Acta Biomaterialia 10 (2014) 4678–4684
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Biophysical inhibition of pulmonary surfactant function by polymeric nanoparticles: Role of surfactant protein B and C Moritz Beck-Broichsitter ⇑, Clemens Ruppert, Thomas Schmehl, Andreas Günther, Werner Seeger Medical Clinic II, Department of Internal Medicine, Justus-Liebig-Universität, Klinikstrasse 33, D-35392 Giessen, Germany
a r t i c l e
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Article history: Received 28 March 2014 Received in revised form 13 June 2014 Accepted 22 July 2014 Available online 1 August 2014 Keywords: Biophysical inhibition Polymeric nanoparticles Pulmonary drug delivery Pulmonary surfactant Hydrophobic surfactant proteins
a b s t r a c t The current study investigated the mechanisms involved in the process of biophysical inhibition of pulmonary surfactant by polymeric nanoparticles (NP). The minimal surface tension of diverse synthetic surfactants was monitored in the presence of bare and surface-decorated (i.e. poloxamer 407) sub100 nm poly(lactide) NP. Moreover, the influence of NP on surfactant composition (i.e. surfactant protein (SP) content) was studied. Dose-elevations of SP advanced the biophysical activity of the tested surfactant preparation. Surfactant-associated protein C supplemented phospholipid mixtures (PLM-C) were shown to be more susceptible to biophysical inactivation by bare NP than phospholipid mixture supplemented with surfactant protein B (PLM-B) and PLM-B/C. Surfactant function was hindered owing to a drastic depletion of the SP content upon contact with bare NP. By contrast, surface-modified NP were capable of circumventing unwanted surfactant inhibition. Surfactant constitution influences the extent of biophysical inhibition by polymeric NP. Steric shielding of the NP surface minimizes unwanted NP–surfactant interactions, which represents an option for the development of surfactant-compatible nanomedicines. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nanotechnology offers a versatility of novel materials, which have found a broad scope of potential applications in medicine [1,2]. As an example, nano-scale materials offered interesting properties for pulmonary drug delivery purposes [3], among them, specified polymeric nanoparticle (NP) formulations, which contributed to the development of more advanced treatment modalities for respiratory diseases [4–8]. However, tolerability assessment of pulmonary applied polymeric NP formulations is currently a subject of intense research [9–12], but scant information is available on the toxicological aspects arising from their interplay with the pulmonary surfactant system [13–15]. Pulmonary surfactant, composed mainly of phospholipids (PL) and surfactant-associated proteins (SP), is a significant element of the alveolar region of the lung, regulating the surface tension of the terminal air spaces and, thus, promoting the gaseous exchange [16,17]. Naturally, factors showing bioadverse impact on the characteristics of pulmonary surfactant might have a severe outcome [18]. Hence, clarification of the extent and mechanisms involved in pulmonary surfactant inhibition by
polymeric NP would facilitate the development of safe nanomedicines causing insignificant interactions with the pulmonary surfactant interface [19]. In this regard, the current study aimed to provide systematic insights into relevant biophysical interactions between polymeric NP and pulmonary surfactant. Therefore, the surface properties of diverse SP-supplemented PL mixtures (PLM) were examined by monitoring the dynamic minimum surface tension (cmin) behavior in the presence of defined sub-100 nm poly(lactide) (PLA) NP formulations. Next, the results from biophysical inhibition experiments were correlated with the impact of polymeric NP on surfactant composition (i.e. depletion of the hydrophobic SP content). Finally, the surface characteristics of the colloidal formulation employed were modified by coating with poloxamer 407 to minimize undesirable interactions with basic surfactant constituents and, thus, biophysical inhibition of the pulmonary surfactant system.
2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel.: +49 641 985 42453; fax: +49 641 985 42359. E-mail address: [email protected] (M. BeckBroichsitter). http://dx.doi.org/10.1016/j.actbio.2014.07.026 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt)
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(POPG), palmitic acid (PA), superparamagnetic iron oxide NP (SPION, size 10 nm), dextran (relative molar mass 100 kDa) and poloxamer 407 were acquired from Sigma-Aldrich (Steinheim, Germany). NaCl and CaCl2 were procured from Carl Roth (Karlsruhe, Germany). VenticuteÒ was obtained from Nycomed (Konstanz, Germany). PLA (ResomerÒ R203H, inherent viscosity 0.25–0.35 dl g1) was purchased from Boehringer Ingelheim (Ingelheim, Germany). Sodium dodecyl sulfate (SDS) was from SERVA (Heidelberg, Germany). Filtrated, double-distilled water was from B. Braun (Melsungen, Germany). All other chemicals and solvents were of analytical grade.
2.2. Methods 2.2.1. Preparation and characterization of NP formulations PLA-NP and SPION-loaded PLA-NP (PLAM-NP) were prepared using a solvent evaporation technique [20]. Briefly, PLA with or without added SPION (10 wt.%) was dissolved in methylene chloride (total solid content 25 mg ml1). Subsequently, the organic phase (2 ml) was transferred to filtrated, double-distilled water (10 ml) containing SDS (0.2 wt.%). After initial pre-mixing of the two phases using a homogenizer, the emulsion was tip sonicated. The organic solvent was removed by rotary evaporation. The resulting colloidal dispersion was transferred to a dialysis bag (molecular weight cut-off of 10 kDa, Spectra/PorÒ 6, Breda, Netherlands) and extensively dialyzed against filtrated, double-distilled water. Following purification, polymeric NP formulations were concentrated by dialysis [21]. Briefly, the nanosuspension was dialyzed (10 kDa) against a counter-dialysis medium composed of filtrated, double-distilled water containing dextran (300 mg ml1), whose volume was at least two times larger than the sample volume. Finally, NP formulations were filtered (5.0 lm; GE Water & Process Technologies, Ratingen, Germany). The actual NP mass concentration and density in suspension was assessed gravimetrically after lyophilization (ALPHA 1–4 LSC, Christ, Osterode, Germany) [22] and using an oscillating density meter (DMA 4100M, Anton Paar, Graz, Austria) [14]. The size and size distribution (i.e. polydispersity index) of NP were measured by dynamic light scattering, and their f-potential was determined by laser Doppler anemometry (Zetasizer NanoZS/ZEN3600, Malvern Instruments, Herrenberg, Germany).
2.2.2. Coating of NP formulations with poloxamer 407 The nanosuspension was incubated with poloxamer 407 (final concentration 0.1 wt.%) for 12 h at 25 °C. After incubation, NP were purified from residual surfactant by repeated centrifugation (Sorvall, DuPont, Bad Homburg, Germany) and redispersion cycles at 4 °C and then filtered (5.0 lm). The desired final NP concentration was adjusted prior to use.
2.2.3. Determination of the adsorbed poloxamer 407 layer thickness on NP The thickness of the adsorbed poloxamer 407 layer (d) on the NP surface was determined using dynamic light scattering [23– 25]. Here, d is derived from the comparison of the size of bare (size0) and coated (sizeads) NP (d = (sizeads size0)/2).
2.2.4. Isolation of surfactant-associated protein B Surfactant-associated protein B (SP-B) was isolated from AlveofactÒ (Lyomark, Oberhaching, Germany) by means of LH60chromatography, as described previously [26–28]. The purity amounted to 95%, as assessed by SDS–polyacrylamide gel electrophoresis [28,29].
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2.2.5. Preparation of surfactant materials VenticuteÒ was supplied as a powder, which contained DPPC (63.4 wt.%), POPG (27.8 wt.%), PA (4.5 wt.%), CaCl2 (2.5 wt.%) and recombinant surfactant-associated protein C (rSP-C, 1.8 wt.%) (Table 1) [30]. rSP-C exhibits the same structure (amino acid sequence) as natural SP-C except for the missing palmitoylation. Moreover, its biophysical activity was reported to be comparable with that of the native protein [31]. Synthetic pulmonary surfactant stock preparations containing 1.8 wt.% of SP-B and SP-B/C (weight ratio SP-B/rSP-C of 1/3) [32] were prepared by supplementing either a PLM (possessing the same composition as VenticuteÒ) or VenticuteÒ with bovine SP-B. The PLM was prepared by dissolving DPPC, POPG, PA and CaCl2 in a mixture of chloroform/ methanol (2/1 (v/v)). Supplementation of the PLM with SP-B (PLMB) was then achieved by adding the hydrophobic SP dissolved in chloroform/methanol [28,31]. Similarly, PLM-B/C was produced by dissolving VenticuteÒ powder in chloroform/methanol followed by addition of organic SP-B solution. The SP content of the synthetic pulmonary surfactant stock preparations was decreased by dilution with organic PLM stock solution. Samples were then dried under nitrogen gas. All surfactant preparations were resuspended in NaCl solution supplemented with Ca2+ by vortexing and brief sonication (SONOREX DIGITEC, BANDELIN, Berlin, Germany) to a final PL concentration of 50 mg ml1. 2.2.6. Incubation of polymeric NP with pulmonary surfactants Synthetic pulmonary surfactant stock preparations and polymeric NP stock suspensions were combined to meet the desired final PL (i.e. 2 mg ml1) and polymeric NP concentration in isotonic NaCl solution containing 2 mM Ca2+. Here, the amounts of added polymeric NP are specified on the basis of total surface area rather than mass (information on added NP mass can be found in the Supplementary data (Figs. S1–S4)). The total surface area introduced was adjusted between 9 cm2 ml1 (i.e. 0.01 mg of PLA-NP per ml) and 4355 cm2 ml1 (i.e. 5.0 mg of PLA-NP per ml). Samples were mixed by vortexing and brief sonication, followed by a 60 min incubation period at 37 °C. 2.2.7. Biophysical studies The surface activity of samples was assessed using the oscillating bubble technique (Electronetics Corp., Amherst, USA) [14,15,31,33,34]. All measurements were performed at a constant PL concentration (2 mg ml1) in isotonic NaCl solution containing 2 mM Ca2+ at 37 °C after the incubation period. Briefly, samples of 35 ll were transferred to the disposable sample chamber, and bubble pulsation was started by sinusoidally oscillating the bubble radius (r) between 0.4 and 0.55 mm. The cycling rate was set to 20 cycles min1. The pressure difference (Dp) across the air/liquid interface was recorded continuously. The surface tension (c) was then calculated using the Young–Laplace equation (c = (Dpr)/2). cmin values were read after 300 s. Dose–effect curve characteristics (i.e. half maximal biophysical inhibitory polymeric NP concentration (IC50 value)) were calculated using a sigmoidal dose–response function (Origin 7.0, OriginLab, Northampton, USA). Physiologically surfactant concentrations are estimated to range between 10 and 20 mg ml1 [18]. Biophysical investigations using the oscillating bubble technique are commonly performed with surfactant concentrations ranging between 1 and 4 mg ml1 [14,15,31,32,34]. These surfactant concentrations allow, on the one hand, the same (low) surface tension values to be reached as observed in vivo (i.e. 10 mg ml1), owing to the increased turbidity of the
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Table 1 Biochemical composition and biophysical characteristics of the synthetic pulmonary surfactant stock preparations. Preparation PLM-B Ò
PLM-C (i.e. Venticute ) PLM-B/C a b
PL (wt.%)
SP (wt.%)
DPPC (63.4) POPG (27.8) DPPC (63.4)a POPG (27.8)a DPPC (63.4) POPG (27.8)
SP-B (1.8) rSP-C (1.8)
a
SP-B (0.45) rSP-C (1.35)
Miscellaneous (wt.%)
cminb (mN/m)
PA (4.5) CaCl2 (2.5) PA (4.5)a CaCl2 (2.5)a PA (4.5) CaCl2 (2.5)
3.3 ± 2.0 2.5 ± 1.7 2.9 ± 1.6
Values were obtained from the literature [30]. Values are presented as the mean ± standard deviation (n = 5).
preparation, which impedes the exact adjustment of bubble size, thereby negating some of the advantages of working at ‘‘dilute’’ surfactant concentrations.
2.2.8. Determination of SP content after challenge of pulmonary surfactant with polymeric NP Depletion of the SP content in the surfactant preparation caused by polymeric NP addition was investigated with pulmonary surfactants containing 1 wt.% of SP-B, rSP-C and SP-B/C, respectively. Stock preparations of synthetic pulmonary surfactants and polymeric NP (i.e. PLAM-NP and PLAM-NP-407) were combined to meet the desired final PL (i.e. 2 mg ml1) and polymeric NP concentration in isotonic NaCl solution containing 2 mM Ca2+. Samples were mixed by vortexing and brief sonication. Following incubation (60 min at 37 °C), NP formulations were magnetically separated (SPHEROÒ FlexiMag Separator, Jr. device, Kisker Biotech, Steinfurt, Germany) from the surfactant samples. The supernatant was carefully discarded, and the SP concentration was determined using the method described by Bradford [35]. Pure surfactant preparations were used for calibration.
2.2.9. Statistical analysis All measurements were carried out in triplicate, and values are presented as the mean ± standard deviation unless otherwise noted. To identify statistically significant differences, one-way ANOVA with Bonferroni’s post t-test analysis was performed (SigmaStat 3.5, STATCON, Witzenhausen, Germany). Probability values of p < 0.05 were considered significant.
3. Results 3.1. Characterization of pulmonary surfactants The biochemical composition and biophysical characteristics of the synthetic pulmonary surfactant preparations employed are outlined in Table 1. Among them, PLM supplemented with 1.8 wt.% of SP-B, rSP-C (i.e. VenticuteÒ ) and SP-B/C (weight ratio SP-B/rSP-C 1/3), respectively, can be found. The PLM consisted of a phosphatidylcholine (i.e. DPPC), a phosphatidylglycerol (i.e. POPG), a fatty acid (PA) and CaCl2. The biophysical properties of the synthetic surfactant preparations revealed cmin values of 3.0 mN m1 (Table 1). Lowering of the SP content in the PLM caused a dose-dependent increase in cmin values (Fig. 1). Here, all SP-based PLM displayed superimposable dose–effect curve characteristics, and cmin values of > 5.0 mN m1 were reached at SP concentrations 64 mN m1) during bubble oscillation (2 mg ml1) (Table 2). 3.3. Biophysical inhibition of pulmonary surfactants by polymeric NP To clarify the extent and mechanisms involved in pulmonary surfactant inhibition by polymeric NP, the current study investigated the sensitivity of synthetic PLM recombined with different amounts (i.e. 0.2–1.5 wt.%) and types (i.e. B, C and B/C) of SP. Surface activity measurements disclosed a dose-dependent loss of dynamic surface tension-lowering properties of the tested preparations upon polymeric NP addition, where the sensitivity of
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M. Beck-Broichsitter et al. / Acta Biomaterialia 10 (2014) 4678–4684 Table 2 Physicochemical characteristics of the NP formulations. Formulation
Size (nm)
PDI
f-Potential (mV)
da (nm)
cmin (mN/m)
PLA-NP PLA-NP-407 PLAM-NP PLAM-NP-407
65.3 ± 3.2 75.9 ± 2.6* 70.5 ± 4.9 83.1 ± 4.1&§
0.073 ± 0.012 0.053 ± 0.008* 0.068 ± 0.016 0.061 ± 0.012
44.3 ± 2.5 23.6 ± 0.8* 44.4 ± 3.1 24.5 ± 2.1&
n.d. 5.3 ± 0.8 n.d. 6.3 ± 0.8
70.7 ± 0.9 64.3 ± 1.6* n.d. n.d.
n.d. = not determined. Values are presented as the mean ± standard deviation (n P 4). Statistically significant differences (p < 0.05): a Adsorbed poloxamer 407 layer thickness. * PLA-NP-407 vs. PLA-NP. & PLAM-NP-407 vs. PLAM-NP. § PLAM-NP-407 vs. PLA-NP-407.
individual preparations was affected by the content and type of employed SP (Figs. 2–4A). Elevation of the rSP-C content improved the biophysical activity of the PLM-C (Figs. 2A and 3). The extent of biophysical inactivation (IC50 values), which was analyzed from the obtained dose– effect curves of individual inhibition experiments, was then plotted against the amount of rSP-C present in the PLM (Fig. 2B). The resulting relationship revealed linear correlation (R2 = 0.962).
Fig. 3. Dose-related effects of PLA-NP on cmin values and PLAM-NP on residual amounts of rSP-C present in the PLM-C. PLM-C containing 1.0 wt.% of rSP-C was used throughout the experiments. Values are presented as the mean ± standard deviation (n P 5).
Fig. 2. Dose-related effect of PLA-NP on cmin values of the PLM supplemented with 0.2 wt.% (light gray circles) and 1.5 wt.% (closed circles) of rSP-C (A). The continuous lines represent sigmoidal dose–response fits of the experimental data. The correlation of rSP-C concentration in the PLM with the PLA-NP-induced biophysical inhibition (i.e. IC50 values calculated from the sigmoidal fits) is illustrated in (B). The straight line depicts a linear fit of the experimental data (R2 = 0.962). The closed diamond in (B) represents the IC50 value for negatively charged poly(styrene) NP (diameter 100 nm) incubated with VenticuteÒ [15]. Values are presented as the mean ± standard deviation (n P 5).
In addition, the biophysical sensitivity of the PLM supplemented with 1 wt.% of SP-B and SP-B/C, respectively, was studied in the presence of polymeric NP (Fig. 4A). IC50 results revealed enhanced biophysical activity for both synthetic surfactant preparations compared with PLM-C (Figs. 2B and 3), but no statistically significant differences were observed between the sample values. To account for the biophysical inactivation potency observed for polymeric NP, synthetic surfactant preparations containing 1.0 wt.% of SP-B, rSP-C and SP-B/C, respectively, were incubated with bare PLAM-NP. Here, an increase in the amount of added polymeric NP led to a dose-dependent depletion of the SP content in the surfactant preparation (Figs. 3 and 4B). Overall, the surface property inactivation and depletion of SP content of synthetic surfactant preparations caused by polymeric NP addition (Figs. 2A, 3 and 4) resembled the surface activity results observed with SP-diluted PLM (Fig. 1). In an attempt to circumvent unwanted surfactant inhibition by polymeric NP, their surface was modified by PEGylation (i.e. poloxamer 407). Surface-decorated polymeric NP formulations were then evaluated for their adverse effect on the biophysical properties and composition of the surfactant preparation employed (Fig. 5). The surface activity of the PLM supplemented with 1.0 wt.% of rSP-C was unaffected by polymeric NP coated with poloxamer 407 over the investigated range of concentrations. Furthermore, substantial higher SP concentrations were detectable after PLM-C incubation with PLAM-NP-407 compared with their bare counterparts (i.e. PLAM-NP) (Fig. 3).
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Fig. 4. (A) Calculated dose–effect curve characteristics (i.e. IC50 value) for the influence of PLA-NP on cmin values of the PLM supplemented with 1.0 wt.% of SP-B and SP-B/C (weight ratio SP-B/rSP-C of 1/3), respectively. IC50 values were calculated from individual inhibition experiments using sigmoidal dose–response fits. (B) The dose-related impact of PLAM-NP on the SP (SP-B (squares) and SP-B/C (weight ratio SP-B/rSP-C of 1/3) (triangles)) content present in the PLM after incubation is illustrated. Values are presented as the mean ± standard deviation (n P 5).
Fig. 5. Dose-related effects of PLA-NP-407 on cmin values and PLAM-NP-407 on residual amounts of rSP-C present in the PLM-C. PLM-C containing 1.0 wt.% of rSP-C was used throughout the experiments. Values are presented as the mean ± standard deviation (n P 5). Statistically significant differences (p < 0.05): ⁄poloxamer 407coated vs. bare polymeric NP (Fig. 3).
4. Discussion Nanomedicine is generally regarded as one of the most promising future technology platforms offering revolutionary novel
material properties of interest for the treatment of numerous severe disorders [1,2]. As an example, tailored polymeric NP revealed considerable potential as controlled drug delivery vehicles to optimize the retention of the encapsulated drug within the respiratory tract and, thus, to advance the therapeutic benefit of the applied medication [3]. Besides the inflammatory potential observed for lung-delivered polymeric NP [10,11], it has become increasingly evident that colloidal drug carriers might have an adverse impact on the biophysical function of the pulmonary surfactant system [14,15]. However, adequate biophysical activity of the surface lining material is essential for maintaining a low surface tension in the deep lung, which prevents collapse of the alveoli, minimizes the work of breathing and, thus, facilitates the gaseous exchange [16–18]. Since the discovery of the hydrophobic SP (i.e. SP-B and SP-C), their crucial role in regulating the surface tension of pulmonary surfactant has been the focus of numerous studies [16,36]. Correspondingly, the presence of SP-B, rSP-C and SP-B/C (weight ratio SP-B/ rSP-C of 1/3) [32] improved the poor surface activity of the simple PLM (Table 1, Fig. 1). Maximum dynamic surface-tension-lowering properties (i.e. cmin values < 5 mN m1) were achieved with SP concentrations exceeding 0.1–0.2 wt.%, which is in general agreement with results from previous studies [31,37]. At these SP concentrations, the cmin values measured in the current study were similar to those observed in vivo [18]. Recently, it was demonstrated that pulmonary surfactant function can be dramatically influenced by inorganic [38], composite [39] and polymeric [14,15] NP formulations. However, scant information is available on the extent and mechanisms of NP-induced surfactant inhibition [13,18]. To close this gap, the present study evaluated the influence of standardized polymeric nanomedicines on the biophysical properties of the pulmonary surfactant system. Model formulations exhibited physicochemical characteristics similar to frequently lung-delivered colloidal drug carriers (Table 2) [4–8,10,11]. In the presence of polymeric NP, a distinct dosedependent biophysical inhibition of SP-supplemented synthetic PLM was noted (Figs. 2–4A). Thus, the total surface area of the applied formulation accounts for the actual surfactant inhibition. Marked differences in polymeric NP-induced surfactant inactivation also became evident upon dose-elevations of rSP-C in the preparation investigated (Figs. 2A and 3). Accordingly, higher susceptibility to biophysical inhibition was noticed for a lower SP content (Fig. 2B). Furthermore, the effect of SP type disclosed a lower susceptibility of SP-B-based and SP-B/C-based compared with rSP-C-based surfactants (Figs. 3 and 4A) [15]. Surfactant dysfunction provoked by inhaled nano-particulate matter is believed to occur through a direct interaction with individual surfactant constituents (e.g. SP) [13–15,38,40]. Hence, the ability of the negatively charged polymeric NP (Table 2) to adsorb hydrophobic, positively charged SP-B and SP-C is one putative mechanism for the observed alteration of surfactant function [40]. As a matter of fact, SP-B and SP-C should reveal different affinity to the NP surface, owing to their difference in molecular weight and charge density [41–43]. These hypotheses were confirmed by the observations made during incubation experiments, which manifested a depletion of SP content upon dose escalations of polymeric NP (Figs. 3 and 4B). Moreover, the results obtained from biophysical inhibition (Figs. 2–4A) and residual SP content (Figs. 3 and 4B) experiments were comparable with surface activity measurements performed with SP-supplemented PLM (Fig. 1), where the SP content was shown to be the major determinant for the observed surface tension-lowering potential [31,37]. Additionally, the present study covered the impact of specified NP characteristics on the bioadverse effect of the pulmonary surfactant system. In this respect, bare polymeric NP were surfacemodified with poloxamer 407, forming a dense brush-like
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poly(ethylene glycol) corona around the NP (Table 2) [23,25,44]. In contrast to bare polymeric NP (Fig. 3), incubation with this formulation revealed negligible effects on the biophysical properties of pulmonary surfactant (Fig. 5). Interestingly, similar results were recently reported in a molecular dynamics simulation study, where coated NP formulations (particle size < 10 nm) were able to penetrate pulmonary surfactant films without perturbation [45]. PEGylation of polymeric NP formulations is generally known to be efficient in preventing unwanted protein adsorption on the colloidal surface [46,47]. Nevertheless, even for poloxamer 407-coated polymeric NP a moderate depletion of SP content was observed (Fig. 5). Overall, the attributes to shield the polymeric NP surface from unwanted protein adsorption and, hence, to minimize their biophysical interactions with the pulmonary surfactant system are favorable for application of nanoparticulate drug carriers to the deep lung. Although the potential of surface-coated colloidal formulations to avoid interactions with individual pulmonary surfactant constituents (i.e. SP) are quite preliminary and speculative at present, these observations should be taken into consideration for the development of safe nanomedicines causing negligible adverse effects on the biological environment present at the target site, which deserves further experimental investigation. 5. Conclusions The present study disclosed experimental findings that account for the extent and mechanisms observed during polymeric NP-induced pulmonary surfactant inhibition. Increasing the SP concentration as well as application of SP-B rather than rSP-C improved the biophysical activity of the synthetic surfactant preparation. Depletion of the surfactant-associated SP content was shown to be regulated by the applied polymeric NP formulation. Hence, shielding of the polymeric NP surface represents a useful strategy for the rational design of colloidal drug delivery vehicles compatible with the essential pulmonary surfactant system present in the respiratory region of the lung. Conflict of interest The authors disclose that no conflicting interests associated with the manuscript exist. Acknowledgements The ‘‘Universitätsklinikum Giessen und Marburg’’ (1/2012 GI) and ‘‘Wirtschafts- und Infrastrukturbank Hessen’’ (Nanosurfact) are gratefully acknowledged for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.0 7.026. References [1] Farokhzad OC, Langer R. Impact of nanotechnology on drug delivery. ACS Nano 2009;3:16–20. [2] Shi J, Votruba AR, Farokhzad OC, Langer R. Nanotechnology in drug delivery and tissue engineering: from discovery to application. Nano Lett 2010;10:3223–30. [3] Beck-Broichsitter M, Merkel OM, Kissel T. Controlled pulmonary drug and gene delivery using polymeric nano-carriers. J Control Release 2012;161:214–24. [4] Beck-Broichsitter M, Gauss J, Packhäuser CB, Lahnstein K, Schmehl T, Seeger W, et al. Pulmonary drug delivery with aerosolizable nanoparticles in an ex vivo lung model. Int J Pharm 2009;367:169–78.
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Fibrinolysisinhibitory capacity of clot-embedded surfactant is enhanced by SP-B and SP-C. Am J Physiol Lung Cell Mol Physiol 2003;284:L69–76. [29] Schaegger H, von Jagow G. Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166:368–79. [30] Spragg RG, Lewis JF, Wurst W, Hafner D, Baughman RP, Wewers MD, et al. Treatment of acute respiratory distress syndrome with recombinant surfactant protein C surfactant. Am J Respir Crit Care Med 2003;167:1562–6. [31] Seeger W, Günther A, Thede C. Differential sensitivity to fibrinogen inhibition of SP-C- vs. SP-B-based surfactants. Am J Physiol Lung Cell Mol Physiol 1992;262:L286–91. [32] Bernhard W, Mottaghian J, Gebert A, Rau GA, von Der HH, Poets CF. Commercial versus native surfactants. Surface activity, molecular
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Biophysical inhibition of pulmonary surfactant function by polymeric nanoparticles: Role of surfactant protein B and C Moritz Beck-Broichsitter ⇑, Clemens Ruppert, Thomas Schmehl, Andreas Günther, Werner Seeger Medical Clinic II, Department of Internal Medicine, Justus-Liebig-Universität, Klinikstrasse 33, D-35392 Giessen, Germany
a r t i c l e
i n f o
Article history: Received 28 March 2014 Received in revised form 13 June 2014 Accepted 22 July 2014 Available online 1 August 2014 Keywords: Biophysical inhibition Polymeric nanoparticles Pulmonary drug delivery Pulmonary surfactant Hydrophobic surfactant proteins
a b s t r a c t The current study investigated the mechanisms involved in the process of biophysical inhibition of pulmonary surfactant by polymeric nanoparticles (NP). The minimal surface tension of diverse synthetic surfactants was monitored in the presence of bare and surface-decorated (i.e. poloxamer 407) sub100 nm poly(lactide) NP. Moreover, the influence of NP on surfactant composition (i.e. surfactant protein (SP) content) was studied. Dose-elevations of SP advanced the biophysical activity of the tested surfactant preparation. Surfactant-associated protein C supplemented phospholipid mixtures (PLM-C) were shown to be more susceptible to biophysical inactivation by bare NP than phospholipid mixture supplemented with surfactant protein B (PLM-B) and PLM-B/C. Surfactant function was hindered owing to a drastic depletion of the SP content upon contact with bare NP. By contrast, surface-modified NP were capable of circumventing unwanted surfactant inhibition. Surfactant constitution influences the extent of biophysical inhibition by polymeric NP. Steric shielding of the NP surface minimizes unwanted NP–surfactant interactions, which represents an option for the development of surfactant-compatible nanomedicines. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nanotechnology offers a versatility of novel materials, which have found a broad scope of potential applications in medicine [1,2]. As an example, nano-scale materials offered interesting properties for pulmonary drug delivery purposes [3], among them, specified polymeric nanoparticle (NP) formulations, which contributed to the development of more advanced treatment modalities for respiratory diseases [4–8]. However, tolerability assessment of pulmonary applied polymeric NP formulations is currently a subject of intense research [9–12], but scant information is available on the toxicological aspects arising from their interplay with the pulmonary surfactant system [13–15]. Pulmonary surfactant, composed mainly of phospholipids (PL) and surfactant-associated proteins (SP), is a significant element of the alveolar region of the lung, regulating the surface tension of the terminal air spaces and, thus, promoting the gaseous exchange [16,17]. Naturally, factors showing bioadverse impact on the characteristics of pulmonary surfactant might have a severe outcome [18]. Hence, clarification of the extent and mechanisms involved in pulmonary surfactant inhibition by
polymeric NP would facilitate the development of safe nanomedicines causing insignificant interactions with the pulmonary surfactant interface [19]. In this regard, the current study aimed to provide systematic insights into relevant biophysical interactions between polymeric NP and pulmonary surfactant. Therefore, the surface properties of diverse SP-supplemented PL mixtures (PLM) were examined by monitoring the dynamic minimum surface tension (cmin) behavior in the presence of defined sub-100 nm poly(lactide) (PLA) NP formulations. Next, the results from biophysical inhibition experiments were correlated with the impact of polymeric NP on surfactant composition (i.e. depletion of the hydrophobic SP content). Finally, the surface characteristics of the colloidal formulation employed were modified by coating with poloxamer 407 to minimize undesirable interactions with basic surfactant constituents and, thus, biophysical inhibition of the pulmonary surfactant system.
2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel.: +49 641 985 42453; fax: +49 641 985 42359. E-mail address: [email protected] (M. BeckBroichsitter). http://dx.doi.org/10.1016/j.actbio.2014.07.026 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt)
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(POPG), palmitic acid (PA), superparamagnetic iron oxide NP (SPION, size 10 nm), dextran (relative molar mass 100 kDa) and poloxamer 407 were acquired from Sigma-Aldrich (Steinheim, Germany). NaCl and CaCl2 were procured from Carl Roth (Karlsruhe, Germany). VenticuteÒ was obtained from Nycomed (Konstanz, Germany). PLA (ResomerÒ R203H, inherent viscosity 0.25–0.35 dl g1) was purchased from Boehringer Ingelheim (Ingelheim, Germany). Sodium dodecyl sulfate (SDS) was from SERVA (Heidelberg, Germany). Filtrated, double-distilled water was from B. Braun (Melsungen, Germany). All other chemicals and solvents were of analytical grade.
2.2. Methods 2.2.1. Preparation and characterization of NP formulations PLA-NP and SPION-loaded PLA-NP (PLAM-NP) were prepared using a solvent evaporation technique [20]. Briefly, PLA with or without added SPION (10 wt.%) was dissolved in methylene chloride (total solid content 25 mg ml1). Subsequently, the organic phase (2 ml) was transferred to filtrated, double-distilled water (10 ml) containing SDS (0.2 wt.%). After initial pre-mixing of the two phases using a homogenizer, the emulsion was tip sonicated. The organic solvent was removed by rotary evaporation. The resulting colloidal dispersion was transferred to a dialysis bag (molecular weight cut-off of 10 kDa, Spectra/PorÒ 6, Breda, Netherlands) and extensively dialyzed against filtrated, double-distilled water. Following purification, polymeric NP formulations were concentrated by dialysis [21]. Briefly, the nanosuspension was dialyzed (10 kDa) against a counter-dialysis medium composed of filtrated, double-distilled water containing dextran (300 mg ml1), whose volume was at least two times larger than the sample volume. Finally, NP formulations were filtered (5.0 lm; GE Water & Process Technologies, Ratingen, Germany). The actual NP mass concentration and density in suspension was assessed gravimetrically after lyophilization (ALPHA 1–4 LSC, Christ, Osterode, Germany) [22] and using an oscillating density meter (DMA 4100M, Anton Paar, Graz, Austria) [14]. The size and size distribution (i.e. polydispersity index) of NP were measured by dynamic light scattering, and their f-potential was determined by laser Doppler anemometry (Zetasizer NanoZS/ZEN3600, Malvern Instruments, Herrenberg, Germany).
2.2.2. Coating of NP formulations with poloxamer 407 The nanosuspension was incubated with poloxamer 407 (final concentration 0.1 wt.%) for 12 h at 25 °C. After incubation, NP were purified from residual surfactant by repeated centrifugation (Sorvall, DuPont, Bad Homburg, Germany) and redispersion cycles at 4 °C and then filtered (5.0 lm). The desired final NP concentration was adjusted prior to use.
2.2.3. Determination of the adsorbed poloxamer 407 layer thickness on NP The thickness of the adsorbed poloxamer 407 layer (d) on the NP surface was determined using dynamic light scattering [23– 25]. Here, d is derived from the comparison of the size of bare (size0) and coated (sizeads) NP (d = (sizeads size0)/2).
2.2.4. Isolation of surfactant-associated protein B Surfactant-associated protein B (SP-B) was isolated from AlveofactÒ (Lyomark, Oberhaching, Germany) by means of LH60chromatography, as described previously [26–28]. The purity amounted to 95%, as assessed by SDS–polyacrylamide gel electrophoresis [28,29].
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2.2.5. Preparation of surfactant materials VenticuteÒ was supplied as a powder, which contained DPPC (63.4 wt.%), POPG (27.8 wt.%), PA (4.5 wt.%), CaCl2 (2.5 wt.%) and recombinant surfactant-associated protein C (rSP-C, 1.8 wt.%) (Table 1) [30]. rSP-C exhibits the same structure (amino acid sequence) as natural SP-C except for the missing palmitoylation. Moreover, its biophysical activity was reported to be comparable with that of the native protein [31]. Synthetic pulmonary surfactant stock preparations containing 1.8 wt.% of SP-B and SP-B/C (weight ratio SP-B/rSP-C of 1/3) [32] were prepared by supplementing either a PLM (possessing the same composition as VenticuteÒ) or VenticuteÒ with bovine SP-B. The PLM was prepared by dissolving DPPC, POPG, PA and CaCl2 in a mixture of chloroform/ methanol (2/1 (v/v)). Supplementation of the PLM with SP-B (PLMB) was then achieved by adding the hydrophobic SP dissolved in chloroform/methanol [28,31]. Similarly, PLM-B/C was produced by dissolving VenticuteÒ powder in chloroform/methanol followed by addition of organic SP-B solution. The SP content of the synthetic pulmonary surfactant stock preparations was decreased by dilution with organic PLM stock solution. Samples were then dried under nitrogen gas. All surfactant preparations were resuspended in NaCl solution supplemented with Ca2+ by vortexing and brief sonication (SONOREX DIGITEC, BANDELIN, Berlin, Germany) to a final PL concentration of 50 mg ml1. 2.2.6. Incubation of polymeric NP with pulmonary surfactants Synthetic pulmonary surfactant stock preparations and polymeric NP stock suspensions were combined to meet the desired final PL (i.e. 2 mg ml1) and polymeric NP concentration in isotonic NaCl solution containing 2 mM Ca2+. Here, the amounts of added polymeric NP are specified on the basis of total surface area rather than mass (information on added NP mass can be found in the Supplementary data (Figs. S1–S4)). The total surface area introduced was adjusted between 9 cm2 ml1 (i.e. 0.01 mg of PLA-NP per ml) and 4355 cm2 ml1 (i.e. 5.0 mg of PLA-NP per ml). Samples were mixed by vortexing and brief sonication, followed by a 60 min incubation period at 37 °C. 2.2.7. Biophysical studies The surface activity of samples was assessed using the oscillating bubble technique (Electronetics Corp., Amherst, USA) [14,15,31,33,34]. All measurements were performed at a constant PL concentration (2 mg ml1) in isotonic NaCl solution containing 2 mM Ca2+ at 37 °C after the incubation period. Briefly, samples of 35 ll were transferred to the disposable sample chamber, and bubble pulsation was started by sinusoidally oscillating the bubble radius (r) between 0.4 and 0.55 mm. The cycling rate was set to 20 cycles min1. The pressure difference (Dp) across the air/liquid interface was recorded continuously. The surface tension (c) was then calculated using the Young–Laplace equation (c = (Dpr)/2). cmin values were read after 300 s. Dose–effect curve characteristics (i.e. half maximal biophysical inhibitory polymeric NP concentration (IC50 value)) were calculated using a sigmoidal dose–response function (Origin 7.0, OriginLab, Northampton, USA). Physiologically surfactant concentrations are estimated to range between 10 and 20 mg ml1 [18]. Biophysical investigations using the oscillating bubble technique are commonly performed with surfactant concentrations ranging between 1 and 4 mg ml1 [14,15,31,32,34]. These surfactant concentrations allow, on the one hand, the same (low) surface tension values to be reached as observed in vivo (i.e. 10 mg ml1), owing to the increased turbidity of the
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Table 1 Biochemical composition and biophysical characteristics of the synthetic pulmonary surfactant stock preparations. Preparation PLM-B Ò
PLM-C (i.e. Venticute ) PLM-B/C a b
PL (wt.%)
SP (wt.%)
DPPC (63.4) POPG (27.8) DPPC (63.4)a POPG (27.8)a DPPC (63.4) POPG (27.8)
SP-B (1.8) rSP-C (1.8)
a
SP-B (0.45) rSP-C (1.35)
Miscellaneous (wt.%)
cminb (mN/m)
PA (4.5) CaCl2 (2.5) PA (4.5)a CaCl2 (2.5)a PA (4.5) CaCl2 (2.5)
3.3 ± 2.0 2.5 ± 1.7 2.9 ± 1.6
Values were obtained from the literature [30]. Values are presented as the mean ± standard deviation (n = 5).
preparation, which impedes the exact adjustment of bubble size, thereby negating some of the advantages of working at ‘‘dilute’’ surfactant concentrations.
2.2.8. Determination of SP content after challenge of pulmonary surfactant with polymeric NP Depletion of the SP content in the surfactant preparation caused by polymeric NP addition was investigated with pulmonary surfactants containing 1 wt.% of SP-B, rSP-C and SP-B/C, respectively. Stock preparations of synthetic pulmonary surfactants and polymeric NP (i.e. PLAM-NP and PLAM-NP-407) were combined to meet the desired final PL (i.e. 2 mg ml1) and polymeric NP concentration in isotonic NaCl solution containing 2 mM Ca2+. Samples were mixed by vortexing and brief sonication. Following incubation (60 min at 37 °C), NP formulations were magnetically separated (SPHEROÒ FlexiMag Separator, Jr. device, Kisker Biotech, Steinfurt, Germany) from the surfactant samples. The supernatant was carefully discarded, and the SP concentration was determined using the method described by Bradford [35]. Pure surfactant preparations were used for calibration.
2.2.9. Statistical analysis All measurements were carried out in triplicate, and values are presented as the mean ± standard deviation unless otherwise noted. To identify statistically significant differences, one-way ANOVA with Bonferroni’s post t-test analysis was performed (SigmaStat 3.5, STATCON, Witzenhausen, Germany). Probability values of p < 0.05 were considered significant.
3. Results 3.1. Characterization of pulmonary surfactants The biochemical composition and biophysical characteristics of the synthetic pulmonary surfactant preparations employed are outlined in Table 1. Among them, PLM supplemented with 1.8 wt.% of SP-B, rSP-C (i.e. VenticuteÒ ) and SP-B/C (weight ratio SP-B/rSP-C 1/3), respectively, can be found. The PLM consisted of a phosphatidylcholine (i.e. DPPC), a phosphatidylglycerol (i.e. POPG), a fatty acid (PA) and CaCl2. The biophysical properties of the synthetic surfactant preparations revealed cmin values of 3.0 mN m1 (Table 1). Lowering of the SP content in the PLM caused a dose-dependent increase in cmin values (Fig. 1). Here, all SP-based PLM displayed superimposable dose–effect curve characteristics, and cmin values of > 5.0 mN m1 were reached at SP concentrations 64 mN m1) during bubble oscillation (2 mg ml1) (Table 2). 3.3. Biophysical inhibition of pulmonary surfactants by polymeric NP To clarify the extent and mechanisms involved in pulmonary surfactant inhibition by polymeric NP, the current study investigated the sensitivity of synthetic PLM recombined with different amounts (i.e. 0.2–1.5 wt.%) and types (i.e. B, C and B/C) of SP. Surface activity measurements disclosed a dose-dependent loss of dynamic surface tension-lowering properties of the tested preparations upon polymeric NP addition, where the sensitivity of
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M. Beck-Broichsitter et al. / Acta Biomaterialia 10 (2014) 4678–4684 Table 2 Physicochemical characteristics of the NP formulations. Formulation
Size (nm)
PDI
f-Potential (mV)
da (nm)
cmin (mN/m)
PLA-NP PLA-NP-407 PLAM-NP PLAM-NP-407
65.3 ± 3.2 75.9 ± 2.6* 70.5 ± 4.9 83.1 ± 4.1&§
0.073 ± 0.012 0.053 ± 0.008* 0.068 ± 0.016 0.061 ± 0.012
44.3 ± 2.5 23.6 ± 0.8* 44.4 ± 3.1 24.5 ± 2.1&
n.d. 5.3 ± 0.8 n.d. 6.3 ± 0.8
70.7 ± 0.9 64.3 ± 1.6* n.d. n.d.
n.d. = not determined. Values are presented as the mean ± standard deviation (n P 4). Statistically significant differences (p < 0.05): a Adsorbed poloxamer 407 layer thickness. * PLA-NP-407 vs. PLA-NP. & PLAM-NP-407 vs. PLAM-NP. § PLAM-NP-407 vs. PLA-NP-407.
individual preparations was affected by the content and type of employed SP (Figs. 2–4A). Elevation of the rSP-C content improved the biophysical activity of the PLM-C (Figs. 2A and 3). The extent of biophysical inactivation (IC50 values), which was analyzed from the obtained dose– effect curves of individual inhibition experiments, was then plotted against the amount of rSP-C present in the PLM (Fig. 2B). The resulting relationship revealed linear correlation (R2 = 0.962).
Fig. 3. Dose-related effects of PLA-NP on cmin values and PLAM-NP on residual amounts of rSP-C present in the PLM-C. PLM-C containing 1.0 wt.% of rSP-C was used throughout the experiments. Values are presented as the mean ± standard deviation (n P 5).
Fig. 2. Dose-related effect of PLA-NP on cmin values of the PLM supplemented with 0.2 wt.% (light gray circles) and 1.5 wt.% (closed circles) of rSP-C (A). The continuous lines represent sigmoidal dose–response fits of the experimental data. The correlation of rSP-C concentration in the PLM with the PLA-NP-induced biophysical inhibition (i.e. IC50 values calculated from the sigmoidal fits) is illustrated in (B). The straight line depicts a linear fit of the experimental data (R2 = 0.962). The closed diamond in (B) represents the IC50 value for negatively charged poly(styrene) NP (diameter 100 nm) incubated with VenticuteÒ [15]. Values are presented as the mean ± standard deviation (n P 5).
In addition, the biophysical sensitivity of the PLM supplemented with 1 wt.% of SP-B and SP-B/C, respectively, was studied in the presence of polymeric NP (Fig. 4A). IC50 results revealed enhanced biophysical activity for both synthetic surfactant preparations compared with PLM-C (Figs. 2B and 3), but no statistically significant differences were observed between the sample values. To account for the biophysical inactivation potency observed for polymeric NP, synthetic surfactant preparations containing 1.0 wt.% of SP-B, rSP-C and SP-B/C, respectively, were incubated with bare PLAM-NP. Here, an increase in the amount of added polymeric NP led to a dose-dependent depletion of the SP content in the surfactant preparation (Figs. 3 and 4B). Overall, the surface property inactivation and depletion of SP content of synthetic surfactant preparations caused by polymeric NP addition (Figs. 2A, 3 and 4) resembled the surface activity results observed with SP-diluted PLM (Fig. 1). In an attempt to circumvent unwanted surfactant inhibition by polymeric NP, their surface was modified by PEGylation (i.e. poloxamer 407). Surface-decorated polymeric NP formulations were then evaluated for their adverse effect on the biophysical properties and composition of the surfactant preparation employed (Fig. 5). The surface activity of the PLM supplemented with 1.0 wt.% of rSP-C was unaffected by polymeric NP coated with poloxamer 407 over the investigated range of concentrations. Furthermore, substantial higher SP concentrations were detectable after PLM-C incubation with PLAM-NP-407 compared with their bare counterparts (i.e. PLAM-NP) (Fig. 3).
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Fig. 4. (A) Calculated dose–effect curve characteristics (i.e. IC50 value) for the influence of PLA-NP on cmin values of the PLM supplemented with 1.0 wt.% of SP-B and SP-B/C (weight ratio SP-B/rSP-C of 1/3), respectively. IC50 values were calculated from individual inhibition experiments using sigmoidal dose–response fits. (B) The dose-related impact of PLAM-NP on the SP (SP-B (squares) and SP-B/C (weight ratio SP-B/rSP-C of 1/3) (triangles)) content present in the PLM after incubation is illustrated. Values are presented as the mean ± standard deviation (n P 5).
Fig. 5. Dose-related effects of PLA-NP-407 on cmin values and PLAM-NP-407 on residual amounts of rSP-C present in the PLM-C. PLM-C containing 1.0 wt.% of rSP-C was used throughout the experiments. Values are presented as the mean ± standard deviation (n P 5). Statistically significant differences (p < 0.05): ⁄poloxamer 407coated vs. bare polymeric NP (Fig. 3).
4. Discussion Nanomedicine is generally regarded as one of the most promising future technology platforms offering revolutionary novel
material properties of interest for the treatment of numerous severe disorders [1,2]. As an example, tailored polymeric NP revealed considerable potential as controlled drug delivery vehicles to optimize the retention of the encapsulated drug within the respiratory tract and, thus, to advance the therapeutic benefit of the applied medication [3]. Besides the inflammatory potential observed for lung-delivered polymeric NP [10,11], it has become increasingly evident that colloidal drug carriers might have an adverse impact on the biophysical function of the pulmonary surfactant system [14,15]. However, adequate biophysical activity of the surface lining material is essential for maintaining a low surface tension in the deep lung, which prevents collapse of the alveoli, minimizes the work of breathing and, thus, facilitates the gaseous exchange [16–18]. Since the discovery of the hydrophobic SP (i.e. SP-B and SP-C), their crucial role in regulating the surface tension of pulmonary surfactant has been the focus of numerous studies [16,36]. Correspondingly, the presence of SP-B, rSP-C and SP-B/C (weight ratio SP-B/ rSP-C of 1/3) [32] improved the poor surface activity of the simple PLM (Table 1, Fig. 1). Maximum dynamic surface-tension-lowering properties (i.e. cmin values < 5 mN m1) were achieved with SP concentrations exceeding 0.1–0.2 wt.%, which is in general agreement with results from previous studies [31,37]. At these SP concentrations, the cmin values measured in the current study were similar to those observed in vivo [18]. Recently, it was demonstrated that pulmonary surfactant function can be dramatically influenced by inorganic [38], composite [39] and polymeric [14,15] NP formulations. However, scant information is available on the extent and mechanisms of NP-induced surfactant inhibition [13,18]. To close this gap, the present study evaluated the influence of standardized polymeric nanomedicines on the biophysical properties of the pulmonary surfactant system. Model formulations exhibited physicochemical characteristics similar to frequently lung-delivered colloidal drug carriers (Table 2) [4–8,10,11]. In the presence of polymeric NP, a distinct dosedependent biophysical inhibition of SP-supplemented synthetic PLM was noted (Figs. 2–4A). Thus, the total surface area of the applied formulation accounts for the actual surfactant inhibition. Marked differences in polymeric NP-induced surfactant inactivation also became evident upon dose-elevations of rSP-C in the preparation investigated (Figs. 2A and 3). Accordingly, higher susceptibility to biophysical inhibition was noticed for a lower SP content (Fig. 2B). Furthermore, the effect of SP type disclosed a lower susceptibility of SP-B-based and SP-B/C-based compared with rSP-C-based surfactants (Figs. 3 and 4A) [15]. Surfactant dysfunction provoked by inhaled nano-particulate matter is believed to occur through a direct interaction with individual surfactant constituents (e.g. SP) [13–15,38,40]. Hence, the ability of the negatively charged polymeric NP (Table 2) to adsorb hydrophobic, positively charged SP-B and SP-C is one putative mechanism for the observed alteration of surfactant function [40]. As a matter of fact, SP-B and SP-C should reveal different affinity to the NP surface, owing to their difference in molecular weight and charge density [41–43]. These hypotheses were confirmed by the observations made during incubation experiments, which manifested a depletion of SP content upon dose escalations of polymeric NP (Figs. 3 and 4B). Moreover, the results obtained from biophysical inhibition (Figs. 2–4A) and residual SP content (Figs. 3 and 4B) experiments were comparable with surface activity measurements performed with SP-supplemented PLM (Fig. 1), where the SP content was shown to be the major determinant for the observed surface tension-lowering potential [31,37]. Additionally, the present study covered the impact of specified NP characteristics on the bioadverse effect of the pulmonary surfactant system. In this respect, bare polymeric NP were surfacemodified with poloxamer 407, forming a dense brush-like
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poly(ethylene glycol) corona around the NP (Table 2) [23,25,44]. In contrast to bare polymeric NP (Fig. 3), incubation with this formulation revealed negligible effects on the biophysical properties of pulmonary surfactant (Fig. 5). Interestingly, similar results were recently reported in a molecular dynamics simulation study, where coated NP formulations (particle size < 10 nm) were able to penetrate pulmonary surfactant films without perturbation [45]. PEGylation of polymeric NP formulations is generally known to be efficient in preventing unwanted protein adsorption on the colloidal surface [46,47]. Nevertheless, even for poloxamer 407-coated polymeric NP a moderate depletion of SP content was observed (Fig. 5). Overall, the attributes to shield the polymeric NP surface from unwanted protein adsorption and, hence, to minimize their biophysical interactions with the pulmonary surfactant system are favorable for application of nanoparticulate drug carriers to the deep lung. Although the potential of surface-coated colloidal formulations to avoid interactions with individual pulmonary surfactant constituents (i.e. SP) are quite preliminary and speculative at present, these observations should be taken into consideration for the development of safe nanomedicines causing negligible adverse effects on the biological environment present at the target site, which deserves further experimental investigation. 5. Conclusions The present study disclosed experimental findings that account for the extent and mechanisms observed during polymeric NP-induced pulmonary surfactant inhibition. Increasing the SP concentration as well as application of SP-B rather than rSP-C improved the biophysical activity of the synthetic surfactant preparation. Depletion of the surfactant-associated SP content was shown to be regulated by the applied polymeric NP formulation. Hence, shielding of the polymeric NP surface represents a useful strategy for the rational design of colloidal drug delivery vehicles compatible with the essential pulmonary surfactant system present in the respiratory region of the lung. Conflict of interest The authors disclose that no conflicting interests associated with the manuscript exist. Acknowledgements The ‘‘Universitätsklinikum Giessen und Marburg’’ (1/2012 GI) and ‘‘Wirtschafts- und Infrastrukturbank Hessen’’ (Nanosurfact) are gratefully acknowledged for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.0 7.026. References [1] Farokhzad OC, Langer R. Impact of nanotechnology on drug delivery. ACS Nano 2009;3:16–20. [2] Shi J, Votruba AR, Farokhzad OC, Langer R. Nanotechnology in drug delivery and tissue engineering: from discovery to application. Nano Lett 2010;10:3223–30. [3] Beck-Broichsitter M, Merkel OM, Kissel T. Controlled pulmonary drug and gene delivery using polymeric nano-carriers. J Control Release 2012;161:214–24. [4] Beck-Broichsitter M, Gauss J, Packhäuser CB, Lahnstein K, Schmehl T, Seeger W, et al. Pulmonary drug delivery with aerosolizable nanoparticles in an ex vivo lung model. Int J Pharm 2009;367:169–78.
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