Each year, the United States spends about $20 billion to treat people who have been infected with antibiotic resistant bacteria. Even so, the development of new antibiotics has slowed considerably since the mid-20th century. As a result, researchers are looking into developing synthetic compounds and materials with antimicrobial activities such as those made by the Schanze and Whitten groups [ACS Appl. Mater. Interfaces 3, 2820 (2011)]. Previously, they have demonstrated that poly(phenylene ethynylene) (PPE) based electrolytes and oligomeric end-only phenylene ethynylene (EO-OPE) based electrolytes possess strong biocidal activity. However, before the PPE and OPE can be used with humans, skin irritation tests are required to ensure their safety. In this work, in vitro skin assays are used to predict in vivo irritation. Tissues were conditioned for 24 h, exposed to test substances for 1 h, and then tested for viability using colorimetric and cytokine assays. Concentrations up to 50 μg/ml were tested. Viability assays and cytokine (IL-1α) assays demonstrated that the two polymers, three symmetric oligomers, and three “end only” oligomers were nonirritants. In addition, electrospun mats consisting of several promising compounds, including poly(caprolactone), were evaluated. Therefore, all test substances are conservatively classified as nonirritants after a 1 h exposure time period.

In the United States, approximately 20 billion dollars are spent each year to treat two million people who are infected with antibiotic resistant bacteria.1 Even so, the development of new classes of antibiotics has slowed substantially since quilonones, gylocopeptides, and streptogramins were introduced in the mid-20th century.2 Naturally occurring antimicrobial peptides were first recognized in 1987. These compounds are part of the innate system response, and they have a potent biocidal activity, giving them great potential as novel therapeutic agents.

More recently, there have been efforts by researchers in developing stable synthetic antimicrobial peptides that will retain their biocidal activity outside of the body. These efforts led to the development of peptodomimetics including α-peptides,3,4 β-peptides,5–7 peptoids (oligo-N-substituted glycines),8,9 and cyclic peptides.10 Facially amphiphillic polymers, developed by the Tew group,11–13 as well as the Whitten and Schanze groups,14 have the amphiphillic feature of antimicrobial peptides and show comparable biocidal action. These synthetic analogs show promise as antibacterial, antifungal and antiviral agents, and thus far have shown no indications of inducing bacterial resistance.

The Schanze and Whitten groups have synthesized, characterized, and developed applications for conjugated phenylene ethynylene-based polymers (PPE) and oligomers (OPE) (reviewed in Ji et al.).14 All of these PPEs and OPEs demonstrate biocidal activity against both Gram-positive and Gram-negative bacteria, including Bacillus anthracis spores, Bacillus atrophaeus, Cobetia marina, and Pseudomonas aeruginosa strain PAO1.15–18 Furthermore, previous research done by our group has shown that in low concentrations, these polymers and oligomers are noncytotoxic to mammalian cells.19 

Potential applications include those in the public sector, such as in antimicrobial gloves and disinfectant wipes, as well as those in the medical sector (such as in wound dressings, hospital curtains, medical examination gloves, etc.). Since it is possible that formulations containing the antimicrobial PPE and OPE will produce contact hazards, evaluating for skin irritation is an important step. ISO 10993-10 provides guidelines for conducting skin and mucosal irritation, eye irritation, and skin sensitization tests.20 Traditionally, these guidelines for skin damage testing involve exposing animals (primarily rabbits and guinea pigs) to test chemicals for a period of time, and then observing them for any redness (erythema) or swelling (edema) that occur as a result of this exposure.20 In 2006, European legislation—specifically the registration, evaluation, authorization, and restriction of chemicals—has called for reducing animal testing on new products.21 

Several in vitro alternatives have been validated in multilaboratory studies.22 In this work, skin irritation is tested in vitro using Epiderm™ tissues to predict in vivo irritation. Epiderm tissues are human-derived products that model the effect of test substances on skin as approved by the European Centre for the Validation of Alternative Methods and approved by the U.S. Department of Health and Human Services via the Organization for Economic Cooperation and Development (OECD) Guidelines TG 439 as one of the three in vitro methods that may be used for identifying potential dermal corrosives.23,24 The Skin Irritation Test Protocol established for Epiderm tissues was used for these experiments.

Previously, we demonstrated that two polymers, six oligomers, and two electrospun mates are nontoxic to bovine aortic endothelial cells and Vero cells.19 In this work, we tested these same samples for skin irritation using an MTT assay and a cytokine assay for secondary confirmation.25,26 We found that all substances can be conservatively identified as nonirritants.

This study uses eight phenyl ethylene compounds including two polymers (mer = phenyl ethylene) [PPEDABCO, or 1,4-diazabicyclo[2.2.2]octane, and PPE-thiophene]; three “end-only” oligomers (mer = phenylene ethynylene) (EO-OPE-1-DABCO, EOOPE-1-Th, and EO-OPE-1-C2); and three symmetric oligomers (mer = phenylene ethynylene) [S-OPE-1(H), S-OPE-2(H), and S-OPE-3(H)]. The synthesis of these compounds has been described in detail previously.27–32 The structures are shown in Fig. 1.

Fig. 1.

Chemical structures of phenylene ethynylene polymers and oligomers, categorized by family.

Fig. 1.

Chemical structures of phenylene ethynylene polymers and oligomers, categorized by family.

Close modal

The polymer and oligomer solutions were made by diluting stock solution and sterile Dulbecco's phosphate-buffered saline (DPBS), a balanced salt solution. Stock solutions were prepared by weighing polymers/oligomers, dissolving them in 100 μl dimethyl sulfoxide (DMSO), a polar solvent, then adding 900 μl of ultrapure water. Electrospun mats were prepared and cut to size using sterile 6-mm biopsy punches. All polymers and oligomers were used “as-is” without any sterilization process. The concentrations used for the first week of testing were 10 μg/ml since this value correlated to observed biocidal activity in several species of bacteria, and it is also an intermediate value in mammalian cytotoxicity. To further test for potential irritation, the concentration of the polymers and oligomers was increased to 50 μg/ml during the second week. In addition, higher concentrations of PPE-DABCO and PPE-Th were tested during the third week due to preliminary cytoxicity testing showing that PPE-DABCO was more cytotoxic to mammalian endothelial cells than PPE-Th at 100 μg/ml.

Tissue kits consisting of 24 tissues per kit were used. Three replicates of Epiderm (Mattek, Ashland) were used to ensure tissue quality. All negative controls, polymer solutions, and oligomer solutions were tested in triplicate. Positive controls to check for interference with the MTT assay and electrospun mats were tested in duplicate or individually. All tissues were conditioned and incubated for an 18-h period at 37 °C, 5% CO2, and 90% humidity prior to testing following EpiDerm test kit's procedures in eight sets of six-well plates. Following the incubation period, tissues were dosed with liquid test substances or solid test substances. For liquid test substances, 30 μl were pipetted on to the skin surface and an 8-mm nylon disc was applied for uniform application. For electrospun mats, 30 μl sterile DPBS were pipetted onto the skin surface and the 6-mm mat was placed with sterile tweezers, an 8-mm nylon mesh disk was applied, and 20 μl of sterile DPBS were pipetted on the mesh. The additional 20 μl of DPBS were applied to fully wet the mat surface. See Fig. 2 for diagram of liquid and solid application. After a 1 h total dose period in the dark, the tissues were rinsed 15 times with sterile DBPS and returned to the incubator. After 24 h, warm assay medium was pipetted into the lower wells of the six-well plates while the medium in the upper wells of the six-well plates was removed for cytokine analysis. Cytokine analysis samples were frozen at −80 °C until time of testing.

Fig. 2.

Diagram comparing application of liquid and solid test substances to EpiDerm tissues. For liquid test substances, 30 μl of liquid were applied to the surface of the tissues followed by a nylon mesh to promote even wetting. For electrospun mats (the solid test substances), 30 μl of DPBS were applied, followed by the mat, the nylon mesh, and an additional 20 μl of DBPS for complete mat wetting.

Fig. 2.

Diagram comparing application of liquid and solid test substances to EpiDerm tissues. For liquid test substances, 30 μl of liquid were applied to the surface of the tissues followed by a nylon mesh to promote even wetting. For electrospun mats (the solid test substances), 30 μl of DPBS were applied, followed by the mat, the nylon mesh, and an additional 20 μl of DBPS for complete mat wetting.

Close modal

The 2 ml MTT concentrate (5 mg/ml) and 8 ml assay medium were warmed and combined to make a 1 mg/ml MTT solution. Tissues that had been incubated for a 24 h period were blotted and transferred to a 300 μl MTT filled 24-well plate. The tissues were then returned to the incubator for 3 h. After 3 h, MTT solution was aspirated from the wells and the wells were rinsed with sterile DBPS. The inserts were blotted and then transferred to a new empty plate. Isopropanol of 2 ml were added by pipetting directly above the skin surface so that the first milliliter fills the insert and the second milliliter overflows into the well, covering both sides of the tissue. The plate was then covered, wrapped in Parafilm, and placed on a plate shaker at a low speed for 2 h. After 2 h, the tissues were pierced with a tweezer to allow the liquid into the insert, the inserts were then removed, and the plate was covered and returned to the shaker for an additional 5 min. At least two aliquots of 200 μl each were made from the remaining 2 ml solution of MTT. At least two aliquots for each well and six isopropanol blanks were taken and added to the plate for immediate analysis. After 10 s of mixing, absorbance readings were taken every 5 nm between 540 and 595 nm.

For this assay, relative viability is based on absorbance (optical density, OD) at 570 nm in this fashion:

OD570(testsubstanceorcontrol,rawdata)OD570(blank,raw)=OD570(testsubstanceorcontrol),
(1)
Relativeviability=[OD570(testsubstance)/OD570(meanofnegativecontrol)]×100%.
(2)

Relative viabilities are calculated with respect to the mean of the negative control tissues of a kit since viabilities are expected to vary slightly among each EpiDerm kit. All polymer and oligomer solutions were tested for interference with the MTT Assay and none displayed a color change.

An interleukin-1α EIA kit (Cayman Chemical, Ann Arbor, MI) was used. Reagents were prepared according to the manufacturer's instructions.33 Media samples that were set aside during skin irritation testing were thawed and diluted with normal maintenance medium (MatTek, Ashland, MA). All samples (except 5% SDS, sodium doecyl sulectropherese, positive controls) were diluted 1:5 using the MatTEK protocol.34 Three assays were performed. Subsequent positive controls were diluted 1:6 because the first assay measured from the positive control (diluted 1:5) showed IL-1α concentrations nearly exceeding the high standard of 250 pg/ml. 1:6 diluted samples of 100 μl of each were added to the plate in triplicate, and 100 μl of eight standards were added to the plate in duplicate. IL-1α AChE Fab′ conjugate of 100 μl was added to each of the wells containing either samples or standards. Two wells were left empty to serve as blanks, and the whole plate was incubated at 4 °C overnight. Prior to plate development, Ellman's reagent from the IL-1α (human) EIA kit was reconstituted with 20 ml of ultrapure water. The plate was removed from the refrigerator, and each well was washed five times with wash buffer. After discarding the washing buffer, 200 μl of Ellman's reagent was added to each well, including those of blanks. The plate was covered with the provided cover sheet, covered with foil, and placed on an orbital shaker at setting 2. At 15, 30, 60, 120, 180, and 360 min, the absorbance at 412 nm (407–417, 1 nm step) was determined for each well on a plate reader. The plate was re-covered and returned to the shaker between each reading.

The threshold to distinguish irritants from nonirritants is 50% relative viability of cells after exposure to a test substance.32 If the relative viability is greater than 50%, then the chemical is considered a nonirritant, and if the relative viability is equal or less than 50%, then the chemical is classified as an irritant. Although cytokine analysis is not included in the OECD TG 439, guidelines for EpiSkin™, a similar human-derived in vitro skin test, can be seen in Table I as these numbers are not available for EpiDerm at this time.24 EpiDerm can be classified as a nonirritant if cell viability is >20% based on OECD's criteria for EpiDerm.24 We will be using the EpiSkin guidelines as they are more clearly stated and are of higher standards than those of the EpiDerm.24 

Table I.

Predictive classification model for two endpoints (MTT and IL-1α).

Relative viability (%)IL-1α release (IU/ml)Classification
≤50 >9 Irritant 
≤50 ≤9 Irritant 
>50 >9 Irritant 
>50 ≤9 Nonirritant 
Relative viability (%)IL-1α release (IU/ml)Classification
≤50 >9 Irritant 
≤50 ≤9 Irritant 
>50 >9 Irritant 
>50 ≤9 Nonirritant 

Previously, we studied cytotoxicity using an MTS assay,19 a colorimetric cell proliferation assay which determines relative cell proliferation and cytotoxicity at a high threshold of 70%. Figure 3 shows the results of this previous study in which epithelial cells (Vero cells) were exposed to each of the eight compounds for 24 h, with the final 50 min in the dark. It can be seen that cells show a decreasing trend in viability as concentrations increase. Figure 3 indicates that 50 μg/ml is the optimal concentration for PPEs and S-OPEs, as this is the highest concentration in which the compounds tested can be classified as nonirritants and nontoxic. Although all three EO-OPEs show a relative viability of less than 50%, indicating they are irritants at this level, 50 μg/ml was still used as the highest concentrations tested in this project as higher concentrations have improved biocidal capabilities, and this is the maximum concentration in which cell viability is ≥70% for PPEs.

Fig. 3.

Relative viabilities. (a) Relative viabilities of (±SD) of two polymers and six oligomers at 10 μg/ml for Vero (epithelial) cells exposed to each of the compounds for 24 h with the final 50 min in the dark. The solid black line indicates 50% relative viability, below which the substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells. (b) Relative viabilities of (±SD) of two polymers and six oligomers at 50 μg/ml for Vero (epithelial) cells exposed to each of the compounds for 24 h with the final 50 min in the dark. The solid black line indicates 50% relative viability, below which the substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells. (c) Relative viabilities of (±SD) of two polymers and six oligomers at 100 μg/ml for Vero (epithelial) cells exposed to each of the compounds for 24 h with the final 50 min in the dark. The solid black line indicates 50% relative viability, below which the substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells.

Fig. 3.

Relative viabilities. (a) Relative viabilities of (±SD) of two polymers and six oligomers at 10 μg/ml for Vero (epithelial) cells exposed to each of the compounds for 24 h with the final 50 min in the dark. The solid black line indicates 50% relative viability, below which the substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells. (b) Relative viabilities of (±SD) of two polymers and six oligomers at 50 μg/ml for Vero (epithelial) cells exposed to each of the compounds for 24 h with the final 50 min in the dark. The solid black line indicates 50% relative viability, below which the substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells. (c) Relative viabilities of (±SD) of two polymers and six oligomers at 100 μg/ml for Vero (epithelial) cells exposed to each of the compounds for 24 h with the final 50 min in the dark. The solid black line indicates 50% relative viability, below which the substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells.

Close modal

Figure 4 compares responses of EpiDerm tissues to the controls, the polymers, and the end-only oligomers. The negative control showed relative viability of 100% as expected, and the positive control showed a low relative viability of approximately 8% as expected. The negative controls had increasing standard deviations across subsequent concentrations. The increasing standard deviations of the negative controls are most likely due to tissue lot number, as each lot is derived from a different source (which is tissue from infant males).

Fig. 4.

Relative viabilities. (a) Relative viabilities (±SD) of two polymers, PPE-DABCO, and PPE-Th, at 10, and 50 μg/ml as determined by MTT assays of EpiDerm tissues 42 h after 1-h exposure (n = 3). The solid black line indicates 50% relative viability, below which substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells. (b) Relative viabilities (±SD) of three end-only oligomers (EO-OPEs) at 10 and 50 μg/ml as determined by MTT assays EpiDerm tissues 42 h after 1-h exposure (n = 3). The solid black line indicates 50% relative viability, below which substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells.

Fig. 4.

Relative viabilities. (a) Relative viabilities (±SD) of two polymers, PPE-DABCO, and PPE-Th, at 10, and 50 μg/ml as determined by MTT assays of EpiDerm tissues 42 h after 1-h exposure (n = 3). The solid black line indicates 50% relative viability, below which substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells. (b) Relative viabilities (±SD) of three end-only oligomers (EO-OPEs) at 10 and 50 μg/ml as determined by MTT assays EpiDerm tissues 42 h after 1-h exposure (n = 3). The solid black line indicates 50% relative viability, below which substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells.

Close modal

Figure 4(a) compares the relative viabilities of tissues exposed to the two polymers at the two concentrations: 10 and 50 μg/ml. PPE-DABCO and PPE-Th show relative viabilities over 100% for both 10 and 50 μg/ml. This high relative viability (similar to negative controls, shown in the same table) indicates that no cell death occurred. There is also an increase in relative viabilities of cells in increasing concentrations of both PPE-DABCO and PPE-Th, though this is not statistically significant. For PPE-DABCO, relative viabilities increased from 112% to 121%; for PPE-Th, relative viabilities increased from 105% to 111%. Therefore, both polymers should be considered nonirritants using the Epiderm skin irritation assay. It is interesting to note that, while not statistically significant, it appears that the viability of cells exposed to PPE-DABCO is higher than those exposed to PPE-Th; the source of this variation will be further explored to determine whether these polymers have beneficial effects on human skin, such as has previously been observed in other in vitro skin irritation assays.23,36

Figure 4(c) compares the relative viabilities of the three end-only oligomers tested at 10 and 50 μg/ml. This figure shows an increase in viabilities with increasing concentrations, though the result is not statistically significant. This increase and those seen in PPE-DABCO and PPE-Th are probably due to the differences between kits as all PPEs and EO-OPEs were tested at 10 μg/ml with the first kit and 50 μg/ml with the second kit in week two. Although, as stated above, it is possible that the source of variation is due to the beneficial effects these polymers have on skin, this possibility will be further explored.23,36 All end-only oligomers show relative viabilities over 100%, indicating a lack of cell death. The relative viabilities of EO-OPE-1-C2 are the highest of all the OPEs at all concentrations. The relative viabilities of EO-OPE-1-DABCO are the lowest of all the OPEs at all concentrations.

Figure 5 shows all polymers and oligomers, as well as their relative viabilities compared at 50 μg/ml. As all compounds have relative viabilities that are well above the 50% mark, none of these compounds are considered irritants. The DABCO-containing compounds appear to have slightly higher viabilities than the thiophene containing compounds, possibly due to inherent variability of samples derived from individual infants, although this trend is not statistically significant.

Fig. 5.

Relative viabilities (±SD) of two polymers (PPEs = blue) and six oligomers (OPES; end only = red, nonend only = green) at 50 μg/ml as determined by MTT assays of Epiderm tissues 42 h after 1 hour exposure (n = 3). The dashed line indicates 50% relative viability, below which substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells.

Fig. 5.

Relative viabilities (±SD) of two polymers (PPEs = blue) and six oligomers (OPES; end only = red, nonend only = green) at 50 μg/ml as determined by MTT assays of Epiderm tissues 42 h after 1 hour exposure (n = 3). The dashed line indicates 50% relative viability, below which substances are classified as irritants. The dashed black line indicates 70% cell viability, above which the substances are nontoxic to cells.

Close modal

The results for cytokine assays are derived from the same tissues that generated relative viability data, and so the controls are the same—tissues treated with DBPS (negative control), and 5% SDS (positive control). Like the viability data, the results for the controls are consistent across all three kits. For the negative controls, IL-1α concentrations range from 21 to 103 pg/ml, with an overall mean of 77 ± 18 pg/ml. For the positive controls, IL-1α concentrations range from 206 to 384 pg/ml, with an overall mean of 307 ± 36 pg/ml.

Table II shows the cytokine assay results for the eight compounds and two electrospun mats assayed at 50 μg/ml compared to those found in the literature.20,23,35 In our tests, IL-1α concentrations range from 20 to 105 pg/ml with a median of 56 pg/ml. All values are below the concentrations for the kit's negative controls (93 ± 16), indicating that the test substances produced no more IL-1α than the negative control (DPBS).

Table II.

Concentration of IL-1α (in pg/mL) from tissues corresponding to test substances. ND = not detected (≤3.9 pg/ml in diluted sample).

Chemical name50 pg/mlLiterature chemical nameIL-1α (pg/ml) ± standard deviation
PPE-DABCO ND Di-propylene glycol 88.38 ± 43.72 
PPE-Th 25 (n = 1) Napthalene acetic acid 35.25 ± 7.02 
EO-OPE-1-DABCO 20 (n = 1) Isopropanol 130.60 ± 116.44 
EO-OPE-1-Th ND Isopropyl myristate 34.27 ± 14.25 
EO-OPE-1-C2 104 (n = 1) Isopropyl palmitate 30.60 ± 16.23 
S-OPE-1(H) 101 ± 40 Heptyl butyrate 97.80 ± 75.94 
S-OPE-2(H) 50 ± 6 Hexyl salicylate 106.20 ± 134.30 
S-OPE-3(H) 56 ± 18 Linalyl acetate 91.25 ± 56.24 
Control ES mat 56 (n = 1) Terpinyl acetate 53.49 ± 26.96 
EO-OPE-Th ES mat 34 ± 1 Di-n-propyl disulphide 91.88 ± 20.15 
Chemical name50 pg/mlLiterature chemical nameIL-1α (pg/ml) ± standard deviation
PPE-DABCO ND Di-propylene glycol 88.38 ± 43.72 
PPE-Th 25 (n = 1) Napthalene acetic acid 35.25 ± 7.02 
EO-OPE-1-DABCO 20 (n = 1) Isopropanol 130.60 ± 116.44 
EO-OPE-1-Th ND Isopropyl myristate 34.27 ± 14.25 
EO-OPE-1-C2 104 (n = 1) Isopropyl palmitate 30.60 ± 16.23 
S-OPE-1(H) 101 ± 40 Heptyl butyrate 97.80 ± 75.94 
S-OPE-2(H) 50 ± 6 Hexyl salicylate 106.20 ± 134.30 
S-OPE-3(H) 56 ± 18 Linalyl acetate 91.25 ± 56.24 
Control ES mat 56 (n = 1) Terpinyl acetate 53.49 ± 26.96 
EO-OPE-Th ES mat 34 ± 1 Di-n-propyl disulphide 91.88 ± 20.15 

Although not statistically significant, the IL-1α release values of PPE-DABCO are lower than those of PPE-Th at 50 μg/ml. However, IL-1α release values of EO-OPE-1-DABCO are higher than those of EO-OPE-1-Th. From these results, it is seen that the trend is not consistent with the DABCO and thiophene functional groups. For the S-OPEs, which vary by the number of repeat units, S-OPE-2(H) had the lowest IL-1α release (50 pg/ml), followed by S-OPE-3(H) (56 pg/ml) and S-OPE-1(H) (101 pg/ml). These results indicate that n = 2 is the optimum number of repeat units for S-OPEs in terms of IL-1α release. For electrospun mats, the control electrospun mat had the lowest IL-1α release (56 pg/ml) followed by the EO-OPE-Th electrospun mat (92 pg/ml).

Literature values were found for IL-1α release for 22 chemicals tested for skin irritation following the same protocol using EpiDerm tissues.33 Ten chemicals that had both in vivo (rabbit) testing and human patch testing are shown on the right-hand side of Table II.25,26 All of these chemicals were classified as nonirritants as determined by the MTT assays for relative viability, with IL-1α release also being used as a secondary confirmation of nonirritation. IL-1α release values for these ten chemicals ranged from 30.6 to 130.6 pg/ml, which is a similar range to those reported for our studies (20–105 pg/ml). Overall, these data support the conclusion that the ten test substances (eight compounds and two electrospun mats) in this work are (on the left-hand side of that same table), should also be considered nonirritants.

Two polymers, three end only oligomers, three symmetric oligomers, and two types of electrospun mats were tested for skin irritation using multilayered tissues based on human epidermal keratinocytes. Viability (MTT) assays and cytokine (IL-1α) assays concluded that all oligomers were nonirritants up to the highest tested concentrations of 50 μg/ml. The poly(caprolactone) (PCL) and PCL/EO-OPE-1-Th ES mats were also found to be nonirritants. As a result, all test substances can be conservatively classified as nonirritants after a 1 h exposure time period. IL-1α results shown in this work are consistent with literature values, supporting the conclusion that the polymers, oligomers, and electrospun mats do not cause tissues to produce additional IL-1α.

The lack of skin irritation for all of these substances, as measured by MTT assay and cytokine assay, helps alleviate initial safety concerns for products based on these polymers and oligomers—both in solutions and as electrospun mats. Further skin irritation testing and eye irritation testing may be needed before these products are used in products such as disinfectant sprays, wipes, paints, wound dressings, and fabrics.

The authors would like to thank Diane Lidke, Debbie Evans, Liping Ding, Eunkyung Ji, Katsu Ogawa, Anand Parthasarath, Yanli Tang, Ying Wang, Zhijun Zhou, Kirsten Cicotte, and Tom Corbitt for their contributions and helpful discussions. This work was funded by the U.S. National Science Foundation (NSF) Partnerships for Research and Education in Materials (UNM/Harvard PREM, DMR-0611616), the NSF Integrative Graduate Education and Research Traineeship (IGERT) in Nanoscience and Microsystems (DGE-0504276), Gates Millennium Foundation, and the U.S. Defense Threat Reduction Agency.

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