Modulatory Effect of Human Plasma on the Internal Nanostructure and

Article pubs.acs.org/Langmuir

Modulatory Effect of Human Plasma on the Internal Nanostructure and Size Characteristics of Liquid-Crystalline Nanocarriers Intan Diana Mat Azmi,† Linping Wu,‡ Peter Popp Wibroe,‡ Christa Nilsson,† Jesper Østergaard,† Stefan Stürup,† Bente Gammelgaard,† Arto Urtti,§,∥ Seyed Moein Moghimi,‡ and Anan Yaghmur*,† †

Department of Pharmacy, Faculty of Health and Medical Sciences and ‡Nanomedicine Laboratory, Centre for Pharmaceutical Nanotechnology and Nanotoxicology, University of Copenhagen, DK-2100 Copenhagen, Denmark § Centre for Drug Research, University of Helsinki, FIN-00014 Helsinki, Finland ∥ School of Pharmacy, University of Eastern Finland, FIN-70211 Kuopio, Finland ABSTRACT: The inverted-type liquid-crystalline dispersions comprising cubosomes and hexosomes hold much potential for drug solubilization and site-specific targeting on intravenous administration. Limited information, however, is available on the influence of plasma components on nanostructural and morphological features of cubosome and hexosome dispersions, which may modulate their stability in the blood and their overall biological performance. Through an integrated approach involving SAXS, cryo-TEM, and nanoparticle tracking analysis (NTA) we have studied the time-dependent effect of human plasma (and the plasma complement system) on the integrity of the internal nanostructure, morphology, and fluctuation in size distribution of phytantriol (PHYT)-based nonlamellar crystalline dispersions. The results indicate that in the presence of plasma the internal nanostructure undergoes a transition from the biphasic phase (a bicontinuous cubic phase with symmetry Pn3m coexisting with an inverted-type hexagonal (H2) phase) to a neat hexagonal (H2) phase, which decreases the median particle size. These observations were independent of a direct effect by serum albumin and dispersion-mediated complement activation. The implication of these observations in relation to soft nanocarrier design for intravenous drug delivery is discussed. Indeed, complement activation and fixation remain central points for the efficient clearance of particulate systems by phagocytic cells. The third complement protein (C3) is central to opsonisation, where its first cleavage product, C3b, acts as an opsonin and becomes covalently bound to the activating nanoparticle surface, thereby aiding nanoparticle binding to phagocytes via complement receptor 1.21 C3b is further degraded to iC3b, C3c, and C3dg, products that serve as ligands for other complement receptors on leukocytes.21 Furthermore, complement activation may significantly affect the integrity of certain lipidic nanoparticles/nanocarriers through insertion of the membrane attack complex, resulting in substantial leakage of the entrapped cargo.21 In contrast to liposomes and oil-in-water (O/W) emulsions, only limited studies have addressed the influence of plasma proteins on the structural integrity of nonlamellar liquidcrystalline dispersions.22,23 For instance, earlier studies suggested that albumin, the most abundant plasma protein, can destabilize monoolein (MO)-based cubosomal dispersions.24,25 In line with these studies and the modulatory role of plasma proteins in controlling the biological performance of nanoparticles, we have now studied the effect of human plasma (and the complement

1. INTRODUCTION The inverted-type liquid-crystalline dispersions comprising cubosomes (aqueous dispersions of lipidic particles with an internal bicontinuous cubic (Q2) phase) and hexosomes (aqueous dispersions of lipidic particles with an internal H2 phase)1−3 are among the emerging platforms for drug solubilization and targeted delivery.4−10 Indeed, owing to their versatility (including biodegradability) and their key properties such as high interfacial area, low viscosity, and the capability to solubilize amphiphilic, hydrophobic, and hydrophilic drugs in their highly ordered self-assembled interiors, there is growing interest in exploiting these structures for the development of injectable nanocarriers.11−14 Furthermore, these assemblies have great potential for biosensing as well as engineering tunable diagnostic/contrast agents for imaging modalities such as singlephoton emission computed tomography/computed tomography and magnetic resonance.15−17 On exposure to blood, nanoparticles and vesicular systems, depending on their curvature, shape, and surface characteristics, attract a wide range of plasma proteins.18−20 Plasma protein deposition is believed to be a dynamic event and may control the biological performance of nanoparticles, including their stability in the blood, in pharmacokinetics, and in tissue distribution.18 For instance, nanoparticles may trigger the complement system, which is the first line of the innate immune system defense, involving more than 30 soluble and membrane-bound proteins.21 © 2015 American Chemical Society

Received: March 5, 2015 Revised: April 7, 2015 Published: April 17, 2015 5042

DOI: 10.1021/acs.langmuir.5b00830 Langmuir 2015, 31, 5042−5049

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Figure 1. SAXS patterns of the PHYT dispersion before and after addition of human plasma. SAXS measurements were performed at a dispersion/ plasma volume of 1:1 and at indicated incubation times. The reflections of the identified internal H2 phase are denoted by the symbol *. The scattering intensity was shifted vertically for better visibility. The right panel shows a schematic illustration of the relevant structural transitions. interrupted by 3 s breaks) at 80% of its maximum power until a stable milky solution was obtained. The samples were directly used as formulated or/and incubated in human plasma. 2.2.2. Incubation with Human Plasma. The incubation of dispersions with human plasma was carried out as previously described.22 Briefly, a series of PHYT-based dispersions were prepared and mixed with human plasma in volume ratios (v/v) of either 1:1 or 1:9. The obtained mixtures were kept in 2.0 mL protein LoBind Eppendorf tubes (Hamburg, Germany) at 37 °C and incubated for up to 17 h in a thermoshaker (MS100, Hangzhou Allsheng Instrument Co. LTD, China). 2.3. Characterization of the PHYT-Based Nanostructured Aqueous Dispersions. 2.3.1. Synchrotron Small-Angle X-ray Scattering (SAXS) Measurements. The structural characterization of PHYT-based dispersions was performed at beamline I911-SAXS (MAX II storage ring, MAX-lab synchrotron facility, Lund University, Lund, Sweden) at an operating electron energy of 1.5 GeV and a wavelength of 0.91 Å. The measurements were made under vacuum to minimize the background scattering during data collection. The detector covered the q range (q = 4π sin θ/λ, where λ is the wavelength and 2θ is the scattering angle) of interest from about 0.12 to 8.50 nm−1. Silver behenate (CH3(CH2)20-COOAg with a d spacing value of 58.4 Å) was used as a standard to calibrate the angular scale of the measured intensity. The scattering patterns were recorded with a 2D image plate detector (Pilatus 1M, Dectris Ltd., Baden, Switzerland) using collection times of 120−180 s. Static and time-resolved studies were performed on the aqueous dispersions before and after incubation in human plasma. For the incubated aqueous dispersions, the characterization study was followed for up to 17 h at different time intervals. The samples were measured in custom-made glass capillaries at 37 °C (±0.1 °C) with the aid of a Peltier element. The lattice parameter of the internal liquidcrystalline nanostructures (the inverted-type cubic (Q2) and hexagonal (H2) phases) of the submicrometer-sized particles was deduced from the reflections with strong intensity by applying standard procedures.3,5 2.3.2. Cryo-Transmission Electron Microscopy (Cryo-TEM). The morphology of the PHYT-based submicrometer-sized particles was investigated in a frozen-hydrated state. Dispersions with a lower total lipid concentration of approximately 3 wt % were characterized. The vitrified samples were prepared as follows: 3−4 μL of an aqueous dispersion or its mixture with human plasma (1:1, v/v) was applied to a lacey carbon 300 mesh copper grid (Ted Pella Inc., California, USA). The grids were first hydrophilized by glow discharge (Leica Inc. EM ACE 200, Germany) to enhance the spreading of the samples. They were then vitrified by being rapidly plunged into liquid-nitrogen-cooled ethane (−180 °C) after quick blotting with filter paper (Fei, Vitrobot, Holland). A Gatan 626 cryoholder (GatanUK, Abingdon, U.K.) was used to observe the samples in a Tecnai G2 20 transmission electron

system) on the integrity of the internal nanostructures and the overall morphology of phytantriol (PHYT)-based nonlamellar crystalline dispersions through a pan-integrated approach involving SAXS, cryo-TEM, and nanoparticle tracking analysis (NTA). PHYT dispersions are less sensitive to biodegradation and lipase attack in plasma compared to MO dispersions,26 which makes them a suitable candidate for such studies. Here, we report a modulatory role of plasma proteins on internal nanostructural transition from a biphasic structure to a neat inverted-type hexagonal (H2) phase, which is independent of serum albumin binding and activation of the complement system. Understanding the role of blood proteins in modulating the physicochemical characteristics of nonlamellar liquid-crystalline dispersions is a prerequisite for the successful development of hexosome- and cubosome-based drug-delivery systems for the intravenous route of administration.

2. MATERIALS AND METHODS 2.1. Materials. Phytantriol (3,7,11,15-tetramethylhexadecane-1,2,3triol) with a nominal purity of >96.4% (from product specifications by gas chromatography) was a gift from DSM Nutritional Products Ltd. (Basel, Switzerland). Monoolein (MO) with a purity ≥90% was obtained from Riken Vitamin Co. (Tokyo, Japan). The lattice parameter of the MO cubic phase with symmetry Pn3m at 37 °C under full hydration conditions was comparable to the previously reported value for highly pure (99%) MO,8 indicating that the impurities do not have a significant effect on the structure. Accordingly, MO was used as received. Pluronic F127 was a gift from BASF SE (Ludwigshafen, Germany). Human plasma was prepared in-house by collecting blood in blood tubes containing lepirudin as an anticoagulant (Roche, Switzerland). Endotoxin-free phosphate-buffered saline (PBS), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), and zymosan A from Saccharomyces cerevisiae were purchased from Sigma-Aldrich (Poole, U.K.). MicroVue SC5b-9 Plus EIA kits were from Quidel (San Diego, CA, USA). All other chemicals were of analytical grade and were used as received. 2.2. Sample Preparation. 2.2.1. Preparation of Nanostructured Aqueous Dispersions. Aqueous dispersions consisting of the ternary PHYT/F127/PBS mixture with a constant weight ratio of 10:1:89 were prepared using an ultrasonication method.5,27 PHYT was first melted at ∼57 °C and then weighed into a glass vial. Polymeric stabilizer F127 and the 67 mM PBS buffer (pH 7.4) were then added to the melted lipid to give 100% total weight of the dispersion. The emulsification process was followed using ultrasonic processor Sonics Vibracell VCX 130 (Sonics & Materials Inc., Newton, CT, USA) for 15 min in pulse mode (8 s pulses 5043


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Figure 2. Cryo-TEM micrographs of the PHYT dispersions before (a, c) and after (e, f) plasma incubation. Micrographs e and f represent dispersions treated in human plasma at a ratio of 1:1 (v/v) for 5 h. Also shown is the fast Fourier transform (FFT) analysis of the dispersions before (b, d) and after (g) incubation in human plasma. microscope (FEI, Eindhoven, The Netherlands) at a high tension of 200 kV at a low-dose rate (∼5 e/Å2s). The images were recorded using an Fei Eagle 4 × 4 k camera (Fei, Holland) at a nominal magnification of 69 000×, resulting in a final image sampling of 0.22 nm/pixel. 2.3.3. Characterization of Size and Relative Light-Scattering Intensity. The size distribution and concentration of PHYT dispersions were assessed by NanoSight LM20 nanoparticle tracking analysis (NTA) equipped with a sample chamber with a 405 nm blue laser and a Viton fluoroelastomer O-ring (NanoSight, Amesbury, U.K.) as described earlier.28 In some studies, the PHYT dispersion was incubated with either human plasma (1:1 v/v) or human serum albumin (35 mg/ mL) at 37 °C for specified time points. Samples were diluted 10 000-fold with PBS and immediately injected into the sample chamber. All measurements were performed at room temperature and repeated at least three times with different preparations. Diluted human plasma (10 000×) was used as a control background in studies concerning the dispersion size profile in plasma. 2.4. Complement Activation. Details of the functional assessment of plasma complement pathways were in accord with our previous studies.29,30 To measure complement activation in vitro, we determined the PHYT dispersion-induced rise in plasma complement activation product SC5b-9 using Quidel’s ELISA kit according to the manufacturer’s protocol as described earlier.29,30 The nonlytic soluble SC5b-9 (C5b-9 bound to protein S or vitronectin) is an established marker of the terminal complement pathway.29 Briefly, complement activation was started by mixing plasma with samples to achieve a plasma concentration of 80% v/v and a final lipid concentration of 0.5 wt %. Following 30 min of incubation at 37 °C, the samples were quickly cooled on ice and diluted appropriately in a diluent solution supplied with the ELISA product with the addition of 25 mM EDTA. After centrifugation, the supernatant was used for the determination and quantification of SC5b-9. Control plasma incubation contained PBS

(the same volume as the lipid dispersion) to assess background levels of SC5b-9. Zymosan (0.2 mg/mL) was used as a positive control for complement activation.31

3. RESULTS AND DISCUSSION 3.1. Structural Investigation of PHYT-Based Aqueous Dispersions in Human Plasma. The results in Figure 1 represent the synchrotron SAXS analysis of PHYT dispersions demonstrating the existence of an internal biphasic nanostructure. The pattern consists of six reflection peaks corresponding to cubosomes with an internal bicontinuous cubic phase with symmetry Pn3m (a diamond type, QD) with a lattice parameter of 63.0 Å in coexistence with traces of an inverted-type hexagonal (H2) phase. The lattice parameter of the identified H2 phase (marked with * in Figure 1) was 46.4 Å. The occurrence of the H2 phase was unexpected because previous studies reported on the formation of PHYT cubosomes with a neat internal nanostructure of the cubic Pn3m phase at ambient temperature.6,15,32 These discrepancies may be due to the presence of impurities in this commercial batch of PHYT, which could account for the formation of the internal biphasic nanostructure as recently indicated.15 The mentioned impurities may include a tiny amount of water (about 0.07%), sulfated ash, heavy metals, and a diastereomer of phytantriol (3,7,11,15-tetramethyl-1,2,3,4-tetrahydroxyhexadecane), which is a compound that differs from PHYT in that it has a hydroxy group in position 4, resulting in five chiral C atoms, as opposed to four chiral C atoms in PHYT. Furthermore, subtle levels of impurities in PHYT have been 5044


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Figure 3. Cryo-TEM micrographs of PHYT dispersions and their fast Fourier transform (FFT) analysis. PHYT dispersions were mixed with human plasma at a ratio of 1:1 (v/v) and incubated for 17 h. Representative micrographs are shown in panels a, c, and d. Panels b and e represent FFT analysis of the internal H2 structure of hexosomes.

the basis of these studies, we further characterized the internal nonlamellar liquid-crystalline nanostructures by fast Fourier transform (FFT). The results in Figure 2a,c represent cryo-TEM micrographs of the PHYT-based dispersion in the absence of human plasma, demonstrating the presence of a heterogeneous population of nanoparticles with distinct well-ordered interior nanostructure of the inverted bicontinuous cubic phase coexisting with nanoparticles enveloping an internal H2 phase (verified by the FFT images, Figure 2b,d). These observations are in agreement with the SAXS analysis. Following a 3 h incubation of PHYT dispersion in human plasma (Figure 2e,f), the FFT analysis (panel g) revealed a clear structural periodicity of the H2 phase that is again consistent with SAXS (Figure 1). However, plasma treatment was associated with an apparent decrease in particle size. This is presumably a phase-transitiondependent phenomenon. After prolonged incubation periods (17 h) in human plasma (Figure 3a,c,d), faceted particles of hexagonal shape with an internal H2 nanostructure were detected (Figure 3b,e). However, there were no apparent further changes in particle dimensions. 3.3. Role of Human Plasma in PHYT Dispersion Size and Concentration. Nanoparticle tracking analysis (NTA) was employed for further analysis of the size distribution and relative scattering intensity28 of the lipidic nanoparticles before and after incubation in human plasma, and the results are summarized in Table 1. PHYT dispersions exhibited mean and median sizes of

suggested to have a significant effect on the temperaturedependent phase behavior of the binary PHYT/water system.33 This dispersion was next incubated in human plasma in a volume ratio of 1:1, and SAXS experiments were performed at different time points. The cubic Pn3m phase started to disappear after 10 min of incubation. A prominent H2 phase started to evolve, and its three characteristic reflections (10), (11), and (20) were clearly visible after 3 h of incubation (Figure 1). The appearance of the H2 phase is most likely attributed to the interaction of a population of submicrometer-sized particles with human plasma components. Furthermore, after prolonged periods of incubation (17 h), a complete transition of the internal biphasic nanostructure to a neat H2 phase (lattice parameter of about 48.4 Å) was detectable, but plasma treatment did not destroy the integrity of the internal nanostructures. Identical structural features were obtained at higher plasma levels (dispersion/plasma volume ratio 1:9, data not shown); however, these conditions resulted in a low signal-to-noise ratio that did not permit accurate data analysis. Recently, it was demonstrated that cubosomes based on monounsaturated monoglyceride MO and Pluronic F127 were unstable (with very fast internal phase transition) in porcine and human plasma.22,34 Bode et al.22 further speculated that the internal nanostructure in the dispersed particles was either an inverse hexagonal (H2) phase or an inverse micellar cubic (I2) phase with symmetry Fd3m, which has often been observed in MO dispersions in the presence of lipophilic additives or lipases.35,36 Leesajakul et al. further indicated that plasma esterases may have been responsible for MO hydrolysis, generating glycerol and oleic acid, thereby resulting in the destabilization and disintegration of the cubosomal particles.24 In contrast to the above-mentioned studies, our results show that the replacement of MO with PHYT can overcome plasmamediated destabilization processes over prolonged periods of time. PHYT comprises of a trihydroxy headgroup and a branched phytanyl tail without the presence of a labile (e.g ester) functionality, which may confer more stability toward enzymatic degradation.26,37,38 Nevertheless, plasma treatment affected the internal nanostructure of the PHYT-based cubosomes with the transition from a biphasic structure to a neat H2 phase. 3.2. Influence of Human Plasma on the Morphological Characteristics of PHYT-Based Nanoparticles. Cryogenic transmission electron microscopy (cryo-TEM) was performed to gain further insight into the effect of human plasma on the morphology of the dispersed liquid-crystalline nanoparticles. On

Table 1. Nanoparticle Tracking Analysis (NTA)-Derived Parameters for PHYT-Based Dispersions before and after Incubation in Human Plasma and Albumin PHYT dispersion

mean size (nm)

mode (nm)

median (nm)

in buffer (10 min)a in undiluted plasma:b 10 min 3h 5h 10 h 24 h in albumin solution:c 10 min 5h

157 ± 22

142 ± 19

148 ± 21

143 ± 25 116 ± 13 111 ± 15 112 ± 6 128 ± 5

118 ± 17 105 ± 13 107 ± 5 104 ± 2 119 ± 10

134 ± 23 111 ± 12 106 ± 14 107 ± 6 123 ± 5

157 ± 8 159 ± 6

138 ± 3 134 ± 6

148 ± 8 149 ± 7

a

Similar patterns were observed after 24 h of incubation. bPHYTbased dispersions were prepared and mixed with human plasma in a volume ratio of 1:1. cThe albumin concentration was 35 mg/mL.

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Figure 4. Typical size distribution profiles (left panel) and 2D plots of relative light-scattering intensity of PHYT dispersions versus the estimate of the particle size (right panel). Panel a represents characteristics of the native PHYT dispersion (PHYT dispersed in excess buffer) whereas panels b−d show time-dependent changes in particle size and concentration attributes on plasma incubation (10 min, 3 h, and 5h, respectively). In panels b−d, dispersions were mixed with human plasma in a volume ratio of 1:1. A similar size distribution and scattering profiles were obtained with separate determinations of the same batch (n = 3) and were reproducible with different batch preparations.

157 ± 22 and 142 ± 19 nm, respectively, and these remained similar on further storage (24 h). In plasma, the particle sizes declined gradually within the first 3 h of incubation, thereafter maintained without a significant change but then increased slightly at 24 h (Table 1). Notably, a comparison of size

distribution profiles before and after plasma incubation indicated the loss of some larger particle subpopulations (≥150 nm), with a concomitant increase in the concentration of smaller species (≤140 nm) within 10 min of plasma treatment, but these changes were more profound with longer incubation periods (Figure 4, 5046


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Langmuir left panels). The scatter plots of relative intensity versus particle size (Figure 4, right panels) further show a rapid shift in the median relative intensity of the PHYT dispersion (from 11 arbitrary units to 4) within 10 min of plasma incubation, which was maintained in further incubation periods. Collectively, these changes are consistent with SAXS and cryo-TEM studies and may represent plasma-mediated phase-transition processes (transformations from dispersion with an internal biphasic nanostructure (cubic Pn3m and H2 phases) to hexosomes with an internal neat H2 phase), where the newly formed hexosomal nanoparticles (≤150 nm in diameter) exhibit lower scattering. Time-dependent plasma-induced size changes are further reflected in the scatter plots by comparing the percentage of the nanoparticle population distribution between the first and the second quadrants, based on the shifted median scattering intensity of 4 arbitrary units. In the absence of plasma, nanoparticles in the second quadrant of the scatter plot (Figure 4a, right panel) show broad scattering, where a small population of nanoparticles scatter at high intensity (20 arbitrary units and above). This may indicate the presence of different nanoparticle species within a typical size bracket in buffer. Some of these particles could have a vesicular nature (coexisting vesicles are typically observed in cubosome and hexosome dispersions)39−43 and disintegrate rapidly (10 min) in plasma. This notion also holds true for some of the larger particles (>150 nm) that accumulate in the fourth quadrant and disappear on plasma incubation (Figure 4, right panels). SAXS analysis (Figure 1) suggested that the transformation to neat hexosomes is completed within 17 h of plasma incubation. In light of this notion, the slight increase in particle sizes at 24 h of plasma incubation compared to incubation between 3 and 12 h periods (Table 1) may be a reflection of irreversible protein adsorption and buildup on the surface of hexosomes. In contrast to earlier studies with MO-based dispersions,22,23 we excluded a possible role for serum albumin in modulating the abovementioned transformations. Indeed, incubation of the PHYT dispersion in the presence of a physiological concentration of human serum albumin (35 mg/mL) generated a similar size distribution profile and scattering to that of the native dispersion consisting of nanoparticles dispersed in excess buffer and without any significant change in the mean and median particle sizes (Table 1). The approximate ellipsoid dimensions of serum albumin are 3.8 × 3.8 × 14.0 nm3. Serum albumin most likely deposits onto the surface of some subpopulations of lipid particles with the side-on mode of adsorption. However, NTA is not a suitable tool for assessing direct protein (albumin) deposition, particularly when applied to polydisperse and heterogeneous systems such as PHYT liquid-crystalline dispersions, where protein-mediated subtle increases in hydrodynamic radii of dispersed particles will be controlled by particle curvature, surface hydration and energetics, and the extent of protein spreading. Next, we studied whether PHYT dispersion-mediated complement activation in plasma may have contributed to morphological transformations. Complement activation requires a surface where the curvature and chemical composition of the surface modulate the systematic assembly of various complement proteins orchestrated by proteolytic steps.21 Such surface manipulations may destabilize the dispersed particles and consequentially affect their internal nanostructure. The results in Figure 5 show that PHYT dispersions are potent activators of the complement system (even when compared to MO-based dispersions), as demonstrated by the significant elevation of

Figure 5. Complement activation by PHYT and MO dispersions. Complement activation is reflected in dispersion-mediated increases in the plasma SC5b-9 levels. The total lipid concentrations in both samples were 0.5 wt %. Complement activation by both dispersions was significantly larger than background levels (p < 0.01). PHYT showed a significantly higher response than MO (*p < 0.01). Zymosan at a final concentration of 0.2 mg/mL generated 15.9 ± 2.9 μg SC5b-9/mL plasma.

plasma SC5b-9 levels above the background. This activation is not due to the possible presence of Pluronic F127 micelles, where the threshold for complement triggering requires a minimum Pluronic F127 level of 1.5 mg/mL.29 Indeed, the final concentration of Pluronic F127 (0.5 mg/mL) in PHYT dispersions during complement measurement studies was below this level. Complement activation is inhibited in the presence of EDTA.29 Interestingly, the size distribution and scattering intensity profiles of PHYT dispersions in plasma were similar in the absence and presence of 20 mM EDTA (data not shown). These observations, therefore, exclude the direct role of complement activation and fixation in plasma-modulated changes in the internal nanostructure and size characteristics of PHYT dispersions. Earlier studies suggested that Pluronic F127-coated surfaces can attract a wide range of apolipoproteins, including apolipoproteins A-I, A-IV, and apo-E.44 Additionally, Pluronic F127 has also been shown to interact with both high- and lowdensity lipoproteins.29 It is therefore plausible that the abovementioned plasma-induced transformations in the internal nanostructure and size profile of Pluronic F127-stabilized PHYT dispersions are brought about through a set of complex interactions with different classes of plasma lipoproteins and/or their apolipoprotein components and, presumably, are independent of calcium and magnesium. These modes of interaction may further aid the transfer of PHYT and Pluronic F127 from the liquid-crystalline dispersions to these lipoproteins, thereby accounting for the observed changes in particle characteristics. These possibilities require further investigation and could be important in identifying plasma components that can perturb inverted-type liquid-crystalline dispersions of different lipid composition and stabilizer concentration.

4. CONCLUSIONS Through an integrated approach involving synchrotron SAXS, cryo-TEM, and NTA, we have shown a modulatory effect of human plasma on biophysical characteristics of PHYT dispersions, which was independent of direct albumin interaction and dispersion-mediated complement activation processes. 5047


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(5) Yaghmur, A.; Glatter, O. Characterization and Potential Applications of Nanostructured Aqueous Dispersions. Adv. Colloid Interface Sci. 2009, 147−148, 333−342. (6) Yang, Z.; Peng, X.; Tan, Y.; Chen, M.; Zhu, X.; Feng, M.; Xu, Y.; Wu, C. Optimization of the Preparation Process for an Oral PhytantriolBased Amphotericin B Cubosomes. J. Nanomater. 2011, 2011, 1−10. (7) Mangiapia, G.; Vaccaro, M.; D’Errico, G.; Frielinghaus, H.; Radulescu, A.; Pipich, V.; Carnerup, A. M.; Paduano, L. Cubosomes for Ruthenium Complex Delivery: Formulation and Characterization. Soft Matter 2011, 7, 10577−10580. (8) Yaghmur, A.; Laggner, P.; Zhang, S.; Rappolt, M. Tuning Curvature and Stability of Monoolein Bilayers by Designer Lipid-Like Peptide Surfactants. PLoS One 2007, 2, e479. (9) Drummond, C. J.; Fong, C. Surfactant Self-Assembly Objects as Novel Drug Delivery Vehicles. Curr. Opin. Colloid Interface Sci. 1999, 4, 449−456. (10) Zhai, J.; Scoble, J. A.; Li, N.; Lovrecz, G.; Waddington, L. J.; Tran, N.; Muir, B. W.; Coia, G.; Kirby, N.; Drummond, C. J.; Mulet, X. Epidermal Growth Factor Receptor-Targeted Lipid Nanoparticles Retain Self-Assembled Nanostructures and Provide High Specificity. Nanoscale 2015, 7, 2905−2913. (11) Malmsten, M. Phase Transformations in Self-Assembly Systems for Drug Delivery Applications. J. Dispersion Sci. Technol. 2007, 28, 63− 72. (12) Malmsten, M. Soft Drug Delivery Systems. Soft Matter 2006, 2, 760−769. (13) Dong, Y. D.; Larson, I.; Barnes, T. J.; Prestidge, C. A.; Allen, S.; Chen, X.; Roberts, C. J.; Boyd, B. J. Understanding the Interfacial Properties of Nanostructured Liquid Crystalline Materials for SurfaceSpecific Delivery Applications. Langmuir 2012, 28, 13485−13495. (14) Thandanki, M.; Kumari, P. S.; Prabha, K. S. Overview of Cubosomes: A Nano Particle. Int. J. Res. Pharm. Chem. 2011, 1, 2231− 2781. (15) Nilsson, C.; Barrios-Lopez, B.; Kallinen, A.; Laurinmäki, P.; Butcher, S. J.; Raki, M.; Weisell, J.; Bergström, K.; Larsen, S. W.; Østergaard, J.; Larsen, C.; Urtti, A.; Airaksinen, A. J.; Yaghmur, A. SPECT/CT Imaging of Radiolabeled Cubosomes and Hexosomes for Potential Theranostic Applications. Biomaterials 2013, 34, 8491−8503. (16) Caltagirone, C.; Falchi, A. M.; Lampis, S.; Lippolis, V.; Meli, V.; Monduzzi, M.; Prodi, L.; Schmidt, J.; Sgarzi, M.; Talmon, Y.; Bizzarri, R.; Murgia, S. Cancer-Cell-Targeted Theranostic Cubosomes. Langmuir 2014, 30, 6228−6236. (17) Moghaddam, M. J.; de Campo, L.; Waddington, L. J.; Weerawardena, A.; Kirby, N.; Drummond, C. J. Chelating OleylEDTA Amphiphiles: Self-assembly, Colloidal particles, Complexation with Paramagnetic Metal Ions and Promise as Magnetic Resonance Imaging Contrast Agents. Soft Matter 2011, 7, 10994−11005. (18) Moghimi, S. M.; Hunter, A. C.; Andresen, T. L. Factors Controlling Nanoparticle Pharmacokinetics: An Integrated Analysis and Perspective. Annu. Rev. Pharmacol. Toxicol. 2012, 52, 481−503. (19) Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F. B.; Dawson, K. A. Physical-Chemical Aspects of Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. J. Am. Chem. Soc. 2011, 133, 2525−2534. (20) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nanotechnol. 2013, 8, 772−781. (21) Moghimi, S. M.; Andersen, A. J.; Ahmadvand, D.; Wibroe, P. P.; Andresen, T. L.; Hunter, A. C. Material Properties in Complement Activation. Adv. Drug Delivery Rev. 2011, 63, 1000−1007. (22) Bode, J. C.; Kuntsche, J.; Funari, S. S.; Bunjes, H. Interaction of Dispersed Cubic Phases with Blood Components. Int. J. Pharm. 2013, 448, 87−95. (23) Lakshmi, N. M.; Yalavarthi, P. R.; Vadlamudi, H. C.; Thanniru, J.; Yaga, G.; K, H. Cubosomes as Targeted Drug Delivery Systems - A Biopharmaceutical Approach. Curr. Drug Discovery Technol. 2014, 11, 181−188.

These studies are fundamentally important and may eventually identify the necessary attributes for the development of pharmaceutically viable cubosomes and hexosomes for drug solubilization and intravenous delivery. However, additional studies are still necessary to assess whether solubilized drug molecules can modulate the internal nanostructure of these nonlamellar liquid-crystalline dispersions. These eventualities are presumably dependent on drug type (amphiphilic, hydrophobic, and hydrophilic molecules) and concentration, a complex set of interactions (mainly lipid−drug and drug−water interactions), and the dispersion composition, which may further affect the dispersion stability in the blood and drug pharmacokinetic parameters. Accordingly, pharmaceutical developments of inverted-type liquid-crystalline dispersions must be evaluated in a case-by-case manner and in line with the desired pharmacological responses for a typical drug (e.g., sustained versus controlled versus rapid drug release). These studies are currently under investigation in this laboratory and in relation to cytotoxic drug delivery for cancer chemotherapy. Finally, we demonstrated that PHYT dispersions could trigger complement activation, a process that may limit their use for intravenous administration as this may initiate infusion-related reactions in sensitive individuals. However, complement activation was significantly milder when PHYT was replaced with MO. Accordingly, through optimization of the lipid core and the introduction of suitable stabilizers it may be possible to overcome complement activation and engineer immunologically safer dispersions.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the Danish Council for Independent Research|Technology and Production Sciences (reference 133500150b to A.Y. and S.M.M.) is gratefully acknowledged. A.Y. further acknowledges financial support from the Danish Natural Sciences Research Council (DanScatt) for SAXS experiments. I.D.M.A. is the recipient of a Ph.D. scholarship award from the Ministry of Higher Education of Malaysia (MOHE). We also thank Tomás S. Plivelic and Ana Labrador (MAX-lab, Lund, Sweden) and Ramon Liebrechts (Core Facility for Integrated Microscopy, University of Copenhagen) for their valuable technical assistance with SAXS measurements and cryo-TEM studies, respectively.

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Modulatory Effect of Human Plasma on the Internal Nanostructure and

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