Surface Chemistry of Photoluminescent F8BT Conjugated Polymer

Jan 15, 2015 - Nanoparticles Determines Protein Corona Formation and. Internalization by Phagocytic Cells. Raha Ahmad Khanbeigi,. †. Thais Fedatto ...
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Surface Chemistry of Photoluminescent F8BT Conjugated Polymer Nanoparticles Determines Protein Corona Formation and Internalization by Phagocytic Cells Raha Ahmad Khanbeigi,† Thais Fedatto Abelha,† Arcadia Woods,† Olivia Rastoin,§ Richard D. Harvey,† Marie-Christine Jones,∥ Ben Forbes,† Mark A. Green,‡ Helen Collins,§ and Lea Ann Dailey*,† †

Institute of Pharmaceutical Science, Franklin-Wilkins Building, King’s College London, 150 Stamford Street, London SE1 9NH, U.K. Department of Physics, King’s College London, Strand, London WC2R 2LS, U.K. § Division of Immunology, Infection and Inflammatory Diseases, Guy’s Campus, King’s College London, 16-15 Newcomen Street, London SE1 1UL, U.K. ∥ Pharmacy and Therapeutics, School of Clinical and Experimental Medicine, University of Birmingham, Robert-Aitken Building, Birmingham, B15 2TH U.K. ‡

S Supporting Information *

ABSTRACT: Conjugated polymer nanoparticles are being developed for a variety of diagnostic and theranostic applications. The conjugated polymer, F8BT, a polyfluorene derivative, was used as a model system to examine the biological behavior of conjugated polymer nanoparticle formulations stabilized with ionic (sodium dodecyl sulfate; F8BT-SDS; ∼207 nm; −31 mV) and nonionic (pegylated 12hydroxystearate; F8BT-PEG; ∼175 nm; −5 mV) surfactants, and compared with polystyrene nanoparticles of a similar size (PS200; ∼217 nm; −40 mV). F8BT nanoparticles were as hydrophobic as PS200 (hydrophobic interaction chromatography index value: 0.96) and showed evidence of protein corona formation after incubation with serum-containing medium; however, unlike polystyrene, F8BT nanoparticles did not enrich specific proteins onto the nanoparticle surface. J774A.1 macrophage cells internalized approximately ∼20% and ∼60% of the F8BT-SDS and PS200 delivered dose (calculated by the ISDD model) in serum-supplemented and serum-free conditions, respectively, while cell association of F8BT-PEG was minimal (200 nm had formed during the emulsification process, and were subsequently removed via filtration. Nanoparticle Characterization: Hydrodynamic Diameter and Zeta Potential. The hydrodynamic diameter (HD) of the nanoparticles was measured using photon correlation spectroscopy (PCS; Nanosizer, Malvern Instruments, UK) at a scattering angle of 173°. Suspensions (2 μg mL−1) of each particle size were prepared in purified water or (Dulbecco’s Modified Eagle’s Medium) DMEM supplemented with 1% sodium pyruvate, 1% penicillin/streptomycin, 1% HEPES buffer and 1% L-glutamine) with or without 10% v/v FBS. The PCS parameters used for measurements in cell culture media were: refractive index of particles = 1.590, refractive index of DMEM/ FBS = 1.337, temperature = 37 °C, dynamic viscosity of DMEM/FBS = 0.74 × 10−3 Pa s and DMEM = 0.72 × 10−3 Pa s. The suspensions were incubated for 24 h at 37 °C with particle size measurements taken every 15 min for the first 4 h, and every 1 h thereafter. The zeta potential was also measured using the Nanosizer in both DMEM and phosphate buffered saline (PBS) with and without FBS at 37 °C to mimic the in vitro experimental conditions. Determination of Quantum Yield (QY). Five dilutions of nanoparticle suspensions and a fluorescein solution were prepared in water (F8BT nanoparticles: 0.3−5 μg mL−1, PS200:0.8−10 μg mL−1 and fluorescein: 0.6−5 μg mL−1). The absorption (494 nm) and

⎛ slope of sample ⎞ (QY)sample = (QY)standard × ⎜ ⎟ ⎝ slope of standard ⎠ where the QY of fluorescein is 93% (Magde et al. 1995). It should be noted that the quantum yield could not be determined reliably in cell culture medium, due to an increased variability in the absorbance measurements performed in the presence of this biofluid. Nanoparticle Quantification and Photostability. To quantify cell-associated nanoparticles in uptake studies, a range of concentrations of nanoparticles were suspended in cell lysis buffer: N-methylpyrrolidone:25 mM Tris buffered-saline pH 8.1 (1:3). Fluorescence was measured using a Cary Eclipse fluorescence spectrophotometer (Varian, UK) in a black 96-well plate at λEx = 468 nm and λEm = 508 nm for PS, λEx = 461 nm and λEm = 530 nm for F8BT nanoparticles. A calibration curve was prepared for each particle type to determine the range of linear fluorescence intensity as a function of nanoparticle mass concentration (Table 1). The calibration curve was considered linear

Table 1. Linear Range of Fluorescence Intensity, LOD, LOQ, and Quantum Yield of Nanoparticles Used in the Studya particle type F8BTPEG F8BTSDS PS200

linear range (μg mL−1)

LOD (μg mL−1)

LOQ (μg mL−1)

quantum yield (%)

0.02−20

0.036 ± 0.020

0.108 ± 0.062

31.0

0.02−20

0.028 ± 0.018

0.085 ± 0.055

30.2

1−275

1.228 ± 1.191

3.720 ± 3.610

21.5

Values represent the mean ± standard deviation of n = 4 individual experiments.

a

when R2 > 0.998. The limit of detection (LOD) was defined as 3.3x(standard deviation of the linear regression/slope), and the limit of quantification (LOQ) was defined as 10 × (standard deviation of the linear regression/slope). F8BT nanoparticle photostability was also measured over a 14 day period in distilled water, DMEM, or DMEM/ FBS at a concentration of 2 μg mL−1. Hydrophobic Interaction Chromatography. Surface hydrophobicity of nanoparticles was assessed using HIC.37,38 Briefly, 250 μL nanoparticle suspension (20 μg mL−1) was prepared in PBS or PBS containing 10% FBS (PBS/FBS). The suspensions were incubated for 18 h at 37 °C in their respective medium. After incubation, loosely bound proteins were removed by centrifugation (13 000 rpm, 15 min) followed by resuspension of the nanoparticle-containing pellet in 1 mL PBS. After three washes the samples were eluted through HiTrap substituted sepharose HIC columns: Butyl FF, Phenyl FF, and Octyl FF. Fractions (1 mL) of elutant were collected and analyzed for particle content via turbidity measurement (SpectraMax 190, λ = 650 nm). Particles were eluted using 7 mL PBS followed by 7 mL 0.1% Triton X-100. The HIC index value was calculated according to the method of Jones and co-workers (2014)38 (Supporting Information Figure S1). Protein Corona Characterization. Characterization of the protein corona was performed using gel electrophoresis as described by Monopoli and co-workers (2011).39 Nanoparticle suspensions (500 μL; 1 mg mL−1) were mixed with 500 μL of DMEM containing 20% FBS to give a final protein content of 10% FBS and an approximately equal particle surface area of 240 cm2/mL. The mixtures were incubated for 18 h at 37 °C on a shaker (110 rpm). After incubation, loosely bound proteins were removed by centrifugation (13 000 rpm, 15 min) followed resuspension of the nanoparticle-containing pellet in 1 mL PBS. This washing procedure was repeated three times and after C

DOI: 10.1021/bm501649y Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 2. Nanoparticle hydrodynamic diameters and count rates were measured over 24 h in (a) DMEM/FBS and (b) DMEM. Zeta potential was measured at 1 h (c) in DMEM ± FBS or PBS ± FBS. Data represent mean ± SD of n = 3 individual measurements. * p < 0.05, **