Nontoxic Carbon Dots Potently Inhibit Human Insulin Fibrillation

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Nontoxic Carbon Dots Potently Inhibit Human Insulin Fibrillation Shanghao Li,† Lingyu Wang,‡ Charles C. Chusuei,§ Valentina M. Suarez,† Patrica L. Blackwelder,∥,⊥ Miodrag Micic,#,∇ Jhony Orbulescu,∇ and Roger M. Leblanc*,† †

Department of Chemistry, 1301 Memorial Drive,University of Miami, Coral Gables, Florida 33146, United States Department of Biology, 1301 Memorial Drive, University of Miami, Coral Gables, Florida 33146, United States § Department of Chemistry, 440 Friendship Street, Middle Tennessee State University, Murfreesboro, Tennessee 37132, United States ∥ University of Miami Center for Advanced Microscopy and Marine Geosciences, 1301 Memorial Drive,University of Miami, Coral Gables, Florida 33146, United States ⊥ Nova Southeastern University Oceanographic Center, 8000 North Ocean Drive, Dania, Florida 33004, United States # Department of Engineering Design Technology, Cerritos College, 11110 Alondra Blvd, Norwalk, California 92650, United States ∇ MP Biomedicals LLC, 3 Hutton Center, Santa Ana, California 92707, United States ‡

S Supporting Information *

ABSTRACT: One prevention and therapeutic strategy for diseases associated with peptide or protein fibrillation is to inhibit or delay the fibrillation process. Carbon dots (C−Dots) have recently emerged as benign nanoparticles to replace toxic quantum dots and have attracted great attention because of their unique optical properties and potential applications in biological systems. However, the effect of C-Dots on peptide or protein fibrillation has not been explored. In this in vitro study, human insulin was selected as a model to investigate the effect of C-Dots on insulin fibrillation. Watersoluble fluorescent C-Dots with sizes less than 6 nm were prepared from carbon powder and characterized by UV−vis spectroscopy, fluorescence, Fourier transform infrared spectrophotometry, X-ray photoelectron spectrometry, transmission electron microscopy, and atomic force microscopy. These C-Dots were able to efficiently inhibit insulin fibrillation in a concentration-dependent manner. The inhibiting effect of C-Dots was even observed at 0.2 μg/mL. Importantly, 40 μg/mL of C-Dots prevent 0.2 mg/mL of human insulin from fibrillation for 5 days under 65 °C, whereas insulin denatures in 3 h under the same conditions without C-Dots. The inhibiting effect is likely due to the interaction between C-Dots and insulin species before elongation. Cytotoxicity study shows that these C-Dots have very low cytotoxicity. Therefore, these C-Dots have the potential to inhibit insulin fibrillation in biological systems and in the pharmaceutical industry for the processing and formulation of insulin.

1. INTRODUCTION Peptide or protein fibrillation in the extracellular space of tissues plays a significant role in the development of several serious human diseases, such as Alzheimer’s disease, type 2 diabetes and Parkinson’s disease.1,2 These peptide or protein fibrils feature well-defined cross-β-sheet structures through misfolding of the native conformations. It is widely accepted that the fibrillation typically follows a nucleation−growth pattern, including initial formation of small nuclei through oligomerization, and then elongation of the fibrils via protofibril formation.3 The formation of nucleation species is the key ratedetermining step, as it is a thermodynamically unfavored process during fibrillation. The intermediate oligomeric species and the mature fibrils have cytotoxicity, provoking the death of related cells.4 Therefore, one prevention and therapeutic strategy for the diseases associated with peptide or protein fibrillation is to inhibit or delay the fibrillation process. Various approaches have been explored to inhibit peptide or protein fibrillation, including small organic molecules,2 functional polymers,5,6 and nanoparticles.7,8 Increasing attention is © 2015 American Chemical Society

being paid to elucidating the effects of nanoparticles on protein fibrillation due to tunable physical/chemical properties of nanoparticles, such as size, components, and surface charge.7−10 Some recent studies have reported the effect of nanoparticles on peptide or protein fibrillation. Protein fibrillation is found to be promoted by carbon nanotubes, cerium oxide nanoparticles, titanium dioxide nanoparticles, and gold nanoparticles.11−13 On the contrary, a few examples of capped quantum dots (QDs) show inhibiting effect on protein fibrillation, such as N-acetyl-Lcysteine capped CdTe QDs, thioglycolic acid-capped CdTe QDs, and dihydrolipoic acid-capped CdSe/ZnS QDs.14−16 But the conjugation with ligands changes the size and surface properties of the QDs. Another major issue of QDs comes from their toxicity due to the heavy metal composition.17 Carbon dots (C-Dots), a new type of nanoparticles in quantum size, have recently emerged as benign nanoparticles Received: December 12, 2014 Revised: February 4, 2015 Published: February 9, 2015 1764

DOI: 10.1021/cm504572b Chem. Mater. 2015, 27, 1764−1771

Article

Chemistry of Materials

dialyzed it with 4 L of deionized water for 5 days; the deionized water was changed every 4−10 h. Then, the solution was concentrated by heating it to 75−85 °C, until about 25 mL remained. Finally, the water was evaporated using a rotovap to yield 27.4 mg of black powder as CDots. 2.3. Characterization of C-Dots. The prepared C-Dots were characterized by UV−vis in a 1 cm cell using Shimadzu UV−2600 spectrometer. The Fourier transform infrared (FTIR) spectrum was recorded on a PerkinElmer Frontier using the solid powder of C-Dots. Fluorescent emission spectra of C-Dots were measured in aqueous solution by a Horiba Jobin Yvon Fluorolog−3 with a slit width of 5 nm for both excitation and emission. X−ray photoelectron spectroscopy (XPS) was performed using a Perkin−Elmer PHI 560 ESCA system with a double-pass cylindrical mirror analyzer operated at 225 W and 12.5 kV using a Mg Kα anode and a hυ = 1253.6 eV photon energy. Core levels of the C 1s and O 1s orbitals were scanned and intensities were normalized according to their respective atomic sensitivity factors.29 Microscopic images of C-Dots were obtained on an Agilent 5420 atomic force microscope using tapping mode and a JEOL 1200X TEM. 2.4. Human Insulin Fibrillation in the Presence of C-Dots. Thioflavin T (ThT) fluorescence was used to monitor the kinetics of human insulin fibrillation. For this experiment, 1 mg/mL of human insulin stock was prepared in hydrochloric acid aqueous solution (pH 1.6) with 0.1 M sodium chloride (NaCl). It is worth noting that physiological conditions such as phosphate buffered saline at pH 7.4 were not used in the experiment, as human insulin was not soluble enough under these conditions. The solution was filtered through a 0.2 μm pore size filter. C-Dots prepared from carbon powder were dissolved in water at a concentration of 1 mg/mL. Then, the final concentration of 0.2 mg/mL of insulin with several concentrations of C-Dots (0, 0.2, 2, and 10 μg/mL) was prepared using 0.1 M NaCl solution at pH 1.6, and incubated at 65 °C. Aliquots of samples were taken every 30 min, and were diluted with ThT (40 μM at pH 1.6, 0.1 M NaCl) to 0.1 mg/mL of protein and 20 μM of ThT. The ThT fluorescence was recorded on a Fluorolog-3 spectrofluorometer at excitation of 440 nm in a 1 cm quartz cuvette with both excitation and emission slit widths at 5 nm. The emission at 485 nm was used to monitor the kinetics of insulin fibrillation. To determine the possible inhibiting effect of 10 μg/mL of C-Dots on insulin fibrillation at the lag phase, C-Dots were added after insulin was incubated at 65 °C for 0, 1, and 2 h, respectively. The samples were then taken at different incubation times and checked by ThT fluorescence under the same conditions as mentioned above. Circular dichroism (CD) spectra were applied to characterize conformational changes of human insulin using a JASCO J-810 spectropolarimeter (Easton, MD). The spectra were recorded between 190 and 260 nm at room temperature using a 2 mm optical path length quartz cell. The spectra were measured using diluted aliquots (to 0.1 mg/mL of insulin) withdrawn at different incubation times from the human insulin or insulin/C-Dots mixture solutions. The fibrillation of 0.2 mg/mL human insulin was also studied in the presence of 40 μg/mL C-Dots. The samples were taken at every 12 h and characterized by CD spectra. Atomic force microscopy (AFM) using tapping mode was applied to observe morphologies during the fibrillation process. The cantilever had a resonance frequency of ∼170 kHz with typical force constant of 7.5 N/m. Aliquots of samples withdrawn at different incubation times were diluted with pure water, drop-coated on a freshly cleaved mica surface, and allowed to dry for at least 1 h. To remove salt in AFM images, in some cases, we further rinsed the sample coated mica slide with water. Solution-based zeta potential analyses were characterized using a Zetasizer Nano ZS System (Malvern, Inc., UK) with irradiation from a standard 633 nm laser. Then, 0.2 mg/mL insulin and 10 μg/mL CDots in a 0.1 M NaCl solution at pH 1.6 were examined to obtain the surface potential. 2.5. Cytotoxicity of C-Dots to Sea Urchin Embryos. We collected gametes from adult sea urchins with ripe gonads. Fresh eggs were washed three times by cold filtered artificial seawater and mixed

with potential to replace heavy metal containing toxic QDs. The potential biological application of C-dots has attracted great attention because of their unique properties, such as excitation wavelength dependent photoluminescence, excellent biocompatibility, low cytotoxicity, and optical stability.18,19 CDots can easily cross cellular membranes and, therefore, have potential applications in bioimaging and theranostics.20−22 For any practical application in biological systems, C-Dots will inevitably contact with peptides and proteins and may change their conformations. However, the effect of C-Dots on peptide or protein conformation and fibrillation has so far been unexplored. In this study, human insulin (∼5.8 kDa) was selected to investigate the effect of C-Dots on its fibrillation at solution level. Insulin fibrils are found in some patients with type 2 diabetes after insulin infusion and repeated injection.23−25 Injected insulin is found to form fibrils irrespective of the site of injection. Insulin amyloids are known to deposit in thighs, shoulders, arms, and abdominal walls.25 In the pharmaceutical industry, insulin is one of the therapeutic proteins with the largest production volume due to its regulation on glucose levels in diabetes patients.26 But its fibrillation is still a challenging problem in production, storage, and delivery of the protein.27,28 Furthermore, it is well accepted that peptides and proteins may share a common molecular mechanism to develop fibrils, regardless of their sources, sequences, and functions.1,3,26 Therefore, this study of C-Dots on insulin fibrillation may provide some insights of their effect on peptide or protein fibrillation, and has potential applications in biological systems as well as pharmaceutical applications.

2. EXPERIMENTAL SECTION 2.1. Materials. Carbon nanopowder (