Programmable Modulation of Membrane Permeability of

Jul 29, 2016 - Membrane permeability is a necessary and overarching attribute for all the hollow microcompartments toward the application as nanoreact...
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Programmable Modulation of Membrane Permeability of Proteinosome upon Multiple Stimuli Responses Pei Zhou, Xiaoman Liu,* Guangyu Wu, Ping Wen, Lei Wang, Yudong Huang, and Xin Huang* MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, 150001, China S Supporting Information *

ABSTRACT: Membrane permeability is a necessary and overarching attribute for all the hollow microcompartments toward the application as nanoreactors or artificial cells. Differing from the widely reported various stimuli models, in this study we describe a way to generate a multistimuli proteinosome capable of being triggered in sequence of temperature, redox species, and pH, thus, showing a continuous modulation on the membrane permeability. Studies showed that the molecular weight cutoff of the constructed proteinosome membrane could be continuously turned up from 78 to 102 kDa and to 142 kDa, and then turned down to 35.2 kDa upon different stimuli. As a proof of concept, such continuous modulated behavior allows a wellcontrolled programmed release upon the encapsulation of a FITC-labeled dextran into proteinosomes. It is anticipated that such designed proteinosomes equipped with programmed modulation of membrane permeability are promising candidates for the further development of artificial model design, such as cellular communication or metabolism in which stuff exchange is required to support in situ procedures.

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could selectively respond to external stimuli and in turn switch the permeability of membrane. However, in most of these cases, the modulation of the permeability of the membrane was focusing on single stimulus or independent multiple stimuli, and there are very few reports to show a combined stimuli with a continuous modulation of the membrane permeability and a concomitant programmable release. In previous work, we showed a type of compartmentalized microarchitecture (proteinosome) constructed based on the interfacial assembly of globular protein−polymer nanoconjugates at the surface of water droplets dispersed in oil.23 The membrane of proteinosomes was delineated by a closely packed monolayer with an outer and inner surface of polymer and protein-rich domains, respectively. Differing from the semipermeable liposomes or less permeable polymersomes which are made of lipids or amphiphilic block copolymers, on the one hand the giant building block (protein−polymer nanoconjugate) of proteinosomes endows the constructed model showing a good permeability which could allow biomolecules with the molecular weight as high as ∼10 kDa to diffuse inside and outside, on the other hand such polymer and protein-rich membrane domain make it possible to realize a continuous modulation of the membrane permeability.

iocompartmentalization represents an extremely efficient organization of membranes and biomolecules that is needed to cope with a complex scenario of metabolic reactions in a confined space.1 It is a prerequisite attribute of all living systems2 and is increasingly recognized as an essential paradigm of synthetic cellularity,3 and an important consideration for developing new strategies to create novel functional ensembles.4−11 Over the past years, strategies toward membranedelineated compartmentalization have been widely developed mainly focusing on the use of a diverse range of amphiphilic building blocks that undergo spontaneous or directed assembly in aqueous solutions or oil/water mixtures to produce microcompartments including liposomes, 12 polymersomes,5,6,13,14 polymer capsules,15−18 colloidosomes,19−21 and protein-based capsules.22−25 Since discovery, they have been frequently utilized to construct drug delivery nanocarriers, nanoreactors, and artificial organelles.5,6,8,26−28 It should be noted that all of these functions are related to the exchange of substances between the outside and inside milieu. The key element for fabrication of the suitable microcompartments is the design of membrane materials which are tailored to undergo a chemical or physical change in response to external stimuli triggering such as pH,29−31 temperature,32 shear-rate,33 light,34,35 redox species,36 enzymes,37 and magnetic and electric fields.30 In general, these can be achieved in various ways by chemical modification of the membrane to create pores,38 directly self-assembling a porous membrane39 or insertion of biopores,40 and reconstitution of channel porins.41 As a consequence, the designed stimuli-responsive microstructure © XXXX American Chemical Society

Received: July 1, 2016 Accepted: July 28, 2016

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ACS Macro Letters Scheme 1a

a (a) Synthetic route towards α-pyridine disulfide activated CTA and the BSA-SH/PNIPAAm conjugating; (b) thiolated procedure of BSA; (c) schematic illustration of the sequential stimuli-triggered behavior of the constructed proteinosome against temperature, reductive agent TCEP and pH, respectively.

synthesized. To realize so, on the one hand, a trithiocarbonate chain transfer agent (CTA) having a pyridine disulfide functionalized R-group was synthesized (Figure S1, Supporting Information) and used to generate a well-defined end-capped pyridine disulfide PNIPAAm by reversible addition−fragmentation chain-transfer (RAFT) polymerization.50 As seen from the 1H NMR spectrum Figure S2 (Supporting Information), there were about 160 repeat unit with a well maintain pyridine group on the α-terminal. On the other hand, the thiolated BSA was synthesized by a two-step procedure. As shown in Scheme 1b, first BSA was cationized by a carbodiimide-activated reaction of aspartic and glutamic acid residues using 1,6hexanediamine. Primary amine titration measurement indicated that cationized BSA (BSA-NH2) consisted of a total number of about 90 surface accessible amines (Figure S3, Supporting Information). Then, the thiolatization of BSA-NH2 was performed by using Traut’s reagent (2-IT, 2-iminothiolane hydrochloride) which was water-soluble and able to react with primary amines quantitatively in the pH range (7−10) of biological solutions. Using Ellman’s test prior to thiolation with BSA-NH2, 0.3 free thiol groups per protein were detected. This value increased to 6 free thiol groups per protein after thiolating with 10:1 molar ratio of 2-IT:BSA (Figure S4, Supporting Information), and then leaving about 80 primary amine groups for the cross-linking step. Lastly, by mixing of the synthesized thiolated BSA (BSA-SH) and end-capped pyridine disulfide PNIPAAm in pH 8.0 aqueous solution, the building block BSASH/PNIPAAm was obtained which was well monitored by the appearance of UV specific absorbance peak of the produced 2-

Herein, we specially designed and synthesized a BSA and poly(N-isopropylacrylamide) (PNIPAAm) conjugate linked via a disulfide bond, which could also self-assemble at the surface of water droplets dispersed in oil (Scheme 1a,b). Given the temperature sensitivity of PNIPAAm, pH induced morphology change of BSA, and the redox species sensitive disulfide bond, the constructed proteinosomes were clearly endowed a multistimuli behavior with a continuous modulation of the membrane permeability. In this studied system, four stages of membrane permeability were distinguished (Scheme 1c): (i) at 40 °C, the aggregation of the hydrophobic PNIPAAm on the surface of the membrane allows proteinosomes to show a low permeability; (ii) while when the temperature cools down to room temperature, the hydrophilic PNIPAAm suspending to aqueous solution allows an increase on the permeability compared with that of at 40 °C; (iii) then with the addition of a reductive agent TCEP, the cleavage of disulfide bond resulting the conjugated PNIPAAm leaving the membrane and shows a further enhancement on the membrane permeability; (iv) finally, adjusting pH of the system to be 3.5, the morphology of BSA deformed from a spherical like structure to an enlongated ellipsoidal structure and results in an obvious decrease on the membrane permeability in contrast. Moreover, such continuous modulation of the membrane permeability was well demonstrated from an investigation of programmed release of encapsulated dextran (MW 70 kDa). A building block, protein−polymer conjugates42−49 based on the coupling reaction between thiolated BSA (BSA-SH) and αpyridine disulfide functionalized PNIPAAm were designed and 962

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ACS Macro Letters mercaptopyridine at 340 nm (Figure S5, Supporting Information). The successful construction of the BSA-SH/ PNIPAAm conjugate was clearly demonstrated from dynamic light scattering measurement. As seen in Figure S6 (Supporting Information), after conjugating with PNIPAAm, there was ∼3.5 nm increase in the mean hydrodynamic diameters at room temperature (6.5 and 10.1 nm for BSA-SH and BSA-SH/ PNIPAAm conjugates, respectively), and such size difference could be further varied when the measurement temperature is increased to 40 °C. The mean hydrodynamic diameters of the BSA-SH/PNIPAAm conjugate increase to be 300 nm, which was due to the fact that when it was above the lower critical solution temperature of PNIPAAm (ca. 32 °C), the increased hydrophobicity of PNIPAAm would induce the aggregation of the conjugates. Also from the zeta potential measurement (Figure S7, Supporting Information), we can see the obvious zeta potential values change after each step reaction, from −2.97 to 10.2 mV to −2.64 to 7.02 mV for native BSA, BSANH2, BSA-SH, and BSA-SH/PNIPAAm conjugates, respectively (Figure S8, Supporting Information), which confirmed the successful conjugation of the PNIPAAm onto BSA as well. Furthermore, from the UV spectra calculation, on average, there were 1.91 conjugated PNIPAAm per BSA molecule (Figure S9, Supporting Information). Subsequently, when mixing an aqueous solution of BSA-SH/ PNIPAAm with 2-ethyl-1-hexanol at an aqueous/oil volume fraction of 0.06, it produced a well dispersion of proteinosomes. After 1 h sediment, the solution gives a turbid water-in-oil emulsion lower phase and a transparent upper oil layer. The generated proteinosomes were observed with the diameters in the range of 20−50 μm (Figure 1a,b), and such spherical structure were stable and remained nonaggregated at room temperature over several weeks. The optical images of partially dried proteinosomes (Figure S10, Supporting Information), scanning electron microscope (Figure 1c), and transmission electron microscopy (Figure 1d) images of the microcompartments indicated clearly that the BSA-SH/PNIPAAm shell consisted of a flexible ultrathin membrane that was structurally robust even when dried under vacuum. The average diameter of the proteinosomes could be also systematically controlled between 10 and 200 μm by changing the concentration of the BSA-SH/PNIPAAm conjugate from 0.2 to 8.0 mg/mL at an aqueous/oil volume fraction of 0.06 (Figure S11, Supporting Information). Moreover, by addition of the cross-linker PEGbis(N-succinimidyl succinate) which could react with the remanent primary amine groups in the conjugates, the crosslinked proteinosomes could be well transferred into aqueous solution (Figure 1e) without loss of structural integrity, as confirmed in the corresponding aqueous fluorescence image with well encapsulated FITC-dextran (MW, 500 kDa) in the core (Figure 1f). With the successful fabrication of the proteinosomes in aqueous solution, considering the temperature sensitivity of the polymer, redox species sensitivity of the disulfide linkage and pH sensitivity of the BSA on the comprised the membrane, the multistimuli behavior of the constructed proteinosome was explored. First, the temperature stimuli behavior was monitored from optical microscopy by the measurements of individual water-dispersed proteinosomes that were thermally cycled from below (25 °C) to above (40 °C) the lower critical solution temperature (ca. 32 °C) of the PNIPAAm (Figure 2a−c). There was about 6% reversible decrease/increase in the diameter of the proteinosome contributed by the phase change

Figure 1. (a) Optical microscope image in oil phase and (b) fluorescence microscope image of BSA-SH/PNIPAAm proteinosomes after encapsulating fluorescein isothiocyanate labeled dextran (FITCDextran MW 500 kDa) in oil phase. (c) Scanning electron microscope (SEM) and (d) transmission electron microscope (TEM) images showing continuous and flexible BSA-SH/PNIPAAm proteinosome membranes. (e) Optical microscope image and (f) fluorescence microscope image of BSA-SH/PNIPAAm proteinosomes after encapsulating FITC-dextran in aqueous solution, respectively. Scale bars in a, e, and f were 50 μm, b was 100 μm, and in c and d were 5 μm.

of PNIPAAm in the membrane (Figure 2d). Obviously, such size change procedure from the shrunk state at 40 °C to normal state at 25 °C will bring an increase on the membrane permeability. While this permeability should be further improved by adding a reductive agent, since the cleavage of the disulfide bonds could result in the leaving of the conjugated polymers from the membrane and, thus, leading to a further increase on the membrane permeability. To confirm so, a fluorescence dye-labeled PNIPAAm (fluorescein O-methacrylate (FOMA)-PNIPAAm) was synthesized by using fluorescein O-methacrylate as a comonomer during the polymerization process. Without adding any reductive agents, the corresponding fluorescence microscopy images showed the presence of green fluorescence associated specifically with the intact microcompartments (Figure. 2e), suggesting that the polymer remained covalently attached to the proteinosome membrane. While, upon the addition of the reductive agent TCEP (tris(2carboxyethyl)phosphine), one can see clearly the leaving of the green fluorescence dye-labeled polymer from the membrane as confirmed by the disappearance of the green fluorescence in the membrane (Figure 2e−g). Lastly, for this bared proteinosome, in contrast, its membrane permeability could be turned down especially considering the pH sensitivity of the BSA. At pH 7.4, BSA has a normal spherical heart-like structure (N-form), while at pH 3.5, it has an expanded cigar-like structure (F-form).51 Such change was also demonstrated from study of the circular dichroism (CD) spectroscopy (Figure 2k), there was a clear 963

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Figure 2. (a−c) In situ optical microscopy images of BSA-SH/PNIPAAm proteinosomes from 25 to 40 °C showing temperature-induced changes in diameter. The detailed data are summarized in (d) for the corresponding proteinosomes (blue line and red line) dispersed in aqueous solution at different temperature. (e−g) Fluorescence microscopy image of an individual proteinosome constructed by BSA-SH/fluorescein-labeled PNIPAAm in aqueous solution in the presence of 1.0 mM of TCEP, the fading of the green fluorescence in the membrane domain indicating the cleavage of the conjugated PNIPAAm. (h) Corresponding intensity profiles for single proteinosome shown in (e−g). (i, j) Optical microscopy images of proteinosomes showing the morphology change before (i) and after (j) incubation in pH 3.5 buffer solution, and the corresponding circular dichroism (CD) spectra (k) indicating the alerted structure of BSA in the proteinosomes.

weight FITC-dextran had been employed to study the membrane permeability, and given the molecular weight cutoff of the proteinosome membrane under different stimuli conditions, a FITC-dextran with molecular weight of a 70 kDa is an ideal stuff in this study. The release kinetics were determined under various stimuli conditions. As summarized in Figure 3c, we can see that a clear programmed controlled release of the encapsulated FITC-dextran was realized. The released rate could be 8× turned up, from 1.8 (Release Percentage per min) at 40 °C to 4.3 (release percentage per min) at 25 °C and to 10.7 (release percentage per min) the highest releasing rate in the presence of TCEP. Then such release could be hindered obviously to be 1.3 (release percentage per min) by adjusting solution pH to 3.5. In all, we should demonstrate a successful design and synthesis of a multistimuli BSA-SH/PNIPAAm building block, which could be used to generate a well-defined proteinosome efficiently with the size ranging from 10 to 200 μm depending on the concentration of the building block in the aqueous solution. The generated proteinosomes showed a clear stimuli responsive behavior against temperature, redox species, and pH, respectively, contributed by the temperature-sensitive PNIPAAm, disulfide bond linkage, and pH-sensitive BSA in the membrane. Significantly, via a sequential triggered stimuli, a continuous modulation of the membrane permeability was realized as shown in a programmed release rate of the encapsulated FITC-dextran. Although our designed modulation is limited for the molecular weight within the range of 10−100 kDa, and more efforts should be given to enlarge the

decrease in pH 3.5 in the characteristic peak intensities at 222 and 208 nm for BSA compared with that in pH 7.4, and the deconvolution of the spectra showed that the level of α-helical and β-sheet were 87.76 and 0.47% in pH 7.4 compared with 10.45 and 29.9% in pH 3.5, respectively. Accordingly, as demonstrated in Figure 2j, this N−F form transition made the proteinosome suffered an obvious loss of the spherical structure, which then lead to the permeability turned down. To further quantitatively evaluate these sequential modulation behaviors, the membrane permeability for each of the abovementioned stages was measured by selectively using different molecular weight FITC-dextran from 10 to 2000 kDa (Figure 3a, Figures S12 and S13, Supporting Information). As anticipated, at 40 °C, the proteinosomes showed a relative low membrane permeability with the molecular weight cutoff of 78 kDa, while this could be continuously turned up to be 102 kDa by cooling down the temperature to 25 °C, then to be 142 kDa upon the subsequent addition of TCEP, and finally, in contrast, this was turned down to be 35.2 kDa by adjusting the solution pH to be 3.5 (Figure 3b). The above results well indicate that the successful modulation of the membrane permeability based on different stimuli. Differing from the most reported independent stimuliresponsive behaviors in microcompartments, definitely, such continuous modulation of the membrane permeability will bring a programmed release of the encapsulated compound. As a step toward showing this goal, a fluorescence labeled dextran was selected as an example and loaded inside the proteinosomes. Through comparison testing, various molecular 964

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Figure 3. (a) Plot of percentage diffusion after 30 min for the proteinosomes incubated in the presence of FITC-dextran of different molecular weight at 40 °C, 25 °C, in the presence of TCEP and pH 3.5, respectively, and (b) the corresponding summary of the molecular weight cutoff of the proteinosome membrane was approximately 78 kDa at 40 °C, 102 kDa at 25 °C, 142 kDa in the presence of TCEP, and 35.2 kDa at pH 3.5. (c) The release kinetics of the encapsulated FITC-dextran (Mw 70 kDa) under the stimuli of temperature, reductive agent, and pH showing a programmed modulation release of the encapsulated stuff.

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ACKNOWLEDGMENTS We thank the Thousand Young Talent Program, NSFC (21474025, 21504020), the Fundamental Research Funds for the Central Universities (HIT.NSRIF. 201632) China Postdoctoral Science Foundation (2015M571400; X.L).

modulation window, as well as the irreversible redox and pH responsive, while in view of the vital role of membrane permeability in the construction of microcompartments toward diverse applications, our demonstrated system is expected to enrich the design of proteinosomes toward protocell models, such as by modulating the reaction intermediates diffusing across the boundary membrane to realize a chemical communication or metabolism procedure control.

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* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00506.

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REFERENCES

Materials, instrumentation, experimental conditions, and experimental results (PDF).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. 965

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