A Cell-Mimicking Structure Converting Analog Volume Changes to

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A Cell-Mimicking Structure Converting Analog Volume Changes to Digital Colorimetric Output with Molecular Selectivity Zijie Zhang, Yibo Liu, Xiaohan Zhang, and Juewen Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04298 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017

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A Cell-Mimicking Structure Converting Analog Volume Changes to Digital Colorimetric Output with Molecular Selectivity

Zijie Zhang, Yibo Liu, Xiaohan Zhang and Juewen Liu* Department of Chemistry, Waterloo Institute for Nanotechnology University of Waterloo 200 University Ave W, Waterloo, ON N2L 3G1 E-mail: [email protected] Phone: 519-888-4567 Ext. 38919

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Abstract: We herein report a three-component cell-mimicking structure with a peroxidase-like iron oxide nanozyme as the nucleus, a molecularly imprinted hydrogel shell as cytoplasm, and a lipid bilayer membrane. The structure was characterized by cryo and negative stain TEM and also by a calcein leakage test. By introducing charged monomers, the gel shell can swell or shrink in response to salt concentration. By lowering the salt concentration, the gradual ‘analog’ gel volume change was reflected in a switch-like ‘digital’ colorimetric output by the burst of membrane and oxidation of substrates such as 3,3’,5,5’-tetramethylbenzidine (TMB). Controlled access was also achieved by using melittin to insert channels cross the membrane, and selective molecular transport was realized by the molecularly imprinted gel. The functions of each component are coupled, and this sophisticated tripartite structure provides a new platform for modular design of new materials. Our cell-mimicking structure is functional and it is complementary to the current protocell work that aims to understand the origin of life.

Keywords: Cell-mimicking; protocells; molecularly imprinted polymers; nanozymes

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The functions of cellular components are highly coupled, enabling complex tasks that define life. Mimicking such functional structures by chemical synthesis is intriguing and inspiring.1, 2 Two directions have been taken to create cell-mimicking structures. One studies reduced physical systems such as nucleic acid encapsulated vesicles (so called protocells) for processes like membrane fusion, compartmentalized biological reactions, and transmembrane communication.38

The other direction constructs cell-mimicking functional structures, which are not necessarily

of biological relevance.9-13 For example, Brinker and coworkers sealed mesoporous nanoparticles (NPs) within lipid bilayers for drug delivery.14, 15 Zhang et al wrapped natural cell membranes on inorganic NPs,16-19 while Mann and coworkers designed artificial cytoskeletal protocells with enzymatic activities.20,

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These structures are sometimes also called protocells. Most cell-

mimicking structures, however, are still quite simple, disallowing coupling of more complex functions. Cells can selectively internalize certain molecules, and respond to external stimuli. The cell membrane defines cellular boundary, the cytoplasm is gel-like with highly crowded biopolymers, and cells also have a nucleus structure. To date, few protocells have such a tripartite structure.22 We reason that as the structure becomes more complex, new materials might be obtained allowing novel applications. In this work, we constructed a tripartite cellmimicking structure with interesting functions such as switch-like colorimetric output sensitive to a very small salt concentration change with molecular selectivity. A tripartite functional cell-mimicking structure. Our tripartite cell-mimicking structure was designed as shown in Figure 1A. Fe3O4 NPs with peroxidase-like activity were chosen as the core material due to its excellent catalytic activity.23-26 In the presence of H2O2, Fe3O4 catalyzes the oxidation of many chromogenic 3 ACS Paragon Plus Environment

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substrates

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(TMB)

and

2,2’-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid) (ABTS) with product absorption peaks at 652 nm and 420 nm, respectively. We prepared Fe3O4 NPs through a hydrothermal method without strong capping ligands to ensure a native oxide surface.27 The average particle size was ~30 nm by TEM (Figure 1B). We then grew a layer of molecularly imprinted hydrogel by mixing a template molecule (e.g. TMB) with a few acrylamide-based monomers and a crosslinker. After washing away TMB, the resulting imprinted cavities in the gel could selectively rebind TMB.28, 29 To make stimuli-responsive gels, we also incorporated cationic DMPA or anionic AMPS as monomers (see Figure S1 in Supporting information for their structures), so that the gel volume was more sensitive to salt concentration. For TMB, three imprinted gels were prepared: uncharged (named T-MIP), negatively charged (T-MIPneg) and positively charged (T-MIPpos). Three more gels were also prepared by imprinting ABTS, and they were named in the same way but started with ‘A-’. From dynamic light scattering (DLS), the non-charged gels (e.g. T-MIP and A-MIP) were 210 ± 15 nm, while the charged gels (e.g. T-MIPneg) were larger (315 ± 22 nm, Figure S2) due to the osmotic pressure and repulsion of the charged polymer chains.30,

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SEM also

confirmed Fe3O4 NPs encapsulated in the gels (Figure 1C),29 and the EDX spectrum indicated the presence of iron (13 ± 2 w%) along with carbon, nitrogen, and oxygen (Figure 1D). Charge directed membrane coating. We then need to coat a lipid bilayer onto the gel surface.32-38 Three types of liposomes were prepared to identify an optimal formulation, each carrying a different charge: DOPC (neutral), DOPS (negative) and DOTAP (positive) (Figure 1E). These extruded liposomes all had a similar size (average of 126 ± 8 nm, Figure S3A). Their ζ-potentials agreed with their expected surface 4 ACS Paragon Plus Environment

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charge (Figure S3B). These liposomes were then respectively mixed with each imprinted gel, resulting in a total of nine combinations.

Figure 1. (A) A scheme of preparing a functionally coupled tripartite cell-mimicking structure. TMB was imprinted on the surface of Fe3O4 NPs by a layer of negatively charged gels (TMIPneg). Then a cationic DOTAP liposome was fused on the gel surface. (B) A TEM micrograph of the free Fe3O4 NPs. (C) A SEM micrograph of negatively charged T-MIPneg gel with Fe3O4 NP core and (D) its EDX spectrum analyzed on the area defined by the red square. (E) The structures of the three lipids used in this work.

We first used ζ-potential to characterize gel/liposome interaction. When each liposome was incubated with the non-charged T-MIP gels, only the anionic DOPS liposome slightly decreased the ζ-potential of the gel (Figure 2A), suggesting adsorption of DOPS. For the two


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charged gels, their ζ-potentials inversed only when incubated with the oppositely charged liposome (Figure 2B and 2C), highlighting the importance of electrostatic attraction. The change of ζ-potential only indicated liposome adsorption, while it did not prove our intended lipid enveloping (Figure 1A). To test this, we designed a liposome leakage assay by encapsulating 100 mM calcein in liposomes resulting in self-quenched fluorescence. If the liposome breaks (e.g. by enveloping the gel), fluorescence is expected to increase by releasing the dye into the whole solution (Figure 2F). Little fluorescence leakage of the DOPC liposomes was observed with all the three gels (Figure 2D, Figure S4A), suggesting either no interaction or simple adsorption. Interestingly, ~81% of calcein leaked from the DOPS liposomes after mixing with the oppositely charged A-MIPpos gel, suggesting formation of the intended cell-mimicking structure (Figure 2E, Figure S4B). To further confirm the liposome/gel interaction, cryo-TEM was performed. After mixing the non-charged DOPC liposomes with the anionic A-MIPneg gel, we observed many intact liposomes associated with darker features assigned to the A-MIPneg gel (Figure 2G), indicating simple adsorption and this was consistent with a lack of calcein leakage (Figure 2D). When the cationic gel was mixed with anionic DOPS liposomes, we could barely find any intact liposome (Figure S5). Since the ζ-potential of this sample inversed and calcein leaked (Figure 2E), DOPS lipids must be on the gel surface. We failed to find the typical supported lipid bilayer feature in the cryo-TEM likely due to a lack of contrast (i.e. over 85% of the gels was water). We then performed negative-stain TEM for the samples. For the DOPC liposome sample, we observed very dark features assigned to dried gels adsorbed on the liposome surface (Figure 2H). While for the anionic DOPS liposomes sample (Figure 2I), the gels appeared inside the liposomes and the contrast of the gel was much lighter. We reason that the lipid layer protected the gel core 6 ACS Paragon Plus Environment

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from being stained by the heavy metal salt, supporting liposome fusion. In the negative stained sample, the gels shrunk when dried on the TEM grid in air allowing us to see the liposome envelope, while in the cryo-TEM sample, the gel retained water disallowing the resolution of the supported lipid bilayer.

Figure 2. The change of ζ-potential of (A) T-MIP, (B) T-MIPneg and (C) A-MIPpos gels after respectively incubated with the three liposomes (excess liposomes washed away before measurement). The percentage of calcein leakage from calcein-loaded (D) DOPC and (E) DOPS liposome after mixing with different gels. (F) A scheme of calcein (Cal) loaded liposome leakage upon fusing the gel surface with increased fluorescence (DOPS), or simple adsorption without leakage (DOPC). (G) A cryo-TEM micrograph of cationic A-MIPpos gel mixed with noncharged DOPC liposomes. The DOPC liposomes adsorbed on the gel surface. Negative-stain TEM micrographs of A-MIPpos gels mixed with (H) non-charged DOPC liposomes and (I) anionic DOPS liposomes. The gel appeared quite dark around the liposomes in (H), while the 7 ACS Paragon Plus Environment

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DOPS contained much lighter gels since the liposome protected the gel from the heavy metal staining agent. The insets in (G) and (I) are the schemes of their liposome/gel products.

After constructing the intended cell-mimicking structure, we then tested its function. The nanozyme core allowed for a colorimetric assay for monitoring molecular diffusion across the membrane and the gel layer. For the negatively charged T-MIPneg gels, adding the cationic DOTAP liposomes decreased its initial oxidation rate by 8.4-fold (from 4.2 ± 0.4 ×10-2 min-1 to 0.5 ± 0.1 ×10-2 min-1), indicating effective lipid sealing, while the other two liposomes had little effect (Figure 3A). The positively charged A-MIPpos gels were also best blocked by the negatively charged DOPS liposomes (Figure 3B). However, the effect of adding liposomes was very small for the non-charged T-MIP gels (Figure S6). These experiments further confirmed membrane sealing onto oppositely charged gels. To further confirm this, we added a surfactant, Triton X-100, to the T-MIPneg@DOTAP sample (i.e. T-MIPneg gels fused by DOTAP liposomes) at 10 min (Figure 3C, purple trace), which immediately rescued the activity to a level similar to that without any lipids (black trace). The A-MIPpos@DOPS (Figure 3D) showed the same behavior. In addition, the free DOPS liposome and the A-MIPpos@DOPS structure had the same leakage profile in response to Triton X-100 concentration (Figure S7). Therefore, we indeed formed the intended functional structure as designed in Figure 1A by mixing the imprinted gels with oppositely charged liposomes, and the membranes could block the access of small substrate molecules.


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Figure 3. The effect of adding various liposomes to (A) T-MIPneg gel and (B) A-MIPpos gel on their kinetics of substrate oxidation. Activity rescue of (C) T-MIPneg@DOTAP and (D) AMIPpos@DOPS by adding Triton X-100 at 10 min indicated by the arrowheads. The gel samples without the lipid layers were also tested for comparison (black traces). All the measurements were in buffer A (20 mM acetate, pH 4, NaCl 50 mM) with 10 mM H2O2. For (A) and (C), 0.5 mM TMB was used as the substrate and monitored at 652 nm. For (B) and (D), 0.5 mm ABTS was used and monitored at 420 nm.

Membrane controlled substrate oxidation. After preparing our cell-mimicking structures, we then tested their responsiveness to ionic strength, which was expected to change its volume.39-41 We first measured the size of our imprinted gels in various concentrations of NaCl (Figure 4A). The size of the non-charged TMIP gel remained constant at 220 ± 25 nm regardless of the NaCl concentration. For the charged T-MIPneg and A-MIPpos gels, the size indeed increased from ~300 nm (with 50 mM NaCl) to 635 ± 42 nm (no NaCl).


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We then tested the salt-dependent reaction of our T-MIPneg@DOTAP. Optimization of the liposome concentration was performed to ensure full surface coverage (Figure S8). As the anionic gel layer swelled upon lowering the salt concentration, the lipid membrane was expected to burst, thus allowing access of TMB to produce a color. From 50 mM to 8 mM NaCl, the samples did not produce much color after 1 h of reaction, and the final absorbance was below ~0.14. Interestingly, when the NaCl concentration was further decreased from 8 mM to 6 mM, the absorbance jumped to 0.8, and it remained at this value for the samples with even lower NaCl concentrations (Figure 4B, blue dots). This suggests that the membrane could no longer withstand the swelling gel at 6 mM NaCl and burst. As a control, the T-MIPneg gels without the DOTAP bilayer remained high activity in all NaCl concentrations (Figure 4B, black dots). In addition, the DOTAP liposome alone did not give any color change as expected (Figure 4B, red dots). The photographs at different NaCl concentrations were also collected (Figure 4D). We further quantitatively measured the reaction kinetics (Figure 4C), also showing a transition between 8 and 6 mM NaCl. Finally, we increased the salt concentration beyond the initial 50 mM NaCl (Figure S9). While the gels shrank slightly, no TMB oxidation was observed. Therefore, the membrane integrity was retained during gel shrinkage (Figure 4E). As a control, we also tested the catalytic activity of the free Fe3O4 NPs alone as a function of NaCl concentration and no effect was observed (Figure S10). Since we used molecularly imprinted gels here, one may expect decreased activity by changing salt concentration since the imprinted sites might be perturbed as the gel swelled by lowering the NaCl concentration. To understand the retained high activity, we measured the binding of MIP gels using ITC in both high salt (low swelling) and low salt (high swelling) conditions (Figure S11). Interestingly, a similar binding was observed regardless of the salt 10 ACS Paragon Plus Environment

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concentration, which might be attributed to the reduced charged screening effect by the lowered salt concentration (note the TMB and the negatively charged gel were oppositely charged). In other words, the adverse effect on the imprinted cavity and the enhanced electrostatic binding might compensate each other. It is interesting to note that the response of the system took place within 2 mM NaCl (e.g. 8 to 6 mM). If we take the first derivative of this transition,42 the half width at half maximum (HWHM) was only 1 mM NaCl, which behaved like a switch (e.g. digital). For comparison, the gel volume change was ‘analog’ (Figure 4A), showing a linear increase as a function of NaCl concentration went from 50 to 0 mM with a HWMW of >25 mM NaCl. Compared to the original gel volume with 50 mM NaCl, the volume expansion to 8 mM NaCl and to 6 mM NaCl was quite similar (Figure 4A). This final slight change reached the elasticity limit of the membrane. Since the substrates used here were chromogenic, this system might serve as a switch in response to a small drop in ionic strength. Our cell-mimicking structure also responded to MgCl2 with an even higher sensitivity (below 100 µM, Figure S12), which is attributed to a higher electric charge density of Mg2+ than Na+. When we increased the ionic strength again after bursting the membrane of cell-mimicking structures, the membranes could not be fully re-sealed (Figure S13). This suggested that some membranes were lost during membrane burst. The radius of the gel increased by ~38% by decreasing the NaCl from 50 mM to 6 mM, while the gel volume swelled expanded by 260% (e.g. qw = 1.383). Therefore, the lipid layer has a strong tolerance to volume expansion before bursting. We also tested swelling of the free liposomes without the gel or the Fe3O4 core as a function of salt concentration using the calcein leakage assay. Upon decreasing the salt concentration, the liposome did not show any sign of breaking (i.e. no calcein release, Figure 11 ACS Paragon Plus Environment

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S14). We also failed to observe much volume change for our free liposomes by decreasing ionic strength (Figure S15). Therefore, in our system, with the same ionic strength change, the volume change of the charged gel was much larger than that of the free liposomes. The charged gel shells thus amplified the ionic strength effect.

Figure 4. The effect of NaCl concentration on (A) the average gel size without lipid membrane, and (B) TMB oxidation activity by T-MIPneg gel, T-MIPneg@DOTAP and free DOTAP liposomes. (C) The kinetics of the T-MIPneg@DOTAP oxidizing TMB at different NaCl concentrations. (D) The photographs after 1 h of the oxidation at different NaCl concentrations for the samples in (B). (E) A scheme of the swelling, shrinkage and breaking of the lipid membrane when the swelling ratio (qw) is greater than 260% in response to varying NaCl concentration. All the measurements were in acetate buffer (20 mM, pH 4.0) with 0.5 mM TMB and 10 mM H2O2.


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It should be noted that depending on lipid composition, some lipid vesicles indeed rupture due to ionic strength change.43-46 It is well-documented that cell membranes are highly sensitive to ionic strength due to water permeability. For example, red blood cells need to be maintained in 0.85% NaCl (~145 mM). Significant hemolysis occurs when the NaCl concentration is below 0.6% (~102 mM), and nearly 100% of the cells burst within 2 min when the NaCl concentration is below 0.4%.47-49 A few studies showed permeability of small ions across the lipid layer even in the absence of transport assistance.50-54 Typical cell membranes also have saturated lipids and cholesterol, which can make the membrane only permeable to water, but not to small salt ions. We reason that our single-component liposomes in the fluid phase are permeable to small ions such as Na+ and Cl- (but not to TMB or calcein).55

Tunable salt sensitivity range. Our cell-mimicking structures prepared with 50 mM NaCl were sensitive to the 8-to-6 mM NaCl transition. We wanted to further test if it is possible to tune the sensitivity range. We prepared a series of T-MIPneg@DOTAP in various initial NaCl concentrations (e.g. 0 to 100 mM; named S0 to S100). When prepared in very low salt concentrations (e.g. 0-10 mM), they were unresponsive to ionic strength and they remained stable at all the tested conditions (Figure 5A, S0, S5 and S10 traces). It is likely that these gels were already highly swelled after preparation and they could only expand by a limited volume that the membrane could tolerate (Figure 5B, S5). However, when prepared in higher NaCl concentrations (e.g. 30-100 mM), the gels were in an initially more shrunk state, and they expanded more significantly such that the membrane could not tolerate (Figure 5B, S100 samples). Therefore, the S30, S50 and S100 structures were all activated by lowering the ionic strength with a sharp transition (Figure 5A). Importantly, the 13 ACS Paragon Plus Environment

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activity transition occurred at a different salt concentration for each sample, and the structure prepared at a lower salt concentration had a lower transition salt concentration, which was consistent with our hypothesis of a critical tolerant limit for volume expansion. Therefore, we successfully tuned the sensitivity range. While all the transitions occurred below 15 mM NaCl, the unique digital response set our system aside from others.40, 54, 56-59

Figure 5. (A) The effect of salt concentration on the activity of the T-MIPneg@DOTAP samples prepared with different initial NaCl concentrations of 0 to 100 mM (named S0 to S100). (B) A scheme showing the swelling of S5 and S100 when decreasing the NaCl concentration to close to zero. The catalytic selectivity for TMB and ABTS oxidation by (C) T-MIPneg gels, and TMIPneg@DOTAP and (D) A-MIPneg gels, and A-MIPneg@DOPS tested in the absence (-Na+) or presence 50 mM NaCl (+Na+). The S50 sample prepared with initial 50 mM NaCl was used. 14 ACS Paragon Plus Environment

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All the measurements were in acetate buffer (20 mM, pH 4) with 0.5 mM TMB or ABTS and 10 mM H2O2 after a 1 h reaction. The absorbance was collected at 652 nm and 420 nm for TMB and ABTS, respectively.

Natural cells have various protein-based channels in the membrane for selective uptake of ions and molecules. With molecular imprinting, our cell-mimicking structure might also mimic such a function but in a different way: we engineered it into the ‘cytoplasm’. For example, if the salt concentration was high, no activity was observed for either TMB or ABTS for the TMIPneg@DOTAP. At a lower NaCl concentration, only TMB was oxidized, while ABTS was left intact (Figure 5C). This indicates that our system could carry out complex logic functions. Similarly, when ABTS imprinted samples were used, only ABTS was oxidized (Figure 5D).29 In a sense, we have produced two types of cell-mimicking samples defined by their cytoplasm. Finally, we tested the response of our system to melittin, a membrane-perturbing amphipathic peptide from bee venom.60 We first measured the percentage of leakage of the calcein-loaded free DOPS liposomes by various concentrations of melittin, confirming its function (Figure 6A). Then we tested A-MIPpos@DOPS with melittin and observed a gradually increased activity suggesting more permeable membranes (Figure 6B). While each protein is different and it is likely that not all membrane proteins can be incorporated with this simple mixing method, this study suggests that other more complex biological structures such as channels might be grafted onto our cell-mimicking structure to further enhance its function.


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Figure 6. (A) Percentage of calcein release from DOPS liposome as a function of melittin in 20 mM HEPES buffer (pH 7.6, NaCl 50 mM). (B) ABTS oxidation activity by A-MIPpos gel, AMIPpos@DOPS structure (abbreviated as Gel@DOPS) and free DOPS liposomes (DOPS Ctrl) as a function of melittin. All the measurements were tested with 0.5 mM ABTS and 10 mM H2O2 after a 1 h reaction in buffer A.

In summary, we demonstrated a tripartite system mimicking the structural features of cells containing a nanozyme core, a molecularly imprinted gel shell as cytoplasm, and a lipid bilayer membrane. Each component had unique functions that were coupled to realize more complex operations. We demonstrated that the elasticity of the lipid membrane can convert the ‘analog’ salt concentration input to a ‘digital’ colorimetric output. In addition, substrate selectivity was harnessed via the imprinted cytoplasm gels. With this reduced physical system, we obtained many useful and new insights into functional cell-mimicking structures. By introducing multiple components with coupled functions, we believe other functional nanostructures can be prepared for controlled release, switching, actuating and biosensing applications. Acknowledgements


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We appreciate Robert Harris at the University of Guelph for assistance in the cryo-TEM experiment and Dr. Biwu Liu for the help in TEM measurement. Funding for this work was from The Natural Sciences and Engineering Research Council of Canada (NSERC). Supporting Information Experimental Section including the chemicals, preparation materials, characterization and nanozyme assays. Supporting data related to DLS, cryo-TEM, ITC, UV-vis and fluorescence spectroscopy (Figure S1-S15). (PDF) This material is available free of charge via the internet at http //pubs.acs.org.

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