Self assembly of amphiphilic C60 fullerene derivatives into nanoscale supramolecular structures
© Partha et al; licensee BioMed Central Ltd. 2007
Received: 26 April 2007
Accepted: 02 August 2007
Published: 02 August 2007
The amphiphilic fullerene monomer (AF-1) consists of a "buckyball" cage to which a Newkome-like dendrimer unit and five lipophilic C12 chains positioned octahedrally to the dendrimer unit are attached. In this study, we report a novel fullerene-based liposome termed 'buckysome' that is water soluble and forms stable spherical nanometer sized vesicles. Cryogenic electron microscopy (Cryo-EM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) studies were used to characterize the different supra-molecular structures readily formed from the fullerene monomers under varying pH, aqueous solvents, and preparative conditions.
Electron microscopy results indicate the formation of bilayer membranes with a width of ~6.5 nm, consistent with previously reported molecular dynamics simulations. Cryo-EM indicates the formation of large (400 nm diameter) multilamellar, liposome-like vesicles and unilamellar vesicles in the size range of 50–150 nm diameter. In addition, complex networks of cylindrical, tube-like aggregates with varying lengths and packing densities were observed. Under controlled experimental conditions, high concentrations of spherical vesicles could be formed. In vitro results suggest that these supra-molecular structures impose little to no toxicity. Cytotoxicity of 10–200 μM buckysomes were assessed in various cell lines. Ongoing studies are aimed at understanding cellular internalization of these nanoparticle aggregates.
In this current study, we have designed a core platform based on a novel amphiphilic fullerene nanostructure, which readily assembles into supra-molecular structures. This delivery vector might provide promising features such as ease of preparation, long-term stability and controlled release.
Nanotherapeutics has become an increasingly important field of research , along with the design and development of novel multifunctional carrier vectors such as nanoparticles [2–4], lipoproteins, micelles, dendrimers , nanoshells , functionalized nanotubes  and polymeric microspheres . Over the past 25 years, conventional phospholipid-based liposomes have been utilized for a variety of biomedical applications ranging from targeted drug delivery , diagnostic imaging , gene therapy  to biosensors . Structural dynamics of the bilayers that constitute liposomal vesicles has been well studied and today, a number of commercially available liposomes are readily used in healthcare applications [13, 14]. Liposomes that mimic biological membranes are typically comprised of glycerol-based phospholipids which contain a hydrophilic/polar head-group and one or two hydrophobic/nonpolar hydrocarbon chains of varying length . However in recent years, many other functional artificial nanostructures such as polymeric micelles have been synthesized that offer an alternative choice to phospholipid based liposomes . Carbon-based nanoparticles such as functionalized single-walled carbon nanotubes (SWNTs) and modified C60 fullerenes have been the subject of great interest in the last decade because of their potential use in materials, electronics, and, most recently, biological systems [17–19]. Water insoluble fullerene lipid membranes have been designed and well characterized by other groups [20, 21].
In this current study we have characterized the self assembly of AF-1 using a variety of techniques such as Cryogenic electron microscopy (Cryo-EM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) under varying pH and solvent conditions. The results indicate that AF-1 self assembles readily into both unilamellar and multilamellar vesicles. Cryo-EM results indicate the formation of bilayer membranes with a width of ~6.5 nm, consistent with molecular dynamics simulations  for amphifullerenes. We also observe the formation of large (400 nm diameter) multilamellar vesicles and smaller unilamellar vesicles in the size range of 50–150 nm in diameter. In addition, complex networks of cylindrical, rod-like aggregates with varying lengths and packing densities are seen. Other, interesting combined morphologies are also occasionally seen which most likely are transient in nature. The vesicle forming AF-1 (buckysomes) can serve as vehicles for encapsulation of drugs and subsequent drug delivery in a manner similar to liposomes, which have been used for controlled release as well as drug stability, solubility, bioavailability, and reduced toxicity. To utilize the potential application of buckysomes for therapeutic drug delivery we have performed cell viability assays on different human cell lines and have observed no remarkable cytotoxicity. We have also studied the uptake of buckysomes by the cells using fluorescent labelled AF-1 and have imaged the cells using fluorescent microscopy. In summary, this is the first detailed study describing the biophysical characterization, cytotoxicity and bio distribution analysis of the globular amphiphile AF-1.
Results and Discussion
Transmission Electron Microscopy (TEM)
Cryogenic Transmission Electron Microscopy (Cryo-EM)
In this case, there is a complex interplay between three major factors namely the (a) charges on the carboxylic acid groups present in the dendrimer which is controlled by the pH, (b) the solvation process (affected by the solvent) and (c)the mode of preparation (sonication or vortexing). These three critical parameters determine whether the end self-assembly structure is a vesicles or a long cylindrical micelle. At pH higher than 7.5 and the presence of HEPES buffer, the cylindrical micelles seemed to be the favoured structure irrespective of the mode of preparation. At pH 7.0 with citrate buffer as the solvent, vesicles are present. Since both the structures are formed from the same AF-1 molecule, the effect of chain length affecting the morphology as described in several papers does not come into play . However, it is well evident that 10 mM citrate in the pH range 7.0–7.4 is necessary for forming the vesicles (Figure 3 &4). When phosphate was added to citrate at the same pH range, mixed morphologies are seen (Figure 5D). In an earlier study, Tour and co-workers reported the effect of solvent polarity as a factor affecting the folding of side-chains resulting in both nanorods and vesicles from the same C60 derivative . The effect of the solvent on the environment around the AF-1 molecule seems to be the key factor governing the formation of different nanostructures at a given pH and preparation methodology. This present study focuses on describing the novel structures observed upon self-assembly of amphifullerenes as well as their biological behaviour. Future studies will be aimed at understanding the driving forces that determine the formation of a specific self assembled structure.
Dynamic Light Scattering (DLS)
Cytotoxicity and cellular localization
Self assembly of molecules in the nano-scale is of great interest due to their potential in biomedical applications. In this present study we have investigated the biological role of a novel globular amphiphile (AF-1) with a fullerene core, a dendrimeric polar head-group and hydrophobic tails mimicking conventional phospholipids. The modified water soluble fullerene core could serve as a template for easy linking of different drug molecules. Currently we are analyzing the conditions needed for the critical tuning of several variables that determine homogenous distribution of selective morphologies. The different factors are pH, sample concentration, temperature, type of dispersant and method of preparation. The results could provide clues for synthetic modifications on the monomer structure to tailor specific nanostructures. In the future, we are planning to perform in vivo experiments of antibody linked buckysomes loaded with contrast agents for targeted diagnostic imaging of vulnerable plaque.
(a) Buckysome Preparation
The globular amphiphile AF-1 was synthesized as previously described . The buckysome preparation was carried out by either one of the four different approaches namely: (a) simple hydration in buffer with occasional shaking to remove clumps, (b) vigorous vortex, (c) sonication for 15 min using a Branson 3510 sonicator and (d) heating followed by extrusion through a mini-extruder (Avanti Polar Lipids, Alabaster, AL) using a 100 nm polycarbonate filter. Extrusion was performed for a total of 21 passes (back and forth). The resulting suspension was analyzed by Cryo-EM, negative stained TEM and DLS.
Buckysomes were coupled to 6-aminofluorescein (Fluka-Sigma-Aldrich, St. Louis, MO) using the following procedure. 400 μL of buckysomes (2 mg/mL) was incubated with 100 μL each of 0.25 M EDC (N-Ethyl-N'- [3- dimethylaminopropyl]carbodiimide) (Fluka) and 0.25 M sulfo-NHS (N-hydroxysulfosuccimide) (Pierce, Rockford, IL) for 2 hrs at room temperature. The pH was adjusted to 7.0 using NaOH. To this solution, 300 μL of 6-aminofluorescein (1 mg/mL prepared in DMSO) was added and incubated overnight at room temperature. The free 6-aminofluorescein was separated from 6-aminofluorescein coupled AF-1 by size exclusion chromatography on Sephadex® G-75 (Sigma-Aldrich, St. Louis, MO) column. The fractions were analyzed by fluorometry (Tecan Systems Inc, San Jose, CA) for 6-aminofluorescein emission at 520 nm.
(b) Transmission Electron Microscopy
The buckysomes were visualized using uranyl acetate negative staining. A 400 mesh Copper grid coated with Carbon film and stabilized with Formvar (Ted Pella Inc, Redding, CA) was coated with poly-L-Lysine prior to the sample staining. The sample was placed on the grid for 5 minutes and excess of sample was blotted with filter paper. The samples were stained with 1% solution of uranyl acetate for 1 minute and allowed to dry. Analysis of the stained grids was performed with a JEOL JEM-1010 Transmission Electron Microscope (Tokyo, Japan) at an accelerating voltage of 80 kV. The images were captured with the AMT Advantage digital CCD Camera system.
(c) Cryo-Electron Microscopy
A 5 μL drop of the buckysome was frozen in liquid ethane on a holey carbon copper grid coated with ultrathin 3 nm carbon (Ted Pella Inc, Redding, CA). Vitrobot™ (FEI, Holland) was used for automated cryo freezing of the grids (1 sec hang time, 1 blot, room temperature). The data were collected with a TVIPS (Gauting, Germany) F415 4 K × 4 K slow-scan CCD camera on a FEI (Eindhoven, Holland) Tecnai G2 TF30 Polara electron microscope operating at 300 kV and at liquid nitrogen temperature by using low-dose protocol. The post magnification value was 1.615 and the CCD pixel size was 15 microns. The micrographs were processed with EMAN v1.7 software (Baylor College of Medicine, Houston, TX).
(d) Dynamic light scattering
Dynamic light scattering (DLS) measurements were performed using a Malvern Nano-ZS zetasizer (Malvern Instruments Ltd, Worcestershire, United Kingdom). The Nano-ZS employs non-invasive back scatter (NIBS™) optical technology and measures real time changes in intensity of scattered light as a result of particles undergoing Brownian motion. The sample is illuminated by a 633 nm Helium-Neon laser and the scattered light is measured at an angle of 173° using an avalanche photodiode. The size distribution of the vesicles is calculated from the diffusion coefficient of the particles according to Stokes-Einstein equation. The average diameter and the polydispersity index of the samples are calculated by the software using CONTIN analysis.
(e) Zeta potential measurements
The zeta potential of liposomes was measured with the Malvern Nano ZS using the technique of Laser Doppler Velocimetry (LDV). In this technique, a voltage is applied across a pair of electrodes at either end of the cell containing the particle dispersion. Charged particles are attracted to the oppositely charged electrode and their velocity was measured and expressed in unit field strength as an electrophoretic mobility. The zeta potential was calculated from the electrophoretic mobility using Henry's equation (Hunter, R. J.Zeta Potential in Colloid Science, Principles and Applications, Academic Press, London, 1981).
(f) Cell Culture
Human Kidney Epithelial cells (CC-2556) and Human Coronary Artery Endothelial cells (CC-2585 were obtained from Cambrex Corp. (Baltimore, MD). Kidney cells were grown in REGM media supplemented with REGM BulletKit® (Cambrex). Endothelial cells were grown in EBM media supplemented with EGM-2 BulletKit® (Cambrex). HepG2 Liver Hepatocellular Carcinoma cells (HB-8065) and Murine Macrophage-like Cells (TIB-67) were obtained from American Type Culture Collection (Manassas, VA). HepG2 cells were grown in Earle's Minimal Essential Media (ATCC) supplemented with 10% fetal bovine serum (Gibco®, Invitrogen, Carlsbad, CA), 2 mM L-glutamine, 100 μg/mL penicillin and 100 U/mL streptomycin (Sigma-Aldrich, St. Louis, MO). Macrophages were grown in Dulbecco's Modified Eagle's Medium (ATCC) supplemented with 10% fetal bovine serum (Gibco®), 2 mM L-glutamine, 100 μg/mL penicillin and 100 U/mL streptomycin (Sigma-Aldrich). All cells were grown at 37°C in 5% CO2.
Murine Macrophage-like cells (MAC, ATCC); HepG2 Liver cells (LIV, ATCC); and Human Kidney Epithelial Cells (HKEC, Cambrex) were exposed to varying concentrations of buckysomes for 18 hrs at 37°C, 5%CO2. Cells were then analyzed for general cytotoxicity using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Lactate Dehydrogenase (LDH) assays from Roche Applied Sciences (Indianapolis, IN) and Promega (Madison, WI) respectively.
Leaking membranes of damaged or dead cells release the cytoplasmic enzyme lactate dehydrogenase (LDH) into the surrounding media. This enzyme can be detected by measuring its catalytic activity and indirectly the conversion of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to another water-soluble formazan dye. Briefly, 2.5 × 104 viable cells were seeded in black-walled Falcon 96 well tissue culture-treated microtiter plates and allowed to attach overnight at 37°C/5%CO2. Cells were then inoculated with appropriate concentrations of AF-1 or control materials and incubated for 18 hrs at 37°C/5% CO2. The LDH assay was performed using the Cyto-Tox ONE™ Membrane Integrity Assay (Promega, Madison, WI) according to the manufacturer's instructions. Results were given as relative values to cells treated with 0.9% Triton-X (vol:vol). Cells only control was treated with equal volumes of Dulbecco's phosphate buffered saline.
For each set, 2.5 × 104 viable cells were seeded into wells of a Falcon 96-well tissue culture-treated microtiter plate (Becton Dickenson, Franklin Lakes, NJ) in triplicate. Cells were treated with the described particle suspensions in a concentration of 50 μg/mL in complete culture medium for 24 hr. Cytotoxicity was determined by measuring the reduction of the water-soluble MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, SIGMA) molecule to water-insoluble MTT-formazan, after incubating in 100 μL solubilization buffer for 24 hr at 37°C/5% CO2. The wells are then measured for absorbance at 550 nm using a Safire2™ plate reader (Tecan Systems Inc, San Jose, CA). The results are given as relative values to cells treated only with equal volumes of Dulbecco's phosphate buffered saline.
(h) Localization of 6-aminofluorescein conjugated AF-1 using Fluorescence Microscopy
Human Coronary Artery Endothelial Cells (Cambrex) were grown in 8-chamber tissue culture slides and exposed to 6-aminofluorescein-buckysomes for 18 hrs at 37°C, 5%CO2. After two washes with Dulbecco's phosphate buffered saline (Gibco®), cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min, and washed twice with Dulbecco's phosphate buffered saline. Chambers were removed and slides were dried. Fixed cells were mounted in ProLong® Gold antifade reagent with DAPI (4',6-diamidino-2-phenylindole) (Invitrogen, Carlsbad, CA). Images of fixed cells were taken with an Olympus IX71 inverted microscope (Olympus America Inc, Center Valley, PA) and Retiga 2000R Camera (Q Imaging, Burnaby, BC, Canada). Images were processed using Compix SimplePCI software (Compix Inc, Sewickley, PA).
This work was supported by grants from NASA (NNJ05HE75A), DoD/TATRC (W81XWH-04-20035T5) AND DoD/TATRC (DAMD17-01-2-0047). We acknowledge Dr. Michael Kellermann in the laboratory of Dr. Andreas Hirsch for synthesis of the globular amphiphile AF-1. We thank Drs Pawel Penczek, Lee Pullan and Angel Paredes at the Structural Biology Research Center, University of Texas Health Science Center, Houston, TX for assistance with the Cryo-Electron Microscope and Mr. Kenneth Dunner, Jr, from The University of Texas M.D. Anderson Cancer Center, Houston, TX (Cancer Center Core Grant CA16671) for help with the Transmission Electron Microscope. We wish to thank Drs. Russ Lebovitz, Don Elrod and Delia Danila for discussions with the manuscript.
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