Monitoring the Collapse of pH-Sensitive Liposomal Nanocarriers and

Article pubs.acs.org/molecularpharmaceutics

Monitoring the Collapse of pH-Sensitive Liposomal Nanocarriers and Environmental pH Simultaneously: A Fluorescence-Based Approach Sören Draffehn and Michael U. Kumke* Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany S Supporting Information *

ABSTRACT: Nowadays, the encapsulation of therapeutic compounds in socalled carrier systems is a very smart method to achieve protection as well as an improvement of their temporal and spatial distribution. After the successful transport to the point of care, the delivery has to be released under controlled conditions. To monitor the triggered release from the carrier, we investigated different fluorescent probes regarding their response to the pH-induced collapse of pH-sensitive liposomes (pHSLip), which occurs when the environmental pH falls below a critical value. Depending on the probe, the fluorescence decay time as well as fluorescence anisotropy can be used equally as key parameters for monitoring the collapse. Especially the application of a fluorescein labeled fatty acid (fPA) enabled the monitoring of the pHSLips collapse and the pH of its microenvironment simultaneously without interference. Varying the pH in the range of 3 < pH < 9, anisotropy data revealed the critical pH value at which the collapse of the pHSLips occurs. Complementary methods, e.g., fluorescence correlation spectroscopy and dynamic light scattering, supported the analysis based on the decay time and anisotropy. Additional experiments with varying incubation times yielded information on the kinetics of the liposomal collapse. KEYWORDS: pH-sensitive liposome, drug carrier system, selective drug release, intracellular pH indicator, time-resolved fluorescence spectroscopy, fluorescence anisotropy, fluorescence correlation spectroscopy

I. INTRODUCTION One approach to increase the chance of cure and improve the administration of drugs (e.g., anticancer) is the application of transport vehicles in which these compounds are encapsulated. During the last decades, medical science, especially the field of drug delivery, was tremendously influenced by nanotechnology, which enabled novel therapeutic as well as diagnostic strategies.1 The encapsulation of drug molecules results in many advantages. The carrier acts as protection as it isolates them from environmental impact and prevents their unwanted degradation, but also enzymatic and chemical inactivation. In turn, this leads to increased duration times in blood circulation as well as improves temporal and spatial distribution of agents.2 However, the embedding reduces side effects as well as the toxicity of the drugs and minimizes the exposure to healthy cells. In cancer therapy liposomal drug delivery is frequently used.2−4 Liposomes are biocompatible as well as biodegradable, and due to their structure, they enable the transport of both hydrophilic water-soluble molecules in the aqueous core and hydrophobic compounds in the lipid membrane. Focused research developed liposomes that are characterized by high mechanical stability, long circulation times, high drug loading, enhanced penetration, and target specificity.3 Although, in some cases, a fast release of the drug is necessary to show its full effect, conventional liposomes are © XXXX American Chemical Society

limited, and the drug may not be completely bioavailable when embedded in the carrier. Hence, the challenge is to improve the control of the drug release and to enable spontaneous full release at the desired site. Therefore, stimuli-responsive liposomes have been introduced, which can release their load triggered by different stimuli, e.g., pH,5 redox potential,6 hyperthermia,7 or (electro)magnetic fields.8,9 Two facts make the application of pH sensitive drug carriers for the targeted and selective release of therapeutic agents in tumor medication favorable. On the one hand, a mildly acidic extracellular environment (pH 6.0−7.0) compared to healthy cells (pH 7.4) characterizes malignant tissues and areas of inflammation or infection.5 On the other hand, following binding to the surface of cells, liposomes can fuse with the cellular membrane or are internalized by endocytosis. In the latter case, during the endocytotic pathway, liposomes will become imprisoned in endosomes that are characterized by descending pH, and hence, pH-sensitive carrier can release their content into cytoplasm by either fusing with or destabilizing the endovacuolar membrane. Received: January 22, 2016 Revised: April 3, 2016 Accepted: April 5, 2016

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DOI: 10.1021/acs.molpharmaceut.6b00064 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

from the transporter would help to distinguish between the distribution of the tracer and the whole transport system. Recently, fluorescence-based techniques were used to observe the drug release, such as FRET between organic dyes,22 or aggregation-induced emission phenomenon of gold nanoclusters.23 To monitor the collapse of pHSLips, we investigated a set of dyes that are very different in their probe properties. Incorporated in liposomes, the probes are located in a hydrophobic microenvironment with a restricted rotational mobility. Upon collapse and the subsequent release, a change to a polar and stronger hydrophilic microenvironment in combination with an increased rotational mobility is expected for the probes in general. Hence, we analyzed chemically different probes regarding their response to changes in the micropolarity as well as microviscosity. On the one side, we investigated dyes that are known to be sensitive toward the polarity of their environment, which results in a change of their photophysical properties (nitrobenzoxadiazol, NBD;24 diphenylhexatriene, DPH).25,26 On the other side, we examined dyes that are not sensitive to the polarity but for which a fluorescence-related response upon changes in their molecular mobility was expected. However, the separation into hydrophilic and hydrophobic dyes offers the opportunity to analyze the influence of the partitioning behavior of the dye moiety. Relying on parameters such as the fluorescence decay time and anisotropy avoids interference from photobleaching or decomposition of the probe since these parameters are determined independently of the dye concentration. In addition, we utilized the well-known intrinsic pH-sensitivity of fluorescein27−30 labeled to palmitic acid, fPA, to monitor the pH of the surrounding media as an additional chemical parameter simultaneously. Complementary to the fluorescence measurements the size of the different pHSLips was investigated with different established methods, like fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS). Since the size of the liposomes plays a very important role in the application as drug carrier,31 in addition to the kinetics of the collapse, we also analyzed the size stability of the liposomes.

Several pH-sensitive transporters for drugs and other therapeutic compounds have been presented so far, including polymer-based nanoparticles, micelles, and liposomes.10 Different strategies emerged to create pH-sensitive liposome (pHSLip)-based delivery systems, e.g., pH triggerable components like polyhistidine residues,11,12 polymers with protonatable groups13,14 or cleavable bonds,15−17 and pH-sensitive lipids.18 However, in most studies presented so far, the detailed characterization of the critical pH value (pHcoll) at which the collapse occurs if the surrounding pH changes is missing. The same is true for the fate of the compounds that constituted the liposome. In this study, we demonstrate a straightforward strategy to monitor the collapse of pHSLips. Among others, we used DOPE-based liposomes that are stabilized by, e.g., cholesteryl hemisuccinate (CHEMS) or oleic acid (OA) as standard system.19,20 Furthermore, we investigated different pH-sensitive lipids that were used in combination with DOPE but also in single component pHSLips. In addition, we performed reference experiments with POPC liposomes as a pH-insensitive liposome under identical conditions. The pH-sensitivity of DOPE-based pHSLips resulted from the molecular shape of these phospholipids and their strong tendency to accumulate in inverse hexagonal phases (HII) instead of lamellar phases like in double layers (Figure 1).5

Figure 1. Scheme of DOPE-based pHSLips. At physiological pH the negatively charged functional group of the stabilizing component creates repulsive forces with the phosphate group of DOPE, which leads to the formation of a lamellar phase and thus liposomes. A decrease in the pH induces the formation of hexagonal (HII) phase and a subsequent collapse of the liposomes.

II. EXPERIMENTAL SECTION II.1. Material and Sample Preparation. The lipids 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-succinate (DOGS), N-(4-carboxybenzyl)N , N - d i m e t h y l - 2 , 3 - b i s ( o l e o y l o x y ) p r o p a n- 1 - a m i n i u m (DOBAQ), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and oleic acid (OA) were purchased from Avanti Polar Lipids (Alabaster, USA) (Figure 2). Also the dye labeled lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7nitro-2−1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (RhB), and 1palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-snglycero-3-phosphoethanolamine (TopFluor) were from Avanti Polar Lipids. 5-Hexadecanoylaminofluorescein (fPA) was obtained from Life Technologies (Darmstadt, Germany). Cholesteryl hemisuccinate (CHEMS), 1,6-diphenyl-1,3,5-hexatriene (DPH), N,N′-Bis(1-hexylheptyl)-perylene-3,4:9,10-bis(dicarboximide) (Per), and other chemicals and solvents were from Sigma-Aldrich (Steinheim, Germany). Liposomes were prepared by dissolving the lipids and optionally the dye in chloroform and drying under nitrogen

Nevertheless, the alliance of DOPE and stabilizing components with negative charged functional groups at physiological pH creates repulsive forces with the phosphate group of DOPE and thus favors the formation of lipid bilayers leading to liposomal constructs. However, the exposure to acidic pH at endosomal stage leads to protonation of the pH-sensitive component accompanied by the loss of repulsion (Figure 1). This will end in the reorganization of DOPE in a HII phase promoting fusion with or destabilization of endosomal membrane, which in turn induces the release of the cargo into the cytoplasm. For the analysis of the distribution and behavior of drug carrier systems, usually radioactive tracers are used, which have the drawback of not responding to the separation of the radiotracer from the transporter.21 Hence, it is not possible to differentiate between bound and separated tracer after administration. A suitable label that responds to the release B


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emission polarizer in vertical (IVV) or horizontal (IVH) position. The subscripts describe the orientation of the polarizers: V, vertical; H, horizontal. The fluorescence anisotropy r was calculated by eq 1.33 Using horizontally polarized excitation light, the instrumental correction factor G was determined. r=

stream as well as at high vacuum overnight. The dry lipid film then was suspended by vortex mixing and 60 min incubation at 50 °C in a citric acid/phosphate buffer of different ratios to adjust the pH (citric acid stock, 21.0 mg/mL; disodium phosphate stock, 35.6 mg/mL).32 The final concentration of lipids was 250 μM for spectroscopic measurements and 500 μM for dynamic light scattering experiments. The suspensions were subjected to five freeze−thaw cycles in liquid nitrogen and were vortexed after each cycle. Afterward, the suspensions were 35 times extruded using a Mini-Extruder (Avanti Polar Lipids, Alabaster, USA) and two-stacked polycarbonate filters with a pore size of 30 nm (GE Healthcare, Little Chalfont, UK). II.2. Steady-State Fluorescence and Depolarization. Steady-state fluorescence as well as fluorescence depolarization measurements were carried out using a Fluoromax3 fluorescence spectrometer (Jobin Yvon). Typical excitation and emission wavelengths are summarized in Table 1. For depolarization experiments, the fluorescence was detected after excitation with vertically polarized light and with the

2

I(t ) = Ibackground + 2

⟨τ ⟩ =

pH-sensitive component

composition

CHEMS OA DOGS DOGS DOBAQ DOBAQ

DOPE/CHEMS 3:2 DOPE/OA 7:3 DOGS DOPE/DOGS 3:2 DOBAQ DOPE/DOBAQ 3:2

⎧

t⎫ ⎬ with i = 1, 2 ⎩ τi ⎭

∑ αi exp⎨− i=1

(2)

2

∑ αiτi/∑ αi with i = 1, 2 i=1

i=1

(3)

II.4. Fluorescence Correlation Spectroscopy (FCS). By fluorescence correlation spectroscopy (FCS) experiments, we determined the diffusion coefficient of the probe fPA. If it is freely diffusing without any hindrance, the determined diffusion coefficient correlates to the probe molecule itself. However, in the case of an incorporated probe, e.g., in liposomes, the observed diffusion corresponds to that macromolecular entity. II.4.1. Experimental Setup. For FCS measurements, we used a confocal fluorescence microscope system MicroTime 200 (PicoQuant) in combination with the inverse microscope IX-71 (Olympus). The samples were excited by a supercontinuum laser source (SC-400-2, Fianium) with a repetition of 20 MHz and an output of (120 ± 20) μW. The excitation light was filtered by an AOTF to λex = 467 nm and then focused into the sample via a dichroic mirror (z467/638rpc, AHF Analysentechnik) and an oil-immersion objective (Zeiss PlanApo, 100×, NA 1.4). Emission light was filtered by the same dichroic

Table 1. Composition of the Investigated pH-Sensitive Liposomes sample

(1)

Based on fluorescence, the rotational mobility of a probe was analyzed by fluorescence depolarization experiments. The more a fluorescent molecule is restricted in its rotation, the less depolarized is the emission compared to the polarized excitation light resulting in a high fluorescence anisotropy value. Probes that are located in intact liposomes experience a strongly hindered rotational mobility due to the close packaging in the liposomal membrane. Thus, the probe shows high anisotropy values. Collapsing liposomes will release the probe, which is no longer constrained in its rotational movement, and hence, it possesses an undisturbed rotation and low anisotropy. The change in anisotropy has been used, for example, to investigate the interaction of different drug molecules with liposomes.34 II.3. Time-Resolved Fluorescence. Time-resolved fluorescence measurements were performed by using a FL920 fluorescence lifetime spectrometer (Edinburgh Instruments), which works in a time-correlated single-photon-counting (TCSPC) mode. For exciting the samples, we used a supercontinuum laser source (SC-400-PP, Fianium), and a multichannel plate (ELDI EM1-132/300; Europhoton GmbH) detected the emission light. For fluorescence decay measurements, the excitation polarizer was set to vertical orientation and the emission polarizer to magic angle condition (54.7°) to avoid anisotropic artifacts. The decay curves were fitted either mono- or biexponentially with αi being the amplitude of the ith component with the corresponding decay time τi (eq 2). In the case of a biexponential fit, we used the average decay time ⟨τ⟩, which is referred to as only “decay time” in the following and was calculated according to eq 3.

Figure 2. Molecular structures of the lipids we used in this study: (A) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE; (B) cholesteryl hemisuccinate CHEMS; (C) 1,2-dioleoyl-sn-glycero-3-succinate, DOGS; (D) N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, DOBAQ; (E) oleic acid, OA; (F) 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine, POPC.

DOPE/CHEMS DOPE/OA DOGS DOPE/DOGS DOBAQ DOPE/DOBAQ

IVV − GIVH I with G = HV IVV + 2GIVH IHH

C


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Molecular Pharmaceutics mirror as well as a 500 nm long pass filter (LP500, AHF Analysentechnik) before passing a 30 μm pinhole and impinging onto two avalanche photodiodes (SPCM-AQR-13 and SPCM-CD-2801, PerkinElmer). The experiments were performed at room temperature (21 ± 2 °C) for 20−30 min. To calibrate the confocal volume, we used fluorescein dissolved in water (c = 1 nM) with a known diffusion coefficient.35 The SymPhoTime software (PicoQuant) was used for data acquisition and calculation of the corresponding autocorrelation curve, which is based on a cross correlation routine using the signal of both photodiodes. II.4.2. Data Analysis. The autocorrelation function G(τ) of free diffusing probes that possess the diffusion coefficient D were analyzed by a 3-dimensional model (eq 4) in which the intensity distribution along the optical axis was assumed to be Gaussian.33

III. RESULTS AND DISCUSSION III.1. Fluorescent Probes for Liposome Collapse Monitoring. Fluorescent probes can monitor the viscosity as well as the polarity of their microenvironment. Consequently, such fluorescent probes are promising candidates to monitor the collapse of micellar or liposomal systems. An evaluation of different probes (vide inf ra) was carried out using DOPE/ CHEMS as a standard pH-sensitive liposomal system. POPCbased liposomes were used as a reference since the POPC system shows no response to pH-alterations. In first screening experiments for fluorescent probes, the liposomes were exposed to either physiological pH (pH 7) or acidic pH (4−5) in order to induce the liposomes to collapse. Since fluorescence intensity-based measurements are prone to variations in the concentration, to leeching effects, and to photobleaching of the dye, the fluorescence decay times and the fluorescence anisotropy were evaluated as preferable detection parameters. III.1.1. Fluorescence Decay Time. Figure 3 shows the fluorescence decay time of the investigated dyes incorporated in

−1 −1/2 ⎛ w0 2 4Dτ ⎞ ⎛ 4Dτ ⎞ ⎟ ⎜ ⎟ + = G D(τ ) = N −1⎜1 + 1 with D 4τD w0 2 ⎠ ⎝ z02 ⎠ ⎝

(4)

N and τD describe the average number and average diffusion time, respectively, of fluorescent molecules that diffuse through the focal volume, expressed by the radial (w0) and axial (z0) radii. In addition to diffusion, other processes may cause fluctuations in the probe fluorescence. If the probe undergoes transitions to triplet states, the autocorrelation function is given by a product of the diffusion part GD(τ) and contribution GT(τ) considering triplet transitions. G(τ ) = G D(τ ) × GT(τ ) −1 −1/2 ⎛ 4Dτ ⎞ ⎛ 4Dτ ⎞ ⎟ ⎟ ⎜1 + = N ⎜1 + w0 2 ⎠ ⎝ z02 ⎠ ⎝ ⎡ ⎧ τ ⎫⎤ AT exp⎨− ⎬⎥ × ⎢1 + ⎢⎣ 1 − AT ⎩ τT ⎭⎥⎦

Figure 3. (Average) Fluorescence decay times of the different analyzed dyes incorporated in DOPE/CHEMS or POPC liposomes and incubated for ∼12 h under physiological or acidic conditions. Shown are the mean values with the standard deviation as error bars. Each experiment was performed at least three times, and statistically significant differences (p < 0.05, determined by Student’s t test) between samples exposed to physiological and acidic pH are marked by an asterisk and ns = not significant. For data, see Table SI1.

−1

(5)

In that equation, τT is the correlation time of the triplet transition and AT is the average fraction of molecules in that triplet state. Applying the Stokes−Einstein equation, from the diffusion coefficient of the liposomes we were able to calculate the hydrodynamic particle size, which is twice the hydrodynamic radius Rh.36 Rh =

kBT 6πηD

pHSLip built from DOPE/CHEMS before (physiologic pH) and after the collapse induced by acidification. The fluorescence decay kinetics of DPH changed from monoexponential under physiological pH to biexponential with a shorter decay time at low pH. Also for NBD, we found a distinct decrease in the fluorescence decay time from 8 to 1.4 ns upon acidification. This change is a result from the polarity dependence of these probes since the release of the probe into the aqueous phase causes a translocation from a hydrophobic to hydrophilic environment. Both dyes were also incorporated in POPC liposomes, which were used as a pH-insensitive reference system. Here, no alteration in the fluorescence decay kinetics of both dyes upon acidification was found (Figure 3). In contrast, the fluorescence probe Per did not show a change in the decay time kinetics in both liposomal systems investigated since this probe is known to be insensitive to the polarity of its molecular environment. As a second group of probes, hydrophilic or charged dyes modified by a “hydrophobic anchor” to accomplish an efficient incorporation into the phospholipid layer of liposomes, were investigated. We used fatty acid or phospholipid labeled dyes to investigate their potential as probes for monitoring the collapse of liposomes. The fluorescence decay kinetics of RhB in both

(6)

kB is the Boltzmann constant, T is the temperature, and η the viscosity of the surrounding medium. Since all experiments were performed with moderately low liposome concentrations, we assumed the viscosity of these solutions were very similar to the viscosity of water and used that value for calculations. II.5. Dynamic Light Scattering (DLS). Using a Zetasizer Nano ZS (Malvern Instruments, Germany) equipped with a He−Ne laser source (633 nm) the size of the liposomes was analyzed by DLS. The detection of the scattered light at 173° supported to reduce artifacts. The sample temperature was adjusted to 25 °C. The liposomes were analyzed at 500 μM (concentration of the phospholipids). The particle sizes shown in this report are number distribution based averaged values as this value mirrors the majority of particles. D


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incorporated in DOPE/CHEMS as well as in POPC liposomes. The anisotropy r was calculated based on fluorescence depolarization measurements (eq 1). We found a distinct decrease in the anisotropy for DPH, Per, and NBD in DOPE/ CHEMS upon acidification (in contrast to POPC liposomes). The observed overall alteration in r was large and is therefore a good indicator to monitor the collapse of liposomes. However, since all three dyes are (almost) not soluble in water, the absolute fluorescence signal intensity decreased dramatically,24,25 and subsequently, the quality of the emission data collected at low pH is poor leading to larger uncertainties in the calculated r-values, which limits the applicability of r in combination with these probes. The dyes TopFluor and RhB did not show a significant change in the fluorescence anisotropy upon acidification, neither in DOPE/CHEMS nor in POPC liposomes. However, the probe fPA showed a distinct decrease in r, which can be attributed to the release when the DOPE/CHEMS liposomes collapsed. Incorporated in POPC liposomes, a slight increase of r was observed under acidic conditions (Figure 4). Therefore, the decrease in r clearly indicates the collapse of the DOPE/ CHEMS liposomes. III.1.3. Concentration Dependency of fPA Signal. From the analysis of the fluorescence decay kinetics as well as the anisotropy, fPA was identified as the probe of choice for further investigations of the pHSLips collapse. Especially because in addition to the collapse response shown by the anisotropy r, both the fluorescence decay time and spectrum of fPA enabled the direct pH determination of the solution, which makes fPA a multiparameter probe (section III.6). In order to exclude artifacts in the analysis of the pHSLips collapse the degree of fPA loading was investigated up to 5 mol % dye content. With increasing fPA loading of liposomes the possibility of dye intermolecular interactions is also increasing. Effects such as reabsorption of fluorescence, homoFRET, or the formation of “dark” dimers may occur and could obscure the probe’s performance. Figure 5 shows the alteration of r and τ with increasing concentration of fPA. Like for the most fluorescent probes, there is an overlap between the absorption and fluorescence emission of fPA, and therefore, reabsorption of emission light as well as the possibility of homoFRET may have to be taken into account at higher concentrations. A comparison of the emission spectra with increasing dye concentration revealed no changes in the spectral intensity

DOPE/CHEMS and POPC liposomes were almost independent of the pH. Only a small hypsochromic shift of about 4 nm in the fluorescence spectrum of RhB due to the collapse of DOPE/CHEMS was observed (data not shown). The fluorescence decay kinetics of TopFluor showed a small but distinct change. The respective fluorescence decay time decreased from 6.3 to 5.4 ns upon acidification, while in the reference system (POPC) no alteration of τ was observed. As a third candidate, carboxyfluorescein labeled palmitic acid (fPA) was investigated. Here, the fluorescein moiety has an additional intrinsic pH-sensitive fluorescence response. As shown in Figure 3, the fluorescence decay time of fPA incorporated in DOPE/CHEMS decreased from 3.9 to 2.9 ns in response to acidification and the subsequent collapse of the DOPE/ CHEMS liposomes. In contrast to the other fluorescent probes investigated, fPA also showed a slight change in the fluorescence decay time upon acidification when incorporated in POPC liposomes. The intrinsic pH-sensitivity of fPA (section III.6) in combination with the localization in the interfacial region of the liposomes are responsible for the observed specific fluorescence response of this probe.27,28 III.1.2. Fluorescence Anisotropy. Figure 4 shows the steadystate fluorescence anisotropy r of the investigated dyes

Figure 4. Fluorescence anisotropy of the different analyzed dyes incorporated in DOPE/CHEMS or POPC liposomes and incubated for ∼12 h under physiological or acidic conditions. Shown are the mean values with the standard deviation as error bars. Each experiment was performed at least three times, and statistically significant differences (p < 0.05, determined by Student’s t test) between samples exposed to physiological and acidic pH are marked by an asterisk and ns = not significant. For data, see Table SI2.

Figure 5. (left) Fluorescence anisotropy values (determined from excitation spectrum for λex = 430−480 nm) and (right) fluorescence decay time of fPA incorporated in DOPE/CHEMS liposomes at physiological pH (7.4). The incubation time was ∼12 h. The amount of incorporated fPA was increased from 0.2 to 4.8 mol % (0.5 to 12 μM). E


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Molecular Pharmaceutics distribution, which indicates that reabsorption is not influencing the measurements (Figure SI1). The fairly strong changes in r and τ point to contributions from homoFRET37,38 (variation in r) and a dynamic self-quenching due to the formation of “dark” complexes (shortened τ).39 Based on the data shown, the concentration of fPA was kept below 0.5 mol % (1.25 μM) in further experiments in order to avoid or at least minimize any contribution from dye−dye interactions. III.2. Size of Liposomes. In the fluorescence experiments, the collapse of the different liposomes investigated was deduced from the changes in the fluorescence parameters r and τ of various fluorescent probes, especially fPA. In order to support these findings, complementary experiments were carried out using techniques, which directly monitor the size of aggregates or particles. Moreover, for a possible application as drug carrier, the size of the liposomes is an important parameter influencing the performance of the carrier system, e.g., the retention time of the transporter. To determine the size of the different liposomes under physiological pH conditions as well as at acidic conditions (to confirm the collapse of the pHSLips), we applied different experimental techniques: fluorescence correlation spectroscopy (FCS) using fPA as probe and dynamic light scattering (DLS). Based on FCS measurements, for DOPE/CHEMS liposomes at physiological pH, we found a particle size of around 90 nm, which agreed fairly well with the size we determined for POPC liposomes (Figure 6). In contrast to pH-insensitive POPC

Figure 7. DLS-based particle sizes of several pHSLips and POPC liposomes after incubation under physiological or acidic pH conditions for ∼12 h. Shown are the mean values with the standard deviation as error bars. Each experiment was performed at least three times, and statistically significant differences (p < 0.05, determined by Student’s t test) between samples exposed to physiological and acidic pH are marked by an asterisk and ns = not significant.

decreases in the particle size after incubation under acidic conditions (except pH-insensitive POPC), which signifies a collapse and hence the pH-response of these pHSLips. III.3. Stability and Collapse Kinetics. In further experiments, we focused on the kinetics of the collapse of DOPE/ CHEMS liposomes following acidification as well as on the stability of DOPE/CHEMS at physiological pH. To investigate the influence of the incubation time on the collapse kinetics or on the size of intact pHSLips, we performed FCS experiments in regular time intervals. This method seems to be appropriate since it allows performing measurements in regular time intervals without time-consuming sample preparation. However, compared to other methods, FCS requires a fluorescent label. Therefore, this method attains selectivity by just observing the labeled molecule/particle of interest. The fluorescence-based approach often was used to study kinetics, e.g., phase separations.40 Figure 8 shows the trend of the particle size of both intact and collapsed liposomes. Particle growth occurred under

Figure 6. Comparison of particle sizes of DOPE/CHEMS and POPC liposomes after incubation for ∼12 h at physiological or acidic pH conditions determined by FCS (black) and DLS (gray). Shown are the mean values with the standard deviation as error bars. Each experiment was performed at least three times, and statistically significant differences (p < 0.05, determined by Student’s t test) between samples exposed to physiological and acidic pH are marked by an asterisk. For data, see Table SI3.

liposomes, DOPE/CHEMS showed a strong decrease in particle size after acidification, which points to a collapse of liposomes. In addition, DLS experiments revealed similar sizes for DOPE/CHEMS liposomes (at physiological pH) and POPC liposomes as well as the collapse of DOPE/CHEMS (Figure 6). These findings support the results based on the fluorescence parameters r and τ and support the application of these as straightforward-parameters to monitor the collapse of pHSLips. Further DLS experiments of other pHSLips showed particle sizes that are in a comparable range to DOPE/CHEMS at physiological pH (Figure 7). Moreover, we found drastic

Figure 8. FCS-based particle sizes of DOPE/CHEMS liposomes after incubation for different duration under physiological or acidic pH conditions.

physiological pH indicated by the increasing size in time. This may be mostly caused by fusion due the higher thermodynamic stability of liposomes of a few hundred nanometers in size and diminishing curvature effects.34 However, also the aggregation of the liposomes (without F


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of pH and a low anisotropy was determined. For pH-sensitive liposomes such as DOPE/CHEMS a sigmoidal trend was measured showing three different regions for the anisotropy. At pH > 6.5 the liposomes were intact and subsequently a high anisotropy was observed, at intermediate pH (6.5 > pH > 4.5) the anisotropy significantly changed with pH and stayed again constant at pH < 4.5 (similar to the anisotropy found for fPA in solutions without liposomes, Figure 9). The collapse of the liposomes is triggered by the protonation of the pH-sensitive component (e.g., CHEMS in the DOPE/CHEMS liposomes) and is indicated by the steep decrease in the anisotropy found in the range 6.5 > pH > 4.5. In the control using POPC (as pHinsensitive liposomes), no such sigmoidal trend was found. For POPC a slight decrease with increasing pH was observed (similar to DOPE/CHEMS at pH > 6.5), which may indicate that either the rotational freedom of fPA is increased or an alteration in the dye−dye interactions occurred (vide supra). Due to the pH-induced change in the protolytic form of fPA, the molecule switched from anionic to dianionic. The higher charge could lead to slightly stronger repulsive forces between the probe molecule and the liposomal double layer, and consequently, fPA has an increased rotational mobility reducing the overall observed anisotropy. III.5. pHcoll Determination of pHSLips. Using the anisotropy values of the pHSLips and POPC as reference, we determined the critical pH value pHcoll at which the collapse of the corresponding pHSLip occurs. In order to remove the intrinsic anisotropy dependency of fPA when incorporated in liposomes, we subtracted the anisotropy value of fPA in POPC liposomes rPOPC from that of fPA in pHSLips rpHSLip resulting in the liposome pH response S of the corresponding pHSLip.

fusion) may be possible, which also leads to a reduced diffusion of the observed system, and thus, bigger particle sizes are calculated. Nevertheless, after an incubation time of approximately 30 h, the particle size seemed to reach a limiting value of about 8 to 10 times the origin value, and no further increase appeared. Nevertheless, the fusion of liposomes is a welldescribed phenomenon, whose mechanism has not been clarified yet.41 That is why interactions between liposomes, e.g., aggregation, fusion, or fission,42 are in the focus of many studies trying to investigate these phenomena by analyzing lipid mixing, content mixing or leakage, or size increase applying different methods, e.g., energy transfer, self-quenching, DLS, or TEM.41,43,44 Incubation of DOPE/CHEMS liposomes under acidic conditions led to the immediate formation of huge aggregates with sizes 2 orders of magnitude higher compared to liposomes under physiological pH values (Figure 8). With elongating incubation time, the apparent particle size decreased, which indicated the release of the probe subsequent to the collapse or disaggregation of pHSLips. The diffusion coefficient of fPA after roughly 36 h of incubation was very similar to that of fPA in the absence of liposomes. In turn, that points to a slow but complete release of the dye molecule over a rather long period. However, the determined particle size using FCS is based on the diffusion coefficient of the fluorescent fPA and thus, we cannot distinguish between a release of the probe from these aggregates and the decomposition of those aggregates. Nevertheless, particle sizes determined by DLS were very similar at long incubation times (Figure 6 and Table SI3). Thus, we concluded that the collapse of pHSLips is accompanied by a formation of huge aggregates and a following slow decomposition of these aggregates. This suggests that FCS results rather point to a slow decomposition, and hence, the release of fPA is the result accompanied by the collapse of the pHSLip. III.4. Monitoring the Collapse of Different pHSLip by fPA. Based on the screening experiments (vide supra) fPA was chosen as fluorescence probe to investigate the collapse of different pHSLip systems in more detail. Figure 9 shows the anisotropy of fPA in the absence and presence of DOPE/ CHEMS or POPC for the pH-range of 2 < pH < 9. In the absence of liposomes, the anisotropy was not influenced by the pH of the solution because fPA is free to rotate independently

S = rpHSLip − rPOPC

(7)

In Figure 10, the extracted liposome pH response S is shown for DOPE/CHEMS. To achieve the corresponding pHcoll, we

Figure 10. Liposome pH response S of DOPE/CHEMS liposomes (black circles) based on analysis of the steady-state fluorescence anisotropy of fPA incorporated in DOPE/CHEMS or POPC liposomes. The red line shows the fit according to eq 10 for the determination of the critical value of pHcoll at which the pHSLip collapsed.

analyzed S for different pHSLips. The analysis was based on the Henderson−Hasselbalch formalism, which assumes a pH depending on equilibrium between a protonated and deprotonated species. Since the pH influences the equilibrium between intact and collapsed liposomes as well, we exploited this formalism to determine pHcoll. The adapted Henderson− Hasselbalch equation (eq 8) describes the relation between pH, pHcoll, and the molar ratio f int of the intact liposomes, which is

Figure 9. Dependency of the steady-state fluorescence anisotropy of fPA incorporated in DOPE/CHEMS (red triangels) or POPC liposomes (black circles) and in the absence of liposomes (w/o liposome, green squares) on the pH. The concentration of fPA was 0.5 mol % (1.25 μM). The samples were incubated for ∼12 h in a buffer system adjusted to certain pH. G


Article


example, the pH decreases gradually during endocytosis and can therefore be used to define the time at which the release of drugs or other therapeutic compounds will occur. III.6. Monitoring pH by fPA. For fluorescein the intrinsically pH-sensitivity is a well-known property and the reason for the frequent application as pH indicator.27−30 Depending on the pH, fPA can exist in four different protolytic species (cationic, neutral, mono-, and dianionic, Figure SI3), which is reflected by alternating fluorescence decay times and spectra.48 To investigate the maintenance of the pH-sensitivity of fPA despite of the incorporation into liposomes, we performed experiments in the absence and presence of different liposomes with varying pH conditions. Both the emission and excitation spectra of fPA revealed changes in response to the alternating pH (Figure SI2). The excitation spectra of fPA revealed characteristics of the four protolytic species, but the emission spectra just showed the change from the dianionic to the anionic form by the presence of a shoulder in the emission spectrum, as those are the only fluorescent species. Furthermore, the decay time indicated a change in the protolytic forms by a shortening due to acidification. Figure 11 shows the decay time of fPA in the

given by the concentration of intact (cint) and collapsed (ccoll) liposomes, f int = cint/(ccoll + cint).45 pH = pH coll + lg

fint 1 − fint

(8)

As the liposome pH response S showed different values for intact and collapsed liposomes, we can describe S as a function of the ratio f int. S = (1 − fint )Scoll + fint Sint

(9)

In that equation, Sint and Scoll are the limiting values of the liposome pH response at very high and very low pH values, respectively. Combining eqs 8 and 9 resulted in eq 10, which gives the liposome pH response S as a function of the pH. S=

(Sint + Scoll ·10 pHcoll − pH) (1 + 10 pHcoll − pH)

(10)

This equation was used to fit the experimental data to obtain pHcoll (Figure 10) and are summarized in Table 2 for different Table 2. Typical Excitation and Emission Wavelengths of Probes Used in This Study probe

excitation wavelength [nm]

emission wavelength [nm]

DPH Per NBD RhB TopFluor fPA

375 484 465 560 500 480

430 526 535 590 520 519

pHSLips. Depending on the pH-sensitive component and the composition of the pHSLips, the critical pHcoll for the collapse of the respective liposomes was in the range of 4.5 < pHcoll < 5.7. Though the typical pKa value of carboxylic acids in water is around 4.8, this value changes when the molecule is incorporated into liposomal membranes and depends on the structure of the membrane.46 For example, molecular dynamics simulations revealed pKa values for OA in different liposomes in the range of 5.4 < pKa < 6.6,47 which agrees fairly well with the finding for DOPE/OA liposomes (5.4 ± 0.1). In addition, this effect also was demonstrated for DOBAQ- and DOGS-based liposomes for which a distinct alteration in the pHcoll values due to the addition of DOPE was found (Table 3); despite of those, liposomes exhibit the same pH-sensitive component. However, having a range of 4.5 < pHcoll < 5.7, a fine-tuning of pHcoll (and subsequently the pH at which the liposomes collapse) is conceivable by adjusting the composition. This information could be useful for the tailoring of drug delivery systems in which the release of a compound from liposomes is planned to be triggered upon an adequate pH change. For

Figure 11. Dependency of the fluorescence decay time of fPA incorporated in DOPE/CHEMS or POPC liposomes and in the absence of liposomes on the pH. The concentration of fPA was 0.5 mol % (1.25 μM). The samples were incubated for ∼12 h in a buffer system adjusted to certain pH.

absence and presence of DOPE/CHEMS as well as POPC. In the absence of liposomes, the decay time decreased by ∼1.5 ns in the investigated range in response to the pH. Also incorporated in DOPE/CHEMS, fPA was still capable of serving as pH sensing probe. The decay times agreed fairly well with those of fPA in the absence of liposomes. Similar to these findings, fPA was also sensitive to pH and showed likewise decay time changes after incorporation into DOPE/OA, DOGS, and DOPE/DOGS (Figure SI4). Noteworthy is that the biggest change in the decay time occurred in the range of 5 < pH < 7. This is very advantageous since during the endocytosis the pH will be reduced from physiological conditions (∼7.4) to values below 5.0,5 which fits the range of the decay time change of fPA very well. In contrast, fPA incorporated in POPC liposomes showed a smaller decrease in the decay time in the range of 5 < pH < 6 (Figure 11). In the case of DOBAQ and DOPE/DOBAQ as pHSLip, the decay time gave a rather similar trend to POPC liposomes instead of the other investigated pHSLips (Figure SI4). This finding points to the presence of either shielding effects that cause less accessibility of fPA for protons or intermolecular interactions between fPA and the corresponding

Table 3. pHcoll Values of the Investigated pHSLips pHSLip DOPE/CHEMS DOPE/OA DOGS DOPE/DOGS DOBAQ DOPE/DOBAQ

pHcoll 5.7 5.4 4.7 5.2 4.5 5.1

± ± ± ± ± ±

0.1 0.1 0.3 0.2 0.2 0.3 H


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Molecular Pharmaceutics phospholipids. However, since we observed distinct pHdependent decay times, we confirmed the maintained pHsensitivity of fPA after incorporation into different liposomes, but with the limitation of having a smaller decay time change in the case of some liposomes. Consequently, we think the analysis of the decay time of fPA is an appropriate method to monitor the endosomal pH. However, since it was reported about dyes that lose their pH-sensitivity when taken up by cells,49 the pH-responding decay times of fPA have to be carefully examined in vivo. Since the fluorescence decay time (as well as the anisotropy) is less prone to photobleaching effects, a decay time-based data evaluation would be also beneficial in imaging experiments.

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Data tables for fluorescence anisotropy values, fluorescence decay time values, and particle sizes (complementary to Figures presented in this publication); additional emission and excitation spectra of fPA at various concentrations and pH values; molecular fraction of protolytic fPA species; decay time values of fPA incorporated in different pHSLips (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +49 331 977 5209. Fax: +49 331 977 5058. Notes

The authors declare no competing financial interest.

IV. CONCLUSION To monitor the triggered drug release of pH-sensitive liposomes (pHSLips), we investigated different fluorescent probes regarding their response to the collapse of these pHSLips. The two groups of probes that were tested containing either hydrophobic dyes (DPH, Per, NBD) or hydrophilic dyes carrying a hydrophobic “anchor” for lipid bilayer incorporation (RhB, TopFluor, fPA). In screening experiments in which DOPE/CHEMS liposomes (and POPC liposomes as pHinsensitive reference) were exposed to either physiological pH or acidic pH (4−5), the fluorescence decay time as well as fluorescence anisotropy were tested as key parameter. Depending on the probe, both parameters could be used equally for monitoring the collapse of the liposomes. Based on the data fPA was chosen as the most suitable probe because it responded to the collapse of pHSLips by a strong anisotropy change, and simultaneously, it offers the possibility to monitor the pH of its microenvironment by using the fluorescence decay time. Equipping the fPA-labeled pHSLips with anticancer drugs would lead to different application in the field of theranostics.50 In addition to the therapeutic effect of the drug, fPA will act as tracer molecule that indicates the mildly acidic environment of tumor tissues and can visualize the drug release. Furthermore, in more detailed experiments (varying the pH in the range of 3 < pH < 9), the anisotropy data were exploited to determine the critical pH value pHcoll at which the pHSLips collapse. Here, we found slightly different values of pHcoll for the investigated pHSLips, which enables the adjustment of the pH-induced release to the needs of the scientific objectives. To verify the fluorescence-based data (decay time and anisotropy), we applied complementary methods that directly allow access to the size of the investigated system. FCS as well as DLS results supported the conclusions made based on the fluorescence decay time and anisotropy, respectively. Further FCS experiments with DOPE/CHEMS liposomes revealed the incubation time as an important parameter. The incubation under acidic conditions caused the formation of huge aggregates and a subsequent but rather slow decomposition. This behavior might be important for the pharmacokinetics of a drug carrier system.

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ACKNOWLEDGMENTS The authors are very grateful for the support provided by H.-G. Löhmannsröben (University of Potsdam) and M. Dathe (Leibniz-Institute for Molecular Pharmacology, FMP). For financial support the authors wish to thank the Federal Ministry for Economic Affairs and Energy (contract number 02E11415F).

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Monitoring the Collapse of pH-Sensitive Liposomal Nanocarriers and

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