Quantitative Monitoring of Microphase Separation Behaviors in

Mar 29, 2016 - Microphase separation behaviors of cationic liposomes have been investigated using a pH-sensitive fluorescent probe with 4-heptadecyl-7...
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Quantitative Monitoring of Microphase Separation Behaviors in Cationic Liposomes Using HHC, DPH, and Laurdan: Estimation of the Local Electrostatic Potentials in Microdomains Keishi Suga,* Kei Akizaki, and Hiroshi Umakoshi* Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyamacho, Toyonaka, Osaka 560-8531, Japan S Supporting Information *

ABSTRACT: Microphase separation behaviors of cationic liposomes have been investigated using a pH-sensitive fluorescent probe with 4-heptadecyl-7-hydroxycoumarin (HHC), 1,6-diphenyl-1,3,5-hexatriene, and 6-lauroyl-2-dimethylaminonaphthalene, and to estimate localized electrostatic potentials. Shifts of the apparent pKa values of HHC were observed in cationic liposomes in proportion to the amount of cationic lipids. Two pKa values were obtained with 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC)/3β-[N(N′,N′dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Ch) liposomes, while only one pKa value was generated with either DOPC/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or DOPC/dimethyldioctadecylammonium-bromide (DODAB) liposomes. The physicochemical membrane property analyses, focusing on membrane fluidity and membrane polarity, revealed heterogeneity among DOPC/DC-Ch liposomes. By analyzing the pH titration curves using sigmoidal fitting, the localized electrostatic potentials were estimated. For DOPC/DOTAP = (7/3), the membrane was in the liquid-disordered phase and the density of cationic molecules was 0.41 cation/nm2. For DOPC/DC-Ch = (7/3), the membrane was heterogeneous and the densities of cationic molecules in liquid-disordered and liquid-ordered phases were 0.25 and 1.24 cation/nm2, respectively. We thereby conclude that the DC-Ch molecules can form nanodomains when these molecules are concentrated to 59%.

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INTRODUCTION Self-assembly of phospholipid molecules is used not only as a biomembrane model but also as cargo and/or carrier of therapeutic molecules in drug delivery. Cationic liposomes, usually composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Ch), and dimethyldioctadecylammonium-bromide (DODAB), are widely applied as nonviral vectors for gene delivery and as fusogenic vesicles.1−6 Liposome−nucleic complexes (lipoplexes) have been studied for delivery of cationic liposomes to target cells through endocytotic or direct membrane fusion pathways.7−12 We have previously reported that the membrane properties (e.g., phase state) of cationic liposomes can be an important factor in controlling membrane−RNA interactions and the efficiency of cell-free gene expression.7 Furthermore, liposomes containing cholesterol (Ch) or its derivatives can form nanosized, ordered domains; such heterogeneous liposomes can be platforms for the performance of emergent functions in biological reactions.7,13−16 The physicochemical properties of such membranes could be key factors in regulatory biological applications, e.g., insertion of proteins and peptides into membranes, determination of rates of membrane fusion, transportation of substances through © 2016 American Chemical Society

membranes, apoptosis, and modulation of the activity of membrane-bound enzymes.17 In particular, the electrostatic potential of the membrane surface is a crucial parameter, especially upon direct interaction with its target (cell membranes, proteins, enzymes, nucleic acids, etc.). In previous reports, electrostatic potential at liposome surfaces has been analyzed using NMR,18 isolation titration calorimetry,19 and zeta potential analysis.20 According to published literature, the surface charge density of a cationic liposome can be designated by controlling the amount of cationic agent (Table 1, Figure S1).1−7,21−25 However, quantitative determination methods for microscopic (or nanoscopic) electrostatic potentials at microdomain (or nanodomain) have not been fully clarified. The lipophilic, pH-sensitive, fluorescent probe 4-heptadecyl7-hydroxycoumarin (HHC) has been studied previously for its ability to determine the chemical stability of the cationic liposomes.26−28 When the hydroxyl group of HHC is deprotonated due to an increase in surrounding pH, its excitation wavelength peak shifts from 320 to 380 nm. At that point, the pH environment can be estimated by monitoring Received: December 22, 2015 Revised: March 29, 2016 Published: March 29, 2016 3630

DOI: 10.1021/acs.langmuir.5b04682 Langmuir 2016, 32, 3630−3636

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round-bottom flask by rotary evaporation under vacuum.35,36 The lipid thin films were kept under high vacuum for at least 3 h and then hydrated with phosphate buffered saline (PBS) at room temperature. The liposome suspension was frozen at −80 °C and then thawed at 60 °C. This freeze−thaw cycle was repeated five times. The liposome suspensions were extruded 11 times through two layers of polycarbonate membrane with mean pore diameter of 100 nm via an extruding device. Liposomes with different compositions were also prepared according to the same method. Deprotonation Degree of HHC (D) Measurements. 10 μL of liposome suspension (20 mM of lipid) and 5 μL of HHC solution (100 μM in ethanol) were diluted in 985 μL of 50 mM PBS buffer (pH 5−13) to achieve a 200:1 molar ratio of lipids and HHC. The pH of sample solution was adjusted by addition of an appropriate amount of concentrated sodium hydroxide or hydrochloric acid. After incubation at 40 °C for 30 min, excitation spectra of HHC were measured by fluorescence spectrophotometer (FP-6500 and FP-8500; Jasco, Tokyo, Japan). The scanning range was from 300 to 400 nm, at an emission wavelength of 450 nm (bandwidths 5 nm). With the increase of deprotonation degree, the excitation wavelength at the maximum fluorescence intensity was shifted from 320 to 380 nm. The fluorescence intensity at the excitation wavelength of 330 nm is known as the pH-independent isosbestic point, which can be an internal reference of the HHC exciting in liposomes.26,27 Therefore, deprotonation degree of HHC (D) can be monitored by the ratio of the excitation fluorescence intensity at 330 and 380 nm:

Table 1. Zeta Potential of Cationic Liposomes liposome DOPE/DC-Ch = (1/1) DODAB DPPC/DODAB = (1/1) DOPE/DOTAP = (1/1) DOPC/DOTAP = (7/3) DOPC/DC-Ch = (7/3)

cationic lipid [%]

buffer (pH)

zeta potential [mV]

ref

50

Tris-HCl (7.4)

42.8 ± 3.9

21

46 ± 6 68 ± 4

23 24

50

water (−) 1 mM NaCl (6.3) PBS (7.4)

37

22

30

water (−)

76.1 ± 1.2

7

30

water (−)

64.5 ± 0.8

7

100 50

excitation spectra. The fluorometric titration of HHC incorporated into monolayers and liposomes facilitates the determination of the interfacial pKa of the probe and the calculation of the variation in electrostatic surface potential (Δψ).29−31 Although zeta potential measurements are one of the many commonly used methods for characterization of the surface electrostatic properties of colloidal materials, no good correlation between amount of cationic lipid and surface charge can be found.24 It has also been reported that the zeta potentials of cetyltrimethylammonium bromide (CTAB) modified liposomes reach a plateau at CTAB concentrations higher than 10 mol %.25 This indicates that the microscopic electrostatic potentials at the liposome membrane surface cannot be precisely determined by zeta potential measurements (Figure S1). In contrast, pH-dependent deprotonation behaviors of HHC can be utilized for electrostatic measurement of cationic liposomes modified with DOTAP and DC-Ch;26 however, the effect of phase separation on local electrostatic potentials has yet to be examined. With regard to the chemical structure of cationic lipids, DOTAP molecules freely diffuse in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, whereas DC-Ch molecules form clusters in DOPC liposomes, as does Ch.32,33 We have also reported that DC-Ch can induce phase segregation in DOPC liposomes using fluorescent probe analysis with 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6lauroyl-2-dimethylaminonaphthalene (Laurdan),5 while the local electrostatic potential for cationic liposome has not been investigated. Consequently, we expected that quantitative analysis of the microphase separation behaviors of cationic liposomes could be investigated by the combined use of lipophilic fluorescent probes. In this study, we investigated the pH dependence of HHC incorporated into various types of liposomes. The phase behaviors of cationic liposomes were revealed by analyzing membrane fluidity and polarity.34 The localized electrostatic potentials at both liquid-ordered (lo) phases and liquiddisordered (ld) phases were calculated using titration curves of deprotonation degree of HHC in heterogeneous cationic liposomes.

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D = I380/I330

(1)

where I380 and I330 represent the peak intensities at 380 and 330 nm, respectively. Calculation of Apparent pKa (pKa(obs)) of HHC. In order to calculate the pKa(obs) values, the D values were fitted by using a sigmoidal function, f(x), as follows:

f (x) =

Dmin − Dmax

+ Dmax

( x −dxx ) 0

1 + exp

(2)

where Dmax and Dmin are the maximum and minimum D values in the presence of liposome. The x and x0 values indicate the pH of bulk solutions and the pH (pKa) values a half of HHC deprotonating, respectively. The dx value represents the slope when bulk pH is pKa. For DOPC/DC-Ch liposomes, two-step sigmoidal fittings were applied as follows:

f1 (x) =

f2 (x) =

D(9) − Dmin 1 + exp

(

x − x01 dx1

)

Dmax − D(9) 1 + exp

(

x − x02 dx 2

)

+ D(9) (pH ≤ 9)

+ Dmax (9 ≤ pH) (3)

where D(9) indicates the D value at pH 9. The details for these calculations are shown in Figure S2. Evaluation of the Membrane Fluidity of Liposomes Using DPH. The inner membrane fluidity of the liposomes was evaluated in a similar manner to previous reports.37−39 10 μL of liposome suspensions (20 mM of lipid) and 5 μL of DPH solution (100 μM in ethanol) were diluted in 990 μL of PBS buffer (pH 7.4): the molar ratio of lipids and DPH was 200:1. The fluorescence polarization of DPH (Ex = 360 nm, Em = 430 nm) was measured using a fluorescence spectrophotometer after incubation at 25 °C for 30 min. The samples were excited with vertically polarized light (360 nm), and emission intensities both perpendicular (I⊥) (0°, 0°) and parallel (I∥) (0°, 90°) to the excited light were recorded at 430 nm. The polarization (P) of DPH was then calculated by using the following equations:

EXPERIMENTAL SECTION

Materials. DOPC, DOTAP, DC-Ch, and DODAB were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Fluorescent probes, DPH and Laurdan, were purchased from Sigma-Aldrich (St. Louis, MO). HHC and other chemicals were purchased from Wako Pure Chemical (Osaka, Japan). All chemicals were used without further purification. Liposome Preparation. A solution of DOPC containing 0−50 mol % DOTAP, DC-Ch, or DDOAB in chloroform was dried in a

P = (I − GI⊥)/(I + GI⊥) 3631

G = i⊥/i

(4)

DOI: 10.1021/acs.langmuir.5b04682 Langmuir 2016, 32, 3630−3636

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usually negative (ψ = −24.2 to −21.8).41,42 The pKa values of negatively charged liposomes were similar to those of zwitterionic liposomes (data not shown). Zuidam and Barenholz reported shifts in the pKa values of HHC incorporated into cationic liposomes;26 a different tendency was observed in DC-Ch liposomes, although a relationship between the pKa and the amount of cationic lipid was apparent. It is notable that zeta potential measurements could indicate nonapproximate electrostatic potentials for cationic liposomes, as we did not find a linear relationship between cationic lipid amount and zeta potential (Figure S1). On the other hand, the pKa(obs) values of HHC incorporated into cationic liposomes could be used to evaluate the local electrostatic environment at the membrane surface. Protonation Behaviors of HHC Incorporated into Cationic Liposomes. Cationic liposomes used in this study were composed of DOPC/cationic lipids, DOTAP, DC-Ch, and DODAB, with molar ratios of 5/5, 6/4, 7/3, 8/2, 9/1, and 10/0. Figure 2 shows the pH-dependent D values of HHC in various cationic liposomes. In the case of DOPC/DOTAP liposomes, the D values fitted to single-step sigmoidal curves, suggesting that DOTAP molecules are homogeneously distributed in liposome membranes (liquid-disordered (ld) phase7). The sigmoidal curves shifted accordingly with an increase in the amount of DOTAP, indicating that the membrane surface had become positively charged in the presence of DOTAP molecules. Shifts in curves were also observed in proportion to DC-Ch amounts. Liposomes including more than 20% of DC-Ch showed twostep sigmoidal curves. It has previously been reported that liposomes, including DC-Ch liposomes forming DC-Ch-rich domains, led to heterogeneous membrane surfaces. Similar deprotonation tendencies of HHC (two-step sigmoidal curves) using DC-Ch/dioleoylphosphoethanolamine = (1/1).26 Considering the heterogeneity in the membrane, the DC-Ch enriched domains are assumed to be more cationic than the membrane regions in which DC-Ch enrichment is poor. DOPC/DODAB liposomes, however, showed patterns in pHdependent D curves similar to those of DOPC/DOTAP liposomes, suggesting membrane homogeneity (ld phases). The pKa values obtained are summarized in Table 2. These results suggest that the phase behaviors of cationic liposomes affect the microscopic electrostatic potentials in membranes.

where i⊥ and i∥ are emission intensity perpendicular to the horizontally polarized light (90°, 0°) and parallel to the horizontally polarized light (90°, 90°), respectively, and G is the correction factor. The membrane fluidity was evaluated based on the reciprocal of polarization, 1/P. Evaluation of the Polarity of the Membrane Surface Using Laurdan. The fluorescent probe Laurdan is sensitive to the polarity around itself, which allows the surface polarity of lipid membranes to be determined.39,40 Laurdan emission spectra exhibit a red shift caused by dielectric relaxation. Thus, emission spectra were calculated by meaning the general polarization (GP340) for each emission wavelength as follows:

GP340 = (I440 − I490)/(I440 + I490)

(5)

where I440 and I490 are the emission intensity of Laurdan excited with 340 nm light. The total concentrations of lipid and Laurdan in the test solution were 200 and 1 μM, respectively.

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RESULTS AND DISCUSSION Evaluation of the Apparent pKa (pKa(obs)) of HHC. The protonation degree D of HHC is sensitive to surrounding pH. Using the nonionic surfactant Triton-X 100, the pH dependence of D values was measured (Figure 1). The obtained data

Figure 1. pH dependence of D values of HHC incorporated in Triton X-100 (a) and in DOPC (b). The D values were calculated according to eq 1. The excitation fluorescence spectra were measured at 25 °C.

were analyzed by fitting them to a single-step sigmoidal curve, and the pKa(obs) of HHC was determined to be 9.1, which corresponded well to previous reports (pKa: 8.8−9.0).17,41 In the case of DOPC liposomes, the pH titration curve indicated a higher pKa(obs) of 11.2, suggesting that the environment within DOPC liposomes was negatively charged. Previous reports reveal that the zeta potentials of zwitterionic liposomes are

Figure 2. pH dependence of D values of HHC incorporated in cationic liposomes: (a) DOPC/DOTAP liposomes, (b) DOPC/DC-Ch liposomes, and (c) DOPC/DODAB liposomes. The excitation fluorescence spectra were measured at 25 °C. 3632

DOI: 10.1021/acs.langmuir.5b04682 Langmuir 2016, 32, 3630−3636

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Langmuir Table 2. Mean Headgroup Area

a

lipid

mean headgroup area [Å2]

ref

DOPC DOTAP DODAB DC-Ch

72 78.4 ± 1.8 80−90 41.9 ± 0.5

43 this studya 44, 45 this studya

Evaluated by π−A isotherm measurements (see Figure S3).

Evaluation of Membrane Fluidity and Polarity. Based on our previous reports, membrane fluidity and polarity, as analyzed using fluorescent probes DPH and Laurdan, can indicate the phase states of different vesicular membranes.7,34 Figure 3 shows a Cartesian diagram of cationic liposomes used in this study, where membrane fluidity and membrane polarity were plotted on x- and y-axes, respectively. DOPC/DOTAP liposomes, DOPC/DODAB liposomes, and DOPC/DC-Ch = (9/1) appeared at similar positions (1st and 4th quadrants) in the diagram, indicating that these liposomes were in liquiddisordered (ld) phases.34 On the other hand, DOPC/DC-Ch liposomes including more than 20% cationic lipids appeared in the 2nd quadrant in the diagram, indicating that these liposomes were in heterogeneous ld + lo phases (lo: liquidordered). These results led us to conclude that the HHC inserted in heterogeneous liposomes could show two independent pKa(obs) values: pKa1 and pKa2 for ld and lo phases, respectively. It has been reported that Ch and DC-Ch molecules can also influence DOPC membrane properties in a similar manner,7 suggesting that the DOPC/DC-Ch liposomes (100 nm) could form nanosized, ordered domains. The mean headgroup area per molecule (A) for each liposome is summarized in Table 2;43−45 for DOTAP and DC-Ch, the headgroups were estimated by π−A isotherm measurement (Figure S3). Considering the phase state of liposomes, the D values in lo phase membranes are higher than those in ld phase membranes. In order to investigate the relationships between the steps of the sigmoidal curves and membrane heterogeneity, the pHdependent D values of HHC in DOPC/DC-Ch = (7/3) were measured with respect to membrane fluidity and polarity at both 25 and 55 °C (Figure 4). When the temperature increased from 25 to 55 °C, the phase state of DOPC/DC-Ch = (7/3) liposomes became homogeneous (ld), and the pH-dependent D curve of HHC in the liposomes switched to a single-step

Figure 4. pH dependence of D values of HHC incorporated in DOPC/DC-Ch = (7/3) liposome. The D values were calculated according to eq 1. The excitation fluorescence spectra were measured at 25 °C.

sigmoid. This suggests that the HHC in homogeneous membranes in ld phases follows a single-step sigmoidal curve, and the pKa(obs) values of HHC decrease in proportion to the cationic lipid molar ratio. In contrast, the HHC in liposomes in the heterogeneous ld + lo phases showed two discrete values of pKa1 and pKa2, whereas significantly lower pKa2 values could be derived from the HHC molecules existing solely in lo phases. HHC can thereby consistently indicate differences between the microscopic electrostatic potentials in heterogeneous, cationic LUVs. Estimation of Local Electrostatic Potentials in Cationic Liposomes. Single-step and two-step sigmoidal curves were fitted on the basis of eqs 2 and 3, respectively (see Figure S2). The relationships between cationic lipid amount and pKa(obs) values are shown in Figure 5. The pKa(obs) values for DOPC/ DOTAP liposomes and DOPC/DODAB liposomes were linear with respect to cationic lipid amount. On the other hand, Sorbal et al. have reported that zeta potentials for 1,2dipalmitory-sn-glycero-3-phosphocholine (DPPC)/DODAB liposomes reach a plateau at DODAB > 10 mol %,24 revealing the difficulty in the quantitative determination for electrostatic potentials at microdomain (or nanodomain) by zeta potential analysis. Based on our membrane property analyses (Figure 3), DOPC/DODAB liposomes were in ld phases and DODAB molecules could freely diffuse in the membrane. Hence, the

Figure 3. Cartesian diagram31 of cationic liposomes at 25 °C: (a) DOPC/DOTAP liposomes, (b) DOPC/DC-Ch liposomes, and (c) DOPC/ DODAB liposomes. The membrane fluidity (1/P) and membrane polarity (GP340) are plotted on x- and y-axes, respectively. Bars show error on individual data (at least n = 3). The total concentrations of lipid, DPH, and Laurdan are 200 μM, 1 μM, and 1 μM, respectively. 3633

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Figure 5. pKa values of HHC incorporated in cationic liposomes at 25 °C.

Figure 6. Schematic illustration of localized cationic molecules in liposome membranes.

pKa(obs) value evaluated by HHC quantitatively provides the local electrostatic potential for cationic liposomes. For the case of DOPC/DC-Ch liposomes, no pKa2 value for DOPC/DC-Ch = (9/1) liposomes was observed, suggesting that DC-Ch molecules ( 20 mol %).

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CONCLUSIONS In the present study, local electrostatic potentials in microphase-separated cationic liposomes were evaluated using HHC, DPH, and Laurdan. The charge density of DOPC/DOTAP and DOPC/DODAB liposomes, which form homogeneous ld phases, increased in proportion to the cationic lipid ratio. On the other hand, DOPC/DC-Ch liposomes including more than 20 mol % DC-Ch showed two-step sigmoidal HHC deprotonation curves, due to a heterogeneity (ld + lo) of phases. These results indicate that the DC-Ch molecules aggregate in the lo phase; by calculation of D values, the positive charge density at the lo phase in heterogeneous DOPC/DC-Ch liposomes was estimated to be 4.9 times higher than that at the ld phase. Our results emphasize that microdomains composed of DC-Ch can form in DOPC/DC-Ch liposomes. Although conventional approaches (e.g., zeta potential measurement) have not been able to discern quantitative local electrostatic potentials in lipid membranes, our method enables monitoring of the formation of microdomains in the presence of high amounts of cationic lipids. Such domains can provide attractive forces within anionic molecules, e.g., DNA, RNA, proteins, enzymes, and negatively charged lipids in cell membranes. In addition, the ordered phase of DC-Ch enriched domains could inhibit denaturation of RNA molecules, as compared to DOTAP liposomes.7 We anticipate that the characterization of phase separation behavior and quantification of local electrostatic potentials in self-assembled membranes will promote the development of a novel platform for accurate regulation of interactions among charged molecules.

Table 3. Localized Electrostatic Potential of Cationic Liposomes density of cationic lipid [unit/nm2] cationic lipid

cationic lipid [%]

DOTAP

10 20 30 40 50 10 20 30 40 50 10 20 30 40 50

DC-Ch

DODAB

a

phase statea ld ld ld ld ld ld ld ld ld ld ld ld ld ld ld

+ + + +

lo lo lo lo

ld phase 0.14 0.27 0.41 0.54 0.67 0.22 0.25 0.25 0.25 0.31 0.25 0.34 0.42 0.51 0.59

lo phase

1.21 1.24 1.24 1.24

■

Determined on the basis of the results in Figure 3.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04682.

charges per unit area of cationic liposomes. The positive-charge density in the lo phase of DOPC/DC-Ch = (7/3) was 4.9 times and 3.0 times higher than that in the ld phase of DOPC/DC-Ch = (7/3) and DOPC/DOTAP = (7/3), respectively (Figure 6). These results suggest that cationic lipids in heterogeneous DOPC/DC-Ch liposomes become concentrated in lo phases,

Literature zeta potentials for cationic liposomes, illustration for the sigmoidal fitting of D values, and π− A isotherms of DOTAP and DC-Ch (PDF) 3634

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AUTHOR INFORMATION

Corresponding Authors

*(K.S.) Tel: +81-6-6850-6286. Fax: +81-6-6850-6286. E-mail: [email protected]. *(H.U.) Tel: +81-6-6850-6287. Fax: +81-6-6850-6286. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Dr. Yukihiro Okamoto (Graduate School of Engineering Science, Osaka University) for his constructive comments and technical support. This work was supported by the Funding Program for Next Generation World-Leading Researchers of the Council for Science and Technology Policy (CSTP) (GR066), JSPS Grant-in-Aid for Scientific Research A (26249116), JSPS Grant-in-Aid for Research Activity Start-up (25889039), and JSPS Grant-in-Aid for Challenging Exploratory Research (T15K142040).

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DOI: 10.1021/acs.langmuir.5b04682 Langmuir 2016, 32, 3630−3636