Characterisation of cytotoxicity and immunomodulatory effects of glycolipid biosurfactants on human keratinocytes.

Skin irritation and allergic reactions associated with the use of skincare products formulated with synthetically derived surfactants such as sodium lauryl ether sulphate (SLES) have encouraged the search for naturally derived and biocompatible alternatives. Glycolipid biosurfactants such as sophorolipids (SL) and rhamnolipids (RL) offer a potential alternative to SLES. However, most studies on the bioactive properties of microbial glycolipids were determined using their mixed congeners, resulting in significant inter-study variations. This study aims to compare the effects of highly purified SL (acidic and lactonic) and RL (mono-RL and di-RL) congeners and SLES on a spontaneously transformed human keratinocyte cell line (HaCaT cells) to assess glycolipids' safety for potential skincare applications. Preparations of acidic SL congeners were 100% pure, lactonic SL were 100% pure, mono-RL were 96% pure, and di-RL were 97% pure. Cell viability using XTT assays, cell morphological analyses, and immunoassays revealed that microbial glycolipids have differing effects on HaCaT cells dependent on chemical structure. Compared with SLES, acidic SL and mono-RL have negligible effects on cell viability, cell morphology, and production of pro-inflammatory cytokines. Furthermore, at non-inhibitory concentrations, di-RL significantly attenuated IL-8 production and CXCL8 expression while increasing IL-1RA production and IL1RN expression in lipopolysaccharide-stimulated HaCaT cells. Although further studies would be required, these results demonstrate that as potential innocuous and bioactive compounds, microbial glycolipids could provide a substitute to synthetic surfactants in skincare formulations and perform immunopharmacological roles in topical skin infections such as psoriasis. KEY POINTS: • Purified glycolipid congeners have differing effects on human keratinocytes. • Compared with SLES, acidic sophorolipids and mono-rhamnolipids have innocuous effects on keratinocytes. • Di-rhamnolipids and mono-rhamnolipids modulate cytokine production in lipopolysaccharide stimulated human keratinocytes.

Glycolipid Biosurfactants in Skincare Applications: Challenges and Recommendations for Future Exploitation.

The 21st century has seen a substantial increase in the industrial applications of glycolipid biosurfactant technology. The market value of the glycolipid class of molecules, sophorolipids, was estimated to be USD 409.84 million in 2021, with that of rhamnolipid molecules projected to reach USD 2.7 billion by 2026. In the skincare industry, sophorolipid and rhamnolipid biosurfactants have demonstrated the potential to offer a natural, sustainable, and skin-compatible alternative to synthetically derived surfactant compounds. However, there are still many barriers to the wide-scale market adoption of glycolipid technology. These barriers include low product yield (particularly for rhamnolipids) and potential pathogenicity of some native glycolipid-producing microorganisms. Additionally, the use of impure preparations and/or poorly characterised congeners as well as low-throughput methodologies in the safety and bioactivity assessment of sophorolipids and rhamnolipids challenges their increased utilisation in both academic research and skincare applications. This review considers the current trend towards the utilisation of sophorolipid and rhamnolipid biosurfactants as substitutes to synthetically derived surfactant molecules in skincare applications, the challenges associated with their application, and relevant solutions proposed by the biotechnology industry. In addition, we recommend experimental techniques/methodologies, which, if employed, could contribute significantly to increasing the acceptance of glycolipid biosurfactants for use in skincare applications while maintaining consistency in biosurfactant research outputs.

Microbial sophorolipids inhibit colorectal tumour cell growth in vitro and restore haematocrit in Apcmin+/- mice.

Sophorolipids are glycolipid biosurfactants consisting of a carbohydrate sophorose head with a fatty acid tail and exist in either an acidic or lactonic form. Sophorolipids are gaining interest as potential cancer chemotherapeutics due to their inhibitory effects on a range of tumour cell lines. Currently, most anti-cancer studies reporting the effects of sophorolipids have focused on lactonic preparations with the effects of acidic sophorolipids yet to be elucidated. We produced a 94% pure acidic sophorolipid preparation which proved to be non-toxic to normal human colonic and lung cells. In contrast, we observed a dose-dependent reduction in viability of colorectal cancer lines treated with the same preparation. Acidic sophorolipids induced apoptosis and necrosis, reduced migration, and inhibited colony formation in all cancer cell lines tested. Furthermore, oral administration of 50 mg kg-1 acidic sophorolipids over 70 days to Apcmin+/- mice was well tolerated and resulted in an increased haematocrit, as well as reducing splenic size and red pulp area. Oral feeding did not affect tumour numbers or sizes in this model. This is the first study to show that acidic sophorolipids dose-dependently and specifically reduces colon cancer cell viability in addition to reducing tumour-associated bleeding in the Apcmin+/- mouse model. KEY POINTS: • Acidic sophorolipids are produced by yeast species such as Starmerella bombicola. • Acidic sophorolipids selectively killed colorectal cells with no effect on healthy gut epithelia. • Acidic sophorolipids reduced tumour-associated gut bleed in a colorectal mouse model.

Biosynthesis of rhamnolipid by a Marinobacter species expands the paradigm of biosurfactant synthesis to a new genus of the marine microflora.

BACKGROUND: In comparison to synthetically derived surfactants, biosurfactants produced from microbial culture are generally regarded by industry as being more sustainable and possess lower toxicity. One major class of biosurfactants are rhamnolipids primarily produced by Pseudomonas aeruginosa. Due to its pathogenicity rhamnolipid synthesis by this species is viewed as being commercially nonviable, as such there is a significant focus to identify alternative producers of rhamnolipids. RESULTS: To achieve this, we phenotypically screened marine bacteria for biosurfactant production resulting in the identification of rhamnolipid biosynthesis in a species belonging to the Marinobacter genus. Preliminary screening showed the strain to reduce surface tension of cell-free supernatant to 31.0 mN m-1. A full-factorial design was carried out to assess the effects of pH and sea salt concentration for optimising biosurfactant production. When cultured in optimised media Marinobacter sp. MCTG107b produced 740 ± 28.3 mg L-1 of biosurfactant after 96 h of growth. Characterisation of this biosurfactant using both HPLC-MS and tandem MS showed it to be a mixture of different rhamnolipids, with di-rhamnolipid, Rha-Rha-C10-C10 being the most predominant congener. The strain exhibited no pathogenicity when tested using the Galleria mellonella infection model. CONCLUSIONS: This study expands the paradigm of rhamnolipid biosynthesis to a new genus of bacterium from the marine environment. Rhamnolipids produced from Marinobacter have prospects for industrial application due to their potential to be synthesised from cheap, renewable feed stocks and significantly reduced pathogenicity compared to P. aeruginosa strains.

Quorum sensing as a potential target for increased production of rhamnolipid biosurfactant in Burkholderia thailandensis E264.

Burkholderia thailandensis E264 is a potential non-pathogenic substitute for producing rhamnolipid biosurfactant, replacing the pathogenic Pseudomonas aeruginosa. However, it has low rhamnolipid production and longer fermentation time. We have earlier suggested that media supplementation with exogenous quorum sensing (QS) molecules could lead to early onset of biosynthesis and increased rhamnolipid yield. Here, we assessed the effect of single, double or triple mutations in the various QS systems of B. thailandensis on rhamnolipid production, with the view to see which system(s) have the most impact on rhamnolipid yield and subsequently use the QS molecule to potentially increase yield in the wild-type B. thailandensis. The triple mutant strain had a rhamnolipid yield of 4.46 ± 0.345 g/l at 240 h of fermentation which was significantly higher than that of the wild type (0.94 ± 0.06 g/l), an unexpected outcome. To gain more insight as to how this might occur, we studied substrate metabolism and energy storage in the form of polyhydroxyalkanoate (PHA) by both the triple mutant and the wild type. We observed increased glycerol metabolism and reduced PHA production in the triple mutant compared with the wild type. Glycerol concentration at 240 h and maximum PHA productivity (g/gDCB) were 8.76 g/l or 16.19 g/l and 21.80% or 31.4% in either the triple mutant or the wild type respectively. Complementation of the triple-mutant cultures with exogenous QS molecules restored rhamnolipid production to similar levels as the wild type. QS therefore is a potential target for increased rhamnolipid production in B. thailandensis.

Natural quorum sensing inhibitors effectively downregulate gene expression of Pseudomonas aeruginosa virulence factors.

At present, anti-virulence drugs are being considered as potential therapeutic alternatives and/or adjuvants to currently failing antibiotics. These drugs do not kill bacteria but inhibit virulence factors essential for establishing infection and pathogenesis through targeting non-essential metabolic pathways reducing the selective pressure to develop resistance. We investigated the effect of naturally isolated plant compounds on the repression of the quorum sensing (QS) system which is linked to virulence/pathogenicity in Pseudomonas aeruginosa. Our results show that trans-cinnamaldehyde (CA) and salicylic acid (SA) significantly inhibit expression of QS regulatory and virulence genes in P. aeruginosa PAO1 at sub-inhibitory levels without any bactericidal effect. CA effectively downregulated both the las and rhl QS systems with lasI and lasR levels inhibited by 13- and 7-fold respectively compared to 3- and 2-fold reductions with SA treatment, during the stationary growth phase. The QS inhibitors (QSI) also reduced the production of extracellular virulence factors with CA reducing protease, elastase and pyocyanin by 65%, 22% and 32%, respectively. The QSIs significantly reduced biofilm formation and concomitantly with repressed rhamnolipid gene expression, only trace amount of extracellular rhamnolipids were detected. The QSIs did not completely inhibit virulence factor expression and production but their administration significantly lowered the virulence phenotypes at both the transcriptional and extracellular levels. This study shows the significant inhibitory effect of natural plant-derived compounds on the repression of QS systems in P. aeruginosa.

Fatty acid synthesis pathway provides lipid precursors for rhamnolipid biosynthesis in Burkholderia thailandensis E264.

Rhamnolipid production was monitored for a period of 216 h using different substrates in Pseudomonas aeruginosa PAO1 and Burkholderia thailandensis E264 which showed comparable crude yields attained by both after 216 h. The crude yield for P. aeruginosa, however, was significantly higher at the early stages of fermentation (72 or 144 h). Additionally, P. aeruginosa produced rhamnolipid with odd and even carbon chain lipid moieties using odd carbon chain fatty acid substrates (up to 45.97 and 67.57%, respectively). In contrast, B. thailandensis produced rhamnolipid with predominantly even carbon chain lipid moieties (up to 99.26). These results indicate the use of the fatty acid synthesis (FAS II) pathway as the main source of lipid precursors in rhamnolipid biosynthesis by B. thailandensis. Isotope tracing using 0.25% stearic acid - d 35 + 1% glycerol as carbon substrate showed a single pattern of deuterium incorporation: with predominantly less than 15 deuterium atoms incorporated into a single Di-C14-C14 rhamnolipid molecule. This further indicates that the FAS II pathway is the main source of the lipid precursor in rhamnolipid biosynthesis by B. thailandensis. The pathogenicity of these strains was also assessed, and results showed that B. thailandensis is significantly less pathogenic than P. aeruginosa with an LC50 at 24 h > 2500, approximately three logs higher than P. aeruginosa using the Galleria mellonella larva model.

Marine derived biosurfactants: a vast potential future resource.

Surfactants and emulsifiers are surface-active compounds (SACs) which play an important role in various industrial processes and products due to their interfacial properties. Many of the chemical surfactants in use today are produced from non-renewable petrochemical feedstocks, while biosurfactants (BS) produced by microorganisms from renewable feedstocks are considered viable alternatives to petroleum based surfactants, due to their biodegradability and eco-friendly nature. However, some well-characterised BS producers are pathogenic and therefore, not appropriate for scaled-up production. Marine-derived BS have been found to be produced by non-pathogenic organisms making them attractive possibilities for exploitation in commercial products. Additionally, BS produced from marine bacteria may show excellent activity at extreme conditions (temperature, pH and salinity). Despite being non-pathogenic, marine-derived BS have not been exploited commercially due to their low yields, insufficient structural elucidation and uncharacterised genes. Therefore, optimization of BS production conditions in marine bacteria, characterization of the compounds produced as well as the genes involved in the biosynthesis are necessary to improve cost-efficiency and realise the industrial demands of SACs.

Adjuvant Antibiotic Activity of Acidic Sophorolipids with Potential for Facilitating Wound Healing.

The sophorolipid class of biosurfactants is finding increasing use in personal care as well as pharmaceutical products and has the potential to disrupt biofilm formation and inhibit the growth of a variety of clinically relevant organisms. In order to investigate potential biomedical applications of sophorolipids derived from nonpathogenic organisms, we fractionated and purified glycolipid biosurfactant sophorolipids produced by the yeast Starmerella bombicola, which yielded nonacetylated acidic C18:1 congeners that were essentially free from other contaminants (>95% purity). These acidic sophorolipids have antimicrobial activities against the nosocomial infective agents Enterococcus faecalis and Pseudomonas aeruginosa, with significant reductions in CFU at concentrations of as low as 5 mg ml-1 In addition, the sophorolipid showed similar effects against the same two bacterial strains when combined with kanamycin or cefotaxime. As a potential use of these sophorolipids is as a component of topically applied creams for the treatment of wound infections, it is clear that they must have no demonstrable adverse effect on wound healing. To assess this, we evaluated mammalian cell toxicity in vitro using viability tests, which revealed no adverse effect on either endothelial or keratinocyte-derived cell lines with sophorolipid concentrations of < 0.5 mg ml-1 In addition, in vivo experiments using a mouse skin wounding assay revealed that the time course of healing wounds was unaffected by the application of sophorolipid-containing creams, and histological examination of regenerated skin tissue confirmed that the healing process was similar to that observed for control animals, with no evidence of inflammation. These results are consistent with the suggestion that acidic sophorolipids can be used as a component of antimicrobial creams to reduce the risk of wound infection during healing.

Adsorption at the Air-Water Interface in Biosurfactant-Surfactant Mixtures: Quantitative Analysis of Adsorption in a Five-Component Mixture.

The composition of the air-water adsorbed layer of a quinary mixture consisting of three conventional surfactants, octaethylene glycol monododecyl ether (C12E8), dodecane-6-p-sodium benzene sulfonate (LAS6), and diethylene glycol monododecyl ether sodium sulfate (SLE2S), mixed with two biosurfactants, the rhamnolipids l-rhamnosyl-l-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoyl, R2, and l-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoyl, R1, has been measured over a range of compositions above the mixed critical micelle concentration. Additional measurements on some of the subsets of ternary and binary mixtures have also been measured by NR. The results have been analyzed using the pseudophase approximation (PPA) in conjunction with an excess free energy, GE, that depends on the quadratic and cubic terms in the composition. The compositions of the binary, ternary, and quinary mixtures could all be fitted to two sets of interaction parameters between the pairs of surfactants, one for micelles and one for adsorption. No ternary interactions or ternary corrections were required. Because the system contains two strongly anionic surfactants, the PPA can be extended, in practice, to ionic surfactants, contrary to the prevailing view. The values of the interaction parameters show that the quinary mixture, SLE2S-LAS6-C12E8-R1-R2, which is known to be a highly effective surfactant system, is characterized by a sequence of strong surface but weak micellar interactions. About half of the minima in GE for the strong surface interactions occur well away from the regular solution value of 0.5.

Microbial rhamnolipid production: a critical re-evaluation of published data and suggested future publication criteria.

High production cost and potential pathogenicity of Pseudomonas aeruginosa, commonly used for rhamnolipid synthesis, have led to extensive research for safer producing strains and cost-effective production methods. This has resulted in numerous research publications claiming new non-pathogenic producing strains and novel production techniques many of which are unfortunately without proper characterisation of product and/or producing strain/s. Genes responsible for rhamnolipid production have only been confirmed in P. aeruginosa, Burkholderia thailandensis and Burkholderia pseudomallei. Comparing yields in different publications is also generally unreliable especially when different methodologies were used for rhamnolipid quantification. After reviewing the literature in this area, we strongly feel that numerous research outputs have insufficient evidence to support claims of rhamnolipid-producing strains and/or yields. We therefore recommend that standards should be set for reporting new rhamnolipid-producing strains and production yields. These should include (1) molecular and bioinformatic tools to fully characterise new microbial isolates and confirm the presence of the rhamnolipid rhl genes for all bacterial strains, (2) using gravimetric methods to quantify crude yields and (3) use of a calibrated method (high-performance liquid chromatography or ultra-performance liquid chromatography) for absolute quantitative yield determination.

Enhanced rhamnolipid production in Burkholderia thailandensis transposon knockout strains deficient in polyhydroxyalkanoate (PHA) synthesis.

Microbially produced rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa, is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa. Multiple knockout strains for the phbA, phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (ΔphbA1, ΔphbB1 and ΔphbC1) with the best enhancement of rhamnolipid production were selected for detailed study. ΔphbB1 produced the highest level of purified RL (3.78 g l-1) compared to the wild-type strain (1.28 g l-1). In ΔphbB1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa, knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway.

Rhamnolipids from Pseudomonas aeruginosa strain W10; as antibiofilm/antibiofouling products for metal protection.

Industrial biofouling-problems associated with the accumulation of microorganisms from flowing water and fluids on processing surfaces can cause severe problems. A Pseudomonas aeruginosa strain W10 was isolated from industrial setting and found to produce predominantly di-rhamnolipids (Rha-Rha-C10-C10) with a yield of around 10 g L-1 and a critical micelle concentration (CMC) of 80 mg L-1 . P. aeruginosa W10 rhamnolipids were able to disrupt up to 99% of 48 h pre-formed biofilms of the Gram-positive organisms Bacillus licheniformis CAN55, Staphylococcus capitis SH6, and a mixed culture (strains CAN55, SH6, and W10), under static conditions, at concentrations of 0.1, 0.5, and 1 mg ml-1 on a stainless steel surface commonly used in industrial process pipelines. CFU measurements and LIVE/DEAD BacLight staining confirmed these observations. Furthermore, a purified di-rhamnolipid fraction was found to be responsible for the microbial inhibition of B. licheniformis strain CAN55. This study provides evidence that rhamnolipids may have valuable applications in preventing biofilms and biofouling in industrial plants and, in a wider context, may also apply to metal medical devices.

In vivo evaluation of biomimetic fluorosurfactant polymer-coated expanded polytetrafluoroethylene vascular grafts in a porcine carotid artery bypass model.

OBJECTIVE: The objective of this study was to evaluate the potential for biomimetic self-assembling fluorosurfactant polymer (FSP) coatings incorporating heptamaltose (M7-FSP) to block nonspecific protein adsorption, the cell adhesive RGD peptide (RGD-FSP), or the endothelial cell-selective CRRETAWAC peptide (cRRE-FSP) to improve patency and endothelialization in small-diameter expanded polytetrafluoroethylene (ePTFE) vascular graft implants. METHODS: ePTFE vascular grafts (4 mm in diameter, 5 cm in length) were coated with M7-FSP, RGD-FSP, or cRRE-FSP by dissolving FSPs in distilled water and flowing solution through the graft lumen for 24 hours. Coatings were confirmed by receding water contact angle measurements on the lumen surface. RGD-FSP and cRRE-FSP grafts were presodded in vitro with porcine pulmonary artery endothelial cells (PPAECs) using a custom-designed flow system. PPAEC coverage on the lumen surface was visualized with epifluorescent microscopy and quantified. Grafts were implanted as carotid artery interposition bypass grafts in seven pigs for 33 ± 2 days (ePTFE, n = 3; M7-FSP, n = 4; RGD-FSP, n = 3; cRRE-FSP, n = 4). Patency was confirmed immediately after implantation with duplex color flow ultrasound and at explantation with contrast-enhanced angiography. Grafts were sectioned for histology and stained: Movat pentachrome stain to outline vascular layers, immunofluorescent staining to identify endothelial cells (anti-von Willebrand factor antibody), and immunohistochemical staining to identify smooth muscle cells (anti-smooth muscle α-actin antibody). Neointima to lumen area ratio was determined to evaluate neointimal hyperplasia. RESULTS: Receding water contact angle measurements on graft luminal surfaces were significantly lower (P < .05) on FSP-coated ePTFE surfaces (M7-FSP, 40 ± 16 degrees; RGD-FSP, 25 ± 10 degrees; cRRE-FSP, 33 ± 16 degrees) compared with uncoated ePTFE (126 ± 2 degrees), confirming presence of the FSP layer. In vitro sodding of PPAECs on RGD-FSP and cRRE-FSP grafts resulted in a confluent monolayer of PPAECs on the luminal surface, with a similar cell population on RGD-FSP (1200 ± 187 cells/mm(2)) and cRRE-FSP (1134 ± 153 cells/mm(2)) grafts. All grafts were patent immediately after implantation, and one of three uncoated, two of three RGD-FSP, two of four M7-FSP, and two of four cRRE-FSP grafts remained patent after 1 month. PPAEC coverage of the lumen surface was seen in all patent grafts. RGD-FSP grafts had a slightly higher neointima to lumen area ratio (0.53 ± 0.06) compared with uncoated (0.29 ± 0.15), M7-FSP (0.20 ± 0.15), or cRRE-FSP (0.17 ± 0.09) grafts. CONCLUSIONS: Biomimetic FSP-coated ePTFE grafts can be used successfully in vivo and have potential to support endothelialization. Grafts modified with the M7-FSP and cRRE-FSP showed lower intimal hyperplasia compared with RGD-FSP grafts.

Characterising rhamnolipid production in Burkholderia thailandensis E264, a non-pathogenic producer.

Burkholderia thailandensis E264 is a rhamnolipid (RL)-producing gram-negative bacterium first isolated from the soils and stagnant waters of central and north-eastern Thailand. Growth of B. thailandensis E264 under two different incubation temperatures (25 and 30 °C) resulted in a significantly higher dry cell biomass production at 30 °C (7.71 g/l) than at 25 °C (4.75 g/l) after 264 h; however, incubation at the lower temperature resulted in consistently higher concentration of RL production throughout the growth period. After 264 h, the concentration of crude RL extract for the 25 °C culture was 2.79 g/l compared to 1.99 g/l for the 30 °C culture. Overall RL production concentration after 264 h was 0.258 g/g dry cell biomass (DCB) for the 30 °C culture compared to 0.587 g/g DCB for the 25 °C culture. Real-time PCR (qPCR) was also used to analyse expression of the RL biosynthesis genes throughout the incubation period at 25 °C showing that the expression of the rhlA, rhlB and rhlC genes is continuous. During the log and early stationary phases of growth, expression levels remain low and are increased upon entry to the late stationary phase. B. thailandensis E264 produces mostly di-RLs and the Di-RL C14-C14 in most abundance (41.88 %). Fermentations were also carried out in small-scale bioreactors (4 l working volume) under controlled conditions, and results showed that RL production was maintained. Our findings show that B. thailandensis E264 has excellent potential for industrial scale RL production.

Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants.

OBJECTIVES: To develop and validate a microdilution method for measuring the minimum inhibitory concentration (MIC) of biosurfactants. RESULTS: A standardized microdilution method including resazurin dye has been developed for measuring the MIC of biosurfactants and its validity was established through the replication of tetracycline and gentamicin MIC determination with standard bacterial strains. CONCLUSION: This new method allows the generation of accurate MIC measurements, whilst overcoming critical issues related to colour and solubility which may interfere with growth measurements for many types of biosurfactant extracts.

Development and validation of an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantitative determination of rhamnolipid congeners.

Rhamnolipids (RLs) are synthesised as a complex mixture of congeners comprising either one or two molecules of rhamnose glycosidically linked to a dimer of 3-hydroxy fatty acids varying in chain length and degree of saturation. Currently, HPLC-MS/MS is the most precise and accurate method for RL determination, while accurate quantification is limited. In this study, a rapid ultra pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the rapid and quantification of individual RL congeners. Increased RLs specificity was achieved using tandem mass spectrometry in multiple reaction monitoring (MRM) mode which was used to quantify RL isomer pairs such as Rha-Rha-C8-C10/Rha-Rha-C10-C8 which are difficult to resolve chromatographically. UPLC showed an 18-fold reduction in retention time for Rha-Rha-C10-C10 (1.07 min) and a 17-fold reduction for Rha-C10-C10 (1.36), the major rhamnolipids present, compared to HPLC, with a total run time less than 2.2 min. The results show that the linear range for the main RL congeners (Rha-C10-C10 and Rha-Rha-C10-C10) is 0.1 to 100 µg/mL. The LOD and LOQ for Rha-C10-C10 is 0.05 and 0.1 µg/mL and for Rha-Rha-C10-C10 is 0.1 and 0.5 µg/mL, respectively. The method was validated for linearity, intra- and inter-day precision and accuracy in accordance with FDA guidelines. The method was applied for the quantification of 14 individual RL congeners produced by Pseudomonas aeruginosa ST5 and comparison of RLs composition on four different carbon sources. Quantification of the individual congeners showed a conserved congener distribution irrespective of carbon source with a preferential selection for C10 ß-hydroxyacids as the lipid component of RLs. The only statistically significant differences detected were between actual RL yields on the various carbon sources.

Influence of calcium ions on rhamnolipid and rhamnolipid/anionic surfactant adsorption and self-assembly.

The impact of Ca(2+) counterions on the adsorption at the air-water interface and self-assembly in aqueous solution of the rhamnolipid biosurfactant and its mixture with the anionic surfactant sodium dodecylbenzenesulfonate, LAS, has been studied using neutron reflectometry and small-angle neutron scattering. The results illustrate how rhamnolipids are calcium tolerant and how their blending with conventional anionic surfactants improves the calcium tolerance of the anionic surfactant. Ca(2+) has relatively little effect upon the adsorption and self-assembly of the monorhamnose, R1, and dirhamnose, R2, rhamnolipids, even at high pH, due to their predominantly nonionic nature. For R1/R2 mixtures the addition of Ca(2+) has little impact upon the adsorbed amount or the surface composition. For R2/LAS mixtures the addition of Ca(2+) results in an increased adsorption and a surface slightly richer in R2. The weak binding of Ca(2+) to R1 and R2 does result in a change to the degree of ionization of the micelles and especially for mixed R1/R2 micelles at R1-rich solution compositions. The stronger binding of Ca(2+) to LAS results in the addition of Ca(2+) having a much greater impact on the self-assembly of R1/LAS and R2/LAS mixtures. For R1/LAS mixtures the addition of Ca(2+) promotes the formation of more planar structures, even at low surfactant concentrations where in the absence of Ca(2+) mixed globular micelle formation dominates. In R2/LAS mixtures, where there is a greater contrast between the high and low preferred curvatures associated with R2 and LAS, the addition of Ca(2+) results in a more complex evolution in micellar aggregation and the degree of ionization of the micelles. This results in variations in Ca(2+) binding that promotes micellar structures in which a spatial segregation of the two surfactant components within the micelle occurs.

Rhamnolipids are conserved biosurfactants molecules: implications for their biotechnological potential.

A range of isolates of Pseudomonas aeruginosa from widely different environmental sources were examined for their ability to synthesise rhamnolipid biosurfactants. No significant differences in the quantity or composition of the rhamnolipid congeners could be produced by manipulating the growth conditions. Sequences for the rhamnolipid genes indicated low levels of strain variation, and the majority of polymorphisms did lead to amino acid sequence changes that had no evident phenotypic effect. Expression of the rhlB and rhlC rhamnosyltransferase genes showed a fixed sequential expression pattern during growth, and no significant up-regulation could be induced by varying producer strains or growth media. The results indicated that rhamnolipids are highly conserved molecules and that their gene expression has a rather stringent control. This leaves little opportunity to manipulate and greatly increase the yield of rhamnolipids from strains of P. aeruginosa for biotechnological applications.

Biomimetic poly(ethylene glycol)-based hydrogels as scaffolds for inducing endothelial adhesion and capillary-like network formation.

The extracellular matrix (ECM) is an attractive model for designing synthetic scaffolds with a desirable environment for tissue engineering. Here, we report on the synthesis of ECM-mimetic poly(ethylene glycol) (PEG) hydrogels for inducing endothelial cell (EC) adhesion and capillary-like network formation. A collagen type I-derived peptide GPQGIAGQ (GIA)-containing PEGDA (GIA-PEGDA) was synthesized with the collagenase-sensitive GIA sequence attached in the middle of the PEGDA chain, which was then copolymerized with RGD capped-PEG monoacrylate (RGD-PEGMA) to form biomimetic hydrogels. The hydrogels degraded in vitro with the rate dependent on the concentration of collagenase and also supported the adhesion of human umbilical vein ECs (HUVECs). Biomimetic RGD/GIA-PEGDA hydrogels with incorporation of 1% RGD-PEGDA into GIA-PEGDA hydrogels induced capillary-like organization when HUVECs were seeded on the hydrogel surface, while RGD/PEGDA and GIA-PEGDA hydrogels did not. These results indicate that both cell adhesion and biodegradability of scaffolds play important roles in the formation of capillary-like networks.