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Highly Stable Artificial Cells from GalactopyranoseDerived Single-Chain Amphiphiles Roberto J. Brea, Ahanjit Bhattacharya, Rupak Bhattacharya, Jingjin Song, Sunil Sinha, and Neal K Devaraj J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09388 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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Journal of the American Chemical Society

Highly Stable Artificial Cells from Galactopyranose-Derived SingleChain Amphiphiles Roberto J. Brea†,§, Ahanjit Bhattacharya†,§, Rupak Bhattacharya‡,§, Jingjin Song‡,⊥, Sunil K. Sinha‡, and Neal K. Devaraj*,† †

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, United States Department of Physics, University of California, San Diego, La Jolla, CA 92093, United States ⊥Department of Materials Science and Engineering, University of California, San Diego, La Jolla, CA 92093, United States ‡

Supporting Information Placeholder ABSTRACT: Single-chain amphiphiles (SCAs) capable

of spontaneously self-assembling into large vesicular structures are attractive components of synthetic cells due to the simplicity of bilayer formation and increased membrane permeability. However, SCAs commonly used for vesicle formation suffer from restricted working pH ranges, instability to free divalent cations, and the inhibition of biocatalysts. Construction of more robust biocompatible membranes from SCAs would have significant benefits. Here we describe the formation of highly stable vesicles from alkyl galactopyranose thioesters. The compatibility of these uncharged SCAs with biomolecules makes possible the encapsulation of functional enzymes and nucleic acids during the vesicle generation process, enabling membrane protein reconstitution and compartmentalized nucleic acid amplification, even when charged precursors are supplied externally.

Lipid vesicles are ubiquitous in fields ranging from membrane protein reconstitution,1,2 drug delivery,3 synthetic biology,4 and origin of life studies.5 While natural lipid membranes primarily consist of dialkyl phospholipids,6 there has been significant interest in the use of single-chain amphiphiles (SCAs) to form vesicles.7–10 SCAs offer several advantages, including simplicity and ease of synthesis.11 Importantly, prior seminal studies have demonstrated that SCAs are more permeable to charged small molecule precursors, enabling internal polymerization of macromolecules such as oligonucleotides.11,12 However, fatty acids, the most common SCAs used to form vesicles, suffer from several drawbacks.13– 18 They have a narrow pH range over which they form stable vesicles.13,14 Moreover, vesicles formed exclusively from fatty acids are not tolerant of divalent cations, such as magnesium and calcium, which are essential in all living system.15–18 Additives such as metal chelators15

or esterified fatty acids17–19 can preserve vesicle integrity in the presence of divalent cations, however, SCAs also have limited compatibility with various biochemical processes. For instance, fatty acids and other SCAs are known to inhibit nucleic acid polymerases,20–22 making DNA/RNA amplification or transcription reactions difficult to perform in such vesicles. Additionally, integral membrane proteins have not been demonstrated to function within membranes consisting solely of SCAs. It would thus be of considerable value to develop artificial cells that enjoy the benefits of SCAs but without the aforementioned limitations. Here we report the serendipitous discovery of oleoyl b-D-1-thiogalactopyranose (OTG), a new class of water-soluble SCA capable of spontaneously forming highly stable micron-sized vesicles (Figure 1A). The uncharged glycolipid can self-assemble into vesicles at sub-millimolar concentrations over a wide pH range and in the presence of high concentrations of free divalent metal cations. The resulting vesicles are compatible with the conditions necessary for enzymatic reactions, such as oligonucleotide polymerization, to function. We initially synthesized OTG by standard coupling of 1-thio-b-galactopyranose with oleic acid (Figures S1A and S2A). Our initial intention was to use this lipid as a detergent, as we expected the amphiphile would form micelles due to the large head group and single alkyl tail. To our surprise, a thin film of the purified OTG, when hydrated, readily formed micron-sized membranebound vesicles (Figure S3). While there are several wellknown examples of non-ionic amphiphiles consisting of two-chains that are capable of assembling to form large vesicles,23–27 it is quite rare for a non-ionic single-chain amphiphile to be capable of forming vesicles. We believe that, apart from the hydrophobic interactions between alkyl chains, hydrogen bonding between galactopyranose head groups plays a crucial role in membrane

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Figure 1. Thiogalactopyranose-based vesicular structures. (A) Schematic representation of the self-assembly of OTG into bilayer membrane vesicles. (B) Phase-contrast microscopy image of membrane vesicles resulting from the selfassembly of OTG. Scale bar denotes 10 µm. (C) Fluorescence microscopy image of membrane-bound vesicles formed by hydration of a thin film of OTG. Membranes were stained using 0.1 mol% Texas Red® DHPE dye. Scale bar denotes 5 µm. (D) TEM image of negatively stained vesicles formed from self-assembly of OTG. Scale bar denotes 500 nm. (E) Spinning-disk confocal fluorescence microscope image demonstrating the encapsulation of GFP in membrane vesicles of OTG. Scale bar denotes 5 µm.

formation, similar to what has been previously observed for other two-chain lipids bearing galactopyranose head groups.23–27 To determine the role of the thioester linkage, we also synthesized oleoyl b-D-1-galactopyranose amide (OGA), an alternative galactopyranose derivative bearing an amide linkage instead a thioester linkage (Figures S1B and S2B). We found that this lipid also self-assembled into vesicles (Figure S4), indicating that the thioester linkage is not essential. The critical vesicle concentration (cvc) of OTG was determined to be 127 µM using dynamic light-scattering (DLS) measurements (Figures S5 and S6).28 The compound was stable to hydrolysis at 37 °C in aqueous solu-

tions at pH 7 over a period of more than 24 h (Figure S7). We performed microscopy studies on hydrated OTG samples to characterize the resulting self-assembled structures (Figure 1B-E, Figures S8 and S9). OTGbased vesicles were initially identified by phase-contrast microscopy (Figure 1B) and fluorescence microscopy using the membrane-staining dye 1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE) (Figure 1C). Transmission electron microscopy (TEM) also confirmed the formation of vesicular structures (Figure 1D). OTG vesicles were capable of encapsulating highly charged fluorescent dyes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and Cy5-azide (Figure S8), as well as GFP (Figure 1E). Fatty acid SCAs assemble into bilayer membranes only when the working pH is near the pKa of the bilayerassociated acid (pH ~ 7-9).13,14 Higher and lower pH favor the spontaneous formation of micelles and oil droplets, respectively. Additionally, divalent metal cations present in the hydration media drives the destruction of the membrane-bound vesicles by causing fatty acid precipitation.15–18 These phenomena are due to the ionizable carboxylic acid head group, which can bind metal ions. In contrast, OTG amphiphiles lack an ionizable headgroup and would not be expected to bind metal cations tightly. We confirmed this by testing the effect of pH on the formation of OTG vesicles. Hydration of thin films of OTG at 37 °C using several aqueous solutions at different pH (2, 5, 7 and 10) resulted in micron-sized vesicle formation (Figure S10). We also explored the effect of free divalent metal ions on the self-assembly of OTG into vesicles. Formation of vesicles at 37 °C was shown for hydrated thin films of OTG in aqueous solutions containing different divalent metal cations (1-10 mM Mg2+ or Ca2+) (Figure S11). OTG spontaneously self-assembles into a multilamellar structure of stacked bilayers when deposited on silicon substrates. This enabled determination of the electron density profile (EDP) of the lipid bilayers via X-ray diffraction (XRD) analysis (Figure 2). Relative humidities (RHs) of the samples were controlled using different saturated salt solutions (K2SO4 ~ 98%, KNO3 ~ 92% and NaCl ~ 75%),29 keeping the temperature fixed at 25 °C, just above the gel-fluid transition of this particular lipid. Figure 2A represents the typical XRD intensity profile of the sample at 75% RH. We observed a distinct set of Bragg peaks in the XRD profile, which indicated long-range ordering in the deposited OTG lipid multilayers. To quantify this further, we calculated the relative EDP of the stacked layers from the XRD intensity profiles data. Figure 2B shows relative electron density (ρ) profile as a function of the distance z. Here the center of each lipid bilayer is considered as the z = 0 plane for obtaining a symmetric profile. The interstitial water layer between the head


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Figure 3. Reconstitution of functional CcO into OTG membrane vesicles. A) Schematic representation of spontaneous reconstitution of CcO during vesicle formation. B) Normalized absorbance at 550 nm of ferrocytochrome c in OTG vesicles with (gold) and without (grey) embedded CcO. Activity of the isolated CcO (orange) is also shown. C) Spinning-disk confocal fluorescence image of a spontaneously reconstituted CcO (labeled with Alexa Fluor® 488)/OTG proteoliposome. Scale bar denotes 5 µm. Figure 2. Physical and structural characterization of an OTG-based multilayer film. (A) X-ray diffraction (XRD) intensity profile of an OTG multilayer film at 25 °C with 75% relative humidity. (B) Relative electron density profile (EDP) of an OTG multilayer film at 75% relative humidity and 25 °C.

groups was calculated to be on the order of 5 Å at 25 °C and its thickness increases at higher relative humidity, likely due to swelling of the membrane. We also estimated the membrane thickness (in terms of the head-tohead distance of the lipid bilayer) to be 30.02 Å at 25 °C, which is less than a corresponding phospholipid bilayer (~40 Å), possibly indicating that the alkyl chains are more disordered or tilted. The EDP also indicated decreased chain overlap in the central region inside the bilayer. Having characterized the OTG membranes, we sought to explore if they are capable of mimicking critical features of biological membranes. Transmembrane proteins are essential membrane components for all cells, regulating transport and signaling. So far, they have not been functionally reconstituted into previously analyzed SCA-based membranes. We tested if functional transmembrane proteins could be incorporated into OTG membranes during vesicle formation (Figure 3A). Cytochrome c oxidase (CcO) was chosen as a test protein because of the existence of well-known methods for assaying its function.2,30–32 We initially exchanged the detergent n-octyl-b-D-glucopyranoside (OGP) for ndodecyl-b-D-maltoside (DDM) present in commercially

available bovine heart CcO preparations by spin filtration. OGP was used as the solubilizing agent for CcO because of its high critical micelle concentration (cmc) and low aggregation number,33 which facilitates the removal of excess detergent during OTG vesicle reconstitution (see Supporting Information for full details and Figure S12). Formation of CcO/OTG vesicles was observed by bright-field microscopy (Figure S13A). When ferrocytochrome c, the substrate of CcO, was added to the CcO/OTG vesicles, we observed a steady decrease in the absorbance at 550 nm over time (Figure 3B), indicating the formation of the oxidized product, ferricytochrome c. A control experiment where ferrocytochrome c was added to OTG vesicles lacking CcO, showed negligible change in the absorbance at 550 nm over same time span (Figure 3B). A small decrease in absorbance at 550 nm was found in the presence of the isolated CcO (CcO in OGP only) (Figure 3B). Finally, we were able to visualize fluorescently labeled CcO reconstituted in OTG membranes by microscopy (Figure 3C, Figure S13B). A possible route toward creating advanced artificial cells would be the coupling of simple vesicular systems with amplification and evolution of nucleic acids. Rolling circle amplification (RCA) is an isothermal DNA amplification technique where a polymerase adds nucleotides unidirectionally to short primers annealed to a single-stranded circular DNA template.34 The robustness of this technique make it an attractive choice for demonstrating nucleic acid replication within vesicles generated by glycolipids (Figure 4). We encapsulated a



Figure 4. Rolling circle amplification (RCA) in thiogalactopyranose-based vesicular microreactors. A) Top, Schematic representation of the RCA process in OTG vesicles, showing the efficient amplification of DNA. Bottom, Fluorescence microscopy images of vesicular structures before (left) and after (right) RCA. Insets show the phase-contrast microscope images of each step, observing that the vesicles were stable during the RCA process. The pink and orange lines represent the section used to make the plot profile histogram (center), which shows the fluorescence intensity of a typical DNA-stained vesicle. EvaGreen® was used as the DNA staining dye. Scale bars denote 5 µm. B) Increment of fluorescence in relative fluorescence units (RFUs) resulting from 24 h of RCA in OTG and DOPC vesicles adding dNTPs inside or outside. Error bars indicate standard deviation (SD; n = 3). C) Microscopy images of OTG vesicles (top) and DOPC vesicles (bottom) in bright-field (left) and fluorescence (right) channels after 24 h of RCA adding dNTPs outside. The amplification was only possible in the case of using OTG vesicles. The images were captured after adding EvaGreen®. Scale bars represent 5 µm.

single-stranded circular M13mp18 DNA template and the RCA machinery [primers, deoxynucleoside triphosphates (dNTPs) and ϕ29 DNA polymerase] into OTG vesicles. The resulting vesicles were treated with proteinase K to remove any residual RCA machinery in the surrounding solution and therefore prevent DNA amplification outside the vesicle. At the start of the experiment, addition of EvaGreen®, a dye used to detect double-stranded DNA, into an aliquot of the reaction mixture showed that the level of double-stranded DNA in vesicles was initially below the level of detection (Figure 4A, Figure S14A). After 21 h of RCA at 29 °C, the addition of the staining dye revealed high fluorescence

levels within the vesicles, indicating that DNA amplification occurred (Figure 4A, Figure S14A). Agarose gel electrophoresis confirmed that DNA had been amplified in the vesicles by RCA (Figure S14B). Moreover, we observed by phase-contrast microscopy that the glycolipid-based vesicles were stable under the RCA conditions over the times scales relevant to the corresponding experiments (Figure 4A, Figure S14A). Previous landmark studies have established that SCAs can have increased permeability to charged small molecules such as nucleotides when compared to two-chain lipids such as phospholipids.17,19 Such permeability can facilitate internal biochemical reactions within synthetic cells, such as oligonucleotide polymerization, even when precursors are supplied externally. Thus, we were interested if OTG vesicles were more permeable to externally added NTPs compared to phospholipid vesicles. Additional experiments showed that OTG vesicles have significantly higher permeability to dNTPs compared to that for two-chain phospholipid vesicles (Figure 4B,C, Figure S15). For instance, we observed that the OTG vesicles could sustain RCA reaction when the dNTPs were solely supplied from the outside, while 1,2dioleoyl-sn-3-phosphocholine (DOPC) vesicles could not under identical conditions (Figure 4B,C, Figure S15). Fluorescence imaging indicated that OTG vesicles retained newly formed oligonucleotides. Therefore, OTG vesicles could be utilized as a semi-permeable artificial cell model where transport of charged solutes can take place without the necessity of special transporters or pore-forming proteins. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Detailed procedures, spectral data and Figures S1-S15 (PDF)

AUTHOR INFORMATION Corresponding Author

*[email protected] ORCID

Roberto J. Brea: 0000-0002-0321-0156 Neal K. Devaraj: 0000-0002-8033-9973 Author Contributions

§These authors contributed equally. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This material is based upon work supported by the National Science Foundation (CHE-1254611; Devaraj) and the US Department of Energy [Biomolecular Materials Program, Division of Materials Sciences and Engineering] (DE-


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SC0018086; Sinha). Roberto J. Brea thanks the Human Frontier Science Program (HFSP) for his CrossDisciplinary Fellowship.

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