Patent Application
20110111012
The present invention relates to wound dressing assemblies prepared from a needleless electrospinning process that comprise single or various, individual layers of material(s). More specifically, the present invention is directed towards wound dressing assemblies that comprise single or various layers of electrospun material(s).
Electrostatic spinning, or electrospinning, has been dependent on spinning of cohesive liquids across a high voltage electric field through hollow needles or capillaries. More recently needleless electrospinning methods (where highly cohesive liquid jets are spontaneously created from liquid surfaces) have enabled improved scaling of the spinning of nanofiber web and sheet. An important example of the needleless spinning approach is where one electrode of a high voltage electric field is a wetted rotating cylinder from whose surface jets of cohesive liquid are propelled by the electric field in the direction of a counter electrode. The spinning electrode forms nanofibers from a polymer solution. The opposite, parallel pole of the voltage source acts as a collecting electrode, wherein the formed nanofibers are propelled towards the collecting electrode and are deposited on a substrate material. The deposited fibers form a linear web of materials upon the substrate. This needleless rotating cylinder approach to the formation of nanofibers provides an important advantage over the capillary or needle approach in that innumerable jets can be formed at one time and hundreds to thousands of grams of nanofibers can be deposited per hour.
Developments in electrospinning processes have generally focused on particular materials that are capable of being spun and research into new materials or compositions that may be spun. Research is still advancing as to the properties and characteristics of these nanomaterials, as well as research into possible uses of these materials.
The present invention provides various nanospun materials and compositions of materials, as well as methods for producing the nanospun materials.
The nanospun materials are incorporated into wound dressing assemblies, wherein the wound dressing assemblies consist of single or multiple layers of nanospun materials. The individual nanospun material layers may each comprise different materials and compositions, with the individual layers being joined or adhered together to form a dressing assembly. Because the individual layers are potentially spun from different materials and compositions, the dressing assembly can be formed and designed to address particular needs, based on a variety of factors, such as hemostasis, fluid handling, drug delivery, antimicrobial and/or other. The present invention also contemplates methods of determining particular materials for each layer of the dressing assembly based on the requirements of the final wound dressing assembly.
The present invention further contemplates electrospinning methods for altering the characteristics of the nanomaterials that are being spun.
The present invention also contemplates various compositions for use as nanospun materials, including various materials that have been cross-linked with each other.
Although the disclosure hereof is detailed and exact to enable those skilled in the art to practice the invention, the physical embodiments herein disclosed merely exemplify the invention which may be embodied in other specific structures. While the preferred embodiment has been described, the details may be changed without departing from the invention.
The present invention contemplates various wound dressing assemblies comprised of nanomaterials and methods of making the wound dressing assemblies. Generally speaking the wound dressing assemblies comprise single or various individual layers of nanomaterial(s) that are combined to form the wound dressing assembly. Depending on the particular uses and requirement for an individual wound dressing assembly, the present invention provides methods for altering and adjusting the electrospinning parameters, as well as methods for determining which compounds and compositions should be incorporated into specific nanomaterial layers.
Still referring to
As opposed to the other layers of material that comprise the dressing assembly 10, the backing layer 26 is not comprised of an electrospun material. As such it should be understood that wound dressing assemblies developed according to the present invention may comprise one or more layers of electrospun material and also one or more layers of materials that comprises a material or materials that are not electrospun. The layers and materials will then be compressed to form the wound dressing assembly 10 as shown in
After the dressing assembly 10 has been applied to the wound 26 for a sufficiently determined time, the backing layer 24 may be removed from the wound 18, as depicted in
The depicted layered dressing assembly 10 provides an assembly and system that provides different materials that have different therapeutic qualities. The assembly 10 comprises different layers of materials joined together in a manner directed by the user, determinative on the specific use required for a layered dressing assembly. As noted, the assembly 10 comprises layers of different materials, with the possibility that one or more of the layers will be of the same material.
Each of the separate layers comprises a separate layer of nanomaterial that can be individually woven and formed, determinative of the specific needs for the dressing assembly.
Needleless electrospinning, as described according to the process described for the present invention, is an efficient way of producing nanofibers of various polymers and polymer blends to form composite fibers. Another advantage of this method is the possibility of adding small insoluble particles to the polymer solution which are subsequently encapsulated in the dry fibers. This enables the addition of soluble drugs or antimicrobial agents to the polymer textiles of nanofibers. In the present invention, the use of nanofibers allows for the delivery of therapeutic material, hemostatic or medicinal materials to a wound site. Further, the present invention provides electrospinning of individual layers of various materials or compositions that can be joined together after the individual layers have been formed with the spinning process.
The polymer solution 118 provides the source of the formed nanofibers 120, which can be one of any number of various types of materials. The spinning electrode 112 is connected to a pole of a high voltage source. For example, the spinning electrode may be connected to the positive pole. The collecting electrode 114 may either be grounded or connected to the opposite pole of that of which the spinning electrode 112 is connected (i.e. the negative pole). The collecting electrode 114 may be formed by a grounded wire, or a rod, or another known arrangement that can act as an electrode. An induced electrostatic field of high intensity is formed between the electrodes 112 and 114, which creates a spinning space 122 for the production of the nanofibers 120. Still referring to
Likewise, by altering the parameters of the electrospinning arrangement, it is possible to alter the positioning of the individual nanofibers 120 as they are deposited on the mat 130. For example, the nanofibers 120 can be deposited in a relatively planar or flat fashion, which is generally stated as being a two-dimensional arrangement (2-D), or the materials can be placed on the mat 130 in a less planar fashion, referred to as a “fluffy” arrangement. The fluffy arrangement can be considered an arrangement wherein the layers are positioned in a three-dimensional (3-D) arrangement. The “fluffy” 3-D arrangement shows potential as having possible advantages by providing more of a volume than a 2-D, such as for increased absorption potential.
The above is exemplary of possible techniques that can be used according to the present invention. Examples of other electrospinning processes can be found in U.S. applications, Pub. Nos. 2008/0284050 and 2008/0307766, which are herein incorporated by reference. Likewise, the electrospinning process could be carried out by using a melt spinning process.
As previously noted, the spinning electrode 112 comprises a cylinder 140, as shown in
Other shapes and forms for the spinning electrode 112 are shown in
Use of the various electrodes will be discussed further in the following sections.
The depicted electrospinning process is considered a “needleless” electrospinning process, which is generally the preferred process of producing wound dressing assemblies according to the present invention. As stated above, it is possible to have a different type of needle for use in collection as shown in
As mentioned above, the various nanofiber mats 130 can be formed of an individual material or a composition of materials.
As discussed previously, the individual layers may be adhered to one another, which may occur during or after the electrospinning process.
After the various nanofiber mats 130 have been formed, either as individual layers or as composite layers, the mats 130 will be used for the layers of the wound dressing assembly of the present invention. If necessary layers of adhesion may be used in between the layers of material. The layers will be positioned as required, as is shown in
The wound dressing assembly 10 is one example of possible dressing assemblies developed according to the present invention.
The dressing assembly 210 contemplates using different layers and, also, different materials. For example, the dressing assembly 210 comprises a first layer 212 consisting of a microdispersed oxidised cellulose material, commonly referred to as MDOC, with the MDOC material being formed by an electrospinning process. Likewise, the second layer 214, which is comprised of a chitosan material, is also formed through an electrospinning process. The interior layer 216 is comprised of a material that has therapeutic qualities, with the interior layer 216 in the form of various types of materials, such as a powder-based material or a nanomaterial that has been cut or chopped into smaller pieces as desired for a particular use. Alternatively, the interior layer 16 could also comprise could comprise another layer of nanospun material. As an example in the wound dressing assembly 10, the interior layer 216 is comprised of powder, which comprises a combination of MDOC and chitosan materials. The wound dressing assembly 210 may be assembled in a similar fashion as discussed with respect to the wound dressing assembly 10.
Alternatively, the layers 212 and 214 would pass through a machine that would allow the bottom layer 212 to be pulled downwardly to form a well for the material 216 to be placed, wherein the layer 214 would then be placed above the material 216 and sealed to the layer 212, as discussed.
The present invention provides layered wound dressing assemblies that are formed from nanospun materials. The wound dressing assemblies are formed according to various prescribed conditions, such as whether the wound dressing assembly is to be hemostatic, antimicrobial, capable of delivering a drug, or a combination of various characteristics. Examples of characteristics that are considered in developing a wound dressing assembly also include regulating the moisture content of the assembly, controlling lateral wicking, and promoting vertical wicking through the assembly. Similarly, the wound dressing assembly may be required to be completely dissolvable or may be required to retain its shape while being used. For each instance, it may be necessary that the wound dressing assembly comprise different layers, as discussed above in section I. The present invention further contemplates methods and processes for determining possible materials to be used in a layered wound dressing assembly.
It should be understood that the processes depicted in
The following section provides further details with respect to some of the various materials noted above for use for individual layers of a wound dressing assembly, including potential uses for the materials.
Wound dressing assemblies according to the present invention can be made from a variety of materials that have various therapeutic, hemostatic, or other efficacious and helpful qualities. To provide various therapeutic regimens, different layers of electrospun materials are provided, with different layers comprising different materials. While the individual layers have been directed at electrospun materials that generally consist of a single material, such as chitosan, MDOC, collagen, carboxymethyl cellulose (CMC), gelatin, hyaluronic acid, etc., the individual spun layers may comprises a composition of more than one compound or material. Various examples of materials that can be electrospun and used according to the present invention include: chitosan compounds, alginate compounds, cellulose compounds, including oxidized cellulose compounds, mircodispersed oxidized cellulose compounds, carboxymethyl cellulose compounds, hydroxyethlyl, cellulose compounds, methyle cellulose compounds, and ethyle cellulose compounds collagen compounds, fibrinogen compounds, albumen compounds, cellulose acetate compounds, nylon compounds, polyurethane compounds, polyurethane ester, ether, urea, and siloxane compounds, polyactic acid compounds, polyglycolic acid compounds, polyhydroxybutyric acid compounds, polyglycoliclactic acid compounds, polyvinyl alcohol and alcohol compounds, polyethylene oxide compounds, gelatin compounds, hyaluronic acid compounds, polycaprolactone compounds, silk compounds, pectin compounds, polyacrylic acid compounds, starch compounds, and combinations thereof.
The following examples discuss various examples of electrospun materials that comprise more than one material or compound, including compounds that may be cross-linked with other compounds.
A. General Examples of Electrospun Materials
Various materials are shown and described in the table shown in
B. Electrospun Chitosan/Polyethylene Oxide (PEO) Matrix
1. Matrix Overview
Chitosan and polyethylene oxide were combined to form a nanofiber matrix. The chitosan material was prepared from solution. As an example, the chitosan selected for processing into the matrix has a viscosity at 25° C. in a 1% solution of acetic acid (AA) with spindle LVI at 30 rpm, which is about 100 centipoise to about 2000 centipoise. More preferably, the chitosan has viscosity at 25° C. in a 1% solution of acetic acid (AA) with spindle LVI at 30 rpm, which is about 125 centipoise to about 1000 centipoise. Most preferably, the chitosan has viscosity at 25° C. in a 1% solution of acetic acid (AA) with spindle LV1 at 30 rpm, which is about 150 centipoise to about 500 centipoise. The chitosan solution could then be combined with the PEO in solution prior to the electrospinning process.
The following results are directed towards an electrospun material having a Chitosan/Polyethylene oxide (PEO) product in the final electrospun fiber with a weight ratio of 90% to 10% respectively. The resultant product was tested and determined to be soluble with a mean fiber diameter of 137±31 nm. An example of a scanning electron microscope (SEM) picture of a Chitosan/Polyethylene (90/10) oxide product at 2 different magnifications (1000× and 20,000×) is shown in
Since the eventual electrospun nanomaterials will be used in wound dressing assemblies and other wound care products and compositions, it is important that sterilization of the product did not have a detrimental effect on the integrity of the sample. That is, it is preferable that sterilization of the materials does not affect the physical parameters and dimensions of the material. Table 1, below, compares a chitosan/PEO nanomaterial matrix that has been sterilized with gamma radiation to a chitosan/PEO nanomaterial matrix that has not been sterilized. Sterilization is generally comprises a gamma radiation between 11-40 kGy, with a preferred gamma radiation being 25 kGy. The tensile strengths of the matrixes were compared. As the results indicate, no detrimental effects occurred from the sterilization process.
The chitosan/PEO matrix was also measured at various fiber diameters, to determine if the physical characteristics would be altered, which potentially could affect the therapeutic qualities of the matrix.
Preferably, the fiber diameter will be consistent, regardless of the basis weight of the fibers, which will provide for more consistent qualities for the chitosan/PEO matrix and analysis of the chitosan/PEO matrix, since the therapeutic qualities will not change significantly if the fiber diameter is altered.
It can be seen in
To form the chitosan/PEO matrix, the chitosan and PEO of the invention should be combined in a suitable fashion. Section 2 below provides possible suitable ways for the compounds to be cross-linked with one another.
2. Cross-Linking of the Chitosan and Poly-Ethylene Oxide
As stated above the nanofiber chitosan product produced by the present invention is soluble in water. This largely renders the product ineffective for use as a hemostat due to the almost immediate dissolution of the product when in contact with blood. One way to convert the soluble nanofiber product to an insoluble product is to crosslink it. Crosslinking is the formation of chemical links between molecular chains to form a three-dimensional network of connected molecules. A substance that promotes or regulates the intramolecular covalent bonding between polymer chains, linking them together to create a connected structure, is called a crosslinking agent. A number of crosslinking agents were used in this project to crosslink the chitosan/PEO nanofiber product. These included glyoxal, gluteraldehyde and genipin. Details of these crosslinking agents can be found in
The most common crosslinking agents, or crosslinkers, used with chitosan are dialdehydes such as glyoxal and glutaraldehyde. These crosslinking agents were used successfully at 130° C. for 10 minutes. The resulting covalently crosslinked fibers were rendered insoluble and were suitable for use as in stabilizing electrospun materials for use in wound dress applications. It should be understood that care must be taken when using these cross-linking compounds, since they are considered toxic above certain concentrations. For example, glutaraldehyde is known to be neurotoxic, and its fate in the human body is not fully understood. Also, glyoxal is known to be mutagenic. Therefore, use of such crosslinking agents requires purification before administration, and potential testing of the presence of free unreacted dialdehydes in the products prior to administration. It should be noted that materials and compositions are routinely heated to elevated temperatures (i.e. 130° C.), with such materials also generally not breaking down due to dissolution. As such, it should be understood that there is the potential that other factors may affect the relationship of the materials, with similar appearances as the crosslinked compounds.
Crosslinkers such as diethyl squarate, oxalic acid or genipin can exhibit direct crosslinking mechanisms, although they remain incompletely elucidated.
The use of genipin is an alternative to dialdehydes as it is a naturally occurring material, which is commonly used in herbal medicine and as a food dye.
Because of potential toxicity limitations of the above described covalent crosslinking approach, other crosslinking processes have also been considered to tend the nanospun materials insoluble. One such process is thermal treatment of the nanospun construct after electrospinning. Thermal treatments of the chitosan/PEO samples were carried out by oven heating the nanofibers at a range of temperatures for 10 minutes. Temperatures from 70° C.-200° C. were examined and the solubility of the samples subsequently analyzed. From this analysis it was concluded that post nanofiber production treatment at 130° C. for 10 minutes gave the optimum sample. SEM images of examples of such products can be seen in
It should be understood that the chitosan/PEO matrix can be formed with or without actual thermal treatment of the chitosan and PEO materials, as well as the fact that matrices at elevated temperatures may not necessarily be rendered insoluble. Depending on the particular uses and the desired requirements of a particular matrix or composition, crosslinked or non-crosslinked, thermally or non-thermally treated, nanospun constructs may be advantageous. The following example discussed thermally treated electrospun chitosan/PEO constructs.
The following example examines chitosan material that has been mixed in a solution with polyethylene oxide (PEO) with subsequent investigation of the effect of thermal-treatment on chitosan/PEO matrices.
The electrospun solution was comprised of a mixed combination of a chitosan solution and a PEO solution. The chitosan solution comprised 7.5 g of chitosan material (Chitosan AK) which was mixed with 60.1 g of deionized water and subsequently mixed with 100 mL of acetic acid and mixed with a magnetic stirrer to form an aqueous solution. The PEO solution was formed by combining 0.7 g of PEO (MW 400,000, purchased from Scientific Polymer Products, Inc., Ontario, Canada) with 24.3 g of deionized water. The chitosan solution was added to the PEO solution to form the solution to be electrospun.
The electrospinning solution was also distributed over polyethylene (PE) foil and allowed to dry. The foils were to be further used in infrared (IR) spectroscopy analysis to further study structural and chemical changes in the chitosan/PEO matrix.
The following parameters were used for the electrospinning process:
Electrode: Small smooth cylinder
Collector: Needles+Fixed spunbond
Fabric speed: 0 m/min.
Electrode spin: 3.2 min−1
The chitosan/PEO solution was spun for approximately one (1) hour, with the obtained materials being orientated in a generally two-dimensional, planar fashion. The spun material was divided into four groups, with each of the groups further treated, as follows:
a) no further treatment;
b) heated to 105° C. for 1 hour;
c) heated to 105° C. for 1 hour, followed by heating to 150° C. for another hour; and
d) heated to 150° C. for 1 hour.
The chitosan powder used for forming the solution and the chitosan foils were treated in the same fashion.
Loss on Drying: Approximately 20% of the volatile compounds (i.e. acetic acid, water) were lost when the materials were dried at 105°, but less than 1% of the volatile compounds further evaporated from the material when the material was further dried to 150° C., indicating that heating to 105° C. is sufficient to remove substantially the volatile compounds. Since acetic acid forms a substantially non-volatile acid salt with chitosan in the absence of water, small amounts of non volatile acetic acid may reside in the chitosan.
Dissolution: The chitosan powder and electrospun samples were tested for dissolution in water and 3% (w/w) acetic acid. All the thermally-treated samples were insoluble in water, while the non thermally-treated samples swelled when placed in water. All of the samples dissolved in the acetic acid, with each of the samples treated to 105° C. dissolving in approximately 5 minutes and the samples heated to 150° C. dissolving in approximately 2 hours. There were no significant differences (in terms of final water solubility) between the samples heated directly to 150° C. and the samples heated to 105° C. and then to 150° C. The significantly slower rate of dissolution in acetic acid of the 150° C. heat treated samples compared to the 105° C. treated samples may be associated with annealing of chitosan structure to a more paracrystalline isoform (possibly with greater hydrogen bonding) which is more resistant to dissolution. Treatment of chitosan acid salts in methanol produces similar annealing effects and dissolution resistance. The fact that the 150° C. sample did eventually dissolve in the acetic acid solution indicated that the thermal treatment did not induce covalent crosslinking in the chitosan
Infrared Spectroscopy:—Infrared spectroscopic analyses of the foils did not reveal any substantial differences between the heat-treated and the non heat treated samples.
Thus, as noted above, the example demonstrates that there is no covalent crosslinking associated with thermal treatment of chitosan/PEO but rather there is volatilization of acid salt and likely annealing of paracrystallinity in the polymer.
The following section contemplates and compares the potential of both thermally treated and untreated chitosan/PEO matrices, with respect to the antimicrobial activity of each. Further, application of thermal treatment is contemplated to affect the antimicrobial activities of the compositions.
3. Enhancement of the Chitosan/PEO Matrix For Increased Antimicrobial Activity
The antimicrobial activity of chitosan and its derivatives against different groups of microorganisms, such as bacteria and fungi, has received considerable attention in recent years. A large number of publications have demonstrated the antimicrobial activity of chitosan. The development of an antimicrobial chitosan wound dressing would be advantageous due to the elimination of the complications in wound healing associated with infection.
The nanofiber Chitosan/PEO matrix discussed above was examined for its antimicrobial activity (see
As can be seen from the results neither non thermally treated nor thermally treated Chitosan/PEO matrixes met with the definition of antimicrobial as defined by the USP28. Although neither had the required antifungal activity it can be seen that the non thermally treated sample was indeed antibacterial. To enhance the antimicrobial qualities of the matrices, two systems were developed. The first system incorporates silver into the Chitosan/PEO product. This initially involved the dispersion of nano silver particles into the PEO component prior to an electrospinning process, such as the electrospinning process discussed previously. After the electrospinning process is performed, a thermal treatment step was further used on the materials.
Silver has been recognized for centuries for its antimicrobial properties (Russell and Hugo, 1994; Klasen, 2000; Lansdown, 2002). References to the use of silver for the treatment of chronic wounds date back to studies on ulcers in the 17th and 18th centuries (Klasen, 2000). Silver acts as a heavy metal by impairing the bacterial electron transfer system and some of its DNA functions (Russell and Hugo, 1994; Cervantes and Silver, 1996). In order to do this the silver ions must be able to enter the cell as the correct concentration in solution (Demling and DeSanti, 2001).
The second system employed to develop an antimicrobial version of the Chitosan/PEO matrix was to use different acids other than acetic acid in the electrospinning process. When subsequent thermal treatment was performed, it was anticipated that these acids would result in the formation of a polycationic structure that would have antimicrobial activity since lactic acid is significantly less volatile than acetic acid.
In comparison, the addition of silver to the chitosan/PEO matrices, both the non thermally treated matrix (
(
In
All of the tested matrices are shown and compared in
Both the systems, silver and acetic acid/lactic acid are described below in further detail.
a. Addition of Silver to the Chitosan/PEO Matrix
Prior to the electrospinning process, approximately 20 ppm of SmartSilver® silver nanoparticles, provided by NanoHorizons, Inc., located in State College, Pa., was added to the chitosan/polyethylene oxide compound. The antimicrobial activity was measured as before and the results of a representative non-crosslinked products are displayed in
The antimicrobial tests performed demonstrated that the product was indeed both antibacterial and antifungal. It met with the USP 28 definition of an antimicrobial composition. Previously it was seen that the non-thermally treated Chitosan/PEO matrix without silver was antibacterial but not antifungal. The addition of silver to the matrix conferred antifungal activity and ability onto the matrix.
The silver containing Chitosan/PEO product was also thermally treated, as previously described and the resulting product was tested for antimicrobial activity.
As demonstrated in
Furthermore, it was also evident from examination of the electrospun chitosan nanofibers that the fibers produced in the presence of silver were of a higher fiber diameter (see Table 2). Therefore a simple investigation was performed with standard electrospun chitosan nanofibers and electrospun silver containing nanofibers under the same conditions at a number of different basis weight. The results can be seen in
The results indicate that the fiber diameter of the electrospun chitosan increases by approximately 100 nm in the presence of silver. With increasing basis weight the diameter of the fibers also increases.
b. Use of Alternative Acids in Producing the Chitosan/PEO Matrix
As previously described, the preferred method for the production of chitosan nanofibers was to dissolve them in acetic before electrospinning. As these samples were not antimicrobial, a number of other acids were also used for dissolving chitosan to form a solution for subsequent electrospinning of the chitosan. Lactic acid, hydrochloric acid and sulphuric acid were attempted for such a process. The greatest success for electrospinning the materials was found when the chitosan materials were dissolved in lactic acid. Lactic acid spun nanofibers of 23.6 g/m2 are shown in
Subsequently, a solution was prepared by dissolving chitosan in acetic acid and adding PEO and 10% (w/w) lactic acid. The solution was electrospun and then thermally treated with the resulting nanofibers presented in
It can be seen that the acetic acid-lactic acid mixed system creates an antimicrobial Chitosan/PEO product. Therefore this process presents an alternate system to the use of silver to create an antimicrobial product.
4. Influence of Relative Humidity on Chitosan/PEO Solution
Including the chemical makeup of a specific solution and the ration of the individual compositions that makeup the solution, the operating parameters can also have a potential effect on the resultant electrospun material. The following example discusses the effect of relative humidity on chitosan/PEO electrospun materials.
A chitosan/PEO solution was prepared, with a chitosan solution and a PEO solution being prepared separately and the combined for a final solution. The composition of the final solution is shown below in Table 9.
The solutions were spun after different periods of being formed. A three day old solution at 30% and 40% relative humidity (RH) was spun, and a ten day old solution at 20%, 30%, 40% RH was spun. The temperature in lab where the spinning took place was held constant at 21° C. In order to observe the impact of the relative humidity to the structure of the spun layer, we didn't use rollers at winding system.
The Electrospinning Equipment was as follows:
Machine: NanoSpider NS-LAB, location Vratislavice, Czech Republic
Electrode: smooth cylinder
Collector: cylinder
Electrode distance: 180 mm
Fabric speed: 0.13 m.min-1
Electrode spin: 4 min-1 HV: 82 kV Number of layer: 5
The results indicated that relative humidity has an influence on the structure of nanofiber layer. It was observed that materials spun at higher relative humidity Had more of a three-dimensional arrangement (i.e. “fluffy”) as compared to the spun materials spun with a lower relative humidity. However, it was also observed that the solutions that were 10 days old appeared to have more of a significant change with respect to the dimensionality of the spun material. As such, while not conclusive, the results indicate that the relative humidity can impact whether the spun material comprises a 2-D or 3-D arrangement.
Absorption capacity depends on basis weight and structure of nanofiber layer. The absorption capacity was also analyzed, and the basis weight results are shown in the graphs of
The initial results, while not conclusive, indicate that the relative humidity may have an effect on whether the electrospun material forms more of a 2-D or a 3-D arrangement. Further, the results indicate that the age of the solution may not have a significant effect on the absorption capacity of the spun material. The following section further describes potential effects on the age of the spun solution.
4. Effects of the Age of the Chitosan/PEO Solution
It has been determined that chitosan/PEO solutions are only capable of being electrospun a certain period of time after preparation. Generally, if the solution is prepared more than about 48 hours prior to the electrospinning process, the solution will lose its spinability, either with a drop of the solutions viscosity or formation of supramolecular agglomerates during storage. Chitosan is more hydrolytically stable than polyethylene oxide, which is known to be hydrolytically unstable. The following section discusses the ability to revitalize older solutions.
A chitosan/PEO solution was prepared by adding 20 grams of chitosan (AK) with 159 g of demineralized water, using a magnetic stirrer (KMO2 (1100)) for approximately 30 minutes. The solution was further shaken for 5 minutes, and then 276.4 g of acetic acid was gradually added. The solution was stirred for an additional 130 minutes. 45.5 g of a 3% PEO solution was further added and stirred for approximately 30 minutes and then allowed to stand for approximately 60 minutes.
The solutions were tested spun with the following parameters:
Collector: Needles with spunbond fixed
Electrode Spin: 3.2 min−1
The solutions were attempted to be spun at daily (D) intervals, D=0 and D=4, to determine the effects of age on the spinning. It was determined that after 4 days of storage (D=4), the solutions were not spinnable, as shown in Table 11.
Four processes four activation of the material were attempted:
1. Mixing the solution with a Turrax stirrer
2. Heating of the solution
3. Heating and mixing with a Turrax stirrer
4. The addition of fresh PEO solution
The results are shown below in Table 12.
The results indicate that reactivation of the solution was capable through the addition of further PEO solution, but heating and stirring did not have an effect on the solution. While possible, it is not certain, however, if the age of the solution will have any effect on the final characteristics of spun materials.
The age of the solution also poses other problems when preparing chitosan solution. For example, acetic acid is often used to prepare the solutions for spinning and to increase the stability of this solution However, this results in a rubberlike substance which has to be mixed for long time to be completely dissolved, usually overnight. As such, the following section discusses whether high-shear mixing (UltraTurrax) could be used for accelerated homogenization of material to minimize issues with stability and spinability in older solutions.
a. Mixing Speeds
A chitosan solution was formed by mixing 10.0 g of chitosan (AK Biotech Co., LTD, China, www.akbio.net.) with 80.0 g of water. The solution was then mixed with 139.0 g of glacial acetic acid, purchased from Fluka Analytcial, in an HDPE flask. Three different solutions were prepared and mixed in three different fashions. The first mixture was mixed using magnetic stirrer, the second by gently mixing using an UltraTurrax 25 mixer and the third vigorously using an UltraTurrax 50 mixer. The two UltraTurrax mixers differ in dispersing elements. The geometry of each, stator and rotor, respectively, are different, as well as diameters of rotor (18 mm and 36 mm, respectively). This results in different shearing forces and different heating during mixing.
The following parameters were used for the electrospinning process:
Equipment: NanoSpider LAB, location TiAnov
Electrode: small smooth cylinder
Collector: needles with spunbond fixed Electrode distance: 130 mm
Fabric speed: −
Electrode spin: 3.2 min−1
HV: variable
After 24 hours, the three solutions were centrifuged (3 min, 3000 min−1) and tested. To determine spinability, 31.8 g of the chitosan solution was first mixed with 3.2 g of 3% PEO in water (w/w, 1.2 g PEO mixed with 38.8 g water) for 30 minutes using magnetic stirrer.
Mixing times and rate are described in Table 13.
Mixing by magnetic stirrer and UT25 results in almost the same increase of temperature (by 4 and 5° C.) and viscosities of both solutions are also comparable. However, mixing with the UT50 mixer causes high increase of solution temperature (by 35° C.) and viscosity of this solution decreases by about 16%. The effect is shown in
b. The Effect of Temperature Increases
A chitosan solution was formed by mixing 10.1 g of chitosan (AK Biotech) was mixed with 79.5 g of water, which was then mixed with 138.1 g of glacial acetic acid (Fluka) in an HDPE flask. The solution was manually agitated for 5 minutes and mixed by magnetic stirrer for 70 min. Part of this solution was heated in water bath to 71.7° C. for 15 minutes and cooled down do room temperature. The original solution and heated solution were centrifuged (3 min, 3000 min−1) and tested. The electrospinning process had the following parameters:
Equipment: NanoSpider LAB, location Tinov
Electrode: small smooth cylinder
Collector: needles with spunbond fixed Electrode
distance: 130 mm
Fabric speed:
Electrode spin: 3.2 min−1
The results indicate that the viscosity of the heated solution (310 mPa·s) was lower by about 36% compared to viscosity of not-heated solution (490 mPa·s). The effect of temperature is also demonstrated in
c. Comparison of Chitosan solution with a Chitosan/PEO solution.
The following example compares chitosan solutions and chitosan/PEO solutions with respect to the age of the spun solution.
A chitosan solution was formed by mixing 10.0 g of Chitosan AK with 79.2 g of water and subsequently mixing with 138.0 g of glacial acetic acid (Fluka) in an HDPE flask. This mixture was manually agitated for 5 minutes and mixed by magnetic stirrer for 70 min. A chitosan/PEO solution was prepared similarly to the chitosan solution, with the chitosan solution prepared as stated and then mixed with PEO solution (0.75 g PEO in 24.3 g of water) using magnetic stirrer for 25 minutes. The solutions were stored at room temperature and stored for approximately 24 hours. The solutions were centrifuged (3 min., 3000 min−1) and tested. The electrospinning process had the following parameters.
Equipment: NanoSpider LAB, location Ti{hacek over (s)}nov
Electrode: small smooth cylinder
Collector: needles with spunbond fixed
Electrode distance: 130 mm
Fabric speed:
Electrode spin: 3.2 min−1
HV: variable
Viscosity was measured, with the results described in Tables 14 and 15 and compared in
The viscosity of both the chitosan solution and the chitosan/PEO solution decreased by about 25% during first day of storage at room temperature. The ageing profile of viscosity is virtually the same for both tested mixtures, as shown in
The Gel permeation chromatograph (GPC) shown in
The chitosan solutions were spinnable when freshly mixed with PEO solution. However, only freshly prepared chitosan/PEO solutions were spinnable. If the chitosan/PEO solution was one or more days old, the spinning process was not satisfactory.
As such, the results indicate that age can have an effect on the spinability of the spun materials.
However, the results do indicate that it is possible to use older solutions for electrospinning in certain situations.
6. Electrospun Chitosan Materials Without PEO
While the focus of the above sections has been on solutions containing chitosan and PEO, the following section discusses electrospinning chitosan solutions without any additional materials. The tested chitosan materials were various chitosan materials, having various levels of deacetylation. The solutions are shown in the Tables 16-20, below.
The results indicate that chitosan materials, without the addition of PEO, are capable of being electrospun, and are also capable of forming both planar and 3-D layers of nanomaterials.
C. Electrospun Microdispersed Oxidized Cellulose (MDOC)/Polyvinyl alcohol (PVA) Matrix
Electrospun microdispersed oxidized cellulose (MDOC) compounds were tested and compared to assess the quantitative and qualitative characteristics of the MDOC compounds. Particularly, the compounds tested were a composition of MDOC and polyvinyl alcohol (PVA) with additives added to the MDOC/PVA matrix. Tested additives included phosphoric acid, glyoxal, and perphosphoric acid. The compositions were also tested with and without sterilization. The various tested compositions are shown in
The compositions were tested and compared to determine how the noted additives affected the MDOC/PVA matrix. The results are discussed in the following sections. For reference, the number of the samples in the legends of the following Figures refers to the number of the compound as listed in
1. Influence of Phosphoric Acid on an Insoluble MDOC/PVA Matrix (8:2 Ratio)
The effect of phosphoric acid on the strength and elongation of the MDCO/PVA matrix was tested. Matrixes 1 and 2 from
While not conclusive, initial results indicated that the presence of phosphoric acid potentially increased the strength of the matrices and did not effect the elongation of the matrices. Strength also increased with increasing basis weight.
Similarly, there results indicated that that sterilization potentially increased the strength of the tested matrices, but the suggested correlation may be based on other factors. However, elongation of the matrices was lower after gamma-irradiation sterilization of the matrices.
It should also be noted that elongation of the matrices increases with decreasing basis weight. For example, in such uses, such as using the nanospun materials in plasters, a basis weight of around 10 g/m2 is preferred, while still maintaining elongation for the matrix.
2. Influence of Phosphoric Acid and Glyoxal on the MDOC/PVA Matrix (3:2 Ratio)
The effect of the addition of phosphoric acid and glyoxal on the strength and elongation of the MDCO/PVA matrix was tested. Matrixes 3, 4, and 10 from
The results for both strength and elongation show no discernible trends, and the addition of either phosphoric acid or glyoxal does not affect the mechanical properties significantly. Because of the weak correlation, it was not possible to determine the positive or negative effect that gamma-irradiation sterilization would have on the matrices.
3. Influence of Phosphoric Acid on a Soluble MDOC/PVA Matrix (8:2 Ratio)
The effect of phosphoric acid on the strength and elongation of the MDOC/PVA matrix was tested. Matrixes 5 and 6 from
The results indicated that the presence of phosphoric acid increased the strength of the sterilized samples, while the strength was not changed significantly by the addition of phosphoric acid for nonsterilized samples. Similar to the previously tested insoluble MDOC matrices, elongation increases with decreasing basis weight.
Strength also increases with increasing basis weight, with the increase being non-linear, and was also increased after sterilization. However, the addition of phosphoric acid indicated that elongation was better prior to sterilization. Sterilization did not appear to have an effect on the matrices that did not contain phosphoric acid.
4. Influence of Phosphoric Acid and Glyoxal on Strength of a Soluble MDOC/PVA Matrix (3:2 Ratio)
The effect of the addition of phosphoric acid and glyoxal on the strength and elongation of the MDCO/PVA matrix was tested. Matrixes 7, 8, and 9 from
Similar to the previously tested insoluble MDOC matrices, the matrices had weak mechanical properties. Strength of the samples appears to increase with increasing basis weight and the trend is non linear. Strength was not changed significantly for both sterilized and non sterilized matrices by addition of phosphoric acid and phosphoric acid and glyoxal.
Elongation of all matrices (7, 8, and 9) is lower after sterilization which means that sterilization worsens the elongation of the matrices. Elongation of the basic mixture without phosphoric acid and glyoxal is higher than elongation of the mixture with mentioned additives, meaning that, in this case, additives have negative influence on elongation and no effect on strength. There is no supposition of any trend of elongation, which may have a connection with difficult measurability.
5. Influence of Phosphoric Acid on Strength and Elongation of a Soluble MDOC/PVA Matrix (8:2 Ratio)
The effect of phosphoric acid on the strength and elongation of the MDOC/PVA matrix were further tested. Matrixes 11 and 12 from
The initial results, while not conclusive, indicate that the presence of phosphoric acid likely did not increase the strength of the sterilized samples, and the strength was not changed significantly by the addition of phosphoric acid for nonsterilized samples. Similar to the previously tested insoluble MDOC matrices, elongation appears increases with decreasing basis weight.
However, the addition of phosphoric acid indicated no effect on elongation prior to sterilization. Sterilization appeared to have a negative effect on the elongation of the samples, but did not appear to follow any trend.
6. Effects of Other Acids on the MDOC/PVA Matrix
PVA and PVA-based nanofiber material can be made insoluble in water by means of heat treatment of material containing phosphoric acid. This methodology has already been used in Vratislavice Lab for cross-linking of MDOC:PVA nanofiber samples. The cross-linking mechanism of PVA with polyfunctional acids is well known. Esters are formed between hydroxyl groups of PVA and acidic groups of polyfunctional acids during heat treatment, resulting in cross-linking. With boric acid, this cross-linking can even be observed in aqueous solution at room temperature.
The following experiment assesses the applicability of other acids and the validity of potential cross-linking mechanisms of these acids, since phosphoric acid could be undesirable for wounds and skin.
The experiment assessed various types of acids, including: hydrochloric acid (strong inorganic monofunctional acid), acetic acid (weak organic monofunctional acid), phosphoric acid (inorganic trifunctional acid), citric acid (weak organib trifunctional hydroxy-acid), lactic acid (weak organic monofunctional hydroxy-acid) and 1,2,3,4-butanetetracarboxylic acid (weak organic tetrafunctional acid). The results are shown in section a, below. Section b, below further discussed the cross-linking of PVA:Citric acid blend, and the effect of acid concentration on the solution for electrospinning. Section c, below, discussed the use of MDOC-H+ as a polyfunctional acid, which could be used instead of phosphoric acid in cross-linking of MDOC:PVA nanofibers.
a. PVA/MDOC Combine With Various Acids
A polyvinyl alcohol solution (PVA) (16±1.5) % m/m, was purchased from Novacke chemicke zavody, Slovakia. The PVA solution was mixed with a predetermined amount of an acid compound (see Table 22), a MDOC compound (batch No. X01032), and deionized water using an UltraTurrax T25 mixture. The ratio of MDOC:PVA in the solution was maintained at 4:6 (unless MDOC was added to a specific solution. Approximately 25 g of each tested solution were placed into a spinning bath and electrospun for 15 minutes under the following conditions:
Equipment: NanoSpider LAB, location Ti{hacek over (s)}nov
Electrode: small cylinder
Collector: Wire with spunbond fixed
Fabric speed: 0, static spunbond
Electrode spin: 3.2 min−1
The formed layers of nanospun material were then treated at 140° C. for 2 hours, with the results shown in Table 22.
The results indicate that the addition of acid to the PVA solutions strongly influences conductivity of solution and pH value. In the MDOC/PVA solutions, the pH values appear to be buffered by the MDOC materials in the solutions.
The results further indicate that the PVA non-treated nanofiber samples were completely soluble in water. nanofiber samples, containing tri- and tetrafunctional acids (phosphoric, citric, butanetetracarboxylic) and heated to 140° C. for 2 hours, were not soluble in water, which suggests a potential cross-linking of the materials within the solutions. Heat treated nanofiber samples, containing only weak organic monofunctional acids (acetic, lactic), were soluble in water, denoting no significant change in structure (no cross-linking). The sample with hydrochloric acid after heat treatment was not soluble in water, which indicates some change in chemical structure, but the mechanism is not certain.
The MDOC/PVA nanofiber samples not heat treated were completely soluble in water, resulting in opaque solution (free H+ form of MDOC is water-insoluble). All other non-treated samples were insoluble, resulting in opaque gel.
The results indicate that there is potential to replace certain acids currently used in electrospinning processes with other acids that may have less deleterious effects when eventually used in wound dressing assemblies. Initial results indicate that bi-, tri- and poly-functional acids can be used for cross-linking of PVA based nanofiber materials, with the formation of esters between hydroxyl groups of PVA and acidic groups of acid believed to be the main mechanism of cross-linking. As such potential cross-linking exists, the following sections discuss in further detail solutions comprising citric acid.
b. Effect of Citric Acid Concentration on the Cross-Linking with PVA
As stated above, it is contemplated that citric acid can be a more suitable alternative to H3PO4 in MDOC/PVA solutions. The MDOC/PVA and PVA solutions were formed as noted in section (a). Citric acid was added to the various samples, as stated in Table 23.
Approximately 25 g of each sample were placed into spinning bath and electrospun for 15 minutes under the following conditions:
Equipment: NanoSpider LAB, location Ti{hacek over (s)}nov
Electrode: small cylinder
Collector: wire with spunbond fixed
Fabric speed: 0, static spunbond
Electrode spin: 3.2 min−1
Layers of nanospun material were then treated at 140° C. for 2 hours. The results are shown in Table 23.
The results indicate that by altering the amount citric acid in the final PVA material (0.5-7.5%), the properties of the resultant nanofiber layer can also be altered. Samples with higher citric acid content (more than 2%) appeared to be insoluble in water, did not swell to gel, and had a high tensile strength and elongation in wet form (which was evaluated only manually/visually). Samples with lower citric acid content were soluble or easily decayed.
The samples containing MDOC swells into a gel when immersed in water and again, higher content of acid in the resultant layer reflects in better stability in water. However, while there is an initial indication of cross-linking within the samples, it is possible that the cross-linking agent could be either citric acid or poly-anhydroglucuronic acid (PAGA), which is produced by reaction of the Na/Ca salt (MDOC) with citric acid. Further, Heat treatment of PVA:Citric acid nanofibers results in material, which is not soluble in water, do not swell and exhibits apparently high tensile strength and elongation in wet form.
b. PVA Cross-Linking with MDOC-H+
The results above indicate that MDOC compounds could have potential cross-linking abilities. Assessment was carried out using MDOC-H+ (free poly-anhydroglucuronic acid (PAGA)) on PVA cross-linking, so that the PAGA would not interfere with the potential results. Because MDOC material is organic as opposed to inorganic, this polyfunctional acid in pure form could be used for cross-linking PVA based materials with its organic nature instead of inorganic (phosphoric) or any other “extrinsic” acids, e.g. in blend with regular. Na/Ca MDOC salt. Limited solubility of MDOC-H+ could affect electrospinning process and especially the microscopic quality of nanofiber layer (macro- and micro-defects). Water-soluble ammonium salt (MDOC-NH4) could be used instead of MDOC-H+. Solubility of this salt could avoid formation of macro and micro defects. Ammonium should dissociate from MDOC-NH4 molecule at higher temperature (heat treatment) forming free acid for cross-linking reaction.
A PVA solution (16±1.5) % m/m was purchased from Novacke chemicke zavody, Slovakia. MDOC-H+ (batch No. 012H+0909) was prepared by dissolving original oxidized cellulose (Synthesia) in sodium hydroxide, which was then re-precipitated into free H+ form by adding nitric acid and washing off residuals. MDOC-H+ was then dried and micronized. The PVA solution was mixed with various amounts of MDOC-H+ as noted in Table 24 (which was previously swollen in water for 16 hours), using UltraTurrax T25. The solutions were centrifuged to remove any undissolved particles. The MDOC-NH4 salt solution was prepared by adding concentrated ammonia into MDOC-H+ suspension (approx 0.4 ml for complete dissolution of 0.56 g MDOC-H+) before mixing with the PVA.
Approximately 25 g of each solution were placed into spinning bath and electrospun for 15 minutes under conditions described below:
Equipment: NanoSpider LAB, location Ti{hacek over (s)}nov
Electrode: small cylinder
Collector: wire with spunbond fixed
Electrode distance: 160 mm
Fabric speed: 0, static spunbond
Electrode spin: 3.2 min−1
Layers of nanospun material were then treated at 140° C. for 2 hours. The results are shown below in Table 24.
The initial results indicate that MDOC-H+ without an additional acid component can be used for cross-linking PVA. A concentration of MDOC-H+ 8% was found to be most effective.
7. Effect of the Type of MDOC on The Mechanical Properties of the Matrix
As previously discussed above in sections III(C)(1-5), MDOC matrices were produced using both soluble and insoluble MDOC compounds, and will be discussed further in the following sections. The insoluble MDOC matrices (1 and 2) from
The initial results, though not conclusive, indicate that the type of MDOC likely does not affect the strength of the matrix, and the addition of phosphoric acid potentially adds a positive effect on the strength of the matrices. Similarly, elongation followed the same trends with both types of MDOC, with elongation being lower after sterilization. Phosphoric acid did not significantly affect the elongation characteristics.
8. Effect of the Ratio of MDOC/PVA on the Mechanical Properties
MDOC/PVA matrices containing 40% MDOC and 60% PVA were compared against one another to assess the mechanical properties of both the insoluble matrices (3, 4 and 10) and the soluble matrices (7, 8, and 9). The strengths were compared in
Comparatively, a decrease in the amount of MDOC in the MDOC/PVA matrix indicated an increase in the mechanical properties of the matrix. Matrices containing 20% MDOC and 80% PVA (1 and 5) were compared to matrices containing 40% MDOC and 60% PVA (3 and 7). The results are shown in
9. Optimization of MDOC/PVA Ratio
The results indicate that there may be an optimal ration of MDOC to PVA. To accomplish this, various MDOC/PVA solutions were mixed and tested. A PVA solution (16±1.5) % m/m was purchased from Novacke chemicke zavody, Slovakia. A 16% m/m dispersion of MDOC was prepared by mixing 64.0 g of MDOC I (Batch No X01 028) with 336.6 g of deionized water using UltraTurrax T25. The MDOC and PVA solutions were mixed in ratio (weight:weight) 3:7, 4:6, 5:5, 6:4 and 7:3 using an UltraTurrax T25 mixer for 1.5 minute, which were then centrifuged (3 min., 3000 min−1) to remove bubbles before electrospinning and measurements.
Approximately 25-26 g of each dispersion was placed into a spinning bath and electrospun for 15 minutes under conditions, as follows:
Equipment: NanoSpider LAB, location Ti{hacek over (s)}nov
Electrode: small smooth cylinder
Collector: wire, with spunbond
Electrode distance: 120 mm
Fabric speed: 0.3 m.min−1
Electrode spin: 3.2 min−1
SEM analyses were completed for assessment of nanofiber quality and are shown in
Each of the various ratios produced acceptable results. The initial results further indicate that a 4:6 ratio of MDOC:PVA produces an electrospun material having the best quality individual nanofibers if any of the tested ratios.
10. Production Capacity
Along with testing the mechanical properties of the various matrices, the matrices described in
The addition of phosphoric acid decreased production capacity, as did the addition of phosphoric acid and glyoxal. Also, insoluble MDOC has lower production capacity than soluble MDOC, and higher MDOC content decreases production capacity.
11. The Effect of Solution Conductivity on PVA Electrospinning
Solution conductivity is reported an important parameter for electrospinning process. The following section attempts to correlate the solution conductivity of a PVA solution with the resultant product. Seven PVA solutions with constant PVA concentration and different concentration of sodium chloride (NaCl) were prepared and electrospun.
A PVA solution (16±1.5) % m/m was purchased from Novacke chemicke zavody, Slovakia. Stock NaCl solution (2 mol/l) was prepared by weighing 23.4 g NaCl, dissolving and diluting to 200 ml by deionized water. This solution was further diluted to 1.64, 1.28, 0.92, 0.56 and 0.2 mol/l. 10.0 g of NaCl solution (or water) was then mixed with 90.0 g of PVA solution (16±1.5) % resulting in approx 14% PVA solution with different conductivity ranging from 0.58 (PVA solution with water) to 13.5 mS/cm. The various solutions are shown in Table 26. The solutions were mixed using an UltraTurrax mixer for 1.5 minute and centrifuged (3 min, 3000 RPM) to remove bubbles before electrospinning and measuring. The various solutions are shown in Table 26.
Approximately 25-26 g of PVA/NaCl solution were placed into a spinning bath and electrospun for 15 minutes under the conditions described, below.
Equipment: NanoSpider LAB, location Tignov
Electrode: small smooth cylinder
Collector: wire with spunbond fixed
Electrode distance: 130 mm
Fabric speed:
Electrode spin: 3.2 min−1
The results are shown in Table 26. A comparison of the conductivity to the area weight is shown in
The results indicate that viscosity of the PVA solutions is slightly influenced by NaCl. It is increased by about 8% for PVA/NaCl solution of the highest NaCl content.
The results also show that the amount of spun material and area weight decrease with increasing conductivity of solution, as shown in
Visual analysis of the spun materials indicated that the quality (lower content of thick fibers and ribbon-like defects) of a nanofiber layer is better for a PVA solution of lower conductivity. However, the initial results indicate that it may be possible to optimize the quality of an electrospun material based on an individual basis.
12. MDOC/PVA/PEO Matrix Further Comprising Chlorhexidine
The following section discusses electrospinning an MDOC/PVA/PEO matrix, which further includes chlorhexidine. Specifically, chlorhexidine hydrochloride solution was added to the matrix, which was calculated on the dry matter content of solution MDOC/PVA/PEO. The dry matter content of MDOC/PVA/PEO solution was 14.16% (with a ratio 40/57/3 wt %, respectively). The tested solutions had concentrations of chlorhexidine dihydrochloride as 1, 2.5, 5, 10, and 20 wt % related to MDOC/PVA/PEO dry matter content.
The electrospinning solution was formed by mixing chlorhexidine dihydrochloride in water. MDOC was added and the formed mixture was agitated using an UltraTurrax T25 for approximately 2 minutes. PVA was then added and the mixture was agitated using an RzR 2041 stirrer for approximately 10 minutes and then was allowed to stand for approximately 12 hours. Finally, a PEO water solution was added and the formed mixture was blended using the RzR 2041 stirrer for approximately 60 minutes.
The electrospinning process was carried out so that each of the tested samples had a relatively similar basis weight. Thus, the electrode distance was changed during the spinning process too keep uniformity with the spun layers. Changing of the electrode distance also allowed for a constant current value for all samples during the process and thereby provide similar basis weight of all prepared samples. It seems that process efficiency decreases with increasing chlorhexidine dihydrochloride concentration. All data of spinning process are listed in Table 27, below.
The matrix comprising MDOC/PVA/PEO with chlorhexidine hydrochloride was successfully spun, in concentrations from 1 to 20 w % chlorhexidine to dry matter content). The process efficiency appeared to with an increased concentration of chlorhexidine dihydrochloride, which was believed to correlate to the insolubility of chlorhexidine material in water. Initial results, while not conclusive, indicate that chlorhexidine materials can be incorporated into an electrospun material, which may provide beneficial results.
D. Gelatin Materials
The following examples discuss the potential of electrospinning gelatin based materials, including gels and other similar materials.
1. Combinations of Gelatin with Various Polymers
Porcine gelatin materials were combined with various non-animal source polymers to determine the spinability of the various combinations. Tested polymers included CMC, PEO, PVA, MDOC and MDOC H+ form.
Gelatin was purchased from Gelita AG, Eberbach, Germany. High molecular weight Gelita gelatin was used for the initial tests, since this type of gelatin can be readily electrospun alone from acetic acid solution, or as a mixture with polymers mentioned above. This gelatin solution has relative low viscosity, so increasing the viscosity by the addition of polymers like CMC did not cause any problem in electrospinning. A 9% gelatin materials in 80% w/w acetic acid solution was heated to 50° C. for approximately 2.5 hours. The solution was then cooled to room temperature, and the desired polymer was added as an aqueous solution to the gelatin solution after cooling down of gelatin solution to laboratory temperature. Compositions of all solutions are mentioned below.
All mixtures were electrospun using the same electrode system (cylinder-cylinder) and NS-Lab machine. For each mixture, the distance between electrodes was changed—current was higher than maximum (1.8 mA) for mixtures gelatin/PEO. Setting of the NS-Lab is, mentioned below in Table 28.
The Solutions and sample data are shown below in Tables 29-34.
Each of the gelatin combinations was capable of being electrospun. Generally, the presence of PEO in gelatin solution significantly increased the electric current during the spinning, and the formed nanolayer of material was visibly wet, which could be a potential combination for use with certain gelatin having lower molecular weight than the GELITA gelatin used in the present example.
Further initial indications seem to show that high molecular porcine gelatin (contrary to low molecular one) can be electrospun directly from CMC and/or MDOC mixtures without any other supporting fiber-forming polymers such as PVA or PEO.
2. Optimization of Gelatin Solution with PEO
As indicated by the previous section, spinning of gelatin materials in combination with other compounds was possible, with PEO being one such compound. The following example further tests an optimal gelatin/PEO combination for electrospinning, using a water-acetic acid solvent system to carry out the testing. The system is applicable at room temperature and does not require a co-polymer.
Several combinations of gelatin/acetic acid concentration and suitable combinations of electrode-collector system for electrospinning of gelatin were tested.
Initial analysis of the effect of gelatin and/or acetic acid concentration on microscopic quality of nanofiber layer indicated that the most suitable solutions was a solution having 9% gelatin dissolved in 80% acetic acid. The effects on the electrode system was further investigated. Three electrodes, cylinder collector and three distance values were set and electrospinning process was performed under HV which was found suitable and sufficient for continuous spinning process.
The gelatin solution was formed by mixing GELITA gelatin: Pigskin gelatin, 300 Bloom, Pharm. grade, purchased from Gelita, with water and left standing for 1 hour. The swollen gelatin was further mixed with acetic acid: 99%, p.a., purchased from Penta, CZ. Suitable amount of gelatin was weighed, using magnetic stirrer for at least 40 minutes for complete dissolution. The time required for complete dissolution depends on the amount of gelatin and concentration of acetic acid.
As an example, 54.0 g of gelatin was weighed, mixed with 109.2 g of water and left standing for 1 hour. Then 437.7 g of acetic acid was added to swollen gelatin and mixed using magnetic stirrer for 2 hours for complete dissolution (a larger amount of gelatin requires a longer time for complete dissolution). Final concentration of acetic acid in diluent is 80% w/w and final concentration of gelatin in solution is 9% w/w. Amount and final concentration of components can be found in Table 35.
Approximately 25 g of gelatin/acetic acid solution were placed into spinning bath and electrospun for 10 minutes under conditions described below. Small amount of fresh polymer blend was added to the bath after each spinning to maintain total weight of solution at 25 g.
Equipment: NanoSpider LAB, location Ti{hacek over (s)}nov
Electrode: small cylinder
Collector: needles with spunbond fixed
Electrode distance: see Table 1
Fabric speed: 0, static spunbond
Electrode spin: 3.2 min−1
82.01)
82.01)
2)
Approximately 450 g of a gelatin/acetic acid solution were placed into spinning bath and electrospun for 15 minutes under conditions described below. Small amount of fresh polymer blend was added to the bath after each spinning cycle to maintain total weight of solution at 450 g.
Equipment: NanoSpider LAB, location Tinov
Collector: cylinder
Electrode distance: see Table 2
Fabric speed: 0, static spunbond
Electrode spin: 3.2 min−1
The results indicated that the use of gelatins in electrospinning processes has potential. Generally, it was visually observed that the lower concentrations, resulted in more “macro defects” in the electrospun materials, while higher concentration of gelatin or acetic acid, however, results in thicker fibers and formation of ribbon-like fibers. As such, it was initially assessed that using 9% gelatin in 80% acetic acid would be a compromise between low number of macro defects (droplets), low number of ribbon-like thick fibers and acceptable fiber diameter. However, the results indicate that other gelatin solutions are spinnable and may have potential uses, depending on the particular requirements or need for a particular use.
E. Electrospun Hyaluronic Solutions
Electrospinning of hyaluronic acid was conducted, as explained below. The hyaluronic acid was supplied from Dermachem Cosmetic Ingredients, LLC, located in Tampa, Fla., and has a high molecular weight (value on the label: 1070 kDa).
An aqueous 5 wt % solution of hyaluronic acid was prepared. As the solution was very viscous, it was diluted to a concentration of 1.71 wt % hyaluronic acid in water. The solution was further blended with 3 wt % PEO in water for further diluting. Thus, the solution was mixed again with a Turrax mixed (2 minutes, 6000-10000 rpm) and electrospinning was reattempted. We used smooth small cylinder and thorn, but process did not start at all.
A 5 wt % solution in 80 wt % acetic acid was prepared. When acetic acid to hyaluronic acid was added to the solution, a precipitation was observed. The precipitation disappeared when other water was added. We used such volumes of acetic acid and water to obtain a concentration around 1.7 wt % of hyaluronic acid in 58.66 wt % acetic acid. We added calcium chloride to the solution in ratio 56:44 (hyaluronic acid: calcium chloride w:w) for decreasing of its viscosity because inorganic salt generally decreases viscosity of HA water solution. As no change in viscosity was observed after the stirring, the solution was heated to 45° C. for 3 hours. After cooling of this solution the viscosity stayed still visually high and solution was not spinnable. The solution was stored without any stirring for one week. The 7 day old solution was spinnable together with 3 wt % PEO in ratio 60:40 HA:PEO in dry matter. The solution was spun both using a thorn and a smooth cylinder. Compositions of all solutions spun are mentioned below in Tables 41-44
The results indicated that the inorganic salt addition had an effect on the process. While 1% HA water solution had a viscosity around 2900 mPa·s, the same solution with NaCl addition (the concentration of NaCl was approximately 0.07 mol/l) had a viscosity of only 980 mPa·s.
Effect of various pH and MDOC addition on viscosity of 1% of HA water solution was also studied. The following-three mixtures were prepared:
1.20 g of 1% water solution was mixed with 0.5 ml of HNO3 (pH<1)
2. 20 g of 1% water solution+0.5 ml of 30% w/w NaOH solution (pH=12)
3. 20 g of 1% water solution+200 mg of MDOC
Viscosity of the 2nd solution dramatically decreased to approximately 2 mPa·s immediately after the alkali addition. The addition of ammonia instead of NaOH was also tested to determine its effect on viscosity. The viscosity of the hyaluronic solution after the ammonia addition did not drop as much as with strong base, but was visually assessed as slightly lower compared to original solution. Viscosity of the solution with nitric acid was more or less comparable with the original solution and presence of MDOC decreased the viscosity to ½. All three solution were then put to a climatic chamber at 40° C. and RH=80% for 5 days. Viscosity of all three solutions significantly decreased during the storage:
solution 1: η25=1.3 mPa·sec
solution 2: η25=1.1 mPa·sec (solution discolored to a yellow hue)
solution 3: η25=97 mPa·sec (grew mold during the storage)
(viscosity of original 1% water solution was approximately 2900 mPa·s)
Solution No 1 and 2 were mixed with 3% PEO in the ratio 60/40 vol/vol. Both solutions willingly spun, but no final nanolayer was obtained due to low concentration of HA in spun solution(s) (only dirty base material was obtained). The results indicate that it may be necessary to increase concentration of hydroaluronic acid in water solution prior to hydrolysis.
Solution 1 and 2 were sampled for GPC analysis and will be compared with a freshly prepared solution.
The 7 day old solution of hyaluronic acid in acetic acid with addition of CaCl2 and PEO (HA090914V01) was successfully spun (i.e. it was obtained a tangible nanolayer. Smooth cylinder as spinning electrode and cylinder as a collector were used. Distance between electrodes was 14-16 cm, rotation of spinning electrode 12-15 Hz and applied high voltage 81.9 kV (with current ca 0.4 mA). This experiment has been done at 25% relative humidity and 21° C. This solution has a tendency to create a generally 3-D, i.e. fluffy, structure.
As such, the result 7 days old solution of HA in acetic acid with CaCl2 addition was spinnable with PEO 3% w/w in ratio 60:40 (this ratio was used for sodium salt of hyaluronic acid in Nanopeutics). Tested type of hyaluronic acid having a molecular weight around 1070 kDa was not spinnable either from water or acetic acid solutions (freshly prepared) due to their high viscosities. The initial results, while not conclusive, indicate that there is a possibility to optimize the ratio HA:PEO (to increase HA content).
F. Electrospun CMC/PVA/PEO Solution with Silver Nanoparticles
The following example was carried out to provide an electrospun CMC/PVA/PEO nanomaterial, that further incorporated silver nanoparticles at concentrations of 100 and 300 ppm. The example attempted to electrospin the materials so that they would form a three-dimensional layer of nanomaterial, or a “fluffy” layer of nanomaterial.
A 6 wt % CMC/PVA/PEO solution was prepared—in ratio 2:1:1 to dry matter content. Each of the CMC—6 wt % solution and the PEO—6 wt % solutions was prepared separately and subsequently mixed together with PVA and water. SmartSilver® silver nanoparticles, supplied by NanoHorizons, Inc., located in State College, Pa., were added to the PEO solution before the final solution blending. Phosphoric acid (8 wt % to dry matter content) was finally added to the solution. Three solutions were prepared (100 ppm silver, 300 ppm silver, and no silver), and each of the solutions was post-treated by heating (due to the samples solubility in water). A comparison of the electrospun materials is shown below in Table 45
The results indicated that the addition of the silver particles to the CMC/PVA/PEO material was negligible on the spinability of the material. That is, each of the samples containing silver particles was able to be spun and produce three-dimension or “fluffy” substrates of nanomaterial. The sample with 100 ppm of silver nanoparticles was nearly identical to the sample without silver, and the sample with 300 ppm was less three-dimensional and more compact than the sample without silver particles. As such, the results indicate that there is the potential to add silver particles to layers of material, if desired.
G. Comparison of Hemostatic Qualities on Various Electrospun Materials
As discussed above, a wide range of materials can be electrospun to form matrices, with the matrices having varying qualities. One of the more important qualities for the matrices is hemostasis, which is, to some extent, a requisite for most wound dressing assemblies. Two tests were carried out to compare hemostatic properties potential wound dressing assemblies. The first test compared various electrospun nanomaterial matrices against one another, while the second test compared electrospun wound dressing assemblies compared to non-electrospun chitosan wound dressing assemblies.
1. Comparison of Hemostatic Qualities of Electrospun Nanomaterials
Five different wound dressing assemblies samples and two of these five wound dressing assemblies in combination with one another were examined for their haemostatic ability. Blood clotting capacity was determined by evaluating activation of the intrinsic pathway of the blood coagulation cascade, this assay was performed by measuring contact activation enzymes (Factor XII and kallikrein) activity. Platelet aggregation assays were employed to determine if wound pads activated platelets in platelet rich plasma (PRP). Also whole blood clotting time assays were used to measure the complete blood clotting capacity of the wound pad samples.
Coleman Whole blood clotting test
Blood was drawn from healthy, non-aspirin-using volunteers using butterfly needles. Subsequently, 0.9 ml aliquots of the non-anti-coagulated blood was rapidly but gently, distributed into reagent-loaded (1.4×1.4 cm wound pad samples presoaked in deionized water) polystyrene tubes. There were three tubes for each determination. The tubes were tilted in a sequential manner and clot times were determined by the obvious formation of clots. The first tube was tilted at regular intervals until clotting was observed. Then the second tube was tilted until clotting was seen. This was repeated for the third tube and the clotting time of this tube was recorded as the clotting time of the hemostat.
Clot times were expressed as the average of the three independent experiments and time zero was taken as the time the first tube was tilted.
To deem whether a sample is haemostatic or not:
For whole blood clotting times, values less than 60.85% are haemostatic and values equal to or less than the positive control are very haemostatic.
The results are shown in
2. Comparison of Electrospun Wound Dressing Assemblies to Other Types of Wound Dressing Assemblies
The hemostatic properties of wound dressing assemblies produced according to the present invention were compared to the hemostatic properties of prior art wound dressing assemblies. Particularly, chitosan-based wound dressing assemblies produced and manufactured by HemCon, Inc. were tested against electrospun wound dressings of the present invention that contained nanomaterial chitosan and nanomaterial MDOC. All samples were examined for their haemostatic ability. Blood clotting capacity was determined by utilizing a range of clotting assays. This gives a detailed account of the modes of blood clotting action of the samples tested. Enzyme activity assays were carried out to determine activation of the contact phase of the intrinsic pathway of the blood coagulation cascade, this assay was performed by measuring contact activation enzymes (Factor XII and kallikrein) activity. Also assays to measure the intrinsic and extrinsic pathways of blood coagulation were utilised. Platelet aggregation assays were employed to determine if samples activated platelets in platelet rich plasma (PRP). Also whole blood clotting time assays were utilised to measure the entire blood clotting capacity of samples.
Nano chitosan samples were prepared at a density of g/m2s and contain 10% PEO and 90% chitosan, as previously discussed above. A non-crosslinked sample and a crosslinked sample (130° C., 10 min in drying chamber) were tested.
Sample 32 is an MDOC nanomaterial dressing assembly containing PVA+008MC1007 (6:4) with 20% of glycerin and 8% of H3PO4 related to the dry mass of the basic blend. 008MC1007 is the MDOC component. As discussed above, MDOC is an oxidized cellulose composing of calcium and sodium salts. Nanofibers are impregnated with MDOC and are arranged into nanopads by spinning fibers to a nano-scale. It forms a very thin layer which does not dissolve in water.
The HemCon wound dressing assembly is a HemCon® Bandage commercially sold and distributed by HemCon, Inc. of Portland, Oreg.
5 mg samples were weighed and added to 1 ml of 100 mM HEPES buffer in an eppendorf tube. Tubes are rotated for 30 minutes. Equal volumes of sample solution and human plasma were mixed and allowed to stand for 10 minutes. 40 ml of this mixture was added to 40 ml of 0.8 mM S-2302 (see below for details). The absorbance change at 405 nm was kinetically measured. This enabled measurement of the activation of the clotting system. HEPES buffer acted as a negative control and 1% m·doc powder made up in HEPES buffer acted as the positive control.
The Factor XII and kallikrein enzymes (clotting factors) become activated in the presence of m·doc·. The plasma kallikrein-like activity catalyses the splitting of p-nitroaniline (pNA) from the substrate H-D-Pro-Phe-Arg-pNA (S-2302). The rate at which the pNA is released was measured photo metrically at 405 nm.
In summary, if the product is haemostatic it will lead to a color change and represent activation of the intrinsic pathway.
The APTTs were determined using sample solution in HEPES buffer as agonists to activate the blood coagulation cascade when the intrinsic pathway was activated. Again HEPES buffer acted as a negative control and 1% m·doc powder made up in HEPES buffer acted as the positive control. The assays were carried out on a 96 well plate with the contents of each well outlined in Table 46.
All the components (excluding the calcium chloride) were added to the wells and mixed once, then after a 10 minute wait the calcium chloride was added using a multichannel pipette directly before the experiment was carried out. The optical density change was recorded over 60 minutes at 405 nm using a microtitre platereader. Readings were taken every minute and samples were also agitated every minute. The assays were carried out in duplicate. To interpret results Microsoft excel was used, the optical density readings taken from each well every minute represent the absorbance of the contents of well. A curve was constructed of absorbance at 405 nm against time and the half max of the curve represents when the clot occurred. These values were used to construct a bar chart of clotting times for the agonists used. The max absorbance values represent the strength of the clot formed.
These prothrombin times were determined using sample solution in HEPES buffer as agonists to activate the extrinsic pathway of the blood coagulation cascade. HEPES buffer acted as a negative control and 1% m·doc powder made up in HEPES buffer acted as the positive control. The assays were carried out in a 96 well plate with the contents of each well outlined in Table 47.
aCephalin (1 μg/ml in TBS)
bThromboplastin was 1 μg/ml in TBS.
All reactants were mixed, added to the wells and allowed to incubate for 10 minutes. The calcium chloride was added last using a multichannel pipette directly before the optical density was read. The optical density change was recorded over 60 minutes at 405 nm using a microtitre platereader. Readings were taken every minute and samples were also agitated every minute. The assays were carried out in duplicate. To interpret results Microsoft Excel was used, the optical density readings taken from each well every minute represent the absorbance of the contents of well. A curve was constructed of absorbance at 405 nm against time and the half max of the curve represents when the clot occurred. These values were used to construct a bar chart of clotting times for the agonists used. The max absorbance values represent the strength of the clot formed.
Blood was drawn from healthy, non-aspirin-using volunteers using butterfly needles. Blood was anti-coagulated with sodium citrate (4%, 1:9, v/v) and centrifuged at 250×g for 10 minutes in capped plastic tubes. The straw colored upper phase was collected and this was the platelet rich plasma (PRP). Aggregation of platelets was performed in 96-well plates. 140 μl of PRP were pipetted per well in 96-well plate. The plate was pre-incubated at 37° C. for 3 minutes. The solution made from 2 mg samples in PBS were added to wells (10 μl) using a multichannel pipette and samples were agitated for 10 seconds. The optical density change was recorded over 30 minutes at 650 nm using a microtitre platereader. During the run the plate was incubated at 37° C. and readings were taken every 20 seconds and samples were also agitated 20 seconds.
Blood was drawn from healthy, non-aspirin-using volunteers using butterfly needles. Subsequently, 0.9 ml aliquots of the non-anti-coagulated blood was rapidly but gently, distributed into reagent-loaded (2 mg samples presoaked in deionized water) polystyrene tubes. There were three tubes for each determination. The tubes were tilted in a sequential manner and clot times were determined by the obvious formation of clots. The first tube was tilted at regular intervals until clotting was observed. Then the second tube was tilted until clotting was seen. This was repeated for the third tube and the clotting time of this tube was recorded as the clotting time of the hemostat. Clot times were expressed as the average of the three independent experiments and time zero was taken as the time the first tube was tilted.
The results are shown and discussed in
As is demonstrated from the above examples, electrospun nanomaterials provide for a wide range of therapeutic qualities, dependent on the chemical composition of the material that will be used in the electrospinning process. These qualities can be used as the basis for which materials may be used in an eventual wound dressing assembly. Depending on the particular requirement for a specific wound dressing, one or more of the various materials may be used in a wound dressing assembly.
Besides changes to the compositions of the electrospun materials, alterations to the parameters of the electrospinning process can also affect the properties of the resultant electrospun matrices. The following section discussed possible adaptations.
The electrospinning process was generally described with respect to
A. Use of an Air Stream During the Electrospinning Process
A hot air stream (105° C.) can also be used as part of the electrospinning process. Depending on requirements the temperature of the air stream can be adjusted for a particular electrospinning process and it is used to increase the number of inter-fiber pores in the nanofiber matrix.
Matrices comprising chitosan and polyethylene oxide (PEO), as discussed above, were produced with and without the use of a hot air stream (105° C.) during the electrospinning process. The resultant Chitosan/PEO products were tested for absorption capabilities at a number of different densities. The same standard Chitosan product was also electrospun with the air stream described turned on. Also, the Chitosan nanofibers were electrospun and collected on a needle collection apparatus both with the air stream on and off. With the air stream off the process runs in an accumulation state with the humidity in the space climbing to some max level. An air stream would provide a steady state environment which will provide more consistent results from run to run. All four Chitosan/PEO samples were analyzed for their absorption capability and the results are presented in
Absorption capacity decreased with increasing area weight. It was also clear that using the airstream approximately doubled the absorption capacity of the produced sample. It is assumed that these interfiber pores can hold and retain fluid better than the normally packed nanofiber matrix. Also, in case of spinning on needle apparatus the absorption capacity is higher when the air stream is used.
The effect on the basis weight of the produced samples with increasing nanofibers layers was also examined. Again this was performed both with and without the airstream. The results are presented in
B. Change of Voltage Used During The Electrospinning Process
As discussed above with respect to
Method
A solution containing 4.26 wt % dry matter, with a ratio of chitosan to polyethylene oxide (PEO) at a ratio of 93.6/6.4 w/w was tested to determine the effect of voltage change on the material. The solution had the parameters as listed in Table 50, below. “Fluffy” chitosan material
The electrospinning device had the following parameters.
Spinning electrode: smooth cylinder—35 cm length
Collector: cylinder
Electrode distance: 130 mm
Electrode spin: 4 rpm
Speed of the substrate: Initially 0.05/min, 2.5-3 cm/min during the spinning process
The solution was weighted before and after electrospinning process and an area of nanofiber sample was measured. The electrospinning process took approximately 24 minutes. Due to the high time consuming process, multiple solutions were prepared to carry out the testing and experiments, which had various viscosities and pH in dependence to temperature. For each combination of high voltage, a one- or two-cycled sample was tested and these samples were either in a treated (“crosslinked”) or non-treated (“non-crosslinked”) form. The one-cycle samples were processed using a new solution added to the reservoir of solution (see reference numeral 116,
Temperature was between 19.6-23° C. and the relative humidity was between 25.7-36% during the testing process. The following high voltages were applied during the testing process: +60/−30, +65/−30, +65/−35, +65/−40, +65/−45 and +65/−50 kV.
Properties of Solution and Nanomaterial
Conductivity, pH, viscosity, and temperature were measured for the tested solutions. There were some changes in viscosity due to different storage temperature. Generally speaking, conductivity and pH values of the solutions were affected by the storage time of the material prior the spinning process. Mixed solutions of chitosan and PEO that were mixed 24 hours prior to spinning and stored at room temperature did not appear to have the requisite conductivity for the spinning process. However, while mixing the chitosan/PEO solution approximately 30 minutes prior to the spinning process did provide the necessary conductivity and had an increased production capacity compared to the earlier prepared solutions, the homogeneity of the spun materials was lacking somewhat. When the storage temperatures of the 24 hour stored material was reduced (approximately to 14° C.), the solution was capable of being processed in the electrospinning process, providing a homogenous material, but with a lower production capacity It was determined that mixing the chitosan/PEO solution approximately 2 hours or so prior to spinning provided a balance between production capacity and homogeneity of the product.
Parameters were also measured for the nanofiber samples: basic area, fiber diameter, absorption capacity and area of nanofiber layer. Two absorption capacities were measured: immediately saturation of the sample (Aci), and after 1 hour leaching in demineralized water when weight of sample was constant (Act). This method includes absorption and swelling capability of the sample.
Parameters were also measured for the electrospinning process: consumption of solution after the electrospinning process (in g/min), various applied high voltages, relative humidity and temperature in laboratory.
Generally, nanofiber diameter decreased with an increase in voltage, as shown in
As the results indicate, the “best combination” is a voltage of +60/−30 up to +65/−40 kV (90 kV to 115 kV). The highest absorption capability was achieved for +60/−30 kV, which was produced with a prepared polymer solution prepared approximately 2 hours before the electrospinning process. For all samples for +60/−30 kV, +65/−35 kV, the preparation time for the solution prior to electrospinning was approximately 2 hours. For the samples having currents of +65/−40 kV, solutions were prepared both at approximately 2 hours before electrospinning and at approximately 20-22 hours prior to electrospinning, with the latter being stored at approximately 14° C.
For the samples having currents of +65/−45 kV, the solution was mixed approximately 20-22 hours prior to electrospinning at a cool temperature (approximately 14° C.). The initial results indicated that the viscosity of the solution possibly increased, while production capability of the process, diameter of nanofibers and water absorption capability decreased. However, it should be understood that the samples were prepared differently, which makes it difficult to make a definitive correlation between the characteristics. That is, while initial indications tend to show a possible correlation, it may be that such a correlation is not prevalent.
The initial results indicate that with higher viscosity of the material, there may be a decrease in the diameter of the nanofibers, as shown in
As such it can be seen that the voltage delivered during the electrospinning process has a potential effect on the produced materials. Depending on the eventual wound dressing assembly the material will eventually be used with, the voltage can be adjusted accordingly to change the absorption capacity or fiber diameter of the materials.
C. Different Electrospinning Methods
As previously discussed, there are alternate electrospinning processes or methods. Generally speaking, electrospinning methods relate to the way the nanomaterials and nanofibers are collected during the electrospinning process. The following section discusses alternate collection processes for the nanomaterials. The first spinning process is a wire collector process, with the spunbond moving passed the collecting electrode at the lowest fabric speed available (Spinning I). The second process alternatively used needle collection with a piece of spunbond material fixed on needles (Spinning II). Both processes were used in a dispersion system of MDOC in polyvinyl alcohol (PVA). The MDOC particles are dispersed in a continuous phase of the PVA solution.
The dispersion mixture for spinning process I was a 16% m/m solution of MDOC and PVA, which was mixed in a ration of 4:6. The 16% solution (16±1.5%) was purchased from Novacke chemicke zavody, Slovakia (http://www.nchz.sk/?str=uovd&lang=en). The MDOC dispersion was prepared with 51.2 g of MDOC mixed with 270 g of deionized water using an UltraTurrax T25. The resulting dispersion was mixed with 479.9 g of the 16% solution and centrifuged for 3 minutes at 4000 rpm to remove any bubbles in the solution prior to electrospinning and testing of the viscosity measurements.
The system parameters were as follows:
Electrode: Big smooth cylinder
Fabric Speed: 0.13 m/min
Electrode spin 3.2 rpm
The dispersion spun for approximately 60 minutes, with a total dispersion mass of 800 g. The spinning time was approximately 1 hour, with samples of the materials taken at approximately every 15 minutes and the results were recorded. The following characteristics were noted:
Temperature and humidity of input and output air were monitored during 60 minutes of spinning using data loggers. HV current was recorded every 1 minute. The following results were noted.
Temperature of dispersion in bath was measured at 0, 15, 30, 45 and 60 min.
Parameters measured for dispersion: viscosity, loss on drying/dry matter; conductivity
Parameters measured for nanospun material: only a thin layer of nanomaterial was spun onto spunbond; hence, only approximate values of area weight are presented. Consequently loss on drying/dry matter was not determined.
The dispersion used for the Spinning II process was the same as describe above for the Spinning I process.
The system parameters were as follows:
Electrode: Big smooth cylinder
Collector: Needles with Spunbond Fixed
Electrode spin 3.2 rpm
The MDOC:PVA dispersion was spun 4 times for 15 minutes. The nanospun material and spunbond was removed after spinning and replaced by new piece of spunbond. The following characteristics were noted:
Temperature, humidity, HV current
Temperature and humidity of input air were monitored during spinning using data loggers. HV current was recorded every 1 minute. The following characteristics were noted:
Parameters measured for nanospun material: area weight, loss on drying/dry matter, fiber diameter (Scanning electron microscope image provided using JEOL 6490 LV, microscope in cooperation with Laboratory of electron microprobe and microscopy, Department of Geological Sciences, Faculty of Science, Masaryk University, Brno, CZ).
The effect of temperature was examined in range 17-25° C. The effect of dry matter in dispersion was_examined in range 14.9-16.7%. The lowest value (14.9%) corresponds to a regular MDOC:PVA dispersion. Higher concentration of polymers in dispersion was provided by partial evaporation of water from regular dispersion using a rotary vacuum evaporator. Dry matter of dispersion was determined experimentally. The following characteristics were noted:
Temperature of dispersion bath decreases as a result of solvent evaporation even when the spinning is switched off. Contribution of spinning process itself to the temperature decrease has a minor effect.
Concentration of polymer(s) in dispersion increases during spinning due to solvent evaporation. Viscosity of dispersion increases as a result of combination of both effects (increasing concentration and drop in temperature). The effect of increasing concentration seems to be more crucial; however, the exact role of both effects simultaneously would have to be investigated in more details to get conclusive results.
Decreasing area weight of nanomaterial can be observed as a result of drop in electrospinning efficacy in time. SEM analysis did not reveal significant differences of nanofiber diameter or morphology during spinning.
The following is a simple scheme of observed effects:
Evaporation of solvent (water)→temperature drop & thickening of dispersion due to evaporation→viscosity increase→decrease of electrospinning efficacy & HV current→decrease in nanomaterial area weight.
Wound dressing assemblies according to the present invention may comprise a wide range of materials, having a wide range of therapeutic and hemostatic properties. The wound dressing assemblies may comprise multiple layers of materials, with each layer of material providing a different quality for the overall wound dressing assembly. Once it is determined what specific qualities are to be used in a wound dressing assembly, the present invention provides guidance in determining individual layers for a dressing assembly, as well as the ability to the electrospinning process to further provide materials having various characteristics.
The foregoing is considered as illustrative only of the principles of the invention. Furthermore, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described. While the preferred embodiment has been described, the details may be changed without departing from the invention, which is defined by the claims.
