasing electroporation strength and DNA concentration. When monolayers were transfected by lipofection with the reporter plasmids, expression and DNA uptake were less than for electroporation. Electroporation was also found to transfect monolayers with siRNA, which resulted in up to 90% inhibition of targeted protein production. Silencing occurred within 24h of transfection and increased with increasing siRNA concentration. These results suggest that electroporation can provide a valuable research tool for transfection of intestinal epithelial monolayers and other differentiated cell systems, and may ultimately be useful for clinical gene therapy applications.

Cell Line↗
PubMed2006

Mechanism of intracellular delivery by acoustic cavitation.

Using conditions different from conventional medical imaging or laboratory cell lysis, ultrasound has recently been shown to reversibly increase plasma membrane permeability to drugs, proteins and DNA in living cells and animals independently of cell or drug type, suggesting a ubiquitous mechanism of action. To determine the mechanism of these effects, we examined cells exposed to ultrasound by flow cytometry coupled with electron and fluorescence microscopies. The results show that cavitation generated by ultrasound facilitates cellular incorporation of macromolecules up to 28 nm in radius through repairable micron-scale disruptions in the plasma membrane with lifetimes >1 min, which is a period similar to the kinetics of membrane repair after mechanical wounding. Further data suggest that cells actively reseal these holes using a native healing response involving endogenous vesicle-based membrane resealing. In this way, noninvasively focused ultrasound could deliver drugs and genes to targeted tissues, thereby minimizing side effects, lowering drug dosages, and improving efficacy.

Acoustics↗
PubMed2006

Measurement and correlation of acoustic cavitation with cellular bioeffects.

Using broadband noise as a measure of cavitation activity, this study determined the kinetics of cavitation during sonication of Optison contrast agent and tested whether cellular bioeffects can be predicted by cavitation dose. Cell suspensions were exposed to ultrasound at varying acoustic frequency, pressure, exposure time, Optison concentration and cell type to obtain a broad range of bioeffects, i.e., intracellular uptake and loss of viability, as quantified by flow cytometry. We found that cavitation activity measured by broadband noise increased and peaked within 20 ms and then decayed with a half-life of tens to hundreds of milliseconds. Intracellular uptake and loss of viability correlated well with the cavitation dose determined by the time integral of broadband noise magnitude. These results demonstrate that broadband noise correlates with bioeffects over a broad range of experimental conditions, which suggests a noninvasive feedback method to control ultrasound's bioeffects in real time.

Albumins↗
PubMed2006

Precise microinjection into skin using hollow microneedles.

Hollow needles of micron dimensions have previously been fabricated and envisioned for use with transdermal patches or infusion pumps to achieve painless delivery of drugs to the skin for local and systemic effects without the need for hypodermic needles. However, little work has been carried out to identify methods to effectively use hollow microneedles for drug delivery. To address this need, we inserted hollow, glass microneedles into hairless rat skin in vivo and human cadaver skin in vitro and then imaged infusion of dye molecules, insulin, polymer microparticles, and cells into the skin by brightfield and fluorescence microscopy. The depth of needle penetration into skin was controlled by inserting needles with a rotary drilling device, which enabled localized injection within the epidermis or dermis with +/-60 microm resolution. Although small quantities of fluid could be injected after needle insertion into skin, partial retraction of the needle by withdrawing back 100-300 microm or vibrating the microneedle array dramatically increased infusion flow rate. We conclude that hollow microneedles can be used for precise microinjection into skin, especially when a single needle is inserted by rotary drilling and then retracted part way before infusion or a microneedle array is inserted by mechanical vibration.

Cell Transplantation↗
PubMed2006

Assessment of trueness of a glucose monitor using interstitial fluid and whole blood as specimen matrix.

BACKGROUND: Interstitial fluid (ISF) is a specimen of increasing interest for glucose measurements because it can be obtained in a minimally invasive manner. Our previous study showed that sufficient ISF can be obtained using microneedles to measure glucose with a conventional electrochemical glucose monitor. The aim of this study was to assess the trueness of this glucose monitor using split-sample comparison with whole blood. We used ISF as specimen and our gas chromatography/mass spectrometry (GC/MS) method as reference. METHODS: We obtained 50 ISF samples and 40 whole blood samples from hairless Sprague- Dawley rats and analyzed for glucose by both methods. RESULTS: For whole blood, a non-significant bias of 5.7% (+/-2 SD: -54.9% to 66.3%) was determined. ISF glucose measurements showed a significant constant bias of 29.5% (+/-2 SD: -85.0% to 144%), which seems to be caused in part by the lack of red blood cells in ISF. The correlation coefficients were 0.782 and 0.679 for whole blood and ISF, respectively. CONCLUSIONS: The assessed electrochemical glucose monitor shows a close agreement with our GC/MS reference method for whole blood, for which this monitor was optimized. When glucose measurements are performed with ISF as matrix, the observed bias needs to be taken into consideration. Further studies are necessary to elucidate the reasons for the wide dispersion of data for ISF.

Animals↗
PubMed2006

Measurement and prediction of lateral diffusion within human sclera.

PURPOSE: Drug delivery via the sclera is a promising approach to retinal disorder treatments that require access to the posterior segment of the eye. To complement existing studies of transverse diffusion across the sclera, this study examined lateral diffusion within the sclera parallel to the scleral surface. METHODS: Using sulforhodamine as a model hydrophilic drug, rates of diffusion were measured in strips of human cadaveric sclera for up to 1 week. Data were analyzed with a mathematical model based on theoretical expressions for one-dimensional diffusion. RESULTS: Measurable amounts of sulforhodamine were detected at distances of 5 and 10 mm from the sulforhodamine donor reservoir at 4 hours and 3 days, respectively. The effective lateral diffusivity of sulforhodamine was determined to be 3.82 x 10(-6) cm2/s, which is similar in magnitude to the transverse diffusivity. The theoretical model agreed with experimental values with an average error of 39%. CONCLUSIONS: This study shows that the lateral diffusion of sulforhodamine in human sclera is slow and localizes to the site of administration.

Biological Transport↗
PubMed2005

Biodegradable polymer microneedles: fabrication, mechanics and transdermal drug delivery.

To overcome the skin's barrier properties that block transdermal delivery of most drugs, arrays of microscopic needles have been microfabricated primarily out of silicon or metal. This study addresses microneedles made of biocompatible and biodegradable polymers, which are expected to improve safety and manufacturability. To make biodegradable polymer microneedles with sharp tips, micro-electromechanical masking and etching were adapted to produce beveled- and chisel-tip microneedles and a new fabrication method was developed to produce tapered-cone microneedles using an in situ lens-based lithographic approach. To replicate microfabricated master structures, PDMS micromolds were generated and a novel vacuum-based method was developed to fill the molds with polylactic acid, polyglycolic acid, and their co-polymers. Mechanical testing of the resulting needles measured the force at which needles broke during axial loading and found that this failure force increased with Young's modulus of the material and needle base diameter and decreased with needle length. Failure forces were generally much larger than the forces needed to insert microneedles into skin, indicating that biodegradable polymers can have satisfactory mechanical properties for microneedles. Finally, arrays of polymer microneedles were shown to increase permeability of human cadaver skin to a low-molecular weight tracer, calcein, and a macromolecular protein, bovine serum albumin, by up to three orders of magnitude. Altogether, these results indicate that biodegradable polymer microneedles can be fabricated with an appropriate geometry and sufficient strength to insert into skin, and thereby dramatically increase transdermal transport of molecules.

Biocompatible Materials↗
PubMed2005

Minimally invasive extraction of dermal interstitial fluid for glucose monitoring using microneedles.

BACKGROUND: Compliance with glucose monitoring by patients with diabetes is poor because of the pain and inconvenience of conventional blood collection using lancets. To improve compliance, and thereby reduce morbidity and mortality associated with poor glucose control, this study sought to develop and test minimally invasive microneedles to extract dermal interstitial fluid (ISF) for glucose monitoring. METHODS: We used a thermal puller to fabricate individual or multi-needle arrays of glass microneedles with tip radii of 15-40 microm to penetrate 700-1,500 microm deep into the skin of anesthetized hairless rats or conscious, normal, adult, human subjects. After applying a vacuum of 200-500 mm Hg for 2-10 min, we extracted ISF and measured glucose concentration. These measurements were compared with glucose levels in blood collected from the tail vein of rats or finger stick on humans. RESULTS: Using this procedure, 1-10 microL of ISF was extracted out of holes punctured in the skin using microneedles. Human subjects generally reported the procedure as painless. ISF glucose concentration correlated well with blood levels based on 140 measurements on 15 rats and six measurements on six human subjects, where 95% of rat data and 100% of human data fell within the clinically acceptable A + B region in Clarke Error Grid analysis. A linear calibration factor was needed to correlate ISF and blood glucose concentrations using our standard procedure. Modifying the procedure to prevent ISF evaporation during extraction provided a one-to-one correlation that eliminated the need for calibration. ISF glucose measurements tracked rapidly changing blood glucose levels following insulin injection with a time lag of less than 20 min. CONCLUSIONS: These results suggest that microneedle devices can be used to extract ISF for painless glucose monitoring.

Animals↗
PubMed2005

Hollow metal microneedles for insulin delivery to diabetic rats.

The goal of this study was to design, fabricate, and test arrays of hollow microneedles for minimally invasive and continuous delivery of insulin in vivo. As a simple, robust fabrication method suitable for inexpensive mass production, we developed a modified-LIGA process to micromachine molds out of polyethylene terephthalate using an ultraviolet laser, coated those molds with nickel by electrodepostion onto a sputter-deposited seed layer, and released the resulting metal microneedle arrays by selectively etching the polymer mold. Mechanical testing showed that these microneedles were sufficiently strong to pierce living skin without breaking. Arrays containing 16 microneedles measuring 500 microm in length with a 75 microm tip diameter were then inserted into the skin of anesthetized, diabetic, hairless rats. Insulin delivery through microneedles caused blood glucose levels to drop steadily to 47% of pretreatment values over a 4-h insulin delivery period and were then approximately constant over a 4-h postdelivery monitoring period. Direct measurement of plasma insulin levels showed a peak value of 0.43 ng/ml. Together, these data suggest that microneedles can be fabricated and used for in vivo insulin delivery.

Animals↗
PubMed2004

Increased permeability of intestinal epithelial monolayers mediated by electroporation.

This study assessed whether electroporation enhances transport across intact intestinal epithelial monolayers that mimic the intestinal epithelium. Confluent Caco-2 monolayers were exposed to electroporation pulses and then monitored over time for transepithelial transport of calcein, a small fluorescent tracer, or fluorescein-labeled bovine serum albumin, a large protein. Cumulative transport of both molecules across the monolayers increased significantly (up to 34-fold) after electroporation and depended on electroporation voltage and pulse length and on molecular size. Increased transport was accompanied by a decrease in the transepithelial electrical resistance of the monolayers. Further analysis of these results suggests that the increase in transport observed after electroporation is due, at least in part, to the killing of a small fraction of cells, which increased transport across "leaky" dead cells that remained adherent and increased transport through small, temporary holes left by dead cells that detached, but appeared to reseal within minutes by monolayer restitution. These findings could form the basis for the development of electroporation as a clinical tool to increase intestinal permeability and, thereby, increase the absorption of poorly absorbed drugs.

Animals↗
PubMed2004

Model of transient drug diffusion across cornea.

A mathematical model of solute transient diffusion across the cornea to the anterior chamber of the eye was developed for topical drug delivery. Solute bioavailability was predicted given solute molecular radius and octanol-to-water distribution coefficient (Phi), ocular membrane ultrastructural parameters, tear fluid hydrodynamics, as well as solute distribution volume (Vd) and clearance rate (Cla) in the anterior chamber. The results suggest that drug bioavailability is primarily determined by solute lipophilicity. In human eyes, bioavailability is predicted to range between 1% and 5% for lipophilic molecules (Phi>1), and to be less than 0.5% for hydrophilic molecules (Phi<0.01). The simulations indicate that the distribution coefficient that maximizes bioavailability is on the order of 10. It was also found that the maximum solute concentration in the anterior chamber (Cmax) and the time needed to reach Cmax significantly depend on Phi, Vd, and Cla. Consistent with experimental findings, model predictions suggest that drug bioavailability can be increased by lowering the conjunctival-to-corneal permeability ratio and reducing precorneal solute drainage. Because of its mechanistic basis, this model will be useful to predict drug transport kinetics and bioavailability for new compounds and in diseased eyes.

Algorithms↗
PubMed2004

Microneedles for transdermal drug delivery.

The success of transdermal drug delivery has been severely limited by the inability of most drugs to enter the skin at therapeutically useful rates. Recently, the use of micron-scale needles in increasing skin permeability has been proposed and shown to dramatically increase transdermal delivery, especially for macromolecules. Using the tools of the microelectronics industry, microneedles have been fabricated with a range of sizes, shapes and materials. Most drug delivery studies have emphasized solid microneedles, which have been shown to increase skin permeability to a broad range of molecules and nanoparticles in vitro. In vivo studies have demonstrated delivery of oligonucleotides, reduction of blood glucose level by insulin, and induction of immune responses from protein and DNA vaccines. For these studies, needle arrays have been used to pierce holes into skin to increase transport by diffusion or iontophoresis or as drug carriers that release drug into the skin from a microneedle surface coating. Hollow microneedles have also been developed and shown to microinject insulin to diabetic rats. To address practical applications of microneedles, the ratio of microneedle fracture force to skin insertion force (i.e. margin of safety) was found to be optimal for needles with small tip radius and large wall thickness. Microneedles inserted into the skin of human subjects were reported as painless. Together, these results suggest that microneedles represent a promising technology to deliver therapeutic compounds into the skin for a range of possible applications.

Administration, Cutaneous↗