Letter pubs.acs.org/macroletters
Fabrication of Multicomponent Multivesicular Peptidoliposomes and Their Directed Cytoplasmic Delivery Soo hyun Kwon and Yong-beom Lim* Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-ro, Seoul 03722, Korea S Supporting Information *
ABSTRACT: A novel self-assembly strategy for the formation of multicomponent and multicompartment vesicles via the hierarchical assembly of the cyclic peptide and lipid building blocks is reported. The primary driving force underlying the formation of dual-component (i.e., peptide and lipid) heteromultivesicular vesicles (hMVVs) is the differential thermostability between the supramolecular building blocks. Furthermore, the combination of the differential thermostability and charge-based separation further enables the fabrication of the hMVVs that incorporate up to four different components (i.e., two different building blocks and two different encapsulated molecules). The quadruple-component hMVVs consist of cyclic peptides, lipids, negatively charged green fluorescent probes (GFPr), and positively charged red fluorescent probes (RFPr). Intracellular delivery study shows that cellular localization of hMVVs is directed by the function of hMVV envelopes, and the nuclear localization signal (NLS) of peptide vesicles appears to use different cellular pathways depending on the site of action (i.e., extracellular space or cytoplasm). This study provides the hierarchical peptide-based hMVVs with sophisticated architectures and cell delivery characteristics, thus making a step toward artificial cells or viruses.
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Previous studies have shown that nanostructures that selfassembled from cyclic peptides have better thermal and conformational stability than those from linear peptides.17,18 Specifically, it has been demonstrated that the cyclic peptides consisting of a charged/hydrophilic segment and a tryptophan (Trp)-rich hydrophobic segment, when appropriately designed, can form self-assembled peptide vesicles.19−21 These cyclic peptide vesicles (cPVs) have been shown to be structurally and thermally more robust than linear peptide vesicles and lipid vesicles (liposomes or LVs). Based on these findings, we hypothesized that if cPVs can maintain their nanostructural integrity during the freeze−thaw cycles of LV formation it might be possible to fabricate hMVVs in which cPVs are entrapped within the LV lumen (aqueous interior). Here, we report the construction of multicomponent and multicompartment self-assembled hMVVs by exploiting the differential physical characteristics between constituent supramolecular building blocks and the charge characteristics of component species. The artificial hMVVs are based on selfassembling peptides and lipids, whose combination has not been reported before to our knowledge. We utilized the differential thermostability between the cyclic peptide and lipid building blocks as the basic mechanism underlying the formation of the peptide−lipid hMVVs, where the selective separation of charged species by ion exchange chromatography further enabled the fabrication of quadruple-component multivesicular vesicles (quad-hMVVs).
he ultimate goal of bottom-up bionanotechnology should be the fabrication of desired nanostructures with sophisticated functions on command. Although many studies have been performed to develop nano-objects with elaborate structural features, the controlled formation of complex and hierarchical self-assembled nanostructures still poses a formidable challenge. The fabrication of multicomponent and multicompartment nanostructures can be a step toward the realization of complex materials with advanced functions.1,2 One of the most interesting and potentially useful nanostructures with a higher level of structural complexity is the multivesicular vesicle (MVV), a multistructural object in which a number of smaller vesicles are entrapped within the aqueous interior of a larger vesicle. In fact, such structures exist in biological systems and are called multivesicular bodies (MVBs). MVBs play important roles in protein sorting (targeting), degradation, and recycling during endocytosis and exocytosis.3,4 The biogenesis and function of MVBs, which are not completely elucidated, are dynamically regulated by the complex interplay of ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery.5 Meanwhile, interest in artificially constructed MVVs or multicompartment systems is growing.6 Most MVVs, including biological MVBs, are composed of lipid molecules.7,8 Furthermore, several studies have shown that MVVs can be fabricated by using polymers9,10 and protein−polymer nanoconjugates.11 The basic mechanisms in the formation of artificial MVVs include a layerby-layer technique,12 the fusion at the water−oil interface,13 emulsion−centrifugation,14 a microfluidic approach,15 and a multistep biphasic system.11,16 Many of these artificial MVVs have been constructed by emulsion-based methods. © XXXX American Chemical Society
Received: January 27, 2017 Accepted: March 14, 2017
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DOI: 10.1021/acsmacrolett.7b00064 ACS Macro Lett. 2017, 6, 359−364
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ACS Macro Letters
Figure 1. Peptide vesicles in a lipid vesicle with fluorescent probes independently encapsulated in each vesicle. (a) Intracellular delivery pathways of quad-hMVVs. (b) Chemical structures of the cyclic peptide (cpBB) and lipid (EYPC) building blocks. EYPC: egg yolk L-α-phosphatidylcholine. A representative structure of EYPC is shown. (c) Fabrication of the quad-hMVV. CPP: cell-penetrating peptide, NLS: nucleus localization signal, HFIP: hexafluoroisopropanol, GFPr: green fluorescent probe (carboxyfluorescein), RFPr: red fluorescent probe (rhodamine B), AEX: anion exchange chromatography, CEX: cation exchange chromatography.
trapped metastable states, cpBB was first dissolved in hexafluoroisopropanol (HFIP) solution (HFIP:water = 30:70, v/v). HFIP strongly induces α-helices in proteins and peptides.25 Thus, combined with the “organic” character of the solvent, the incubation of cpBB in HFIP solution would unfold nonspecific aggregates and weaken hydrophobic interactions between cpBB molecules. The sample was then lyophilized to preserve the unfolded and disassembled state as much as possible,26 was redissolved in water, and was vigorously sonicated to facilitate the formation of molecular assemblies at thermodynamic equilibrium. Investigation by transmission electron microscopy (TEM) showed that cpBB assembled into spherical nanostructures with typical sizes of several tens of nanometers to about a hundred nanometers (Figure 2a). Considering the molecular length of cpBB in the fully extended state (∼4−6 nm), the observed spherical objects are likely to be vesicles (cPVs) rather than micelles. Atomic force microscopy (AFM) studies performed on a different substrate (i.e., mica) corroborated the formation of spherical objects from cpBB (Figure S4). Encapsulation of fluorescent probes within cPVs further supports the vesicular structure (vide inf ra). To address the question of cPVs’ thermostability, we performed the temperature-dependent
The designed cyclic peptide building block (cpBB) consists of a charged/hydrophilic segment, a Trp-rich hydrophobic segment, and a hydrocarbon tail segment (Figure 1). The charged/hydrophilic segment consists of 14 amino acids and is derived from the arginine-rich motif (ARM) of human immunodeficiency virus (HIV) type-I Rev protein.22,23 This Rev ARM peptide can function as both a cell penetration peptide (CPP) and a nucleus localization signal (NLS).22,24 In this design, the hydrocarbon tail block was incorporated anticipating that the flexible alkyl chain would reinforce the structural robustness of the self-assembled state and assist the formation of more closely packed molecular assemblies by filling structural defects, thus providing leak-proof nanocontainers when vesicles are formed. We then asked whether cpBB could form molecular aggregates at an appropriate concentration range by determining the critical aggregation concentration (CAC). The CAC was determined by measuring the concentration-dependent changes in Trp fluorescence (Figure S3).19 The calculated CAC of cpBB was 1.5 μM, indicating a relatively strong propensity to aggregate. All of the following studies were performed at concentrations above the CAC. To facilitate the formation of more homogeneous molecular assemblies, as well as to minimize the generation of kinetically 360
DOI: 10.1021/acsmacrolett.7b00064 ACS Macro Lett. 2017, 6, 359−364
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ACS Macro Letters
Figure 2. Self-assembly of cpBB into vesicles (cPVs). (a) Negative-stain TEM image of cPVs at different focal planes. Bar = 100 nm. (b) Temperature-dependent CD spectra of cPVs. Right: forward scan. Left: backward scan. [cpBB] = 20 μM.
repeated FTCs of cPVs (Figure 3d). This result suggests the nanostructural integrity and the permeability to small molecules remain intact in cPVs, even after the harsh thermal perturbation. Based on the finding that cPVs retained their structural integrity after FTCs, we set out to examine the possibility of fabricating sophisticated dual-component hMVVs (dualhMVVs). Preformed cPVs in an aqueous solution were added to a thin film of egg yolk L-α-phosphatidylcholine (EYPC), and the mixture was subjected to FTCs. A melting cycle of FTCs was performed at 55 °C, which is higher than the glass transition temperature (Tg) of EYPC (−15 to −7 °C). The morphological state of the mixture investigated by AFM showed the presence of LVs with diameters of several hundreds of nanometers and of the smaller cPVs with diameters of several tens of nanometers (Figure S5b). Small bumps were also observed on the top of the LVs whose sizes corresponded well to those of cPVs. This morphological state suggests the entrapment of the smaller cPVs within the larger LVs. Removal of free cPVs will be critical in preparing dualhMVVs that are free from cPV impurities. The corona of cPVs is positively charged, whereas that of LVs is neutral; thus, we expected that the two differently charged species might be
circular dichroism (CD) measurements with cPVs. As shown in Figure 2b, CD spectra remained fairly constant with a thermal ramp up to 94 °C, indicating the high conformational stability of cpBB molecules in cPVs against heat denaturation. We next evaluated whether cPVs are structurally and thermally more stable than LVs. To compare the structural stability, cPVs and LVs entrapping a fluorescent probe (calcein) were prepared. The free dye molecules that were not entrapped within the vesicles were separated using size exclusion chromatography (SEC). To the calcein-loaded vesicles, the detergent Triton X-100 was added to disrupt the vesicular membranes. As shown in Figure 3a−c, the entrapped calcein was released from LVs in less than 2 min, whereas as much as 30 min was required for the complete release of calcein from cPVs. Thus, the results indicate that cPVs are structurally more stable than LVs against membrane disruption from the detergent. Next, the calcein-loaded cPVs were subjected to the freeze−thaw cycles (FTCs) to examine their thermal stability. Freeze−thaw is a widely used method of liposome preparation.27,28 The FTC ruptures and refuses liposomes, during which time the liposomes themselves fuse and become larger. In contrast to the findings for typical liposomes or LVs, the leakage of entrapped calcein was not noticeable even after the 361
DOI: 10.1021/acsmacrolett.7b00064 ACS Macro Lett. 2017, 6, 359−364
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Figure 3. Comparison of cyclic peptide vesicles (cPVs) and lipid vesicles (LVs). Leakage assays for (a) LVs and (b) cPVs encapsulated with calcein following treatment with Triton X-100 (1%, v/v) and (c) a plot of the calcein fluorescence intensity as a function of time. (d) Thermostability of the cPV. Calcein-loaded cPVs were subjected to 5 freeze−thaw cycles (FTCs; − 196 and 55 °C).
Finally, we compared similarities and differences in cell internalization characteristics of cPVs, LVs, and quad-hMVVs. HeLa cells were treated with the vesicles for 4 h, and the intracellular distribution of the fluorescent probes was visualized with CLSM. As expected, green fluorescence in cells treated with cPV (FAM) localized predominantly within the nucleus because of the CPP and NLS functions of the Rev ARM peptide corona (Figure 5a). Cells treated with LV (RDB) showed the preferential localization of red fluorescence in the cytoplasm (Figure 5b). Interestingly, fluorescence signals were predominantly detected in the cytoplasm of cells treated with quadhMVVs (Figure 5c,d). The yellow signals indicated the colocalization of green and red fluorescence, suggesting that some of the quad-hMVVs remained intact within the cells. The destruction and release of payloads from quad-hMVVs were evident as evidenced by the presence of green and red fluorescence. Considering the NLS activity of the Rev ARM peptide and the result from Figure 5a, the predominant cytoplasmic localization of green fluorescence from cPV is unexpected. The released cPVs from quad-hMVVs could be guided toward the nucleus as indicated by the dense green fluorescence around the perinuclear region but could not get across the nuclear membrane (Figure 5d). Thus, although the exact mechanism is currently unclear, the Rev ARM peptide corona of cPVs appears to use a different cellular localization pathway depending on the site of action (i.e., extracellular space or cytoplasm), and the NLS function of Rev ARM can be partially inactivated when released from inside of the cells (Figure 1a).
separated using cation exchange chromatography (CEX). Indeed, we verified the preparation of pure dual-hMVVs after the CEX process (Figure 4a). The AFM image shows the presence of spherical nanostructures of several tens of nanometers to about a hundred nanometers inside the larger vesicles of several hundreds of nanometers to about a micrometer. We reconfirmed the formation of dual-hMVV nanostructures by using cryo-TEM (Figure 4b). We then addressed the possibility of fabricating the quadruplecomponent heteromultivesicular vesicles (quad-hMVVs). The quad-hMVVs entrap differently colored fluorescent probes within each subcompartment of the dual-hMVVs (Figure 1c). First, a negatively charged green fluorescent probe (carboxyfluorescein; FAM) was entrapped within cPVs. The free FAM molecules could be removed by using anion exchange chromatography (AEX). Second, the solution of cPVs loaded with FAM [cPV (FAM)] was added to the thin film of EYPC. Subsequently, a positively charged red fluorescent probe (rhodamine B; RDB) was added, and the mixture was subjected to FTCs. Next, the quad-hMVVs [cPVs (FAM) in an LV (RDB)] could be separated from free RDB by using CEX. The quad-hMVV consists of four componentscpBB, EYPC, FAM, and RDB. We investigated this preparation using confocal laser scanning microscopy (CLSM). We observed both green and red fluorescence localized within the large vesicular structure, indicating the simultaneous confinement of cPVs (FAM) and RDB within an LV (Figure 4c). Taken together, the results indicate that this self-assembly strategy enables differential entrapment in the segregated subcompartments of hMVVs. 362
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Figure 4. Fabrication of dual-hMVVs (cPVs in an LV) and quad-hMVVs. (a) AFM images of dual-hMVVs. Left: height image. Right: phase image. A line profile is shown above. (b) Cryo-TEM images of dual-hMVVs. The cyan and yellow lines denote the circumferences of LVs and cPVs, respectively. Bar = 100 nm. (c) Fabrication of quad-hMVVs. CLSM images of quad-hMVVs. Bg: background, Gr: green, Rd: red. Gr + Rd: overlay of green and red channels. Green channel: the background fluorescence appears at the outside of the vesicles because of the long exposure time and is not the real fluorescence. Bar = 10 μm.
In summary, we established a self-assembly strategy for the formation of peptide-based dual-hMVVs and more sophisticated quad-hMVVs by exploiting the differential thermosensitivity and the charge state of supramolecular building blocks and nanostructures. We discovered that NLS functions might use a differential cellular pathway and machinery depending on the site of action. We envision that the peptide-based multicomponent hMVVs can be a novel model system that can mimic the complicated behavior of biological vesicles and cells. Moreover, the multicomponent hMVVs can be developed as materials that can perform complex and unusual functions. For example, hMVVs can be developed as artificial nanostructures that mimic enveloped viruses. Enveloped viruses have lipid bilayer envelopes covering their protein capsids.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00064.
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Figure 5. Differential intracellular delivery modes. Overlay of brightfield and fluorescence images of cells treated with (a) cPV (FAM), (b) LV (RDB), and (c) quad-hMVVs. Nuclei are marked with dotted lines. (d) Overlay of green and red fluorescence signals in cells treated with quad-hMVVs. Inset: an enlarged image.
Reaction scheme, HPLC and MALDI data, CAC determination, and AFM images (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 363
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(20) Jeong, W. J.; Lim, Y. B. Macrocyclic Peptides Self-Assemble into Robust Vesicles with Molecular Recognition Capabilities. Bioconjugate Chem. 2014, 25, 1996−2003. (21) Kwon, S. H.; Jeong, W. J.; Choi, J. S.; Han, S.; Lim, Y. B. Nanomorphological Diversity of Self-Assembled Cyclopeptisomes Investigated via Thermodynamic and Kinetic Controls. Macromolecules 2016, 49, 7426−7433. (22) Pollard, V. W.; Malim, M. H. The HIV-1 Rev Protein. Annu. Rev. Microbiol. 1998, 52, 491−532. (23) Fang, X.; Wang, J.; O’Carroll, I. P.; Mitchell, M.; Zuo, X.; Wang, Y.; Yu, P.; Liu, Y.; Rausch, J. W.; Dyba, M. A.; Kjems, J.; Schwieters, C. D.; Seifert, S.; Winans, R. E.; Watts, N. R.; Stahl, S. J.; Wingfield, P. T.; Byrd, R. A.; Le Grice, S. F.; Rein, A.; Wang, Y. X. An Unusual Topological Structure of the HIV-1 Rev Response Element. Cell 2013, 155, 594−605. (24) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B. The Design, Synthesis, and Evaluation of Molecules That Enable or Enhance Cellular Uptake: Peptoid Molecular Transporters. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 13003−13008. (25) Hirota, N.; Mizuno, K.; Goto, Y. Cooperative Alpha-Helix Formation of Beta-Lactoglobulin and Melittin Induced by Hexafluoroisopropanol. Protein Sci. 1997, 6, 416−421. (26) Rey, L. Free Drying/Lyophilization of Pharmaceutical and Biological Products, 3rd ed.; Informa Healthcare: 2010. (27) New, R. R. C. Liposomes: A Practical Approach; IRL press: 1990. (28) Costa, A. P.; Xu, X.; Burgess, D. J. Freeze-Anneal-Thaw Cycling of Unilamellar Liposomes: Effect on Encapsulation Efficiency. Pharm. Res. 2014, 31, 97−103.
Yong-beom Lim: 0000-0001-6590-7373 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by grants from the National Research Foundation (NRF) of Korea (2014R1A2A1A11050359) and Yonsei University Future-leading Research Initiative.
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