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Compatibility of PEGylated polymer nanoparticles with the biophysical function of lung surfactant Moritz Beck-Broichsitter Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03818 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017
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Compatibility of PEGylated polymer nanoparticles with the biophysical function of lung surfactant Moritz Beck-Broichsitter*,† Medical Clinic II, Department of Internal Medicine, Justus-Liebig-Universität, Giessen, Germany Institut Galien, Faculté de Pharmacie, Université Paris-Sud XI, Châtenay-Malabry, France
Abstract In order to minimize an unwanted interference of colloidal drug delivery vehicles with the biophysical functionality of lung surfactant, the surface of polymer nanoparticles was modified with poly(ethylene glycol) (PEGylation). Plain poly(lactide) nanoparticles provoked a statistically relevant decrease in surface activity of the naturally-derived lung surfactant Alveofact®. By contrast, the extent of lung surfactant inhibition induced by PEGylated polymer nanoparticles was significantly attenuated. Here, escalations of the PEG coating layer thickness (>3 nm, with a chain-to-chain distance of ≤4 nm) on the colloidal surface were capable of circumventing bioadverse effects. Accordingly, polymer nanoparticles equipped with PEG chains with a molecular weight above 2-5 kDa were compatible with the biophysical function of Alveofact®. Overall, PEGylation of polymer nanoparticles presents a promising approach for the development of inhalation nanomedicines revealing negligible effects on the surface activity of the lining layer present in the deep lungs.
Keywords Biophysical inhibition; drug delivery; inhalation; lung surfactant; PEGylation; polymer nanoparticles
Introduction
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Polymer nanoparticles have been praised for the therapy of numerous lung diseases.1-3 So far, the development of colloidal drug delivery vehicles was mainly focused on controlled release and targeting properties4 as well as absence of toxicity towards cells and tissue following inhalation administration.5 However, toxicological aspects of lung-delivered polymer nanoparticles can also originate from their interplay with the lining layer present in the deep lungs (i.e., lung surfactant).6 Scant information is available describing the impact of nanoscale matter on the essential biophysical function of lung surfactant.7 Material properties (surface charge and hydrophobicity) and the applied dose (surface area) were shown to contribute to inhibition of lung surfactant function in vitro.8-11 Simultaneously, interest accrued in the interaction of nanoscale matter with individual components of the lung lining layer.12-15 Said studies disclosed an affinity of surfactant-associated proteins (SP) to the colloidal surface. The adsorption process could then deplete the content of said relevant components16 and consequently, lead to an unwanted dysfunction of lung surfactant.11,17 Hu et al. and Raesch et al. reported the behavior of PEGylated polymer nanoparticles with respect to cytotoxicity towards respiratory epithelial cells and protein adsorption from bronchoalveolar lavage fluid.13,18 However, these authors made no attempt to correlate the auspicious findings (i.e., low toxicity of and decreased adsorption of SP to PEGylated formulations) with a potential impact on the surface activity of the lining layer present in the deep lungs. To close this gap, the current work investigated the compatibility of surface-modified polymer nanoparticles with the biophysical function of lung surfactant. Therefore, a panel of poly(lactide)-block-poly(ethylene glycol) (PLA-b-PEG) copolymers with varying chain length of the PEG block were synthesized and transformed to polymer nanoparticles by nanoprecipitation. After thorough physicochemical characterization of the colloidal formulations, the surface activity of a naturally-derived lung surfactant (Alveofact®) was examined in the presence of plain and the diverse PEGylated polymer nanoparticles by monitoring the surface tension behavior in a pulsating bubble surfactometer. The lung surfactant was further investigated for the remaining SP content following the challenge with the diverse colloidal formulations. Accordingly, the present results could provide better understanding of the polymer nanoparticle-lung surfactant interplay and thus, facilitate for a development of nanomedicines, which are compatible with the biophysical function of lung surfactant.
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Experimental section Materials. PLA, (intrinsic viscosity: ~0.2 dl/g) was acquired from Polysciences (Germany). Sodium and calcium chloride, distilled water and Alveofact® were from Carl Roth (Germany), B. Braun (Germany) and Lyomark (Germany), respectively. All other chemicals and solvents were obtained from Sigma-Aldrich (Germany) in analytical grade and used as received.
Synthesis of PLA-b-PEG copolymers. The synthesis of PLA-b-PEG was carried out by ring opening polymerization of D,L-lactide from PEG monomethyl ether (number-average molecular weight (Mn): 0.75, 2, 5 and 10 kDa (given by the supplier)).19 Briefly, lactide (70 mmol), PEG monomethyl ether (0.2 mmol), the catalyst stannous octoate (0.2 mmol) and 10 ml of dry toluene were polymerized in a dried Schlenk round bottom flask with continuous stirring at 130 °C under an argon atmosphere. The reaction was stopped at ~60 % monomer conversion. After evaporation of toluene, the product was purified by consecutive precipitations into cold diethyl ether and water. The copolymers were then vacuum-dried (yield: 50-70 %). The synthesized copolymers are hereafter abbreviated as PLA-b-PEGx, with x displaying the Mn (in kDa) of the PEG block. Proton
nuclear
magnetic
resonance
(1H-NMR)
spectroscopy.
1
H-NMR
spectroscopy was performed in CDCl3 at 25 °C and a copolymer concentration of 1020 mg/ml (300 MHz; Avance 300, Bruker, UK). The chemical shift scale was corrected on the basis of the solvent peak. The Mn of the PLA block was determined by a comparison of the integration from the methyl group of the PLA segment to the methylene protons of PEG. The 1H-NMR spectra of PLA and PLA-b-PEG5 are provided in Figure S1 (Supporting Information).
Gel permeation chromatography (GPC). Samples were dissolved in chloroform (34 mg/ml). After filtration (0.2 µm; Acrodisc®, Germany), 100 µl of the sample solution was injected into the system, consisting of two columns (300×7.5 mm, bead diameter: 5 µm; PLgel MIXED-D, Varian, USA) and a differential refractive index detector (SpectraSystem RI150, Thermo Electron Corp., USA). The elution was performed at 30 °C with chloroform at a
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flow rate of 1 ml/min (toluene as flow-rate marker). Poly(methyl methacrylate) standards (Polymer Laboratories, UK) were used for calibration.
Preparation of polymer nanoparticles. Polymer nanoparticles were prepared by a nanoprecipitation technique.20-22 Briefly, PLA, PLA-b-PEG0.75, PLA/PLA-b-PEG2 (blend of 1/1 (w/w)), PLA/PLA-b-PEG5 (blend of 4/1 (w/w)) and PLA/PLA-b-PEG10 (blend of 9/1 (w/w)) were dissolved in acetone. The resulting (co)polymer solutions were subsequently injected into a magnetically stirred aqueous phase (i.e., distilled water containing 0.1 % of sodium cholate for PLA and PLA-b-PEG0.75 and distilled water for PLA/PLA-b-PEG2, PLA/PLA-b-PEG5 and PLA/PLA-b-PEG10). The organic solvent was removed by rotary evaporation. Nanosuspensions were then purified by repeated centrifugation/re-dispersion cycles and finally filtered (5.0 µm). Magnetic PLA and PLA-b-PEGx nanoparticles were prepared by nanoprecipitation as outlined above after addition of superparamagnetic iron oxide nanoparticles (~10 nm) to the organic polymer solution.23 The physicochemical properties of the magnetic PLA and PLA-bPEGx nanoparticles are displayed in Table S1 (Supporting Information). The utilized polymer nanoparticles are hereafter abbreviated as PLA(y)/PLA-bPEGx(z), with y and z displaying the respective mass of PLA and the copolymer employed during the fabrication process.
Characterization of polymer nanoparticles. The size and size distribution (polydispersity index (PDI)) of polymer nanoparticles were measured by dynamic light scattering (non-invasive back scatter technology, λ = 633 nm, scattering angle of 173°), and their ζ-potential was determined in 1 mM NaCl solution by laser Doppler velocimetry (Zetasizer NanoZS/ZEN3600, Malvern Instruments, Germany). The morphology of polymer nanoparticles was observed using a scanning electron microscope (JSM-7500F, JEOL, Germany) after sample coating with a platinum layer (Alto 2500, Gatan, Germany). Scanning electron micrographs of PLA and PLA(4)/PLA-b-PEG5(1) nanoparticles are presented in Figure S2 (Supporting Information). The thickness of the PEG layer on the PLA core was assayed by means of ζ-potential measurements as a function of the NaCl concentration (fixed aqueous layer thickness).24 ζPotentials are defined as the electrostatic potentials at the position of the slipping plane, which is thought to occur just outside the fixed aqueous layer of the coated polymer nanoparticles.
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ln(|ζ-potential|) was plotted against 3.33·c1/2, where c is the molality of the electrolyte. Doing so, the slope of the obtained regression line equaled the shell thickness (δ) of the PEG coating. To determine the coating density of the PEG chains on the surface of the polymer nanoparticles, the diverse formulations were centrifuged and the obtained pellets were freezedried (Alpha 1-2 LDplus, Christ, Germany). Specified amounts were then investigated for their PEG content by a colorimetric assays based on the Stewart principle (complex formation with ammonium ferroisothiocyanat in organic solution).25 Finally, the surface (S) that was occupied by a single PEG chain on the nanoparticle (S = π·dh2/n, with n as the number of PEG chains on one nanoparticle) and the chain-to-chain distance (D = 2·(S/π)1/2) were calculated.26
Determination of phospholipid content. Lipids were extracted from the surfactant preparations according to Bligh and Dyer.27 Phospholipids were then quantified by means of a colorimetric phosphorus assay.28 Alveofact® contained >90 % of phospholipids.29
Determination of SP-B and -C content. Surfactant preparations were assayed for the SP-B and -C content using respective enzyme-linked immunosorbent assays according to the manufacturer’s protocol (MyBioSource, San Diego, USA). Alveofact® contained 5 ± 1 and 14 ± 3 µg SP-B and -C per µmol of phospholipid, respectively.29 Incubation of polymer nanoparticles with lung surfactant. Alveofact® was resuspended in aqueous sodium chloride solution supplemented with Ca2+ by brief sonication (Sonorex Digitec; Bandelin, Germany) at a final phospholipid concentration of 50 mg/ml. Alveofact® stock suspension and polymer nanoparticle stock suspensions were then combined to meet the desired final phospholipid (i.e., 2 mg/ml) and polymer nanoparticle concentration in isotonic saline solution containing 2 mM Ca2+. Samples were mixed by vortexing and brief sonication, followed by 30 min incubation (without shaking) at 37 °C.
Biophysical studies. The surface activity of samples was assessed on a pulsating bubble surfactometer (Electronetics Corp., USA) at 37 °C.8,11,17,30 Briefly, samples (composition as outlined above) of 35 µl were transferred to the disposable sample chamber, and the adsorption behavior (γads) was measured. Therefore, a bubble of minimal radius (r; 0.4 mm) was created and while maintaining the bubble at that minimal size without pulsation, the pressure difference (∆p) across the air/liquid interface was monitored. Next, pulsation was started by sinusoidally oscillating the bubble (r between 0.4 and 0.55 mm, 20 cycles/min) to
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determine the dynamic surface tension at the minimum bubble radius (γmin). ∆p across the air/liquid interface was recorded continuously. The surface tension (γ) was then calculated using the Young-Laplace equation (γ = (∆p·r)/2). γads and γmin values were read after 12 and 300 s, respectively. Separation of polymer nanoparticles from Alveofact®. Nanoformulations were magnetically separated (SPHERO® FlexiMag Separator, Jr. device, Kisker Biotech, Steinfurt, Germany) from the lung surfactant preparation.11,17,30 The supernatant was sampled and subsequently assayed for the remaining phospholipid, SP-B and -C concentration as outlined above.
Statistics. All measurements were carried out in quadruplicate and values are presented as the mean ± SD unless otherwise noted. To identify statistically significant differences, one-way ANOVA with Bonferroni’s post t-test analysis was performed (SigmaStat 3.5; STATCON, Germany). Probability values of p
