Interaction between Albumin and Pluronic F127 Block Copolymer

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Interaction Between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling Maria Victoria Neacsu, Iulia Matei, Marin Micutz, Teodora Staicu, Aurica Precupas, Vlad Tudor Popa, Athanasios Salifoglou, and Gabriela Ionita J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b02199 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 17, 2016

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The Journal of Physical Chemistry

Interaction between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling

Maria Victoria Neacsu,† Iulia Matei,† Marin Micutz,†,‡ Teodora Staicu,‡ Aurica Precupas,† Vlad Tudor Popa,† Athanasios Salifoglou,§ and Gabriela Ionita*†

†

“Ilie Murgulescu” Institute of Physical Chemistry of the Romanian Academy, 202 Splaiul Independentei, Bucharest 060021, Romania

‡

Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bd. Regina Elisabeta 4-12, Bucharest 030018, Romania

§

Department of Chemical Engineering, Laboratory of Inorganic Chemistry and Chemistry of Advanced Materials, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece

* Corresponding author. E-mail: [email protected]; Tel: +40213188595

ABSTRACT The interaction of human serum albumin (HSA) with amphiphilic block copolymer Pluronic F127 has been investigated by several physical methods. Interest in studying this system stems from a broad range of bioactivities involving both macromolecules. Serum albumins constitute a significant class of proteins in the circulatory system, acting as carriers for a wide spectrum of compounds or assemblies. Pluronic block copolymers have revealed their capacity to ferry a variety of biologically active compounds. Circular dichroism, rheological measurements and differential scanning microcalorimetry (µDSC) were employed to get insight into the interaction betweeen the two macromolecules. The results reveal that Pluronic F127 induces conformational

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changes to albumin if it is organised in a micellar form, while albumin influences the selfassembly of Pluronic F127 into micelles or gels. F127 micelles, however, induce smaller conformational changes compared to ionic surfactants. The µDSC thermograms obtained for HSA and/or F127 show that HSA shifts the critical micellar temperature (cmt) to lower values, while concurrently the HSA denaturation behavior is influenced by F127, depending on its concentration. Rheological measurements on solutions of F127 17% have shown that a sol-to-gel transition occurs at higher temperatures in the presence of HSA and the resulting gel is weaker. The global profile on HSA/F127 systems was complemented by local information provided by EPR measurements. A series of X-band EPR experiments was performed with spin probes 4(N,N’-dimethyl-N-hexadecyl)ammonium-2,2’,6,6’-tetramethyl-piperidine-1-oxyl

iodide

(CAT16) and 5-doxyl stearic acid (5-DSA). These spin probes bind to albumin sites and are sensitive to phase transformations in Pluronic block copolymer solutions. For a given F127 concentration, the spin probe binds only to HSA below cmt and migrates to the F127 micelles above cmt. The collective data suggest soft interactions between the macromolecules, with the emerging results projecting potential applications linked to reaching optimal conditions for certain drug formulations.


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INTRODUCTION Triblock copolymers poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), generically named Pluronics, have been intensely studied for their thermoreversible selfassembly properties in solution.1-3 The presence of oxyethylene groups is responsible for the greatly enhanced solubility of Pluronics in water, although the oxypropylene groups are also hydrated. At the same time, self-assembly is controlled by the degree of hydration of each type of macromolecular unit. At low concentration and temperatures below the critical micellar temperature (cmt), block copolymers of the Pluronic family exist in solution as monomers. By increasing the temperature, the monomers begin to aggregate into micelles, with the nonhydrated polypropylene block (PPO) as hydrophobic micellar core and flanking hydrated polyethylene (PEO) blocks representing the micellar corona.1-3 Depending on their hydrophobic/hydrophilic balance, under suitable conditions of concentration and temperature, Pluronic solutions undergo a micelle-to-gel transition due to progressive dehydration of PEO blocks.1 This common behavior for Pluronics, together with their low toxicity and high biodegradability, emphasize the broad range of their applications. In this regard, solutions of Pluronics are used as non-ionic surfactants to generate various molecular vehicles for drug delivery and targeting,4-6 as templates in the synthesis of nanostructured materials7 or in food processing.8 The behavior of Pluronic F127 to form gels at temperatures closer to the physiological temperature, at relatively low concentration (16%)9 justifies its wide use in drug formulation.10 The amphiphilic character of this copolymer determines its ability not only to host drug molecules, but also to interact with biomacromolecules, hydrophobic surfaces and biological membranes.11 At the same time, albumins are well-known proteins acting as carriers for drug



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delivery, although as host molecules they themselves cannot encapsulate at once a high content of active molecules. The present study focuses on the investigation of Pluronic F127/human serum albumin (HSA) systems, aiming at probing into local interactions occurring in these binary systems in solution, in connection with further applications in designing efficient drug formulations. Our approach combines a series of physicochemical methods providing global information on a system (differential scanning microcalorimetry (µDSC), oscillatory rheology, circular dichroism), with electron paramagnetic resonance (EPR) spectroscopy, a method wellrecognized for its versatility, describing local properties for a non-homogeneous system at nanoscale level. We have previously reported two EPR studies on a) the interaction of albumins with ionic surfactants (sodium dodecyl sulphate and cetyltrimethylammonium bromide) and β-cyclodextrin, as a function of different factors such as the surfactant/cyclodextrin concentrations12 and the temperature,13 and b) the ability of a polymeric gel, containing cyclodextrin, to remove the surfactant from its complex with the protein.13 In light of those results, demonstrating the usefulness of EPR spectroscopy in providing information on ionic surfactant/protein interactions and monitoring denaturation12,13 or purification13 processes of proteins, we were prompted to apply this spectroscopic technique in the investigation of interactions in Pluronic F127/HSA solutions at the nanoscale level, by monitoring changes in the EPR parameters of two spin probes (Figure 1) as a function of the F127 concentration and temperature. Such an experimental approach is expected to provide a detailed view on the transfer of low molecular mass molecules between the artificial drug carrier, represented by Pluronic assemblies, and the natural drug carrier represented by HSA.


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N

O

.

O

O H3C

OH

(CH2)12

H3C(H2C)15

5-DSA

N+

N

.

O

CAT16 Figure 1. Molecular structures of the spin probes.

EXPERIMENTAL Materials. Pluronic F127 and HSA, fatty acid free (Aldrich), were used as received. The spin probes 5-DSA and CAT16 were purchased from Sigma and Molecular Probes, respectively. Sample preparation. All samples investigated were prepared in doubly distilled water at room temperature, by dissolving HSA and/or F127 to yield a final protein concentration of 20 mg/ml and concentrations of Pluronic F127 5% and 17%. These solutions were used for EPR, µDSC and rheological measurements. The samples were left to equilibrate for 12 h prior to any measurement. For CW-EPR measurements, an appropriate volume of 10-2 M ethanol solution of 5-DSA or CAT16 was added into a vial followed by solvent evaporation under a stream of inert gas. Then, the solid spin probe was re-dissolved in appropriate volumes of solutions containing HSA and/or F127. The final concentration of each spin probe in these solutions was approximately 2×10-4 M. For EPR experiments aiming to investigate the effect of temperature on the spin probe dynamics, the measurements were carried out in the resonator cavity of the spectrometer. In each case, the



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solution containing the spin probe previously transferred to a capillary was brought to a specific temperature (in the range 273–333 K) and kept for 10 min prior to recording the spectrum. For circular dichroism measurements, the concentration of HSA was 2 mg/ml, while the F127 concentrations were 2%, 5% and 17%. Instrumentation. EPR spectra were recorded on a JEOL FA 100 spectrometer, equipped with a cylindrical type resonator TE011, operating at a frequency modulation of 100 kHz, microwave power of 0.998 mW, sweep time of 480 s, modulation amplitude of 1 G, time constant of 0.3 s, and a magnetic field scan range of 100 G. The instrument was equipped with a temperature control unit, allowing cooling/heating of the samples. Rotational correlation times of the spin probe showing an isotropic dynamic regime were determined using equation 1:

τ c = 6.51 × 10

−10

 h  12  h 1/ 2  ∆H 0  0  +  0  − 2  h−1    h+1   

(1)

where ∆Ho is the peak-to-peak width (in Gauss) of the central line, and h−1, h0 and h+1 are the heights of the low, central, and high field lines, respectively.14 Additional EPR parameters, used in this study, include the order parameter, S, describing the EPR spectra of 5-DSA in micellar solutions of F127 and defined according to equation 2: S = (All–A┴)/[Azz– (Axx+Ayy)/2]

(2)

where Azz, Axx, Ayy are the principal components of the A tensor, in the absence of molecular motion, and All and A┴ are obtained from experimental spectra. This specific parameter provides information about the order degree of the amphiphilic Pluronic F127 in systems emerging through self-assembly. Since this parameter depends on polarity, the values used for the tensors Azz, Axx and Ayy were 33.5 G, 6.3 G and 5.8 G, respectively.15,16


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EPR spectral simulations. In simulating slow motion spectra, with some of them showing two component features, the program developed by Budil et al.,17 based on non-linear least–squares NLLS fits, was used. Circular dichroism spectra were recorded on a JASCO J-815 CD spectropolarimeter, at room temperature, in 1 cm path length cuvettes, at 4 s response time, 100 nm/min scan speed, 1 nm bandwidth and 1000 millidegrees (mdeg) sensitivity. The characteristic HSA signals in the 200– 300 nm wavelength range were averaged over five scans and the blank spectrum was subtracted. The results were expressed in ellipticity, θ (in mdeg). The HSA secondary structure content, in the absence or presence of F127 at different concentrations, was determined using the DichroWeb online server.18,19 The α-helix, β-sheet and random coil contributions were estimated employing the K2D deconvolution algorithm.20 Normalized root mean square deviations (NRMSD)21 lower than 0.222 were obtained for all fits. Differential scanning microcalorimetry (µDSC). µDSC measurements were performed on a SETARAM µDSC VII Evo instrument. The sample batch cell (Hastelloy®) was filled with 0.6 ml of the degassed solution. The reference cell was filled with water in such a way as to ensure that an identical mass of solution was present in both the sample and reference cells. The µDSC thermograms were recorded using a scanning rate of 1.0 K min−1 in the temperature range 273−363 K. Sample and reference cells were placed inside their individual compartments of the microcalorimeter and equilibrated at the initial temperature for 1 h before the onset of the heating process. The three heating/cooling cycles performed for each sample allowed for discrimination between native (1st cycle) and denatured (2nd and 3rd cycle) protein influence on Pluronic micellization.



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The Calisto v1.077 software package was used for data acquisition, baseline integration, and export to Excel. Heat values obtained were further processed in Origin. Assuming contribution of two components, the endothermic peak attributed to HSA thermal denaturation was decomposed using PeakFit v.4.12 software with Haarhoff-Van der Linde built-in function. Detailed description of data processing is given elsewhere.23,24 Dynamic rheological measurements were carried out using a Micro Fourier Rheometer MFR 2100 (GBC, Australia), equipped with a temperature control system connected to a circulating Lauda E100 water bath. Rheological measurements were performed in the temperature range 283–333 K. Dynamic rheometry experiments for the samples with composition of 17% F127 were run on the same set of parameters for all systems investigated: gap between the upper and the bottom plates 300 µm, frequency range 0.5–120 Hz, displacement amplitude 0.03 µm (to fall into linear viscoelasticity behavior).

RESULTS AND DISCUSSION Pluronic block copolymers have been known to pass through various phases, i.e. from sol to micellar and gel phase, depending on the prevailing physical parameters (concentration, temperature, solvent composition). Micellization of Pluronics is initiated at a fixed temperature by increasing the concentration.1 Proteins are bio-macromolecules adopting a variety of conformations, which also depend on physico-chemical parameters. Circular dichroism spectra have revealed conformational changes of albumin in the presence of ionic surfactants even at concentrations below the critical micellar concentration (cmc).12 In such processes, the presence of ionic surfactants might have a protective role in preserving the helicity content following thermal denaturation.25 Aiming to probe the influence of F127, possessing properties of a non-


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ionic surfactant, on HSA conformation, circular dichroism spectra were recorded in the absence and presence of F127 at 293 K (Figure 2). The concentrations of F127 were 2%, 5% and 17%. At this temperature, Pluronic molecules are in a micellar form only at a concentration of 17 %.

0 -20 -40 θ (mdeg)

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-60 -80

F127 0% F127 2% F127 5% F127 17%

-100 -120 -140 200

210

220

230

240

250

260

λ (nm)

Figure 2. Circular dichroism spectra of 2 mg/ml HSA in the absence and presence of different F127 concentrations at 293 K. The error bars for the spectrum in blue indicate the standard deviation in the measurements.

Analysis of the circular dichroism spectra provided the different conformational contributions (α-helix, β-sheet and random coil) of HSA. The values can be found in Table 1. It can be seen that the presence of F127 in the monomer form has a negligible effect on the protein conformation corresponding to a 2-5 % loss of α-helix with respect to native HSA. At high concentration of F127 (17%), allowing micelle formation at 293 K (vide infra), there is an appreciable decrease in the α-helical content of HSA although the circular dichroism spectrum preserves the features characteristic to a highly helical protein. It can be noted that changes in αhelical contribution, similar to those observed in the presence F127 micelles, were reported for



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ionic surfactants at concentrations bellow their corresponding cmc. For example, in the case of 1 mM SDS (cmc = 8.2 mM),26 the α-helical content drops to 0.43%, while in the presence of 0.1 mM CTAB (cmc = 1 mM),26 the α-helicity contribution represents 0.39%.12 The lower effect of F127 on protein helicity, compared to that induced by ionic surfactants, is not surprising. The protein itself exhibits a negatively charged surface, due to the presence of carboxylate groups, which promotes interactions with ionic surfactants. Various studies have outlined the importance of the hydration shell, which seems to play a determining role in the dynamic behavior of the protein.27,28 Obviously, the perturbing action of ionic and non-ionic surfactants is different, as in the first case the electrostatic interactions are predominant, whereas in the second case the hydrophobic interactions may play a substantial role. This particular aspect of protein hydration, which also influences the ligand binding, has been investigated by different techniques including EPR,29-31 NMR,32,33 Overhauser dynamic nuclear polarization NMR spectroscopy,28,30,31 time-resolved ﬂuorescence upconversion,34,35 and two-dimensional infrared spectroscopy.27,36

Table 1. Secondary structure content of HSA in the absence and presence of non-ionic F127 CF127 (%)

α-helix

β-sheet

random coil

0

0.58

0.08

0.34

2

0.56

0.11

0.33

5

0.53

0.10

0.37

17

0.43

0.13

0.44


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On the other hand, the macromolecular chains of Pluronics are soluble in water due to the hydrogen bonding network established between hydrogen atoms of water and ether-type oxygen atoms of the copolymer. The ethylene oxide and propylene oxide groups have different hydrophobicities. As a result, the degree of hydration is not uniform through the monomer chain or through different assemblies of the copolymer in micelles and gels. The equilibrium between the hydration shell of the copolymer and bulk water molecules is a function of copolymer concentration and temperature. Thus, PEO chains are more hydrated than PPO chains and during micellization the hydration water of PPO is eliminated, thus facilitating hydrophobic interactions between PPO fragments, ultimately leading to the formation of a PPO-rich micellar core.37,38 As the concentration of F127 reaches a certain value (>16%), increasing the temperature of the F127 solution affects the dehydration process in the PEO chains. This, in turn, forces the micelle-to-gel transition, due to the entanglement of the dehydrated PEO chains.37 The temperature corresponding to the micelle-to-gel transition known as cgt is not only determined by the concentration of Pluronic, but is also sensitive to the presence of other species that can reduce or increase the phase transition temperature. In this respect, salts such as sodium chloride, calcium chloride or sodium alginate, lower the cgt value,39-41 whereas the presence of long polyethylene oxides (MW > 1000)42 or cyclodextrins43 increases the value of cgt. In view of the fact that in both cases of HSA and F127 the water of hydration influences the conformation and assembly, respectively, it is expected that, in turn, the parameters characterizing phase transitions occurring in F127 block copolymer systems will be sensitive to the presence of HSA. Rheological measurements provide information on both cgt and gel strength. Figure 3 shows the temperature dependence of the storage modulus G’ for F127 17% with and without HSA. The micelle-to-gel transition is indicated by the sharp change in the



mechanical properties (expressed as storage modulus) at the temperature denoted as cgt. In the presence of HSA, it can be observed that the storage modulus beyond cgt decreases, thereby indicating that a) HSA leads to the formation of a weaker gel, and b) the micelle-to-gel transition is not accompanied by a steep increase of the G’ modulus. This is a clear sign that in the presence of a voluminous protein, entanglement of PEO chains is not favored. As stated above, formation of gel phase in Pluronics is driven by dehydration of PEO chains. This dehydration is clearly demoted by the presence of the protein, which may act by means of its own hydration shell. The variation of the storage modulus (G’) with temperature indicates not only a delay in gel formation (the cgt is 301.6 K in the absence and 308.7 K in the presence of HSA), but also a broader interval for the transition to gel phase corresponding to the HSA/F127 system. The rheological behavior of the HSA/F127 system in the temperature range corresponding to the gel phase is an indication that the protein molecules interfere in the architecture of the Pluronic gel network.

1200

Storage modulus (Pa)

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1000 800

301.6 K 600

308.7 K

400 200

HSA/F127 17% F127 17%

0 280

290

300

310

320

330

340

T (K)

Figure 3. The storage modulus, G’, of F127 17% (circles) and HSA/F127 (squares) as a function of temperature.


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The µDSC technique provides useful information on individual and interactive processes taking place in HSA/F127 systems. Depending on the solution composition, the DSC thermograms exhibit a peak corresponding to either F127 micellization or HSA denaturation or both. The DSC thermograms obtained from µDSC measurements for F127 5% and 17%, HSA (20 mg/ml), and corresponding to the first heating cycle of HSA/F127 mixtures are shown in Figure S1. Both peaks associated with micellization and denaturation are broad, although the intensities and hence the thermal effect(s) are different. The DSC thermograms reflect the large endothermic effect associated with the PPO chain dehydration of F127 at lower temperatures, and a significantly smaller effect associated with denaturation of the protein at temperatures over 333 K. Figure 4 shows the partial DSC thermograms (1st cycle) exhibiting the peaks corresponding to F127 micellization in the absence and in the presence of HSA in native form.

0 -1

Heat flow (mW)

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-2 -3 -4

F127 5% HSA/F127 5% F127 17% HSA/F127 17%

-5 -6 280

290

300

310

320

T (K)

Figure 4. The partial µDSC thermograms displaying the effect of native HSA on F127 micellization: F127 5% (black), HSA/F127 5% (red), F127 17% (blue) and HSA/F127 17% (magenta).



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The thermodynamic parameters describing the micellization of F127 are summarized in Table 2.

Table 2. Thermodynamic parameters of the micellization process in solutions of F127 5% and F127 17%, in absence and presence of HSA Sample

Tonset (K)

Tpeak (K)

∆ H (kJ/mol)

F127 5%

293.9

297.4

343.92

HSA/F127 5%

293.6

296.9

351.86

F127 17%

287.2

290.4

311.48

HSA/F127 17%

286.6

289.8

315.60

The temperature corresponding to micelle formation in Pluronic solutions (cmt) is indicated by the onset temperature (Tonset) of the endothermic peak.42-44 Both in the case of F127 5% and F127 17% the micellization temperature decreases slightly in the presence of HSA. The peak temperature (Tpeak), indicating the temperature at which micellization reaches the maximum rate is influenced in the same manner, decreasing in the presence of HSA. Pluronic F127 experiences two different transitions, from sol-to-gel and micelle-to-gel (only in the case of F127 17%) in the temperature interval in which HSA is not thermally denaturated. While the first event can be evidenced by µDSC measurements, the second one is not detectable by this method.45 The effect of HSA on the micellization of F127 is, however, less evident than the effect of HSA on the micelle-to-gel transition, as revealed by rheological data. This might be an indication that PEO blocks are more involved in the interaction with HSA than PPO blocks. Our results show that the micellization enthalpies of F127 are slightly higher in the presence of HSA,


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and increased concentration of Pluronic favors micelles formation. These observations support the hypothesis that interactions between Pluronic F127 and HSA involve, in the first instance, a hydrophilic interaction, including hydrogen bonding reorganization, whereas a hydrophobic interaction contribute less. In fact, some studies reporting on the effect of polyethylene glycols on the secondary structure of albumins underline both the contribution of hydrophilic as well as hydrophobic forces.46,47 Calorimetric measurements provide thermodynamic parameters on the denaturation process of albumins, i.e. the denaturation temperature (TD) and enthalpy of denaturation (∆HD).48,49 The structure of HSA is represented by three homologous domains (named I, II and III) which are predominantly α-helical. Each of these domains is made up of two separate helical subdomains (A and B), which are connected through a random coil.50-52 The peak attributed to HSA denaturation was decomposed assuming contributions from two components: the first component of HSA denaturation with a lower denaturation temperature corresponds to domain III and a large portion of domain II of albumin, while the second component includes domain I and a small portion of domain II.53,54 Thermal denaturation of HSA involves rupture of disulfide and hydrogen bonds, with each of these types of bonds contributing to the overall enthalpy with 25 kcal/mol and 4 kcal/mol, respectively.48 The shape of the µDSC peaks corresponding to the denaturation process depends on the concentration of the protein, the presence of other species in solution and measurement conditions. Under certain experimental conditions, conformational changes in the protein, induced by heating, determine protein gelation.55 Like thermal denaturation leading to protein aggregation at higher temperatures, protein gelation is driven by non-specific interactions between the hydrophobic parts of albumin, with contributions from hydrogen bonding and ionic interactions



and disulphide cross linking between polypeptide chains.55-59 Albumin gelation has also been reported in one of our previous studies on the interaction of bovine serum albumin with ionic surfactants.12 The presence of ionic surfactants in submicellar concentration favors gelation, whereas in the case of surfactant concentration above cmc this process was not observed. Barone et al.60 have previously reported an overlap of a small exothermic peak corresponding to protein gelation with an endothermic peak corresponding to thermal denaturation. The experimental µDSC thermograms corresponding to protein denaturation shown in Figure 5 do not exhibit a sharp exothermic peak associated with protein gelation, although the gel state was macroscopically observed at the end of calorimetric experiments in protein containing systems.

0.00 -0.02

Heat flow (mW)

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-0.04 -0.06 -0.08 HSA HSA/F127 5% HSA/F127 17%

-0.10 -0.12 320

330

340

350

T (K)

Figure 5. The µDSC thermograms characteristic to HSA denaturation, in absence and presence of F127. PeakFit analyses of the endothermic parts of the thermograms shown in Figure 5 provide the values of the temperature of denaturation, TD1 and TD2 (peak temperatures), and two different values of the unfolding enthalpy of HSA corresponding to the two peaks (∆H1 and ∆H2). These


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values were determined in the error limit of ±0.1 K for temperatures of micellization or denaturation and in the limit of ± 1 kJ/mol for enthalpies. The decomposed thermograms are presented in Figures S2-S4. PeakFit thermodynamic parameters thermodynamic parameters of HSA denaturation are summarized in Table 3.

Table 3. Thermodynamic characteristics of HSA denaturation in the absence and in the presence of F127 Sample

∆H,

∆H 1,

∆H 2,

(kJ/mol)

(kJ/mol)

(kJ/mol)

39.82

248.15

149.33

98.81

55.93

44.07

507.91

284.07

223.83

77.01

22.98

585.04

450.54

134.50

Tpeak

TD1

TD2

% A1

(K)

(K)

(K)

HSA

337.7

334.8

339.8

60.18

HSA

339.7

335.8

342.6

335.9

334.8

340.8

%A2

5% F127 HSA 17%F127

The contributions of the two peaks in the overall denaturation process are different in solutions of HSA/F127 compared to solutions of HSA. In the case of HSA, the ratio of these contributions is roughly 3:2, whereas in the presence of F127 this depends on the Pluronic concentration. The temperature shift of the two peaks of the overall denaturation process observed in solution of F127 5% supports the hypothesis that F127 micelles stabilize the protein conformation and delay the denaturation process. At higher concentration of F127 (17%), the overall denaturation peak is shifted to lower temperatures; the contribution of the first component of the denaturation process increases, whereas the contribution of the second one decreases compared to the denaturation process of HSA in the absence of the Pluronic. Upon heating of a HSA/F127 17% solution, Pluronic F127 exhibits a transition from micelle to gel. As



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the denaturation process occurs in a temperature interval overlapping only partially with the gel state of F127, it is expected that reorganization of Pluronic micelles influences the denaturation of the protein. The endothermic change of the enthalpy during albumin denaturation increases considerably in the presence of F127. This proves the interaction between F127 and albumin, reflecting a change in the hydration shell of the protein that influences the denaturation process. Taking into account the energy contribution of the of hydrogen bond rupture (16-17 kJ/mol)46 it is possible to estimate the additional numbers of hydrogen bonding type interactions involved in the denaturation process in the presence of F127: these numbers are approximately 15 for F127 5% and 20 for F127 17%, respectively. Figure 6 displays the marked difference between the micellization endotherms of the first heating cycle and the following ones for HSA/F127 5% and HSA/F127 17% systems. Thermal signals are practically identical for the 2nd and 3rd heating cycles, but their onset temperatures and enthalpies are lower than the corresponding ones corresponding to the 1st heating cycle. This indicates an enhanced effect of denaturated HSA (irreversible denaturation takes place after the 1st heating cycle) on the F127 micellization compared to a similar effect produced by the protein in its native form. This effect may be ascribed to the formation of a protein gel following denaturation (1st heating) that captures a sizable amount of water molecules resulting in a more concentrated solution of the other component, F127. Micellization temperatures (Table 4) and enthalpies of the latter are thus lowered as compared to the initial (nominal) values of F127 concentrations (5% and 17%, respectively).


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0.0

0.0 -0.5

F127 5% -1.0

HSA/F127 5% st 1 heating cycle nd 2 heating cycle rd 3 heating cycle

-1.5 (A)

-2.0

285

300

315

Heat flow (mW)

-1.0

Heat flow (mW)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


330

-2.0

F127 17%

-3.0

HSA/F127 17% st 1 heating cycle nd 2 heating cycle rd 3 heating cycle

-4.0 -5.0 (B) -6.0 280

290

T (K)

300

310

320

T (K)

Figure 6. Native vs. denatured HSA influence on the (A) F127 5% and (B) F127 17% micellization process. After the first heating, cycle the protein in irreversible denatured.

Table 4 Tonset values for F127 micellization System

Tonset (K)

System

Tonset (K)

F127 5%

293.92

F127 17%

287.25

F127 5%_2nd heating cycle

293.70

F127 17%_2nd heating cycle

287.17

F127 5%_3rd heating cycle

293.72

F127 17%_3rd heating cycle

287.17

F127 5%_HSA_1 st heating cycle

293.57

F127 17%_HSA_1st heating cycle

286.62

F127 5%_HSA_2nd heating cycle

292.28

F127 17%_HSA_2nd heating cycle

286.06

F127 5%_HSA_3rd heating cycle

292.21

F127 17%_HSA_3rd heating cycle

286.03

The effect of multiple heating cycles on the Pluronic micellization temperatures is practically negligible compared to the same effect in the presence of HSA. Figure S5 represents the thermal behavior of Pluronic F127 aqueous (protein free) solutions subject to several heating cycles, which demonstrates the aforementioned minor changes in temperature and thermal effect.



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Circular dichroism, rheology and µDSC measurements offer global information on the HSA/F127 systems, providing evidence on how specific properties of HSA and F127 are influenced by each other in the mixture. These systems are non-homogeneous at the nanoscale level, thereby pointing toward EPR spectroscopy, with its spin probe capability representing a potential source of information on local changes induced by the variation of physical parameters such as temperature or concentration.

Local physicochemical profiling resulting from EPR studies Using the spin probe method of EPR spectroscopy, it was possible to gain insight into the processes occurring at the nanoscale level in solutions of HSA and/or F127 as a function of temperature and copolymer concentration. EPR methods are recognized as valuable tools in investigating co-polymer assemblies as a function of temperature.16,61-64 By introducing spin probes with affinity for both macromolecules involved it is possible to monitor such interactions through line shape analysis of the EPR spectra. The spin probes 5-DSA and CAT16 (Figure 1) have similar structures bearing a paramagnetic moiety attached to a hydrophobic tail. Due to its ionic character, CAT16 binds preferentially to protein sites exposed on the protein surface,12,13 whereas 5-DSA binds to hydrophobic pockets on HSA.13,51 The spin probes CAT16 and 5-DSA are probably targeting different locations in the Pluronic micelles as well, as their EPR spectra exhibit different motions. Thus, in micellar media, CAT16 has a motion still in the fast regime, whereas in the case of 5-DSA slowing down of the motion can be observed and further attested by the order parameter S, described by equation 2. These observations are illustrated in the spectra of CAT16 and 5-DSA recorded in solutions of F127 5% at 293 K (Figure 7).


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Figure 7. EPR spectra of (a) CAT16, and (b) 5-DSA recorded in solutions of F127 5% at 293 K. In spectrum b, points indicated define All and A┴ used in the calculation of the order parameter S (equation 2).

The EPR spectra of CAT16 and 5-DSA were recorded in the temperature interval 273– 303 K in solutions of F127 5%, F127 17%, HSA 20 mg/ml and in the corresponding HSA/F127 mixtures. The EPR spectra of CAT16 and 5-DSA in the system of F127 17% are shown in Figure 8A and 8B, respectively. It can be seen that the EPR spectra of CAT16 reflect a relatively fast regime, while in the case of 5-DSA there is a clear change in the motion regime from a relatively fast motion at temperatures below cmt to a restricted motion characterized by the order parameter S as micelles form. The EPR spectra of CAT16 and 5-DSA exemplify the micellization process in the temperature interval 285–303 K. Thus, in both cases, once micelles form in solution at a certain temperature (288 K for CAT16 and 285 K for 5-DSA), a second



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component appears in the EPR spectra, which becomes the sole component once micellization reaches completion (see EPR spectra recorded at 303 K, Figure 8A and 8B).

(A)

(B)

Figure 8. EPR spectra of (A) CAT16 and (B) 5-DSA in F127 17% in the temperature interval 278–303 K.

The hyperfine splitting constant aN represents an EPR parameter indicating the polarity of the microenvironment around the paramagnetic moiety. In the case of the EPR spectra of CAT16, the aN value is directly derived from the spectra, whereas in the case of the EPR spectra of 5-DSA corresponding to micellar solutions, this parameter is extracted through equation 3:16 aN = 1/3(All+A┴)

(3)

Figure 9 shows the variation of aN for both spin probes as a function of temperature. It can be observed that in the case of 5-DSA there is a steep variation of aN with temperature around the micellization temperature, while in the case of CAT16 the change in the slope is visible but less pronounced. The polarity inside the F127 micelles is not homogeneous, with the arising curves supporting the conclusion that the two spin probes are located in different regions of the micelle, in view of the fact that the observed variations are not similar.


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CAT16 5-DSA

17 16 15

aN(G)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14 13 12 11 275

280

285

290

295

300

305

T (K)

Figure 9. Temperature dependence of aN for CAT16 (squares) and 5-DSA (circles) in F127 17% solution.

The EPR spectra recorded in the same temperature interval for the spin probes in a solution of F127 5% are presented in Figure S6. The variation of aN exhibits a similar trend in solution of F127 5% (Figure S7), with the steep change of aN around the temperature of micellization corresponding to this concentration. Furthermore, a correlation between the information provided by DSC measurements and EPR spectroscopy emerges evident. The EPR spectra of CAT16 and 5-DSA in HSA solutions have been previously analyzed.12 The experiments reveal a faster motion of CAT16 in the complex with HSA than in the case of 5-DSA. The spectra are similar to those observed in BSA solutions,12,13 supporting the similarities between the binding sites of these albumins. In fact, the spectrum of CAT16 emerges in the relatively fast regime, while 5-DSA is immobilized in the complex with HSA (Figure S8). Figure S8A shows a faster motion for CAT16 in solutions of HSA as the



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temperature rises. In the case of spin probe 5-DSA (Figure S8B), immobilization of the spin probe in the protein complex persists although the temperature rises up to 333 K. This behavior clearly demonstrates strong binding of 5-DSA to albumin that is not influenced by rising temperatures. At 293 K, aN for CAT16 in F127 is 16.3 G, whereas for HSA is 16.54 G, suggesting that in the complex with the protein, this spin probe experiences a less hydrophobic environment than in the case of F127 micelles. The spectra of CAT16 and 5-DSA recorded in solutions of HSA/F127 in the temperature interval 273–303 K are illustrated in Figure 10. Both spin probes experience a migration between HSA and F127, depending on the self-assembly process of F127. Thus, at temperatures below cmt for F127, the spin probe forms a complex with the protein. Once micelles form, the spin probe gradually migrates to the F127 micelles. This is clearly evident in the case of 5-DSA, which experiences a more pronounced dynamic change than CAT16 in similar systems. The analysis of the EPR spectra shown in Figure 10A reveals that the transfer of CAT16 from its complex with HSA to the F127 micelle is accompanied by smooth changes of the EPR parameters, indicating that this spin probe is placed in the hydrated region represented by the micellar corona. The experimental spectra of CAT16 in mixed solution have been simulated in order to determine their parameters and, by comparison with the parameters obtained for HSA or F127, to clearly establish the distribution of the spin probe. For instance, in Figure S9 are presented the experimental and simulated spectra of CAT16 at different temperatures for a solution F127 17%. The corresponding EPR parameters are shown in Table S1. Figure S10 presents the experimental and simulated spectra of CAT16 in HSA, F127 17 % and HSA/F127 17% recorded at 288 K. The values of the rotational correlation times, τc, and those of aN for CAT16 derived from the simulation parameters reveal that the spin probe at 288 K is located in


Page 25 of 38

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the micelle (Table S2). In micellar solution, aN slightly decreases compared to HSA, this being the parameter that differentiates between different environments for the analyzed systems. At 288 K, the τc values are similar in all three solutions. At 293 K, the EPR spectra of CAT16 in F127 17% and in the mixture HSA/F127 17% are characterized by the same aN value (16.33 G). The values of τc for CAT16 are 1.85×10-10, 1.80×10-10, and 2.95×10-10 s in solution of F127 (17%), F127/HSA and HSA, respectively. These values demonstrate that, at 293 K, the spin probe CAT16 in HSA/F127 solution is located in the F127 micelles, as both aN and τc values are similar to those observed in F127 17 %.

(B)

(A)

Figure 10. EPR spectra of (A) CAT16 and (B) 5-DSA in HSA/F127 17% in the temperature interval 278–303 K. The two components feature of 5-DSA EPR spectrum is clearly evident at 288 K in solution of HSA/F127 17%. The simulations of the EPR spectra of 5-DSA in similar solutions and temperature intervals were performed and the results are shown in Figures S9 and S10, with the EPR parameters summarized in Tables S1 and S2.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Careful examination of the values of the order parameter S (shown in Table 5), calculated for 5-DSA in solutions of F127 17% and HSA/F127 mixtures at various temperatures, suggests that the presence of HSA does not influence the local environment in the F127 micelle, where the spin probe is hosted. This result makes sense as 5-DSA is located more probably in the micellar core.

Table 5. The values of order parameter, S, corresponding to 5-DSA in micellar systems F127 17% and HSA/F127 17% T (K) 285

S F127 17 % 0.55

S HSA/F127 17% 0.56

288

0.55

0.55

293

0.51

0.51

303

0.44

0.44

313

0.39

0.39

323

0.35

0.35

Figure 11 shows the variation of the order parameter S for 5-DSA in F127 5% and 17% at temperatures above cmt. This figure demonstrates that packing of F127 chains in the micelle is independent of the overall concentration of the copolymer.


Page 26 of 38

Page 27 of 38

0.54

F127 17 % F127 5 %

0.52 0.50 0.48 0.46 0.44

S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.42 0.40 0.38 0.36 0.34 290

295

300

305

310

315

320

325

T

Figure 11. Temperature dependence of the order parameter, S, in micellar solutions of F127 5% (circles) and F127 17% (squares).

This information, resulting from the analysis of the S parameter characterizing the EPR spectra of 5-DSA, supports the hypothesis that the interaction between HSA and F127 micelles involves the hydration shell of the protein and the hydrated corona of Pluronic micelles. .

CONCLUSIONS The work presented here a) pertains to the investigation of interactions between the amphiphilic block copolymer F127 and HSA, and b) assesses the impact of polymer selfassembly on ligand–protein interactions perused by techniques poised to provide both global and local information. All techniques employed reveal that the interaction between F127 and HSA



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influences the properties of the participants, i.e. the protein and the copolymer. Thus, cmt and cgt for F127 are influenced by the presence of HSA, with the denaturation temperature of the protein being also dependent on the self-assembly of the copolymer molecules. This reciprocal influence observed at the macroscopic scale is also noted at the nanoscale level. The spin probe method of EPR spectroscopy has been proven to be a most suitable tool demonstrating the emerging effects. In particular, changes in the EPR spectra of 5-DSA and CAT16 in solutions of F127, HSA as well as mixtures thereof, testify to the migration of spin probes between HSA and F127 micelles. The spin probes are released from the complex with the protein, once the F127 chains assemble into micelles. The release of the spin probes commences at the cmt. Thus, EPR spectroscopy can be used in the determination of the physical conditions optimally efficient for transfer between exogenous drug carriers (in this case F127) and indigenous drug carriers (albumin). Collectively, the results of the study support potential applications in optimizing certain drug formulation processes involving Pluronic block copolymers.

Supporting Information Available: Figures: The µDSC thermograms of F127 5%, 17%, HSA, HSA/F127 5% and HSA/F127 17%; PeakFit decomposition of µDSC thermograms of HSA thermal denaturation, HSA/F127 5% and HSA/F127 17%; The EPR spectra of CAT16 and 5DSA in F127 5% at different temperatures; Temperature dependence of aN for CAT16 and 5DSA in F127 5%; The EPR spectra of CAT16 and 5-DSA in HSA solution at different temperatures; Experimental and simulated EPR spectra of CAT16 and 5-DSA in F127 17% solution at various temperatures. Experimental and simulated EPR spectra of CAT16 and 5-DSA in different media at 288 K. Tables: The EPR parameters of the spin probes in F127 17%


Page 29 of 38

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solution, at different temperatures; The EPR parameters of the spin probes in different media, at 288 K. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS: This work was supported by a grant from the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN-II-ID-PCE-2011-30328. G.I. gratefully acknowledges the sponsorship of COST Action CM1201. The authors gratefully acknowledge the support of the EU (ERDF) and the Romanian Government for approving acquisition of the research infrastructure under POS-CCE O 2.2.1 project INFRANANOCHEM – Nr.19/01.03.2009.

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Interaction between Albumin and Pluronic F127 Block Copolymer

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