Double Charge Inversion in Polyethylenimine-Decorated Liposomes

Jun 19, 2012 - and Félix Sarmiento. †. †. Biophysics & Interfaces Group, Department of Applied Physics, Universidade de Santiago de Compostela, 1...
1 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Langmuir

Double Charge Inversion in Polyethylenimine-Decorated Liposomes Juan Sabín,*,† Carmen Vázquez-Vázquez,†,‡ Gerardo Prieto,† Federico Bordi,§ and Félix Sarmiento† †

Biophysics & Interfaces Group, Department of Applied Physics, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain ‡ Colloid Chemistry Group, Departamento de Química Física, Universidade de Vigo, 36310 Vigo, Spain § Dipartimento di Fisica, Università di Roma “La Sapienza” and CNR-IPCF, Piazzale Aldo Moro 5, I-00185 Rome, Italy S Supporting Information *

ABSTRACT: The study of the interaction of a cationic polymer as PEI with phospholipids membranes is of special relevance for gene therapy because the PEI is a potential nonviral vector to transfer DNA in living cells. We used light scattering, zeta potential, and electron transmission microscopy to characterize the interaction between DMPG and DOPC liposomes with PEI as a function of the charge molar ratio, pH, temperature, initial size of the liposomes, and headgroup of the lipids. Unexpectedly, a double charge inversion and two different ranges of PEI−liposome concentrations where an aggregation occurs were found, when the proper pH and initial size of the liposomes were chosen. The interaction is analyzed in terms of the interaction potential proposed by Velegol and Thwar for colloidal particles with a nonuniform surface charge distribution. Results show a remarkable dependence of the stability on pH and the initial size of the liposomes, which explains the low reproducibility of the experiments if no special care is taken in preparing the samples. Comparatively small changes in the pH or in the liposomes size lead to a completely different stability behavior.

1. INTRODUCTION Polyethylenimine (PEI) is a cationic polymer well-known in the chemical industry, where it has been used for decades in water purification, the recycling of paper, shampoo manufacturing, and food technology.1 In 1995, it was used for the first time for the delivery of DNA and oligonucleotides in vitro and in vivo experiments, becoming the second polymeric transfection agent, after poly(L-lysine).2 Since then, many different kinds of PEI and other cationic polymers have been studied as potential nonviral vectors in gene therapy.3−6 The interest to improve and understand the mechanism of transfection of these cationic polymers into cells also prompted the study of their interaction with lipid membranes.7,8 Particularly, the study of the interaction between liposomes and polymers of oppositely charged polyelectrolytes has attracted much attention in the past decade due to the peculiar consequences of the adsorption of these polymers on the liposome surface. The combined effect of the electrostatic attraction between the polymer and the membrane and the repulsion between the like-charged polymers leads to a “patchy” adsorption, where the electrostatic surface potential of the complexes alternatively changes from positive to negative depending on whether that part of the liposome surface is covered or not by the polymer.9 The liposome surface hence appears “decorated” by the adsorbed polymer chains. This correlated adsorption of the polymer chains leads to a nonuniform distribution of the surface charge of the complexes, which works as a “key−lock” mechanism in their mutual interaction: i.e., an attractive force emerges between the © 2012 American Chemical Society

oppositely charged patches of different particles. The surface charge nonuniformity has been proven more relevant in colloidal systems than a priori was expected. Velegol et al. have shown that the nonuniformity of the surface charge is the reason why many colloidal systems need special dispersant when they are used for industrial application, even when the classical DLVO theory predicts an electrostatic barrier 100− 1000 times higher than KBT.10 Nonuniform surface charge distribution was used to explain the aggregation of 1,2dioleoyloxy-3-trimethylammoniumpropane (DOTAP) liposomes decorated by sodium polyacrylate, an anionic synthetic polyelectrolyte frequently used to mimic DNA molecules.11 Quemeneur and co-workers also studied the stability of giant and nano liposomes of DOPC in the presence of chitosan and hyaluronan and concluded that the decoration of the liposome surfaces by polymers drives the aggregation.12 Recent experiments carried out with light scattering and AFM show that “patchlike” interactions control the stability of latex particle coated by different oppositely charged polyelectrolytes.13 Aggregation of DMPC small unilamellar vesicles interacting with PEI also was reported by Sikor and co-workers.14 However, the disparity of results obtained in different experimental conditions requires a systematic study to obtain clear conclusions on the stability of nonuniformly charged colloids. In the present paper we use light scattering techniques Received: February 17, 2012 Revised: June 13, 2012 Published: June 19, 2012 10534

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542

Langmuir

Article

critical aggregation concentration of DMPG liposomes with La3+ ions in water17 and in the presence of 10 mM HEPES buffer18 reveal that the adsorption of buffer molecules to the liposomes surface does not modify significantly the electrostatic charge of the liposomes. At biological pH the PEI is estimated to have a degree of protonation of 0.5.19 Figure 7 shows that at pH = 7 the zeta potential value of PEI is ζPEI = +15 mV. Figure 1 shows the zeta potential and size of the PEIdecorated DMPG liposomes as a function of the PEI

and zeta potential measurements to study the effect of pH, initial size of liposomes, charge of the lipid headgroups, and temperature on the stability of different liposomes decorated by PEI. Results are discussed in terms of the interaction potential proposed by Velegol and Thwar for a nonuniform distribution of the surface charge.

2. MATERIALS AND METHODS 2.1. Materials. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-ditetradecanoyl-sn-glycero-3-phospho-1′-rac-glycerol sodium salt (DMPG) were purchased from Avanti Polar Lipids. Highly branched polyethylenimine (PEI) with high charge density, low molecular weight (Mw = 1800 g/mol, PDI = 1.14), and primary, secondary, and tertiary amine groups in a ratio of approximately 1/2/1 was purchased from Polysciences Inc. The following buffers were used to fix the pH of the solutions: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic sodium salt (HEPES), aminacetic acid (glycine), and sodium acetate (NaOAc) were purchased from Sigma; sodium diacid phosphate (Na2HPO4) was purchased by Panreac. All products were used without further purification. 2.2. Liposomes Preparation. Multilamellar large liposomes were prepared following the freeze−thaw−vortex protocol.15 This procedure entails freezing the solution by immersion into liquid nitrogen, followed by thawing by immersion into 50 °C water and further followed by thorough vortexing. Large unilamelar liposomes were prepared by the extrusion method. The details of the extrusion process have been described elsewhere.16 The dispersion was preextruded at 50 °C by forcing it once through polycarbonate membrane filters (Millipore) with a nominal pore diameter of 800 nm held in an extruder (Northern Lipids) and using dry nitrogen at high pressure. The liposome suspension was then extruded at least 10 times through two polycarbonate membrane filters with a nominal pore diameter of 100 nm. The liposomes solutions used to study the effect of the initial size of the liposomes on the stability were extruded through filters with pore diameters of 600, 400, and 100 nm. 2.3. Dynamic Light Scattering. The size of liposomes and clusters was measured by dynamic light scattering (DLS), at 25 °C, using an Autosizer 4800 spectrometer from Malvern Instruments. This instrument is equipped with a Uniphase 75 mW Ar laser operating at 488 nm with vertically polarized light. Time correlation was analyzed by a digital autocorrelator PCS7132 from Malvern Instruments and using the CONTIN algorithm. Samples were prepared by mixing a given amount of liposomes with PEI solutions of increasing concentration. Measurements were carried out 15 min after the mixing at a scattering angle of 90°. The time evolution of the aggregation was monitored measuring the size of the cluster every 10 s and beginning just after the mixing. 2.4. Zeta Potential Measurements. Zeta potentials (ζpotentials) of the liposomes and of the PEI−liposome aggregates were measured using a ZEN2600 Zetasizer Nano ZS equipped with a 633 nm HeNe laser (Malvern Instruments). The measurements were performed with a universal “Dip” cell, also from Malvern Instruments. For all samples, an average of five measurements was taken 15 min after the mixing of a given amount of liposomes with PEI solutions of increasing concentration. 2.5. Transmission Electron Microscopy. Transmission electron microscopy (TEM) micrographs were taken using a CM-12 Philips microscopy. Samples containing liposomes with 0.1 mg/mL of DMPG or DOPC were stained with 2% w/v phosphotungstic acid and placed on copper grids with Formvar films.

Figure 1. Zeta potential and diameter of the PEI-decorated DMPG liposomes in HEPES buffer as a function of the PEI concentration and molar charge ratio. The DMPG concentration was 0.1 mg/mL, and the radius of the liposomes was 60 nm. Red line shows the theoretical result predicted by using the Velegol potential eq 1. Black line shows the results of adding the van der Waals potential (eq 4) to eq 1 with a stability limit (see text) fixed at 1 KBT. Blue lines shows the same results but considering the stability limit at 10 KBT. The values at the top show the increase of the pH as the PEI concentration increases (see text for details).

concentration and the molar charge ratio (R± = [PEI]+/ [DMPG]−) between the polymer and the liposomes. All the samples were prepared independently, by mixing proper amounts of liposome suspension and of the PEI solution in a single step. As suggested by Felgner et al.,20 R± is calculated assuming that the DMPG membrane has the same number of lipids in the inner as in the outer layer and that all the amine groups of the PEI are protonated. We assumed this nomenclature because it is commonly used in the characterization of polyplexes for gene therapy.21 Figure 1 shows two “charge inversions” on the zeta potential values as the PEI concentration increases. There are also two regions of aggregation at the PEI concentrations where the zeta potential values are lower than 10−15 mV in absolute value (i.e., for 2
R± < 10 000). Summarizing, there are two separate ranges of PEI concentrations where the DMPG liposomes decorated by PEI aggregate and three regions where the system is stable. Despite that we are using a buffer solution the pH was not constant in the solutions with high PEI concentrations. Different concentrations of HEPES buffer were used to control the pH of the PEI solution, but even at 100 mM HEPES the pH raised when the PEI concentration exceeded 10 mg/mL. We hence decided to adopt the 10 mM HEPES buffer throughout our study because this is the commonly used buffer in gene therapy experiments. The values at the top of Figure 1 show the real pH of the solution measured after adding the PEI solution to the liposomes. We will discuss this effect in detail in the following sections. In both the two ranges of PEI concentrations where the system aggregates, it is also possible to distinguish a region where the complexes keep growing until flocculation occurs and another range of PEI concentrations where the aggregation ceases when the clusters reach a certain size which depends on the PEI/liposome concentration ratio. Black symbols in Figure 2 show the time evolution of the aggregation of DMPG

other) and of the mismatch of the charge modulation patterns on the surface and on the adsorbing macroions. The second charge inversion appears as an indirect consequence of the specific features of the polymer. It is known that PEI has buffer capacity and high tendency to capture protons, thus increasing the pH of the solution.27 Figure 3a shows that PEI drastically increases the pH of the

Figure 3. (a) Effect of the PEI concentration on the pH of water. (b) Schematic illustrations showing the “proton sponge” effect. Adapted from ref 4.

solution for concentrations larger than 10−4 mg/mL. When a highly concentrated PEI solution is mixed with DMPG liposomes and the liposomes surface is saturated, the excess polymer in the bulk significantly increases the pH of the solution, even if a buffer is present. In turn, the increase of the pH tends to decrease the charge of the PEI adsorbed on the liposomes surface and the overall zeta potential of the complexes. In other words, the excess of PEI molecules in the bulk tend to capture the protons of the PEI adsorbed on the liposomes surfaces. At high enough concentration, this effect is so pronounced that the PEI−liposome complexes invert again the sign of their zeta potential reaching a new aggregation region when the overall zeta potential is lower than 15 mV. Figure 4b shows a TEM picture of the aggregates in the second charge inversion region. For PEI concentrations higher than 20 mg/mL (R± = 4 × 103) the electrostatic repulsion is high enough for the system to become stable again. The strong influence of the PEI on the pH (that makes it difficult to control the pH in highly concentrated PEI solutions), far from being a disadvantage, makes PEI highly attractive for its use as an efficient nonviral vector to transfer DNA into cells.28 When polyplexes built up with PEI interact with the external membrane of a living cell, they are easily captured by an endosome.29 During the intracellular trafficking, the capacity of the PEI to adsorb protons is coupled with the influx of protons and chloride ions mediated by the endosomal V-ATPase pumps to decrease the pH.30 The result is a drastic

Figure 2. Time evolution of the size of the clusters of PEI-decorated DMPG liposomes in HEPES buffer at PEI concentrations in the range of the first charge inversion (●, 7.5 μg/mL; ▲, 9.5 μg/mL; ■, 25 μg/ mL) and in the range of the second charge inversion (○, 1.2 mg/mL; △, 5 mg/mL). The DMPG concentration was 0.1 mg/mL.

liposomes at different concentrations of PEI in the range of the first charge inversion; open symbols show the data for PEI concentrations in the second charge inversion. Both sets of data confirm the dependence of the size of the clusters on the PEI concentration. The first charge inversion was also observed in other liposome systems interacting with oppositely charged polymers.12,22,23 Charge inversion or “overcharging” is expected when highly charged polyelectrolytes adsorb on the surface of oppositely charged colloidal particles. The phenomenon of charge overcompensation occurs when more polyelectrolyte adsorbs on an oppositely charged surface than is needed to neutralize it, so that eventually the sign of the net charge of the “decorated” surface is opposite to that of the original “bare” surface. As it has been shown by different authors,24−26 this rather counterintuitive phenomenon can be described in terms of the correlated adsorption of the polyelectrolyte chains on the surface (that are attracted by the surface but repelled each 10536

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542

Langmuir

Article

Figure 4. TEM images of (a) stable DMPG liposomes without PEI, (b) PEI-decorated DMPG liposomes interacting with PEI polymers at R± = 2 × 103 in HEPES buffer, and (c) PEI-decorated DOPC liposomes in a Na2HPO4 buffer with initial pH = 6.3 at R± = 0.3 and (d) R± = 10.

increase of the ionic strength inside the endosome that induces an osmotic swelling and provokes its rupture, with the consequent release of the polyplexes to the cytoplasm. This phenomenon is called “proton sponge” effect and is the “trick” that polyplexes use to enter the cell.4 Figure 3b shows a sketch of this mechanism. 3.2. The Nonuniform Charge Distribution Model. One of the most interesting features of the PEI−liposomes complexes is the formation of stable clusters at PEI concentrations around the two charge inversions. This stabilization of the clusters is hardly explained by the classical DLVO theory. On the basis of the study of the formation of stable clusters observed previously in other colloid−polymer complexes,12,23 we suggest that also in this case the origin of the formation of stable cluster lies on the nonuniform distribution of charge on the surface of PEI-decorated liposomes. To describe the interaction between two nonuniformly charged colloid particles, Velegol and Thwar31 have proposed an interaction potential that combines a net charge dependent monopole term, which for particles having the same sign the net charge is repulsive, and a multipole term, arising from the charge heterogeneity, which is in any case attractive.11 For two identical particles this potential is

The repulsive and attractive terms of this interparticle potential have different interaction ranges. Their combination results in a potential barrier whose height, relative to the thermal energy (KBT), controls the stability of the complexes. Colloidal systems with a repulsive potential barrier comparable with the thermal energy have a certain probability of aggregating. However, as the barrier height increases so that, at a given temperature, the aggregation process stops when a sufficient height of the barrier is reached. Assuming the identity of the electrostatic potential (ψ) and of the measured zeta potential, from the values measured at the different PEI concentrations we can estimate a degree of coverage (Π) of the liposomes by the adsorbed PEI as ψ = ΠζPEI + (1 − Π)ζDMPG

(2)

with ζPEI and ζDMPG being the zeta potential values of pure PEI and pure DMPG liposomes, Π is interpreted as the probability of the liposome surface to be covered by the polymer. The standard deviation of the surface potential (σ) can be then estimated by σ=

Π(ζPEI − ψ )2 + (1 − Π)(ζDMPG − ψ )2

(3)

This simple calculation allows us to evaluate the interaction potential described by eq 1 for different PEI concentrations and different sizes of the clusters. The red line in Figure 1 shows the theoretical diameters of the final stable clusters for each PEI concentration. To calculate these theoretical values, we initially evaluated the interaction potential of eq 1 assuming R = 60 nm, using the corresponding zeta potential values for each PEI concentration as the average surface potential and calculating the standard deviation using eq 3. At PEI concentrations where the initial interaction potential has a repulsive barrier higher than the thermal energy (Vmax > 1 KBT), the system was considered stable and the final size of the system was kept at 60

⎡ ⎤ ⎛ ⎛ κH ⎞ ⎞ V = επR ⎢2ζ 2 ln⎜coth⎜ ⎟⎟ + (ζ 2 + σ 2) ln(1 − e−2κH )⎥ ⎝ 2 ⎠⎠ ⎝ ⎣ ⎦ (1)

where H is the distance between the surfaces of the two approaching particles, ε is the permittivity of the medium, κ is the inverse of the Debye length, R is the radius of the colloidal particles, and ψ and σ are the values of the electrostatic surface potentials averaged over the whole surface of the particles and of its standard deviations, respectively. 10537

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542

Langmuir

Article

nm. For those PEI concentrations with potential barrier lower than 1 KBT, we searched for the smallest size at which the height of the interaction potential overcomes the thermal energy. Notably, from Figure 1 it appears that this model predicts the aggregation in a range of PEI concentrations which is narrower than that defined by the experimental data. In a previous work,32 we already discussed the importance of combining the interaction potential of eq 1 and the van der Waals forces to predict the aggregation and the size of the stable clusters. The van der Waals forces are present in all colloidal systems and are especially relevant at short distances. For liposomes systems, the potential has the form33 VA =

AR ⎡ 1 2 1⎤ A − + ⎥− ⎢ 12 ⎣ 2d + H d+H H⎦ 6 2 ⎡ ⎛ d ⎞⎤ ⎟ ⎥ ln⎢1 − ⎜ ⎝d + H ⎠ ⎦ ⎣

(4)

Here R is the radius of the liposomes, d is the thickness of the bilayer, assumed equal to 4.5 nm,34 and A is the Hamaker constant, chosen to be 0.5 × 10−20 J in accordance with the bibliography.35 The addition of the van der Waals forces to the interaction potential results in the lowering of the repulsive barrier (Figure 5c). Figure 5a shows the interaction potentials, calculated combining eqs 1 and 4, as a function of the distance between two liposomes for all the PEI concentrations under study. Using this more realistic potential, three well-defined regions of aggregation (colored in red and brown) and two regions of stability (in blue) clearly appear. The black line in Figure 1 represents the predicted sizes of the clusters adding the van der Waals forces to eq 1 and considering again the stability condition Vmax > 1 KBT. Results show a broadening of the aggregation region but still not enough to agree with the experimental data. By using Monte Carlo simulations, Truzzolillo et al.36 showed that for a system of nonuniformly charged particles interacting, as in our case, through a Velegol and Thwar potential, the radius of the aggregates stops increasing and tends to become constant for further Monte Carlo steps (time) when the barrier becomes ∼10 KBT. According to this study, we obtain a better fitting of our experimental data when 10 KBT is chosen as the stability limit of the potential built combining eqs 1 and 4 (blue line in Figure 1). It has been proposed that the van der Waals forces not only decrease the height of the repulsive barrier but also promote the formation of a secondary minimum in the interaction potential whose depth increases as the cluster size grow.11,37 Based on this hypothesis, complexes with moderate zeta potential aggregate in the primary minimum so that the clusters stop growing when the repulsive barrier becomes high enough due to the increasing size, but for a low enough zeta potential the complexes can aggregate in the secondary minimum that was formed in front of the barrier so that they keep growing and eventually flocculate. Figure 5c shows this secondary minimum and also shows that, as the clusters grow, the minimum becomes deeper, promoting the continuous aggregation. The aggregation in the first charge inversion, even at those PEI concentrations where flocculation occurs, is not reversible under dilution. Actually, dilution did not have any effect on the dynamic of the aggregation (data not shown). This behavior is

Figure 5. (a) Interaction potential obtained by adding the van der Waals forces (eq 4) to the potential proposed by Velegol and Thwar for nonuniformly charged colloids (eq 1) as a function of the distance between liposomes for different PEI concentrations (here R = 60 nm). Red color indicates the values of the potentials greater than 1 KBT, while blue indicates ranges of PEI concentration and distances where the interaction potential becomes increasingly negative. (b) Interaction potentials of PEI-decorated DMPG liposomes with [PEI] = 2 mg/mL (R± = 500) and ζ = 15 mV calculated using the Velegol and Thwar potential (eq 1) for colloidal systems with diameters of 60, 120, 200, 300, 400, 600, and 800 nm as a function of the distance. The black arrow indicates the increasing of the diameter. (c) The interaction potential obtained by adding the van der Waals potential (eq 4) to eq 1 for the same diameters.

usually attributed to aggregation in the primary minimum.38−40 In any case, whether the aggregation takes place in the primary or in the secondary minimum seems clear that the attractive van der Waals forces have to be taken into account to describe the aggregation of these nonuniformly charged colloids. The aggregation corresponding to the second charge inversion is also irreversible, but the aggregation process can be modified by dilution. Figure 6 shows the time evolution of the aggregation of DMPG liposomes in the presence of PEI concentration in the range where the second charge inversion occurs. The concentrations of PEI chosen to promote the aggregation are 2.5 and 25 mg/mL of PEI at the edges of the 10538

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542

Langmuir

Article

parameter in the aggregation. We studied the stability of PEI decorated DMPG liposomes at 10, 25, 30, and 60 °C without observing any change in the PEI concentrations that triggers the aggregation. However, 24 h after the aggregation starts, samples aggregating in the second charge inversion at 10 and 25 °C were turbid, at 30 °C big clots of clusters were visible, and at 60 °C the clusters were completely precipitated. This behavior suggests that clusters grow larger when the thermal energy is higher. Despite the polydispersity of the clusters when aggregation occurs, dynamic light scattering also shows significant bigger clusters in the second charge inversion when temperature was increased (see Supporting Information). These observations are consistent with previous studies of the aggregation of polyelectrolyte decorated DOTAP liposomes at different temperatures.41 3.4. Effect of the pH on the Stability. As previously shown with DMPG liposomes, the stability of the PEIdecorated liposomes is not only controlled by the charge ratio (R±), but also by the pH of the solution. DMPG lipids are anionic lipids that form liposomes whose zeta potential does not depend on the pH. However, most of the lipids used as liposome gene carriers, as well as most of the lipids composing biological cell membranes, are zwitterionic, and they form liposomes whose zeta potential strongly depends on pH. Figure 7 shows the dependence of the zeta potential on the pH for

Figure 6. Time evolution of the size of the cluster of PEI-decorated DMPG liposomes aggregating at (□) [PEI] = 2.5 mg/mL (R± = 550) and (○) [PEI] = 25 mg/mL (R± = 7 × 103) in HEPES buffer. The DMPG concentration was 0.1 mg/mL. Buffer solution was added at the time indicated by the black arrows keeping the molar charge ratio R± constant but diluting the PEI concentration and decreasing the pH of the solution.

second aggregation peak in Figure 1. At these PEI concentrations, the decorated liposomes have a moderate zeta potential (+10 and −10 mV, respectively), and they form stable clusters with a diameter around 400 nm. Black arrows in Figure 6 indicate the time where the solutions were diluted to one-half of the original concentrations. When the sample with 2.5 mg/ mL of PEI was diluted, the clusters did not disaggregate but the aggregation ceased. The cease of the aggregation cannot be explained by simple dynamic stabilization because when a sample with the same amount of liposomes and with 25 mg/ mL of PEI was diluted to one-half, the clusters become bigger and the aggregation speeds up. Dilution in the first charge inversion does not affect the aggregation because the aggregation is irreversible and the R± is kept constant. However, when dilution takes place in the second charge inversion, R± is still constant but the pH of the solution decreases. Figure 1 shows that samples with 1.25 mg/mL and pH = 8 are more stable than samples with 2.5 mg/mL and pH = 9. This is consistent with the observations in Figure 6. These measurements confirm that the size of the stable clusters of DMPG liposomes decorated by PEI is controlled not only by the PEI concentration and the molar charge ratio but also by the pH of the solution. 3.3. Effect of the Temperature on the Stability. Since the stability of the PEI-decorated liposomes is controlled by the height of the interaction potential barrier compared with the thermal energy, temperature is expected to be an important

Figure 7. Zeta potential of DOPC liposomes (■) and PEI polymers (●) as a function of pH. Lines indicate the pH values where the study of the stability of Figure 8 was carried out. The DOPC and PEI concentrations were 0.1 and 5 mg/mL, respectively.

unilamelar DOPC liposomes and PEI polymer. According to previous studies where PEI of different molecular weights were used,42,43 the zeta potential of the PEI also decreases as the pH increases, as is shown in Figure 7. Because of this strong dependence of the zeta potential of both PEI19 and DOPC liposomes on the pH, also the interaction between them and the stability of the PEI-decorated liposomes is expected to depend on the pH. In fact, we distinguished three different regions in Figure 7: a low pH region where both the PEI and the liposomes are positive, the range 6 < pH < 10 where the PEI is positive and the DOPC liposomes are negative, and a further region, for pH > 10, where both the PEI and the DOPC liposomes are negative. Different buffers were used to study the interaction between PEI and DOPC liposomes. Despite the buffers are not strong 10539

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542

Langmuir

Article

regions. When DOPC liposomes are mixed with PEI polymer at sufficiently low concentrations in a buffer with pH = 5, there is no interaction between them because they are charged with the same sign. As the PEI concentration increases, the pH raises, changing the zeta potential of both the polymer and the liposomes. Since the DOPC liposomes charge decreases, they start to interact with the polymers forming PEI-decorated liposomes that eventually aggregate at 10 mg/mL of PEI. 3.5. Effect on the Stability of the Initial Size of the Liposomes. Another interesting experimental test of the hypothesis that the aggregation is driven by a Velegol-like potential is to study the effect of the initial size of the colloidal particles on the aggregation process. This is an important test because the strong dependence of the potential on the size of the colloidal particles plays a key role in the formation of stable clusters when patchy electrostatic interactions are involved. We chose DOPC liposomes to carry out these experiments because with DOPC lipids are easier to prepare liposomes with different sizes by using filters with different pore size in the extrusion process. On the contrary, DMPG liposomes resulted to have the same final size regardless the pore size of the filter. All the batches of DOPC liposomes were initially filtered through an 800 nm pore size filter and then through filters of decreasing sizes. Liposomes filtered through 600 nm filters resulted with an average diameter of 190 ± 6 nm; the batches filtered through 400 nm filters had an average diameter of 170 ± 5 nm; and the ones filtered through filters with a pore size of 100 nm resulted with a diameter of 120 ± 3 nm. The zeta potential of the three batches of liposomes follows the same behavior with the PEI concentration as the red points of Figure 7. Despite the small differences in the diameter of the DOPC liposomes, the aggregation is drastically affected. Figure 9 shows the size of the

enough to keep the pH constant at high PEI concentrations, we used 10 mM NaOAc, 10 mM Na2HPO4, 10 mM HEPES, and 10 mM glycine buffer to control the initial charge of the PEI and DOPC liposomes at pH = 5, 6.3, 7, and 10.5, respectively. Figure 8 summarizes the stability of the four systems.

Figure 8. Zeta potential and size of the PEI-decorated DOPC liposomes as a function of the PEI concentration and the molar charge ratio in NaOAc with initial pH = 5 (☆), Na2HPO4 with initial pH = 6.3 (□), HEPES with initial pH = 7 (○), and glycine with initial pH = 10.5 (△). The DOPC concentration was 0.1 mg/mL.

For pH > 10, PEI and DOPC liposomes have like-charge surfaces of the same sign and no electrostatic attraction is expected to occur. The size and zeta potential of the DOPC liposomes remain constant over the whole range of PEI concentrations studied, suggesting that in this condition the PEI does not adsorb on the liposomes. At pH = 7 the PEI becomes positively charged and adsorbs on the negative liposome surface in a correlated way, i.e., forming patches, due to the electrostatic repulsion between the PEI polymers. At pH = 6.3 the DOPC liposomes have a lower negative zeta potential, but the interaction with the positive PEI polymer still is strong enough to form decorated liposomes. At pH = 6.3 the initial zeta potential values of the bare liposomes is so small (only −6 mV) that the adsorption of the PEI provokes a first charge inversion and the change of the pH at high PEI concentration induces a second charge inversion in a similar way as we showed with DMPG liposomes in Figure 1. As a consequence of the two charge inversions, the PEI-decorated liposomes aggregate forming stable clusters in two PEI ranges of concentrations. Figure 4c,d shows TEM pictures of clusters of PEI-decorated DOPC liposomes in the two charge inversion

Figure 9. Size of the PEI-decorated DOPC liposomes in a HEPES buffer as a function of the PEI concentration and molar charge ratio (R±) for liposomes with initial diameter of (□) 190, (△) 170, and (○) 120 nm.

PEI−liposome complexes as a function of the PEI concentration for the three batches of liposomes with different initial diameters. The bigger particles with a diameter of 190 nm did not aggregate in the whole range of PEI studied. Liposomes with 170 nm in diameter did aggregate in a very narrow range of PEI concentrations when the zeta potential reaches its lower absolute value. Finally, the smallest particles, with a diameter of 10540

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542

Langmuir 120 nm, did aggregate in a wide range of PEI concentrations even when the zeta potential has quite high values. These results support the hypothesis that the bigger the particles are, the higher is the potential barrier due to the nonuniform charge distribution potential which controls the particles aggregation.

■

REFERENCES

(1) Amiji, M. M. In Polymeric Gene Delivery: Principles and Applications; CRC Press: New York, 2004; p 704. (2) Boussif, O.; Lezoualch, F.; Zanta, M.; Mergny, M.; Scherman, D.; Demeneix, B.; Behr, J. A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in-Vivo Polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (3) Honore, I.; Grosse, S.; Frison, N.; Favatier, F.; Monsigny, M.; Fajac, I. Transcription of Plasmid DNA: Influence of Plasmid DNA/ polyethylenimine Complex Formation. J. Controlled Release 2005, 107, 537−546. (4) Behr, J. The Proton Sponge: A Trick to Enter Cells the Viruses did Not Exploit. Chimia 1997, 51, 34−36. (5) Lungwitz, U.; Breunig, M.; Blunk, T.; Gopferich, A. Polyethylenimine-Based Non-Viral Gene Delivery Systems. Eur. J. Pharm. Biopharm. 2005, 60, 247−266. (6) Wang, D.; Narang, A.; Kotb, M.; Gaber, A.; Miller, D.; Kim, S.; Mahato, R. Novel Branched Poly(Ethylenimine)-Cholesterol WaterSoluble Lipopolymers for Gene Delivery RID G-6195−2010. Biomacromolecules 2002, 3, 1197−1207. (7) Zaliauskiene, L.; Bernadisiute, U.; Vareikis, A.; Makuska, R.; Volungeviciene, I.; Petuskaite, A.; Riatiba, L.; Lagunavicius, A.; Zigmantast, S. Efficient Gene Transfection using Novel Cationic Polymers Poly(Hydroxyalkylene Imines). Bioconjugate Chem. 2010, 21, 1602−1611. (8) Zintchenko, A.; Konak, C. Interaction of DNA/polycation Complexes with Phospholipids: Stabilizing Strategy for Gene Delivery. Macromol. Biosci. 2005, 5, 1169−1174. (9) Sennato, S.; Bordi, F.; Cametti, C. Correlated Adsorption of Polyelectrolytes in the ″Charge Inversion″ of Colloidal Particles. Europhys. Lett. 2004, 68, 296−302. (10) Jones, J.; Holtzer, G.; Snyder, C.; Yake, A.; Velegol, D. Charge Nonuniformity Light Scattering. Colloids Surf., A 2005, 267, 79−85. (11) Bordi, F.; Sennato, S.; Truzzolillo, D. Polyelectrolyte-Induced Aggregation of Liposomes: A New Cluster Phase with Interesting Applications. J. Phys.: Condens. Matter 2009, 21, 203102. (12) Quemeneur, F.; Rinaudo, M.; Maret, G.; Pepin-Donat, B. Decoration of Lipid Vesicles by Polyelectrolytes: Mechanism and Structure. Soft Matter 2010, 6, 4471−4481. (13) Hierrezuelo, J.; Sadeghpour, A.; Szilagyi, I.; Vaccaro, A.; Borkovec, M. Electrostatic Stabilization of Charged Colloidal Particles with Adsorbed Polyelectrolytes of Opposite Charge. Langmuir 2010, 26, 15109−15111. (14) Sikor, M.; Sabin, J.; Keyvanloo, A.; Schneider, M. F.; Thewalt, J. L.; Bailey, A. E.; Frisken, B. J. Interaction of a Charged Polymer with Zwitterionic Lipid Vesicles. Langmuir 2010, 26, 4095−4102. (15) Mayer, L.; Hope, M.; Cullis, P. R.; Janoff, A. Solute Distributions and Trapping Efficiencies Observed in Freeze-Thawed Multilamellar Vesicles. Biochim. Biophys. Acta 1985, 817, 193−196. (16) Patty, P.; Frisken, B. The Pressure-Dependence of the Size of Extruded Vesicles. Biophys. J. 2003, 85, 996−1004. (17) Sabin, J.; Prieto, G.; Ruso, J. M.; Messina, P.; Sarmiento, F. Aggregation of Liposomes in Presence of La3+: A Study of the Fractal Dimension. Phys. Rev. E 2007, 76, 011408. (18) Sabin, J.; Prieto, G.; Sarmiento, F. Studying Colloidal Aggregation using Liposomes. Methods Mol. Biol. 2010, 606, 189−198. (19) Ballif, J. B.; Lerf, C.; Schlapfer, C. W. The Proton Affinity Spectrum of Polyethylenimine. Chimia 1994, 48, 336−340.

ASSOCIATED CONTENT

S Supporting Information *

Size of the PEI-decorated DMPG liposomes as a function of the PEI concentration at 10, 25, 30, and 60 °C in a HEPES buffer. This material is available free of charge via the Internet at http://pubs.acs.org.

■

ACKNOWLEDGMENTS

This work was supported by the Spanish “Ministerio de Economiá y Competitividad” (Project MAT2011-26330) and by the “European Regional Development Fund (ERDF)” and from “Xunta de Galicia” (Project INCITE08PXIB206030PR). J.S. is supported by the “Á ngeles Alvariño” Program of the “Xunta de Galicia”. We also thank Dr. M. Alatorre for his valuable comments in the preparation of the PEI solutions.

4. CONCLUSIONS The study of the interaction of a cationic polymer as PEI with phospholipid membranes is of special relevance for gene therapy because the PEI is a potential nonviral vector to transfer DNA in living cells. Besides, the PEI−liposomes complexes present a stability behavior not predicted by the classical DLVO theory for colloidal systems. In this work we presented a systematic study of the stability of different PEI-decorated liposomes as a function of the headgroup of the lipid, the size of the initial liposomes, pH, temperature and the charge ratio R±. Results were analyzed using the interaction potential proposed by Velegol and Thwar,31 which takes into account the effect of the nonuniform distribution of the electric charge on the surface of the polyelectrolyte decorated liposomes. The strong buffer capacity of the PEI alters the dependence of the zeta potential of the complex with the PEI concentration. We have demonstrated that using the proper buffer and the proper initial liposome size, a double charge inversion of the PEI-decorated liposomes built up with zwitterionic and anionic lipids can be observed. In such cases, the complexes present a very rich stability behavior with three different ranges of PEI concentration where the system is stable and two ranges where a phase separation occurs. In the range of PEI concentrations where aggregation occurs we can also distinguish regions where the aggregation leads to stable clusters and other regions where the clusters grow continuously. Experiments carried out with DOPC liposomes show that small changes of only 30% in the diameter of the liposomes lead to a rather different stability behavior of the PEI− liposomes complexes. This strong dependence of the interaction potential on the initial size gives further support to the hypothesis that nonuniform charge distribution interactions govern the stability of the complexes. An overview of our results shows a remarkable dependence of the stability on the pH and the initial size of the liposomes which explains the low reproducibility of the experiments if no special care is taking in the preparation of the samples. Small changes in the pH or in the liposomes diameter lead to a completely different stability behavior.

■

■

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Fax +34 881814112; Ph +34 881814048. Notes

The authors declare no competing financial interest. 10541

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542

Langmuir

Article

(40) Jeffrey, G.; Ottewill, R. Reversible Aggregation part I. Reversible Flocculation Monitored by Turbidity Measurements. Colloid Polym. Sci. 1988, 266, 173−179. (41) Sennato, S.; Truzzolillo, D.; Bordi, F.; Cametti, C. Effect of Temperature on the Reentrant Condensation in PolyelectrolyteLiposome Complexation. Langmuir 2008, 24, 12181−12188. (42) Adamczyk, Z.; Michna, A.; Szaraniec, M.; Bratek, A.; Barbasz, J. Characterization of Poly(Ethylene Imine) Layers on Mica by the Streaming Potential and Particle Deposition Methods. J. Colloid Interface Sci. 2007, 313, 86−96. (43) Griffiths, P. C.; Paul, A.; Stilbs, P.; Petterson, E. Charge on Poly(Ethylene Imine): Comparing Electrophoretic NMR Measurements and pH Titrations. Macromolecules 2005, 38, 3539−3542.

(20) Felgner, P.; Barenholz, Y.; Behr, J.; Cheng, S.; Cullis, P.; Huang, L.; Jessee, J.; Seymour, L.; Szoka, F.; Thierry, A.; Wagner, E.; Wu, G. Nomenclature for Synthetic Gene Delivery Systems. Hum. Gene Ther. 1997, 8, 511−512. (21) Alatorre-Meda, M.; Taboada, P.; Hardl, F.; Wagner, T.; Freis, M.; Rodriguez, J. R. The Influence of Chitosan Valence on the Complexation and Transfection of DNA the Weaker the DNAChitosan Binding the Higher the Transfection Efficiency. Colloids Surf., B 2011, 82, 54−62. (22) Volodkin, D.; Mohwald, H.; Voegel, J.; Ball, V. Coating of Negatively Charged Liposomes by Polylysine: Drug Release Study. J. Controlled Release 2007, 117, 111−120. (23) Bordi, F.; Cametti, C.; Sennato, S.; Truzzolillo, D. Strong Repulsive Interactions in Polyelectrolyte-Liposome Clusters Close to the Isoelectric Point: A Sign of an Arrested State. Phys. Rev. E 2007, 76, 061403. (24) Grosberg, A.; Nguyen, T.; Shklovskii, B. Colloquium: The Physics of Charge Inversion in Chemical and Biological Systems RID B-1956−2009. Rev. Mod. Phys. 2002, 74, 329−345. (25) Dobrynin, A.; Deshkovski, A.; Rubinstein, M. Adsorption of Polyelectrolytes at Oppositely Charged Surfaces. Macromolecules 2001, 34, 3421−3436. (26) Nguyen, T.; Shklovskii, B. Model of Inversion of DNA Charge by a Positive Polymer: Fractionalization of the Polymer Charge RID B1956−2009. Phys. Rev. Lett. 2002, 89, 018101. (27) Akinc, A.; Thomas, M.; Klibanov, A.; Langer, R. Exploring Polyethylenimine-Mediated DNA Transfection and the Proton Sponge Hypothesis. J. Gene Med. 2005, 7, 657−663. (28) Ho, Y.; Liao, Z.; Panda, N.; Tang, D.; Yu, S.; Mi, F.; Sung, H. Self-Organized Nanoparticles Prepared by Guanidine- and DisulfideModified Chitosan as a Gene Delivery Carrier. J. Mater. Chem. 2011, 21, 16918−16927. (29) Remy, J.; Abdallah, B.; Zanta, M.; Boussif, O.; Behr, J.; Demeneix, B. Gene Transfer with Lipospermines and Polyethylenimines RID A-9821−2011. Adv. Drug Delivery Rev. 1998, 30, 85−95. (30) Jeong, J. H.; Kim, S. W.; Park, T. G. Molecular Design of Functional Polymers for Gene Therapy. Prog. Polym. Sci. 2007, 32, 1239−1274. (31) Velegol, D.; Thwar, P. K. Analytical Model for the Effect of Surface Charge Nonuniformity on Colloidal Interactions. Langmuir 2001, 17, 7687−7693. (32) Sabin, J.; Prieto, G.; Blanco, E.; Ruso, J. M.; Angelini, R.; Bordi, F.; Sarmiento, F. Interaction of Gadolinium with Phospholipids Bilayer Membranes - Photon Correlation Spectroscopy and DSC Study. J. Therm. Anal. Calorim. 2007, 87, 199−203. (33) Tadmor, R. The London-Van Der Waals Interaction Energy between Objects of various Geometries. J. Phys.: Condens. Matter 2001, 13, L195−L202. (34) Kucerka, N.; Kiselev, M.; Balgavy, P. Determination of Bilayer Thickness and Lipid Surface Area in Unilamellar Dimyristoylphosphatidylcholine Vesicles from Small-Angle Neutron Scattering Curves: A Comparison of Evaluation Methods. Eur. Biophys. J. Biophys. Lett. 2004, 33, 328−334. (35) Loosleymillman, M.; Rand, R.; Parsegian, V. Effects of MonoValent Ion Binding and Screening on Measured Electrostatic Forces between Charged Phospholipid-Bilayers. Biophys. J. 1982, 40, 221− 232. (36) Truzzolillo, D.; Bordi, F.; Sciortino, F.; Cametti, C. Kinetic Arrest in Polyion-Induced Inhomogeneously Charged Colloidal Particle Aggregation. Eur. Phys. J. E 2009, 29, 229−237. (37) Sennato, S.; Truzzolillo, D.; Bordi, F.; Sciortino, F.; Cametti, C. Colloidal Particle Aggregates Induced by Particle Surface Charge Heterogeneity. Colloidd Surf., A 2009, 343, 34−42. (38) Molina-Bolivar, J.; Galisteo-Gonzalez, F.; Hidalgo-Alvarez, R. Colloidal Aggregation in Energy Minima of Restricted Depth. J. Chem. Phys. 1999, 110, 5412−5420. (39) Sabin, J.; Prieto, G.; Ruso, J. M.; Sarmiento, F. Fractal Aggregates Induced by Liposome-Liposome Interaction in the Presence of Ca2+. Eur. Phys. J. E 2007, 24, 201−210. 10542

dx.doi.org/10.1021/la3019259 | Langmuir 2012, 28, 10534−10542