Characterization of Cell-Penetrating Lipopeptide Micelles by

Article pubs.acs.org/JPCB

Characterization of Cell-Penetrating Lipopeptide Micelles by Spectroscopic Methods Sören Gehne,† Karl Sydow,‡ Margitta Dathe,‡ and Michael U. Kumke*,† †

University of Potsdam, Institute of Chemistry (Physical Chemistry), Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany Leibniz Institute for Molecular Pharmacology (FMP), Robert-Roessle-Str. 10, 13125 Berlin, Germany

‡

ABSTRACT: The transport of bioactive compounds to the site of action is a great challenge. A promising approach to overcome application-related problems is the development of targeting colloidal transport systems, such as micelles which are equipped with uptake mediating moieties. Here, we investigated a set of novel lipopeptides which exhibit a surfactant-like structure due to attachment of two palmitoyl chains to the Nterminus of cationic or anionic amino acid sequences. We analyzed the association behavior of these lipopeptides by using 5(6)-carboxyfluorescein (CF)-labeled derivatives as a fluorescent probe and different spectroscopic methods such as fluorescence anisotropy and fluorescence correlation spectroscopy (FCS). The photophysical properties as well as the diffusion and rotational movements of the CF-labeled lipopeptides were exploited to determine the cmc and the size of the micelles consisting of lipopeptides. We could distinguish cationic and anionic lipopeptides by their association behavior and by studying the interactions with mouse brain capillary endothelial cells (b.end3). The cationic derivatives turned out to be very strong surfactants with a very low cmc in the micromolar range (0.5−14 μM). The unique combination of micelle-forming property and cell-penetrating ability can pave the road for the development of a novel class of efficient drug carrier systems. targets.8 Recently, we developed a lipopeptide that combines the advantages of micelle formation,9,10 uptake mediating properties of CPPs,11−13 and target specificity.14,15 Dipalmitoylation (P2) of the N-terminal amino acid of a highly cationic tandem dimer (A2) of the binding sequence of apolipoprotein E (ApoE) for the low density lipoprotein receptor (LDL) conferred surfactant-like properties upon a peptide called P2A2 (see Figure 1).16,17 A2, P2A2 micelles and peptide-modified liposomes efficiently entered endothelial cells of different blood vessels, but the uptake of micelles was specific for endothelial cells of the blood−brain barrier.18,19 We suggest that these findings open a promising way to impprove drug administration to the brain. The structural basis of the selectivity in terms of particle size, surface charge, peptide sequence, and structure remains to be elucidated. Therefore, three P2A2-derived lipopeptides rich in arginine (R), lysine (K), or glutaminic acid (E) were synthesized (P2R, P2K, P2E). Labeling with the dye 5(6)-carboxyfluorescein (CF) yielded analogues (P2fA2, P2fR, P2fK, P2fE) which allowed the spectroscopic characterization of lipopeptide micelles of P2R, P2K, and P2E, respectively. One disadvantage of micelles is the fact that their effectiveness depends upon the critical micelle concentration (cmc). The cmc determines the concentration of spontaneous association of the monomers. Dilution of micellar formulations

I. INTRODUCTION Many potent drug candidates and potential diagnostic tools fail in further formulation development because of limited solubility, low stability, or high toxicity. To overcome these problems, research has been focused upon the development of nanoparticulate drug carrier systems (DCS).1 The spectrum of such carriers reaches from polymers and colloids over nanocrystals, solid lipid nanoparticles and liposomes to dendrimers and micellar dispersions.1−4 Micelles formed by self-assembly of amphiphilic molecules have been used for decades to solubilize poorly soluble pharmaceuticals.5 The hydrophobic core acts as a microreservoir for encapsulated compounds, and the hydrophilic shell interfaces the biological environment.2,6 Depending on the building units, micelles are rather uniform and small spherical particles with 5−20 nm in diameter. Incorporation of drugs in these carriers results in reduced toxicity, enhanced translocation across physiological barriers, and favorable drug distribution. Micellar preparations for a variety of applications have been suggested (for a review, see ref 2). Thus, micelles demonstrate spontaneous penetration into tissues with a leaky vasculature such as tumors. Variations in the chemical structure of the building units may be used to improve incorporation of drugs with different physicochemical properties or to make the micelles responsive to external stimuli, thereby enhancing and controlling drug delivery.2,7 Attachment of cell recognizing and membrane translocating molecules such as receptor ligands and/or cell penetrating peptides (CPPs) confers specificity upon the carrier and makes bioactive molecules rapidly accessible to cytoplasmic © 2013 American Chemical Society

Received: June 19, 2013 Revised: November 1, 2013 Published: November 4, 2013 14215

dx.doi.org/10.1021/jp406053g | J. Phys. Chem. B 2013, 117, 14215−14225

The Journal of Physical Chemistry B

Article

II. EXPERIMENTAL SECTION II.1. Material and Sample Preparation. The synthesis of the dipalmitoylated peptide P2A2 and the 5(6)-carboxyfluorescein (CF)-labeled compound P2fA2 has been described in detail elsewhere.17−19 Lysine (K)-, arginine (R)-, and glutaminic acid (E)-rich lipopeptides and their CF-bearing analogues (P2fR, P2fK, and P2fE, respectively) were prepared following the same protocol (see Table 1). The N-terminally Table 1. Characteristics of Investigated Lipopeptides molecular mass (g/mol) lipopeptide (abbreviation)

calculated

determined

HPLC purity (%)

P2A2 P2R P2K P2E P2fA2 P2fR P2fK P2fE

3532.79 2853.67 2517.51 2528.79 4019.27 3340.15 3003.99 3015.27

3531.02 2852.42 2516.35 2527.72 4016.69 3338.84 3002.77 3013.05

79.5 73.5 81.6 89.3 99.7 85.2 96.3

dipalmitoylated (P2) peptides have the structure palmitoylWK(palmitoyl)GX with X representing an apolipoprotein Ederived sequence (see Figure 1), a sequence of oligo-arginine (P2R), oligo-lysine (P2K), or anionic glutamic acid residues (P2E). Palmitoyl chains of the CF analogues were attached to the α- and ε-amino groups of an additional N-terminal K, and CF was introduced into the side chain of K at position three (see Figure 1). The purification procedure of the peptides has also been described.18 The molecular masses were determined by mass spectrometry using a ACQUITY UPLC-MS system (Waters). The results correspond to the theoretical values (see Table 1). As P2fE was difficult to purify, no additional purification has been performed after manual coupling of the palmitoyl chains to the peptide sequence. However, the molecular mass of the main peak of the chromatographic analysis reflected the theoretical mass. To investigate the association behavior of the lipopeptides, we performed experiments at various lipopeptide concentrations (P2A2, P2R, P2K, or P2E, respectively). In the experiments, the concentration of the corresponding CFlabeled lipopeptide was kept constant (typically at a concentration of 100 nM, lower in fluorescence correlation spectroscopy). Hence, we could be certain that differences in the spectroscopic properties of the CF-labeled lipopeptides were related to the association behavior of the lipopeptides and reflected the formation of micelles. The lyophilized lipopeptides were dissolved in PBS to ensure biological conditions and were vortexed for 30−60 min to allow for the formation of micelles (dependent on the overall lipopeptide concentration) and to achieve equilibrium between unlabeled and CF-labeled lipopeptides. Solutions containing free CF were prepared as a control. II.2. Absorption and Emission Measurements. The absorption and steady-state emission measurements were performed on a Cary 500 UV/vis spectrometer (Varian) and a Fluoromax3 fluorescence spectrometer (Jobin Yvon), respectively. The fluorescence kinetics measurements were carried out using an FL920 fluorescence lifetime spectrometer (Edinburgh

Figure 1. Structures of P2A2 and P2fA2 (peptide sequences in singleletter amino acid code). Palmitoyl chains were coupled to the Nterminal amino group and a lysine side chain.

after administration will result in release and precipitation of incorporated material as soon as the concentration decreases below the cmc. According to the available literature, the cmc of micelles for pharmaceutical application has to be at least in the lower micromolar range, assuring intact micelles after i.v. administration and dilution during the circulation in the bloodstream.2 Thus, determination of the cmc is an essential step in the development of efficient micellar DCSs. A broad variety of techniques has been used to determine the cmc (e.g., titration calorimetry,20 capillary electrophoresis,21 UV/visspectroscopy, or electrical conductivity method22), but due to their limits in quantification, they are less suitable for compounds forming micelles at or below micromolar concentrations.23 Here we investigated the association behavior of the lipopeptides (P2A2 and its derivates) by sophisticated fluorescence based techniques which are very sensitive to changes in the molar environment caused by self-association. The methods allow the determination of cmc in the micromolar range. We not only analyzed changes in the photophysical properties of CF-labeled lipopeptides by timeresolved and steady-state emission spectroscopy but also analyzed changes in microscopic properties which are due to the association of the lipopeptides. Moreover, fluorescence correlation spectroscopy (FCS) allowed studying the diffusion of fluorescent molecules. Furthermore, we performed timeresolved fluorescence depolarization (TRFD) experiments allowing the analysis of rotational movements of the aggregates as well as of the fluorescence probe CF itself. Both methods, FCS and TRFD, directly monitor microscopic parameters (diffusion coefficient and rotational correlation time, respectively) of the micelles and are of outstanding sensitivity. 14216



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cells (b.end3) and uptake experiments using the method of confocal laser scanning microscopy (CLSM) have been described in detail elsewhere.14,16,18,19 b.end3 cells were cultured on poly-L-lysine coated coverslips positioned in 35 mm plastic culture dishes. After 1 day, the cells were exposed to 1 μM peptide in Dulbecco’s phosphatebuffered saline (DPBS-G) for 1 h at 37 °C. CLSM pictures were taken using an LSM 510 inverted confocal laser scanning microscope equipped with a Plan-Neofluar 100×/1.3 oil objective (Carl Zeiss, Germany). CF was exited at λex = 488 nm with an argon ion laser (200 mW), and the emitted light was filtered using a BP 505-530 band-pass filter. The pinhole was set to 300 μm. A droplet of 0.5% trypan blue in PBS was added to each coverslip to prove cell viability. The dye was excited at 633 nm with a helium−neon laser (15 mW) at 80% intensity, and emission was recorded using a LP650 cutoff filter. II.6. Data Analysis. Fluorescence Anisotropy Decay r(t). Coupling of a chromophore to macromolecules (e.g., peptide, lipopeptide) may alter its photophysical properties, e.g., the fluorescence decay time, as well as the rotational correlation time.29,30 A well established model for the r(t) analysis of dyes like CF is the “wobble-in-a-cone” model (eq 3).25

Instruments) which was operated in the time-correlated singlephoton-counting (TCSPC) mode. The CF and CF-labeled lipopeptides were excited by a supercontinuum laser source (SC-400-PP, Fianium) at λex = 497 nm and a repetition rate set to 20 MHz. The fluorescence was detected at λex = 521 nm in a right-angle configuration (relative to the excitation beam) by a multichannel plate (ELDI EM1-132/300; Europhoton GmbH). To avoid anisotropy artifacts in the measured fluorescence decay curves, the emission polarizer was set to magic angle conditions (54.7°). II.3. Time-Resolved Fluorescence Depolarization (TRFD). Samples containing CF and the CF-labeled lipopeptides were excited by vertical polarized excitation light, and the fluorescence decays I(t) were measured with emission polarizers set to a vertical (IVV(t)) or horizontal (IVH(t)) position. The orientations of the polarizers in the excitation and emission beam are denoted by the subscripts V (vertical) and H (horizontal), respectively. The resulting fluorescence anisotropy decay r(t) was calculated from the fluorescence decays according to eq 1,24 and the G-factor was determined by fitting the synthesized fluorescence decay curve IVV(t) + 2·G·IVH(t).25 r (t ) =

IVV(t ) − GIVH(t ) IVV(t ) + 2GIVH(t )

⎧ ⎫ ⎪ t ⎪ ⎬ + r∞ − r(t ) = (r0 − r∞) exp⎨ ⎪ ⎪ ⎩ φdye ⎭

(1)

Here, I(t) represents the fluorescence intensity at a preset polarizer configuration (V, vertical; H, horizontal) and G is the instrumental correction factor. II.4. Fluorescence Correlation Spectroscopy (FCS). For the FCS measurements, a confocal fluorescence microscope system (MicroTime 200 and Pico-Harp 300 PC-board, Picoquant) equipped with an inverse microscope (IX-71, Olympus) was used. The samples were excited using an AOTF filtered output of a supercontinuum laser source (SC400-2, Fianium) at λex = 467 nm with a repetition rate of 20 MHz and an excitation power of 120 ± 20 μW. The excitation light was directed through a dichroic mirror (z467/638rpc, AHF Analysentechnik) into the microscope, and the laser beam was focused into the sample by an oil-immersion objective (Zeiss PlanApo, 100×, NA 1.4). The resulting fluorescence was collected by the same objective, filtered by a dichroic mirror in combination with a 500 nm long pass filter (LP500, AHF Analysentechnik) to cut off the excitation light, and guided through a 30 μm pinhole. For the detection, two avalanche diodes (SPCM-AQR-13 and SPCM-CD-2801, Perkin-Elmer) were used. The measurements were performed for 60 min at room temperature (21 ± 2 °C), and the data acquisition as well as the calculation of the autocorrelation curves G(τ)24,26,27 were carried out by the instrument software (SymPhoTime 5.3.2.2, PicoQuant, Germany). The autocorrelation curves G(τ) were calculated by cross-correlating photons from both photodiodes. By measuring CF in water (cCF = 1 nM) as the calibration standard with a known diffusion coefficient (D = 4.25 cm2/s at 25 °C28), the focal volume (described by an optical plane w0 and an optical axis z0) was determined. To account for the temperature dependence of the diffusion coefficient D, eq 2 (derived from the Stokes−Einstein equation) was applied: D(T1) = D(T2) ·

T1 η(T2) · T2 η(T1)

(3)

Here, r0 denotes the fundamental anisotropy and r∞ the limiting anisotropy. φdye denotes the rotational correlation time of the dye. The extent of motional restriction can be expressed by the parameter A∞ and is given by eq 4 with the semi cone angle θc. A∞ =

r∞ ⎤2 ⎡1 = ⎢ cos θc(1 + cos θc)⎥ ⎦ ⎣2 r0

(4)

In most cases, the consideration of the overall rotation of the macromolecule (here lipopetide) or of the micelle in addition to the fast (but restricted) motion of the dye itself is recommended. Consequently, r(t) is a result of both rotational motions and eq 3 is modified to eq 5.24 ⎡ ⎤ ⎧ ⎫ ⎧ t ⎫ ⎪ t ⎪ ⎬ + r∞⎥ exp⎨− ⎬ − r(t ) = ⎢(r0 − r∞) exp⎨ ⎪ ⎪ ⎢ ⎥ ⎩ φm ⎭ ⎩ φdye ⎭ ⎣ ⎦ ⎪

⎪

⎪

⎪

(5)

Here, φdye and φm represent the rotational correlation times for the restricted rotation of the dye and for the overall rotation of the macromolecule (or aggregate), respectively. Depending on the sample composition, φm describes the rotation of either monomeric lipopeptides or micelles and consequently φm corresponds to φLP or φmic, respectively. FCS-Autocorrelation Function. The autocorrelation functions G(τ) can be fitted by using a three-dimensional diffusion model (eq 6) assuming a Gaussian intensity distribution along the optical axis.31,32 −1 −1/2 ⎛ 4Dτ ⎞ ⎛ 4Dτ ⎞ ⎟ ⎜ ⎟ G D(τ ) = N −1⎜1 + 1 + w0 2 ⎠ ⎝ z02 ⎠ ⎝

D=

(2)

w0 2 4τD

with

(6)

Here, D is the diffusion coefficient of the fluorescent molecule, N is the average number, and τD the average diffusion time of

II.5. Confocal Laser Scanning Microscopy (CLSM). The cultivation of immortalized mouse brain capillary endothelial 14217



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the fluorescent molecules in the focal volume which is described by the radial (w0) and axial (z0) radii. In addition to diffusion processes for CF as the fluorescent probe, the transition to a triplet state has to be considered.33 The autocorrelation function G(τ) was fitted by eq 7, which is a product of the two contributions GD(τ) and GT(τ) for diffusion and triplet transitions, respectively. G(τ ) = G D(τ ) × GT(τ ) −1 −1/2 ⎛ 4Dτ ⎞ ⎛ 4Dτ ⎞ ⎟ ⎜ ⎟ 1 = N −1⎜1 + + w0 2 ⎠ ⎝ z02 ⎠ ⎝ ⎡ ⎧ τ ⎫⎤ AT exp⎨− ⎬⎥ × ⎢1 + ⎢⎣ 1 − AT ⎩ τT ⎭⎥⎦

(7) Figure 2. Fluorescence spectra of CF and P2fA2 in the absence and presence of P2A2 micelles (λex = 470 nm, cCF = 1 μM, cP2fA2 = 0.1 μM, with micelles: cP2A2 = 6.1 μM).

τT reflects the correlation time of the triplet transition, and AT describes the fraction of molecules in the triplet state. Determination of Critical Micelle Concentration (cmc). We analyzed the dependency of spectroscopic as well as microscopic properties of CF-labeled lipopeptides on the overall lipopeptide concentration cLP (labeled + unlabeled). The experimental data were fitted by the sigmoidal-Boltzmann equation34 (SBE, eq 8). y0 − y∞ + y∞ y= c − cmc 1 + exp LPΔc

{

LP

}

component τF,1 increased compared to P2fA2 alone (see Table 2). Using the differences in the fluorescence spectra as well as in the fluorescence decay kinetics of P2fA2 in the absence and presence of P2A2 micelles, the transition from free lipopeptides to a lipopeptide−micellar system was monitored. In Figure 3a, the maxima of the fluorescence spectra of P2fA2 are depicted at different overall lipopeptide concentrations cLP (cP2A2 + cP2fA2). The emission maximum changed in a sigmoidal manner, and data analysis based on SBE (eq 8) gave a cmc of 1.1 μM (see Tables 2 and 3). A global fit of the decay curves (with different P2A2 concentrations) by a biexponential model yielded τF,1 = 1.3 ns and τF,2 = 4.1 ns with various relative fractions depending on cLP (see Table 2). Depiction of these fractions against cLP showed a steep decrease or rise at 1.0 μM, respectively (see Figure 3b and Table 3). P2R, P2K, and P2E. Accordingly, two cationic lipopeptides (P2R and P2K) as well as an anionic analogue (P2E) which contained as P2A2 two palmitoyl chains were analyzed. In reference experiments, the spectroscopic properties of the CFlipopeptide constructs (P2fR, P2fK, and P2fE) showed spectroscopic properties similar to P2fA2 (see Table 2). Variation of cLP resulted in changes of the fluorescence maximum as well as fluorescence decay time of P2fR and P2fK (see Table 2). Evaluation of steady-state and timeresolved fluorescence data yielded the cmc for each lipopeptide. Similar to P2A2, P2R and P2K exhibit a cmc in the lower micromolar range (see Table 3). In contrast, for the anionic lipopeptide P2E, no alterations in the fluorescence parameters upon variation of cLP were observed. P2fE changed neither the fluorescence spectrum nor the fluorescence decay kinetics as a function of cLP. III.2. Time-Resolved Depolarization (TRFD) Measurements. P2A2. On the basis of TRFD measurements and the subsequent calculation of the fluorescence anisotropy, we were able to study the rotational movements of the CF-labeled lipopeptides in the absence and presence of micelles. Small and fast rotating molecules like an organic dye such as CF show a fast depolarization. With increasing molecular size, the depolarization process slows down. This relation became obvious already when comparing the fluorescence depolarization of free CF and CF bound to P2A2 (P2fA2, see Figure 4). Upon coupling CF to the lipopeptide, the fluorescence anisotropy decay was decelerated. Due to the interaction of

(8)

Here, y corresponds to the cLP dependent value of the experimental parameter (e.g., diffusion coefficient) and the indices “0” and “∞” refer to the initial and final values of the respective parameter. The cmc is the critical micelle concentration, and ΔcLP describes the concentration range in which the change of value y occurs.

III. RESULTS III.1. Steady-State and Time-Resolved Fluorescence Experiments. P2A2. In life sciences, the xanthene dye CF and its derivatives are widely used as fluorescent probes.35−37 Here, we marked the lipopeptide P2A2 with CF (P2fA2), to probe the self-association of the lipopeptide. First, we analyzed the changes of the spectroscopic properties of CF due to covalent attachment to the lipopeptides. Second, effects of the incorporation of free CF and P2fA2, respectively, into P2A2 micelles were investigated at P2A2 concentration well above the expected cmc.17 A comparison of the fluorescence spectra of CF and P2fA2 in PBS showed only slight differences in the respective spectra. Emission maxima at λmax = 516 nm and λmax = 515 nm were found for CF and P2fA2, respectively. In the presence of P2A2 micelles (cP2A2 = 6.1 μM) for both CF and P2fA2, a bathochromic shift of the emission maximum λmax was found (Δλ ≈ 8 nm; see Figure 2 and Table 2). Similar effects were observed for fluorescein isothiocyanate (FITC) in CTAB micelles.38 Time-resolved fluorescence experiments for CF showed a monoexponential decay with a decay time of τF,2 = 4.4 ns. In contrast, P2fA2 showed biexponential fluorescence decay kinetics with τF,2 similar to CF and a shorter second decay time of τF,1 = 1.3 ns (see Table 2). In the presence of P2A2 micelles, both CF and P2fA2 showed biexponential decay kinetics with τF values corresponding to the pure P2fA2 in solution. However, the relative fraction a1 of the decay 14218



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Table 2. Spectroscopic Parametersa of CF and CF-Labeled Lipopeptides in the Absence and Presence of Correspondingb Lipopeptide Micelles without micelles (cLP < cmc)

with micelles (cLP > cmc)

parameter

CF

P2fA2

P2fR

P2fK

P2fE

CF

λmax (nm) τF,1c (ns) τF,2c (ns) a1 (τF,1) a2 (τF,2)

516 1.3 4.4 0.00 1.00

515 1.3 4.1 0.10 0.90

517 1.1 4.0 0.00 1.00

520 1.3 4.1 0.07 0.93

519 1.3 4.0 0.20 0.80

523

0.14 0.86

P2fA2

P2fR

524 528 see cLP < cmc see cLP < cmc 0.54 0.69 0.46 0.31

P2fK 526

0.66 0.34

λmax, maximum of fluorescence spectrum (λex = 470 nm); τF,1/2, fluorescence decay time (λex = 497 nm, λem = 521 nm); a1/2, relative fraction of τF,1/2. bExperiments were performed as a function of the corresponding unlabeled lipopeptide; for CF as a function of cP2A2. cFluorescence decay times were determined by a global fit of the fluorescence decays measured at different lipopeptide concentrations.

a

Figure 3. (a) Emission maxima of the fluorescence spectra (λex = 497 and 470 nm) and (b) relative fractions of the two decay times of P2fA2 at different concentrations of P2A2 (λex = 497 nm and λem = 521 nm, cP2fA2 = 0.1 μM, cLP = 0.2−6.2 μM) shown with the corresponding sigmoidal fit (according to SBE (eq 8), solid line).

Table 3. Determined cmc of the Cationic Lipopeptides Derived from Analyzing Steady-State Emission Spectra (Emission Maximum λmax), Fluorescence Decay Times (Rel. Fraction a1/2 of Decay Time τF,1/2), TRFD (Rel. Fraction α/ β of Rotational Correlation Time φLP/mic), and FCS (Diffusion Coefficient D) cmca (μM) analyzed parameter

P2A2

P2R

P2K

emission maximum λmax rel. fraction a1/2 of decay time τF,1/2 rel. fraction α/β of rotational correlation time φLP/mic diffusion coefficient D

1.1 ± 0.1 1.0 ± 0.5 1.5 ± 0.1

6.8 ± 2.0 4.7 ± 0.2 14.0 ± 1.7

3.1 ± 1.0 1.9 ± 0.9 3.9 ± 0.1

2.9 ± 0.5

12.1 ± 0.2

2.4 ± 0.4

a

Value ± experimental error.

Figure 4. Comparison of fluorescence anisotropy decays of CF and P2fA2 in the absence and presence of P2A2 micelles (λex = 497 nm, λem = 521 nm, cCF = 1 μM, cP2fA2 = 0.1 μM, with micelles: cP2A2 = 6.1 μM).

CF or P2fA2 with P2A2 micelles, the size of the observed overall system is distinctly increased and subsequently the fluorescence anisotropy decay is further decelerated (see Figure 4). In the case of a free rotating dye (e.g., CF), the time dependence of the depolarization can be analyzed using a monoexponential decay law with the rotational correlation time φdye. For CF, a rotational correlation time of φdye = 0.3 ns was found (see Table 4). Similar results were found for structurally comparable organic dyes, e.g., rhodamine B.39 In the case of P2fA2, the motion of the dye itself becomes restricted and the rotation of the lipopetide has to be considered (see eq 6). For

P2fA2, a fast correlation time of the dye itself of φdye = 0.5 ns and a correlation time attributed to the lipopeptide rotation of φLP = 2.0 ns was determined. In contrast, in the presence of P2A2 micelles, both CF and P2fA2 showed similar anisotropy kinetics. The fast rotation of the dye molecule itself was found nearly unchanged, and in addition, a long rotational correlation time attributed to the rotational movement of the micelles of about ∼18.5 ns was determined (see Table 4). For P2fA2, no contribution from the rotational motion of the lipopeptide itself 14219



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Table 4. Parametersa of the Fluorescence Anisotropy Analysis of CF and Different CF-Labeled Lipopeptides in the Absence and Presence of Correspondingb Lipopeptide Micelles (according to eq 9) without micelles (cLP < cmc)


parameter

CF

P2fA2

P2fR

P2fK

P2fE

φdyec (ns) φLPc (ns) φmicc (ns) α(φLP) β(φmic) A∞ θc

0.3

0.5 2.0 16.8 0.99 0.01 0.10 64°

0.3 2.0 23.7 1.00 0.00 0.05 70°

0.8 1.8 17.2 1.00 0.00 0.27 50°

0.5 3.0

20.1

0.00 90°

1.00 0.00 0.20 55°

CF

0.87 18°

P2fA2

P2fR

P2fK

see cLP < cmc see cLP < cmc see cLP < cmc 0.01 0.03 0.99 0.97 0.80 0.87 22° 18°

0.07 0.93 0.94 11°

a φdye/LP/mic, rotational correlation times attributed to dye/lipopeptide/micelle rotation; α/β, fractional amount of correlation time φLP/mic; A∞, order parameter. bExperiments were performed as a function of the corresponding unlabeled lipopeptide; for CF as a function of cP2A2. cRotational correlation times were determined by a global fit of the fluorescence anisotropy decays measured at different lipopeptide concentrations.

(free in solution or lateral motion in micelles) could be resolved when incorporated into micelles. Monitoring the fluorescence depolarization of P2fA2 with increasing P2A2 concentration, the relative contribution of the rotational correlation times of P2fA2 attributed to the free and micelle-bound lipopeptide changed. The fractional amounts α and β for the rotational correlation times φLP and φmic were calculated according to eq 9, respectively. These parameters describe the fraction of the different rotational movements to the overall depolarization.

was determined on the basis of the depolarization measurements (see Table 3). Formation of micelles not only changed the rotational correlation time but also restricted the rotation of the dye molecule attached to the lipopeptide more. The extent of the restriction is described by the order parameter A∞ which can be calculated by dividing the limiting anisotropy r∞ by the fundamental anisotropy r0 (see eq 4). A value close to 1 means a very strong restriction. Subsequently, due to the micelle formation, the order parameter increases (see Table 4). Assuming the dye rotation is restricted to a cone-like volume, A∞ allows calculating the semi cone angle θc based on eq 4. Due to the incorporation of P2fA2 into the micelles, this angle decreased from 64 to 22° (see Table 4). This finding indicates that the mobility of the dye molecule is distinctly restricted to a smaller volume. P2R, P2K, and P2E. Comparable to P2fA2, TRFD experiments showed alterations in the fluorescence anisotropy decay of P2fR and P2fK with increasing cLP (see Table 4). These findings corroborate the results of the fluorescence experiments (vide supra) and point toward an association of the lipopeptides. Data fitting based on eq 9 provided cmc values in the lower micromolar range (see Table 3), similar to P2A2 and in good agreement with the results obtained from steady-state and time-resolved fluorescence measurements. Also, the CF-dye moiety was more restricted in micelles compared to free lipopeptide in solution (see Table 4). Compared to the other lipopeptides, pure P2fE in solution gave a different rotational correlation time (3 ns for P2fE vs ∼2 ns for other lipopeptides). Moreover, no changes in the fluorescence anisotropy decay could be observed upon increasing the overall lipopeptide concentration cLP. III.3. Fluorescence-Correlation Spectroscopy (FCS). P2A2. In FCS experiments, the diffusion coefficient of CF and P2fA2 in the absence and presence of P2A2 micelles was determined by fitting the respective autocorrelation curves using eq 7. Complementary to TRFD, FCS can monitor changes in the size of the system under investigation. For free CF, a diffusion coefficient of D = 3.9 × 10−6 cm2/s was determined which matches the literature value33 (see Figure 6 and Table 5). Upon dye coupling to P2A2 (yielding P2fA2), only a slightly reduced diffusion coefficient was found (D = 3.7 × 10−6 cm2/s). However, for CF or P2fA2 incorporated in P2A2 micelles, the diffusion coefficient distinctly decreased to D = 1.2 × 10−6 cm2/s. FCS data were acquired for different cLP, and the determined diffusion coefficients are depicted in Figure 7. The diffusion coefficient changed in a sigmoidal manner, and

⎡ ⎤ ⎧ ⎫ ⎪ t ⎪ ⎬ + r∞⎥ − r(t ) = ⎢(r0 − r∞) exp⎨ ⎪ ⎪ ⎢ ⎥ ⎩ φdye ⎭ ⎣ ⎦ ⎡ ⎧ t ⎫⎤ ⎧ t ⎫ ⎬ + β exp⎨− ⎬⎥ × ⎢α exp⎨− ⎢⎣ ⎩ φLP ⎭ ⎩ φmic ⎭⎥⎦ ⎪

⎪

⎪

⎪

⎪

⎪

⎪

⎪

(9)

As a result of the global fit of the P2A2 concentration dependent fluorescence anisotropy decays, a rotational correlation time of φdye = 0.5 ns for dye rotation, φLP = 2.0 ns for lipopeptide rotation, and φmic = 16.8 ns for P2A2− micelle rotation was found (see Table 4). In Figure 5, the calculated parameters α and β are shown for different overall lipopeptide concentrations. From these data, a cmc of 1.5 μM

Figure 5. Relative fractional amounts α (black dots) and β (red dots) of the two rotational correlation times φLP and φmic (with corresponding fit according to SBE (eq 8) as solid line) plotted against the overall concentration of lipopeptides cLP (cP2fA2 = 0.1 μM, cLP = 0.2−6.2 μM). 14220



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Figure 7. Diffusion coefficient D of P2fA2 (and corresponding fit according to SBE (eq 8) as red solid line) plotted against the overall concentration of lipopeptides cLP (cP2fA2 = 2.3 nM, cLP = 2.3 nM to 1 mM).

Figure 6. Comparison of FCS autocorrelation curves of CF and P2fA2 in the absence and presence of P2A2 micelles (with corresponding fit according to eq 7, cCF = 1 nM, cP2fA2 = 2.3 nM, with micelles: cP2A2 = 1 mM).

photophysical properties of CF as well as of the CF-labeled cationic lipopeptides was observed in the presence of the respective micelles. The spectral properties as well as decay kinetics of CF (free or labeled) were altered. In the emission spectra, a bathochromic shift was found and the decay kinetics were distinctly changed, showing altered relative fractions of the two decay times. Here, as for free CF, we suggest interionic interactions with cationic peptide sequences. The CF-labeled lipopeptides will incorporate into the corresponding micelles forced by the lipid part of the constructs. The bathochromic shift but even more so the altered fluorescence decay kinetics indicate that the intramolecular interactions between the CF moiety and parts of the lipopeptide (e.g., amino acids) are present either due to electronic interactions and/or due to the formation of weak bonds (e.g., H-bond between amino acid and CF). As a consequence, the dye molecule is located in a different and more restricted microenvironment. However, the emission of CF is sensitive to oxygen40 as well as to environmental parameters like microviscosity, pH, and ionic strength.41 Due to coupling to the lipopeptide and further micelle incorporation, the dye molecule becomes more shielded and consequently less accessible for oxygen. Due to incorporation into micelles, the surrounding media experienced by the CF moiety will be changed as well (e.g., due to the close proximity of surrounding amino acids and alkyl chains), leading to the observed changes in the photophysical parameters. Both fluorescence anisotropy and FCS demonstrated slower rotational as well as translational motion of the CF−lipopeptide constructs compared to free CF. The reason is the large overall size of the molecular assembly compared to the free dye. For both CF and the CF−lipopeptide constructs, constructive

data analysis based on SBE (eq 8) yielded a cmc of 2.9 μM (see Table 3). P2R, P2K, and P2E. Performing FCS measurements, an increased diffusion coefficient could be observed also for P2R and P2K when cLP is raised (see Table 5 for diffusion coefficients). By plotting the respective diffusion coefficients over cLP, a sigmoidal trend could be observedsimilar to P2A2and a fit yielded a cmc of 12.1 μM for P2R and 2.4 μM for P2K (see Table 3). In contrast, for P2E, no self-association could be deduced from the FCS data. Independent of the concentration of P2E (cP2E = 1.5 nM to 0.5 mM) for P2fE, a constant diffusion coefficient of D = 2.5 × 10−6 cm2/s was determined. Interestingly, the diffusion coefficient is slightly smaller than that of the other lipopeptides (see Table 5).

IV. DISCUSSION IV.1. CF-Labeled Lipopeptides. Lipopeptides are potential building units of DCS because of their ability to form micelles and deliver poorly soluble molecules as needed.17,18 Here, we labeled lipopeptides with the organic dye CF and analyzed the photophysical properties of the CF-labeled cationic lipopeptides P2fA2, P2fR, and P2fK as well as the anionic compound P2fE. At first, we analyzed the spectroscopic properties of CF after coupling to the lipopeptides. The spectral properties (absorption and emission spectra) did not change, but a weak alteration in the fluorescence decay kinetics was observed (see Table 2). No difference between the cationic and anionic lipopeptides was found with respect to the change in the fluorescence decay kinetics. A more pronounced effect on the

Table 5. Diffusion Coefficientsa D and Corresponding Diffusion Times τD of CF and CF-Labeled Lipopeptides in the Absence and Presence of Correspondingb Lipopeptide Micelles without micelles (cLP < cmc)


parameter

CF

P2fA2

P2fR

P2fK

P2fE

CF

P2fA2

P2fR

P2fK

D (10−6 cm2/s) τD (10−2 ms)

3.9 2.8

3.7 3.2

3.4 3.1

3.1 3.6

2.5 4.6

1.2 9.0

1.2 9.2

0.7 14.2

0.9 12.9

FCS measurements were performed at room temperature (21 ± 2 °C), and D is calculated for 21 °C according to eq 2. bExperiments were performed as a function of the corresponding unlabeled lipopeptide; for CF as a function of cP2A2. a

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the order parameter A∞ and the subsequent calculation of the semi cone angle θc (see eq 4), we showed that the CF molecule is more strongly hindered in its rotation due to the micelle formation (see Table 4). Unlike the cationic lipopeptides of P2fA2, P2fR, and P2fK, no changes in the spectroscopic properties of the anionic P2fE could be found by varying the overall lipopeptide concentration. These observations show that either (i) P2fE does not change the spectroscopic properties of the attached CF upon incorporation into micelles or (ii) P2E does not form micelles. Due to the fact that the analysis of the diffusion as well as the rotational properties of the CF moiety in P2fE showed no changes with increasing cLP, we concluded that the anionic lipopeptide does not form micelles in the investigated concentration range. This is surprising, since the stability of micelles is dominated by hydrophobic interactions of the lipophilic molecule moiety, as documented for a number of surfactants. Additionally, for P2fE, we found a slightly larger rotational correlation time as well as a smaller diffusion coefficient compared to the cationic sequences. This points to a larger hydrodynamic radius of P2fE (see Table 6), which leads to the assumption that P2E exists in a different and more bulky structure, subsequently preventing self-association. Additionally, we investigated the interaction between the lipopeptides and brain capillary endothelial cells (b.end3). Cationic P2fA2 micelles were efficiently internalized into the cells (see Figure 8, left). The green fluorescence in the

interactions with the corresponding micelles could be evidenced by a significant decrease in the diffusion coefficient and increase in the rotational correlation time (see Tables 4 and 5). Moreover, the results from fluorescence anisotropy (especially the increased order parameter) support the fact that the microviscosity increases due to the incorporation of CF and CF-labeled lipopetides into micelles (vide supra). Additionally, our data showed that fluorescence anisotropy is very sensitive to changes in the size of the observed system. The difference between the rotational correlation times of the CF moiety (which was equal to the free CF) and the overall CF−lipopeptide construct molecule was clearly resolved in the fluorescence anisotropy data. In contrast, the determined diffusion coefficients of CF and CF−lipopeptide constructs revealed only small (or no) differences. Here, the alterations in the translational motion of CF and CF−lipopeptide constructs were too small to be clearly resolved by FCS. IV.2. Self-Association of Lipopeptides. Because P2A2 and P2fA2 are of the same lipopeptide structure, P2fA2 is rapidly incorporated into the micellar assembly formed by P2A2. The same holds true for the other lipopeptide constructs investigated in this study. On the basis of changes in the spectroscopic (e.g., λem, τF) and microscopic properties (e.g., D, φ) of the CF-labeled lipopeptide constructs, the association of the lipopeptides was proven. The analysis of the emission maxima as well as the relative fractions of the fluorescence decay times (a1, a2) of the cationic CF-labeled lipopeptides at different cLP values showed a shift of λmax as well as a change in the value of a1 and a2 (see Figure 3 for P2A2). We attributed the step-like changes of the spectroscopic parameters to the formation of micelles. This was supported by TRFD as well as FCS experiments. By calculating the fluorescence anisotropy decay from TRFD data, the rotation of the fluorescent probe was interrogated and a rotational correlation time φ was determined. By FCS, we analyzed the diffusion of the fluorescent probe at different cLP values. Upon formation of micelles, the rotational correlation time as well as the diffusion coefficient of the cationic lipopeptides distinctly changed. An increasing φ and a decreasing D with rising cLP point to the formation of micelles and corroborate the results of the fluorescence data. Using the Stokes−Einstein equation, we calculated the hydrodynamic radius Rh of the lipopeptides and the corresponding micelles. The hydrodynamic radius increased due to the self-association up to 2−3 nm for the micelles (see Table 6). The radii are in good agreement with the calculated radius based on the molecular mass of the micelles (3.4 nm for P2A2).17 An in-depth analysis of the fluorescence anisotropy yielded further insight on the alteration of the microenvironment of the CF moiety. Upon micelle formation, the rotational freedom became more strongly restricted. From the analysis of the data, the accessible cone-like volume was estimated. On the basis of

Figure 8. CLSM images of mouse brain capillary endothelial cells (b.end3) incubated with P2fR (top left) and P2fE preparations (top right) for 1 h at 37 °C. The integrity of the cell membrane is reflected by staining with trypan blue (bottom). (CF, green; trypan blue, red).

cytoplasm, which is not observed in experiments performed at 4 °C (data not shown) points to an endocytotic uptake. Unlike the cationic peptide micelles, no uptake of P2fE into cells was observed, but the anionic lipopeptide strongly accumulates in the cell membrane (see Figure 8, right). This is likely driven by insertion of the hydrophobic lipopeptide chain into the lipid matrix of the membrane which may be related to the dissociation of P2fE structures. Hence, also in uptake experiments, the behavior of the anionic and cationic lipopeptides differs. Consequently, from our results, we conclude that in contrast to the cationic lipopeptides the

Table 6. Hydrodynamic Radius Rh of CF-Labeled Lipopeptides in the Absence and Presence of Correspondinga Lipopeptide Micelles without micelles (cLP < cmc)


parameter

P2fA2

P2fR

P2fK

P2fE

P2fA2

P2fR

P2fK

Rh (nm)

0.6

0.7

0.7

0.9

2.0

3.0

2.5

a

Experiments were performed as a function of the corresponding unlabeled lipopeptide. 14222



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time of the micelles roughly φ = 20 ns (see Tables 2 and 4). Thus, during the excited state of the dye, the micelle only rotates by one-eighth of its size (∼45°), so accurate determination of φmic is difficult. In contrast, FCS is not limited by the electronic properties of the probe. The diffusion can be well analyzed independent of the decay time. Moreover, in addition to rotational movements, there are several processes affecting the depolarization, e.g., fluorescence quenching, and resonance energy transfer (RET). Due to the small Stokes shift of CF (and CF-labeled lipopeptides), reabsorption and homoRET between two CF molecules may occur.42 Considering a Förster distance of R0 = 4.9 nm for homo-RET between two CFs, this distance is nearly twice the hydrodynamic radius of the micelles (Rh ≈ 2.5 nm). Thus, in micelles with two CFlabeled lipopeptides, a homo-RET is easily conceivable. By FCS, various processes can be analyzed simultaneously if the correlation times according to the certain processes are different. Comparing diffusion (∼μs to ms time range) and photophysical processes (∼ns to μs time range) in the present case, this condition applies. Therefore, the diffusion coefficient can be identified without any disturbances due to photophysical processes. In summary, by using different analytical methods, various values for the cmc may be obtained. Because FCS seems to be less prone to cross sensitivities, we propose that this method provides the most reliable resultsespecially for compounds with very low cmc’s.

anionic P2E behaves very different (no micelle formation, no internalization into b.end3). Further studies are required to elucidate the physicochemical basis of the different properties of the positively and negatively charged lipopeptides. Here, the influence of the surface potential on the physicochemical properties is of special interest. IV.3. cmc of Lipopeptides. A set of experimental parameters representing different molecular properties was analyzed as a function of cLP. Fitting by SBE (eq 8) yielded an inflection point which was interpreted as the cmc. Depending on the method and the analyzed parameter, we found only very slight differences in the cmc values for the individual cationic lipopeptides (see Figure 9 and Table 3). For P2A2 and P2K,

V. CONCLUSION Here, we characterized micelles composed of cationic lipopeptides as potential targeted DCSs. Recently, we demonstrated the target specificity and uptake mediating ability of micelles of the cell-penetrating and receptor recognizing lipopeptide P2A2.17−19 Now we demonstrate that the high stability of these micelles is indicated by the very low cmc in the lower micromolar range. We show that fluorescence-based methods are very well suited to investigate the association behavior of surfactants in concentration ranges too low for commonly used methods (e.g., surface tension, light scattering, or capillary electrophoresis). Especially with FCS, it is even conceivable to decrease the limit of quantification down to the sub-nanomolar range, which subsequently would allow the determination of a cmc in the nanomolar range. The very low cmc denotes the occurrence of strong hydrophobic interactions between the palmitoyl chains of the cationic lipopeptides. Compared to common surfactants like sodium dodecyl sulfate (cmc of about ∼8 mM34), their cmc values are orders of magnitudes lower. However, to use micelles as DCS, the cmc should be at least in the lower micromolar range in order to maintain long-term stability under physiological conditions. The very low cmc’s of the lipopeptides investigated in this study are of crucial advantage. When applied as drug carriers, the lipopeptides are able to form micelles in highly diluted solution and thus guarantee the transport of drugs to the point of need. On the basis of this criterion, P2A2, the dipalmitoylated receptor-binding and cellpenetrating sequence derived from apolipoprotein E, seems to be most promising in the development as a micellar carrier. Hence, the next steps are the investigation of the interaction between this DCS and possible drug compounds. The exchange kinetics of drugs in P2A2 micelles and its surrounding is of particular interest in this context.

Figure 9. Comparison of cmc determined by different analytical approaches for the lipopeptides investigated.

the differences are very small and cmc values in the range between 1.0−2.9 μM and 1.9−3.9 μM were found, respectively. For P2R, a cmc of 4.7−14 μM was calculated. The small differences in the determined cmc are related to the fact that each method monitors properties of the probe (e.g., electronic properties vs size) which are characterized by different interdependencies and cross sensitivities. On the one hand, the electronic (spectroscopic) properties of CF were influenced by the microenvironment of the dye molecule. Here, parameters like polarity, viscosity, pH, and the refractive index have to be considered. However, also intermolecular interactions with neighboring amino acids (vide supra) and the restriction of the dye molecule influenced the electronic properties. By attaching the CF to the lipopeptides, the decay kinetics were altered (see Table 2). The changes in the emission spectra as well as the decay time can be related to a combination of several effects and subsequently can be less sensitive to small changes. In addition to monitoring the electronic properties of the fluorescence probe (CF), size-related molecular properties such as rotational and translational motion proved to be very sensitive for the determination of micellar properties. By TRFD experiments, only rotational movements characterized by rotational correlation times comparable to the time window defined by the fluorescence decay time of the probe can be analyzed. The averaged decay times of the CF-labeled lipopeptides were τF = 2.3 ns and the rotational correlation 14223



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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors want to thank Prof. Dr. H. G. Löhmannsröben for his encouragement and Dr. Thomas Ritschel for a lot of support and assistance in theoretical calculations (both University of Potsdam). Heike Nikolenko (FMP) is thanked for her excellent work in cellular uptake experiments. The research work was partially supported by the DFG grant DA 324/9-1.

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