Impact of Select Sophorolipid Derivatives on Macrophage Polarization

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Impact of Select Sophorolipid Derivatives on Macrophage Polarization and Viability Patricia Diaz-Rodriguez, Hongyu Chen, Joshua D Erndt-Marino, Fei Liu, Filbert Totsingan, Richard A Gross, and Mariah S. Hahn ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00799 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 3, 2019

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ACS Applied Bio Materials

Full Title: Impact of Select Sophorolipid Derivatives on Macrophage Polarization and Viability

Running Title: Sophorolipid Derivatives and Macrophage Polarization and Viability

Authors: Patricia Diaz-Rodrigueza†, Hongyu Chena†, Joshua D. Erndt-Marinoa, Fei Liub, Filbert Totsinganb, Richard A. Grossb and Mariah S. Hahna *

†P.D.R. and H.C. contributed equally to this work.

Affiliations: aDepartment

of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180,

United States bDepartment

of Chemistry and Biology, Rensselaer Polytechnic Institute, Troy, NY,12180,

United States

*Corresponding Author: Mariah S. Hahn, PhD Professor, Biomedical Engineering Rensselaer Polytechnic Institute Tel: 518-276-2236 Email: [email protected]

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ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT A major limitation of many biomaterials is the induction of a host response that challenges the integrity and overall efficacy of the implanted material. Emerging literature suggests that the resolution of inflammation is essential for proper healing and restoration of homeostasis. Macrophages are highly plastic immune cells that play a variety of critical roles throughout the duration of the host response. Specifically, the transition from a pro-inflammatory, M1 phenotype to an anti-inflammatory/wound healing, M2 macrophage phenotype is a central feature in the resolution of inflammation. The long-term goal of this work is to incorporate natural or modified sophorolipids (SLs), a class of glycolipids, as novel drug-loading or bioactive coating candidates to facilitate resolution of biomaterial-induced inflammation. Toward this goal, the diacetylated lactonic SL (L) and seven SL-esters (modified to present methyl (M), ethyl (E), propyl (P), butyl (B), pentyl (Pent), hexyl (H) or octyl (O) groups) were compared with respect to macrophage viability and phenotype to identify promising SL-esters for biomaterial applications. An initial viability screen showed that certain SL-ester structures (L, Pent and O) have relatively higher toxicity. Macrophage phenotypic assessments also revealed that most SLesters suppressed the M1 profile in lipopolysaccharide stimulated macrophages (M(LPS)). However, only two SL-ester candidates (E and B) were also capable of increasing the M2 profile in M(LPS), largely by enhancing the production of the vascular endothelial growth factor A. Cumulatively, these results suggest that further investigation of SL-esters E and B for facilitating biomaterial-induced inflammation resolution is warranted.

Key Words: sophorolipids; sophorolipid esters; macrophage polarization; biomaterial-mediated inflammation


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1. INTRODUCTION As one of the key factors in tissue engineering strategies, biomaterials are used in a wide range of clinical applications, including osteochondral regeneration, cardiovascular tissue repair, drug release, and more1-2. Broadly speaking, one of the most critical issues associated with biomaterial implantation is the host response elicited by the presence of these foreign materials3-5. The host response consists of an initial, acute inflammatory response in which immune cells attempt to remove the material via phagocytosis and secretion of reactive oxygen species5-6. The persistence of inflammation due to the continued presence of the implant may not only accelerate biomaterial degradation by oxidative stress, but may also contribute to fibrous capsule formulation surrounding the implant7-8. Together, these processes ultimately compromise the efficacy of many implanted biomaterials. Therefore, proper resolution of the inflammatory response to promote normal wound healing and limit fibrosis is an important design consideration for a successful biomaterial. Macrophages are myeloid immune cells present in nearly every tissue throughout the body. They are also primary responders and direct effectors to foreign materials5,

9-10.

Macrophages are involved in different stages of inflammation including initiation, progression and resolution11-12. In addition, macrophages regulate wound healing by secreting cytokines and growth factors critical to extracellular matrix production13-14. The multitude of macrophage functions are enabled by their plasticity, taking on a variety of activation states depending on environmental stimuli. Generally, activated macrophages can be categorized into classically activated (M1, pro-inflammatory) and alternatively activated (M2, wound healing/antiinflammatory) phenotypes15. Macrophages initially take on a M1 phenotype upon biomaterial implantation as they try to degrade and phagocytose the foreign material. Although M1



macrophages are needed for the initial inflammatory response, positive correlations between inflammation and fibrosis exist. Ideally, M1 macrophages transition toward a M2 phenotype after phagocytosis, signifying the resolution of inflammation and promotion of wound healing. Therefore, the transition from a M1 to a M2 macrophage phenotype is likely both necessary and beneficial for reducing inflammation and promoting proper wound healing processes after biomaterial implantation16-18. Such an approach is supported by other groups which have demonstrated reduced, albeit not completely absent, fibrotic outcomes in select model systems when molecules that promote M1M2 transition were incorporated into the biomaterial19-22. Sophorolipids (SLs), an important class of glycolipid biosurfactants, consist of a sophorose (glucose disaccharide) polar head group that can be acetylated at the 6'- and/or 6"positions. The sophorose moiety is β-glycosidically linked to a sub-terminal hydroxylated fatty acid that is either free or internally esterified at the 4" sophorosehydroxyl site23. SLs are naturally produced by several non-pathologic yeast species, with no known natural functions in humans. SLs have been widely researched for potential anti-microbial, anti-inflammatory and anti-cancer properties24-25. Therapeutically, research has shown that SLs may exhibit anti-cancer activity26-28 and also inhibit the lethal effect of septic shock in rats attributed, in part, to suppression of M1 macrophage markers29. Even though SLs have been suggested to be specifically toxic toward malignant cells26, there are also studies indicating evident cytotoxicity in rats when systemically injected at high concentrations (375 mg/kg)30. Unfortunately, these previous investigations have been limited to only 1 or 2 SL structural types and also did not consider other important functions of macrophages including wound healing. Consequently, it is unknown whether natural or modified SLs can promote the M1M2 transition. Separate from SLs, small molecules with anti-inflammatory properties are often loaded into biomaterials for immunomodulation31-32, and


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lipids have been used as coatings for a diverse array of biomaterial surfaces because of their inherent biocompatibility33. We envision that the potential immunomodulatory properties of SLs may position them as unique drugs for incorporation into biomaterials and/or for surface coatings to facilitate resolution of the host response and promote normal wound healing following biomaterial implantation. In the present manuscript, we utilize molecular editing – the systematic chemical modification of natural products34 – to manipulate the structure of the SL skeleton to generate SL-esters with open-ring structures where the ester extension of the hydrophobic chain can be modified to gradually increase in length. We then assessed the effects of the associated molecular modifications on macrophage viability and phenotype. Generally, the mechanisms via which SLs affect cells are still unclear and may be application-specific. Given the complexity of potential underlying mechanisms of SL bioactivity, the present work did not seek to test a specific hypothesis regarding increasing chain length of SLs and resulting macrophage behavior. In contrast, this work was largely characterization-based and goal-driven as a prerequisite before examining observed phenomena in more depth. The goal of the present study was to perform an extensive characterization of diacetylated lactonic SL and derivative SL-esters with respect to macrophage viability and phenotype to identify promising SL drugs for biomaterial applications. Following SL-ester synthesis and characterization, we assessed the dose and time dependent effect on macrophage viability as an initial screening tool to determine non-toxic concentrations and SL structures. Lastly, the capacity of candidate SLs to transition macrophage activation from a M1 toward a M2 phenotype was assessed utilizing protein level markers associated with each phenotype.



2. MATERIALS AND METHODS 2.1 Sophorolipids synthesis and characterization The synthesis of naturally derived lactonic diacetate and other SL alkyl esters was performed according to our previous work. Briefly, diacetylated lactonic SL (L) was synthesized by fermentation35 and separated from crude fermentation products by recrystallization from ethyl acetate/hexane (1:1). The crude mixture from fermentation was dried under vacuum and the resulting solid (100 g) was dissolved in hot ethyl acetate (0.5 L). After filtration to remove solid suspensions, hexane (0.5 L) was added under magnetic stirring and the mixture was kept at 4 °C for 2 h. SL-esters were prepared by ring-opening lactonic SL with various n-alkyl sodium alkoxides, using the corresponding alcohol as solvent, following previously reported procedures 36-38.

The desired SL ester products were isolated by recrystallization from ethanol/cold water

(1:10) or by flash chromatography using a chloroform/methanol mixture. The obtained white solid was filtered off and dried under vacuum for 24 h. Specifically SL methyl ester (M), SL ethyl ester (E), SL propyl ester (P), SL butyl ester (B), SL pentyl ester (Pent), SL hexyl ester (H), and SL octyl ester (O) were prepared. Both natural lactonic SL diacetate and SL alkyl esters were characterized by nuclear magnetic resonance (1H NMR) and liquid chromatography–mass spectrometry (LC-MS). Data can be found in our previously published studies36-39. Lactonic SL diacetate (L) has the ringclosed structure (Figure 1A). Conversely, the methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl and n-octyl SL-esters (M, E, P, B, Pent, H and O, respectively) possess open-ring structures where the ester extension of the hydrophobic chain gradually increases in length (Figure 2-8 A). 2.2 Macrophage expansion and activation


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A vial of cryopreserved Raw 264.7 (ATCC) murine macrophages was thawed and expanded in monolayer culture. Macrophages were maintained at 37 °C/5% CO2 in cell culture medium, which consisted of Dulbecco’s Modified Eagle’s Medium (DMEM, Cellgro) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). Macrophages were seeded at 6,500 cells/cm2 in 75 cm2 cell culture flasks (Corning), cultured for 24 h and activated for 4 days with 1 µg/ml lipopolysaccharide (LPS) from Salmonella enterica serotype enteritidis (Sigma Aldrich). Medium was changed every day. LPS was chosen to polarize macrophages towards a pro-inflammatory, M1 phenotype40-42, denoted hereafter as M(LPS). M(LPS) were utilized in this initial screening study because they closely mimic the macrophage phenotype often associated with initial biomaterial-implantation9, 43

and are therefore likely the most relevant for studying acute cell-material interactions. We

have previously validated this activation regimen for M1 macrophage activation44. 2.3 Sophorolipid treatment and endpoint analyses M(LPS) were seeded in 96-well plates at 150,000 cells/cm2 with assay medium (AM: DMEM (without phenol red) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin). After overnight culture without SL treatment, the medium in each well was replaced by AM containing various concentrations of SLs. The tested concentrations included 50 µM, 100 µM, 200 µM and 400 µM for each SL-ester. Concentrations for L were limited to 12.5 µM, 25 µM and 50 µM. Different concentration ranges were examined for SL-esters relative to the parent L due to differences in 1) their solubility and 2) the concentrations at which they are biologically active, with the SL-esters generally requiring higher concentrations to achieve biological effects26, 45-46. The upper limits of concentrations employed herein for the synthetic SL-esters are generally higher than any other study investigating these polymers whereas the



lower limits are comparable to similar studies performed with SL-esters45. The concentrations tested herein for L are relatively lower but consistent in magnitude compared to in vitro studies performed to support in vivo findings29-30, 47. The use of the diacetylated lactonic form of L, rather than the ring-opened deacetylated form (ie. acidic SL), was chosen for two reasons. First, it is the most widely studied SL and including it gives us a basis to incorporate our results with existing literature. Second, previous reports suggest that the acidic SL has low biological activity when compared to both the natural L SL and no treatment controls27, 46. As indicated throughout the study, SL effects on macrophage viability and phenotype were determined for cultures at various time points using the Vybrant cell metabolic assay and multiplex immunoassays, respectively. Fluorescence outcomes from the Vybrant cell metabolic assay are routinely utilized to assess cell viability48-50. Vybrant™ Cell Metabolic Assay: The influence of SL derivatives on macrophage viability was measured with the Vybrant™ Cell Metabolic Assay Kit (ThermoFisher) following the manufacturer’s protocol. Briefly, VybrantTM cell metabolic activity reagent, C12-resazurin, was prepared at 20 μM in cell culture medium. Then, 100 μl of the resulting assay reagent was added to each well of a 96-well plate seeded with macrophages and containing 100 μl of lactonic SL or SL-ester solution, followed by mixing. Cell viability was measured at 6, 16 and 24 h after SL application by monitoring the fluorescence (excitation/emission 530/590 nm). A calibration curve was obtained by performing serial dilutions of resorufin (metabolite of C12-resazurin) from 20 µM to 0.3125 µM. Wells containing blank medium (no C12-resazurin) were used as background controls. For each time point, cell viability was calculated with the corresponding M(LPS) activated macrophage controls being defined as 100% viability.


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Multiplex Immunoassays: Secretion of a select panel of cytokines and growth factors by treated macrophages was quantitatively assessed with multiplex immunoassays. After 24 h of culture in the presence various SL derivatives, conditioned medium from each sample was collected and processed using a mouse cytokine/chemokine MILLIPEX® MAP kit (Millipore) for TNF-α, interleukin-6 (IL-6), IL-1β, monocyte chemotactic protein-1 (MCP-1), IL-10 and vascular endothelial growth factor A (VEGF) per the manufacturer’s protocol. Briefly, kit standards were reconstituted using 250 µl deionized water to prepare the highest standard, and serial dilutions (1:5) were performed using assay buffer to prepare an additional 5 standards with lower concentrations. To account for cell medium proteins, blank cell culture medium was used as background standard and was subtracted from all sample readings. In a 96-well plate (Greiner), 25 µl per well of undiluted sample/cell medium, kit assay buffer, and beads were added (75 µl total volume) and incubated overnight at 4 °C with shaking. The beads were then washed (2x) and resuspended in 25 µl of detection antibodies and incubated for 1 h at room temperature. Then, 25µl of streptavidin-phycoerythrin was added per well. After 30 min of incubation at room temperature, beads were washed (2x) and re-suspended in 150 µL drive fluid. Median fluorescence intensity (MFI) of each analyte was read using a MAGPIX system (Luminex). Concentrations of proteins of interest were calculated using the MFI and the standard curve of each analyte. The resulting values were presented in both “individual” and “pooled” formats. In the “individual”, protein concentrations from the multiplex analyses were normalized by their respective sample DNA content, assessed with the PicoGreen assay per the manufacturer’s instructions (Life Technologies). Each treatment group was then further normalized to notreatment M(LPS) controls for the purpose of comparison.



In the “pooled” format, M1 marker data and M2 marker data were each presented in a “pooled” format. Specifically, the panel of markers for macrophage phenotype evaluation was selected according to commonly designated classifications for either M1 (TNF-α, IL-1β, IL-6 and MCP-1) or M2 (IL-10, VEGF) phenotypes14-15, 51-55. All markers belonging to the M1 or M2 classification were pooled into a single metric (rather than several separate proteins for each). For a given marker (eg. TNF-α), the concentration determined via multiplex analysis was normalized first to DNA concentration and then to the average concentration for that marker (eg. TNF-α) across all samples. These normalization steps enable: 1) concentrations for a given marker (eg. TNF-α) to be expressed on a per cell basis (DNA normalization) and 2) comparison between proteins that are normally expressed at different absolute amounts (eg. TNF-α vs. MCP1). The resulting normalized values for TNF-α, MCP-1, IL-1β, and IL-6 or for VEGF and IL-10 were averaged to yield values representing the M1 and M2 profile, respectively. A strength of this method is that it simplifies complex, marker dependent data into a more understandable and relatable format. However, it assumes that all included proteins have equal weights in terms of their contribution to the M1 or M2 phenotype/functions, which may or may not be the case in vivo. Pooling data also may overlook the nuance of cell-biology. As such, we reasoned that presenting the data in both individual and combined formats was appropriate. That said, it is important to note that pooling markers together is an accepted method of analyzing macrophage phenotype56-59. 2.4 Statistical analyses All results are reported as the mean ± standard deviation. To assess the effect of concentration and time of SL treatment on macrophage viability, means were compared using a two-way ANOVA (n = 3-6 samples per group). A two-way ANOVA is the appropriate statistical


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test to determine significance resulting from main effects of either independent variable (i.e. SL concentration or treatment time) or significance resulting from the interaction between these two variables. To assess phenotypic differences, means of each marker were compared using an independent-samples, Students t-test between macrophages treated with a given SL formulation or untreated macrophages. For all tests, a p-value < 0.05 was considered significant and SPSS software was utilized.

3. RESULTS Following presentation of results for the L SL formulation, which retains the largest literature support, the remaining results for the synthetic SL-esters are presented in order of increasing chain length. 3.1 SL-lactonic diacetate (L): Due to the relatively low solubility of L, the range of concentrations investigated was 12.5 - 50 µM. Relative to control M(LPS), treatment with 12.5 or 25 µM of L (Figure 1A) did not affect viability at any time point (Figure 1B). In contrast, macrophage viability was significantly reduced at every time point when treated with 50 µM of L (p < 0.001, Figure 1B). Given the viability results, a concentration of 25 µM of L was selected for M1M2 transition assessment. Moreover, despite being relatively more toxic than the SL-esters studied, macrophage assessment was still performed in order to compare our results with previous reports, which are generally limited to only L23, 26. Relative to untreated M(LPS), macrophages treated with L (M(LPS, L)) produced significantly decreased levels of the M1 markers IL-6 (p = 0.007) and TNF-α (p = 0.029) and lower levels of MCP-1, although this did not reach statistical significance (p = 0.056). Consequently, the corresponding “pooled” M1 profile was significantly



reduced in M(LPS, L) (p = 0.006; Figure 1C) relative to M(LPS). In contrast to M1 marker data, the M2 marker VEGF was significantly elevated in M(LPS, L) relative to M(LPS) controls (~5.2-fold, p = 0.012; Figure 1D). Despite this favorable change, IL-10 was significantly decreased (p = 0.006) in M(LPS, L) in comparison to M(LPS). Thus, the overall “pooled” M2 profile was unchanged for M(LPS, L) (Figure 1D). Cumulatively, these data suggest that 24 h treatment with L: 1) does not adversely affect macrophage viability up to 25 µM, 2) suppresses M1 polarization and 3) does not promote M2 polarization in M(LPS). 3.2 SL-methyl ester (M): The structure of M is presented in Figure 2A. Relative to untreated M(LPS) controls, there was no toxic effect on macrophage viability when treated with 50, 100, 200, or 400 µM of M (Figure 2B). In fact, macrophage viability was significantly enhanced when treated with 400 µM of M (p < 0.001, Figure 2B). A concentration of 200 µM of M was selected for M1M2 transition assessment to be consistent with other SL-ester formulations, allowing us to control for concentration as a potential factor driving macrophage phenotypic differences. Relative to the untreated M(LPS), macrophages treated with M (M(LPS, M)) produced significantly lower levels of the M1 marker IL-6 (p = 0.003) and lowered TNF-α and MCP-1 production, although these differences did not achieve statistical significance (p = 0.096 and 0.064, respectively; Figure 2C). Consequently, the corresponding “pooled” M1 profile was significantly reduced in M(LPS, M) relative to M(LPS) (p = 0.031; Figure 2C). In contrast to M1 marker data, a significant increase in the M2 marker VEGF was noted in M(LPS, M) relative to M(LPS) (~2.8-fold, p = 0.026; Figure 2D). However, IL-10 was significantly decreased (p = 0.046) in M(LPS, M) in comparison to M(LPS). Thus, as for M, the “pooled” M2 profile was unchanged in M(LPS, M) relative to M(LPS) (Figure 2D).


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Cumulatively, these data suggest that 24 h treatment with the SL-ester M: 1) has either a limited or a positive effect on macrophage viability up to 400 µM, 2) suppresses M1 polarization and 3) does not promote M2 polarization in M(LPS). 3.3 SL-ethyl ester (E): Relative to untreated M(LPS), treatment with 50, 100 or 200 µM of E (Figure 3A) did not affect macrophage viability (Figure 3B). However, treatment with 400 µM of E significantly reduced M(LPS) viability at all investigated time points (p < 0.001, Figure 3B). Given the viability results, a concentration of 200 µM of E was selected for M1M2 transition assessment. Relative to control M(LPS), macrophages treated with E (M(LPS, E)) exhibited significantly decreased levels of the M1 marker MCP-1 (p = 0.034). Although other M1 markers (TNF-α, IL-1β, and IL-6) were unchanged, the reduction in MCP-1 was sufficient to render the “pooled” M1 profile significantly reduced in M(LPS, E) relative to M(LPS) (p = 0.047; Figure 3C). In terms of M2 markers, VEGF was significantly increased in M(LPS, E) (~22.5fold, p = 0.023) but IL-10 was significantly reduced relative to M(LPS) (p = 0.025, Figure 3D). Overall, the “pooled” M2 profile of M(LPS) was significantly increased in M(LPS, E) (p = 0.034). Cumulatively, these data suggest that 24 h treatment with the SL-ester E: 1) has a limited effect on macrophage viability with concentrations up to 200 µM, 2) suppresses M1 polarization and 3) promotes M2 polarization in M(LPS). 3.4 SL-propyl ester (P): The structure of P is shown in Figure 4A. Relative to untreated controls, macrophage viability was significantly enhanced when treated with 50, 100, 200 or 400 µM of P (p < 0.042; Figure 4B). In terms of M1M2 transition assessments, treatment with 200 µM of P for 24 h was analyzed to be consistent with other formulations. Relative to control M(LPS), macrophages



treated with P (M(LPS, P)) displayed significantly decreased levels of IL-6 (p = 0.003) (Figure 4C). However, all other M1 and M2 markers and the “pooled” M1 and M2 profiles were not affected by P (Figure 4C, 4D). Cumulatively, these data suggest that 24 h treatment with SLester P: 1) has a positive effect on macrophage viability, 2) does not suppress M1 polarization and 3) does not promote M2 polarization in M(LPS). 3.5 SL-butyl ester (B): Relative to untreated M(LPS) controls, exposing activated macrophages with B (Figure 5A) had no adverse effect on viability at 50, 100, or 200 µM concentrations of B (Figure 5B). However, treatment with 400 µM of B significantly reduced macrophage viability at every time point investigated (p < 0.001, Figure 5B). Given the viability results, a concentration of 200 µM of B was selected for M1M2 transition assessment. Relative to control M(LPS), macrophages treated with B (M(LPS, B)) produced significantly decreased levels of IL-6 (p = 0.003) and lower levels of MCP-1, although this difference did not reach statistical significance (p = 0.098; Figure 5C). Despite other M1 markers (TNF-α and IL-1β) being relatively unaffected, the overall “pooled” M1 profile was significantly reduced in M(LPS, B) relative to M(LPS) (p = 0.009; Figure 5C). In contrast to M1 marker data, the M2 marker VEGF was significantly elevated in M(LPS, B) relative to M(LPS) (~3.2-fold, p = 0.002; Figure 5D), although IL-10 was relatively unaffected. Consequently, the overall “pooled” M2 profile was significantly elevated in M(LPS, B) in comparison to M(LPS) (p = 0.043, Figure 5D). Cumulatively, these data suggest that 24 h treatment with the SL-ester B: 1) has no adverse effect on macrophage viability up to 200 µM, 2) suppresses M1 polarization and 3) promotes M2 polarization in M(LPS). 3.6 SL-pentyl ester (Pent):


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Although 50 and 100 µM concentrations of the SL-ester Pent (Figure 6A) did not adversely affect macrophage viability, treatment with 200 and 400 µM of Pent significantly reduced cell viability relative to untreated M(LPS) controls (p < 0.001; Figure 6B). Due to this relatively high cytotoxicity for a SL-ester and the need to assess the effects of SL-esters on macrophage polarization at similar concentrations, the Pent formulation was not further assessed. 3.7 SL-hexyl ester (H): Similar to E, there was no change in macrophage viability when treated with 50, 100 or 200 µM of H (Figure 7A) relative to untreated M(LPS) (Figure 7B). However, viability was significantly reduced when 400 µM of H was applied (p = 0.015, Figure 7B). Given the viability results, a concentration of 200 µM of H was selected for M1M2 transition assessment. Relative to the control M(LPS), macrophages treated with H (M(LPS, H)) were associated with significantly decreased levels of the M1 marker IL-6 (p = 0.009) and lower levels of IL-1β, and MCP-1 although these did not reach statistical significance. TNF-α expression remained unchanged between M(LPS) and M(LPS, H). The overall “pooled” M1 profile was significantly reduced (p = 0.019) in M(LPS, H) in comparison to M(LPS) (Figure 7C). In contrast to M1 marker data, the M2 markers VEGF and IL-10 were unchanged in M(LPS, H) relative to control M(LPS) (Figure 7D), although the trend for VEGF was increasing. Consequently, there was no statistical difference between the overall “pooled” M2 profile between M(LPS) and M(LPS, H) (Figure 7D). Cumulatively, these data suggest 24 h treatment with the SL-ester H: 1) has a limited effect on macrophage viability at concentrations up to 200 µM, 2) suppresses M1 polarization and 3) does not promote M2 polarization in M(LPS). 3.8 SL-octyl ester (O):



With the exception of 50 µM, treatment with all concentrations of SL-ester O (Figure 8A) significantly decreased macrophage viability at every time point investigated relative to untreated M(LPS) controls (p < 0.001; Figure 8B). Due to this relatively high cytotoxicity for a SL-ester and the need to assess the effects of SL-esters on macrophage polarization at similar concentrations, the O formulation was not further assessed.

4. DISCUSSION The long-term goal of this work is to utilize SLs as novel drugs that can be loaded into biomaterials and/or used as coatings to promote the resolution of inflammation and normal wound healing. In the present study, a series of SL derivatives, including diacetylated lactonic SL (L) and open-ring SL-esters (M, E, P, B, Pent, H and O) with systematic variation in the ester n-alkyl group, were prepared. M1 macrophages treated with these various modified SLs were compared in terms of viability and the M1M2 phenotypic transition. We focused on characterizing the macrophage response to these modified SLs due to the presence of macrophages in nearly every tissue, and the fact that these cells play several key roles in the host-response to foreign objects9, 43, 60. In terms of cytotoxicity, many SL formulations (L, E, B and H) demonstrated a negative effect only at the highest concentration tested: 50 µM for L (Figure 1B) or 400 µM for E, B, and H (Figures 3B, 5B, 7B). Importantly, below these concentrations, these SL formulations did not negatively influence viability at any time point investigated. In contrast, the SL-ester Pent and O demonstrated toxicity at relatively lower dosages than other ring-opened SL-esters (Figure 6B, Figure 8B). On this basis, SL-ester Pent and O were excluded for further examination.


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There are few studies assessing the effects of SLs on cell viability, and they are limited to only a subset of the SL structures (L, B and M) investigated herein. For example, one study recently concluded that 30 µM of B did not affect LPS-induced mouse peritoneal macrophage viability, consistent with our results for this SL-ester45. Other studies have found significant cytotoxic effects of L on a variety of cell types61, including normal colonic epithelial cells and various colorectal cancer cell lines at 0.03 µM or 0.1 µM, respectively. Lastly, Fu and colleagues found that L had greater toxicity than M when tested on human pancreatic carcinoma cells26, consistent with our data. Specifically, L exhibited cytotoxicity on M(LPS) at 50 µM while M did not negatively affect viability up to 400 µM. Beyond confirming some of these general viability trends, to our knowledge, this study is the first to examine the impact of a range of different SL formulations, concentrations, and time points on macrophage viability. In doing so, we have expanded the potential use of SL-esters and their respective concentrations that may be utilized in biological applications. After cytotoxicity evaluation, a subset of SL candidates was investigated for their potential to resolve biomaterial-induced inflammation. Specifically, we investigated their capacity to transition pro-inflammatory M(LPS) towards a wound healing, M2 phenotype, as this transition is a central feature in the resolution of acute inflammation62-63. In general, L, M, E, B and H exhibited an inhibitory effect on the overall “pooled” M1 profile of M(LPS) while only E and B significantly promoted overall M2 polarization. Therefore, E and B exhibit the strongest potential as drug-loading and/or coating candidates for biomaterial-based applications. Future work will extend upon and confirm these observations. Aside from the overall “pooled” profile analysis, many of the SL compounds exhibited specific effects on individual cytokines, both in terms of marker-type and magnitude.



Specifically, M, B and H significantly reduced IL-6 expression; E significantly reduced MCP-1 expression; and L significantly reduced both IL-6 and TNF-α expression. In terms of M2 markers, nearly all the SLs promoted VEGF production. The increase in VEGF varied quite extensively in terms of magnitude, ranging from ~22.5-fold in E to ~2.8-fold in M. It would be interesting to test if these effects render certain SL-esters suitable for desired applications in future work. For example, when utilized as an antibiotic loaded into a topical cream, Lydon found no negative effect on wound healing with the acidic SL formulation64. It is possible that the E formulation could enhance wound healing in this application while retaining antibiotic properties. At present, we have no explanation for the differential macrophage viability and induction of inflammatory cytokines and growth factors elicited by the various SL derivatives. This is consistent with existing literature in which the proposed mechanisms of action of SLs are complex and remain to be elucidated. For example, SLs performing as antimicrobials are hypothesized to insert into and disrupt the bacterial membrane via charge-charge properties, increasing membrane permeability29, 65 and cell death, and decreasing the probability of bacteria to acquire antibiotic resistance due to spontaneous mutations66. By increasing ester n-alkyl chain length, it may be possible to alter the bioactivity of SLs, but this has yet to be demonstrated. Indeed, with increasing chain length, physicochemical studies on SLs have revealed a complex, biphasic behavior with respect to critical micelle concentration, a decrease of minimum surface tension37, and an increase in hydrophobicity. Cumulatively, these properties could alter how these amphiphilic molecules interact with the membrane. Thus, with respect to the current study of changing macrophage response, the mode of action is unknown, but may be related to their ability to insert into cell membranes.


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That said, no clear relationship with increasing ester n-alkyl chain length was found for either macrophage viability or the M1M2 transition. For instance, the most toxic esterformulations were Pent and O, which correspond to chain lengths of 4 and 7, respectively. Formulations that were toxic only at the highest concentrations included E, B, and H (n = 1, 3, and 5) whereas M and P (n = 0 and 2) were not toxic. This potentially loose association between increasing chain length and reduced viability is similar to what has been observed in human pancreatic carcinoma cells26 and sperm46, but seemingly opposite to that observed in vaginal epithelial cells46. In terms of the M1M2 transition, while all formulations exhibited specific effects in terms of marker-type and magnitude, none of the changes appeared related to chain length. For example, M, B, and H all reduced IL-6 but their chain lengths (0, 3, and 5) were scattered across the range of lengths investigated herein. The lack of a clear structure-function relationship with increasing chain length of SLs may be related to a complex interplay between their resulting physicochemical properties and their likely multifaceted interaction with macrophage membranes. Performing systematic biological studies to study their interactions with cells, and the underlying cellular pathways that are altered to yield the observed secretome changes would be required to more definitively determine potential relationships between LSester chain length and the macrophage cell response. Comparing our macrophage data to previous work is challenging due to the general lack of reports with these molecules. To our knowledge, immunomodulatory properties of the L and B on macrophage phenotype have only been documented in the contexts of sepsis29, 47, 67 and spinal cord injury45. Bluth and colleagues showed that treatment with natural SLs (L makes up the highest proportion23) significantly reduced nitric oxide production from LPS-activated RAW 264.7 cells, consistent with the reduction of pro-inflammatory markers IL-6 and TNF-α noted



with L herein29. In contrast, the same authors observed an increase in IL-1β and TNF-α, but no change in IL-6 gene expression in a LPS-activated rat alveolar macrophage cell line exposed to SLs for 36 h. Moreover, Ziemba and colleagues found that treatment of peritoneal macrophages with B (3 nM to 30 µM for 24 h) did not affect gene expression of either M1 markers (nitric oxide synthase and IL-12p40) or M2 markers (arginase-1 and IL-10)45. These results are different from the general reduction in the M1 profile and enhancement of the M2 profile noted here with B treatment. The above discrepancies for L and SL-ester B may potentially be explained by a variety of factors, including differences in: 1) end-point assessments (i.e. gene vs protein expression and marker type), and/or 2) culture parameters (i.e. time and dosage). Since we noted that the SL-ester B was one of the more promising structures based on more definitive protein level assessments, resolving these inconsistencies, in addition to further characterizing a wider range of dose-response relationships, may allow us to proceed with future development of B with greater confidence. Comparing our results more broadly to alternate approaches to promote M1M2 is difficult due to a variety of reasons, many of which are listed above. Generally, several reports have characterized this transition by demonstrating a decrease in selected M1 markers and increase in selected M2 markers19-21, consistent with the effects of certain SL formulations noted herein. Importantly, these reports also demonstrated a reduced fibrotic response in their respective applications, suggesting the anti-fibrotic potential of SLs. Moreover, in contrast to these approaches, we both profiled and demonstrated formulation-dependent modulation of markers (ie. MCP-1 and VEGF). Such control may be important given the tissue- and contextdependent nature of the inflammatory response.


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Several limitations of the present study merit comment. First, the investigated macrophage activation markers were by no means exhaustive, especially for M2 markers where only VEGF and IL-10 were included. However, our marker selection is generally equivalent or more extensive than related studies, all of which chose 2-4 markers for M1 and 1-2 markers for M2 assessments29, 47. Furthermore, none of the previous studies investigated VEGF production, which plays a critical role in angiogenesis and wound healing14, 68. Second, only a single time point with a limited range of potential concentrations with a murine macrophage cell line was utilized for M1M2 transition assessments. Macrophage phenotype and function are known to vary according not only with source but also with time69-70. Since we were testing the influence of several SL-esters and L, these experimental settings (RAW 264.7 cells, 24 h treatment) were selected as a starting point to be consistent with previous SL-based macrophage research29, 45. Future work will temporally profile different tissue-resident and infiltrating macrophage populations with a more complete spectrum of phenotypic markers. This information will yield deeper insight into potential tissue-specific effects of natural and modified SLs with implications for biomaterial-based tissue engineering strategies. Lastly, in addition to further developing these SLs as drug-loading candidates for biomaterials, future work will seek to incorporate the promising formulations identified herein (E and B) as surface coatings. Such work would enable another layer of engineering control (ie. cell-biomaterial interactions) for implementing SLs as novel immunomodulatory therapeutics.

5. CONCLUSION In summary, the present work synthesized, characterized, and investigated the effects natural SL L and seven SL-esters on macrophage viability and activation profiles. Among these



eight SL structures, five (M, E, P, B, and H) had limited effects on effects on macrophage viability whereas L, Pent and O have relatively higher toxicity levels. Five of the SLs (L, M, E, B and H) suppressed M1 polarization and two (SL-esters E and B) also promoted M2 polarization in M(LPS). Cumulatively, our results expand the potential SL-esters for use in biological applications and suggest that certain structures (E and B) possess the potential to transition M(LPS) from a M1 to a M2 phenotype. This property may be beneficial for implementing SLs as novel loading drugs and/or coatings to improve the resolution of inflammation upon biomaterial implantation.

6. ACKNOWLEDGEMENTS This work was supported by NSF Division of Materials Research (NSF-DMR) Biomaterials (BMAT) Grant 1508422 to R.A.G. and M.S.H, and by NIH National Institute on Deafness and Other Communication Disorders (NIDCD) Grant R01DC013508 to M.S.H.

7. CONFLICTS OF INTEREST The authors declare no competing financial interest.


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9. FIGURE LEGEND Figure 1. (A) Chemical structure of natural lactonic sophorolipid diacetate (L). (B) Relative macrophage viability after treatment with L at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 25 µM of L. For (B), * denotes a significant main effect of L relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. Figure 2. (A) Chemical structure of sophorolipid methyl ester (M). (B) Relative macrophage viability after treatment with M at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of M. For (B), * denotes a significant main effect of M relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. Figure 3. (A) Chemical structure of sophorolipid ethyl ester (E). (B) Relative macrophage viability after treatment with E at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of E. For (B), * denotes a significant main effect of E relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. Figure 4. (A) Chemical structure of sophorolipid propyl ester (P). (B) Relative macrophage viability after treatment with P at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200


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µM of P. For (B), * denotes a significant main effect of M relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. Figure 5. (A) Chemical structure of sophorolipid butyl ester (B). (B) Relative macrophage viability after treatment with B at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of B. For (B), * denotes a significant main effect of B relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. Figure 6. (A) Chemical structure of sophorolipid pentyl ester (Pent). (B) Relative macrophage viability after treatment with Pent at various times and concentrations. * denotes a significant main effect of Pent relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. Figure 7. (A) Chemical structure of sophorolipid hexyl ester (H). (B) Relative macrophage viability after treatment with H at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of H. For (B), * denotes a significant main effect of H relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. Figure 8. (A) Chemical structure of sophorolipid octyl ester (O). (B) Relative macrophage viability after treatment with O at various times and concentrations. * denotes a significant main



effect of O relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA.


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Figure 1. (A) Chemical structure of natural lactonic sophorolipid diacetate (L). (B) Relative macrophage viability after treatment with L at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 25 µM of L. For (B), * denotes a significant main effect of L relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. 101x76mm (300 x 300 DPI)



Figure 2. (A) Chemical structure of sophorolipid methyl ester (M). (B) Relative macrophage viability after treatment with M at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of M. For (B), * denotes a significant main effect of M relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. 101x76mm (300 x 300 DPI)


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Figure 3. (A) Chemical structure of sophorolipid ethyl ester (E). (B) Relative macrophage viability after treatment with E at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of E. For (B), * denotes a significant main effect of E relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. 101x76mm (300 x 300 DPI)



Figure 4. (A) Chemical structure of sophorolipid propyl ester (P). (B) Relative macrophage viability after treatment with P at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of P. For (B), * denotes a significant main effect of M relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. 101x76mm (300 x 300 DPI)


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Figure 5. (A) Chemical structure of sophorolipid butyl ester (B). (B) Relative macrophage viability after treatment with B at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of B. For (B), * denotes a significant main effect of B relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. 101x76mm (300 x 300 DPI)



Figure 6. (A) Chemical structure of sophorolipid pentyl ester (Pent). (B) Relative macrophage viability after treatment with Pent at various times and concentrations. * denotes a significant main effect of Pent relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. 101x38mm (300 x 300 DPI)


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Figure 7. (A) Chemical structure of sophorolipid hexyl ester (H). (B) Relative macrophage viability after treatment with H at various times and concentrations. (C) Relative protein production of M1 and (D) M2 macrophage phenotypic markers after 24 h of treatment with 200 µM of H. For (B), * denotes a significant main effect of H relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. For (C, D), * denotes a significant difference relative to M(LPS) determined by a Student’s t-test. 101x76mm (300 x 300 DPI)



Figure 8. (A) Chemical structure of sophorolipid octyl ester (O). (B) Relative macrophage viability after treatment with O at various times and concentrations. * denotes a significant main effect of O relative to LPS-stimulated macrophages (M(LPS)) determined by a two-way ANOVA. 101x38mm (300 x 300 DPI)


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TOC Graphic 114x76mm (300 x 300 DPI)


Impact of Select Sophorolipid Derivatives on Macrophage Polarization

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