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Mass Transport in Coacervate-based Protocell Coated with Fatty Acid under Non-Equilibrium Conditions Hairong Jing, Ya'nan Lin, Haojing Chang, Qingwen Bai, and Dehai Liang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00470 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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Mass Transport in Coacervate-based Protocell Coated with Fatty Acid under Non-Equilibrium Conditions Hairong Jing, Ya’nan Lin, Haojing Chang, Qingwen Bai, Dehai Liang*

Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Polymer Chemistry and Physics, College of Chemistry and Molecular Engineering, Peking University, Beijing, China, 100871

*Corresponding author: [email protected]

Abstract

ACS Paragon Plus Environment

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Construction of protocell models from prebiotically plausible components to mimic the basic features or functions of living cells is still a challenge. In this work, we prepare a hybrid protocell model by coating sodium oleate on the coacervate droplet constituted by poly(L-lyine) and oligonucleotide, and investigate the transport of different molecules under electric field. Results show that sodium oleate forms a layered viscoelastic membrane on the droplet surface, which is selectively permeable to small, polar molecules, such as oligolysine. As the droplet is stimulated at 10 V cm-1, the oleate membrane slips along the direction of electric field while maintaining its integrity. Most of the molecules are still excluded under such conditions. As repetitive cycles of vacuolization occur at 20 V cm-1, all molecules are internalized and sequestrated in the droplet through their specific pathways except enzyme, which anchors in the oleate membrane and is immune to electric field. Cascade enzymatic reactions are then carried out and the product generated from the membrane exhibits a time-dependent concentration gradient across the droplet. Our work makes a step towards the nonequilibrium functionalization of synthetic protocells capable of biomimetic operations.


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Introduction Protocell model provides a practical approach to fabricate molecular assemblage capable of compartmentalization, metabolism, and replication, which is conducive to the illustration of origin of life.1-4 Such protocellular systems are based predominantly on the fatty acid vesicles for their prebiotic availability5-6 and resemblance to biological cells,7-8 although other membrane-delineated structures, such as phospholipid vesicle,9-10 polymersomes,11-12 colloidosomes,13-14 protein-based capsules,15-16 and polyelectrolyte capsules,17 have also been taken into consideration. These models have been systematically explored and engineered to achieve various archetypal properties of cells, such as molecular encapsulation,18-19 spatially controlled reactions,20-22 and stimulusinduced growth and divisions.23-25 Alternatively, membrane-free protocell models, including coacervate droplet constituted by electrostatically mediated complexation of polycations and polyanions,26-28 have received much recent attention due to their relevance to intracellular membrane-less organelles.29-30 These droplets are featured by


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the molecularly crowded, chemically enriched, and highly dynamic viscoelastic interiors, which enable the protocells to sequester exogenous solutes,31-32 enhance enzymatic reactivity,33-35 and be energized by electric field.36-37 Although the protocell models aforementioned exhibit distinct features and advantages, they account for only one structural feature of the biological counterparts, either plasma membrane or cytosol environment, which restricts their further complication and functionalization. Therefore, a new class of hybrid protocell model, based on the selfassembly of amphipathic molecules (block copolymers or fatty acids, etc.) on the surface of coacervate microdroplets, have been developed.38-40 These hybrid protocell models, which integrate the chemical barrier of membrane with polymer-crowded interior, not only resemble living cell in architecture, but also provide more potential for bio-functionality. Cells consume energy to selectively uptake nutrients and release wastes which are crucial to metabolism and life sustainment.41-42 The engagement of such non-equilibrium mass transport in a protocell model, however, has yet to be achieved. It’s known that direct current electric field is ubiquitous in living cell.43-44 Our previous study demonstrated that the coacervate droplets composed of single-stranded oligonucleotide (ss-oligo) and


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poly(L-lysine) (PLL), once excited by electric field, exhibited repetitive cycles of vacuolization and mass circulation,45 both of which had the capacity to regulate transport of materials in and out of the droplet. It is also reported that fatty acid can assemble into vesicle structures under controlled pH values.8 Since the fatty acid vesicle is prone to grow and change shapes, allowing only certain molecules to pass through the membrane, it is proposed that fatty acid is a plausible membrane components of primordial cell.5 Therefore, a hybrid protocell model based on coacervate droplet and fatty acid coating should resemble the cellular morphology and be able to achieve complex “living” features under electric field. To test this hypothesis, we coat the PLL/oligo droplet with sodium oleate (SO) and investigate its uptake and sequestration of different molecules under electric field. The hybrid protocell only allows small, polar molecules to enter the droplet without electric field. Once excited by electric field, the oleate membrane slips along the field direction with no damage on its integrity. Only the uptake of negatively charged oligonucleotide is enhanced under such conditions. As the vacuolization, which is triggered at higher electric field strength disintegrates, the membrane, all molecules manage to enter the droplet and generate their specific distributions. Surprisingly, protein


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is well-protected by the oleate membrane and immune to electric field, which is used to mimic the behavior of membrane proteins. Cascade enzymatic reaction is carried out in the hybrid protocell model by anchoring the enzymes on the oleate membrane. The product exhibits a time-dependent concentration gradient across the droplet.

Materials and Methods Materials. Single-stranded DNA molecules (12-mer and 21-mer, both are random sequence) with or without Cy5 fluorescence labelled at the 5’-terminus were purchased from Invitrogen Inc. Polylysine (PLL, Mw = 30-70 kDa), FITC-tagged polylysine (FITCPLL, Mw = 30-70 kDa), FITC-tagged dextran (Mw = 4 kDa), glucose oxidase (GOx, Mw = 160 kDa, dimer), horseradish peroxidase (HRP, Mw = 40kDa), sodium oleate (SO, CH3(CH2)7CH=CH(CH2)7COOH, pKa = 7-9), calcein and rhodamine 6G were all purchased from Sigma-Aldrich (St. Louis, MI, USA). The lipid membrane soluble dye BIDIPY 558/568 C12 was purchased from Invitrogen. The peptides FAM-K3L8K3 (FAMAhx-KKKLLLLLLLLKKK-NH2)

and

FITC-K10

(FITC-Ahx-KKKKKKKKKK-NH2)

were

purchased form GL Bitchem Ltd (Shanghai, P. R. China). Polyvinylpyrrolidone (Mw = 30


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kDa) was from Sinopharm Chemical Reagent Co. Ltd. Fluorescence labelling of enzymes was conducted using a previously reported protocol.36 All chemicals were used as received without further purification. All aqueous stock solutions of above-mentioned chemicals except sodium oleate and BODIPY 558/568 C12 were prepared by directly dissolving the samples in phosphate buffer (0.025 M, pH 7.5). Sodium oleate was dissolved in deionized water (pH = 12, 20 mM) and diluted to required concentration with phosphate buffer before use. BODIPY 558/568 C12 stock solution was obtained in ethanol at a concentration of 0.025 mg/ml (52.7 uM). Preparation of fatty acids-coated coacervate microdroplets in microfluidic channel. The polylysine/oligonucleotides coacervate microdroplets in microfluidic device were prepared by following a procedure previously described.36 In brief, 21-mer ss-oligo and PLL aqueous solutions were loaded into opposite wells in a microfluidic channel device (channel dimensions: 80 μm width × 25 μm depth), which was washed beforehand in sequence by NaCl (1 M), NaOH (1 M), HCl (1 M), deionized water, and 1% (w/w) polyvinylpyrrolidone. The flowing solutions driven by gravity were mixed in the central


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channel of the chip and formed discrete microdroplets automatically. As the droplets grow to 10-15 m in diameter, residual aqueous ss-oligo and PLL solutions were replaced by phosphate buffer. For the preparation of SO-coated microdroplets, the ss-oligo and PLL solution were replaced by phosphate buffer, followed by SO solution. After incubated for certain time, mostly 5 min, the residual SO solution was replaced by phosphate buffer. Electric field-mediated mass transport experiments. BODIPY 558/568 C12 was chose to monitor the changes of oleate membrane under the direct current (DC) electric field. Others were used as model molecules to conduct the energy-mediated mass transport experiments. When the distribution of these molecules was stable, a DC electric field with the intensity of 10-20 V cm-1 was applied along the microfluidic channel by using a power supply (DYY-16D, Beijing Liuyi Instrument), and the changes were recorded by using laser scanning confocal microscopy (LSCM). Membrane-mediated enzymatic reactions. As coacervate droplets were prepared, a mixture containing 2 mM SO, 40 g ml-1 GOx (glucose oxidase from Aspergillus niger), and 20 g ml-1 HRP (horseradish peroxidase) was loaded into channel wells to coassemble on the droplet surface. 0.01 mg ml-1 amplex red (Sigma-Aldrich, ref: A12222,


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substrate for HRP) together with 0.36 mg ml-1 β-D-glucose (substrates GOx) in phosphate buffer were injected into the channel wells to replace the original buffer and initiate the membrane-mediated reaction. The fluorescence intensity of the product resorufin was measured using LSCM (543 nm excitation and 580 nm emission).

Results and discussion Construction of fatty acids-coated coacervate microdroplets. Mixing of PLL and 21-mer ss-oligo in the microfluidic channel produced a string of discrete droplets with the size of 10-15 m. The coating of fatty acids on the polyelectrolyte-dense coacervate surface was undertaken at pH 7.5 by incubating oleate sodium with preformed droplets for 5 min. SO, at the concentration of 2.0 mM, self-assembled into a continuous shell on the positive charged surface of droplets (+/- = 2.0) , as demonstrated by the distribution of lipid-soluble dye molecule

46

BODIPY 558/568 C12 (Figure 1). We interpreted it as a multilayered

membrane as reported in literature.40, 47 At 1.0 mM, the amount of SO was not enough to form a uniform coating layer. When the SO concentration raised to 5.0 mM, the surfactant molecules formed vesicles in aqueous solution, leading to no coating but the absorption


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of vesicle into the droplet (Figure S1). The surface charge on the droplet also affected the coating efficiency of SO. For the droplet at +/- =4.0, SO accumulated on the positivecharged surface due to the strong PLL-SO electrostatic interaction, while at negativecharged droplet at +/- = 0.5, SO could only form a weak and non-uniform coating (Figure S1). Consequently, the droplet at +/- =2.0 coated by SO at 2.0 mM for 5 min was used in the following experiments.

A

B

C

Figure 1. Formation of PLL / ss-oligo coacervate microdroplets coated with sodium oleate: (A) schematically showing the microfluidic channel. (B) SO-coated droplets, the green, orange, and red color denote the trace of PLL, SO, and ss-oligo, respectively. (C)


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3D confocal fluorescence microscopy reconstruction of (from left to right) the shell bottom, the shell, and the overlay of the shell and the infilled ss-oligo. Scale bar, 10 m.

Molecular sequestration/exclusion in hybrid protocells. To reveal the effect of oleate membrane on the propensity of droplets to sequester or exclude substances, we replaced the phosphate buffer with aqueous solutions containing different model molecules. These molecules were readily partitioned into the milieu of uncoated, positively charged microdroplets due to hydrophobic and/or electrostatic interactions (Figure 2A’ to 2H’). The negatively charged oleate membrane, however, made things different (Figure 2A to 2H). Positively charged molecules with certain degree of hydrophobicity, such as rhodamine 6G (Rhd-6g) (Figure 2A) and bola-typed peptide K3L8K3 (Figure 2E), formed a layer on the surface because of their strong interaction with the oleate membrane. Protein enzymes such as horseradish peroxidase (HRP), containing both positive and negative charges, also belonged to this category (Figure 2D). Hydrophilic molecules that are negatively charged (calcein, Figure 2B) or electric neutral (dextran, Figure 2F) were completely excluded from the SO-coated droplet. Small, highly-charged biomolecules,


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such as oligolysine (K10, Figure 2C) and cy5-12-mer oligonucleotides (oligo12, Figure 2G), could pass through the membrane and be sequestered into the droplet, demonstrating that the coated SO layer on the irregular droplet surface was permeable. For similar solutes but with larger size, the oleate membrane dramatically lowered their permeation rate inside the droplet, as demonstrated by the distribution of oligo21 (Figure 2H and Figure S2).


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Figure

2.

Confocal

fluorescence

microscope

images

showing

the

sequestration/exclusion of different molecules by PLL/ss-oligo coacervate droplets (+/- = 2.0) coated with (A to H) and without (A’ to H’) sodium oleate. Scale bar: 10 m.

Electric energy-mediated mass transport. The response of SO-coated coacervate microdroplets to electrical stimulation was investigated by applying a DC electric field to the system. At 10 V cm-1, a lateral slip of oleate membrane along the direction of the electric field was observed by tracing the fluorescence changes of dye molecule BODIPY 558/568 C12 distributed inside the oleate membrane (Figure 3A and Supplementary Movie S1). The fluorescence of the surfactant layer partially recovered after powering off (Figure S3). Control experiments exhibited that the BODIPY 558/568 C12 labelled oleic acid, which aggregates at pH = 6,48 underwent a fast and complete slip on the uncoated surface of droplet but in the opposite direction under the same electric field (Figure 3C and Supplementary Movie S2), with no fluorescent recovery was observed after power off. We attributed the slip on the droplet surface to viscous flow correlated with the electric field-induced motion of electrical double layer (electro-osmotic flow),49-50 whose moving


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direction was determined by the surrounding counterions. Since the SO coating reversed the surface charge of the droplet from positive to negative, it slipped along opposite direction under electric field (Figure 3B and 3D). The layered membrane behaved as an entity and exhibited certain elasticity, which accounted for its extremely slow slipping rate and partial recovery after power off. The internal crowded environment prevented the elastic membrane from migrating inside the droplet at 10 V cm-1. Therefore, only the initial slipping stage instead of the whole circulation process was observed under such conditions.


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Figure 3. PLL/ss-oligo coacervate droplet excited by an electric field. (A and B) droplet coated with oleate membrane and excited at 10 V cm-1; (C and D) droplet without oleate membrane and excited at 10 V cm-1; (E) the droplet coated with oleate membrane and excited at 20 V cm-1, repetitive cycles of vacuolization occur under such conditions, as shown in corresponding snapshot in (F). BODIPY 558/568 C12 is used as the tracer in (A, C, E). The green arrows in B and D show the moving direction of counterions under electric field. Scale bar, 10 m.


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Repetitive cycles of vacuolization (Figure 3F) occurred in the SO-coated droplet as the electric field increased to 20 V cm-1. The transport of external aqueous water phase was driven by osmotic pressure generated by an elevated local ion concentration inside the droplets as the PLL/ss-oligo complex was dissociated under electric field.36 The volume of the droplet experienced corresponding changes during vacuolization. The oleate membrane lost its integrity as the droplet enlarged but recovered when shrank (Figure 3E and 3F, Supplementary Movie S3), demonstrating the viscoelasticity of the SO layers. The slip of the membrane was negligible, indicating that vacuolization dominated the dynamics of the SO-coated droplet once it was initiated at an elevated electric field.


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Figure 4. Confocal fluorescence microscope images show the uptake of (A) oligo21, (C) Rhd 6g, and (D) HRP enzyme by SO-coated coacervate droplet at 10 Vcm-1. (B) shows the fluorescence profile along the vertical line in A. Scale bar, 10 m.

The oleate membrane kept its integrity at 10 V cm-1, thus preventing the initially excluded molecules, such as calcein and dextran, from entering the droplet even under electric excitation (Figure S4). Oligo21, which was initially located inside the membrane and had the same charge as SO, permeated into the droplet by following a similar trajectory as BODIPY 558/568 C12 (Figure 4A, 4B and Supplementary Movie S4). In contrast, Rhd 6g had a strong interaction with the oleate coating via both electrostatic and hydrophobic interaction. This changed the structure of oleate membrane, leading to a violent slip without the migration of the dye molecules into the droplet (Figure 4C and Supplementary Movie S5). While HRP, which initially accumulated on the droplet surface, was immune to electric field at 10 V cm-1. No prominent change in distribution was observed under the studied conditions (Figure 4D).


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The vacuolization initialized at 20 V cm-1 controlled the dynamics of the SO-coated coacervate droplet, thus changed the permeability of the surfactant layers. The fierce water exchange facilitated the internalization of oligo21 and led to a homogenous distribution in the non-vacuole area inside the droplet (Figure 5A and Supplementary Movie S6). The initially excluded calcein, which had a strong affinity for water, was repetitively internalized following the pattern of vacuolization (Figure 5B and Supplementary Movie S7). Only trace amount of dextran entered the droplet during the vacuolization process owing to its larger size (Supplementary Figure S5 and Movie S8). And in the case of Rhd 6g, its hybrid membrane with SO was more waterproof under such conditions, resulting in a suppression of the vacuolization that was driven by osmotic pressure. On this account, the lateral slip of the membrane along the direction of the electric field was still dominant. The Rhd 6g/oleate membrane was forced into the droplet from the top following a circulation pattern under such conditions45 (Figure 5C and Supplementary Movie S9). Interestingly, HRP still firmly bounded to the oleate membrane during the vacuolization process at 20 V cm-1. The relatively positions of HRP were even fixed during the expansion-shrinkage cycles (Figure 5D and supplementary Movie S10).


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It was well-established that charged surfactants, such as sodium dodecyl sulfonate, can wrap around protein to form stable structures.51 In our case, the multilayered oleate membrane strongly interacted with HRP in the surrounding medium. The membrane could reorganize to encapsulate HRP into discrete domains until all the charges on the membrane were neutralized, by which the responsiveness to the electric field was lost.


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Figure 5. Confocal fluorescence microscope images show the uptake of (A) oligo21, (B) calcein, (C) Rhd 6G, and (D) HRP by SO-coated coacervate droplet at 20 Vcm-1. Scale bar, 10 m.

Oleate membrane-mediated enzymatic reactions. Inspired by the anchoring effect of enzyme on the oleate membrane, we explored the possibility of achieving localized enzymatic reactions on membrane in an attempt to mimic the activity of membrane proteins. Speciﬁcally, GOx and HRP were premixed with sodium oleate, followed by the incubation with preformed droplets to integrate and co-assemble into membrane. Afterwards, a mixture of β-D-glucose (substrate for GOx) and amplex red (substrate for HRP) was added into the channel to initiate the reactions at the droplet surface. The final product resorufin, a fluorescent molecule, was used to monitor the enzyme catalytic progress. At the early stage, the resorufin-characteristic fluorescence mainly appeared on the droplet surface (Figure 6A and Figure S6), indicative of the enzymes’ localization inside the oleate membrane with biological activity. The resorufin preferred to stay inside the droplet because of its hydrophobicity. Therefore, the fluorescence intensity in the


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center region of the droplet showed a time-dependent increase (Figure 6B) and even higher than that in the border after 15 min when the resorufin generation rate decreased due to the reduction of substrates in the environment. The diffusion of resorufin out of the droplet occurred simultaneously and would dominate when the concentration inside the droplet reached a certain value. At this point, the fluorescence intensity decreased in both the boundary and the center region of the droplet.

Figure 6. Oleate membrane-mediated enzymatic reactions at the droplet surface. (A) confocal fluorescence microscope images show the distribution of products (resorufin) at


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different times. (B) Time-dependence of the fluorescence intensity in the center and on the boundary of the droplet as well as their differences. Scale bar, 10 m.

Conclusion By integrating sodium oleate with PLL/ss-oligo coacervate droplet, we prepare a hybrid protocell model coated with a viscoelastic membrane. The multi-layered oleate membrane contains hydrophobic domains and negatively charged surface, which expels neutral or single-charged hydrophilic molecules, but traps hydrophobic molecules and oppositely charged large molecules on the membrane. Since the membrane on the droplet surface is not as homogeneous and smooth as that in the fatty acid vesicle, it still selectively permeates polar solutes with small size, such as oligolysine and oligonucleotide. The hybrid protocell model experiences circulation and vacuolization under electric field. The circulation regulates internal mass transports. Only the molecules unevenly distributed inside the droplet, oligo21 in our case, is affected. The repetitive cycles of vacuolization facilitate the uptake of almost all molecules but by different pathways. For example, calcein is internalized and expelled with the same frequency of


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vacuolization cycle, while Rhd 6g is dragged into the droplet together with the oleate membrane. Enzymes show a different behavior as they are encapsulated inside the oleate membrane and immune to electric field. By taking advantage of this anchoring effect, we achieve enzymatic cascades reactions specifically in the membrane. The product prefers to stay inside the coacervate, generating a time-dependent distribution profile across the droplet. Even though the oleate membrane-coated coacervate droplet is still primitive, the hybrid protocell operating under electric field can successfully regulate the molecular transport via a specific pattern related to its properties. In this regard, our work highlights the connection between fatty acids and complex coacervate, both of which are thought to contribute to the origin of life, and offers a practical approach towards synthetic protocells capable of achieving complex living functions and processes.

Associated content


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Supporting Information. Additional figures (Fig. S1 to S6) and supporting movies (Movie S1 to S10). The Supporting Information is available free of charge on the ACS Publications website. Author information Corresponding Author. *E-mail: [email protected]. Notes. The authors declare no competing financial interest. Acknowledgment Financial support from the National Natural Science Foundation of China (21774002, 21574002) is greatly acknowledged.

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Mass Transport in Coacervate-based Protocell ... - ACS Publications

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