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Biophysical activity of impaired lung surfactant upon exposure to polymer nanoparticles Moritz Beck-Broichsitter Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02893 • Publication Date (Web): 17 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016
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Biophysical activity of impaired lung surfactant upon exposure to polymer nanoparticles Moritz Beck-Broichsitter*,† 5 Medical Clinic II, Department of Internal Medicine, Justus-Liebig-Universität, Giessen, Germany
Abstract 10 Colloidal drug carriers could improve the therapy of numerous airway diseases. However, it remains unclear to what extent nano-scale particulate matter affects the biophysical function of the essential surface-active lining layer of the lungs, especially under pre-disposed conditions of airway diseases. Accordingly, the current study investigated the 15
impact of defined polymer nanoparticles on impaired lung surfactants. Admixtures of plasma proteins (albumin and fibrinogen) to Curosurf® led to a controllable decrease in surface activity (i.e., adsorption and minimal surface tension of >25 and >5 mN/m, respectively), which served as models for dysfunctional lung surfactants. Next, Curosurf® pre-incubated with plasma proteins was challenged with negatively- and positively-
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charged poly(lactide) nanoparticles. Negatively-charged nanoparticles significantly perturbed the biophysical function of impaired Curosurf® in a dose-dependent manner, most-likely due to a binding of essential surfactant components. By contrast, addition of positively-charged nanoparticles led to no further loss of surface activity, but a remarkable depletion of plasma protein content. Once adsorbed to the surface of polymer nanoparticles, plasma proteins were
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hindered to displace relevant surfactant components from the air/liquid interface. Overall, the current study indicated that, depending on their physicochemical properties, colloidal drug carriers could compromise the biophysical function of impaired lung surfactants. Notably, a positive surface charge represents a parameter for the rationale design of polymer nanomedicines causing negligible adverse events on an impaired surface-
30
active lining layer in the lungs.
Keywords
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Biophysical inhibition; impaired lung surfactant; plasma proteins; polymer nanoparticles; protein adsorption
Introduction 40 The deep lungs are coated by a highly surface-active lining layer composed of phospholipids (PL) and surfactant-associated proteins (SP)1,2. Lung surfactant causes a drastic decrease in surface tension at the air/liquid interface3-5, which prevents collapse of the alveolar region during breathing. However, various airway diseases such as asthma6, 45
pneumonia7-9, acute respiratory distress syndrome7,9 and cystic fibrosis8,10, can be associated with relevant surfactant dysfunction. In particular, leakage of plasma proteins (PP) into the lining layer is known to perturb a proper biophysical function of lung surfactant11. Meanwhile, polymer nanoparticles (NP) have been identified as potential drug carriers for the treatment of diverse airway diseases12-14, allowing for a prolonged drug release in the
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desired organ15. Despite the beneficial therapeutic performance of nano-scale drug delivery vehicles, the safety assessment of inhaled nanomaterials is currently a subject of intense research16-18, especially with respect to the interplay with the lung surfactant system19. As an example, diverse inorganic and polymeric nanomaterials were shown to interact with individual surfactant components in vitro, thereby interfering with the essential function of
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lung surfactant20-26. However, the impact of polymer NP should be further considered under pre-disposed conditions of airway diseases, which can be associated with significant dysfunction of the lung surfactant. This study aimed at addressing the question whether a provoked dysfunction of a lung surfactant is amplified in the presence of polymer NP. Therefore, highly surface-active lung
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surfactant from a porcine source (i.e., Curosurf®) was pre-incubated with PP (i.e., albumin and fibrinogen) leading to a controllable decrease in surface activity. Standardized polymer NP were then admixed and the activity of the impaired lung surfactant preparation was analyzed by the oscillating bubble technique. Next, the content of PP was determined in the presence of polymer NP to account for the obtained results from surface tension
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measurements. The current observations could be used to compile suggestions for the design
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of safe nanomedicines causing insignificant interactions with impaired lung surfactant of diseased subjects.
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Experimental section Materials. The porcine lung surfactant Curosurf® was obtained from Chiesi (Hamburg, Germany). NaCl, CaCl2 and albumin (from bovine serum, fraction V, ≥98 %) were acquired from Carl Roth (Karlsruhe, Germany), while fibrinogen (from bovine plasma,
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type I-S, 65-85 %), fluorescein isothiocyanate-labeled albumin (≥7 mol FITC/mol albumin), benzalkonium chloride (≥95 %), superparamagnetic iron oxide NP (SPION, size ~10 nm) and dextran (relative molar mass ~100 kDa) were procured from Sigma-Aldrich (Steinheim, Germany). FITC-labeled fibrinogen (5 mol FITC/mol fibrinogen) was prepared as described by Xia et al.27. Sodium dodecyl sulfate (≥99 %) was from SERVA (Heidelberg, Germany).
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Biodegradable poly(lactide) (PLA; Resomer® R203S, inherent viscosity 0.25–0.35 dl/g) was purchased from Boehringer Ingelheim (Ingelheim, Germany). Distilled water and isotonic saline were from B. Braun (Melsungen, Germany). All other chemicals and solvents were of analytical grade and used without further purification. Preparation and characterization of surfactant material. Curosurf® was supplied
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a
suspension,
which
contained 28,29
dipalmitoylphosphatidylcholine) 28
ratio SP-B/SP-C of 1/2-3)
~80
mg/ml
of
phospholipids
(~50-70
%
and ~1 mg/ml of hydrophobic SP-B and SP-C (weight
as main components. The surfactant preparation was re-
suspended in saline solution supplemented with Ca2+ (pH 7.0) by vortexing and brief 90
sonication (SONOREX DIGITEC, BANDELIN, Berlin, Germany). The exact PL content was determined by a colorimetric phosphorus assay30 after lipid extraction according to Bligh and Dyer31.
Preparation and characterization of NP. Negatively-charged PLA-NP with a 95
nominal size of 100 nm (PLA100-), SPION-loaded PLA100-, positively-charged PLA-NP with a nominal size of 100 nm (PLA100+) and SPION-loaded PLA100+ were prepared by a solvent evaporation technique24,32. Briefly, an organic phase containing PLA with or without added SPION was emulsified in distilled water containing sodium dodecyl sulfate and
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benzalkonium chloride, respectively. The organic solvent was removed by rotary evaporation. 100
Next, the resulting colloidal dispersions were purified by dialysis. Following purification, the nanosuspensions were further dialyzed against a counter-dialysis medium containing dextran for concentration purpose33. Finally, polymer NP were filtered (5.0 µm; GE Water & Process Technologies, Ratingen, Germany). The actual NP concentration in suspension was assessed gravimetrically after lyophilization (ALPHA 1-4 LSC, Christ, Osterode, Germany)34.
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The size and size distribution (i.e., polydispersity index (PDI)) of NP were measured by dynamic light scattering (DLS), and their ζ-potential was determined by laser Doppler anemometry (Zetasizer NanoZS/ZEN3600, Malvern Instruments, Herrenberg, Germany)35. Scanning electron microscopy (SEM) was performed on a JSM-7500F (JEOL, Eching, Germany) as previously described32.
110 Incubation of lung surfactant with PP and polymer NP. Curosurf® stock preparations, PP stock solutions and polymer NP stock suspensions were combined to meet the desired final PL (i.e., 4 mg/ml), PP and polymer NP concentration in isotonic saline containing 2 mM Ca2+ (pH 7.0). Samples were mixed by vortexing and brief sonication, 115
followed by a 60 min incubation period (without shaking) at 37 °C.
Biophysical studies. The surface activity of the samples was assessed on a pulsating bubble surfactometer (PBS; Electronetics Corp., Amherst, USA) at 37 °C24,25,36,37. This technique provided read-outs of the surface tension after film adsorption (γads, static 120
measurement) and at a minimum bubble radius during film oscillation (γmin, dynamic measurement). Briefly, after the incubation period, samples of 35 µl were transferred to the disposable sample chamber, and the adsorption rate was measured. Therefore, a bubble of minimal radius (0.4 mm) was created and while maintaining the bubble at that minimal size without pulsation, the pressure difference across the air/liquid interface was monitored. Next,
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pulsation was started by sinusoidally oscillating the bubble radius between 0.4 and 0.55 mm. The cycling rate was set to 20 cycles/min. The pressure difference across the air/liquid interface was recorded continuously and used to calculate the surface tension according to the Young-Laplace equation. γads and γmin values were read after 12 and 300 s, respectively. Biophysical investigations using the pulsating bubble technique are commonly
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performed
with
“dilute”
surfactant
preparations
(e.g.,
PL
concentrations
of
2-
4 mg/ml)24,25,36,37. These surfactant concentrations allow, on the one hand, for the same (low) surface tension values to be reached as observed in vivo (i.e.
