Patent Application
20230270599
Incorporated herein by reference in its entirety is the Sequence Listing being concurrently submitted as a XML file named SeqList, created Apr. 7, 2023, and having a size of 3,748 bytes.
This application relates to the fields of nanofiber structures. More specifically, this invention provides nanofiber swabs, methods of synthesizing, and methods of use thereof.
Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
In late 2019, the emergence of the COVID-19 pandemic revealed the importance of swabs in diagnostic testing. The overwhelming shortage of flocked nasopharyngeal swabs led to innovation and repurposing of several new swab types (Vermeiren, et al., J. Clin. Microbiol. (2020) 58: e00669-20; Péré, et al., J. Clin. Microbiol. (2020) 58: e00721-20; Freire-Paspuel, et al., Front. Cell. Infect. Microbiol. (2020) 10:365; Wyllie, et al., N. Engl. J. Med. (2020) 383:1283-1286). Notably, advances in 3D printed swabs of different geometries and repurposing of cotton swabs addressed some of the more immediate shortage needs (Freire-Paspuel, et al., Front. Cell. Infect. Microbiol. (2020) 10:365; Decker, et al., Clin. Infect. Dis. (2021) 73(9): e3027-e3032; Arjunan, et al., J. Mech. Behav. Biomed. Mater. (2021) 114:104175; Ford, et al., 3D Print. Med. (2020) 6:21; Chen, et al., Matter (2020) 3:1589-1600; Callahan, et al., J. Clin. Microbiol. (2020) 58: e00876-20; Williams, et al., Med. J. Aust. (2020) 213:276-279). However, an alarming rate of false negative SARS-CoV-2 tests highlighted the need to not only create more swabs but improve their sample collection yield and recovery to improve testing sensitivity (Pan, et al., Clin. Chem. (2020) 66:794-801; Xiao, et al., J. Med. Virol. (2020) 92(10):1755-1756; West, et al., Mayo Clin. Proc. (2020) 95:1127-1129). Swab performance is largely determined by absorption and release of samples with flocked swabs consistently outperforming cotton and 3D printed swabs in both metrics (Zasada, et al., AMB Express (2020) 10:46; Brownlow, et al., J. Forensic Sci. (2012) 57:713-717; Viviano, et al., Int. J. Women's Health (2018) 10:229-236; Wigger, et al., J. Microbiol. Methods (2019) 157:47-49). Therefore, designing a highly absorptive swab capable of releasing a large percent of the absorbed specimen is a necessity for improved SARS-CoV-2 detection and COVID-19 test sensitivity. Similarly, swabs with higher absorption and release can have direct implications in bacteria, cell, and protein identification and detection, biological sample collection and harvesting, and DNA analysis for forensic applications (Brownlow, et al., J. Forensic Sci. (2012) 57:713-717; Wigger, et al., J. Microbiol. Methods (2019) 157:47-49; Rock, et al., Infect. Control Hosp. Epidemiol. (2018) 39:1257-1261; Probst, et al., Appl. Environ. Microbiol. (2010) 76:5148-5158; Bruijns, et al., J. Forensic Sci. (2018) 63:1492-1499; Webb, et al., J. Mater. Chem. C (2014) 2:10446-10454; Schulz, et al., J. Forensic Sci. (2010) 55:492-498).
In accordance with the instant invention, nanofiber swabs are provided along with methods of producing the nanofiber swabs and methods of use. In certain embodiments, the method for producing a nanofiber swab comprises a) fixing (e.g., thermally and/or chemically) at least one point of a nanofiber mat (e.g., a side of a nanofiber mat), b) expanding the nanofiber mat by exposing the nanofiber mat to gas bubbles (optionally by more than one exposure), thereby producing an expanded nanofiber structure, and c) attaching the expanded nanofiber structure to an end of a handle, thereby producing the nanofiber swab. In certain embodiments, the nanofiber mat comprises electrospun nanofibers. In certain embodiments, the expanded nanofiber structure has a rounded geometry (e.g., cylindrical or capsular). In certain embodiments, the expanded nanofiber structure has a rectangular geometry (e.g., rectangular or cubic). In certain embodiments, the gas bubbles are generated as a product of a chemical reaction (e.g., the hydrolysis of sodium borohydride). In certain embodiments, the gas bubbles are generated by a physical releasing (e.g., depressurized CO2). In certain embodiments, the nanofiber mat is expanded by exposing the nanofiber mat to a subcritical fluid (e.g., subcritical CO2) and depressurizing (e.g., within a container). In certain embodiments, the nanofiber mat comprises a plurality of aligned nanofibers, random nanofibers, and/or entangled nanofibers. In certain embodiments, the method further comprises synthesizing the nanofiber mat by electrospinning prior to step a). In certain embodiments, the method further comprises cutting and/or trimming the nanofiber mat (e.g., prior to step b) into a desired shape (e.g., rectangle). In certain embodiments, the nanofiber mat is frozen (e.g., with liquid nitrogen) prior to cutting and/or trimming. In certain embodiments, the nanofiber mat comprises polycaprolactone (PCL) and, optionally, a poloxamer (e.g., poloxamer 407 or poloxamer 188). In certain embodiments, the method further comprises coating the expanded nanofiber structure (e.g., before and/or after attachment of the handle) with a substance such as a hydrogel (e.g., gelatin, gelatin methacryloyl (GelMA), and/or chitosan). In certain embodiments, the method further comprises crosslinking the expanded nanofiber structure and/or coating substance (e.g., hydrogel). In certain embodiments, the crosslinking is achieved by contacting and/or exposure to a crosslinker (e.g., glutaraldehyde) or a thermal treatment. In certain embodiments, the handle is attached to the expanded nanofiber structure by an adhesive (e.g., an epoxy).
In accordance with the instant invention, nanofiber swab comprising an expanded nanofiber structure and a handle are provided. In certain embodiments, the expanded nanofiber structure is attached to the terminus of the handle. In certain embodiments, the nanofiber swab is produced by a method of the instant invention.
In accordance with the instant invention, methods of collecting a sample from a surface or subject are provided. In certain embodiments, the method comprises contacting a nanofiber swab with a site in or on the subject. In certain embodiments, the sample is collected from the site via the expanded nanofiber structure. In certain embodiments, the sample is a biological sample. In certain embodiments, the sample comprises a bacteria, a virus, and/or a cell. In certain embodiments, the sample comprises a nucleic acid and/or a polypeptide.
In accordance with the instant invention, the expanded nanofiber structures can be used as wipes (e.g., to clean a surface). In certain embodiments, the expanded nanofiber structure lacks a handle. In certain embodiments, the expanded nanofiber structure is attached to a handle. In certain embodiments, the expanded nanofiber structure is used to remove dust, virus, bacteria, etc. from a surface.
Following the COVID-19 outbreak, swabs for biological specimen collection were thrust to the forefront of healthcare materials. Swab sample collection and recovery are vital for reducing false negative diagnostic tests, early detection of pathogens, and harvesting DNA from limited biological samples. Herein, a new class of nanofiber swabs are provided which are tipped with hierarchical 3D nanofiber objects produced by expanding electrospun membranes with a solids-of-revolution-inspired gas foaming technique. Nanofiber swabs significantly improve absorption and release of proteins, cells, bacteria, DNA, and viruses from solutions and surfaces. Implementation of nanofiber swabs in SARS-CoV-2 detection reduces the false negative rates at two viral concentrations and identifies SARS-CoV-2 at a 10× lower viral concentration compared to flocked and cotton swabs. The nanofiber swabs will improve test sensitivity, leading to timely and accurate diagnosis of many diseases.
In accordance with the instant invention, nanofiber swabs, methods of synthesizing nanofiber swabs, and method of using nanofiber swabs are provided. As described herein, the nanofiber swabs of the instant invention have improved collection efficiency over other swabs including traditional cotton and flocked swabs. In certain embodiments, the nanofiber swab is a specimen collection swab. The swabs of the instant invention can be used to collect specimen from anywhere, including any part of a subject. In certain embodiments, the nanofiber swab is a nasal swab. In certain embodiments, the nanofiber swab is a nasopharyngeal swab. In certain embodiments, the nanofiber swab is an oropharyngeal swab.
These nanofiber swabs of the instant invention may be used to collect biological and/or non-biological specimens including but not limited to: fluids (e.g. blood, saliva, urine, serum, plasma, etc.), exudates, bacteria, viruses, cells and/or subcellular materials (e.g., nucleic acid molecules, DNA, RNA, proteins, peptides, polypeptides, etc.). The nanofiber swabs of the instant invention may be used in medicine. The nanofiber swabs of the instant invention may be used, without limitation, in clinical, veterinarian, forensic, agriculture, and/or any non-clinical settings. The nanofiber swabs may be used to collect a specimen from a non-biological entity such as a surface at a crime scene.
Generally, the nanofiber swabs of the instant invention comprise an expanded, nanofiber structure comprising a plurality of nanofibers. The expanded, nanofiber structure of the instant invention (e.g., the head or tip of the nanofiber swab) may be attached to a handle. The expanded, nanofiber structure will typically be at the end or tip of the swab handle. In certain embodiments, the expanded, nanofiber structure covers or encompasses the end or tip of the swab handle. In certain embodiments, the nanofiber swab comprises an expanded, nanofiber structure at each end of the handle (i.e., the nanofiber swab comprises two expanded, nanofiber structures). When the nanofiber swab comprises two expanded, nanofiber structures, the first and second expanded, nanofiber structures can be the same or different (e.g., different in size, and/or different polymer, etc.).
The swab handle may be made from a variety of materials. For example, the swab handle may comprise, without limitation: wood, plastics and other polymers, paper (e.g., rolled paper) and paper related materials, and/or metal. In certain embodiments, the swab handle comprises plastic. Generally, the swab handle is rounded or has a rounded geometry (e.g., a cylinder, cylindrical, tubular, etc.).
The nanofiber material may be assembled directly onto the swab handle or the nanofiber material may be attached to the swab handle (e.g., via an adhesive agent). For example, the swab handle may be inserted into the expanded, nanofiber structure (e.g., in to the middle (e.g., into a cavity in the middle created by the expansion process)). The swab handle may be attached or fixed to the expanded, nanofiber structure. The swab handle may be attached or fixed by any means. In certain embodiments, the expanded, nanofiber structure is mechanically attached or fixed to the end of the swab handle. In certain embodiments, an adhesive (e.g., epoxy, glue, cement, paste, binder, etc.) is used to attach the expanded, nanofiber structure to the swab handle. In certain embodiments, the expanded, nanofiber structure is attached to the swab handle by an epoxy.
The swab handle can be any length. In certain embodiments, the swab handle is less than about 25 cm in length, less than about 22 cm in length, less than about 20 cm in length, less than about 18 cm in length, less than about 16 cm in length, less than about 14 cm in length, less than about 12 cm in length, less than about 10 cm in length, or less than about 8 cm in length. In certain embodiments, the swab handle is more than about 4 cm in length, more than about 6 cm in length, more than about 8 cm in length, more than about 10 cm in length, more than about 12 cm in length, more than about 14 cm in length, more than about 16 cm in length, more than about 18 cm in length, more than about 20 cm in length, more than about 22 cm in length, or more than about 24 cm in length. In certain embodiments, the swab handle is about 6 cm to about 25 cm in length, about 6 cm to about 22 cm in length, about 8 cm to about 20 cm in length, about 10 cm to about 20 cm in length, about 12 cm to about 20 cm in length, about 12 cm to about 18 cm in length, or about 14 cm to about 16 cm in length. In certain embodiments, the swab handle is about 1 mm to about 10 mm, about 1 mm to about 5 mm, about 2 mm to about 5 mm, or about 2 mm to about 4 mm in thickness (e.g., average diameter).
The nanofiber swabs of the present invention can be formed and manufactured into any shape, size, and/or thickness. For example, the nanofiber swab may have a three dimensional shape such as, without limitation: a capsule, cylinder, tube, cone, sphere, dome, rectangle, or cube. In certain embodiments, the nanofiber swab has a rounded geometry. For example, the nanofiber swabs may be spherical, cylindrical, or capsular in shape. Generally, a capsular shape approximates the shape of a capsule (e.g., a geometric shape consisting of a cylinder with hemispherical ends). In certain embodiments, the expanded nanofiber structure has a rectangular geometry (e.g., rectangular or cubic).
The nanofibers of the instant invention can be fabricated by any method. For example, the nanofiber material may be manufactured using a variety of methods including but not limited to electrospinning, phase separation, centrifugal force spinning, hypersonic spinning, and aerogels. In certain embodiments, the nanofiber mat or structure is synthesized by electrospinning. In certain embodiments, the expanded, nanofiber structures comprise electrospun nanofibers. The expanded nanofiber structure may comprise aligned fibers (e.g., uniaxially aligned), random fibers, and/or entangled fibers. In certain embodiments, the expanded nanofiber structure comprises aligned fibers (e.g., uniaxially, radially, vertically, or horizontally). In certain embodiments, the expanded nanofiber structure comprises radially aligned nanofibers, vertically aligned nanofiber, or a combination thereof. While the application generally describes nanofibers (fibers having a diameter less than about 1 m (e.g., average diameter)) structures and the synthesis of three-dimensional nanofibrous structures, the instant invention also encompasses microfibers (fibers having a diameter greater than about 1 m (e.g., average diameter)) structures and the synthesis of three-dimensional microfibrous structures.
In certain embodiments of the instant invention, the methods comprise fixing at least one point, edge, end, or side—or a portion thereof—of a nanofiber mat (sometimes referred to as 2D structure herein) and then expanding the nanofiber mat into an expanded nanofiber structure (sometimes referred to as a 3D scaffold herein). In certain embodiments, a whole or entire side of the nanofiber mat is fixed. In certain embodiments, one or more sections or portions of the nanofiber mat is fixed (e.g., the top and bottom corners on one side may be fixed). The nanofiber mat may be fixed by any means. For example, the nanofiber mat may be thermally fixed or chemically fixed. In certain embodiments, the nanofiber mat is thermally fixed.
In certain embodiments, the nanofiber mat is fixed by exposing at least one point, edge, end, or side—or a portion thereof—of the nanofiber mat to elevated temperatures. In certain embodiments, the nanofiber mat is exposed to temperatures at or above the melting temperature of the nanofibers. In certain embodiments, the nanofiber mat is fixed by exposing at least one point, edge, end, or side—or a portion thereof—of the nanofiber mat to a temperature of at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., or higher. To avoid excess fixation and/or damage to the remainder of the nanofiber mat, the exposure to elevated temperatures may be brief (e.g., less than 10 seconds, less than 5 seconds, or for about 1 second). In certain embodiments, the heat is applied perpendicularly to the nanofiber mat. In certain embodiments, the thermal fixing comprises exposing at least one point, edge, end, or side—or a portion thereof—of a nanofiber mat to about 75° C. to about 95° C., particularly about 85° C. (e.g., for less than 5 seconds, particularly about 1 second).
In certain embodiments, the nanofiber mat is chemically fixed, for example, by exposure to a chemical, solvent, or crosslinker. In certain embodiments, a chemical or solvent based method is used to fix the nanofiber mat. The chemical or solvent can be, without limitation: dichloromethane (DCM), dimethylformamide (DMF), dichloroformamide, acetone, and other organic solvents. In certain embodiments, the nanofiber mat is fixed by exposure to a crosslinker. In certain embodiments, the nanofiber mat is chemically fixed by exposing at least one point, edge, end, or side—or a portion thereof—of the nanofiber mat to a chemical, solvent, or crosslinker with minimal or no exposure the remainder of the nanofiber mat to the chemical, solvent, or crosslinker.
The methods of the instant invention may further comprise synthesizing the nanofibrous structure (e.g., mat) prior to expansion (e.g., exposure to gas bubbles). In certain embodiments, the nanofiber mat is synthesized using electrospinning. In certain embodiments, the nanofiber mat comprises aligned fibers (e.g., uniaxially), random fibers, and/or entangled fibers.
The nanofiber mat may be cut, trimmed, or shaped prior to expansion. The nanofiber mat may be cut, trimmed, or shaped prior to fixation or cut, trimmed, or shaped after fixation. In certain embodiments, the nanofiber mat is cut, trimmed, or shaped under cryogenic or frozen conditions (e.g., in liquid nitrogen). The nanofiber mat can be cut, trimmed, or shaped into any desired shape such as, without limitation: rectangles, squares, triangles, quadrangles, pentagons, hexagons, circles, ovals, semicircles, L's, C's, O's, U's, and arches. While the application generally describes nanofiber mats as the 2D structure prior to expansion, the instant invention also encompasses any nanofibrous structure which can be expanded by the methods provided herein (e.g., structures other than a mat or 3D structures which can be further expanded).
In certain embodiments, the nanofiber mat is synthesized (e.g., electrospun) as a rectangle or square. In certain embodiments, the nanofiber mat is cut into a rectangle or square. In certain embodiments, the nanofiber mat is rectangular. In certain embodiments, the width (e.g., average width) of the nanofiber mat is less than 10 mm, less than 9 mm, less than 8 mm, less than 7 mm, less than 6 mm, less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, or less than 1 mm. In certain embodiments, the width (e.g., average width) of the nanofiber mat is more than 1 mm, more than 2 mm, more than 3 mm, more than 4 mm, more than 5 mm, more than 6 mm, more than 7 mm, more than 8 mm, or more than 9 mm. In certain embodiments, the width (e.g., average width) of the nanofiber mat is about 0.5 mm to about 10 mm, about 0.5 mm to about 8 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 5 mm, about 1 mm to about 5 mm, about 1 mm to about 4 mm, about 2 mm to about 4 mm, or about 2 mm to about 3 mm. In certain embodiments, the length (e.g., average length) of the nanofiber mat is more than about 3 mm, more than about 5 mm, more than about 6 mm, more than about 7 mm, more than about 8 mm, more than about 9 mm, more than about 10 mm, more than about 11 mm, more than about 12 mm, more than about 13 mm, more than about 14 mm, more than about 15 mm, more than about 16 mm, more than about 17 mm, more than about 18 mm, more than about 19 mm, more than about 20 mm, more than about 21 mm, more than about 22 mm, more than about 23 mm, more than about 24 mm, more than about 25 mm, more than about 26 mm, more than about 27 mm, more than about 28 mm, more than about 29 mm, more than about 30 mm, or more than about 35 mm. In certain embodiments, the length (e.g., average length) of the nanofiber mat is less than about 40 mm, less than about 35 mm, less than about 30 mm, less than about 29 mm, less than about 28 mm, less than about 27 mm, less than about 26 mm, less than about 25 mm, less than about 24 mm, less than about 23 mm, less than about 22 mm, less than about 21 mm, less than about 20 mm, less than about 19 mm, less than about 18 mm, less than about 17 mm, less than about 16 mm, less than about 15 mm, less than about 14 mm, less than about 13 mm, less than about 12 mm, less than about 11 mm, less than about 10 mm, less than about 9 mm, less than about 8 mm, less than about 7 mm, less than about 6 mm, less than about 5 mm, or less than about 3 mm. In certain embodiments, the length (e.g., average length) of the nanofiber mat is about 3 mm to about 40 mm, about 5 mm to about 30 mm, about 5 mm to about 25 mm, about 10 mm to about 30 mm, about 10 mm to about 20 mm, about 12 mm to about 20 mm, about 12 mm to about 18 mm, or about 12 mm to about 15 mm.
In certain embodiments, the area (e.g., surface area) of the nanofiber mat is about 10 mm2 to about 200 mm2, about 10 mm2 to about 100 mm2, about 10 mm2 to about 75 mm2, about 20 mm2 to about 75 mm2, about 20 mm2 to about 50 mm2, about 25 mm2 to about 55 mm2, or about 25 mm2 to about 45 mm2. In certain embodiments, the area (e.g., surface area) of the nanofiber mat is less than about 200 mm2, less than about 150 mm2, less than about 125 mm2, less than about 100 mm2, less than about 90 mm2, less than about 80 mm2, less than about 70 mm2, less than about 60 mm2, less than about 50 mm2, less than about 40 mm2, or less than about 30 mm2. In certain embodiments, the area (e.g., surface area) of the nanofiber mat is more than about 1 mm2, more than about 5 mm2, more than about 10 mm2, more than about 15 mm2, more than about 20 mm2, more than about 25 mm2, or more than about 30 mm2.
In certain embodiments, the thickness (e.g., average thickness) of the nanofiber mat is about 0.01 mm to about 10 mm, about 0.05 to about 10 mm, about 0.1 to about 10 mm, about 0.1 to about 5 mm, about 0.1 to about 3 mm, about 0.5 to about 3 mm, about 0.5 to about 2 mm, about 0.75 to about 1.5 mm, or about 0.75 to about 1.25 mm. In certain embodiments, the thickness (e.g., average thickness) of the nanofiber mat is more than about 0.01 mm, more than about 0.025 mm, more than about 0.05 mm, more than about 0.75 mm, more than about 0.1 mm, more than about 0.2 mm, more than about 0.3 mm, more than about 0.4 mm, more than about 0.5 mm, more than about 0.6 mm, more than about 0.7 mm, more than about 0.8 mm, more than about 0.9 mm, more than about 1 mm, more than about 2 mm, more than about 3 mm, more than about 4 mm, or more than about 5 mm. In certain embodiments, the thickness (e.g., average thickness) of the nanofiber mat is less than about 20 mm, less than about 17.5 mm, less than about 15 mm, less than about 12.5 mm, less than about 10 mm, less than about 9 mm, less than about 8 mm, less than about 7 mm, less than about 6 mm, less than about 5 mm, less than about 4 mm, less than about 3 mm, less than about 2 mm, or less than about 1 mm.
In certain embodiments, the nanofiber mat is expanded into an expanded nanofiber structure by exposing the nanofiber mat to gas bubbles. In certain embodiments, the nanofiber mat is expanded radially and/or around a fixed axis (e.g., as defined by the fixed portion of the nanofiber mat). The bubbles can be generated by chemical reactions or physical manipulations. For example, the nanofiber mat can be submerged or immersed in a bubble/gas producing chemical reaction or physical manipulation. Generally, the longer the exposure to the bubbles, the greater the thickness and porosity of the expanded nanofiber structure increases. The nanofiber mat may also be expanded within a mold (e.g., a metal, plastic, or other material that does not expand in the presence of gas bubbles) to assist in the formation of a desired shape. The nanofiber mat may be treated with air plasma prior to exposure to gas bubbles (e.g., to increase hydrophilicity). The nanofiber mat may be exposed to gas bubbles more than once (e.g., repeatedly).
After exposure to the bubbles, the expanded nanofiber structure may be washed and/or rinsed in water and/or a desired carrier or buffer (e.g., a pharmaceutically or biologically acceptable carrier). Trapped gas bubbles may be removed by applying a vacuum to the expanded nanofiber structure. For example, the expanded nanofiber structure may be submerged or immersed in a liquid (e.g., water and/or a desired carrier or buffer) and a vacuum may be applied to rapidly remove the gas bubbles. The process may be repeated one or more times. After expansion (e.g., after rinsing and removal of trapped gas), the expanded nanofiber structure may be placed in storage in cold solution or lyophilized and/or freeze-dried.
The gas bubbles of the instant invention can be made by any method known in the art. The bubbles may be generated, for example, by chemical reactions or by physical approaches. Electrospun nanofiber mats can be expanded three dimensionally using a gas foaming based expansion method (example methods may be found in WO 2016/053988; WO 2019/060393; and WO 2020/124072, each incorporated herein by reference). Electrospun nanofiber mats can be expanded in the third dimension with ordered structures using gas bubbles generated by chemical reactions in an aqueous solution (see, e.g., WO 2016/053988; WO 2019/060393; Jiang et al. (2018) Acta Biomater., 68:237-248; Jiang, et al. (2015) ACS Biomater. Sci. Eng., 1:991-1001; Jiang, et al. (2016) Adv. Healthcare Mater., 5:2993-3003; Joshi, et al. (2015) Chem. Eng. J., 275:79-88; each of the foregoing incorporated by reference herein). In certain embodiments, the chemical reaction or physical manipulation does not damage or alter or does not substantially damage or alter the nanofibers (e.g., the nanofibers are inert within the chemical reaction and not chemically modified). As explained hereinabove, the nanofiber mat may be submerged or immersed in a liquid comprising the reagents of the bubble-generating chemical reaction. Examples of chemical reactions that generate bubbles include, without limitation:
NaBH4+2H2O═NaBO2+4H2
NaBH4+4H2O=4H2(g)+H3BO3+NaOH
HCO3−+H+=CO2+H2O
NH4++NO2=N2+2H2O
H2CO3=H2O+CO2
2H++S2−═H2S
2H2O2=O2+2H2O
3HNO2=2NO+HNO3+H2O
HO2CCH2COCH2CO2H=2CO2+CH3COCH3
2H2O2=2H2+O2
CaC2+H2O═C2H2
Zn+2HCl=H2+ZnCl2
2KMnO4+16HCl=2KCl+2MnCl2+H2O+5Cl2
In certain embodiments, the chemical reaction is the hydrolysis of NaBH4 (e.g., NaBH4+2H2O═NaBO2+4H2). In certain embodiments, CO2 gas bubbles (generated chemically or physically) are used (e.g., for hydrophilic polymers).
Examples of physical approaches for generating bubbles of the instant invention include, without limitation: 1) create high pressure (fill gas)/heat in a sealed chamber and suddenly reduce pressure; 2) dissolve gas in liquid/water in high pressure and reduce pressure to release gas bubbles; 3) use supercritical fluids (reduce pressure) like supercritical CO2; 4) use subcritical gas liquid (then reduce pressure) (e.g., liquid CO2, liquid propane and isobutane); 5) fluid flow; 6) apply acoustic energy or ultrasound to liquid/water; 7) apply a laser (e.g., to a liquid or water); 8) boiling; 9) reduce pressure boiling (e.g., with ethanol); and 10) apply radiation (e.g., ionizing radiation on liquid or water). The nanofiber mat may be submerged or immersed in a liquid of the bubble-generating physical manipulation.
In certain embodiments, the nanofiber mats are expanded using a subcritical or supercritical fluid or liquid (e.g., CO2, N2, N2O, hydrocarbons, and fluorocarbons). In certain embodiments, the nanofiber mats are expanded by exposure to depressurized CO2. In certain embodiments, liquid CO2 is utilized. For example, nanofiber mats may be expanded by exposing to, contacting with or being placed into (e.g., submerged or immersed) a subcritical liquid/fluid (e.g., subcritical CO2) and then depressurized. The cycle of placing the nanofibrous structures into subcritical CO2 and depressurizing may be performed one or more times. Generally, the more times the expansion method is used the thickness and porosity of the nanofibrous (or microfibrous) structure increases. For examples, the cycle of exposure to subcritical CO2 and then depressurization may be performed one, two, three, four, five, six, seven, eight, nine, ten, or more times, particularly 1-10 times, 1-5 times, or 1-3 times. In certain embodiments, the cycle of exposure to subcritical CO2 and then depressurization is performed at least 2 times (e.g., 2-10 times, 2-5 times, 2-4 times, or 2-3 times). In certain embodiments, the method comprises placing the nanofibrous mat and dry ice (solid CO2) in a sealed container, allowing the dry ice to turn into liquid CO2, and then unsealing the container to allow depressurization.
The nanofiber mat and subcritical fluid (e.g., subcritical CO2; or solid form of subcritical fluid (e.g., dry ice)) may be contained in any suitable container (e.g., one which can withstand high pressures). For example, the subcritical fluids and the nanofiber mat may be contained within, but not limited to: chambers, vessels, reactors, and tubes. In certain embodiments, the equipment or container used during the methods of the present invention will have a feature or component that allows control of the depressurization rate of the subcritical fluid. Depressurization of the subcritical fluid can be done using a variety of methods including but not limited to manually opening the container to decrease pressure or by using some type of equipment that can regulate the rate of depressurization of the reaction vessel.
The nanofibers of the instant invention may comprise any polymer. In certain embodiments, the polymer is biocompatible. The polymer may be biodegradable or non-biodegradable. In certain embodiments, the polymer is a biodegradable polymer. The polymer may by hydrophobic, hydrophilic, or amphiphilic. In certain embodiments, the polymer is hydrophobic. In certain embodiments, the polymer is hydrophilic. The polymer may be, for example, a homopolymer, random copolymer, blended polymer, copolymer, or a block copolymer. Block copolymers are most simply defined as conjugates of at least two different polymer segments or blocks. The polymer may be, for example, linear, star-like, graft, branched, dendrimer based, or hyper-branched (e.g., at least two points of branching). The polymer of the invention may have from about 2 to about 10,000, about 2 to about 1000, about 2 to about 500, about 2 to about 250, or about 2 to about 100 repeating units or monomers. The polymers of the instant invention may comprise capping termini.
Examples of hydrophobic polymers include, without limitation: poly(hydroxyethyl methacrylate), poly(N-isopropyl acrylamide), poly(lactic acid) (PLA (or PDLA)), poly(lactide-co-glycolide) (PLG), poly(lactic-co-glycolic acid) (PLGA), polyglycolide or polyglycolic acid (PGA), polycaprolactone (PCL), poly(aspartic acid), polyoxazolines (e.g., butyl, propyl, pentyl, nonyl, or phenyl poly(2-oxazolines)), polyoxypropylene, poly(glutamic acid), poly(propylene fumarate) (PPF), poly(trimethylene carbonate), polycyanoacrylate, polyurethane, polyorthoesters (POE), polyanhydride, polyester, poly(propylene oxide), poly(caprolactonefumarate), poly(1,2-butylene oxide), poly(n-butylene oxide), poly(ethyleneimine), poly(tetrahydrofurane), ethyl cellulose, polydipyrolle/dicabazole, starch, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polydioxanone (PDO), polyether poly(urethane urea) (PEUU), cellulose acetate, polypropylene (PP), polyethylene terephthalate (PET), nylon (e.g., nylon 6), polycaprolactam, PLA/PCL, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), PCL/calcium carbonate, and/or poly(styrene).
Examples of hydrophilic polymers include, without limitation: polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poly(ethylene glycol) and poly(ethylene oxide) (PEO), chitosan, collagen, chondroitin sulfate, sodium alginate, gelatin, elastin, hyaluronic acid, silk fibroin, sodium alginate/PEO, silk/PEO, silk fibroin/chitosan, hyaluronic acid/gelatin, collagen/chitosan, chondroitin sulfate/collagen, and chitosan/PEO.
Amphiphilic copolymers or polymer composites may comprise a hydrophilic polymer (e.g., segment) and a hydrophobic polymer (e.g., segment) from those listed above (e.g., gelatin/polyvinyl alcohol (PVA), PCL/collagen, chitosan/PVA, gelatin/elastin/PLGA, PDO/elastin, PHBV/collagen, PLA/hyaluronic acid, PLGA/hyaluronic acid, PCL/hyaluronic acid, PCL/collagen/hyaluronic acid, gelatin/siloxane, PLLA/MWNTs/hyaluronic acid).
Examples of polymers particularly useful for electrospinning are provided in Xie et al. (Macromol. Rapid Commun. (2008) 29:1775-1792; incorporated by reference herein; see e.g., Table 1). Examples of compounds or polymers for use in the fibers of the instant invention, particularly for electrospun nanofibers include, without limitation: natural polymers (e.g., chitosan, gelatin, collagen type I, II, and/or III, elastin, hyaluronic acid, cellulose, silk fibroin, phospholipids (Lecithin), fibrinogen, hemoglobin, fibrous calf thymus Na-DNA, virus M13 viruses), synthetic polymers (e.g., PLGA, PLA, PCL, PHBV, PDO, PGA, PLCL, PLLA-DLA, PEUU, cellulose acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP), blended (e.g., PLA/PCL, gelatin/PVA, PCL/gelatin, PCL/collagen, sodium alginate/PEO, chitosan/PEO, Chitosan/PVA, gelatin/elastin/PLGA, silk/PEO, silk fibroin/chitosan, PDO/elastin, PHBV/collagen, hyaluronic acid/gelatin, collagen/chondroitin sulfate, collagen/chitosan), and composites (e.g., PDLA/HA, PCL/CaCO3, PCL/HA, PLLA/HA, gelatin/HA, PCL/collagen/HA, collagen/HA, gelatin/siloxane, PLLA/MWNTs/HA, PLGA/HA). In certain embodiments, the nanofiber comprises polymethacrylate, poly vinyl phenol, polyvinylchloride, cellulose, polyvinyl alcohol, polyacrylamide, PLGA, collagen, polycaprolactone, polyurethanes, polyvinyl fluoride, polyamide, silk, nylon, polybennzimidazole, polycarbonate, polyacrylonitrile, polyvinyl alcohol, polylactic acid, polyethylene-co-vinyl acetate, polyethylene oxide, polyaniline, polystyrene, polyvinylcarbazole, polyethylene terephthalate, polyacrylic acid-polypyrene methanol, poly(2-hydroxyethyl methacrylate), polyether imide, polyethylene glycol, poly(ethylene-co-vinyl alcohol), polyacrylnitrile, polyvinyl pyrrolidone, polymetha-phenylene isophthalamide, gelatin, chitosan, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starch-acrylonitrile co-polymers, and/or combinations of two or more polymers. In certain embodiments, the polymer comprises polycaprolactone (PCL). In certain embodiments, the polymer comprises polycaprolactone (PCL) and gelatin (e.g., at a 1:1 ratio).
In certain embodiments, the nanofiber, nanofiber mat, and/or expanded nanofiber structure may further comprise at least one amphiphilic block copolymer comprising hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO). In certain embodiments, the nanofiber mat and/or expanded nanofiber structure comprises a poloxamer or an amphiphilic triblock copolymer comprising a central hydrophobic PPO block flanked by two hydrophilic PEO blocks (i.e., an A-B-A triblock structure). In certain embodiments, the amphiphilic block copolymer is selected from the group consisting of Pluronic® L31, L35, F38, L42, L44, L61, L62, L63, L64, P65, F68, L72, P75, F77, L81, P84, P85, F87, F88, L92, F98, L101, P103, P104, P105, F108, L121, L122, L123, F127, 10R5, 10R8, 12R3, 17R1, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25R5, 25R8, 31R1, 31R2, and 31R4. In certain embodiments, the nanofiber, nanofiber mat, and/or expanded nanofiber structure comprises poloxamer 188. In certain embodiments, the nanofiber, nanofiber mat, and/or expanded nanofiber structure comprises poloxamer 407 (Pluronic® F127). The amphiphilic block copolymer (e.g., poloxamer) may be added in various amounts to the polymer solution during the synthesis process (e.g., electrospinning). In certain embodiments, about 0% to about 20%, about 0% to about 15%, about 0% to about 10%, about 0.1% to about 5%, about 0.5% to about 2%, or about 0.1% to about 1.0% (e.g., w/v) of the polymer solution is an amphiphilic block copolymer (e.g., a poloxamer (e.g., poloxamer 407 and/or poloxamer 188)). In certain embodiments, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20% (e.g., w/v) of the polymer solution is polymer (e.g., PCL).
In certain embodiments, the polymer solution comprises about 10% polymer (w/v) (e.g., PCL) and about 0.5% poloxamer 407 (w/v) (Pluronic® F127). In certain embodiments, the polymer solution comprises PCL and poloxamer 407 in a ratio (e.g., by weight) of about 200:1 to about 2:1, about 100:1 to about 4:1, about 50:1 to about 8:1, about 40:1 to about 10:1, about 30:1 to about 15:1, or about 20:1.
In certain embodiments, the nanofibers and/or nanofiber structures are coated with additional materials to enhance their properties. In certain embodiments, the expanded nanofiber structure, optionally attached to the handle, is coated with the additional material. For example, the nanofibers and/or nanofiber structure can be coated with a material to help reinforce the structure and increase fluid absorption. In certain embodiments, the nanofibers and/or nanofiber structure may be coated with proteins, collagen, fibronectin, collagen, a proteoglycan, elastin, or a glycosaminoglycans (e.g., hyaluronic acid, heparin, chondroitin sulfate, or keratan sulfate). In certain embodiments, the nanofibers and/or nanofiber structures comprise a material that enhances the nanofiber structure's ability to absorb fluids, particularly aqueous solutions (e.g., blood), and/or allow for the 3D shapes/structures of the expanded nanofiber structure to be recoverable after compression. In certain embodiments, the nanofibers comprise a polymer and the material which enhances the absorption properties.
In certain embodiments, the nanofibers and/or nanofiber structures are coated with the material which enhances the absorption properties. The term “coat” refers to a layer of a substance/material on the surface of a structure. Coatings may, but need not, also impregnate the nanofiber structure. Further, while a coating may cover 100% of the nanofibers and/or nanofiber structure, a coating may also cover less than 100% of the surface of the nanofibers and/or nanofiber structure (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more the surface may be coated). Materials which enhance the absorption properties of the expanded nanofiber structures include, without limitation: gelatin, gelatin methacryloyl (GelMA), alginate, chitosan, collagen, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starch-acrylonitrile co-polymers, other natural or synthetic hydrogels, and derivatives thereof (e.g., del Valle et al., Gels (2017) 3:27). In certain embodiments, the material is a hydrogel (e.g., a polymer matrix able to retain water, particularly large amounts of water, in a swollen state). In certain embodiments, the material is gelatin, gelatin methacryloyl (GelMA), and/or chitosan. In certain embodiments, the material is gelatin. In certain embodiments, the expanded nanofiber structure is coated with the coating material (e.g., gelatin) by submersion into a solution of the coating material (e.g., gelatin). In certain embodiments, the expanded nanofiber structures are coated with about 0.05% to about 20%, about 0.1% to about 20%, about 0.1% to about 10%, or about 0.1% to about 1% coating material (e.g., gelatin) (e.g., by weight or w/v). In certain embodiments, the coating material (e.g., hydrogel) is freeze-dried after coating. In certain embodiments, the coating material (e.g., hydrogel) is crosslinked (e.g., by glutaraldehyde) after coating (e.g., after freeze-drying).
In certain embodiments, the nanofiber structures of the instant invention are crosslinked (e.g., before or after expansion). In certain embodiments, the expanded nanofiber structure is crosslinked after coating. Crosslinking may be done using a variety of techniques including thermal crosslinking, chemical crosslinking, UV-crosslinking, and photo-crosslinking. For example, the nanofiber structures of the instant invention may be crosslinked with a crosslinker such as, without limitation: formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, a photocrosslinker, genipin, and natural phenolic compounds (Mazaki, et al., Sci. Rep. (2014) 4:4457; Bigi, et al., Biomaterials (2002) 23:4827-4832; Zhang, et al., Biomacromolecules (2010) 11:1125-1132; incorporated herein by reference). The crosslinker may be a bifunctional, trifunctional, or multifunctional crosslinking reagent. In certain embodiments, the crosslinker is glutaraldehyde. In certain embodiments (e.g., instead of crosslinking or in addition to crosslinking), the nanofiber structure is thermally treated (e.g., at a temperature close to, but below, the melting point of the nanofibers; e.g., about 50° C.).
The expanded nanofiber structures and/or nanofiber swabs of the instant invention may also be sterilized. For example, the expanded nanofiber structures can be sterilized using various methods (e.g., by treating with ethylene oxide gas, gamma irradiation, or 70% ethanol). In certain embodiments, the expanded nanofiber structure and/or nanofiber swab are sterilized by treating with ethylene oxide.
The instant application also encompasses the nanofiber swabs synthesized by the methods of the instant invention. Compositions comprising the nanofiber swabs synthesized by the methods of the instant invention and at least one pharmaceutically or biologically acceptable carrier are also encompassed by the instant invention.
In accordance with the instant invention, nanofiber swabs are provided. In certain embodiments, the nanofiber swab comprises an expanded, nanofiber structure, optionally, with a handle, wherein the expanded, nanofiber structure is attached to (e.g., covers and/or encompasses) the handle. In certain embodiments, the expanded, nanofiber structure of the nanofiber swab has an average diameter of about 3 mm to about 7 mm. In certain embodiments, the expanded, nanofiber structure of the nanofiber swab has an average length of about 10 mm to about 17 mm. In certain embodiments, the expanded, nanofiber structure is as described herein. In certain embodiments, the handle is as described herein.
In accordance with the instant invention, methods of collecting a sample (e.g., a biological sample) from or within a surface, object, or subject are provided. In certain embodiments, the method comprises contacting a nanofiber swab or expanded, nanofiber structure of the instant invention with a site in or on the surface, object, and/or subject. The site may be contacted more than one time with the nanofiber swab or expanded, nanofiber structure of the instant invention. The sample is collected from the site of the surface, object, and/or subject via the expanded nanofiber structure of the nanofiber swab. In certain embodiments, the sample comprises a cell. In certain embodiments, the sample comprises bacteria. In certain embodiments, the sample comprises a virus (e.g., a SARS-CoV-2 virus). In certain embodiments, the sample comprises a nucleic acid molecule (e.g., DNA, RNA, mRNA, etc.). In certain embodiments, the sample comprises a polypeptide, protein, or peptide. In certain embodiments, the site is within the subject's head (e.g., into a cavity (e.g., nasal cavity, nasopharynx cavity, or oropharynx). In certain embodiments, the site is contacted more than one time with the nanofiber swab. In certain embodiments, the contacting of the site comprises rotating and/or wiping the nanofiber swab on and/or around the site. In certain embodiments, the method further comprises placing the expanded nanofiber structure in a container (e.g., a tube). In certain embodiments, the container comprises a carrier. In certain embodiments, the carrier preserves the sample dislodged from the expanded nanofiber structure. In certain embodiments, the sample obtained from the surface, object, or subject is analyzed (e.g., identified).
In accordance with the instant invention, methods of cleaning a surface or object are provided. In certain embodiments, the method comprises contacting a nanofiber swab or expanded, nanofiber structure of the instant invention with a site in or on the surface or object. The site may be contacted more than one time with the nanofiber swab or expanded, nanofiber structure of the instant invention. In certain embodiments, the method removes a cell, bacteria, dust, and/or a virus from the site. In certain embodiments, the contacting of the site comprises rotating and/or wiping the nanofiber swab on and/or around the site. In certain embodiments, the method further comprises placing the expanded nanofiber structure in a container (e.g., a tube). In certain embodiments, the container comprises a carrier. In certain embodiments, the carrier preserves the sample dislodged from the expanded nanofiber structure. In certain embodiments, the sample obtained from the surface, object, or subject is analyzed (e.g., identified).
The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “electrospinning” refers to the production of fibers (i.e., electrospun fibers), particularly micro- or nano-sized fibers, from a solution or melt using interactions between fluid dynamics and charged surfaces (e.g., by streaming a solution or melt through an orifice in response to an electric field). Forms of electrospun nanofibers include, without limitation, branched nanofibers, tubes, ribbons and split nanofibers, nanofiber yarns, surface-coated nanofibers (e.g., with carbon, metals, etc.), nanofibers produced in a vacuum, and the like. The production of electrospun fibers is described, for example, in Gibson et al. (1999) AlChE J., 45:190-195.
“Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., TrisHCl, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington.
As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind.
“Hydrophobic” designates a preference for apolar environments (e.g., a hydrophobic substance or moiety is more readily dissolved in or wetted by non-polar solvents, such as hydrocarbons, than by water). In certain embodiments, hydrophobic polymers may have aqueous solubility less than about 1% wt. at 37° C. In certain embodiments, polymers that at 1% solution in bi-distilled water have a cloud point below about 37° C., particularly below about 34° C., may be considered hydrophobic.
As used herein, the term “hydrophilic” means the ability to dissolve in water. In a particular embodiment, polymers that at 1% solution in bi-distilled water have a cloud point above about 37° C., particularly above about 40° C., may be considered hydrophilic.
As used herein, the term “amphiphilic” means the ability to dissolve in both water and lipids/apolar environments. Typically, an amphiphilic compound comprises a hydrophilic portion and a hydrophobic portion.
As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.
The term “hydrogel” refers to a water-swellable, insoluble polymeric matrix (e.g., hydrophilic polymers) comprising a network of macromolecules, optionally crosslinked, that can absorb water to form a gel.
The term “crosslink” refers to a bond or chain of atoms attached between and linking two different molecules (e.g., polymer chains). The term “crosslinker” refers to a molecule capable of forming a covalent linkage between compounds. A “photocrosslinker” refers to a molecule capable of forming a covalent linkage between compounds after photoinduction (e.g., exposure to electromagnetic radiation in the visible and near-visible range). Crosslinkers are well known in the art (e.g., formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, etc.). The crosslinker may be a bifunctional, trifunctional, or multifunctional crosslinking reagent.
The following example illustrates certain embodiments of the invention. It is not intended to limit the invention in any way.
PCL (Mw=80 kDa), gelatin (porcine), Pluronic® F-127, and NaBH4 were all purchased from Sigma-Aldrich (St. Louis, Mo.). Dichloromethane (DCM) and N, N-dimethylformamide (DMF) were purchased from Oakwood Chemical (Estill, S.C.). Plastic sticks were purchased from Amscan Inc. (Elmsford, N.Y.) and Z-poxy was purchased from Pacer Technology, Inc. (Ontario, CA). Cotton swabs (6 in, Cotton-Tipped Applicators STERILE, wood shaft) were purchased from McKesson (Irvington, Tex.) and flocked swabs (6 in, PurFlock ULTRA®, Sterile Flocked Collection Devices) were purchased from Puritan Medical Products (Pittsfield, Me.). Dye (Assorted Food Color & Egg Dye) was purchased from McCormick & Company (Baltimore, Md.). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (PenStrep) were all purchased from Invitrogen (Carlsbad, Calif.). HEK293 cells were purchased from ATCC (Manassas, Va.). Blood was collected from sacrificed Sprague Dawley rats, which were purchased from Charles River Laboratories (Wilmington, Mass.). DNA quantification kits were QIAamp® DNA Investigator Kit which was purchased from Qiagen (Venlo, Netherlands). MRSA USA300 LAC was obtained in-house at the University of Nebraska Medical Center (UNMC). Columbia CAN with 5% sheep blood agar medium was purchased from Remel (Lenexa, Kans.) and tryptic soy broth (TSB) bacterial medium was purchased from Thermo Fisher Scientific Inc. (Waltham, Mass.). SARS-CoV-2 (Strain: BEI_USA-WA1/2020) was obtained from BEI Resources. Phosphate Buffered Saline (PBS) was purchased from Fisher Scientific (Hampton, N.H.). QIAamp® Viral RNA Mini Kit was purchased from Qiagen (Venlo, Netherlands). RT-PCR (2X) Master Mix and SuperScript™ III Platinum Taq Mix was purchased from Invitrogen (Carlsbad, Calif.). Primers and Probe for RT-PCR was purchased from Integrated DNA Technologies (Coralville, Iowa). QuantStudio™ 3 instrument and software was purchased from Applied Biosystems (Foster City, Calif.). A vacuum (Black & Decker Dustbuster®) for vacuumed mass loss was purchased from Black & Decker (New Britain, Conn.).
PCL nanofiber mats were generated via electrospinning (Chen, et al., Appl. Phys. Rev. (2020) 7(2):021406; Chen, et al., Nano Lett. (2019) 19(3):2059-2065; Chen, et al., Biomaterials (2018) 179:46-59; Chen, et al., Acta Biomater. (2020) 108:153-167; Chen, et al., Adv. Mater. (2020) 32(43):2003754). PCL pellets (10% w/v) and Pluronic® F-127 (0.5% w/v) were dissolved in a 4:1 (v/v) DCM:DMF solution. The resulting PCL/F-127 solution was electrospun at 16 ml/hour at 25° C., 55% RH, and under 32 kV applied voltage using an LE-100 electrospinning machine with a circular 20 emitter array equipped with 21 gauge needles (such that each needle had a flow rate of 0.8 ml/hour). The nanofibers were collected on a high-speed drum collector until an approximately 1 mm thick mat was obtained.
The nanofiber mat was removed from the drum and cut into different sized rectangles while submerged in liquid nitrogen, with the width of the rectangle serving as the radius of the swab. In this case, for each study, rectangles were cut measuring roughly 2.5 mm wide by 13.5 mm length. Mats were completely soaked in the liquid nitrogen before cutting to ensure no fusion could occur between fibers. Next, rectangles were removed from the liquid nitrogen and thermally fused along one of the long sides (applying heat perpendicular to the face of the rectangle) by melting the edge on an 85° C. hot plate. Once the rectangles cooled, they were added to a 1 M NaBH4 solution and left overnight to expand into cylinders. After expansion, the cylinders were washed with distilled water three times expanded under vacuum at 200 Pa three times. After the third vacuum, all water was removed and the cylinders were freeze-dried. Once dry, swab tips were removed and submerged in a 0.75% gelatin solution and allowed to soak for 20 minutes. Gelatin-coated swab tips were directly freeze-dried again. Once dry, swab tips were separated and crosslinked in a glutaraldehyde (GA) chamber for 24 hours. After crosslinking, plastic sticks were inserted into the middle of each cylinder and bound with epoxy. Finally, before use, each swab was sterilized with ethylene oxide (EtO).
Each swab was imaged using a camera and scanning electron microscope (SEM) (FEI, Quanta 200, Oregon). SEM images of each swab were taken in sections and combined using Adobe Photoshop's merge function. Merged edges were smoothed and the composite image was set on a black background. SEM images were taken at 200× and 1556×. The diameters of each swab fibers were measured using ImageJ. The dimensions of each swab were measured using a digital caliper (Mitutoyo, Kanagawa, Japan). Porosity of each nanofiber was estimated based on the volume difference between the whole swab and the raw materials of the swab, which is summarized in the following equation:
η=(Vst−Vbm)/Vst×100% (1)
Where η is porosity, Vst the volume occupied by the swab tip, and Vbm is the volume occupied by the bulk material comprising the swab tip. Bulk volume of each swab is assumed to be Vbm=mbm/ρbm, where mbm is the mass of the bulk swab tip material, and ρbm is the density of the bulk swab tip material. Both flocked and nanofiber swabs were assumed cylindrical, while cotton swabs were assumed egg-shaped in generating Vst. Density values were assigned based on the listed material composition of each swab tip. A summary of measurements and material values are provided in Table 1.
st= swab tip.
bm= bulk material.
In order to ascertain inhalation of nanofibers from nanofiber swabs did not pose as a risk, two different mass change studies were carried out. First, initial swab masses were taken. Next, swabs were placed directly into a small opening in a handheld Black & Decker Dustbuster® vacuum and suctioned at approximately 18 airwatts for 5 seconds each and final volume was recorded. Mass change was expressed as a mass change percent of the initial swab mass. Additionally, each swab type was subjected to 50 and 100 abrasive cycles with twists. One abrasive cycle was considered as one full back-and-forth swab on a flat surface. Masses were taken before abrasion, after 50 cycles, and after 100 cycles, and percent mass change was expressed as a percent of the initial swab mass.
Blue dye was added at a 1:10 ratio to distilled water to create a stock dye solution. Six serial 10× dilutions were performed and absorbance measured at 620 nm using a Synergy H1 Hybrid MicroPlate Reader (BioTek, Winooski, Vt.) to create a standard curve using a final volume of 2 ml. Next, initial swab masses were taken, swabs were immersed in stock dye solution, and a final weight was taken. Absorption ratio was calculated as:
Absorption Ratio=(mf−mi)/mi×100% (2)
Each swab was submerged in 100×diluted dye solution for 10 seconds, removed, and tip placed into a 5 ml conical tube with 2 ml of water and vortexed for 10 seconds. The suspension was plated and measured on the microplate reader at 620 nm. To determine the amount of released dye, the maximum absorbable volume for each swab tip was determined with cotton absorbing the smallest volume near 120 μl. Subsequently, 50 μl of the 100× diluted dye solution was pipetted directly onto the swab tips (experimental) or directly into 2 ml water (standard). The tips were then placed in 5 ml tubes with 2 ml of distilled water, vortexed for 10 seconds, and absorbance measured on the microplate reader. The released dye was expressed as:
Released Dye=abssw/absstd×100% (3)
Where, abssw is the absorbance measured from the dye solution released from the swab tips and absstd is the absorbance measured from the 50 μl of dye solution, both of which were diluted into 2 ml of water.
Stock Bovine Serum Albumin (Pierce BCA Protein Assay Kit, Pierce, Rockford, Ill.) was used as a model protein for both surface and solution swabbing. During wet extraction (solution swabbing), each swab tip was immersed in a 1500 μg/μl solution of BSA and distilled water for 10 seconds with gentle stirring. After removal, swab tips were added to 5 ml conical tubes with 2 ml of working reagents and incubated following the BCA Protein Assay Kit protocol. Absorbance values were measured using a microplate reader and compared to a standard curve generated from known concentrations of BSA protein. The extraction rate was measured as the ratio of total protein detected after swabbing:total protein in solution. To measure surface protein recovery, 500 μl of 1000 μg/μl BSA solution was pipetted onto a glass slide and allowed to dry. Swab tips were moistened by dipping in distilled water and the dried BSA was directly swabbed. Swab tips were added to a 5 ml conical tube with 2 ml working reagent, incubated, and analyzed with a microplate reader. Recovered protein was measured as the ratio of detected protein from the swabs to the known protein amount in the dried solution.
Human embryonic kidney cells (HEK 293) were cultured in completed DMEM+10% FBS+1% PenStrep until confluent. After reaching confluence, cells were trypsinized and resuspended in fresh media which was subsequently aliquoted into individual 50 ml tubes. Each swab was submerged in the cell suspension, moving back and forth for 10 seconds. After removal, the tips of each swab were added directly to 10 ml of fresh media. A small sample from each resuspension was stained with trypan blue and counted using a hemocytometer, from which the total cell count was estimated. Similarly, HEK 293 cells were grown to confluence on a petri dish. Media was removed from each petri dish and the cell monolayer was directly swab using each swab type. The tips of each swab were added directly to 10 ml of fresh media and vortexed for 10 seconds. Resuspended cells were stained with trypan blue and counted using a hemocytometer, from which the total cell count was estimated. Small volumes of cell suspension from the solution and surface swabbing were imaged using a field microscope.
Fresh blood was collected from Sprague Dawley rats and kept on ice until ready for use. Known volumes of blood were added to 2 ml microcentrifuge tubes and allowed to dry completely. DNA was isolated using a Qiagen DNA Investigator Kit and quantified using a NanoDrop™ OneC Spectrophotometer (Thermo Fisher Scientific, Waltham). Next, 25 μl, 50 μl, and 100 μl of blood was pipetted onto a glass surface and allowed to dry. Swab tips were moistened in distilled water and dried blood was directly swabbed. Swab tips were vortexed in buffer for 10 seconds and DNA isolation and quantification was carried out as described. Additionally, blood absorption was also measured by weighing dry swab mass, immersing the swab tips in blood (35° C.) for 10 seconds, and measuring the final mass of the swabs with absorbed blood. The equation used to generate blood absorption percent is given in Equation 2. Released DNA was measured by pipetting 50 μl of blood directly onto each swab tip, isolating and measuring the DNA, and expressing the release as the ratio of detected DNA from the swabs to total DNA from 50 μl of blood.
An initial stock of SARS-CoV-2 with a titer of 1.30×105 plaque forming units per ml (pfu/ml) was serially diluted in PBS by single log dilution down to 1.30×100 pfu/ml. A copy of each dilution was created for each swab type in 50 ml conical tubes for a total of 3 tubes per dilution. Each swab was dipped into its dilution tube and pressed against the insides of its respective 1.5 ml microcentrifuge tube already containing 500 μl of PBS for sample collection. Each 1.5 ml microcentrifuge tube was vortexed for 10 seconds to ensure thorough sample mixture into the PBS. The sample collection was conducted in triplicates for each swab type and dilution. A negative control for each swab type consisted of the 50 ml conical tube containing PBS only while repeating the same sample collection method. 140 μl of each collected sample was taken for RNA isolation via Qiagen QIAamp® Viral RNA Mini Kit. The viral RNA was analyzed by RT-PCR. The RT-PCR was performed on the QuantStudio™ 3. The RT-PCR reactions underwent an initial condition of 55° C. for 10 minutes then a 94° C. for 4 minutes followed by 45 cycles of 94° C. for 15 seconds and 58° C. for 30 seconds. The RT-PCR blank sample control resulted in no amplification. Each RT-PCR reaction consisted of 5.6 μl nuclease free water, 12.5 μl Invitrogen (2×) Master Mix, 0.4 μl MgSO4, 1.0 μl Primer/Probe Mix (0.15 μl Forward Primer (100 μM stock), 0.2 μl Reverse Primer (100 μM stock), 0.05 ηl Probe (100 μM stock), 0.6 μl TE Buffer), 0.5 μl SuperScript™ III Platinum Taq Mix, and 5.0 μl extracted Sample RNA. *E gene target primers and probe:
All experiments and measurements were done with a minimum of three replicates. Data is expressed in tables and graphs as the mean±standard deviation. All physical measurements were taken on a calibrated digital caliper and all photographic measurements were made using ImageJ. Ordinary one-way ANOVAs, paired and unpaired t-tests, two-way ANOVAs (displaying significance within-group), and frequency distributions were used where appropriate. Post-hoc testing (Tukey's), residual distribution, and normality were checked following each analysis. Definitive outliers were identified and removed using Iterative Grubbs' testing. Interpretation of the SARS-CoV-2 data included within group comparisons except at 101 and 100 pfu/ml, where no virus was detected. Each data set was analyzed and charted using GraphPad Prism Version 9.0.0. Significance was denoted as ns≥0.05, 0.01<*p<0.05, 0.01<** p<0.001, 0.001<***p<0.0001, ****p<0.0001. All graphics are original and were prepared using BioRender.
Superabsorptive 3D nanofiber matrices with layered structures can be synthesized by expanding electrospun nanofiber membranes using an innovative gas foaming technique (Chen, et al., Biomaterials (2018) 179:46-59). By applying the solids-of-revolution concept to the gas foaming, various nanofiber objects (e.g., spheres, cylinders, and cones) can be generated from expanding electrospun nanofiber membranes around a thermally fixed axis (Chen, et al., Nano Lett. (2019) 19:2059-2065). Herein, novel nanofiber swabs were synthesized that improve sample collection and release and test sensitivity in a variety of applications. More specifically, radially aligned, 0.75% w/v gelatin-coated PCL nanofiber objects with cylindrical shape were generated and then bonded to plastic swab sticks. The performance of the novel nanofiber swabs was compared in analyzing different biological materials including bovine serum albumin (BSA), HEK293 cells, DNA from blood, Methicillin-resistant Staphylococcus aureus (MRSA), and SARS-CoV-2 (
Ultimately, swab performance is directly related to their morphology. The three swab types including cotton, flocked, and nanofiber were characterized and compared. Cotton swabs (sterile 6 in, McKesson) have approximately 1 cm long ovular tips while flocked swabs have roughly 2 cm long cylindrical tips. Nanofiber swabs used in each experiment were cylindrical and approximately 1 cm long (
To examine the collecting efficacy, blue dye solutions (water with blue food coloring (McCormick & Company, Md.)) were swabbed and colorimetric changes were compared (
Detecting proteins from surfaces and wet samples is commonplace in food safety and medical diagnostics (Lahou, et al., Int. J. Environ. Res. Public Health (2014) 11:804-814; Keeratipibul, et al., Food Control (2017) 77:139-144; Topkas, et al., Clin. Chim. Acta (2012) 413:1066-1070). To evaluate and compare the efficacy of nanofiber swabs at detecting protein from surfaces and solutions, BSA was used as a model protein. A BSA solution of a known concentration was either directly swabbed (wet extraction) or a known volume was added to a glass slide and allowed to dry before swabbing with a premoistened swab (dry extraction) (
Next, the swabs were used to both extract HEK293 cells from a suspension and physically remove them from a plate-adhered tissue monolayer (
Swabs are heavily used in forensic science for DNA extraction from surfaces and aqueous solutions and are often used for patient, perpetrator, or victim identification, making them a vital tool in forensics (Schulz, et al., Forensic Sci. Int. (2002) 127: 128-130; Turingan, et al., Int. J. Legal Med. (2020) 134: 863-872; Kitayama, et al., Leg. Med. (2020) 46: 101713; Katilius, et al., Forensic Sci. Int.: Genet. (2018) 35: 9-13). To investigate the potential of nanofiber swabs in harvesting DNA, known volumes of blood were pipetted onto glass slides and allowed to dry completely. Premoistened swabs were used to extract DNA from the dried blood and analyzed using NanoDrop (
Identification of pathogens by swabbing is, without question, the most critical role swabs play in medicine. Whether identifying bacteria or viruses, ensuring ultrahigh sensitivity is critical for reducing false negative test rates, particularly in patients with low titers or bacterial counts (Pan, et al., Clin. Chem. (2020) 66:794-801; Xiao, et al., J. Med. Virol. (2020) 92(10):1755-1756; Palavecino, E. L., Rapid Methods for Detection of MRSA in Clinical Specimens. In Methicillin-Resistant Staphylococcus Aureus (MRSA) Protocols; Ji, Y., Ed.; Methods Mol. Biol. Humana Press: Totowa, N.J., 2014; pp 71-83; Roth, et al., PLoS One (2016) 11: No. e0159667; Davis, et al. medRxiv (2020) medrxiv.org/content/10.1101/2020.06.09.20124008v4). Decreased false negative rates via increased swab sensitivity can help identify pathogens and increase the accuracy of viral contact tracing and viral RNA identification. Methicillin-resistance Staphylococcus aureus is one of the most fiscally burdensome bacterial infections in clinics and contributes significantly to increased hospitalization time and decreased survival rates (Roth, et al., PLoS One (2016) 11: No. e0159667; Issler-Fisher, et al., Burns (2015) 41:1212-1220). Nasal swabbing with cotton or flocked swabs is a point-of-care method to rapidly identify a MRSA infection (Palavecino, E. L., Rapid Methods for Detection of MRSA in Clinical Specimens. In Methicillin-Resistant Staphylococcus Aureus (MRSA) Protocols; Ji, Y., Ed.; Methods Mol. Biol. Humana Press: Totowa, N.J., 2014; pp 71-83; Schulz, et al., Lab Chip (2020) 20:2549-2561). The relative sensitivity of nanofiber swabs was determined using MRSA as a model bacterium. Serial dilutions of stock MRSA were prepared and directly swabbed with cotton, flocked, and nanofiber swabs. Swabs were then used to directly inoculate an LB agar plate using a spreading method and colonies were counted after an overnight incubation (
Similarly, identification of viral pathogens commonly occurs through nose, mouth, or throat swabbing, where sensitive swabs are needed to collect as much virus-containing droplets as possible (Fisher, et al., J. Clin. Virol. (2019) 115:43-46; Abu-Diab, et al., J. Clin. Microbiol. (2008) 46:2414-2417; Tunsjø, et al., APMIS (2015) 123:473-477). Increased sensitivity of SARS-CoV-2 tests result in a decrease in false negative test rates, which is vital in allowing time for treatment and early identification (Woloshin, et al., N. Engl. J. Med. (2020) 383: e38). To compare sensitivity of nanofiber swabs to cotton and flocked swabs, a serial dilution of SARS-CoV-2 was made and each dilution was swabbed by each swab type (
In summary, a new type of swab tipped with cylindrical nanofiber objects produced by gas-foam expanding electrospun membranes around a fixed axis has been developed. Because of the small diameters and hierarchical porous structures, nanofiber swabs showed ultrahigh absorption, far surpassing the absorptive capabilities of cotton and flocked swabs. Additionally, the improved ability of the nanofiber swabs to detect samples from surfaces as well as release absorbed samples is demonstrated. The shape-tunable and morphologically hierarchical swabs showed improved collection and identification of proteins, cells, DNA, bacteria, and viruses. Nanofiber swabs would increase the diagnostic accuracy and assessment of population infection rates during the COVID-19 pandemic. Production of nanofiber swabs can occur at an industrial level, as industrial electrospinning and lyophilization are common, thus making large-scale production of such swabs feasible. These nanofiber swabs have far-reaching potential in medical and forensic fields and serve as an important step toward increasing diagnostic test sensitivity. Although the demand for highly sensitive COVID-19 tests may decrease over time, the need to accurately identify pathogens or proteins in contaminated food samples or identify persons of interest from blood or DNA at a crime scene will always exist. Improving the outcome of specimen identification will help in treatment and prevention of foodborne illnesses, reduce false incarcerations from insensitive DNA analysis, and help forensic teams identify persons of interest from often miniscule biological specimens.
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/299,110, filed Jan. 13, 2022. The foregoing application is incorporated by reference herein.
This invention was made with government support under Grant No. R01 GM123081 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63299110 | Jan 2022 | US |
