A detailed analysis of the influence of β-cyclodextrin derivates on the thermal denaturation of lysozyme

Accepted Manuscript A detailed analysis of the influence of β-cyclodextrin derivates on the thermal denaturation of lysozyme Tatiana Starciuc, Nicolas Tabary, Laurent Paccou, Ludovic Duponchel, Yannick Guinet, Bernard Martel, Alain Hédoux PII: DOI: Reference:

S0378-5173(18)30803-2 https://doi.org/10.1016/j.ijpharm.2018.10.060 IJP 17878

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

7 August 2018 24 October 2018 26 October 2018

Please cite this article as: T. Starciuc, N. Tabary, L. Paccou, L. Duponchel, Y. Guinet, B. Martel, A. Hédoux, A detailed analysis of the influence of β-cyclodextrin derivates on the thermal denaturation of lysozyme, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.10.060

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A detailed analysis of the influence of β-cyclodextrin derivates on the thermal denaturation of lysozyme Tatiana Starciuc1, Nicolas Tabary1, Laurent Paccou1, Ludovic Duponchel2, Yannick Guinet1, Bernard Martel1, Alain Hédoux1. Université Lille F-59000 Lille France 1FST UMET UMR 8207, F-59655 Villeneuve d’Ascq France 2FST LASIR UMR 8516, F-59655 Villeneuve d’Ascq France ABSTRACT The influence of HPβCD on the thermal denaturation of lysozyme was analyzed mainly from microcalorimetry and Raman investigations carried out in the molecular fingerprint and the low-frequency regions. It was shown that Raman spectroscopy investigations performed on a wide spectral range give the opportunity to describe the influence of HPβCD on the mechanism of protein denaturation. Using D2O as solvent allowed us to show that HPβCD mainly destabilizes the tertiary structure of lysozyme by enhancing the protein flexibility and thus inducing the destabilization of the secondary structure. Principal components analysis (PCA) was used for spectra treatment, providing important information about inclusion complex formation between protein hydrophobic residues and CDs molecules. Combining PCA and classical technics (curve fitting) of data analysis allowed a better understanding of the influence of HPβCD on the protein denaturation that seems to be related to the CDs capacity to form inclusion complex. It was observed that these interactions prevent the formation of new strong H-bonds between β-sheet structures thereby inhibiting protein aggregation. This study reveals that CDs are promising systems for inhibiting protein aggregation without protein denaturation, only if designing derivative CDs is carefully controlled. Keywords

:

cyclodextrins;

protein

aggregation;

protein

denaturation;

Raman

spectroscopy; Principal Component Analysis.

INTRODUCTION Biotechnology advances have allowed the development and the large-scale production of complex polymers of amino acids for the treatment of genetic and cancerous diseases. A major concern of the pharmaceutical industry is related to the

drug stability, especially for therapeutic proteins, since these biomolecules may lose their functionality in solution before administration. Various opportunities can be used for improving the stability of the proteins native state, depending on the storage time. For long-term storage, proteins are preferentially converted to a stable solid by freezedrying. However, aggregation often occurs during powder reconstitution probably because of structural distortion induced by the freeze-drying process. In order to inhibit protein aggregation and to improve solubility, different excipients (surfactants, polymers, proteins) including cyclodextrins (CDs) (Aachmann et al., 2003; Serno et al., 2011) are added into the protein formulations. CDs can be described as cyclic, water soluble olygosaccharides, composed of 6 – 8 glucose units (α – 6 units, β – 7 units and γ – 8 units) and have a hydrophobic cavity (Szejtli, 1990). The specificity of CDs is their ability to form inclusion complexes with a hydrophobic guest (Blanchemain et al., 2011; Serno et al., 2011; Valentini et al., 2015) thereby making these materials interesting drug delivery systems (DDS) (Loftsson and Duchêne, 2007; Singh et al., 2002). β-CDs are most accessible and useful compared to α- and γ-CDs, due to an optimal cavity diameter adapted for complexation of hydrophobic amino-acids (Aachmann et al., 2003; Otzen et al., 2002; Rajabzadeh et al., 2011). Previous investigations (Cooper, 1992; Otzen et al., 2002) on the action of α- and γ -CDs on various proteins revealed notable destabilizing effects at elevated temperatures which could be a factor limiting their use. CDs are generally used as building blocks. Designing derivative CDs allows to control their solubility. It was observed that CDs-derivates bioprotective effectiveness depends on : i) the nature of substituents (Otzen et al., 2002; Tavornvipas et al., 2006), ii) the substitution degree (Tavornvipas et al., 2004; Yoshida et al., 1988) and iii) the CDs concentration (Anchordoquy et al., 2001; Iwai et al., 2007; Izutsu et al., 1994). It was shown, that Hydroxy-Propyl-β-CD (HPβCD) has systematically a higher bioprotective effect on the protein stability than other CD-s derivates (Otzen et al., 2002; Sah, 1999; Serno et al., 2011). On the other hand, methyl-β-cyclodextrin (Me-βCD) is known as promising DDS agent (Blanchemain et al., 2011; Taha et al., 2013). The

influence of CDs on protein stability deserves significant consideration, in order to be efficiently used as both DDS and bioprotective agent. The aim of this paper is to analyze the lysozyme stability (model protein) in presence of various concentrations of HPβCD and Me-βCD, using Raman spectroscopy. It was previously shown that this is a suitable technique for deciphering the mechanisms of protein denaturation (Hédoux et al., 2006) and stabilization (Hédoux et al., 2014; Starciuc et al., 2017). This technique allows the monitoring of both the protein secondary structure (amide I band region) and the H-bond network of the solvent (low-frequency region). Additionally Raman spectroscopy can be also used to detect the complexation of amino acids substituents within CDs cavities (Valentini et al., 2015).

MATERIALS AND METHODS 1. Sample preparation Lyophilized chicken egg white lysozyme (Lys) was purchased from Sigma Aldrich (purity minimum 90%). Kleptose HPB® and Kleptose HP® Hydroxypropyl-Cyclodextrins with molar substitution (MS) 0.85 and 0.62 respectively called HPβCD_0.85 and HPβCD_0.62) and Crysmeb® Methyl-β-Cyclodextrin (Me-βCD) were provided by Roquette & Frères (France). Lysozyme and HPβCD was dissolved in double distillated (H2O) or heavy water (D2O), with a 99,99% isotopic purity atom D, purchased from Sigma Aldrich (France). In order to determine the influence of CDs content on protein stability during heating, different protein/excipient weight ratio were analyzed in this work (1:0, 1:1, and 1:2 w/w protein/CDs). The averaged molar weights of CDs derivatives used in this work are similar (~ 1380 – 1480 for HPβCD and ~ 1310 for MeβCD). Solutions were agitated in an Ependorf agitator at room temperature for 1h before analyzing.

2. Instruments Raman investigations in the 600 – 1800 cm-1 spectral range were carried out using a Renishaw InVia Raman spectrometer (Renishaw plc, Wottonunder-Edge, Gloucestershire, UK). The 785 nm laser beam was focused via a ×50 long working distance Leica objective (Leica microsystemes, SAS, Nanterre, France), which allows to

analyze a volume of about 150 µm3 (the spot size being about 1 μm in diameter and 150-200 μm in depth) within the sample. The spectra were collected in backscattering geometry, with a resolution of 2 cm-1, in the 600 – 1800 cm-1 spectral range. Heating runs were performed using THMS600 Linkam temperature controlled chamber (Linkam Scientific Instruments, Guildford, Surrey, UK). Low frequency Raman investigations in the 10 – 600 cm-1 spectral range were performed in backscattering geometry to obtain non polarized light-scattering spectrum under a scattering angle θ = 180°, using a mixed argon-krypton coherent laser at 514.5 nm. The very high-dispersive XY Dilor spectrometer is composed of three gratings. The choice of experimental conditions, i.e. 514.5 nm laser line, the width of entrance and exit slits opened at 200 μm, allows a rejection of the exciting line down to 5 cm-1, and leads to a resolution slightly lower than 2 cm-1 in the low-frequency region. Solutions (~ 0.4 cm3) were loaded in spherical Pyrex cell and hermetically sealed. The sample was placed under the regulated nitrogen flux of an Oxford device. Low and high – frequency Raman spectra were collected using both spectrometers, with an acquisition time of 60 s during heating ramps at 0.5 K/min. In these conditions a spectrum was collected every 0.5 K. Differential Scanning Calorimetry (DSC) measurements were carried out on a very sensitive microcalorimeter microDSC III (Setaram Instrumentation, Caluire, France). Typical sample mass of 400 mg was used for lysozyme formulations. Only heating runs were performed with a scanning rate of 0.5 K/min. The enthalpy of denaturation was determined using Calisto Preprocessing software (Setaram, Instrumentation, Caluire, France), by integration of the heat flow after subtraction of the sigmoidal baseline tangent to the heat flow trace above and below the endotherm of denaturation.

3. Description of methods for the analysis of Raman Data Figure 1a shows the Raman spectra in the molecular fingerprint region of lysozyme aqueous solutions at 0 and 10 % of HPβCD, compared with that of cyclodextrin (CDs) solution. It is clearly observed that the protein and CDs bands are

overlapping over a wide spectral range, except the AI region. Consequently, two different methods have been used for analyzing the amide I band and the rest of the fingerprint region between 600 and 1600 cm-1. 3.1. Monitoring of Protein unfolding from the analysis of amide I band The amide I (AI) band is detected in an isolated spectrum region between 1600 and 1800 cm-1. In this spectral range, Figure 1a shows that there is no overlapping with Raman bands of other chemical species, which could pollute the spectrum of the protein, except the bending vibration of H2O which has a low intensity. The AI band mainly arises from C=O stretching vibrations with a minor contribution of C – N stretch and C-N-H in-plane bend (Surewicz et al., 1993; Williams and Dunker, 1981). This latter is responsible for the sensitivity of the AI band to NH/ND isotopic exchanges. Figure 1b represents the AI region of lysozyme dissolved in H2O (LWh) and D2O (LWd) at room temperature. The downshift of the AI band position in LWd sample, with respect to the AI band position in LWh, results from isotopic exchanges between D2O and NH groups exposed to the solvent in the native state of the protein. Using D2O for dissolving lysozyme has two important interests. First, the spectrum of AI band is not polluted by the contribution of the bending band in H2O (~ 1600 cm-1) and second, isotopic exchanges between solvent and proteins provide important information about the mechanism of protein denaturation (Hédoux et al., 2014, 2006). The temperature dependence of the AI region in the LWd sample, plotted in Figure 1b, clearly shows a frequency downshift by heating up to 70 °C, explained by the enhancement of isotopic exchanges OD → NH. This phenomenon was interpreted as corresponding to the solvent penetration in the protein interior, in a more flexible tertiary structure with intact secondary structure (Hédoux et al., 2014). The AI frequency upshift and band broadening, observed above 70 °C were associated to the unfolding of the secondary structure. Consequently, the AI band probes changes in tertiary and secondary structures of the protein by using D2O as solvent. In order to determine conformational changes of protein structure the position and the shape of AI band was widely analyzed (Belton and Gil, 1994; Hedoux et al., 2013; Hédoux et al., 2014). In the present study, the AI peak position was determined by fitting the AI bandshape to Gaussian functions (Figure 1b) using the Wire software (v4.1,

Renishaw plc, Wottonunder-Edge, Gloucestershire, UK) and PeakFit software (v4.12, Systat Software Inc, USA). The temperature dependence of AI band position provides a denaturation curve characterized by a sigmoidal shape well described by : 𝜈=

(

)

ν𝐹 ‒ ν𝑈

(

1 + 𝑒𝑥𝑝

𝑇 ‒ 𝑇𝑚 ∆𝑇

)

(1) + ν𝑈

where ν𝐹 and ν𝑈 correspond to the AI position in the folded (F) and the unfolded (U) states, 𝑇𝑚 is the midpoint temperature and ∆𝑇 the temperature range where the unfolding process occurs (Hédoux et al., 2006). 3.2. Monitoring of the CDs-protein interactions using Principal Component Analysis (PCA) The Raman spectroscopy is a well-adapted technique for detecting complex formation and identifying encaged molecular groups. Table 1 and figure 2 represent the Raman bands corresponding to the internal vibrations of protein hydrophobic residues, i.e. tyrosine, tryptophan and phenylalanine, likely to be encaged within CDs cavities (Couling et al., 1998; Harada et al., 1986; Herrero et al., 2010; Milan-Garces et al., 2013; Miura et al., 1988; Siamwiza et al., 1975; Takeuchi and Harada, 1986; Unno et al., 2010),. Changes in the temperature behavior of these Raman bands parameters, detected by adding CDs into lysozyme solution, will reveal the complex formation between hydrophobic protein residues and CDs molecules. Considering the overlapping of Raman bands of lysozyme and HPβCD observed in figure 1a, PCA was considered as a well-adapted method for detecting and identifying the protein-CDs complex formation in the 600 – 1600 cm-1 region. The spectra collected in these two samples (LWh and LWhHPβCD), during a heating ramp from 25 to 100 °C were analyzed by PCA as a single data set using the PLS Toolbox (Eigenvector Research Inc. WA) for Matlab environment (version 2015; Mathworks). The data preprocessing was performed in two steps. In a first step, all spectra were baseline corrected using polynomial function of degree 3. In a second step all data were normalized by the integrated intensity in the 1500 – 1800 cm-1 spectral region, distinctive of the protein. All data were centered by removing the mean offset

from each variable. In order to compare the influence of the CDs molar substitution on the protein-CDs interactions, PCA of preprocessed data was performed on two data sets containing spectra collected in lysozyme solutions without CDs (LWh) and with HPβCD_0.85 or HPβCD_0.62, respectively. 3.3.

Low-frequency analysis

Raman spectra collected during the heating ramp at 0.5 °C/min were plotted in figure 3a, after conversion of the Raman intensity (𝐼𝑅𝑎𝑚𝑎𝑛(𝜔,𝑇)) into reduced intensity (𝐼𝑟 (𝜔)) according to : 𝐼𝑟(𝜔) = 𝐼𝑅𝑎𝑚𝑎𝑛(𝜔,𝑇) ([𝑛(𝜔) + 1]𝜔) with 𝑛(𝜔) = [1 ‒ 𝑒𝑥𝑝(ℏ𝜔 𝑘𝑇)]

‒1

(2)

, in order to obtain low-frequency spectra free of band-

shape distortion due to thermal population effects. The low-frequency spectrum of a protein solution is typical of a highly disordered molecular system (Hédoux, 2016), with overlapping of the relaxational and the vibrational contributions within the 10 – 70 cm-1 region. These two contributions can be separated by fitting procedure described in figure 3b. The relaxational contribution, also called quasielastic intensity (𝐼𝑄𝐸𝑆) is well described by a Lorentzian shape centered at ω = 0. The temperature dependence of 𝐼𝑄𝐸𝑆 can be obtained by plotting the integrated intensity of the Lorentzian function or by plotting the integrated intensity of 𝐼𝑟(𝜔) in the 10 – 30 cm-1 region, where the vibrational contribution gives a negligible and temperature independent contribution to the spectrum. The vibrational contribution (𝐼𝑣𝑖𝑏) is described by a lognormal function which can be obtained from the 𝐼𝑟(𝜔)-spectrum by subtracting the Lorentzian function. The Raman susceptibility is then obtained according to : 𝜒"(𝜔) = 𝜔.𝐼𝑣𝑖𝑏(𝜔) =

𝐶(𝜔) 𝐺(𝜔) 𝜔

(3)

where 𝐶(𝜔) and 𝐺(𝜔) correspond respectively to the light-vibration coefficient and the vibrational density of states (VDOS). Comparing inelastic neutron scattering and lowfrequency Raman spectra has shown that the Raman susceptibility is a representation

very close to the VDOS (Frontzek et al., 2016; Hédoux et al., 2001). 𝜒"(𝜔)-spectra are plotted in the inset of figure 3b in the native (25 °C) and denatured (95 °C) states of lysozyme dissolved in D2O. These spectra are composed of two broad bands. The lowfrequency band (I) is mainly related to the protein dynamics, while the high-frequency band (II) corresponds to the intermolecular O – H stretching vibrations in the H-bond network of water (Hédoux et al., 2014). It was shown that the low-frequency spectrum was sensitive to the rigidity of the predominant secondary structure, thereby providing information on the structural changes associated with the protein denaturation (Perticaroli et al., 2014).

RESULTS AND DISCUSSION Figure 4 represents the contribution and the complementarity of different measurements performed in the analysis of various protein formulations. The DSC trace, plotted in figure 4a clearly reveals an endotherm distinctive of the protein denaturation, characterized by three parameters: (i) the midpoint temperature Tm, (ii) the enthalpy of denaturation ∆H and (iii) the Cp jump ∆Cp. These parameters are reported in tables 2 and 3 for lysozyme dissolved in H2O and D2O, in absence and in presence of HPβCD (MS = 0.85 and 0.62) and MeβCD. The temperature dependence of the AI band position, also plotted in figure 4a, points out two important features in the denaturation mechanism. The frequency downshift of the AI band was attributed to enhanced isotopic exchanges inherent to the solvent penetration in the protein interior. The frequency minimum was related to the molten globule (MG) state, corresponding to a very flexible tertiary structure with an intact secondary structure. The MG state is systematically observed in globular proteins (Hédoux et al., 2014, 2009; Seo et al., 2010), prior to the unfolding process of the secondary structure, characterized by the frequency upshift of the AI band. U ) is rigorously corresponding to the The half-transformation temperature (Tm

midpoint of the unfolding process, while Tm determined by DSC corresponds to the midpoint temperature of the whole protein denaturation process (Hédoux et al., 2006), including the transformation into the MG state and the unfolding of the secondary

structure. The temperature dependence of the quasielastic intensity determined in the very low-frequency (10 – 30 cm-1) range is plotted in figure 4b. The change in this temperature dependence is clearly detected at the temperature corresponding to the minimum of the AI band position, i.e. to the MG state.

1. Analysis of microcalorimetric data DSC traces for lysozyme formulations 0, 10 and 20 % of MeβCD and HPβCD_0.62 and HPβCD_0.85 are plotted in figure 5. The heat-induced protein denaturation in solution is characterized by three thermodynamic parameters reported in tables 2 (powders dissolved in H2O) and 3 (powders dissolved in D2O). It can be observed, that dissolving lysozyme in D2O slightly increases Tm, in agreement with previous studies performed on various proteins (Hédoux et al., 2009, 2006; Seo et al., 2010). This behavior was related to the temperature dependence of intermolecular O…H/D interactions in the hydrogen-bond network of water (Hédoux et al., 2006). The comparison of figures 5a, 5b and 5c reveals a significant shift towards lower temperatures of the denaturation endotherm recorded during heating of protein solutions containing 10 % of HPβCD_0.85 or MeβCD. This phenomenon is explained by a clear destabilizing effect of these two CDs derivates, more marked for MeβCD. By contrast, figure 5b reveals that HPβCD_0.62 has no significant effect on the endotherm midpoint temperature, in agreement with table 3. At high CDs concentration (wt 20 %), the protein stability decreases during heating (figure 5a and 5b). To summarize, the main information arising from these experiments is the significant influence of the molar substitution and the concentration of HPβCD on the protein stability, as it was expected (Branchu et al., 1999; Cooper, 1992; Cueto et al., 2003).

2. Analysis of Raman data Taking into account the strong destabilizing effect of MeβCD at low concentration (wt 10%), our attention was focused on the influence of both HPβCD compounds, on the

protein stability. To get a better insight to the influence of HPβCD on the phase transformations associated to the lysozyme thermal denaturation, Raman investigations were carried out on lysozyme solutions in absence and in presence of 10 and 20% of HPβCD in the molecular fingerprint region (600 - 1800 cm-1) and low-frequency range (10 – 300 cm-1). 2.1.

Analysis of the Amide I region (1500 – 1800 cm-1)

The spectral region 1500 – 1800 cm-1 is mainly composed of Raman bands corresponding to internal modes of lysozyme dominated by the AI band centered at about 1655 cm-1 (native state). The position of this band reflects the population of folded and unfolded conformations in a two-state denaturation process. For proteins dissolved in D2O, the position of the AI band is sensitive to isotopic exchanges making possible the detection of the solvent penetration into the protein interior, as shown in figure 1. Consequently, the position of the AI band was determined with a fitting procedure using Gaussian functions, widely described in previous studies (Hedoux et al., 2013; Hédoux et al., 2014, 2012, 2009, 2006; Starciuc et al., 2017). Figure 6a represents the position of the AI band evolution during heating, for lysozyme dissolved in D2O, at 0, 10 and 20 % of HPβCD_0.85. As observed in figure 4, figure 6a firstly reveals a downshift of the AI band position, reflecting enhanced NH/ND isotopic exchanges, induced by the penetration of the solvent in the protein interior. This phenomenon corresponds to the transformation of the native (N) state into the molten globule (MG) state, characterized by a highly flexible tertiary structure with an intact secondary structure. The downshift of the AI band can be fitted using a sigmoidal function similar to eq. (1) to determine 𝑇𝑀𝐺 𝑚 , the temperature of half transformation between the N and MG states, reported in table 4.It is worth noting that the downshift of the AI band is detected at lower temperatures in presence of HPβCD. Moreover, the onset temperature of the AI band downshift decreases with increasing the HPβCD concentration. This indicates that the addition of HPβCD promotes the flexibility of the tertiary structure and thereby inducing the penetration of the solvent within the protein. The unfolding of the secondary structure is observed via the upshift of the AI band mainly inherent to the C=O…H(D) H-bond breaking within helices. The unfolding of 𝑈 , the temperature rigorously the secondary structure is characterized by 𝑇𝑚

corresponding to the half transformation between MG and U (unfolded) states, at which the population of folded and unfolded states are equivalent. This temperature was determined by fitting the temperature dependences of AI band position curve using eq. (1). Table 4 summarize the obtained results. The analysis of the AI band is in agreement with calorimetric analysis, confirming that the addition of HPβCD_0.85 destabilizes the (N) state of lysozyme. However, Raman data provide subtle additional information i.e. HPβCD promotes the N → MG transformation, inducing by this way the secondary structure unfolding. Interestingly, it is clearly observed in figure 6a and in table 4, that 20 % wt of HPβCD destabilizes 𝑈 significantly the tertiary structure (𝑇𝑀𝐺 𝑚 decreases), but not the secondary structure (𝑇𝑚

being similar for 10 and 20% of HPβCD). Therefore, it can be assumed that HPβCD_0.85 has a stabilizing effect on the MG state. Figure 6b represents the temperature dependence of the AI band position for lysozyme dissolved in D2O, in absence and in presence 10 % of HPβCD_0.85 HPβCD_0.62. The addition of HPβCD, low molar substitution (0.62), has no significant effect on the protein denaturation, since the denaturation curves, described by the AI band position, in LWd and LWd-HPβCD_0.62 samples are almost superimposed. In this context, it is worth noting that the substitution degree of the CDs derivatives has an important effect on the stability of the native protein conformation. 2.2. Analysis of the Amide III band In order to obtain more information about the interactions between lysozyme and HPβCD, the 600 – 1600 cm-1 spectrum, plotted in figure 7a, was investigated. Only samples dissolved in H2O was analyzed in this part of the work, in order to avoid frequency shifts of some Raman bands inherent to NH/ND isotopic exchanges. The Raman band localized around 1220 and 1260 cm-1 was assigned to amide III band corresponding to β sheet and α helix structures respectively (Frontzek et al., 2016; Hédoux et al., 2011; Kocherbitov et al., 2013). It was observed that during heating of a protein solution, the AIII-β band shifts toward the low frequencies and its intensity

increases. These changes were attributed to the formation of new and stronger H-bonds between β-sheets (Ikeda and Li-Chan, 2004). To quantify these spectral modifications, the integrated intensity in the 1220 – 1260 cm-1 range was calculated in each spectrum. Figures 7b and 7c represent the results obtained from the analysis of spectra recorded during heating of three samples. It can be observed that the increase in intensity is larger in LWd sample and is detected at lower temperatures in (~ 40 °C) than in presence of CDs (~ 66 °C). Additionally, the comparison of figures 7b and 7c indicates that the intensity increase for the sample containing HPβCD_0.85 is two times larger than for HPβCD_0.62. In this context, it can be assumed that the CDs derivatives reduce the H-bonds formation between β-sheets, responsible for the protein aggregation phenomenon (Azakami et al., 2005), this effect being intimately dependent on their molar substitution. 2.3. Analysis of molecular fingerprint 600 – 1600 cm-1 using PCA The principal components analysis (PCA) is a powerful method to reveal subtle spectral changes and was used to characterize the CDs-protein interactions. The first PCA was realized, after data preprocessing, as described in section 3.2 of Materials and Methods section, on a data matrix containing spectra recorded during a heating ramp of two lysozyme solutions containing 0% (LWh) and 10% of HPβCD_0.85 (LWhHPβCD0.85), respectively. PCA results lead to considering four significant principal components (PCs). The second PCA was realized on a data matrix containing spectra recorded during a heating ramp of two lysozyme solutions containing 0% (LWh) and with 10% of HPβCD_0.62 (LWhHPβCD-0.62). Figures 8a represent the loading of the first principal component (PC1), while 8b and 8c describe the temperature evolution of PC1 corresponding scores in LWh, LWhHPβCD_0.85 and LWh-HPβCD_0.62 samples. The first principal component express ~ 95 % of the total variance of the collected spectra. Figure 8a shows that PC1 loading (black solid line) can be superimposed with the spectrum difference between spectra recorded in LWh-HPβCD and LWh samples at 25 °C. This spectrum difference (pink solid line) is distinctive of the CDs contribution. This indicates that the CDs structure is not affected by the presence of lysozyme in the formulation. On the other hand, it can be

observed that all scores corresponding to LWh sample are negative, contrasting with all positive values in LWh-HPβCD samples (figure 8b and 8c). This indicates that the variance corresponding to PC1 is attributed to the presence of CDs in this sample. It is known that each principal component explains a part of the total variance of the data set (Hotelling, 1933; Pearson, 1901). This variance decreases from PC1 to PC2, PC1 describing the maximum variance of the total variance and PC2 explains the maximum variance of the total residual variance, etc. In this context, a PCA applied on a single dataset containing spectra recorded in two samples, with and without CDs, allows removing the CDs contribution (PC1) in the spectra, making it possible to detect proteinCDs interactions from other PCs. Figures 9a represent the loading of the second principal component (PC2), while 9b and 9d the PC2 corresponding scores. The second principal component express only 3 % of the total variance of the collected spectra. Nevertheless, this variance has a real physical meaning. The variance corresponding to PC2 is generated by changes of Raman bands highlighted in figure 9a. Most of these Raman bands are assigned to Trp internal motions (table 4) and to amide bands (1200 – 1300 and 1600 – 1700 cm-1). Figure 9b and 9d represents the temperature dependence of PC2 scores compared to the temperature dependence of the AI band position (figure 9c and 9e), corresponding to the unfolding curves of the protein structure of the same formulations (LWh, LWhHPβCD_0.85 and LWh-HPβCD_0.62). It can be observed that the evolutions of scores and AI band position values are similar. All values slightly increase in the 25 – 72 °C temperature range, drastically increase from 72 to 86 °C and remain constant from 86 to 100 °C. A comparison between evolutions of scores values in LWh and LWh-HPβCD points out several important differences. In the sample containing HPbCD_0.85, PC2 scores are significantly lower than in LWh sample, in the 25 – 72 °C temperature range (figure 9b). Additionally, in the same temperature range, the temperature dependences of the AI band position in LWh and LWhHPβCD samples, are superimposed (figure 9c). Consequently, the differences observed between the evolution of PC2 scores values, from 25 to 72°C, in LWh and LWh-HPβCD-0.85, were interpreted as representative of the

protein – CDs interactions. PC2 scores strongly increase above 72 °C in both analyzed samples (LWh and LWhHPβCD). It is worth noting that this phenomenon and the AI frequency upshift, i.e. protein secondary structure unfolding, are simultaneously observed in the same temperature range. This indicates that PC2 scores behavior, in this temperature range, is associated to the unfolding process of the protein secondary structure. This is confirmed by the same temperature shift observed between both types of curves (AI position and PC2 scores) induced by addition of CDs. The lower quality of spectra in this dataset, leads to considering only two significant PCs. It can be observed in figure 9a that the two PC2 loading curves, obtained after the first and second PCA are superimposed, indicating that the variance corresponding to PC2 in both cases, is identical. The temperature dependences of PC2 scores and AI band position are plotted in figure 9d and 9e, respectively. Contrarily to HPβCD_0.85, it is clearly observed that in this case, there is no difference identified at room temperature between PC2 scores determined in formulations with and without HPβCD_0.62. Additionally, the two types of curves obtained in presence and in absence of CDs are superimposed in the 72 – 100 °C temperature range. Consequently, it can be concluded that CDs – protein interactions are detected in a lesser extent in the sample characterized by lower molar substitution (MS = 0.62) and the complex formation is strongly related to the thermal stability of lysozyme. The third principal component (figures 10a and 10b) corresponds to ~ 0.3 % of total variance and is associated to spectral changes of Raman bands observed at 759 cm-1 (Trp), 850 cm-1 (Tyr), 1003 cm-1 (Phe), 1008 cm-1 (Trp) highlighted in figure 10a. The PC3 scores temperature evolution, calculated in LWh and LWhHPbCD_0.85 samples are represented in figure 10b. It can be observed that all scores increase from room temperature to 70-72 °C and decrease drastically above this temperature. The first part of the score evolution curve is similar for two analyzed samples (LWh and LWhHPβCD-0.85). By contrast, above 70-72 °C a shift of the scores toward the low temperatures for the sample containing CDs can be observed. A comparison between figure 10b and figure 9c indicates that the unfolding of the secondary structure begins at 72 °C. In this context it can be concluded that PC3 can be associated with the thermal

denaturation process of the protein, from the native state to the molten globule state (N → MG) and from the molten globule state to the unfolded state (MG → D). The last significant principal component corresponds to 0.06 % of the total variance and is associated to spectral changes of Raman bands highlighted in figure 10c. Most of these Raman bands are associated to internal vibrational motions of hydrophobic amino-acids (Trp, Tyr and Phe) and of CDs ring (953 cm-1) (Valentini et al., 2015). PC4 scores in LWh and LWhHPβCD-0.85 are plotted in figure 10d indicating opposite behavior of these two samples. In absence of CDs, the scores are nearly constant from room temperature to 55 – 60 °C and increase with further heating up to about 85 °C. The temperature region (55 – 60 °C) corresponds to the detection of changes in the tertiary structure of the protein, as observed on figure 6a. In this state, hydrophobic amino-acids buried in the tertiary structure become accessible to the solvent, inducing spectral changes in regions where vibrations of these hydrophobic residues are Raman active. By contrast, in presence of CDs, the PC4 scores are significantly higher at room temperature, decrease by heating up to 72°C, and remain nearly constant above this temperature. This opposite behavior indicates that hydrophobic amino-acids have a different environment at room temperature and during all heating process. It is noticeable, that the variance corresponding to PC4 is generated by spectral changes of the Raman band at 953 cm-1, associated to CD ring vibration, which is very sensitive to the presence of a host molecule. In this context, it can be concluded that protein hydrophobic amino-acids are included in the hydrophobic cage of CDs molecules. The decrease of the PC4 scores indicates the low stability of the protein-CDs complexes upon heating. To summarize, it can be concluded that HPβCD_0.85 has a higher ability to form inclusion complex with lysozyme than HPβCD_0.62. As expected, the CDs –protein complex formation, decreases the thermal stability of lysozyme. It was observed earlier (figure 6a) that CDs derivate at MS = 0.85, improve the protein tertiary structure denaturation. The important affinity of hydrophobic amino-acids to the CDs hydrophobic cage facilitate the exposure of the buried hydrophobic residues into the tertiary structure, inducing by this way a higher flexibility of the protein structure and promoting the protein secondary structure denaturation.

2.4.

Analysis of the low-frequency spectrum

Ir(ω)-spectra were calculated using eq. (2) and the temperature dependences of the integrated intensity (IQES) are plotted in figure 11 for LWd and LWdHPβCD10 (MS = 0.85 and 0.62) solutions. It is firstly observed that the three IQES(T) curves are similar, and those corresponding to formulations containing CDs are almost superimposed and shifted with respect to the lysozyme aqueous solution, toward high IQES-values. However, IQES is slightly lower for HPβCD_0.62 around room temperature. These observations can be interpreted as reflecting a higher flexibility of the protein in presence of CDs, slightly more marked for higher molar substitution of CDs. This phenomenon is probably induced by the protein – CD interactions. A break is observed at temperatures corresponding to the minimum position of the AI band, i.e. to the MG state (figure 6). This temperature behavior of IQES reveals a change in the protein dynamics entering in the anharmonic regime from the MG state. As expected from the temperature dependence of the AI band position plotted in figure 6, the change in the slope of IQES(T) is detected at lower temperature in the formulation containing HPβCD_0.85. This analysis shows that CDs – protein interactions enhance the protein flexibility thereby promoting the N → MG transformation of the protein. The influence of HPβCD on the vibrational dynamics of water and lysozyme can be analyzed through the Raman susceptibility (χ″(ω)) obtained from the vibrational contribution of Ir(ω) according to eq. (3). The Raman susceptibility is considered as a close representation of the vibrational density of states (VDOS). In a first step χ″(ω) spectra of binary mixtures (water – CDs) were plotted in figure 12, for various weighted concentration of HPβCD (0, 10 and 20 %) in D2O to determine the influence of CDs on water. The analysis was performed for both molar substitutions of HPβCD (0.85 and 0.62). No difference can be detected between the low-frequency spectra corresponding to these two MS-values, and then spectra were only plotted for MS = 0.85. The spectrum of neat water is composed of two broad bands interpreted as corresponding to intermolecular vibrations of water molecules within the cage formed by their neighbors (Lerbret et al., 2011) (band I) and to collective intermolecular O – H …O stretching vibrations (Lerbret et al., 2011) (band II). It is observed that addition of HPβCD induces

a slight shift of the band (I) towards the low frequencies reflecting that the local environments experienced by water molecules are slightly softer in presence of CDs. By contrast, the influence of HPβCD is stronger on the second band (band II), inducing a significant intensity decrease accompanied with a shift towards low frequencies of the band. This indicates the break of the H-bond network of water probably responsible for the softening of the cage. The Raman susceptibility spectra of lysozyme solutions without and with 10, and 20 wt % of CDs (MS = 0.85) are plotted in figure 13a in the native state of lysozyme at room temperature. χ″(ω)-spectra obtained for the same concentrations of HPβCD_0.62 are strictly identical and was not shown. In absence of HPβCD, the spectrum is also composed of two broad peaks, which have the same meaning that those observed in the spectrum of neat water. In the spectra of lysozyme solutions, band (I) is related to the protein dynamics and the protein – solvent interactions (Lerbret et al., 2009), while band (II) mostly reflects the dynamics of the H-bond network of water (Lerbret et al., 2009). Addition of HPβCD slightly changes the shape and position of band (I) on the highfrequency side, and induces a significant decrease of the intensity of band (II), as observed in absence of protein in figure 12. From the analysis of the vibrational properties of lysozyme aqueous solutions, HPβCD has a H-bond breaker effect on water and does not influence significantly the vibrational dynamics of the protein and the protein – solvent interactions. The spectra of the same solutions, in the denatured (D) state of lysozyme (~ 100 °C), are plotted in figure 13b. The spectra collected in absence and in presence of 10 wt% of HPβCD_0.85 are superimposed, while in presence of 20 wt% of HPβCD_0.85, the spectrum is only different from the two others in the region of band (II). Figure 13c shows a very subtle difference between spectra recorded in the sample containing 10% of HPβCD for MS = 0.85 and MS = 0.62, only observed in the region of band (II). To summarize the low-frequency analysis, the addition of HPβCD has a main influence on the rapid motions of protein residues detected in the very lowfrequency region (below 50 cm-1) and on the dynamics of the solvent. The destructive action of HPβCD on the H-bond network of water enhances the protein flexibility and promotes the transformation of the tertiary structure of lysozyme.

CONCLUSION The in-situ Raman analysis of the molecular fingerprint region of lysozyme solutions with and without CDs, have shown that the influence of CDs on the protein stability is dependent on the derivative, the molar substitution and the concentration of CDs. MeβCD has a strong destabilizing effect on the protein structure, while HPβCD has a destabilizing effect for high molar substitution and for high concentrations. It was shown that PCA performed in the molecular fingerprint region makes it possible to detect protein – CDs interactions and to identify protein residues encaged within CD cavities. Low-frequency analyzes have revealed that addition of CDs enhanced the protein flexibility by interacting with hydrophobic protein residues and by breaking the H-bond network of water. The enhancement of protein flexibility favors the transformation of the native protein tertiary structure into the MG state, precursor of protein secondary structure unfolding. In the best case, HPβCD has no stabilizing effect on the secondary structure of the protein. However, the presence of HPβCD systematically prevents the formation of new H-bonding between β-sheet structures, thereby inhibiting the protein aggregation. Consequently, CDs can be considered as promising drug delivery systems inhibiting protein aggregation by encaging amino acids of relatively small peptides (Valentini et al., 2015). This study shows that caution must be taken on the choice of the molecular architecture of the material within the wide variety of CDs, to avoid protein denaturation. Similar investigations must be performed with different kinds of proteins, since it was shown that the effect of CDs on proteins are also depending on the protein structure (Aachmann et al., 2003; Serno et al., 2011). REFERENCES Aachmann, F.L., Otzen, D.E., Larsen, K.L., Wimmer, R., 2003. Structural background of cyclodextrin-protein interactions. Protein Eng 16, 905–12. https://doi.org/10.1093/protein/gzg137 Anchordoquy, T.J., Izutsu, K.I., Randolph, T.W., Carpenter, J.F., 2001. Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freezedrying. Arch. Biochem. Biophys. 390, 35–41. https://doi.org/10.1006/abbi.2001.2351 Azakami, H., Mukai, A., Kato, A., 2005. Role of amyloid type cross β-structure in the formation of soluble aggregate and gel in heat-induced ovalbumin. J. Agric. Food Chem. 53, 1254– 1257. https://doi.org/10.1021/jf049325f Belton, P., Gil, A.M., 1994. IR and Raman spectroscopic studies of the interaction of trehalose with hen egg white lysozyme. Biopolymers 34, 957–61.

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Figures

Figure 1 : Raman spectra of lysozyme formulations with D2O solvent a) in the molecular fingerprint region, in absence and in presence of 10% of HPβCD compared to the spectrum of HPβCD dissolved in D2O b) in the AmI region, in absence of HPβCD, at different stages of the denaturation process: the native state (25 °C), the molten globule state (70 °C) and the denatured state (95 °C). The AmI spectrum of lysozyme dissolved in D2O (LWd) is compared to that of lysozyme dissolved in H2O (LWh) at 25 °C. Arrows highlight frequency shifts of AI band.

Figure 2 : Raman spectra of lysozyme formulations dissolved in D2O, in the molecular fingerprint region. Arrows localize the Raman band assigned to the hydrophobic amino-acids internal vibrations described in Table 1.

Figure 3 : Low-frequency Raman spectra of lysozyme dissolved in D2O, plotted in the reduced intensity representation using (2) a) spectra collected during a heating ramp b) fitting procedure for separating relaxational and vibrational contributions; Raman susceptibilities in the native and denatured states are plotted in the inset.

Figure 4 : Detailed description of the thermal lysozyme denaturation process obtained using D2O as solvent a) from the DSC trace compared to the temperature dependence of the AmI position b) and from the temperature dependence of the quasielastic intensity

Figure 5 : Microcalorimetry analyzes of the thermal denaturation of lysozyme in absence (LWd) and in presence of : a) 10 % (LWdHPβCD10) and 20 % (LWdHPβCD20) of HPbCD with a high molar substitution (MS = 0.85) b) 10 % (LWdHPβCD10) and 20 % (LWdHPβCD20) of HPbCD with a low molar substitution (MS = 0.62) c) 10 % of HPβCD with a low (LWdHPβCD-0.62) and high molar substitution (LWdHPβCD-0.85) and of MeβCD (LWdMeβCD)

Figure 6 : temperature dependence of the AI band position a) in absence (LWd) and in presence of 10% (LWdHPβCD10) and 20% (LWdHPβCD20) of HPβCD (MS = 0.85) dissolved in D2O.

b) in absence (LWd) and in presence of 10% HPβCD, MS = 0.85 (LWhHPβCD_085) and HPβCD, MS = 0.60 (LWhHPβCD_060) dissolved in D2O Lines correspond to the fitting procedures using (1)

Figure 7 : Classical analysis of the AIII band region : (a) Raman spectra in the molecular fingerprint region of lysozyme dissolved in H2O in absence (LWh) and in presence (LWhHPβCD10) of 10% of HPβCD in the native (N) and denatured (D) states. The frame localizes the spectral range where the integrated intensity was calculated.

(b) Temperature dependence of the integrated intensity calculated in the 1220 – 1260 cm-1 range, of Raman spectra collected in solutions of lysozyme dissolved in H2O in absence (LWh) and in presence of 10% of HPβCD with MS = 0.85. (c) Temperature dependence of the integrated intensity calculated in the 1220 – 1260 cm-1 range, of Raman spectra collected in solutions of lysozyme dissolved in H2O in absence (LWh) and in presence of 10% of HPβCD with MS = 0.62. Dashed lines are only guides for the eyes to detect a change in the temperature dependences

Figure 8 : The loading (a) and scores (b, c) of the first principal component obtained after two PCA performed on a unique data set containing spectra recorded in three protein formulations, in absence (LWh) and in presence of HPbCD MS = 0.85 (LWhHPbCD_0.85) and HPbCD MS = 0.62 (LWhHPbCD_0.62), during a heating procedure.

Figure 9 : Loading (a) and scores (b, d) of the second principal component compared with AI position evolution (c, e) determined on spectra recorded during heating of three protein solutions

: in absence (LWh) and in presence of CDs at high (LWhHPbCD_0.85) and low (LWhHPbCD_0.62) molar substitution.

Figure 10 : Loadings (a, c) and scores (b, d) of the third and fourth principal components determined on spectra recorded during heating of two protein solutions : in absence (LWh) and in presence of CDs at high (LWhHPbCD_0.85) molar substitution.

Figure 11: temperature dependence of the quasielastic intensity of lysozyme dissolved in D2O without CD (LWd) and with 10% of HPβCD with two molar substitution 0.85 (LWdHPβCD_085) and 0.6 (LWdHPβCD_062).

Figure 12: Raman susceptibility of D2O-HPβCD mixtures for 0% (Wd), 10% (Wd-HPβCD10), and 20% (Wd-HPβCD20) of cyclodextrin. The arrows show the shift of band (I) and the intensity decrease of band (II) with addition of HPβCD.

Figure 13: Raman susceptibility of lysozyme dissolved in D2O without (LWd) and with 10% (LWdHPβCD10), and 20% (LWdHPβCD20) of HPβCD_0.85, and with HPβCD_0.60 a) In the native state (~ 20 °C); spectra of LWdHPβCD for MS = 0.85 and 0.62 are superimposed, and then only spectra corresponding to MS = 0.85 are plotted b) In the denatured state (~ 100 °C) c) In the denatured state for both formulations composed of 10% of HPβCD characterized by a degree of substitution of 0.85 and 0.62 Stars localize laser lines in Raman spectra. Table 1 : Raman bands corresponding to the internal vibrations of protein hydrophobic amino-acids Frequency in H2O (cm-1)

759.5

836.5

Frequency in D2O (cm-1)

757.5

833.5

857.3

852.8

875.7

873.1

1003.8

1003.0

Vibrations

Indole ring breathing The doublet is due to Fermi resonance between the ringbreathing vibration and the overtone of an out-of-plane ringbending vibration Mixed mode of the benzene 12like vibration and N-H motion Phenyl ring breathing

Intensity

Molecule

Strong Tryptophan

Strong in D2O and weak in H 2O

Tyrosine

References (Milan-Garces et al., 2013; Takeuchi and Harada, 1986; Unno et al., 2010)

(Couling et al., 1998; Siamwiza et al., 1975)

(Combs et al., 2005; Miura et al., 1988)

Strong Tryptophan

(Herrero et al., 2010)

Strong Phenylalanine

1011.8

1011.4

Benzene ring breathing

(Takeuchi and Harada, 1986)

Strong Tryptophan

1340 1360

1380

Due to Fermi resonance between skeletal stretching and out of plane vibrations of the indole ring

Strong Tryptophan

(Harada et al., 1986; Miura et al., 1988)

1553

Pyrole ring breathing

1552

(Milan-Garces et al., 2013; Takeuchi and Harada, 1986)

Strong Tryptophan

Table 2 : Thermodynamic parameters distinctive of lysozyme thermal denaturation for lysozyme dissolved in H2O in the absence of CD (LWh) and in the presence of 10% (LWhHPβCD10), 20% (LWhHPβCD20) of HPβCD with different molar substitution (0.62 and 0.85) and 10 % of MeβCD (LWhMeβCD10)

Tm [°C]

ΔH [J/g]

ΔCp [J/(°C·g)]

77.7 75.7

3.51 3.15

0.056 0.045

Tm [°C]

ΔH [J/g]

ΔCp [J/(°C·g)]

LWdHPβCD_0.85

75.2 73.4

3.43 3.44

0.051 0.057

LWdHPβCD_0.62

75.0

3.58

0.062

LWh

Tabl e3:

Ther mod LWhHPβCD10(0.85) yna mic 3.20 0.047 LWhHPβCD20(0.85) 73.5 para mete 3.43 0.029 rs LWhHPβCD10(0.62) 77.3 disti 3.50 0.046 nctiv LWhHPβCD20(0.62) 76.8 e of 72.3 3.09 0.050 lyso LWhMeβCD10 zym e thermal denaturation for lysozyme dissolved in D2O in the absence of CDs (LWd) and in the presence of 10% of HPβCD high molar substitution (LWdHPβCD_0.85) and low molar substitution (LWdHPβCD_0.62)

LWd

Tabl e4: Tem pera tures disti nctiv e of

two transformations identified from Raman spectroscopy (1) 𝑇𝑀𝐺 𝑚 (°𝐶) : midpoint temperature of (N) → (MG) 𝑈 (°𝐶) : midpoint temperature of (MG) → (D) transformation identified via the isotopic exchanges and (2) 𝑇𝑚 transformation where « U » means Unfolded state of the secondary structure

𝑻𝑴𝑮 𝒎 (°𝑪) LWd LWdHPβCD10 LWdHPβCD20

69.4 ± 0.2 67.7 ± 0.2 67.1 ± 0.2

𝑼 𝑻𝒎 (°𝑪)

80.9 ± 0.2 79.5 ± 0.2 79.1 ± 0.2

ΔT (°C) 11.5 ± 0.2 11.8 ± 0.2 12.0 ± 0.2