PEGylated Perylenemonoimide-Dithienylethene for Super-Resolution

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PEGylated Perylenemonoimide-Dithienylethene for Super-Resolution Imaging of Liposomes Jun-Xia Liu, Bo Xin, Chong Li, Nuo-Hua Xie, Wen-Liang Gong, Zhen-Li Huang, and Ming-Qiang Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15076 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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PEGylated Perylenemonoimide-Dithienylethene for Super-Resolution Imaging of Liposomes Jun-Xia Liu‡, Bo Xin‡, Chong Li, Nuo-Hua Xie, Wen-Liang Gong, Zhen-Li Huang, and Ming-Qiang Zhu* Wuhan National Laboratory for Optoelectronics, College of Optical and Electronic Information, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China. ABSTRACT. We have designed and synthesized an amphiphilic photoswitchable fluorophore, PEGylated perylenemonoimide-dithienylethene (PEG-PMI-DTE), which exhibits evident bistable photochromism, fluorescence switching and fatigue resistance. The fine nanostructures of liposomes can be observed directly under super-resolution fluorescent microscopy by aid of the amphiphilic photoswitchable fluorophore as a staining agent, with an optical resolution of 30 nm. KEWORDS: dithienylethene, perylenemonoimide, PEGylated, liposomes, super-resolution fluorescent microscopy.

Liposomes are spherical vesicles and promising candidates of drug delivery due to their amphiphilic nature and large aqueous core as well as excellent biocompatibility.1-5 Generally, liposomes are classified as multilamellar vesicles (MLV, 400-5000 nm), large unilamellar vesicles (LUV, diameter 100-400 nm), and small unilamellar vesicles (SUV, diameter < 100 nm), depending on the number of layers and the diameter.6,7 As is well known, liposomes can interact with cells by different mechanisms such as adsorption, endocytosis,8 and membrane fusion.9 The interactions essentially depend on the liposomal characteristics.10 Liposomes with size larger than 200 nm are internalized more readily than smaller liposomes, 11 while the upper size limit for extravasations into tumors is about 400 nm.12 It was also proved that liposomes smaller than 70 nm are more rapidly cleared from the circulation than larger ones and the efficiency of cellular uptake decreases along with the liposome size ranging from 100 nm to over 1 µm. 12-14 Therefore, optical visualization of fine nanostructures of liposomes with different sizes is essential to understand the liposomal biological events. Scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), and atomic force microscopy (AFM) act as conventional imaging methods to visualize the morphology of dry liposomes with invasive, high-vacuum or time-consuming drawbacks.15-17 Conversely, fluorescence microscopy is a non-invasive imaging method and has been applied to visualize the spatial distribution of nanoparticles such as liposomes,18,19 whereas the diffraction limit defines the spatial resolution.20 Remarkably, super-resolution fluorescence microscopy based on the single molecule localization using photoswitchable probes, such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM), has broken the optical diffraction limit and been applied in bioimaging.21,22 Hochstrasser R. M. used Nile Red 23 and Merocyanine 540 24 for PAINT microscopy of LUVs, which shows advantages that the object to be imaged need not be labeled. However, the PAINT approach in the aqueous cytoplasm needs to be explored. 25 Meanwhile, many molecular probes have

significantly contributed to understand of lipids in cell.26 Reinhard Jahn imaged the clusters stained for PIP2, using super-resolution stimulated-emission depletion (STED) microscopy for PHPLCd–citrine-stained membrane sheet of PC12 cells.27 As the alternatives to staining proteins, smaller-size fluorescent probes are desirable for super-resolution imaging.28 Therefore, a series of photochromic fluorescent dyes, such as spiropyrans,29 hexaarylbiimidazole30 and dithienylethenes (DTEs) 31, 32 have been designed by integrating photochromic materials and fluorescent dyes to achieve novel super-resolution imaging probes. Currently the photochromism of DTEs33-35 has also been investigated in liposomes while there are no investigations of DTE probes in super-resolution imaging of liposomes. DTE with excellent fatigue resistance and thermal irreversibility is one kind of the most promising photochromic materials.32,36,37 In previous work, we have explored the super-resolution imaging of lysosomes in Hela cells using the polymer probe based on dithienylethene-naphthalimide (DTE-NI) as the photoswitchable fluorophore. 31 Here we designed and synthesized a novel type of amphipathic and fluorescence-switchable probe, i.e., PEGylated perylenemonoimide-dithienylethene (PEG-PMI-DTE). The structure and schematic photoswitching processes are presented in Scheme 1a with PEG in green, PMI in red and DTEs in blue. The detailed synthetic procedures of compounds and liposomes are described in Supporting Information (SI). PEG-PMI-DTE is obtained by an efficient click reaction between water-soluble polymer MPEG5000-N3 and compound 8 (Scheme S1) which has a PMI fluorophore and two fluorescence-switchable DTE subunits. It was prepared with a high yield and characterized by NMR and GPC (Figure S1). Hydrophilic PEG-PMI-DTE is not completely water-soluble probably due to the large hydrophobic fraction PMI and DTE, although PEG has a very good water-solubility. Liposomes were prepared with the film dispersion method, 38 which is presented in Scheme 1b and Figure S2. This work aims at the optical visualization of liposomes with sub-wavelength

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resolution utilizing photoswitchable fluorescent probe PEG-PMI-DTE. On the basis of fluorescent switching of PEG-PMI-DTE, the microphase structures of liposomes become visualized using super-resolution imaging (Scheme 1c). Scheme 1. The PEG-PMI-DTE-fused liposomes for super-resolution imaging. (a) Schematic photoswitching of PEG-PMI-DTE. (b) Components and structure of PEG-PMI-DTE-fused liposomes. (c) Super-resolution imaging of liposomes containing PEG-PMI-DTE.

We designed this amphiphilic photoswitchable probe PEG-PMI-DTE on the basis of previous researches.32, 39 PEG chain was incorporated to endow hydrophilicity and biocompatibility of the probe. And the introducing long alkyl chain would increase the solubility of the molecule, which greatly reduce the difficulties in synthesizing. On the other hand, the long alkyl chain might provide better intermiscibility in the staining of liposomes. DTE doesn’t exhibit any fluorescence itself and only shows photochromism ability unless integrated together with the PMI fluorophore. As a result, the fluorescence of PMI is reversibly controlled via photoswitchable fluorescence resonance energy transfer (pFRET) to the fluorescence quencher DTEs (Scheme 1a). The PMI modified by more than one DTE has been proved high fluorescence switching speed, high fluorescence on/off ratio and provides prospective opportunity in super-resolution imaging.26 PEG-PMI-DTE has a very good solubility in common organic solvents, whereas cannot be directly soluble in pure water. The optical properties of PEG-PMI-DTE in organic solvents are shown in Figure S3 and summarized in Table S2. Figure 1 shows the absorption and fluorescence spectra of PEG-PMI-DTE upon irradiation with 302 nm UV light in THF for 10 s and in liposome suspension for 30 s. As depicted in Figure 1a and Figure S4, the open form of PEG-PMI-DTE has an intense absorption peak at 293 nm belong to DTE subunits and another intense absorption band at about 521 nm assigned to the PMI subunit, whereas the photoswitchable characteristic bands at 324 nm and 600 nm in long wavelength region after UV irradiation are assigned to the closed form DTE. Upon 302

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nm UV-light irradiation, the peak 293 nm of DTE in the open form decreases and the 324 nm and 600 nm of DTE in the closed form increases, which corresponds to the photochemical conversion from the open form to the closed form of DTE. Meanwhile, the “increase” of absorption located around PMI subunit is because of the absorption overlap between DTE in the closed form (500-750 nm) and PMI (430-575 nm) (Scheme 1a). Compared with in organic solvents, the photochromism speed in THF/water becomes a little slower (Figure S5). It takes about 30 s to attain photo stationary state (PSS) of PEG-PMI-DTE in THF/water. The a little slower photochromism speed may be result from that the water is a poor solvent for PMI-2DTE unit and a good solvent for PEG chain, which both hinder the motion of photochromism unit PMI-2DTE. The more interesting result about the fluorescence switching of PEG-PMI-DTE in THF has been disclosed. After 2 s of irradiation, the FL intensity around 520 nm drops by more than 90% in comparison to the initial intensity (Figure 1b). The fluorescence intensity reaches saturate after 4-6 s of irradiation, indicating the reversible photoisomerization between the open-from and closed-form of PEG-PMI-DTE in THF attains dynamic equilibrium at ambient temperature and 302 nm irradiation environment. The fluorescence quantum yields (FLQYs) of PEG-PMI-DTE in THF were calculated to be 0.85 at open form whereas 0.005 at close form. The maximum fluorescence intensity on/off ratio is 149 in THF. Correspondingly, the FLQYs in THF/water were estimated to be 0.27 at open form and 0.002 at close

Figure 1. The optical properties of PEG-PMI-DTE. (a) UV-Vis absorption and (b) emission spectra upon 302 nm irradiation of PEG-PMI-DTE in THF at the concentration of 0.01 mg/mL. (c) UV-Vis absorption and (d) emission spectra upon 302 nm irradiation of liposome suspension containing the fluorescence switchable probe PEG-PMI-DTE at the amount 0.5 mol % of totally molar lipid mixture. (e) Absorbance (monitored at 601 nm) and (f) emission intensity (monitored at 625 nm) between two states of PEG-PMI-DTE in THF/water (1/9, v/v), as photoswitching is alternating between 302-nm irradiation (8 s) and ≥ 490 nm (10 min) illumination, respectively. Excitation: 514 nm.

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form, respectively, indicating a fluorescence intensity on/off ratio of 357 (Table S2). Though the FLQYs of PEG-PMI-DTE are reduced in polar solvent, the fluorescence intensity on/off ratio is still considerable. It is discovered that the optical properties of PEG-PMI-DTE efficiently retain in liposome suspension (Figure 1c and 1d) and the fluorescence intensity on/off ratio is 97, which is qualified for super-resolution imaging. More clearly, we can see the rapidly declining line in the fluorescence intensity against real-time FL on/off ratio curves, which indicates efficient fluorescence quenching of PEG-PMI-DTE occurs in THF and liposome suspension (Figure S6). The conversion efficiencies of PEG-PMI-DTE at PSS were measured about 98% according to the monitored 1H NMR spectra before and after 302 nm irradiation (Figure S12). By monitoring time-resolved spectroscopic of the forward and backward photochromic interconversions between open and closed forms of PEG-PMI-DTE (Figure S7), the photocyclization quantum yield is 0.45 and the cycloreversion quantum yields is 0.008. Upon UV-light irradiation, the open form PEG-PMI-DTE can be converted to the closed form isomer quickly. Inversely, visible light illumination would cause gradual decreasing of photochromic band absorbance, which is resulting from the back-conversion from closed form to open form. The photoswitching cycles of PEG-PMI-DTE assemble solution in THF/water upon alternating 302 nm and visible light (≥ 490 nm) irradiation, which are presented in Figure 1e and 1f, were investigated by monitoring the absorbance changes at 601 nm and the fluorescence intensity changes at 625 nm. The absorbance at 601 nm appears and increases in 8 seconds upon 302 nm irradiation. Subsequently, it disappeared after a 10 minutes visible light (≥ 490 nm) irradiation. Inversely, the strong fluorescence emission at 625 nm were quenched sharply upon 302 nm irradiation while returned to the original level after a following visible light irradiation. The photochromic process and the fluorescence photoswitching of the probe can be repeated many times (more than 10 cycles), which indicate that the probe has a good fatigue resistance. Thus, PEG-PMI-DTE can be reasonably used in super-resolution imaging. There are a long hydrophilic PEG chain and hydrophobic fluorescence photoswitching group in the amphiphilic probe PEG-PMI-DTE, which is proposed to promote the probe to be located in the hydrophobic-hydrophilic interface of liposomes. Liposomes stained by PEG-PMI-DTE were prepared by film dispersion method. Their morphology was characterized by TEM, dynamic light scattering (DLS), AFM, fluorescence microscopy imaging and super-resolution microscopy which are described in SI. Our intention is to demonstrate that PEG-PMI-DTE works well in the super-resolution imaging of liposomes including LUVs, MLVs and SUVs. In conventional TEM, as shown in Figure 2a, it is observed that liposomes exist in LUV form with diameter about 140-300 nm, while it may be present in MLV form with diameter larger than 300 nm. The size of SUVs ranges from 30 to 70 nm (for example: the red line region in Figure 2a). From the TEM image, the mean diameter of these vesicles is calculated to be about 186 nm ranging from 26 nm to 342 nm, which shown in Figure 2b. The mean size of liposomes in suspension is estimated to be 187.5 nm with PDI 0.213 by DLS and shown in Figure 2c. Meanwhile, we also studied the morphology of liposomes in the suspension using AFM (Figure 2d and 2e). The mean size of

the liposomes is calculated to be about 149 nm ranging from 67 nm to 341 nm in AFM image (Figure 2d), which is shown in Figure 2f. The 3D AFM image (Figure 2e) presents triangular pyramidal structure which indicate that these particles are spherical. The morphology of liposomes in AFM images provided an additional evidence that the successful formation of liposomes.

Figure 2. Morphology of the liposomes stained by PEG-PMI-DTE. (a) TEM image. (b) Diameter distribution of particles in a using Nanomeasure 1.2 (50 selected particles totally). (c) Diameter distribution of liposome suspension from DLS. (d) and (e) AFM images; (f) Diameter distribution of particles in d using Nanomeasure 1.2 (150 selected particles).

We obtained typical liposomes and imaged them using the fluorescence-switchable probe PEG-PMI-DTE. We investigated and compared fluorescence microscopy imaging and super-resolution imaging of liposomes in an identical selected area, utilizing the amphiphilic photoswitchable PEG-PMI-DTE as staining agent. Conventional fluorescence imaging by irradiation with a 473 nm laser reveals that the existing of nanoparticles as emissive dots, but the fine structure of liposomes smaller than 200 nm cannot be evidently visualized. Super-resolution imaging succeeds in addressing this problem, which is based on photoswitchable probe and conducted by alternate irradiation with a 473 nm laser and 302 nm UV light source. The detail imaging processes were given in SI. The reconstructed high resolution images of the liposome are presented in Figure 3 and Figure S8. The measured liposomes size in super-resolution images is comparable with that of TEM and AFM. As seen in Figure 3a, conventional fluorescence microscopic images can only reveal that these liposomes are in hundreds of nanometer in aqueous dispersion. Due to optical diffraction limit, the mean diameter of liposomes is almost 500 nm in conventional fluorescence image, which is much larger than the diameter obtained from TEM and AFM. The sub-200 nm morphological details of liposomes are unavailable using the conventional fluorescence imaging, even though in the magnified images (Figure 3c, 3e and 3g). PEG-PMI-DTE in liposomes exhibits excellent photoswitchable fluorescence upon alternating UV and visible light irradiation and thus can serve well as the super-resolution imaging agent. The much finer nanostructures are observed by super-resolution imaging, which reveals that these liposomes in the selected area are LUVs and SUVs (Figure 3). The super-resolution fluorescent images in Figure 3b indicate that the diameter of liposomes, which is statistically calculated by Nanomeasure 1.2, is about 50-655 nm (Figure S8). The sizes of most liposomes (about 63%) in the selected region are smaller than 200 nm. Considering the limitation of conventional fluorescent

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microscopy, the optical visualization of small vesicles using super-resolution imaging is meaningful. The statistical result about photoswitching of PEG-PMI-DTE in liposomes demonstrates the mean photon counts of 2.4 x 103 and the median value of 1.6 x 103 per molecule per switching (Figure S9).

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intensity 1.64 W/cm2 basing on the researches of changed intensities of 473 nm (Figure S11). These comparable details of liposome morphologies and sizes in super-resolution imaging and TEM indicate that super-resolution imaging based on photoswitchable fluorescent probe PEG-PMI-DTE is a promising approach to characterize microstructures of liposomes although there is certain gap between the achieved optical resolution (~30 nm) and the real thickness of vesicular bilayers (below 10 nm). However, it is the first example of super-resolution imaging of liposomes using a DTE probe to achieve the clear images of several typical liposomes defined by size and layers. We believe that the real thickness of liposomal vesicles as well as cell membranes can be measured providing that more super-resolution imaging probes with high-performance fluorescence and higher photoswitching ratio are developed. In conclusion, we have designed and synthesized a novel amphiphilic photoswitchable fluorescent probe PEGylated Perylenemonoimide-Dithienylethene (PEG-PMI-DTE), which exhibits excellent photochromism, fluorescence switching and fatigue resistance upon UV-light and visible light irradiation. The staining of probe enables the optical visualization of fine structures of liposomes (MLVs, LUVs and SUVs) under super-resolution optical microscope. This work provides a new optical probe and optical approach to investigate microstructure using photoswitchable fluorescent probes in super-resolution imaging.

ASSOCIATED CONTENT Supporting Information. Materials and methods. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions ‡

Figure 3. Optical imaging of liposomes. (a) Fluorescence image. (b) Reconstructed super-resolution images. Scale bar: 1um. (c) and (d) are the expansion of the blue box in a and b respectively. (e) and (f) are the expansion of the green box in a and b respectively. (g) and (h) are the expansion of the purple box in a and b respectively. (i) Cross-sectional profiles of along the solid lines in super-resolution image d, f and h. (j) Fourier ring correlation curve calculated from (a) and (b). Scale bar of c-h: 300 nm.

Moreover, the full-width at half-maximum (FWHM) of the localized bilayer of liposomes in Figure 3d, 3f and 3h was determined from Gaussian deconvolution as shown in Figure 3i. The FWHM of a typical LUV bilayer (Figure 3f) is 40.4 nm. We also obtained the more complex liposomes such as Figure 3d with the FWHM of a liposomal bilayer at 29.3 nm. The size of a typical SUV (Figure 3h) is 31.7 nm, which is proposed to be the sum of two bilayers of a LUV. In addition, Fourier ring correlation (FRC) is developed as one of the resolution criterions for super-resolution microscopy.40, 41 Using the relevant plugin40, we computed directly from the image Figure 3j to get the resolution in optical nanoscopy of liposomes was 29.17±1.01 nm. The super-resolution imaging of multilamellar vesicles (MLVs) with the size above 400 nm and the FWHM 42.7 nm are also presented in Figure S10. All these fluorescence microscopy images were obtained at the

These authors contributed equally (J.-X.L and B.X).

ACKNOWLEDGMENT This work was supported by the 973 Program of China (2015CB755602 and 2013CB922104), NSFC (21474034, 51673077 and 51603078). We also thank Analytical and Testing Center of Huazhong University of Science and Technology and the Center of Micro-Fabrication and Characterization (CMFC) of WNLO for use of their facilities.

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