Diffusion cells and related methods

A method of performing a diffusion test includes clamping a membrane to a body such that a first surface of the membrane is in fluid communication with an interior chamber of the body and a second surface of the membrane is exposed to ambient air, flowing a substance through the ambient air such that at least a portion of the substance lands on the second surface while the membrane is vertically oriented, and determining a concentration of the substance in the interior chamber after some of the substance has diffused through the membrane.

TECHNICAL FIELD

This disclosure relates to diffusion cells and associated methods of performing diffusion tests, such as diffusion tests for examining the movement of airborne substances (e.g., sprayed or aerosolized substances) through materials.

BACKGROUND

Development of substances used in a variety of applications often requires an understanding of how the substances move through materials. For example, an ability of a substance (e.g., drugs, chemicals treatments, and various particulates) to diffuse through a semi-permeable material construct can provide insight into an effectiveness or a toxicity of the substance, as well as characteristics of the material construct. In some implementations, diffusion cells can be used to examine such parameters.

SUMMARY

In general, this disclosure relates to diffusion cells and methods of using the diffusion cells to perform diffusion tests, such diffusion tests that examine the movement of airborne substances through semi-permeable membranes. The diffusion cells are advantageously configured and, accordingly, particularly useful for in vitro examination of diffusion characteristics of airborne substances, which may not be adequately examined using conventional diffusion cells that include liquid carrying donor chambers. An open, accessible configuration of the disclosed diffusion cells provide an air-solid interface to which a volume of an airborne substance can be delivered with a substantially even distribution across an area of a membrane secured to the diffusion cells. Accordingly, the configuration of the diffusion cells facilitates experimental acquisition of representative data that reflects a realistic application of a substance.

Furthermore, the configuration of the diffusion cells and a horizontal experimental arrangement of the diffusion cells advantageously permit examination of an airborne substance using only a small volume of a substance, which may be beneficial when the substance is only available in limited amounts or is obtained at a high cost. Additionally, the horizontal experimental arrangement and a flat donor structure of the diffusion cells prevents an airborne substance from settling and pooling on a membrane, which may otherwise occur with conventional experimental arrangements or conventional diffusion cells.

In another aspect, a method of performing a diffusion test includes clamping a membrane to a body such that a first surface of the membrane is in fluid communication with an interior chamber of the body and a second surface of the membrane is exposed to ambient air, flowing a substance through the ambient air such that at least a portion of the substance lands on the second surface while the membrane is vertically oriented, and determining a concentration of the substance in the interior chamber after some of the substance has diffused through the membrane.

In some embodiments, the method further includes preventing the substance from pooling on the membrane.

In certain embodiments, the method further includes arranging the body in a horizontal orientation prior to flowing the substance through ambient air.

In some embodiments, the method further includes assembling a splash guard with the body to prevent an airborne flow of the substance from contacting the body.

In certain embodiments, clamping the membrane to the body includes providing an air-solid interface.

In some embodiments, flowing the substance through the ambient air includes evenly distributing the substance across the second surface of the membrane.

In certain embodiments, the method further includes distributing a volume of about 2 μL/cm2 to about 20 μL/cm2 of the substance across the second surface of the membrane.

In some embodiments, the method further includes flowing a heat transfer fluid through an exterior chamber of the body that surrounds the interior chamber of the body.

In certain embodiments, the method further includes delivering a fluid buffer to the interior chamber of the body.

In some embodiments, the method further includes introducing the fluid buffer into a port located above the interior chamber of the body.

In certain embodiments, the method further includes withdrawing a sample of the fluid buffer from the interior chamber of the body at multiple predetermined times after at least the portion of the substance has landed on the second surface of the membrane.

In some embodiments, determining the concentration of the substance in the interior chamber includes determining respective concentrations of the substance in the fluid buffer following the multiple predetermined times.

In certain embodiments, the method further includes determining one or more diffusion parameters associated with one or both of the substance and the membrane based on the respective concentrations.

In some embodiments, flowing the substance through the ambient air includes flowing an aerosolized substance towards the membrane.

In certain embodiments, flowing the substance through the ambient air includes spraying the substance towards the membrane.

In some embodiments, flowing the substance through the ambient air includes flowing nanoparticles towards the membrane.

In certain embodiments, flowing the substance through the ambient air includes flowing a drug towards the membrane.

In some embodiments, flowing the substance through the ambient air includes flowing a chemical that is toxic to animals towards the membrane.

In certain embodiments, clamping the membrane to the body includes securing a construct including one or both of an artificial tissue and a natural tissue to the body.

In another aspect, a diffusion cell includes a body defining an interior chamber and an adjustable clamp configured to secure a membrane to the body across an open end of the interior chamber, wherein the adjustable clamp defines a beveled edge configured to prevent pooling of a substance on the membrane.

In some embodiments, the diffusion cell includes one or more seals located between the body and the adjustable clamp.

In certain embodiments, the diffusion cell includes a guard configured to engage one or both of the body and the adjustable clamp to block the flow of the substance.

In some embodiments, the diffusion cell provides an air-solid interface.

In some embodiments, the body defines an exterior chamber that surrounds the interior chamber.

In certain embodiments, the exterior chamber provides a liquid jacket for heating and cooling the interior chamber.

In some embodiments, the body defines an inlet port and an outlet port for flowing a heat transfer fluid through the exterior chamber.

In certain embodiments, the body defines one or more sample ports for delivering a fluid buffer to and withdrawing a fluid buffer from the interior chamber.

In some embodiments, the adjustable clamp includes one or more threaded fasteners for securing a frontal plate of the adjustable clamp to the body.

In certain embodiments, the frontal plate defines the beveled edge.

In some embodiments, the beveled edge surrounds an opening in the frontal plate through which the substance can flow to the membrane.

Other aspects, features, and advantages will be apparent from the description, the drawings, and the claims.

DETAILED DESCRIPTION

FIGS. 1-5illustrate various views of a diffusion cell100(e.g., a Munt-Dash diffusion cell) used for examining movement of a substance (e.g., a permeant) through a membrane101(e.g., for examining penetration of the substance into and movement of the substance through the membrane101). The diffusion cell100can be used for measuring parameters such as flux (e.g., an amount of permeant that crosses a membrane per unit area per unit time), accumulation (e.g., an amount of permeant that crosses a membrane within a certain time period), diffusivity (e.g., a measure of how easily a permeant penetrates a membrane, as an area per unit time), a permeability coefficient (e.g., a rate of permeant penetration per concentration, expressed as a distance per unit time), and a lag time (e.g., a time required for a permeant to permeate through a membrane and into a receptor fluid and to reach a steady state of diffusion).

The membrane101is a selective, semi-permeable barrier that allows passage of some components (e.g., molecules, ions, and small particles) and that prevents passage of other components based on pore sizes of the membrane101. Example applications for which the diffusion cell100can be used include transdermal drug testing of patches, ointments, and other topical formulations (e.g., ultra violet (UV) radiation protection in a sunscreen), ophthalmic drug formulations, and membrane suitability as a vapor barrier, as will be discussed in more detail below. The diffusion cell100includes a main body102and a clamp104that secures the membrane101to the main body102.

FIG. 6illustrates a perspective view of the main body102. The main body102defines an interior chamber106that serves as a receptacle (e.g., a receptor chamber) for a fluid buffer (e.g., a receptor solution) and three sample ports108,110,112that provide access to the interior chamber106for introducing the fluid buffer to or removing the fluid buffer from the interior chamber106. The interior chamber106can be hermetically sealed by the clamp104at an open end114and at the sample ports108,110,112by any of caps, syringes, tubing, and septa, depending on a flow configuration and a sampling technique utilized for a diffusion test. The main body102also defines an interior recess116in which a magnetic stir bar (not shown) can be placed for mixing the fluid buffer within the interior chamber106.

The main body102also defines an exterior chamber118that surrounds the interior chamber106and portions of the sample ports108,110,112such that the sample ports108,110,112extend through a wall of the exterior chamber118. The exterior chamber118provides a liquid jacket (e.g., a water jacket) through which liquid can be flowed to transfer heat to or transfer heat away from the interior chamber106. In this regard, the main body102defines an inlet port120located near the open end114of the interior chamber106and an outlet port122located near an opposite end of the main body102. The inlet and outlet ports120,122can be connected to liquid flow lines. The main body102further defines an exterior recess124along a circumference of the main body102and along which the clamp104is secured, as will be discussed in more detail below.

The main body102may be made of one or more chemically robust materials that are non-corrosive, that can withstand temperatures of up to about 50° C., that are transparent or translucent, that are non-reactive, and that have good thermal conductance, such as glass and polymers like polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), and acrylic. The interior chamber106has a generally cylindrical shape. The exterior chamber118has a generally annular cross-sectional shape along a portion surrounding the interior chamber106and a generally cylindrical shape along a remaining portion. The main body102has a wall thickness of about 1 mm to about 5 mm (e.g., about 2 mm) along the interior and exterior chambers106,118. The interior chamber106has a length of about 2 cm to about 1] cm (e.g., about 2.5 cm) and an internal diameter of about 6 mm to about 100 mm (e.g., about 11.5 mm), such that the interior chamber106defines an interior volume of about 0.5 mL to about 1000 mL (e.g., about 3.5 mL) and defines a diffusion area of about 0.3 cm2to about 80 cm2(e.g., about 1.04 cm2). The exterior chamber118has a length of about 3 cm to about 15 cm (e.g., about 4.5 cm) and a maximum internal diameter of about 18 mm to about 120 mm (e.g., about 24 mm), such that the exterior chamber118defines an internal volume of about 7.6 mL to about 1700 mL (e.g., about 20 mL).

The main body102has a wall thickness of about 1 mm to about 5 mm (e.g., about 1.5 mm) along the sample ports108,110,112and the inlet and outlet ports120,122. The sample ports108,110,112have a length of about 0.5 cm to about 5 cm (e.g., about 1 cm) and an internal diameter of about 0.2 cm to about 1 cm (e.g., about 0.4 cm). The sample port108is located about 1.5 cm to about 5 cm (e.g., about 2.2 cm) from the open end114of the interior chamber106, and the sample ports110,112are located about 1.8 cm to about 7 cm (e.g., about 2.8 cm) from the open end114of the interior chamber106. The sample port108is spaced apart from each sample port110,112by an angle of about 5 degrees to about 45 degrees (e.g., about 15 degrees) around the wall of the exterior chamber118. The inlet port120has a length of about 1.5 cm to about 10 cm (e.g., about 3 cm), an internal diameter of about 0.3 cm to about 2 cm (e.g., about 0.4 cm), and an outer diameter of about 0.5 cm to about 2.5 cm (e.g., about 0.7 cm). The inlet port120is located about 0.2 cm to about 14.5 cm (e.g., about 0.2 cm) from the open end114of the interior chamber106. The outlet port122has a length of about 1.5 cm to about 10 cm (e.g., about 3 cm) and an internal diameter of about 0.3 cm to about 2 cm (e.g., about 0.4 cm). The outlet port122is located about 0.2 cm to about 14.5 cm (e.g., about 3.7 cm) from the open end114of the interior chamber106. The inlet port120is spaced apart from the outlet port122by about 90 degrees to about 180 degrees (e.g., about 180 degrees) around the wall of the exterior chamber118.

The exterior recess124has an outer diameter of about 1.5 cm to about 11.5 cm (e.g., about 1.9 cm). The exterior recess124is located about 0.7 cm to about 5 cm (e.g., about 1.2 cm) from the open end114of the interior chamber106. The interior recess116has a diameter of about 0.3 cm to about 2.5 cm (e.g., about 0.4 cm) and a depth of about 0.1 cm to about 2.5 cm (e.g., about 0.2 cm), such that the interior recess116defines a volume of about 0.03 mL to about 13 mL (e.g., about 0.1 mL).

FIG. 7illustrates a rear perspective view of the clamp104, andFIG. 8illustrates cross-sectional view of a portion of the clamp104. In addition to securing the membrane101to the main body102, the clamp104serves as a donor port through which the substance can be delivered to the membrane101. Referring toFIGS. 1-8, the clamp104includes a frontal plate126, two gaskets128disposed adjacent the frontal plate126, a rear plate130engaged with the exterior recess124of the main body102, and two fastener assemblies132for adjusting the frontal plate126with respect to the rear plate130to secure the clamp104to the main body102.

The frontal plate126has four beveled lateral edges148and defines a central opening134through which the substance can access the membrane101. The opening134is surrounded by a beveled circular edge136within a wall of the frontal plate126. The beveled circular edge136reduces a thickness of the frontal plate126that extends at a right angle from the central opening134, while still allowing a surrounding portion of the frontal plate126to maintain a maximal strength. An inward narrowing of the beveled circular edge136serves to minimize the amount of material of the frontal plate126near the membrane101(e.g., to minimize a thickness of the frontal plate126near the membrane101) to prevent the substance from pooling on the membrane101along an edge of the central opening134and to cause excess droplets of the substance on the frontal plate126to run off of the frontal plate126rather than settling in a place on the membrane101or on the frontal plate126where such settling could alter the study. The beveled circular edge136is oriented at an angle of about 10 degrees to about 45 degrees (e.g., about 30 degrees). The frontal plate126also defines two lateral openings138through which the fastening assemblies132extend toward the rear plate130. The frontal plate126has a length of about 3 cm to about 16 cm (e.g., about 5.1 cm) and a width of about 2.2 cm to about 16 cm (e.g., about 3.8 cm). The frontal plate126has a thickness of about 1 mm to about 5 mm (e.g., about 2 mm). The central opening134of the frontal plate has a diameter of about 0.6 cm to about 10 cm (e.g., about 1.15 cm) and equals an inner diameter of the beveled circular edge136. The beveled circular edge136has an outer diameter of about 0.8 cm to about 12 cm (e.g., about 1.4 cm). The openings138have a diameter of about 2 mm to about 15 mm (e.g., about 5 mm).

The rear plate130is generally u-shaped and is positioned within the exterior recess124of the main body102. Accordingly, an inner curved (e.g., semi-circular) portion of the rear plate130has a radius of about 0.9 cm to about 5.8 cm (e.g., about 1.1 cm). The rear plate130also defines two lateral openings140through which the fastening assemblies132extend toward the frontal plate128. An outer profile of the rear plate130generally has the shape of an outer profile of the frontal plate126, such that the rear plate130has a length and a width that are equal to the length and the width of the frontal plate126. The rear plate130has a thickness of about 1 mm to about 5 mm (e.g., about 2 mm). The openings140have a diameter of about 2 mm to about 15 mm (e.g., about 5 mm).

In operation of the diffusion cell100, the frontal plate126can be tightly secured to the main body102using the fastening assemblies132. Each fastening assembly132includes a screw142that abuts the rear plate130, a spring144that surrounds a shaft of the screw142, and a threaded knob146that can be adjusted along the shaft of the screw142to force (e.g., push) the frontal plate126towards the spring144. The shafts of the screws142have a length of about 1.6 cm to about 5 cm (e.g., about 2.5 cm). The components of the fastening assemblies132, the frontal plate126, and the rear plate130may be made of one or more chemically robust materials that are non-corrosive, can withstand temperatures of up to about 50° C., that are non-reactive, that are non-fragile, and that have minimal (<1%) deformation under a working load, such as stainless steel, anodized aluminum, PTFE, PVDF, and other rigid polymers of sufficient thickness to inhibit deformation.

The gaskets128have an annular shape and provide seals between the clamp104and the main body102. The frontal gasket128is located adjacent the frontal plate126, and the rear gasket128is located adjacent the main body102. The gaskets128are separated by the membrane101. The gaskets128have an outer diameter of about 2 cm to about 12 cm (e.g., about 2.9 cm) and an inner diameter of about 0.6 cm to about 10 cm (e.g., about 1.15 cm). The gaskets128have a thickness of about 0.25 mm to about 2 mm (e.g., about 0.6 mm). The gaskets128may be made of one or more chemically robust materials that provide suitable sealing functionality, that are non-reactive, that are non-adsorbing to permeant, and that are pliable, such as PTFE foam, solid silicone, PVDF foam, silicon coated polyurethane foam, or other pliable waterproof synthetic or natural polymers.

In some implementations, a guard can be attached to a frontal region of the diffusion cell100to prevent fluids from splashing rearward onto the diffusion cell100. For example,FIG. 9illustrates a splash guard150attached to the diffusion cell100, andFIG. 10illustrates a rear perspective view of the splash guard150. The splash guard150may be a unitary structure that includes a frontal plate152and a base plate154that extends perpendicularly from the frontal plate152.

The frontal plate152has a generally rectangular shape and defines a central opening156that aligns concentrically with the central opening134of the frontal plate126of the clamp104such that the central opening156helps to guide the substance towards the membrane101. Accordingly, the central opening156has a diameter of about 0.7 cm to about 10.2 cm (e.g., about 1.2 cm). The frontal plate152also defines two lateral openings158that align concentrically with the lateral openings138of the frontal plate126of the clamp104to allow passage of the knobs146. The lateral openings158have a diameter of about 0.5 cm to about 2.5 cm (e.g., about 1.2 cm). The frontal plate152has a length of about 4 cm to about 20 cm (e.g., about 6.1 cm), a width of about 4 cm to about 20 cm (e.g., about 5 cm), and a thickness of about 0.25 mm to about 2 mm (e.g., about 0.7 mm).

The base plate154has a generally rectangular shape and includes two tabs160that flank the main body102of the diffusion cell100to centrally position and lock the splash guard150with respect to the diffusion cell100. The base plate154has a length of about 1.7 cm to about 5 cm (e.g., about 2 cm), a width of about 3 cm to about 12 cm (e.g., about 4.5 cm), and a thickness of about 0.25 mm to about 2 mm (e.g., about 0.7 mm). The tabs160have a length of about 0.5 cm to about 5 cm (e.g., about 1 cm), a height of about 5 cm to about 0.5 cm (e.g., about 5 cm), and a thickness of about 0.25 mm to about 2 mm (e.g., about 0.7 mm). The size and shape of the splash guard150can prevent an overspray of fluid from contaminating the sample ports108,110,112of the diffusion cell100.

As discussed above, the diffusion cell100can be employed to examine (e.g., measure, compute, observe, visualize, or otherwise examine) diffusion characteristics of a substance through the membrane101. The membrane101may be made of one or more natural or synthetic materials, such as celluloses (e.g., regenerated cellulose, nitrocellulose, or cellulose esters), regenerated or synthetic keratin membranes, lipid infused synthetic membranes (e.g., such as that used in a parallel artificial membrane permeability assay), PTFE, PVDF, nylon, other synthetic polymer membranes, live human or animal skin, dead human or animal skin (e.g., cadaver skin), or other live or dead tissue portions (e.g., lung or corneal tissue). In some embodiments, the membrane101may be formed from or in part from an in vitro cell or tissue culture. In some embodiments, the membrane101may have a bulk elastic modulus in a range of about 1 kPa to about 1000 kPa. For example, in some embodiments, the membrane101may be made of human skin that has a Young's Modulus in a range of about 25 kPa to about 450 kPa, depending upon a study, an age, a temperature, and a site from which the skin was taken. In some embodiments, the membrane101may have pore sizes ranging from a nominal size of about 0.04 nm (100 Daltons) to about 1 μm. The membrane101is sized to be contacted along a peripheral edge by the gaskets128, which are in contact with the frontal plate126of the clamp104and the main body102. Accordingly, the membrane101typically has a diameter of about 1 cm to about 12 cm (e.g., about 2.5 cm).

In some implementations, the substance may be an aerosolized substance (e.g., a spray, vapor, gas, or mist formulation) or a non-aerosolized substance (e.g., provided in a powder, cream, or gel formulation) that is examined to determine diffusion characteristics that relate to on an effectiveness, a toxicity, or a contamination profile of the substance, or that is examined for interactions with the membrane101, such as surface binding of tanners, sunscreens, and insect repellants. Example aerosolized substances include inhaled drugs (e.g., nebulizer solutions), airborne nanoparticles used in various applications, cigarette smoke, and other environmental insults, such as pesticides, chemical fumes or vapors, environmental pollutants, electromagnetic insults (e.g., light, radiation, etc.), and other chemical insults. Other example substances included topical drugs (e.g., skin formulations) and other topical chemical compounds, such as skin absorbed toxic substances (e.g., topical VX nerve agent use), and modification materials (e.g., testing of penetration enhancers).

FIG. 11illustrates a side view of a diffusion system1000(e.g., including the diffusion cell100and the splash guard150) as arranged to carry out a diffusion study of an airborne substance103. The substance103can be prepared in a dosage source105that delivers fixed doses, such as a syringe pump sprayer (e.g., as shown inFIG. 11), or another metered dose device. The membrane101is equilibrated (e.g., via submersion or exposure in a fixed relative humidity chamber) in a volume of a fluid buffer107that will be used as a receptor solution to prevent shifting of the membrane101that may otherwise occur due to a sudden exposure to the fluid buffer107. The fluid buffer107may be water-based or organic and may be a liquid or a gas. Example fluid buffers107include biological mediums (e.g., plasma, serum, blood, cerebrospinal fluid, aqueous humor, etc.), other buffers (e.g., phosphate buffer, phosphate buffered saline, Hank's balanced salt solution, Krebs buffer, cell culture media, etc.), and modifications thereof, which may include surfactants, such as dipalmitoylphosphatidylcholine (DPPC), polysorbate (tween), or other surfactants. A stir bar disposed within the interior recess116is activated during the equilibration of the membrane101. The membrane101is placed between the two gaskets128of the clamp104, and the clamp104is secured to the main body102using the knobs146to fix the membrane101in place across the open end114of the interior chamber106.

The splash guard150may be optionally assembled with the diffusion cell100, and the diffusion cell100is placed in a horizontal orientation atop a surface109(e.g., a balance, a heater, a table, a magnetic stir plate, or an automated sampling robot apart from the dosage source105. The fluid buffer107is delivered to the interior chamber106using a syringe or another delivery device (e.g., a pipette, a fluid pump, or an automated sampling robot, not shown) via one or more of the sample ports108,110,112. An initial volume of about 0.5 mL to about 1000 mL may be delivered to the interior chamber106. Any resulting bubbles in the fluid buffer107may be removed from the interior chamber106by tilting and/or tapping the diffusion cell100or by flushing solution through lines connected to the sample ports108,110,112until the bubbles are removed (e.g., in the case of a flow through study). Any open sample ports108,110,112are closed with a syringe or another closure device (not shown). The fluid buffer107and the membrane101may then be equilibrated to a selected temperature of about 20° C. to about 50° C. by flowing a heat transfer fluid through the inlet port120, the exterior chamber118, and the outlet port122. Example heat transfer fluids include water, 10-50% (v/v) propylene glycol or ethylene glycol (e.g., 30% ethylene glycol results in only approximately a 1% loss of thermal conductance in water, a minimal change in viscosity, and acts as a deterrent to fungus, bacteria, and algae growth), and other fluids. The heat transfer fluid is typically flowed for a period of about 5 min to about 1 day, continuously for a duration of the experiment.

Once the fluid buffer107and the membrane101achieve a selected temperature, the diffusion cell100is positioned such that the membrane101is located at a distance of about 5 cm to about 32 cm from an exit port111of the dosage source105and such that a central axis of the main body102of the diffusion cell100is vertically displaced from a center of the exit port111by about 0 cm to about 3 cm. A central axis of the main body102may be centrally aligned with or vertically offset from a central axis of a spray of the substance103, depending on a distance between the membrane101and the dosage source105. For example, the central axis of the main body102is approximately aligned with the central axis of the spray at a distance of about 8 inches, whereas the central axis of the main body102is vertically offset from the central axis of the spray by about 1 cm to about 2 cm at a distance of about 12 inches. In some implementations, the diffusion cell100is then placed into a fitted metallic or polymer holder atop the surface109. In some examples, the use of polymer holders with low thermal conductivity improves the efficiency of the water jacketing system provided by the exterior chamber118. In some implementations, 3-4 prong system can be used to hold the diffusion cell in place atop the surface109.

The substance103is ejected (e.g., sprayed) from the dosage source105towards the membrane101in an aerosolized form, which is determined by experiment-specific parameters and will vary based upon solvent properties. For example, a fixed volume of about 50 μL to about 500 μL of the substance103is ejected from the dosage source over a period of about 0.25 s to about 1 s such that about 2 μL/cm2to about 20 μL/cm2(e.g., about 10 μL/cm2) of the substance achieves contact with the membrane101. An upper limit to an amount of substance103per area that can be delivered to the membrane101depends upon the properties of the solvent and the membrane101. About 1% to about 100% of the ejected volume may contact the membrane101, depending on a size of the membrane101, the distance, a solvent, and dosage source characteristics. In some implementations, about 5% to about 60% of the ejected volume may contact the membrane101with an even distribution, according to the particular experimental parameters discussed herein. After the substance103is delivered to the membrane101, the splash guard150may be optionally removed from the diffusion cell100.

A sample volume of about 10 μL to about 5000 μL of the fluid buffer107is removed from the interior chamber106with a syringe or another removal device at selected intervals that may vary between about 0.5 min and about 240 min over a period of about 5 min to about 1 day (e.g., about 300 min). In some implementations, a volume of the fluid buffer107equal to the volume of the fluid buffer107that was removed may be delivered to the interior chamber106via the same one or more sample ports108,110,112through which the fluid buffer107was removed (e.g., sequentially following the removal) to replenish the fluid buffer107, and the one or more sample ports108,110,112are then closed. In some implementations, a volume of the fluid buffer107equal to the volume of the fluid buffer107that was removed may be delivered to the interior chamber106via one or more sample ports108,110,112that are not used for removal of the fluid buffer107, while the fluid buffer107is being removed from the interior chamber106(e.g., in a parallel, flow-through configuration), to replenish the fluid buffer107, and the sample ports108,110,112are then closed.

Concentrations of the substance103in the samples of the fluid buffer107are measured using various analytical techniques following removal of the samples from the interior chamber. Example analytical techniques include photometric methods, such as UV/Visible spectrophotometry, spectrofluorophotometry, and luminomitry. Such methods may be used alone, such as a plate reader or individual cuvettes, or as part of a chromatographic separation, such as HPLC or UPLC. Such methods may also use electrochemical detection. Mass spectrophotometery may also be used coupled with LC (LC/MS) or Gas chromatography (GC/MS). The concentrations and the area of the membrane101may be used to compute parameters including suitability of the membrane101for certain objectives, a flux, an accumulation, a diffusivity, a permeability coefficient, and a lag time.

The diffusion cell100is advantageously configured and, accordingly, particularly useful for in vitro examination of diffusion characteristics of aerosolized substances, which may not be adequately examined using conventional diffusion cells (e.g., Franz diffusion cells, side-by-side diffusion cells, and Valia Chien diffusion cells) that include liquid carrying donor chambers and that accordingly provide liquid-liquid or liquid-membrane-liquid interfaces. For example, in some cases, a substance that is typically aerosolized in its useful form may only be studied using a conventional diffusion cell in a different, non-aerosolized form, such that data obtained from the studies may not accurately represent behaviors of the substance in its useful form. In contrast, an open, accessible configuration of the clamp104(e.g., owing in part to the configuration of the central opening134and the beveled circular edge136) of the diffusion cell100provides an air-solid interface (e.g., an interface defined by the solid membrane101and an ambient air environment) to a which volume of aerosolized substance can be delivered with a substantially even distribution across an area of the membrane101. In some examples, an even distribution of a substance across a membrane is taken as an underlying assumption for diffusion cell experiments, such that results obtained from the experiments may not accurately represent a true behavior of the substance in a realistic application if the substance is not evenly distributed across the membrane. The configuration of the diffusion cell100advantageously facilitates such an even distribution and thereby allows realistic, representative data to be obtained from experiments.

Furthermore, the configuration of the diffusion cell100and the horizontal experimental arrangement of the diffusion cell100advantageously permit examination of an aerosolized substance using only a small volume per unit area of the membrane101, which may be beneficial when the substance is only available in limited amounts or is obtained at a high cost. For example, the methods discussed herein may achieve a distribution of about 10 μL/cm2when ejecting a sample volume of about 200 μL the substance. In contrast, conventional diffusion cells typically require a significantly larger sample volume in order to achieve an even distribution of substance on a membrane. Additionally, the horizontal experimental arrangement of the diffusion cell100and the flat surface of the frontal plate126can prevent an aerosolized substance from settling and pooling on the membrane101, which may otherwise occur with vertical experimental arrangements or conventional donor chamber structures employed for conventional diffusion cells. Another significant benefit of the diffusion cell100and the associated methods discussed above is drying of the small volume of the substance103on the surface of the membrane101. A loss of solvent due to the drying can drastically change the diffusivity of the test substance103.

FIG. 12illustrates an example process200for performing a diffusion test using the diffusion cell100. In some implementations, the process includes clamping a membrane (e.g., the membrane101) to a body (e.g., the main body102) such that a first surface of the membrane is in fluid communication with an interior chamber (e.g., the interior chamber106) of the body and a second surface of the membrane is exposed to ambient air (202). In some implementations, the process further includes flowing a substance through the ambient air such that at least a portion of the substance lands on the second surface while the membrane is vertically oriented (204). In some implementations, the process further includes determining a concentration of the substance in the interior chamber after some of the substance has diffused through the membrane (206).

FIGS. 13A-13Fprovide a set of graphs that illustrate permeation as a function of time for a drug substance tested with a liquid spray application using the diffusion cell100(Spray Chamber), the same drug substance tested with a bulk liquid application using a conventional Franz diffusion cell (Franz Hi), and the same drug substance diluted 1:10 in deionized water, tested with a bulk liquid application using a conventional Franz diffusion cell (Franz Low). In the example experiments, diclofenac sodium was allowed to diffuse through a snake skin model (e.g., a membrane made of shed snake skin) for an outermost epidermis layer (e.g., stratum corneum) over 5 hours at room temperature (FIGS. 13A, 13B, and 13C) and at 32° C. (FIGS. 13D, 13E, and 13F). The diclofenac was tested in three different formulations that each contained approximately 4% (w/v) diclofenac sodium. The first formula (FIGS. 13A and 13D) consisted of 4% (w/v) aqueous diclofenac sodium. The second formula (FIGS. 13B and 13E) was a generic diclofenac formulation consisting of diclofenac sodium (aqueous 4% (w/v), isopropanol (25% v/v), propylene glycol (1.5% w/v), and soy lecithin (HLB 7, 1% w/v). The second formula was made by first dissolving soy lecithin into water. Isopropanol and propylene glycol were then added to the solution and mixed to homogeneity. Diclofenac sodium was weighed into a volumetric flask and brought to volume using the soy lecithin solution. This was then ultrasonicated in a bath sonicator (35 watt Fisher Scientific) for 30 minutes to dissolve the diclofenac immediately prior to use. The third formula (FIGS. 13C and 13F) was the commercially available formulation Voltaren (4% w/v diclofenac). Due to the difference in applied substance volume (100 μL) in the Franz chambers, versus 10 μL for the diffusion cell100, the three systems were examined using solutions of the same concentration (40 mg/mL diclofenac), and the same total amount of drug was applied (4 mg/mL diclofenac).

Spray volume calculations showed that during the experiments using the diffusion cell100, 10.09±1.22 μL/cm2(n=10) and 5.06±1.87 μL/cm2(n=6) of the diclofenac sodium formulation was delivered at a substantially even distribution to the membrane when 200 μL and 100 μL of the diclofenac sodium formulation, respectively, were sprayed from 3 mL syringes at a distance of about 8 inches from the membrane, illustrating a regularity of the system. Such delivery volumes meet standard, accepted guidelines of 10-12 μL per cm2of skin surface for finite dose in vitro testing of skin absorption/permeability. In contrast, the high and low concentration Franz diffusion cells required delivery of about 100 μL of the diclofenac sodium formulation to the membrane to achieve an even distribution across the membrane. Accordingly, the diffusion cell100was able to be utilized with about ten times less the amount of substance as required for the conventional Franz diffusion cells.

As shown in the graphs ofFIGS. 13E and 13F, the conventional Franz diffusion cells could not distinguish the permeability of diclofenac sodium between the generic formulation and the commercial formulation, whereas the diffusion cell100did show a difference in permeability of diclofenac sodium between the generic formulation and the commercial formulation. In this regard, the diffusion cell100can distinguish certain differences that cannot be distinguished by the Franz diffusion cells. Accordingly, the diffusion cell100can be used to test a substance via a regular, useful aerosol delivery in a biologically relevant range for topical skin applications.

A number of embodiments and implementations have been described above. However, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure to accommodate various substance characteristics or other requirements (e.g., a low substance availability, a high substance or membrane cost, a micro-dialysis-based receiver chamber, a low substance detectability, etc.).

For example, while the diffusion cell100and the splash guard150have been described and illustrated as including certain dimensions, shapes, and material formulations, in some embodiments, diffusion cells and splash guards that are similar in one or both of construction and function to the diffusion cell100and the splash guard150may include one or more components that have different dimension, shapes, and/or material formulations.

While the diffusion cell100has been described and illustrated as including the three sample ports108,110,112, in some embodiments, a diffusion cell that is similar in construction and function to the diffusion cell100may include a different number or a different type of sample ports.

While the diffusion cell100has been described and illustrated as including the exterior chamber118as a water jacket, in some embodiments, a diffusion cell that is similar in function to the diffusion cell100may not include an exterior chamber.

While the frontal plate126of the adjustable plate has been described and illustrated as a flat plate with a flat central opening134, in some embodiments, a diffusion cell includes an adjustable clamp that has a curved frontal plate that may have a curved opening for specialized biological or synthetic membranes, such as a cornea or a contact lens. Such diffusion cells may also include a body that has a corresponding curved central opening.

In some embodiments, a diffusion cell that is similar in construction and function to the diffusion cell100can be used to perform diffusion tests on airborne substances, such as the methods described above with respect to the diffusion cell100. For example,FIG. 14is a side schematic view of a diffusion cell300that includes a main body302, an adjustable clamp304including a cover plate326, an interior chamber306, an exterior chamber318, an inlet port320, an outlet port322, a sample port308, and silicon gaskets328.FIG. 15provides a graph that illustrates accumulation of a substance (e.g., Blue #1 dye in water) through a 2 kDa molecular weight cut-off cellulose ester dialysis membrane. During the test, 3.1±0.91 μL/cm2(n=10) was delivered to the membrane.

FIG. 16illustrates an alternative splash guard450that is similar in construction and function to the splash guard150and that can be assembled with the diffusion cell100or with another diffusion cell to block contamination of the sample area of the body of the diffusion cell. The splash guard450is similar in construction and function to the splash guard150, except that the splash guard450has a narrower, shorter frontal plate452that does not include lateral openings for engaging fastening assemblies of a diffusion cell.

While the methods of using the diffusion cell100have been described and illustrated with the diffusion cell100arranged in a horizontal orientation, in some implementations, the diffusion cell100is arranged in a vertical orientation to perform a diffusion test.

While the methods of using the diffusion cell100have been described with respect to certain volumes, time points, and ordered sequences of events, in some implementations, the diffusion cell100is used to perform diffusion tests including different volumes, times points, and sequences of events.

Additionally, other embodiments and implementations are within the scope of the following claims.