Combined electrostatic and air driven electrospinning for biomedical applications

Electrospinning (ES) is an additive manufacturing process that produces micro-to-nanoscale polymer fibers with extremely high surface-area-to-volume ratios. Electrospun polymer fibers are used in a wide range of applications, including biomedical,^1,2 energy transfer and storage,³ water filtration,^3,4 and material strengthening.⁵ ES devices can be used to deposit fibers directly onto conductive surfaces in the traditional setup or onto nonconductive surfaces by placing the substrate between a conductive spinneret and electrode. ES hardware typically includes large, table-top equipment that electrically isolates the device for safety, a syringe pump to move polymer into the system, and a high-voltage power supply. Deposition is limited to flat, noncomplex substrates placed directly in the path of the spinneret. Deposition of fibers onto geometrically diverse objects is extremely difficult with traditional ES units due to interrupted electrostatic forces and lack of maneuverability.

To address the limitations of traditional ES, portable ES devices have been developed. Portable ES devices enable maneuverability during deposition onto complex surfaces. Existing portable ES devices essentially consist of the same components as a table-top unit, with the exception of the electrical isolation box and syringe pumps being miniaturized to fit in a hand-held or relatively smaller device. These devices have shown results similar to table-top ES units and are capable of ES directly onto human skin.^6–8 The devices previously reported require the use of an electrode attached to the target surface during the ES process. Such a configuration grounds the surface and creates a voltage differential between the spinneret and the surface, thus allowing for ES and subsequent deposition onto the grounded substrate to occur. For biomedical applications, it is problematic and dangerous to place living tissues into the unprotected high-voltage electric field during ES. Additionally, by placing noncharged substrates into the ES electric field, a barrier is presented that shields the electric field and leads to unpredictable effects. ES onto a conductive substrate occurs at the position of the highest electrostatic field strength. When an uncharged object is placed between the spinneret and the conductive surface, electrostatic field lines wrap around the obstruction and terminate on the obscured conductive surface. Such an effect leads to unpredictable fiber deposition, and ES of conductive polymer materials results in a complete electrical circuit from the spinneret to the conductive surface, resulting in arching in open air and destruction of the resultant fibers.

Here, a portable ES device with a completely encased electric field is presented. The portable device described uses electrostatic force for fiber creation inside a quick-connect barrel, and an airflow deposition system that overcomes the electrostatic force pulling fibers toward a ring electrode, alternately driving the fibers onto an uncharged surface beyond the end of the barrel. In this system, the deposited substrate and fiber materials are inherently protected from the damaging results that come with the high-voltage environment present in traditional ES. The portable, combined electrostatic and air driven (EStAD) ES device provides a 40× reduction in volume when compared to a traditional table-top unit. The EStAD device is battery operated and uses a spring-loaded syringe pump, eliminating the dependency on plug-in power and associated hardware and allowing free movement during ES to uniformly coat compound surfaces. The EStAD device can be used as a traditional ES unit with a grounded target surface as well as in the ungrounded configuration, allowing for versatile operation of the equipment for on-demand deposition onto wound sites or creation of bandages for later use.

Table-top ES devices have been used to manufacture polymer dressings that can be used as bandages, and drug-delivering fibers have been shown to kill and limit the growth of harmful bacteria in wounds.^9,10 While bandages made using traditional ES methods require stationary manufacturing areas and sterile storage environments, portable ES devices allow drug-delivery materials to be placed directly on wound sites, simplifying the bandage production process and decreasing the time to treatment delivery. To test the capabilities of drug-delivery bandages deposited with the EStAD device, biocompatible polymers doped with antibiotics were manufactured and tested on Staphylococcus aureus (S. aureus), a bacterium known to cause severe skin infections.

The EStAD device manufactured bandages using two types of polymer solutions. All polymers used in ES experiments were purchased from Sigma-Aldrich. The first polymer solution used polyethylene oxide (PEO) with 200k average molecular weight (MW) dissolved into de-ionized (DI) water at 14 wt. %. The polymer was added directly into DI water and blended at 30 °C for 1 h until dissolved.

The second polymer solution was a blend of cellulose diacetate (CDA) with 30k MW and PEO with 100k MW. The polymer to polymer weight ratio of the blend was 35:65 CDA to PEO at 7 wt. % in chloroform. The polymers were added directly into chloroform, heated to 30 °C, and mixed with a stir bar for 2 h until dissolved.

The EStAD device produces bandages using two manufacturing methods: direct and transitional bandage deposition. With direct bandage deposition, bandages are electrospun directly onto wound sites. Transitional deposition uses traditional ES methods to produce a bandage onto a collection surface to be removed and stored to be placed onto a wound site at a later time. Both methods were tested using the EStAD device and 14 wt. % PEO solution.

To demonstrate direct deposition, a gloved hand was placed 10 cm from the exit of the flow chamber for 3 min (Fig. 3). During direct ES deposition, the air velocity was set at 7.25 m/s, the spinneret used was 20 gauge, and the voltage supplied to the spinneret was 10 kV. In the previous work, it was found that stable ES with the EStAD device occurred between 4 and 16 cm separation distance to ensure reliable deposition onto the substrate.¹¹ Previous studies also showed that reliable ES with the portable system occurred between 8 and 10 kV and with airflow between 6.90 and 7.25 m/s.¹¹ No external electrodes were used. During transitional ES deposition, airflow, the spinneret used, and the voltage supplied to the spinneret were the same values used for direct ES deposition. Transitional deposition was directed onto a parchment paper held 6 cm from the end of the barrel. The parchment paper used was attached to a grounded electrode.

Manufacturing drug-delivery bandages through direct and transitional deposition with the EStAD device was demonstrated by the release of antibiotics onto bacterial lawns. Vancomycin hydrochloride suspended in dimethyl sulfoxide with an effective drug concentration of 100 mg/ml was purchased from Sigma-Aldrich and used as the antibiotic for the drug release trials. The vancomycin was blended with the 14 wt. % 200k MW PEO polymer solution, giving the drug an effective concentration of 4.4 mg/ml. The S. aureus (PS88 strain, ATCC® 33742) used for these tests was grown from frozen stock on 3% tryptic soy agar plates supplemented with 25 ml/l of a 20% dextrose solution and 1 mM citrate (TSA⁺⁺) and grown for liquid culture in tryptic soy broth with 25 ml/l of 20% dextrose solution and 1 mM citrate (TSB⁺⁺). Liquid cultures were prepared by picking a single colony and placing it in 3 ml of TSB⁺⁺ overnight at 37 °C and 225 rpm. Plates prepared for testing antibacterial bandages were TSA⁺⁺ that were streaked with a loop containing a single bacterial colony lifted straight from a TSA⁺⁺ plate or by dipping the loop in liquid culture and then streaking the TSA⁺⁺ plate. Immediately after preparing the streak plate, direct and transitional deposition methods were used to deposit the vancomycin + PEO polymer fiber mats or PEO-only control fiber mats onto the streak plates. After deposition, bacterial plates were placed in an incubator at 37 °C for 24 h.

The gold colloid solution used was sterile and contained 20 ppm (0.02 mg/ml) gold nanoparticles suspended in DI water and was purchased from Purest Colloids. The gold colloid solution was used as a substitute for standard DI water. For this polymer solution, 200k MW PEO was added directly into the gold colloid solution to create a 14 wt. % polymer solution. The mixture was heated to 30 °C and mixed with a stir bar until the polymer was dissolved. CDA + PEO blend polymer fibers were also electrospun for observation via a scanning electron microscope (SEM). The polymer solution for these fibers was prepared by mixing gold colloid purchased from Sigma-Aldrich into the CDA + PEO polymer blend to achieve a gold nanoparticle concentration of 0.002 mg/ml.

Bandages were electrospun using transitional deposition from the gold colloid + PEO polymer solution and neat PEO polymer solution. The bandages were manufactured using a polymer flow rate of 0.5 ml/h, 10 kV applied voltage, 6.9 m/s air velocity, 20 gauge spinneret, and 6 cm separation distance between the end of chamber and the deposition surface. An electrospun 35:65 CDA to PEO fiber mat was placed in DI water for ∼24 h and removed. The fiber mat was then dried in an environment held at 30 °C until the retained moisture was completely driven off. Once dry, a sample was cut, placed on a sample stub, and sputter coated with gold for SEM and energy dispersive x-ray spectroscopy (EDS) analysis.

The dissolved gold colloid + PEO bandage solutions were examined via a Micromanipulator probe station equipped with an optical microscope and an Ocean Optics USB 4000 spectrometer. Droplets of the solution containing dissolved bandages were placed on aluminum foil and positioned within the focal region of the optical microscope. Aqueous samples from a dissolved gold colloid + PEO bandage, a dissolved neat PEO bandage, and the pure gold colloid were analyzed.

Bandages were also made with the 35:65 CDA to PEO and gold colloid solution using the EStAD device and analyzed via a Tescan Mira 3 SEM with a field emission electron gun equipped with an EDAX Octane Elect EDS system. These bandages were manufactured with transitional deposition using the following operating parameters: a polymer solution flow rate of 1.5 ml/h, 10 kV applied voltage, 7.25 m/s air velocity, and 8-cm separation distance between the end of chamber and the deposition surface. After deposition and removal of the bandages, small sections were cut from each bandage and placed on aluminum sample stubs. Samples were sputter coated with gold for ∼1 min before SEM-EDS analysis.

Both direct and transitional ES were used to deposit materials for this work. While direct deposition allows for the ease of fabrication and decreases the treatment delivery time, transitional ES can be used when the fabricated material is intended to be stored for treatment delivery at a later time. In Fig. 1, direct deposition is demonstrated onto a gloved hand. In this setup, no external electrode is required and, therefore, safety and predictability of the fiber mat deposited is improved as compared to a traditional table-top ES unit. In Fig. 2, the transitional deposition method is shown.

Various substrate materials were tested to demonstrate satisfactory deposition onto a variety of noncharged and nonflat substrates with the EStAD device. Polymer fiber mats made from PEO were deposited onto a gloved hand as shown in Fig. 1, as well as onto produce samples (Fig. 3), and porcine skin samples (Fig. 4). PEO was selected for the ES solution in this trial, as it is an established biocompatible and biodegradable polymer.¹² PEO is a desirable polymer for drug-delivery bandages where rapid treatment release is needed. The highly soluble nature of PEO in aqueous environments allows bandages to dissolve into wound sites quickly and release the encapsulated drug.

Produce was used as a testing surface because of the characteristic of having a dry exterior and moist interior, which resembles human physiology. Fibers were visibly more likely to adhere to materials that contained internal moisture as compared to completely dry materials, likely due to changes in charge storage and dissipation. An apple was damaged for a wound site simulation, and electrospun fibers were observed bridging the damaged surface. The watermelon substrate demonstrated a similar fiber adhesion and resulting fibers covered a large area of the surface.

In addition to fruit substrates, ES was tested on various nonconductive, natural skin samples to further examine the capability of the device for biomedical treatment of a mammalian tissue. Figure 4(a) shows a wounded porcine skin containing a 6.5 cm incision. The EStAD device was used to directly deposit PEO fibers across the wound site and is shown in Fig. 4(b). The EStAD device demonstrated nearly complete coverage of the porcine skin wound site. The upper section of the incision was covered completely with a thin layer of PEO fibers, while the lower half had a much thicker deposited fiber mat. During this test, the device was kept stationary when depositing the bandage, resulting in a noncentralized fiber mat concentration. When the EStAD device is held by a user and directed across the entire wound as the bandage forms, fiber placement can be better controlled and result in a more uniform bandage.

Antibiotic + PEO bandages manufactured with the EStAD device were delivered to bacterial plates using both direct deposition and transitional deposition (Fig. 5). Effectiveness of the antibiotic bandages deposited was determined by observed death zones on the bacterial lawns indicating antibiotic release.

Direct deposition of a water-soluble bandage was difficult to observe because the fibers immediately dissolved when in contact with the moist surface (Fig. 5). To be consistent, trials were timed for 5 min of continuous ES. Direct deposition using the EStAD device was conducted with both antibiotic-doped PEO and neat PEO, which acted as a control to ensure the polymer itself was not killing bacteria. Figure 6 shows the results from the direct deposition tests.

When compared to the control bacteria plate [Fig. 6(a)], the neat polymer solution did not affect the growth of the bacteria on its respective plate [Fig. 6(b)]. Alternatively, a large circular death zone was observed on the plate, which was electrospun onto with the antibiotic + PEO polymer solution. These results are promising that the EStAD device can provide direct deposition onto human tissues to prevent bacterial growth.

To manufacture antibiotic bandages using the transitional method, antibiotic + PEO fibers were deposited onto a parchment paper that was laid on top of an electrode in order to decrease adhesion after deposition so that the bandage could be removed. The bandages produced using transitional deposition were placed on S. aureus bacterial lawns immediately after streaking and then left in the incubator for 24 h before observations were made (Fig. 7).

The bandage containing antibiotic prevented bacteria growth in the area that it was placed. The death zone approximately matches the area where the bandage dissolved into the plate. In the case of the PEO-only bandage, the opposite result was observed, and bacteria growth increased in the area where the bandage dissolved. The most likely cause for the increase in bacteria growth is that the liquid polymer from the dissolved bandage acted as a transport mechanism for bacteria and spread growth from the initial streak. Although transitional deposition with the EStAD device is less novel than direct deposition, it speaks to the versatility of the device and broadens the applications for which it can be used.

Nanoparticles have been used in electrospun fiber meshes to prompt treatment release when interrogated with light,¹³ to supplement antibiotic therapies such as phage therapy,¹⁴ and even to provide antibacterial activity themselves upon release.¹⁵ Due to the breadth of biomedical applications related to electrospun materials containing antimicrobial particles, the EStAD device was used to deposit gold nanoparticle-containing electrospun bandages. For this demonstration, a CDA + PEO polymer blend and PEO were used as the carrier polymers. PEO is a well-known biocompatible polymer. Previous research has shown that a 2:1 weight ratio of CDA to PEO polymer blend can act as a delayed release mechanism for drug delivery.¹⁶ Individual fibers swell and rupture when introduced to an aqueous environment, thus releasing encapsulated drugs. The CDA to PEO blend was used and modified to fit the EStAD device by lowering the total weight percent of the polymer and increasing the amount of PEO. The airflow within the chamber of the EStAD device increased the evaporation rate of the solvent, consequently hardening the solution at the spinneret. The increase of solvent in the blend counteracted the high evaporation rates, making ES of fiber mats possible. Increasing the amount of PEO in the blend ultimately increased the overall solubility of the bandage and resulting release rate.

Figure 8 shows CDA + PEO blend fibers containing gold colloid before and after being placed in an aqueous environment for 24 h. After 24 h in an aqueous environment, CDA + PEO fibers appeared to lose fiber morphology and exhibited swelling. The noticeable beading on the fibers in Fig. 8 is a known occurrence during ES with both PEO and CDA; increasing polymer concentrations and applied voltage will decrease beading of produced fibers.^17,18 Transitionally, electrospun CDA + PEO fiber bandages containing gold nanoparticles were also analyzed with EDS and the data are shown in Fig. 9.

To monitor antibiotic release from PEO fibers, ultraviolet-visible (UV-Vis) spectroscopy was used. Gold nanoparticles exhibit a maximum plasmonic response at 522 nm. Results from the PEO + gold colloid fibers are shown in Fig. 10.

The gold colloid had a reflectance response between 550 and 600 nm and increased over this range. The PEO + gold colloid bandage demonstrated the same response as the pure gold colloid, leading to the conclusion that effective nanoparticle release was obtained after dissolving the PEO + gold colloid bandage. This suggests that the EStAD device has the capability to manufacture bandages that release biomedically relevant nanoparticles into wound sites for antimicrobial purposes.

ES is an additive manufacturing process that produces micro-to-nanoscale polymer fibers with extremely high surface-area-to-volume ratios for a wide range of applications from biomedical to energy transfer and storage. Drawbacks to traditional electrospinning include deposition directly on a charged electrode and a lack of modularity of the ES hardware. Deposition of fibers onto compound surfaces with arbitrary electrical properties is made possible with the EStAD device, which is inherently safe for fiber deposition onto patients and nondamaging for fibers with high electrical conductance.

The EStAD device provides a 40× reduction in volume when compared to a traditional table-top unit and has a built-in portable power system. Fiber formation occurs in an isolated barrel before airflow forces the fibers beyond the system and onto an uncharged surface beyond, completely isolating the deposition surface from the electric field. Such a configuration also prevents the damaging results that come with the high-voltage environment required in traditional ES. The EStAD device can be used as a traditional ES unit with a grounded target surface as well as in the ungrounded configuration, allowing for versatile operation of the equipment for on-demand deposition (direct deposition) onto wound sites or creation of bandages for later use (transitional deposition).

Traditional ES has been used for a plethora of biomedical applications. To demonstrate feasibility of the EStAD device, several biomedical materials were fabricated by the EStAD device and subsequently characterized. Results showed that both direct deposition onto bacterial growth and manufacture and subsequent transport of antibiotic polymer fiber bandages onto bacterial growth (transitional deposition) resulted in effective antibiotic drug release and bacterial death.

Metallic nanoparticles have been used for various drug-delivery applications from electrospun fibers, and blending of hydrophilic and hydrophobic polymers has been used to control release of treatment over time. Here, we showed successful deposition by the EStAD device of polymer blend fibers containing gold nanoparticles. In addition, successful gold nanoparticle-releasing PEO fibers were fabricated and successful nanoparticle release was monitored by UV-Vis spectroscopy.

The EStAD device successfully demonstrated the fabrication of several biomedically relevant electrospun materials that can be deposited directly or fabricated and stored for later use. The versatile nature of the EStAD will allow deposition of these materials safely and predictably on charged and uncharged surfaces that are flat or complex, further broadening the applications of ES and providing the ease of access to nanomanufacturing of electrospun materials.

Research was sponsored by the Combat Capabilities Development Command Army Research Laboratory and was accomplished under Cooperative Agreement No. W911NF-15-2-0020. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Combat Capabilities Development Command Army Research Laboratory or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation herein. Research was also sponsored by the gracious support of the Slater Family Research Trust. In addition, the authors would like to acknowledge the Montana Tech Nanotechnology Laboratory, Sydney Jennings, and M. Katie Hailer for their support and contributions in conducting this work.