Article pubs.acs.org/Langmuir

Association between Cationic Liposomes and Low Molecular Weight Hyaluronic Acid Antonio A. M. Gasperini,† Ximena E. Puentes-Martinez,†,‡ Tiago Albertini Balbino,§ Thais de Paula Rigoletto,∥ Gabriela de Sá Cavalcanti Corrêa,§ Alexandre Cassago,⊥ Rodrigo Villares Portugal,⊥ Lucimara Gaziola de La Torre,*,§,# and Leide P. Cavalcanti*,†,# †

Brazilian Synchrotron Light Laboratory, CNPEM, Caixa Postal 6192, CEP 13.083-970, Campinas, São Paulo, Brazil Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo, Rua Talim, 330, CEP 12.231-280, São José dos Campos, São Paulo, Brazil § School of Chemical Engineering, University of Campinas, UNICAMP, PO box 6066, CEP 13.083-970, Campinas, São Paulo, Brazil ∥ Centro Universitário das Faculdades Associadas de Ensino UNIFAE, Largo Engenheiro Paulo de Almeida Sandeville, 15, CEP 13.870-377, São João da Boa Vista, São Paulo, Brazil ⊥ Brazilian Nanotechnology National Laboratory, CNPEM, Caixa Postal 6192, CEP 13.083-970, Campinas, São Paulo, Brazil ‡

S Supporting Information *

ABSTRACT: This work presents a study of the association between low molecular weight hyaluronic acid (16 kDa HA) and cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2dioleoyl-3-trimethylammonium-propane (DOTAP). The cationic liposome/HA complexes were evaluated to determine their mesoscopic structure, average size, zeta potential, and morphology as a function of the amount of HA in the system. Small angle X-ray scattering results revealed that neighboring cationic liposomes either stick together after a partial coating of low concentration HA or disperse completely in excess of HA, but they never assemble as multilamellar vesicles. Cryo-transmission electron microscopy images confirm the existence of unilamellar vesicles and large aggregates of unilamellar vesicles for HA fractions up to 80% (w/w). High concentrations of HA (> 20% w/w) proved to be efficient for coating extruded liposomes, leading to particle complexes with sizes in the nanoscale range and a negative zeta potential.

1. INTRODUCTION Liposomes have been widely investigated for several decades in the cosmetic, pharmaceutical, and food fields as sophisticated delivery vehicles with target and release control.1,2 Liposomes are colloidal aggregates composed of amphiphilic lipids; in excess water, these lipids self-assemble into vesicles containing an inner aqueous core.3,4 Advancements in the fields of gene therapy and nucleic acid delivery5−7 have led scientists to develop cationic carriers for specific intracellular delivery.8 Among the myriad of cationic nanoparticles, cationic liposomes have emerged as part of a promising strategy for nucleic acid delivery.9−11 When cationic liposomes are employed, the driving force for intracellular delivery is electrostatic because cellular membranes present anionic characteristics. The cationic liposome/nucleic acid complex can be delivered to different organs or cells.12,13 In this case, the delivery of nucleic acids into cells will occur without specific targeting. To gain selectivity in cell delivery, different strategies can be employed via coupling to molecules that specifically interact with receptors in cell membranes. One © 2015 American Chemical Society

example is the electrostatic coating of poly(ethylenimine) (PEI)/pDNA complexes with folic acid (FA) to decrease the nanoparticle cytotoxicity and also to target the FA-specific receptor (FR) in the melanoma cell line (B16−F10 cells).14 The association between hyaluronic acid (HA) and cationic liposomes is another example of a cell-targeting strategy. HA is a biopolymer that naturally occurs in living organisms. Different properties confer a unique importance to this biopolymer, such as its natural mucobioadhesion and biocompatibility.15,16 It also plays an important role in cell signaling via CD44 and RHAMM (receptor for hyaluronan-mediated motility, CD168),17−19 and HA receptors (CD44) are expressed in different tumors and stem cells.20−22 The coupling of HA to the surface of liposomes can be accomplished through two routes, the chemical derivatization of molecules to incorporate HA in the phospholipids and the Received: November 24, 2014 Revised: February 23, 2015 Published: March 2, 2015 3308

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In this work, we investigate the association between cationic liposomes (EPC/DOTAP/DOPE) and HA. We used low molecular weight HA because it has not been previously investigated in association with liposomes and because of our hypothesis that we could obtain particles with sizes in the nanoscale range for the complexes with the cationic liposomes. We evaluated the influence of low molecular weight HA on the final structural and physicochemical properties, aiming to understand the main parameters that govern the association between cationic liposomes and HA for the future development of new strategies for drug and gene delivery.

coating of cationic nanoparticles with HA. For liposomes, chemical derivatization can be performed by covalently coupling HA molecules to the polar headgroup of the lipid. Liposome−HA chemical conjugates can be produced using conventional procedures for liposome production. The HA− 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (HA−DOPE) chemical conjugate was successfully used to produce cationic liposomes ([2-(2,3-didodecyloxypropyl)hydroxyethyl]ammonium bromide (DE) and HA-DOPE) to deliver the plasmid DNA pCMV-luc.23,24 Lipoplexes containing HADOPE could efficiently deliver the plasmid DNA to CD44expressing cell lines in culture. Qhattal and Liu25 produced HA−liposomes that were chemically conjugated after liposome preparation by chemically conjugating HA to 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysulfosuccinamide (NHSS). The HA−liposome cell uptake correlated with the HA molecular weight (5−8 < 10−12 < 175−350 kDa), among other parameters. The photo-crosslinking technique could also provide efficient HA chemical conjugation of drug-loaded nanoparticles, enhancing the colloidal stability and drug release for in vivo tumor targeting.26 The electrostatic interaction between cationic liposomes and HA can be used to develop surface-modified liposomes. It is noteworthy that only a few studies have focused on the electrostatic interaction between HA and cationic liposomes with the aim of forming HA-coated liposomes. Sagristá et al.27 investigated surface-modified liposomes that were coated with HA. The authors demonstrated the capability of HA-coated liposomes to hold encapsulated drugs. In 2008, Esposito et al.28 reported proof-of-concept cationic liposomes that were functionalized with HA (103−104 kDa) and loaded with a magnetic resonance imaging (MRI) contrast agent. According to the authors, the noncovalent route is straightforward and simple; however, the final complex size depends on the HA concentration. The HA−liposome complexes showed affinity for cells (the C6 line, which expresses high levels of the CD44 receptor), and simple electrostatic interactions produced an efficient and specific MRI contrast agent with cell-targeting ability and low toxicity. The formation of an electrostatic complex between cationic liposomes and HA can be considered a polymer-vesicle system, and investigating the behavior of this colloid is important for further in vivo or in vitro applications.29 Mutual interactions, such as electrostatic bridging and hydrophobic and hydrogenbond interactions, control the polymer−vesicle association, leading to segregated or associated systems.30 To extend the promising results from the complexation of cationic liposomes and HA, systematic studies regarding the association between HA and cationic liposome are necessary to fully understand the mechanism of these electrostatic interactions. These studies can also contribute to a better understanding of polymer−vesicle associations. Our research group has studied liposomes composed of the lipids egg phosphatidylcholine (EPC), DOPE, and 1,2-dioleoyl3-trimethylammonium-propane (DOTAP) to deliver the DNAhsp65 plasmid as a single-dose tuberculosis vaccine via intranasal administration,31,32 and we evaluated the system in vitro in transfected HeLa cells.33 Recently, a nuclear localization signal peptide for nuclear targeting was incorporated into the liposome structure for in vivo tuberculosis treatment.34 The possibility to specifically target EPC/DOTAP/DOPE liposomes may be an important strategy for further developments in tuberculosis vaccine and treatment.

2. MATERIALS AND METHODS 2.1. Materials. Sodium hyaluronate with a size of 16 kDa was purchased from Lifecore. The lipids egg phosphatidylcholine (EPC) (96% purity), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (99.8% purity), and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) (98% purity) were purchased from Lipoid and used without further purification. We used water that was purified using a Milli-Q-plus, and the water was deionized for a resistivity of 18.2 MΩ cm and filtered (0.22 μm). All chemicals were USP grade. 2.2. Sample Preparation. 2.2.1. Extruded Cationic Liposome (ECL). The liposomes were prepared according to Bangham’s method.35 Briefly, the required amounts of all lipid stock solutions in chloroform (EPC/DOPE/DOTAP 50/25/25% molar) were mixed and dried to a thin film using a rotary evaporator in a 650 mmHg vacuum for 1 h. The dried lipid film was hydrated to 16 mM with water at 30 °C, which is above its phase transition temperature.31,32 The liposomes were extruded through two-stacked polycarbonate membranes (100 nm nominal pore) 15 times under 12 kgf/cm2 nitrogen pressure, generating ECLs. 2.2.2. Electrostatic Complexes Containing HA and Liposome. The HA/ECL complexes were obtained by adding varying amounts of 0.3% w/v sodium hyaluronate solution to the ECL dispersion in three steps under vortex in an ice bath (4 °C). All the samples had the same lipid concentration prior to the complexation. The HA fraction in the final solution (HA(%)) was determined by considering the weight ratio between the amount of HA and the total mass of lipid and HA (w/w). In the present work, all the samples studied had a maximum HA concentration of 0.3% w/v, related to the pure HA solution, and we did not see any significant changes on viscosity among samples at different HA fractions. Even at high HA(%) conditions, the dispersions of liposomes/HA complexes were quite diluted, and we considered plausible to assume the differences in the studied conditions on the rheological behavior as being negligible. 2.3. Dynamic Light Scattering (DLS) and Zeta Potential. The average hydrodynamic diameter and size distribution were measured using the dynamic light scattering (DLS) technique and a Malvern Zetasizer Nano ZS. The mean diameter and the particle size distribution were estimated using the CONTIN36 algorithm. The diameter values reported from now on are the standard z average diameter (which is intensity-weighted) given by the Malvern software version 4.0. The zeta potential was measured in water at 25 °C using the same equipment. 2.4. Langmuir Monolayer. A ternary lipid monolayer composed of EPC/DOTAP/DOPE (50:25:25 mol %) was prepared by spreading 50 μL of 1 mM chloroform solution on the surface of a pure water subphase or on HA aqueous solutions in a Langmuir trough (Insight, Brazil, total area of 216 cm2), resulting in an initial surface pressure of zero. Approximately 10−15 min was allowed for surface pressure stabilization and solvent evaporation. The monolayers were then compressed by moving the lateral barriers at 0.42 cm2 s−1 until collapse occurred. To study the interaction of HA with EPC/DOPE/DOTAP mixed monolayers, isotherms were recorded as a function of the average lipid molecular area both on pure water and on different concentrations of HA solutions. The experiments were performed at 25.0 ± 0.5 °C, which is above the main phase transition temperature of 3309

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Langmuir the lipids, and always-fresh solution was used to avoid chemical modification. The isotherms were recorded at least in triplicate, and the maximum experimental error was 2 Å2/molecule. 2.5. Small Angle X-ray Scattering (SAXS). Structural information, such as the bilayer size and electronic density profile (EDP), was obtained using small-angle X-ray scattering (SAXS). The experiments were performed at the SAXS-1 beamline at the Brazilian Synchrotron Light Laboratory (LNLS) using an 8 keV beam energy (λ = 1.55 Å) and a sample-to-detector distance of 1590 mm. The measured scattered intensity was normalized for the beam intensity and absorption from the sample, and it is displayed as a function of the scattering vector modulus q = 4π sin(θ)/λ, where θ is the scattering angle and λ is the radiation wavelength. The typical q range was from 0.007 to 0.23 Å−1. The scattered intensity from pure water was used as a standard for background subtraction. The liposome dispersion exhibits a liquid-crystalline character; the dispersion has both a form factor, which is related to the EDP across the lipid bilayer, and a structure factor, which is related to the periodicity of the lipid bilayers. The models used in this work for the lipid bilayer form factors are described in detail elsewhere.33,37,38 In this work, we propose a modified form factor for the lipid bilayer; this form factor takes into account the bonded HA between the lipid bilayers. We also used a simplified polymer model (generalized Gaussian polymer coil) to simulate the scattering of the free HA of the system; this model fits the experimental data well. 2.6. Cryo-Transmission Electron Microscopy (Cryo-TEM). For cryo-TEM analysis, liposome concentrations ranging from 1 to 6 mM were used in combination with 0 to 80% (w/w) HA. Cryo-TEM grids were prepared using an automated vitrification system (Vitrobot Mark IV, FEI, The Netherlands). Specimens were prepared in a controlled environment with the temperature and humidity set to 22 °C and 100%, respectively, which prevented sample evaporation sample during the preparation. Before the application of the sample, the grids were subjected to a glow discharge treatment using an easiGlow discharge system (Pelco) with 15 mA current for 25 s in air atmosphere to make them hydrophilic. A 3 μL sample droplet was deposited on a 300 mesh lacey carbon-coated cooper grid (Ted Pella) and prepared with a blot time of 3 s, blot force of approximately −5, and 20 s of waiting time before blotting. Specimens were analyzed in low dose condition, with a defocus range of −2 to −4 μm, using a Jeol JEM-2100 instrument operating at 200 kV. Images were acquired using an F-416 CMOS camera (TVIPS, Germany). Sample preparation and data acquisition were performed at the Electron Microscopy Laboratory (LME)/Brazilian Nanotechnology National Laboratory (LNNano).

Figure 1. Top: average hydrodynamic diameter (z-average, intensityweighted). Bottom: zeta potential. The asterisk (*) corresponds to the average diameter and zeta potential of the liposomes (without HA). The error bars correspond to the standard deviation of three independent experiments; the straight lines are just a visual guide.

when the zeta potential curve crosses zero, as shown in the graph in Figure 1. The negatively charged HA molecule plays the role of an electrostatic glue between cationic liposomes. As the amount of HA increases up to 20%, large aggregates are formed, leading to phase separation. Above 20% HA, the size of the complexes decreases to the original size of the pure ECL. Our hypothesis is that when a large amount of HA is added to the system, individual cationic liposomes are rapidly coated, favoring the dispersion of the liposomes instead of the agglomeration. 3.2. Cryo-TEM of ECL/HA Complexes. Figure 2 shows the cryo-TEM images for pure ECL and three different concentrations of HA in ECL/HA complexes. The cryo-TEM samples were prepared within 1 day of extrusion and complexation. Pure ECLs are seen in Figure 2a with dimensions ranging from 40 to 200 nm. The vesicles of the pure ECL are mostly dissociated from each other. The images of the ECL/ HA complexes exhibit vesicles attached to each other with a well-defined interface. For a low HA concentration, such as 0.25% (w/w) shown in Figure 2b, we can observe individual vesicles and vesicles that stick together, with the latter being more frequent for 3% and 10% HA (Figure 2c and d, respectively). For concentration of 20% HA (Figure 2e), large agglomerates of liposomes were observed with most of the vesicles sticking together. For higher concentrations, the presence of juxtaposed liposomes became less frequent than observed for 20% HA, resembling the observations made for lower concentrations of HA. For concentration of 60% HA (Figure 2f and g), the presence of individual and associated vesicles were observed. In Figure 2g, we can observe the difference of juxtaposed liposomes that are glued with HA (white and hollow arrows) and liposomes with no contact (black and hollow arrows). Neighbor liposomes with no

3. RESULTS AND DISCUSSION 3.1. Physicochemical Properties of ECL/HA Complexes. The ECL/HA complexes were obtained for different HA/liposome ratios (w/w): 0.25, 0.5, 1, 3, 10, 20, 40, 60, and 80%. The average hydrodynamic diameter and zeta potential values as a function of HA fraction are presented in Figure 1. The samples were measured in triplicate in the same day of the complexation. Size and zeta potential are important liposome characteristics for monitoring the shelf life stability: smaller liposomes with higher electrical surface potential are often more stable macroscopically. The pure ECL had a size of 114 ± 4 nm and zeta potential of 55 ± 20 mV. Figure 1 shows how the size and zeta potential change as a function of HA content. The size and zeta potential remained nearly unaffected for 0 to 1% HA. The size started to increase at 3% HA, but it could not be measured for 5 and 10% HA because the mean size of the complexes increased to values beyond the reliable limit for the equipment. We can clearly see precipitates using an optical microscope for the samples containing 10% HA (data not shown). The precipitation occurs in the region of isoneutrality, 3310

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Figure 2. Cryo-TEM images of ECL/HA complexes for different HA fractions: (a) pure ECL; (b) 0.25% HA; (c) 3% HA; (d) 10% HA; (e) 20% HA; (f, g) 60% HA; (h, i) 80% HA (w/w). White solid arrows indicate the presence of double bilayered liposomes. White hollow arrows indicate juxtaposed single bilayer liposomes. Black arrows indicate dispersed liposome, with no contact.

Figure 3. Left: SAXS intensity of ECL/HA for systems with different HA fractions. The model does not consider q values smaller than 0.035 Å−1, which are in the gray shadowed area. Right: cartoon of the complexation stages as a function of HA content showing the single bilayer liposome at low HA concentration, double bilayer formed by juxtaposed liposomes from 3 to 20% and the dispersion at high concentrations containing free HA.

together in small clusters. Few double-bilayer liposomes were observed inside big vesicles; also very rare multilayer vesicles were observed, with less than 10 objects in about 500 pictures

contact might be completely coated by HA leading to electrostatic repulsion among them. For 80% HA (Figure 2h and i), most of the vesicles observed were individual or sticking 3311

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Figure 4. Left: scattering intensity profiles simulated for the three phases presented in the ECL/HA systems (single bilayer, double bilayer and free HA). Right: SAXS intensity of ECL/HA systems after subtracting the free HA contribution calculated from eq 3.

well. In the next paragraphs, we will describe more in detail these three phases (single lipid bilayer, double lipid bilayer and free HA). Figure 4 (left) shows a representation of the phases correlating the structure to their scattering profiles. A similar model was found in our previous work on the complexation of plasmid DNA with liposomes,33 in which the plasmid DNA played the role of an electrostatic glue between two neighboring unilamellar liposomes, at molar charge ratios from 3 up to 9, that is, low DNA content. In the present work, we found that the negatively charged HA molecule plays the same role of an electrostatic glue that facilitates adhesion between two neighboring cationic single bilayer liposomes. This result is consistent with the results obtained from the DLS analysis because the increase in the number of juxtaposed liposomes increases the size of the multivesicle aggregates without changing the number of stacked bilayers. The correlation peaks are most pronounced when the HA content is 10%, and they decrease as the HA content increases, suggesting a decrease in the coexistence of the double bilayer phase. At high HA concentrations (i.e., 60 and 80% HA), the scattering profiles are typical of those for the coexistence of lipid phases (single and double bilayer) and the free HA phase characterized by the peak at q ∼ 0.035 Å−1. In summary, the studied ECL/HA system presents at least three different phases, as determined by SAXS scattering patterns, that is, (1) single lipid bilayer, (2) double lipid bilayer with bonded HA, and (3) free HA in aqueous media. Combinations of these phases can coexist depending on the amount of HA in the system. The free HA scattering (last curve in green) was fitted using a “generalized Gaussian polymer coil” model as form factor combined with a structure factor can simulate the correlation peak. We used the best-fit parameters of this case as the input parameters to model the coexistence of the free HA phase with other phases in other samples. This model gives an excellent curve fit for q values greater than 0.02 Å−1, which is the region of interest for studying liposome bilayers. To improve the

(see the Supporting Information). Despite having taking several representative images before selecting those that we present here, this study was not intended to be statistical. The main finding of the cryo-TEM study was the clear identification of the objects that we used for building the model to analyze the SAXS data that we present next: the single bilayer liposome and the juxtaposed single bilayer liposome. The occurrence of double bilayer as seen in one liposome like in Figure 2d (white and solid arrows) or from neighbor liposomes, as seen in Figure 2c (white and hollow arrows), cannot be distinguished by SAXS results; both objects will have the same scattering pattern. However, we have seen through cryo-TEM that the double bilayer from neighbor liposomes are predominant. Multilayer liposomes are so rare that, even having a much higher scattering intensity compared to single or double bilayer liposome, they were not detected by SAXS results presented next. 3.3. Synchrotron SAXS of the ECL/HA Complexes. The SAXS intensities of the ECL/HA complexes for different HA contents were measured and compared with those of pure ECL (without HA) and free HA. The samples were measured 1 day after preparation. Figure 3 shows the scattering intensities and their best-fit curves for ECL/HA with different HA contents (0, 10, 20, 40, 60, 80, and 100% w/w HA). The SAXS experiment was performed to obtain information about the molecular structure of the systems through a proper modeling of the scattering data. The scattering profile of the free HA (last curve, in green) presents a scattering profile characteristic of a rod-like particle, with a correlation peak at q ∼ 0.035 Å−1 and an intensity that drops as q−1 for high values of the scattering vector. The pure ECL system (first curve in dark blue) corresponds to the typical form factor of a single lipid bilayer or unilamellar vesicle system.37,38 As HA is added to the system, a new scattering profile is observed. From 0 to 10% HA, we see a slow transition from single bilayer to double bilayer (data shown in the Supporting Information). For HA contents from 10 to 40% w/w, the systems present the structure factor that is typical for a double lipid bilayer.37,38 The model fits the curve 3312

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previous approach (36 vs 20), which makes the results more reliable. The scattering intensity Ii(q) for each system “i” (i = 10, 20, 40, 60, and 80% HA) using this model is given as follows:

model so that it can describe the scattering for the low q region, one should take into account the secondary and tertiary structure of the HA and the curvature of the lipid membrane, which we are not showing in the present work. To describe the SAXS scattering of the single bilayer and the multiple bilayer, we proposed a model composed of (1) a form factor, which carries information about the shape of the bilayer and its electronic contrast and (2) a structure factor, which provides information about the system periodicity and number of correlated layers. For the form factor, we used the square modulus of the Fourier transform of a sum of equally spaced Gaussian functions, which can model the EDP across the lipid bilayer.38 To model the bilayer stacking, we tested two different approaches. In the first approach, we used a modified Caillé structure factor to describe the bilayer stacking. We used also a single form factor for each of the five HA−liposome complexes, and we assumed that each system was composed of a fraction of single bilayer vesicles and a fraction of multiple bilayer vesicles; thus, for each system, the scattering intensity I(q) is written as I(q) = P(q) Seff (q) + fHA IHA (q)

i i I(q) = NSC {f Si PS(q) + f Di PD(q)} + f HA IHA(q)

(2)

where f iS and f iD are the single and double bilayer phase fractions, respectively, for the ith system (f S + f D = 1), PS(q) and PD(q) are the form factors of the single and double bilayer phases, respectively (each form factor is the same for all systems), f iHA is the fraction of free HA present in the ith system, IHA(q) is the scattering intensity of free HA, which is obtained from the fitting of the pure HA system, and NiSC is a scaling parameter. Several assumptions were made to reduce the number of variables for the fitting: (1) the interaction between the HA and liposomes can change the EDP only at the position of the polar head groups in the lipid bilayer (i.e., the electronic density in the tail region does not change with complexation; thus, this electronic density is the same in both single and double lipid bilayers); (2) the molecular structure of the free HA in aqueous media does not change from that of the free HA system; and (3) the fraction of free HA in the systems was estimated using a simple model that assumes that each HA molecule complexed in the lipid membrane occupies an average area A of the lipid membrane in the single bilayer phase and that it occupies the same area A for each bilayer (thus, 2A) when packed into the double lipid bilayer phase. For this model, the equation for f iHA can be written as

(1)

where Seff(q) = f SB + f MBSMCT(q), in which f SB is a parameter related to the fraction of single bilayers, f MB gives the fraction of multiple bilayers, SMCT(q) is a modified Caillé structure factor, and P(q) is the bilayer form factor. The f HAIHA(q) term is the contribution to the scattering related to the fraction f HA of free HA present in the system (i.e., not complexed with the ECL). IHA(q) is the scattering intensity of the free HA and is obtained from the fitting of the pure HA system. As in our previous work,33 the average number of correlated layers was always ∼2 for all samples. This result suggests the existence of domains of juxtaposed single bilayer liposomes, as seen in the cryo-TEM images in Figure 2. These domains of double bilayers also exist at the interface between the liposomes of large multivesicle aggregates. Although this model could describe the position and intensity of the correlation peaks in the scattering profiles, it could not provide a good fit for all curves and for all fitting ranges (the calculated intensity of this fitting is not shown here). The model also did not provide a good estimate of the Caillé parameter because the scattering range probed by our SAXS experiment is not able to provide information about higher order peaks and the number of correlated bilayers is very low. The main limitation of this first approach was the use of a single EDP to describe both the single and multiple bilayer phases for the same system; the use of this single EDP could not mimic the scattering contrast of the HA between the two types of bilayer phases. To overcome this limitation, we proposed a second approach. In this approach, we use a combination of three phases to describe all five curves: (1) a single lipid bilayer phase, (2) a double lipid bilayer phase, in which the bilayers are bonded by the HA between the two consecutives bilayers, and (3) free HA (not bonded in the HA−liposome complexes) in aqueous media. In this model, we simultaneously fit all five ECL/HA scattering curves shown in Figure 3. Using this model, we achieved a better fitting result compared with that for the previous model mainly for the following reasons: (1) this model allows for differences among the single and double bilayer EDPs in the same system and (2) this model permits a simulation of the electronic density of the bonded HA between two consecutive bilayers, which was not possible using eq 1. This model also uses much fewer variables than does the

i i f HA = (1 − (1 + f Si )/(1 + f Sref )(Facref )/(Faci ))(c HA ) free /(c HA )

(3)

where f S is the fraction of the single lipid bilayer phase, Fac is the ratio i/(100 − i) (i =10, 20, 40, 60, and 80), and cHA is the HA concentration (w/w) in the system. The superscript “ref” indicates the values for a standard sample. For the standard sample, the zeta potential reaches a negative constant value, but the system with the standard sample has no free HA. We have chosen the 10% HA sample as the reference. Although it is simple, this model gives an excellent approximation to the free HA fraction, but it has limitations because the complexation dynamics most likely changes with the HA amount. Nevertheless, the main error source in this equation is the choice of reference sample. In total, we used 20 variables to fit all five ECL/HA scattering curves: five scaling factors and five single/double bilayer phase fractions (10 in total), nine independent variables to describe the single bilayer (PS(q)) and double bilayer (PD(q)) form factors (two exclusively for the single bilayer, three exclusively for the double bilayer, two shared by both, and two variables to describe the electronic contrast of the HA between the two bilayers). The last variable we used was a variable that corrected the f HA value at 80% because the use of eq 3 overestimates the free HA content in this system. The value found after several fittings was close to 0.96 (i.e., 96% of the calculated value); thus, we fixed it to 0.96 f (80%) HA . Figure 4 on the left shows the simulation of the scattering intensity used to fit all five ECL/HA systems (10, 20, 40, 60, and 80% of HA) curves shown in Figure 3. The simulation curves are related to the three phases found in the system: free HA, single lipid bilayer and double lipid bilayer. Figure 4 on the right shows the scattering curves for all five ECL/HA systems of Figure 3 fitted 3313

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the final structure of the complex (see section 2 of the Supporting Information for the in situ complexation experiment). To increase the double bilayer phase (and, consequently, the size of the aggregates), a cationic liposome should complex with a liposome that is partially coated with HA. For low HA content (up to 3%), this condition is easily fulfilled. The size of the aggregates increases with the HA amount, as shown in Figure 1, and the zeta potential is positive for the value that corresponds approximately to that of pure liposome system. For intermediate HA content (between approximately 3% and 20%, according to Figure 1), the positive and negative charges from the lipids and HA molecules, respectively, compensate one another, and the probabilities of finding HA-coated and uncoated liposomes are comparable. The electrostatic attraction between them and the complexes formed by them makes very large final aggregates. The zeta potential for this HA fraction depends strongly on the charge balance, and a negative zeta potential value means that the complexes are coated by HA molecules. For the high HA concentration (>20% HA), the probability that an uncoated liposome will encounter a coated liposome before the liposomes become homogeneously coated by HA decreases as the HA content increases. Thus, the size of these aggregates decreases with HA content, as shown in Figure 1. In this concentration range, the final complexes are already coated by HA molecules, as shown by the zeta potential measurements. In our previous work,33 when the system reached the region near the inversion of the zeta potential sign near R ± 1, the high concentration of plasmid DNA in the complexes disrupted the lipid membranes, and the membranes stacked together, forming a multilamellar complex. In this work, HA 16 kDa behaves as a weak acid, and it is not able to disrupt the membranes. The excess of HA molecules remain free in water, even for high concentration. In order to study the influence of the HA molecular weight on the final structural properties of the complexes, we studied two other HA chain sizes: 6 kDa (HA6) and 100 kDa (HA100). Figure 5 shows the SAXS patterns for the ECL/HA100 complexes for different HA100 content. The graph shows the SAXS intensity for pure ECL, the same one shown in the graph of Figure 3, and for pure HA100. The intermediate curves are related to different ratio of these two systems, but contrary to the results shown in graph of Figure 3, we cannot say that we have a combination of the scattering intensity from both systems, it is clear that there is no bilayer structure on the systems ECL/HA100 (10−90% w/w), the same happening with ECL/HA6 (72−80% w/w, data not shown here). This result suggests that the HA100 would play as a strong tensioactive on ECL system, which is in accordance with the obtained by Pasquali-Ronchetti et al.39 for high HA molecular weights. Pasquali-Ronchetti et al.39 investigated the interaction between unilamellar and multilamellar vesicles (DPPC, 1,2dipalmitoyl-sn-glycero-3-phosphocholine) and HA with a molecular weight ranging from 170 to 740 kDa. The complexation was performed at 50 °C (above DPPC melting temperature), and the suspension was mechanically stirred for 1 h to 4 days. The authors did not observe any difference between the unilamellar and multilamellar liposomes. They used TEM images to demonstrate that, after 48 h, there were no isolated vesicles, and the material was organized into a linear interconnected network with regular spacing that were 12 nm wide. The differences between the study by Pasquali-Ronchetti

when the contribution from the free HA scattering in each system was subtracted. The figure shows that the inclusion of HA changes the fractions of the single and double lipid bilayer phases, but these phases are the same for different HA amounts. The five curves on the right are very similar indicating that the local order, the structure of the bilayer, changes very little as a function of HA content in the system. This is not observed for HA 100 kDa, which was also studied in this work, as shown in Figure 5. The fitting parameters are shown in Table 1.

Figure 5. Experimental SAXS patterns for ECL/HA 100 kDa for different HA100 fraction.

Table 1. Parameters Obtained from a Simultaneous Fitting of the Scattering Curves phase fraction induced by HA presence total HA%

free HA fractiona

0 10 20 40 60 80 100

0c 0.25 0.54 0.76 0.83d 1c

single bilayer 1 0.83 0.90 0.91 0.91 0.94

± ± ± ± ±

0.02 0.02 0.01 0.01 0.01

double bilayer (period = 66.4 ± 0.3 Å) 20% w/w). 3.4. Langmuir Monolayers of EPC/DOTAP/DOPE in the Presence of HA. In our previous work,40 the same composition of EPC/DOTAP/DOPE lipids in Langmuir monolayers was evaluated, and we observed that miscibility was energetically favored, producing homogeneous and stable monolayers. In the present work, we added HA to the subphase of the Langmuir lipid monolayers at several concentrations in the range of 0.25 to 30% w/w to characterize the interaction between the molecules. The Langmuir monolayer is a flat surface; thus, there is no effect of the curvature of the membrane as there is in vesicles. However, to a first approximation, the HA molecules in the subphase interact with the polar headgroups of the lipids in the same manner as the HA molecules in the aqueous media interact with the vesicle membrane. Figure 4 presents the isotherms of the EPC/ DOTAP/DOPE monolayer in the presence of different amounts of HA in the subphase. The profiles are characteristic of expanded liquids and lack noticeable phase transitions at this resolution. The HA interacts strongly with the membrane because we do not observe the coexistence of pure lipid phases. The isotherm in the presence of HA presents a higher surface compressional modulus (Cs−1 = −dπ/d ln A); in other words, in the presence of HA, the monolayers are more rigid. For all the curves, we observe a shift to the left (decrease in the area/molecule) as we increase the HA concentration, indicating that the presence of HA in the subphase allows the lipids in the monolayer to be closer. The electrostatic interactions between the positive charges from the DOTAP polar headgroup and the negative charges from the HA biopolymer neutralize the electrostatic repulsion among the lipids from the monolayer. The packing is denser (shift to the left) with HA, and we do not observe a saturation or limit of the packing for this range of HA concentration up to 30% w/w. It is known that HA molecules present secondary and tertiary structures. The primary HA structure is an unbranched linear chain with the monosaccharides linked together. The secondary structure is related to the hydrophobic faces formed by the axial hydrogen atoms and CH groups, allowing the formation of a tertiary structure as a result of molecular aggregation.15,41 The HA/lipid proportion might not correspond to that studied in the liposome system.

Figure 6. Surface pressure−area isotherms for monolayers of EPC/ DOTAP/DOPE with different amounts of HA (16 kDa) in the subphase.

indicating full coating of HA and the size decreases back to the order of 120 nm as indicated by our DLS and zeta potential data. (3) The SAXS results revealed that the aggregates are a simple juxtaposition of neighboring liposomes but not multilamellar vesicles. The model for the SAXS data with the best fit proposes single lipid bilayer and double lipid bilayer objects; the latter is a double membrane between neighboring unilamellar vesicles (or in rare cases double-bilayered liposomes, as shown in the Supporting Information). We propose a way to estimate the fraction of the scattering intensity due to each different structure in the system. In this way, we observed that at high concentrations of HA the scattering intensity is mainly coming from single bilayers, indicating that the system is well dispersed. (4) Cryo-TEM images confirm the existence of objects like single and double bilayer vesicles and large aggregates of single bilayer vesicles, all objects that were used in SAXS modeling. Also we observed that large aggregates were mostly found in systems with HA fractions up to 20% w/w. We conclude that a low concentration of HA (20% w/w) produces a homogeneous coating that promotes better dispersion of the unilamellar vesicles. Low molecular weight HA (16 kDa) proved to be adequate for forming complexes with extruded liposomes and generating structures in the nanoscale size range with a negative zeta potential. These findings contribute to the understanding of cationic liposomes and HA interactions and to the development of new strategies for gene delivery and vaccine therapy for systems based on the electrostatic association between polymers and vesicles.

4. CONCLUSION We have studied the formation of an electrostatic complex between cationic liposomes and low molecular weight (16 kDa) HA and analyzed the structural and physicochemical properties of the system. We observed the following: (1) The negatively charged HA biopolymer has good miscibility with the studied cationic blend of EPC/DOTAP/ DOPE lipids according to our Langmuir monolayer results. (2) Varying the ratio of HA to lipid from 0.25 to 80% (w/w), we found that between 3 and 20% the system is in the isoneutrality region and large aggregates appear, but at high concentration of HA (80%) the surface potential is negative

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ASSOCIATED CONTENT

S Supporting Information *

Electronic density profile; SAXS patterns of ECL/HA complexation kinetics; double bilayer liposome (commented cryo-TEM images); multilayered liposome (commented cryo-TEM images). This material is available free of charge via the Internet at http://pubs.acs.org. 3315

DOI: 10.1021/la5045865 Langmuir 2015, 31, 3308−3317

Article

Langmuir

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AUTHOR INFORMATION

Corresponding Authors

*(L.P.C.) E-mail: [email protected]. *(L.G.d.L.T.) E-mail: [email protected]. Author Contributions #

L.G.d.la.T. and L.P.C. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. * Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Brazilian agency FAPESP for financial support (Grant No. 2011/19952-6 and 2010/02203-8). X.E.P.-M. thanks the Brazilian agency CAPES for financial support. A.C. and R.V.P. thank CNPq for financial support (Grant No. 400796/2012-0). The SAXS experiments were performed at the SAXS-1 beamline at the Brazilian Synchrotron. The cryoTEM experiments were performed at the Electron Microscopy Laboratory (LME) in the Brazilian Nanotechnology National Laboratory (LNNano). We thank Prof. M. H. Santana for fruitful project ideas and lab infrastructure support at Unicamp and Prof. M. E. Zaniquelli for precious contributions to the revision of this work.

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