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Dipole Orientation Matters: Longer-circulating Choline Phosphate than Phosphocholine Liposomes for Enhanced Tumor Targeting Shuya Li, Feng Wang, Xiaoqiu Li, Jing Chen, Xiaohan Zhang, Yucai Wang, and Juewen Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017
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Dipole Orientation Matters: Longer-circulating Choline Phosphate than Phosphocholine Liposomes for Enhanced Tumor Targeting Shuya Li,3,+ Feng Wang,1,2,+,* Xiaoqiu Li,3,4 Jing Chen,3 Xiaohan Zhang,2 Yucai Wang,3,* and Juewen Liu2,* 1. School of Biological and Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China 2. Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada 3. The CAS Key Laboratory of Innate Immunity and Chronic Diseases, School of Life Sciences and Medical Center, University of Science & Technology of China, Hefei, Anhui, 230027, PR China 4. Department of Oncology, the First Affiliate Hospital of Anhui Medical University, Hefei, Anhui 230022, China + S. Li and F. Wang contributed equally to the work. Supporting Information Placeholder
ABSTRACT: Zwitterionic phosphocholine (PC) liposomes are widely used for drug delivery since they are highly biocompatible and have a long blood circulation time. We herein report that by flipping the direction of the PC dipole, the resulting choline phosphate liposomes (named CPe) have an even longer circulation time, as confirmed at both cellular and animal model levels. Even when 33% cholesterol was included in the lipid formulation with a polyethylene glycol (PEG) layer, the CPe liposome still had a longer blood circulation time. Isothermal titration calorimetry (ITC) indicates a lack of protein adsorption or PC membrane attachment for the CPe liposomes. This is different from the previously reported adhesion of CP polymers to PC lipid membranes, which may be attributed to the different ways of displaying the CP headgroup. With a longer blood circulation time, the CPe liposomes accumulated in tumors more easily than PC liposomes, likely due to the enhanced permeation and retention (EPR) effect and tumor cell uptake. This study provides key insights into zwitterionic biointerfaces for biomedical, analytical, and materials applications.
and also resisted protein adsorption.24 Based on their cell membrane adhesion property, such polymers were also employed to facilitate cellular uptake,25-27 and to design stimuli-responsive materials.28 The strong binding of CP polymers to cells was attributed to the CP dipole interacting with the PC dipole in the lipid headgroups of the cell membrane.24 Inspired by this model, fundamental polymer self-assembly studies were also carried out with PC and CP mixed polymers.29 Aside from dendritic polymers, liposomes represent another convenient way to display CP. Liposomes and polymers are quite different in flexibility and density of functional groups. While CP is rarely found in natural lipids, lipids with the PC headgroup are the main component of the outer membrane of eukaryotic cells.30 The structure of a dioleoyl-sn-glycero-3phosphocholine (DOPC) lipid is shown in Figure 1A and its headgroup flipped CP lipid with an ethyl cap (DOCPe) is in Figure 1B.31 CP lipids have been used for drug delivery,32 and for preparing supported lipid bilayers.33, 34 Herein, we report a quite surprising and useful finding: the CPe liposomes are more resistant to cellular uptake with an even longer blood circulation time than PC liposomes (Figure 1E).
Keywords: drug delivery, biointerfaces, liposomes, adsorption, choline phosphate. Introduction Nanomaterials with a long blood circulation time have always been sought for drug delivery.1-4 Some general strategies have been developed for this purpose, including using noncharged hydrophilic polymers such as polyethylene glycol (PEG),5-8 zwitterionic molecules,9 and their various combinations. Phosphocholine (PC) is a zwitterion, and PC surfaces have excellent antifouling properties.10 PC has been extensively used in liposome formulations,11-13 drug delivery,14-16 and in coating nanomaterials for improving their biocompatibility and ligand conjugation.17-22 An interesting design is to flip the PC dipole to make choline phosphate (CP), which still keeps its zwitterionic property. Brooks and co-workers prepared a dendritic polymer terminated by CP, which adhered strongly to the cell membrane,23
Figure 1. The structures of a (A) DOPC and (B) DOCPe lipid. Cryo-TEM micrographs of (C) DOPC and (D) DOCPe liposomes. (E) A scheme highlighting that DOCPe liposomes are resistant to protein, PC membrane, and cell membrane adsorption, allowing even longer blood circulation than DOPC.
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Materials and Methods Chemicals. All the phospholipids, including DOPC and DOCPe were purchased from Avanti Polar Lipids (Alabaster, AL). -Mercaptoethanol (BME), 4-morpholineethanesulfonic acid (MES), 3,3'-deioctadecyloxacarbocyanine perchlorate (DiO), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), 4',6-diamidino-2-phenylindole (DAPI) and fluoromount aqueous mounting medium were from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488 phalloidin was from Invitrogen (Carlsbad, CA). Cholesterol-rhodamine B bioconjugate (Chol-RhoB) was synthesized using a previously reported method.35 Antibodies including APC-labeled anti-CD11b, PElabeled anti-Ly6G, PercpCy5.5-labeled anti-Ly6C and PECy7labeled anti-F4/80 were from Biolegend (San Diego, CA). Milli-Q water was used to prepare all the buffers and solutions. Preparation of liposomes. Liposomes were prepared using the standard extrusion method. DOPC, DOCPe, DOPG, DOPC/DOTAP (1:1, w/w), DOPC/Chol (2:1), DOCPe/Chol (2:1) DOPC/Chol/DSPE-PEG2000 (2:1:0.16) or DOCPe/Chol/ DSPE-PEG2000 (2:1:0.16) with a total mass of 2.5 mg were respectively dissolved in 100 L of chloroform and then dried by N2 as previously described (molar ratio for samples containing cholesterol).34 To prepare liposomes, the dried lipid films were hydrated with 0.5 mL buffer A (100 mM NaCl, 10 mM HEPES, pH 7.4) and extruded through two stacked polycarbonate membranes (pore size = 100 nm) for 21 times. Rhodamine-labeled liposomes were prepared by including 1% (w/w) Rh-PE lipid in chloroform before drying. Rh-labeled DOCPe/MPB-PE liposomes were prepared by adding 1% RhPE and 5% MPB-PE. For liposome stability test, liposomes were prepared by including DiO and Chol-RhoB (lipids:DiO:Chol-RhoB = 500:5:18 (w:w:w)). For flow cytometry analysis, DiO-labeled liposomes were prepared by including 0.25 wt% DiO of total mass of lipids. For pharmacokinetics and bio-distribution studies, DiR-loaded liposomes were prepared by including 0.25 wt% DiR of total mass of lipids.36 To encapsulate calcein, the above prepared lipid films were hydrated with 100 mM disodium calcein solution overnight and then extruded. Free calcein was removed by passing 50 μL of the samples through a Pd-10 column using buffer A for elution. The first 600 μL of the fluorescent fraction was collected. Cell culture. The RAW264.7 murine macrophage cells and the MDA-MB-231 human breast cancer cells from American Type Culture Collection (ATCC, Manassas, Virginia) were cultured in Dulbecco's Modified Eagle's medium supplemented with 10% FBS and 1% penicillin/ streptomycin at 37 C using a humidified 5% CO2 incubator. HeLa cell line obtained from the ATCC was cultured in DMEM/F12 medium. Animals and xenograft tumors. ICR and BALB/c nude female mice were purchased from Beijing HFK Bioscience Co., LTD (Beijing, China) and used at 7 weeks (ICR mice) and 6 weeks (BALB/c nude mice) of age. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Use Committee of University of Science and Technology of China. To generate xenograft tumors, 100 L MDA-MB-231 cells (3×106) diluted in 20% Matrigel (BD Biosciences, Franklin Lakes, NJ) were implanted orthotopically into the mammary fat pad of the second left nipple of BALB/c nude mice. After approximately one week, the sizes of tumors reached 150-180 mm3.
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Flow cytometry. The RAW264.7 murine macrophages were seeded at 8×104 cells per well in 24-well plates for 24 h. The media was replaced with fresh media containing Rh-DOPC, Rh-DOCPe or Rh-DOPC/DOTAP (1/1, w/w) liposomes (70 g mL-1) for 1, 3 or 6 h. After designated incubation time, the cells were washed twice with cold PBS, harvested, and suspended in 200 μL of PBS for analysis using fluorescence activated cell sorting (FACS, Calibur flow cytometer, BD Biosciences, Bedford, MA). Cells with PBS treatment were used as a control. Rh fluorescence in RAW264.7 cells was then analyzed using FlowJo Software 7.6 (data plotted as means ± SEM). NIR fluorescence imaging of tumor-bearing mice. To evaluate the biodistribution of DOPC and DOCPe liposomes in mice bearing cancer tumor, DiR-loaded DOPC or DOCPe liposomes were administrated intravenously into the tail vein at an equivalent dose of 1.5 mg lipid per mouse (n = 5 for each liposome) when the tumor size reached 150-180 mm3. Whole-body fluorescence images were recorded at predetermined time intervals (0.41, 1, 2 4, 6, 8, 12, 24 h) after intravenous administration using IVIS Spectrum (Perkin Elmer, Waltham, MA). The mice were placed in the ventral position to obtain whole-body optical images. All images were acquired with the following parameters: exposure time =auto; binning=8; f/stop=2. Filter sets for DiR: excitation at 745 nm and emission at 800 nm. The acquired images were analyzed with Living Image @ 4.5.1 Software. The region of interest (ROI) was drawn around the whole body and tumor to quantify at the ventral position. The distribution of DiR in the whole body and tumor was quantified by average radiant efficiency, total photons per second per square centimeter per steradian in the irradiance range (microwatts square centimeter): [p/sec/cm2/sr]/[μW/cm2] (data were plotted as means ± SEM). Additional methods are in the SI.
Figure 2. (A) Confocal fluorescence micrographs of macrophage RAW264.7 cells after incubation with Rh-labeled DOPC, DOCPe or DOTAP (with 50% DOPC) for 24 h at 37 C. (B) Flow cytometric analysis of uptake after 6 h incubation. (C) Kinetics of liposomal uptake (n = 3, **P < 0.01, ***P < 0.001).
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Results and Discussion DOCPe liposomes are more resistant to cellular uptake than DOPC liposomes. Our DOPC and DOCPe liposomes were prepared by the extrusion method yielding an average size of ~100 nm as characterized by dynamic light scattering (DLS, Figure S1A). Cryo-TEM microscopy confirmed their liposomal structure (Figure 1C, D). With an ethyl group capping the phosphate, DOCPe is also essentially charge neutral (Figure S1B), like that of DOPC. Both liposomes were structurally stable over 150 h in 10% fetal bovine serum (FBS) as confirmed by a fluorescence resonance energy transfer (FRET) assay (Figure S2) and by DLS (Figure S3). Their stability in terms of membrane integrity was further studied by a calcein dye leakage assay (Figure S4). While neither liposome leaked in buffer, a very slow leakage was observed in the presence of 10% FBS. Adding cholesterol suppressed the leakage for both liposomes. For typical drug delivery applications, cholesterol is often included to increase the stability of lipid membranes. Overall, the stability of DOCPe and DOPC liposomes was similar. To track cellular uptake, these liposomes were labeled with 1% rhodamine-phosphoethanolamine lipid (Rh-PE). We first compared their uptake by RAW264.7 macrophage cells using confocal fluorescence microscopy. The macrophages were chosen since they are responsible for clearing foreign materials in the body. After incubation for 24 h, no red fluorescence indicative of liposome uptake was observed with the DOCPe liposome (Figure 2A), a weak fluorescence was observed with the DOPC liposome, and very strong uptake occurred with the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, which served as a positive control. For quantitative measurement, flow cytometry was also performed. The mean fluorescence of the cells incubated with the DOCPe liposomes was similar to the untreated cells (Figure 2B), while the DOPC signal was indeed stronger. Suppressed DOCPe uptake was observed at each time point (Figure 2C, Figure S5). Therefore, while DOPC is resistant to uptake, DOCPe is even more so. Since we already confirmed the structural stability of the liposomes (Figure S2, S3), the difference between DOPC and DOCPe liposomes is likely due to their headgroup structure. This is a quite interesting and surprising observation, since by simply flipping the dipole orientation, we can affect their interactions with cells. DOCPe liposomes have longer blood circulation time. Encouraged by this observation at the cell culture level, we next injected 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine (DiR, a near IR fluorophore)-labeled liposomes into mice and measured the fluorescence in plasma at different time points (Figure 3A). The DOCPe liposomes indeed circulated longer. To quantify the results, we made a calibration curve for each liposome (Figure S6), and calculated their concentrations in blood (Figure 3B). The DOCPe concentration in blood was over twice of that of DOPC from 30 min to 8 h, consistent with the greater resistance to DOCPe uptake by the macrophages. Since phagocytosis is primarily responsible for clearing foreign particles from blood, we next performed a detailed analysis of related immune cells in different organs after injecting the liposomes. Multi-color flow cytometry was used to identify the phagocytes including monocytes, neutrophils, and macrophages that may uptake liposomes in blood, the liver, and the
spleen (Figure 3C-E and Figure S7-S9). The monocytes generally took up a higher amount of DOPC than DOCPe liposomes, while the macrophages in blood internalized more DOPC liposomes, and no significant difference was observed for uptake by neutrophils. These results suggest that DOCPe liposomes are resistant to phagocytes for the prolonged blood circulation. For practical drug delivery applications, pure DOPC or DOCPe liposomes are unlikely to be used due to in vivo stability concerns (Figure S4). Normally, cholesterol and PEGylated lipids are also included in the lipid formulation to enhance lipid membrane rigidity and to further increase the blood circulation time by forming stealth liposomes. Since the main goal of this study is to compare cellular uptake of DOPC and DOCPe liposomes and fundamental surface chemical interactions, we conducted most experiments with pure lipid systems (e.g. without cholesterol or PEG) to simplify chemical understanding. To have a preliminary test of the practical aspect, we also prepared DOPC (or DOCPe) liposomes mixed with 33% cholesterol and DSPE-PEG2000 (2:1:0.16). In this case, the overall circulation time increased consistent with the expected property of stealth liposomes. The DOCPe-based liposome still circulated slight longer with a blood half-life of 14.8 h compared to that of DOPC-based liposomes (10.8 h, Figure 3B). DOCPe liposomes do not adhere to DOPC liposomes. Our observations for CP liposomes are quite different to what was reported for CP polymers, which instead adhere to cells. The adhesion of CP polymers to cells was attributed to CP interacting with the PC lipids in cell membrane.23, 25 Note that PC lipids are the main component of the outer leaflet of the eukaryotic cell membrane. The inhibited (instead of enhanced) uptake of our CPe liposomes led us to explore its fundamental surface interactions with PC headgroups in lipid membranes. Instead of using live cells, we prepared DOPC liposomes as a model membrane to test the CP/PC interactions. First, we mixed DOCPe liposomes and DOPC liposomes, and followed the size of these liposomes using DLS. The size of the samples stayed at ~100 nm for all the tested DOCPe-to-DOPC liposome ratios ranging from 0.2 to 100 (Figure 4A, black trace), suggesting the lack of interaction. A control experiment was performed by adding cationic DOTAP liposomes to anionic DOPG liposomes, showing obvious aggregation with maximal size >1 μm at a ratio of 1:1 (Figure 4A, red trace). The above experiments were performed in 100 mM NaCl to mimic the physiological ionic strength. We further repeated the experiment without NaCl to reduce charge screening (Figure S10), but still no size change was observed for the DOCPe/DOPC samples. Next, isothermal titration calorimetry (ITC) was performed to follow the heat released upon titrating DOCPe liposomes into DOPC liposomes (Figure 4B). Again, we failed to observe any heat release, while the control experiment of mixing DOTAP and DOPG liposomes released a significant amount of heat (Figure 4C). Therefore, both DLS and ITC argued against the adhesion of DOCPe liposomes to DOPC liposomes. Using live cells, we also failed to observe DOCPe liposomes sticking to the cell membrane at 4 C (Figure S11). The lack of cell binding at 4 C was also observed in a CP-terminated micelle.37 Therefore, DOCPe liposomes cannot bind to PC lipids either in model membranes or in cell membranes.
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Figure 3. Pharmacokinetics of DiR- or DiO-loaded DOPC and DOCPe liposomes in mice. (A) In vitro fluorescence images of the plasma extracted at different time points after injecting the two liposomes and (B) its quantification. Liposomes containing 33% cholesterol and PEGylated lipids were also tested. Flow cytometry following liposome uptake by neutrophils (neut), monocyte (mo), and macrophage (mf) of (C) blood, (D) liver and (E) spleen by measuring the geometry mean fluorescence intensity (GMFI) of DiO fluorescence (n = 5, data are means ± SEM, *P
