Thermoresponsivity of poly(N-isopropylacrylamide) microgels in water-trehalose solution and its relation to protein behavior
Benedetta Petra Rosi, Letizia Tavagnacco, Lucia Comez, Paola Sassi, Maria Ricci, Elena Buratti, Monica Bertoldo, Caterina Petrillo, Emanuela Zaccarelli, Ester Chiessi, Silvia Corezzi
aa r X i v : . [ c ond - m a t . s o f t ] F e b Thermoresponsivity of Poly(N-isopropylacrylamide) microgels in water-trehalosemixture: an experimental and simulation study
Benedetta Petra Rosi a,1 , Letizia Tavagnacco b,c,1 , Lucia Comez d , Paola Sassi e , Maria Ricci e , Elena Buratti b,c , MonicaBertoldo f,g , Caterina Petrillo a , Emanuela Zaccarelli b,c , Ester Chiessi h, ∗ , Silvia Corezzi a, ∗ a Dipartimento di Fisica e Geologia, Universit`a di Perugia, I-06123 Perugia, Italy b CNR-ISC, Sapienza Universit`a di Roma, I-00185 Roma, Italy c Dipartimento di Fisica, Sapienza Universit`a di Roma, I-00185 Roma, Italy d CNR-IOM, Dipartimento di Fisica e Geologia, Universit`a di Perugia, I-06123 Perugia, Italy e Dipartimento di Chimica, Biologia e Biotecnologie, Universit`a di Perugia, I-06123 Perugia, Italy f Dipartimento di Scienze Chimiche, Farmaceutiche ed Agrarie, Universit`a di Ferrara, I-44121 Ferrara, Italy g CNR-ISOF, Area della Ricerca, I-40129 Bologna, Italy h Dipartimento di Scienze e Tecnologie Chimiche, Universit`a di Roma “Tor Vergata”, I-00133 Roma, Italy
Abstract
In this work we use a multi-technique approach to gain new insights into the effects of trehalose on the volume phasetransition of poly(N-isopropylacrylamide) (PNIPAM) microgels, a model synthetic biomimetic system. By dynamiclight scattering the temperature-dependent microgel hydrodynamic volume is monitored, while Raman spectroscopy andmolecular dynamics simulations explore at the molecular scale changes of solvation and dynamics across the transition.At 0.72 M trehalose concentration the phase transition temperature is lowered by ≈
10 K, with a concomitant decrease oftransition enthalpy not compensated by a corresponding reduction in transition entropy. In water-trehalose solution themicrogel particles are ≈
20% more expanded than in pure water, keeping unchanged their thermal contraction, swellingcapacity, and the amount of absorbed water. Strongly hydrated trehalose molecules mainly develop water-mediatedinteractions with PNIPAM, thus preserving the polymer hydration state both before and after the transition. However,the presence of trehalose deeply impacts on the dynamics of the system, with a drastic slowing down of PNIPAM motionsand of its hydration shell. Our investigation reveals a coherent picture in which trehalose sustains hydration and favors,both thermodynamically and kinetically, the collapsed conformation of the microgel, by inhibiting local motions ofpolymer residues and water.
Keywords:
Poly(N-isopropylacrylamide) (PNIPAM), microgels, trehalose, lower critical solution temperature (LCST)sep volume phase transition (VPT), cosolvents and cosolutes, biomimetic material, bioprotection, hydration water
1. Introduction
Poly( N -isopropylacrylamide) (PNIPAM) is the best-known amphiphilic polymer that, dissolved in water orin aqueous media, undergoes a reversible transition froma coil to globule chain conformation, leading to aggrega-tion and phase separation, in response to external stimulisuch as temperature, pressure, ionic strength, mechanicalforces, magnetic and electric fields, presence of cosolvents[1]. This transition, occurring at the lower critical solutiontemperature (LCST), is mainly driven by the entropy gainfor the reduction of excluded volume to solvent moleculeswhich compensates, at the critical temperature, the en-thalpy cost for the partial dehydration of amide groups[2, 3]. In pure water, the LCST is around 305 K. The ∗ Corresponding author
Email addresses: [email protected] (Ester Chiessi), [email protected] (Silvia Corezzi) These authors contributed equally sensitivity to environmental changes of the polymer chainconformation reflects on the behavior of PNIPAM micro-gels, which are crosslinked polymer networks of colloidalsize [4]. These discrete particles drastically reduce theirvolume, passing from a swollen hydrated state to a col-lapsed state at a critical temperature very close to thepolymer LCST, called the volume phase transition tem-perature (VPTT). In the collapsed state, at sufficientlyhigh concentration, microgel particles tend to self-assembleand phase separate. The ability of PNIPAM microgels todrastically modify their inner structure and behavior uponslight environmental changes, along with their capacity toretain a great amount of water, makes them valuable forseveral applications in biotechnology and pharmaceutics,such as drug delivery, biocatalysis, water purification, cellculture, and tissue engineering among others [4–8].Due to this responsivity, which is capable to reproducein a simple homopolymeric structure many aspects of thefolding/unfolding transition typical of proteins [2, 9, 10],PNIPAM also constitutes an ideal model system for gain-ng new insights into biomimetic behaviors and for study-ing the effect of specific cosolvents/cosolutes used for bi-ologically relevant actions (e.g. cryoconservation, biopro-tection, stabilization, denaturation, drug preparation). In-deed, the addition of organic solvents, salts, and osmolytes,strongly affects the conformational stability in solution ofproteins, and of PNIPAM as well.Cosolvents and cosolutes typically originate new regionsin the PNIPAM phase diagram, with the addition of up-per critical solution temperature boundaries and the oc-currence of cononsolvency [11–17]. A main consequenceof the presence of additives is a shift of the LCST. Withonly few exceptions [12, 18–20], most of neutral and ioniccosolvents at low molar fractions decrease the LCST [21–23]. Also, they have an effect on the hydration shell ofPNIPAM and on the properties of bulk water in the mix-ture [21, 24]. The impact of a given cosolvent or cosoluteon the polymer solubility specularly reflects on the micro-gel volume phase transition, by shifting the VPTT and/orby affecting the particle’s swelling ability [13, 16, 25].Sugars represent an interesting class of additives, asthey help stabilize proteins and membranes under biop-reservation protocols that include cryogenic temperaturesor dehydration conditions. Among bioprotective agents,trehalose has a special place. This natural disaccharide,formed by two glucose units linked by an α, α − (1 →
1) gly-cosidic bond, is known to be more effective than others topreserve the functional conformation of biomacromoleculesunder physico-chemical stress conditions that would nor-mally promote their lability or denaturation [26]. Indeed,the production of trehalose is used in nature by some plantand animal cells to survive under extreme conditions ofdehydration, allowing dried microorganisms to resuscitatefrom a dormant state when the moisture content is re-stored to physiological levels [27]. The protective capabil-ities of trehalose can also be induced through its artificialaddition, and used in cryopreservation [28, 29], in main-taining biological activity in the presence of chemicals [30],or in increasing the thermal stability of delicate proteinsand enzymes in solution [31, 32], making this sugar veryattractive for biotechnological purposes and food preser-vation technologies.Several molecular mechanisms, often complementary,have been proposed to explain the remarkable effective-ness of trehalose in preserving biomacromolecules. Thehypotheses range from trehalose that is supposed to sub-stitute water in the protein hydration shell by formingdirect hydrogen bonds (HBs) with the hydrophilic sitesof the biomolecule ( water replacement [33]), to trehalosethat is supposed to maintain the protein hydration andnative structure by confining water molecules close to thebiomolecule surface ( water entrapment [34–36]). Thesemechanisms are often associated with preferential adsorp-tion or exclusion of cosolute from the protein hydrationshell [37]. In this regard, it has been proposed that coso-lute preferential exclusion may lead to protein stabilizationby non-specific interactions, such as excluded volume ef- fects [21]. Since biological activity relies on the motionof biomolecules, which is strictly interrelated with the sol-vent mobility [38–40], considerable attention has also beengiven to the influence of trehalose on the protein dynam-ics and the motion of surrounding water molecules. Inthis respect, the capabilities of trehalose glassy matrixesto immobilize embedded biomolecules in the presence ofresidual water by suppressing fast motions that would pro-mote conformational changes [41], have been recognizedand increasingly used as a way of preserving proteins, orbiological systems such as living cells at room tempera-ture, without the need for freeze drying [42]. Moreover, aslowing down of protein hydration water induced by tre-halose, detected by numerical [35, 43] and experimental[36] studies, has been proposed to play a key role in thecryoprotectant action of the sugar, as it may inhibit iceformation during freezing, facilitating vitrification. De-spite the large efforts, however, understanding the originof the bioprotective properties of trehalose remains unclearin many respects.In this context, it is worth studying the specific ef-fect of this biologically relevant sugar on the collapseof PNIPAM-based architectures. The PNIPAM-water-trehalose ternary system is much less investigated bothexperimentally and numerically as compared to any otherternary system of the same polymer, including those con-taining saccharides [44–49]. Experimental investigationsusing cloud point measurements, differential scanning andisothermal titration-microcalorimetry [44, 46], report a sig-nificant decrease of LCST of PNIPAM chains in aqueoussolution on increasing mono- and disaccharide concentra-tion, finding a good correlation of the saccharide hydrationwith the decrease of LCST, and hence with the promotionof the globular compact state of the polymer. The ef-fect of saccharides has also been studied on another typeof PNIPAM-based system, i.e. hydrogels, by evaluatingthe influence on the VPTT and swelling behavior, alsoin this case with a focus on the water-saccharide interac-tions [45, 47, 49]. To our best knowledge, however, onlyvery few studies refer to trehalose. In particular, the de-crease of LCST of PNIPAM chains on increasing the con-centration of trehalose has been found significantly largercompared to other saccharides and polyols [46, 48], whilethe influence of trehalose on the corresponding swellingbehavior of PNIPAM microgels is still unexplored. Alsoinformation from molecular dynamics simulation are miss-ing, with the exception of the work of Narang et al. [48],where the numerical investigation is limited to the effecton the PNIPAM-water interaction induced by the additionof a single trehalose molecule in a system containing onePNIPAM chain (35-mer) and 4370 water molecules. Thissolvent composition corresponds to a very low trehaloseconcentration ( ≈ .
012 M), unable to change the polymerLCST compared to that in pure water (Table S1 of ref.[48]). The simulation was carried out at 300 K, whichis below the LCST in this highly diluted trehalose solu-tion, contrary to what stated by the authors [48]. There-2ore, the numerical study only revealed trehalose-inducedchanges in the hydration shell and intramolecular bondingof the PNIPAM chain in the coil conformation, leavingunexplored the effects produced across the coil-to-globuletransition. Furthermore, simulations to address the influ-ence of trehalose on the dynamics of the polymer solvationshell are not present in literature.With this background, the present work aims at achiev-ing a molecular description of the transition of PNIPAMin a water-trehalose mixture having a significant trehalosecontent, by exploring in particular the conformationalchanges of the polymer across the transition in relationto changes that occur in the structure and dynamics ofthe polymer solvation shell. For our study we consid-ered the binary solvent with trehalose to water mole ratio1 : 65 .
3, that corresponds to 0.72 M trehalose concentra-tion, able to significantly lower the polymer LCST withrespect to that in water. Experiments were carried out onPNIPAM microgels. The larger surface-to-volume ratio,providing rapid particle collapse, and the colloidal sta-bility make these systems particularly advantageous forthe experimental investigation of the solution behavior ofPNIPAM, as compared to its macrogel and linear chaincounterparts [4]. Microgels were synthesized with a lowcross-linking degree to provide a high swelling ratio acrossthe transition [50], and with a small size, less that 50 nmin hydrodynamic radius at room temperature, to ensurean almost homogeneous internal structure of the parti-cles [51, 52]. We used dynamic light scattering (DLS)to monitor the PNIPAM solubility changes in water andwater-trehalose solution through the variation of the mi-crogel hydrodynamic size on heating across the VPTT. Tocorrelate effects observed on a mesoscopic scale with lo-cal effects concerning the PNIPAM hydration pattern, themicrogel suspensions in the two solvent media were inves-tigated by means of Raman spectroscopy. Experimentalinformation were complemented by numerical simulationswith an atomistic detail. To ensure a proper comparisonbetween experimental and computational results, the en-vironmental conditions of the experiments (both temper-ature and solvent composition) were carefully reproducedin the simulation. Extended analysis of the trajectoriesallowed us to explore (i) the influence of the addition oftrehalose on the water affinity for PNIPAM, (ii) the spe-cific and selective interactions, if any, of the sugar with thepolymer, and (iii) how the presence of trehalose affects themobility of polymer and water in the solvent medium.
2. Experiments
The monomer N -isopropylacrylamide (NIPAM)( M w =113.16, Sigma-Aldrich, 97 % purity) andthe crosslinker N , N ’-methylene-bis-acrylamide (BIS)( M w =154.17, Eastman Kodak, electrophoresis grade)were purified by recrystallization from hexane and Recipe for microgel particles preparation.
NIPAM (g) BIS (g) SDS (g) KPS (g)24.162 ± ± ± ± methanol, respectively, dried under reduced pressure(0.01 mmHg) at room temperature and stored at 253 Kuntil use. The surfactant sodium dodecyl sulphate (SDS)( M w =288.372, 98% purity), the initiator potassium per-sulfate (KPS) ( M w =270.322, 98% purity) and all solvents(RP grade) were purchased from Sigma-Aldrich and usedas received. Ultrapure water (resistivity: 18.2 MΩ/cmat 298 K) was obtained with Millipore Direct-Q ® , 3.0 % wt) and ethylenediaminetetraacetic acid(EDTA, 0.4 % wt) at 343 K for 10 min, rinsed in distilledwater at 343 K for 10 min and finally in fresh distilledwater at room temperature for 2 h.PNIPAM microgels were synthesized by precipitationpolymerization in a 2000 mL four-necked jacked reactorequipped with condenser and mechanical stirrer. Properamounts of NIPAM, BIS and SDS, as reported in Ta-ble 1, were dissolved in 1560 mL of ultrapure water andtransferred into the reactor, where the solution was deoxy-genated by bubbling nitrogen for 1 h and then heated at343 ± Lyophilized PNIPAM microgels were re-suspended inultrapure water and in water-trehalose solution. D-(+)-trehalose dihydrate ( M w =378.33, ≥ .
3, or equivalently,a trehalose molar fraction x tr = 0 . µ m pore size.For DLS measurements, PNIPAM microgel suspensions inthe two solvent media were prepared at high dilution (mi-crogel concentration ≈ − − − g/mL). Samples forRaman scattering measurements and for thermal analysiswere prepared at microgel concentration of 18 wt% and 3wt%, respectively. All samples were stored at 275–277 Kfor at least two days before use. Both water and water-trehalose solution used in thepreparation of microgel suspensions were characterized bydensity, viscosity and refractive index over the entire tem-perature range investigated by DLS measurements. Forthe water-trehalose solution, the temperature dependenceof kinematic viscosity η kin ( T ) was measured by using amicro-Ubbelhode viscosimeter, and that of mass density ρ ( T ) by means of an Anton Paar DMA 5000 M densito-meter. The dynamic viscosity η ( T ) was then calculated as η = ρη kin . According to the Lorentz-Lorenz equation, themass density ρ and the refractive index n D are related by r = ρ − ( n D − / ( n D + 2), where the specific refractivity r is rather temperature-independent. The value of r was ob-tained using n D = 1 .
368 measured at 25.2 ◦ C by means ofa NAR-1T Liquid Abbe refractometer, and ρ = 1 . − independently measured at the same temperature.The temperature dependence of the refractive index wasthen obtained by n D ( T ) = p (1 + 2 rρ ( T )) / (1 − rρ ( T )).As to pure water, data of mass density, refractive indexand viscosity as a function of temperature are availablefrom the literature. We used ρ ( T ) and n D ( T ) from ref.[55]. Kinematic viscosity measurements were repeated,providing dynamic viscosities η ( T ) in perfect agreementwith literature data [56]. Comparison of the temperaturedependence of density, viscosity and refractive index of thetwo solvent media is shown in Fig. S1. DLS measurements were performed with a commercialsetup equipped with a Brookhaven BI-9000AT correla-tor using a solid state laser source of wavelength λ =532nm. The monochromatic beam was focused on the sampleplaced in a cylindrical VAT for index matching and tem-perature control. The temperature was regulated within0.1 ◦ C by a thermostatic circulator. The scattered lightwas collected at an angle θ = 90 ◦ , that corresponds toa scattering wave vector q = (4 πn/λ ) sin( θ/ n the refractive index of the sample at the incident wave-length ( ≈ n D ). The intensity autocorrelation function G (2) ( q, t ) ≡ h I ( q, I ( q, t ) i was measured as a function oftemperature on heating across the VPTT (from 288 to 320K for the sample in water, from 278 to 320 K for the samplein water-trehalose). For each temperature, the sample wasequilibrated for 10 min and measurements were repeated at least in three different points of the sample. The resultsare reported as the mean ± SD of the values obtained inthese repetitions.The measured G (2) ( q, t ) is related to the autocorre-lation function of the scattered field, G (1) ( q, t ) ≡h E ∗ ( q, E ( q, t ) i , by G (2) ( t ) = A (cid:2) β | G (1) ( t ) | (cid:3) , where A is a measured baseline, and β is the coherence factor,an instrumental parameter of the order of unit. For amonodisperse suspension, G (1) ( t ) decays as a single ex-ponential function, giving (cid:2) G (2) ( t ) − A (cid:3) / ≡ C ( t ) ∝ exp( − Γ t ), where the decay time τ ≡ Γ − is related tothe translational diffusion coefficient D of the particles as τ = 1 / ( Dq ). For a polydisperse suspension, G (1) ( t ) doesnot decay exponentially and its deviation from a single ex-ponential behavior contains information about the diffu-sion coefficient distribution. In this case, C ( t ) can be rep-resented as a superposition of exponential contributions,each related to a population of particles with a character-istic diffusion coefficient, and weighted by the populationscattering intensity, i.e. C ( t ) = A Z ∞ I (Γ)e − Γ t d Γ , (1)with A a proportionality constant, Γ = Dq , and I (Γ) thenormalized distribution function of the scattered intensityfrom particles with diffusion coefficient D . The quantityin equation (1) can be analyzed by the method of cumu-lants [57], according to which the logarithm of C ( t ) can beexpanded in a power series in t ln C ( t ) = ln A − K t + 12! K t − K t + ..., (2)where K n is called the n th cumulant. These coefficientsare related to the moments of the diffusion coefficient dis-tribution. The explicit form of the first two cumulantsin equation (2) is: K = R ∞ I (Γ)Γ d Γ = < Γ > z = q
P DI = < ( δD ) > z / < D > z . It must benoted that the mentioned analysis only applies if solventmolecules are far less efficient scatterers of light than mi-crogel particles, and if microgels move much more slowly4han solvent molecules so that the contribution of the sol-vent to the decay of the correlation function is character-ized by a time scale smaller than the experimental timewindow. While these conditions are fulfilled for the mi-crogel suspension in water, it is not the case for the sam-ple in water-trehalose solution. In this sample, in fact,the relaxation dynamics of trehalose molecules is not suffi-ciently temporally separated from the motion of microgelparticles, thus entering the time window of the autocor-relation function and contributing with an additional ex-ponential decay to G (1) ( t ) at small times (Fig. S2). Suchcontribution has to be carefully subtracted before apply-ing a cumulant analysis to the light scattering signal ofthe microgel suspension. An example of this preliminarytreatment of DLS spectra in the presence of trehalose isillustrated in Fig. S3. Note that, without such treatment[48] the analysis of the autocorrelation function at smalltimes would have no relation with the diffusion propertiesof the suspended microgel. With this premise, a cumu-lant analysis was performed for both samples, applyingthe same criteria, and the values of R h and P DI at eachtemperature were calculated from the coefficients K and K of a fourth-order polynomial fit of ln C ( t ), with C ( t )only the contribution to the autocorrelation function dueto the Brownian motion of microgel particles. The temper-ature dependence of R h is shown in Fig. S4. The resultsfor the suspension in pure water were validated by the cu-mulant analysis performed by means of the Brookhavencommercial software. Raman spectra were acquired at 5 cm − resolution, us-ing the 532 nm emission of a solid state laser OXXIUS,mod. LMX (100 mW laser power on the sample). A back-scattering geometry was realized using the 50x long work-ing distance objective of an OLYMPUS microscope MODBX40. The scattered radiation was analyzed by an iHR320imaging spectrometer Horiba Jobin-Yvon, equipped with adigital camera HORIBA, mod. Syncerity. The signal wasdispersed by a 1800 grooves/mm grating that allowed spec-tra acquisition in the 750-1800 cm − range. The spectrom-eter was calibrated to the Raman lines of a polystyrenefilm. PNIPAM microgel samples in water and in water-trehalose solution were measured at three different tem-peratures, namely, 283, 300 and 318 K. Temperatures werechosen in relation to the volume-phase-transition in thetwo samples, recognized by visual inspection: at 283 Kboth suspensions are transparent, i.e., before the tran-sition; at 318 K both suspensions are milky white, i.e.,after the transition; at 300 K microgels in water and inwater-trehalose are found, respectively, below and abovethe transition temperature. The temperature was con-trolled by using a FTIR600 Stage by Linkam ScientificInstruments, equipped with a Linkam pump system usingliquid nitrogen as coolant. Spectra were collected by cu-mulating several repetitions corresponding to 2 hours of acquisition for each temperature. Spectra of the two sol-vent media, water and water-trehalose, were also acquiredunder the same experimental conditions. For each tem-perature, the solvent spectrum was then subtracted fromthat of the corresponding PNIPAM binary and ternarysystem, as much as to avoid negative differences in the so-obtained solvent-free spectrum. Notably, in the trehalose-containing system this operation resulted in the completeremoval of the peak at about 800 cm − , the only trehalosesignal with no superposition with those of PNIPAM. Moredetails about the solvent subtraction procedure are pro-vided in Fig. S5 and S6. The volume-phase-transition of microgel particles in wa-ter and water-trehalose solution was analyzed by using adifferential scanning calorimeter SII DSC 7020 EXSTARSeiko, equipped with liquid nitrogen as cooling agent. Theinstrument was calibrated with Indium, Zinc and heptaneas standards. For each sample, 20–25 mg of PNIPAMmicrogel suspension were hermetically sealed in a 60 µ Lstainless steel pan equipped with rubber O-ring for oper-ative pressure up to 24 bar, taking care not to exceed thetransition temperature before measurement. The thermalanalysis was carried out on heating the sample from 0 to60 ◦ C, using a scan rate of 5 ◦ C/min.
3. Molecular dynamics simulation
Simulations were performed on a PNIPAM linear chainin diluted regime both in water and in water-trehalosebinary mixture at the same trehalose molar fraction( x tr =0.015) used in the experiments. The polymer chainwas made of 30 repeating units in atactic configuration tomimic the experimental condition of the microgel network.PNIPAM and trehalose were described, respectively, withthe OPLS-AA force field [58] in the implementation of Siuet al. [59] and with the OPLS-AA carbohydrates force field[60]. The Tip4p/Ice model was used for water [61], as itwas shown to properly reproduce the PNIPAM behavior inaqueous solution over a wide temperature range includingthe coil-to-globule transition [3] and the low temperatureregion [53, 62, 63].The polymer chain in an extended conformation wascentered in a cubic box of side 8.2 nm, and oriented alonga box diagonal to maximize the distance between peri-odic images. Then, the chain was solvated with 22849 wa-ter molecules for the PNIPAM-water model, while for thesystem in water-trehalose mixture 15598 water moleculesand 239 trehalose molecules were added randomly aroundthe polymer. Energy minimization with tolerance of 100kJ mol − nm − was carried out, and the resulting con-figurations were used to start simulations at two differenttemperatures, namely, 283 K and 318 K. The mass density5f the water-trehalose model at 283 K and 318 K was, re-spectively, 1092( ± · m − and 1086( ± · m − ,consistent with the measured values of 1097 kg · m − and1085 kg · m − at the same temperatures. Numerical simu-lations were carried out in the NPT ensemble for 350 nsat each temperature. Trajectories were acquired with theleapfrog integration algorithm [64] with a time step of 2 fs.Cubic periodic boundary conditions and minimum imageconvention were applied. The length of bonds involving Hatoms was constrained by the LINCS procedure [65]. Thetemperature was controlled by using the velocity rescal-ing thermostat coupling algorithm, with a time constantof 0.1 ps [66]. The pressure of 1 atm was maintained bythe Parrinello-Rahman approach, with a time constant of2 ps [67, 68]. The cutoff of non-bonded interactions wasset to 1 nm and electrostatic interactions were calculatedby the smooth particle-mesh Ewald method [69]. The last 75 ns of each trajectory were considered foranalysis, sampling 1 frame every 5 ps. The radius of gy-ration of the polymer, R g , was calculated as R g = s P i m i s i P i m i (3)where m i is the mass of the i -th atom and s i its distancefrom the center of mass of the polymer chain.The water accessible surface area (WASA) of PNIPAMis defined as the surface of closest approach of watermolecules to the solute molecule, where both solute andsolvent are described as hard spheres. Numerically, thisquantity was calculated as the Van der Waals envelope ofthe solute molecule extended by the radius of the solventsphere about each solute atom centre. A spherical probewith radius of 0.14 nm was used and the values of the Vander Waals radii were taken from the literature [70, 71].The distribution of WASA values were calculated with abin of 0.1 nm .Water molecules in the first hydration shell of PNIPAMwere defined as molecules having the oxygen atom at adistance from oxygen, nitrogen, or methyl carbon atoms ofPNIPAM lower than the first minimum distance of the cor-responding radial distribution function. This cutoff valuecorresponds to 0.35 nm for nitrogen and oxygen atomsand 0.55 nm for methyl carbon atoms. Water molecules inthe first hydration shell of trehalose were selected for hav-ing their oxygen within 0.35 nm from a trehalose oxygenatom. Finally, trehalose molecules in the first solvationshell of PNIPAM were defined by having their anomericoxygens within 0.6 nm from PNIPAM atoms. The proper-ties of bulk solvent molecules were evaluated by samplingmolecules at a distance larger than 3 nm from the poly-mer.The trehalose concentration was monitored as a functionof the distance from the PNIPAM surface by calculating the trehalose molar fraction x tr , measured by the ratio be-tween the number of anomeric oxygen atoms of trehaloseand the sum of the number of trehalose anomeric and wa-ter oxygen atoms, in shells 0.5 nm thick.The number of PNIPAM-PNIPAM, PNIPAM-water,PNIPAM-trehalose and water-trehalose HBs was also eval-uated. The hydrogen bonding interaction was identified byadopting the geometrical criteria of an acceptor-donor dis-tance (A · · · D) lower than 0.35 nm and an angle Θ (A · · ·
D–H) lower than 30 ◦ , irrespective of the AD pair. The life-time of PNIPAM-water HBs, τ P W − HB , was also estimatedby calculating the intermittent HB autocorrelation func-tion and by taking the time at which this function is de-cayed of the 63% of its amplitude.The diffusion coefficient of PNIPAM and trehalose hydro-gen atoms was calculated from the long-time slope of themean square displacement (MSD) D = 16 lim t →∞ ddt h| r ( t ) − r (0) | i (4)where r ( t ) and r (0) represent the position vector of thehydrogen atom at time t and 0, respectively, and h· · · i denotes an average performed over both time origins andhydrogen atoms. In addition, the MSD of oxygen atomsfor bulk water and for PNIPAM and trehalose hydrationwater was calculated in time windows of 500 ps.The exchange time of water and trehalose molecules in thePNIPAM first hydration shell, τ F HS , was estimated fromthe time evolution of the number fraction of molecules inthe first solvation shell and defined as the time at whichthe autocorrelation function is decayed of the 63% of itsamplitude.Trajectory acquisition and analysis were carried out withthe GROMACS software package (version 2016.1) [72].The molecular viewer software package VMD was used forgraphic visualization [73].
4. Results and discussion
DLS measurements reveal that both size and distribu-tion of PNIPAM microgels are affected by the solventmedium in which the particles are suspended. Fig. 1 showsthe temperature dependence of the hydrodynamic volume,calculated from the hydrodynamic radius as V h = πR h ,and the polydispersity index P DI of the microgel particlessuspended in water and in water-trehalose solution.The value of V h shows a drastic drop on increasing thetemperature within a narrow range, revealing the occur-rence of the volume phase transition in both environments.From a qualitative comparison of the data in Fig. 1, sev-eral differences with respect to the pure water suspensionemerge in the presence of trehalose at the investigated con-centration. Namely, (i) the transition occurs at a tempera-ture lower more than about 10 K, (ii) the particles in theirthermodynamically stable state, either below or above the6
80 290 300 310 3200.1
PN/w PN/w/t V h ( n m ) T (K) (a) (a) Hydrodynamic volume, V h , on a logarithmic scale as a function oftemperature, T , for PNIPAM microgels in water and water-trehalosesolution. Solid lines are obtained by fitting the data with the two-state transition model (equation (5)); arrows indicate the transitiontemperature T c in the two suspensions. Open circles are obtaineddividing by a factor 1.2 the data in water-trehalose solution. Thedashed straight lines show that in trehalose the particle volume is a20% higher than in pure water at the same temperature. (b) Tem-perature dependence of the polydispersity index, P DI , for PNIPAMmicrogels in water and water-trehalose solution. The range of val-ues for particles in the swollen state is highlighted in color, that forcollapsed particles in gray. transition, remain sensibly more expanded in the presenceof trehalose, and (iii) the sample polydispersity is higherwhen the particle swelling occurs in the presence of tre-halose.Going into a more quantitative, yet model-independentevaluation, we find that the increase of size in trehalose isproportional to the size the particle would have in water atthe same temperature, the difference being ≈
20% both inthe swollen and the collapsed state, as shown in Fig. 1a. Itis worth noting that the ratio between the measured den-sity of water-trehalose and pure water (Fig. S1) is constantover the whole temperature range here investigated andimplies a constant ratio ≈ [74] and the volume of a watermolecule is about 30 ˚A , one trehalose molecule approxi-mately occupies 18% the volume of 65.3 water molecules,1:65.3 being the trehalose to water molar ratio of our bi-nary mixture. Consequently, the ≈
20% increase of hydro-dynamic volume detected when the microgel is immersedin water-trehalose solution could be explained by assuminga solvent-cosolute ability to enter/exit the microgel parti-cles across the volume phase transition maintaining themolar composition of the external medium. This roughestimate also suggests that the same amount of water per-meates the polymer network independently of the pres-ence of trehalose, both in the swollen and the collapsedstate. Our observation is consistent with the hypothesisadvanced in ref. [46], based on data for PNIPAM chains,that trehalose in the presence of PNIPAM remains pref-erentially hydrated and mainly develops water-mediatedinteractions with the polymer.Concerning polydispersity, Fig. 1b reveals that the par-ticle size distribution is broader when microgels swell in thetrehalose-containing solvent. Indeed, PDI values around0.3 are found in pure water and around 0.5-0.6 in water-trehalose solution. Above the transition, instead, particlesin the collapsed state give in both samples almost monodis-perse suspensions, with PDI values around 0.1. Changesof polydispersity have in general been observed in watersuspensions of microgels synthesized with different hydro-dynamic size, where polydispersity was related to microgelstructure [52]. In the present case, on the contrary, parti-cles have the same internal structure and a higher polydis-persity in trehalose has to be regarded as a solvent-inducedeffect. These findings therefore suggest that polydispersityis not just an intrinsic property of the polymer network,but is rather a sample feature dependent on the microgel-solvent interactions. Moreover, it is worth noticing an in-teresting correlation with the transition region, i.e., thetemperature interval ∆ T t where the hydrodynamic size ofmicrogels deviates from the extrapolation from both sidesof the transition. Fig. 1a shows that this interval is about9 K wide in pure water and about 14 K in water-trehalosesolution. Comparing with Fig. 1b reveals that the widerthe particle size distribution in the swollen state the wider7he transition region.To obtain more quantitative information and to derivethermodynamic parameters associated to the transition,we apply a van’t Hoff analysis to the temperature depen-dent data of hydrodynamic volume, V h . In analogy to thetreatment of protein folding-unfolding reaction [10], themodel considers a microgel as consisting of independent‘cooperative units’ and the transition in each of these unitsis regarded as a temperature-induced all-or-none transitionfrom one state (swollen state of the cooperative unit) toanother state (collapsed state of the cooperative unit). Atany temperature T of the swollen ⇆ collapsed thermody-namic equilibrium, the measured value of V h is the valueaveraged over the two cooperative unit populations, i.e., V h ( T ) = V s f s ( T ) + V c f c ( T ), where V s and V c are the con-tribution to V h from cooperative units in the swollen andcollapsed states, while f s and f c denote the mole fractionof swollen and collapsed units, with f s + f c = 1. The equi-librium constant of the reaction, K eq = f c /f s , is expressedby K eq = exp( − ∆ G T /RT ), where R is the gas constant, T the absolute temperature, and ∆ G T = ∆ H T − T ∆ S T is thestandard free-energy change accompanying the transitionat temperature T . It is reasonably assumed [75–77] thatthe enthalpy and entropy changes ∆ H T and ∆ S T at tem-perature T negligibly differ from the values ∆ H and ∆ S in the middle point of the transition, i.e., at temperature T c defined by the condition K eq ( T c ) = 1. This conditionis equivalent to ∆ G ( T c ) = 0, implying ∆ S = ∆ H /T c .Combining these relations, gives V h ( T ) = V s + V c exp h ∆ H R (cid:16) T c − T (cid:17)i h ∆ H R (cid:16) T c − T (cid:17)i (5)Due to negative thermal expansion in molecular NIPAM[78, 79], the microgel network is expected to contract onheating. This temperature dependence in the volume offully swollen and fully collapsed particles (i.e., composedof cooperative units all in the swollen or collapsed state) istaken into account by introducing a thermal contractioncoefficient for each state ( α s and α c ), and writing intoequation (5), V s = V cs [1 − α s ( T − T c )] for fully swollenparticles and V c = V cc [1 − α c ( T − T c )] for fully collapsedparticles, with V cs ≡ V s ( T c ) and V cc ≡ V c ( T c ) representingthe hydrodynamic volume extrapolated to T c from bothsides of the transition.The curves obtained by fitting the experimental datawith equation (5), shown in Fig. 1a, indicate that theswelling-to-collapse transition of microgels in both sol-vent media is perfectly described by the two-state transi-tion model. The corresponding best-fit parameters are re-ported in Table 2, and provide valuable information aboutthe trehalose effect on the microgel properties.Addressing the ability of microgels to shrink in responseto temperature changes, we here separate the thermal ef-fect that occurs without change of the particle thermody-namic state (i.e., outside the transition region) from the effect produced by conformational rearrangements of thepolymer network due to the phase transition. First, wenote that when the particles are far from the transitionregion (in either swollen or collapsed state), their contrac-tion coefficient is the same, within the uncertainty, in thetwo solvent environments. Since this coefficient is reason-ably related to the elasticity of the polymer network, ourfinding suggests that the addition of trehalose does notappreciably affect this property. We stress that, althoughthe contraction coefficient of the collapsed microgel is sig-nificantly lower than (about one half) that of the swollenparticle, however its value remains different from zero, con-firming a residual shrinking ability even in the collapsedstate [80, 81]. The other relevant aspect to consider is themicrogel ability to shrink across the transition, which wequantify by calculating the ratio r ∗ ≡ V cs /V cc of particlevolume at T c extrapolated from both sides of the transi-tion. Notice that this evaluation differs from that reportedin literature studies, where the swelling ratio is referred tothe particle hydrodynamic radius well below and above T c , without disentangling the contribution due to thermalcontraction. The fit with the van’t Hoff equation providesthe values r ∗ = 5 . ± . . ± .
1, respectively in waterand water-trehalose solution, which are equal within theuncertainty. This result and the one concerning the ther-mal contraction coefficient are consistent with our previousmodel-independent observation, further demonstrating thephysical suitability of the van’t Hoff two-state model to de-scribe the microgel transition process.As expected, the swelling-to-collapse transition is an en-dothermic process, with a positive value of transition en-thalpy ∆ H per mole of cooperative unit undergoing thetransition, and also occurs with an increase of entropy(∆ S > H and ∆ S is observed in the presence of trehalose. To derive micro-scopic information from these data it is necessary to es-timate, in the two samples, the number of monomers percooperative unit. This number can be calculated divid-ing the transition enthalpy ∆ H obtained from the van’tHoff analysis by the transition enthalpy per mole of PNI-PAM residues obtained from calorimetric measurements.In this respect, several calorimetric studies have measuredthe transition enthalpy per mole of PNIPAM residues inwater [9, 10, 18, 21, 52, 82–85], but no determination isavailable in water-trehalose solution at the present concen-tration. From our thermal analysis we obtain 4.8 kJ mol − in water and 3.1 kJ mol − in water-trehalose. Notice thatthe value we measured in water is in agreement with thenumerous literature data, that are distributed with an av-erage value of 5 . ± . − , and in particular is veryclose to the results reported for microgels prepared withthe present synthesis protocol [52, 82]. With these valueswe estimate 170 ±
30 and 140 ±
30 PNIPAM residues percooperative unit, respectively in water and water-trehalosesolution, indicating a slight reduction of cooperativity inthe presence of trehalose despite the large experimental un-certainty of our estimate. As an increased protein stability8 hermodynamic parameters obtained by fitting with equation (5) the hydrodynamic volume V h of PNIPAM microgels in water and water-trehalose solution: volume-phase-transition temperature ( T c ); enthalpy (∆ H ) and entropy of transition (∆ S ) per mole of cooperative units;hydrodynamic volume extrapolated to T c , from low temperatures before the transition ( V cs ) and from high temperatures above the transition( V cc ); thermal contraction coefficient in the swollen ( α s ) and collapsed state ( α c ). Sample T c (K) ∆ H (kJ mol − ) V cs (nm ) V cc (nm ) α s (K − ) α c (K − )PN/w 304 . ± . ±
60 (1 . ± . (3 . ± . (3 . ± . − (1 . ± . − PN/w/t 293 . ± . ±
50 (2 . ± . (4 . ± . (3 . ± . − (1 . ± . − is found to be accompanied by a reduced cooperativity ofthe unfolding transition [86, 87], our findings may reflectthe ability of trehalose to stabilize biomolecules againstdenaturation.Using the number of monomers per cooperative unit, wecalculate the entropy change per mole of PNIPAM residuesas 15.4 and 10.5 J K − mol − , respectively in water andwater-trehalose solution, revealing a significantly lower in-crease of entropy associated to the microgel collapse inthe presence of trehalose. On the other hand, the mea-sured values of transition enthalpy per mole of PNIPAMresidues give a heat absorption 1.7 kJ mol − lower in thepresence of trehalose. Such a difference is very small com-pared to the typical enthalpy of formation/rupture of hy-drogen bonds and then reflects the energy balance result-ing from variations of several interactions. More generally,one should observe that these quantities provide informa-tion about a global effect, whose details at a microscopiclevel remain hidden. As a demonstration, we may comparethe cases of aqueous PNIPAM solution with trehalose andethanol, the latter being an alcohol that directly exposesthe hydrophobic moiety to the surrounding. The litera-ture [21] reports that there exists a water-ethanol compo-sition ( x et ≈ . x tr ≈ . − . Therefore, in order to get a microscopic pictureof the mechanism of transition of PNIPAM in the presenceof trehalose, analyses at a local/atomistic scale constitutean essential further step of investigation. Raman spectra are used to get insight into the hydra-tion pattern of PNIPAM microgels in the two solvents,at a molecular level. Fig. 2a shows the effect of tempera-ture across the volume phase transition on the solvent-freespectra of PNIPAM in water and water-trehalose solution.Fig. 2b compares the solvent-free spectra of the two sam-ples at each temperature. In the analysis, we will focus onthe amide I band, mainly arising from the C=O stretch-ing vibration ( ≈ ≈
800 1000 1200 1400 1600 (a)
283 K 300 K 318 K
C-C stretch C a H b amide III C-C skeletal stretch PN/w CH scissor def amide I PN/w/t
Raman shift (cm -1 )
800 1000 1200 1400 1600
T=318 KT=300 KT=283 K
PN/w PN/w/t (b)
Raman shift (cm -1 ) (a) Raman solvent-free spectra of PNIPAM microgels in water andwater-trehalose solution at three different temperatures, as indicatedin the legend. The assignment of the principal vibrational modes ofPNIPAM are indicated by arrows. (b) Comparison between solvent-free spectra of PNIPAM microgels in water and water-trehalose solu-tion at fixed temperature. In both panels, the spectra are presentednormalized to the intensity of the isopropyl group’s CH deformationband at ≈ − .
283 K
PN/w (a) I n t en s i t y ( a . u . )
318 K283 K
PN/w/t I n t en s i t y ( a . u . ) Raman shift (cm -1 )
318 K
Raman shift (cm -1 ) (a) Fit of the Raman solvent-free spectra of PNIPAM microgels inwater and water-trehalose solution performed in the 1530-1740 cm − frequency range, below ( T = 283 K) and above ( T = 318 K) the vol-ume phase transition. The amide I band is deconvoluted into low(sub-band 1), intermediate (sub-band 2) and high-frequency (sub-band 3) pseudo-Voigt components. The three components have thefollowing parameters, the same for both samples and independentof temperature: center 1627 ±
2, 1656 ±
2, 1695 ± − ; full-width-at-half-maximum 36 ± − . An additional pseudo-Voigtfunction (black dashed line) is used to reproduce the adjacent bandaround 1556 cm − . The red solid line represents the total fittingcurve. (b) Molar fraction of C=O groups involved in intra-PNIPAMHBs, f ( C = O · · · HN ), and C=O groups involved in HBs with wa-ter, f ( C = O · · · HO ), for microgels in water (blue) and in water-trehalose solution (green), plotted against temperature. Colored barsindicate the respective values of T c obtained from the van’tHoff anal-ysis of hydrodynamic volume data (equation (5)). collapsed state, the amide I band does not exhibit differ-ences in the two samples, differently from what observedat 300 K where the two microgels are in a different state.More importantly, the solvent-free spectra at 283 and 318K are well superimposed in the two samples over the wholefrequency range investigated which reveals, within the ex-perimental sensitivity, that the signals of solvent and poly-mer are not frequency shifted. This suggests that directbinding of PNIPAM to trehalose is negligible, or rather,that it cannot be reliably distinguished from that of PNI-PAM to water. Quantitative information about changes inthe hydration state across the transition is obtained by acurve fitting analysis of the Raman signal in the 1530-1740cm − frequency range. The amide I profile is reproducedassuming the presence of three sub-bands which are con-stant in position and shape and only change in intensityupon temperature variation. The low-frequency compo-nent is attributed to C=O groups hydrogen bonded towater, the intermediate component to C=O groups hy-drogen bonded to N-H of another amide group [88], andthe high-frequency component to unbonded C=O groups[89]. This testifies that the amide I band is highly sen-sitive to changes in both PNIPAM hydration and intra-PNIPAM interactions. The observed frequency upshiftacross the transition reflects a change in the intensitybalance among the three sub-bands, with a higher rela-tive increase of the intermediate-frequency contribution.Three pseudo-Voigt functions are used to describe the low( ≈ − ), intermediate ( ≈ − ) and high-frequency ( ≈ − ) amide I sub-bands, and anotherterm to describe the adjacent band around 1565 cm − .The total fit curves together with their individual con-tributions are reported in Fig. 3a. Assuming that theRaman cross sections of the different C=O species is ap-proximately the same, as expected for poly-amide systems[89, 90], the mole fraction of unbonded C=O is calculatedas the relative area of the sub-band around 1695 cm − (sub-band 3 in Fig. 3a), i.e. f unbonded = A /A tot ,where A tot = A + A + A is the sum of peakareas of the three components. It is worth noticing thatthis small fraction is temperature and slightly solvent de-pendent. In fact, in the swollen microgels (at 283 K) itis about 3 and 7% in the absence and presence of tre-halose, respectively; in the collapsed microgels (at 318 K)it vanishes in both samples. On the other hand, the rela-tive area f ( C = O · · · HN ) = A /A tot of the sub-bandat 1656 cm − (sub-band 2 in Fig. 3a) provides the molefraction of C=O groups involved in intra-PNIPAM HBsand the relative area f ( C = O · · · HO ) = A /A tot ofthe sub-band at 1627 cm − (sub-band 1 in Fig. 3a) pro-vides the fraction of hydrated C=O groups, whose val-ues are plotted as a function of temperature in Fig. 3b.Even in the swollen phase a non-negligible amount of C=Ogroups, around 16%, is engaged in intra-PNIPAM bond-ing, and it increases by 14% in the collapsed phase, reach-ing a value around 30%. Concerning hydration, the dataindicate that most (around 80%) of the C=O groups are10
50 100 150 200 250 300 3501.01.52.0280 290 300 310 3201.01.5
283 K 318 K R g ( n m ) t (ns) (a) PN/w PN/w/t R g ( n m ) T (K) (b)
283 K 318 K PN/w PN/w/t W ASA ( n m ) R g (nm) (c) (a) Time evolution of the radius of gyration, R g , of PNIPAM in water-trehalose at 283 K and 318 K. The time interval used for calculatingthe averaged properties (last 75 ns of the trajectory) is highlighted in yellow. (b) Average radius of gyration as a function of T for PNIPAM inwater and water-trehalose solution. (c) Correlation between WASA and radius of gyration. Data calculated for PNIPAM in water are shownwith blue/magenta circles at 283K/318K, while those for PNIPAM in water-trehalose are shown with light blue/pink circles at 283K/318K. hydrogen bonded to water in the swollen phase, but onlya small fraction, around 10%, of these groups dehydrateduring the collapse transition, and there is still a lot ofwater retained in the collapsed particles. This behaviorfavourably compares with previous experimental and nu-merical evidence for PNIPAM microgels [89] and linearchains in water [3, 88, 91]. However, a quantitative com-parison with literature results is prevented since several f values are reported, suggesting a possible effect due topolymer topology and/or concentration. Nonetheless, thecomparison in Fig. 3b is not affected by this issue, andthe absence of any difference, within the experimental er-ror, between the values of f ( C = O · · · HO ) for the samemicrogel particles in the two solvent media indicates thattrehalose does not interfere with the ability of C=O groupsto sustain their HBs with water. These findings support,with information at the molecular scale, observations de-rived from hydrodynamic volume data (sec. 4.1), and fur-ther suggest that trehalose-induced effects on PNIPAMstability are water-mediated. To have information at a molecular level on the rela-tionship between conformation and solvation pattern ofPNIPAM in water-trehalose mixture, as compared to thesystem in pure water, we performed MD simulations of anatactic PNIPAM 30-mer in diluted regime at 283 and 318K, respectively below and above the volume phase transi-tion of microgels in both solvent media, as experimentallyrevealed. The time evolution of the polymer radius of gy-ration, R g , reported in Fig. 4a for the trehalose-containingsystem, shows the occurrence of the chain collapse at 318 K, while extended conformations are mainly populated at283 K. Fig. 4b shows the comparison between the averagevalue of R g in water-trehalose and pure water as a solvent.In both systems, the value is significantly lower at 318 K,as compared to 283 K. However, the size of the chain in thepresence of trehalose is larger than in water, both in thecoil and the collapsed state. The water accessible surfacearea (WASA), a property strictly correlated to the chainsize in aqueous environment, confirms what found for R g ,as shown in Fig. 4c. The associated distributions of R g and WASA are reported in Fig. S7 and S8. The increaseof chain size in the presence of trehalose, both below andabove the transition, finds a qualitative correspondencewith the higher hydrodynamic volume of PNIPAM micro-gels in water-trehalose as compared to that measured inwater (see Fig. 1a).To evaluate whether the difference in chain size betweenwater and water-trehalose arises from steric effects due toa preferential adsorption of trehalose, we analyzed the lo-cal concentration of trehalose as a function of the distancefrom the PNIPAM surface, as shown in Fig. 5. We findthat the molar fraction of trehalose in close proximity tothe chain is lower than that in the bulk of the solution, thisfeature being more pronounced at 283 K, thereby exclud-ing preferential adsorption on PNIPAM. This result forPNIPAM is quite remarkable, since in protein aqueous so-lutions trehalose is generally found to be preferentially ex-cluded from the protein hydration shell, similarly to whatdetected in the current simulations. Preferential exclusionis thought to be linked to the increase in the water surfacetension induced by trehalose, implying that the cosolute ispreferentially depleted from the water surface layer. Due11 .0 0.5 1.0 1.5 2.00.0050.0100.0150.020 283 K 318 K x t r Distance (nm)
Molar fraction of trehalose, x tr , as a function of the distance fromthe surface of the PNIPAM chain, in the coil (283 K) and globule(318 K) conformation. to this effect, the compact state of the protein is favouredsince less hydrophobic surface is exposed [31, 37].On average, only one out of ten PNIPAM residues formsHBs with trehalose, irrespective of temperature (Fig. 6a).The extent of this interaction is negligible as compared tothat of the polymer with water, as discussed in the follow-ing. However, by considering that the size of a trehalosemolecule is about three or four times that of a PNIPAMresidue, the proximity of even very few trehalose moleculescan favor more extended conformations. To highlight thissituation, we calculated the radial distribution functionsbetween the anomeric oxygen of trehalose and nitrogen,oxygen and methyl carbon atoms of PNIPAM, shown inFig. S9. The most accessible PNIPAM atoms are oxy-gens, for which a well defined first coordination shell oftrehalose anomeric oxygens is visible at 283 K, with afirst minimum distance of about 0.6 nm. At 318 K thisshell is broader, but with a similar thickness. A repre-sentative snapshot of PNIPAM conformation at the twoinvestigated temperatures is reported in Fig. 7a and 7b,where the trehalose molecules having the anomeric oxygenat a distance lower than 0.6 nm from oxygen, nitrogen ormethyl carbon atoms of PNIPAM and the water moleculesof the first hydration shell are included (vide infra). Forcomparison, we display in Fig. 7c and 7d a correspond-ing snapshot from the simulations of PNIPAM in water.Overall, Fig. 7 illustrates how the presence of trehalosein the surrounding of PNIPAM, even if at a concentrationlower than that in the bulk of the solution, induces moreextended conformations than in pure water. However, thisdifference does not affect the intra-PNIPAM HBs, whosenumber is the same in the two solvent media, in both thecoil and collapsed state (Fig. 6b). The increase of intra-chain hydrogen bonding across the transition is in qualita-tive agreement with the behavior observed in the Ramanexperiment (Fig. 3b), although the number of bonds per repeating unit obtained by simulations is lower than thevalue estimated experimentally. This discrepancy is at-tributable to the much higher polymer concentration ofthe experimental sample, which makes contacts betweenPNIPAM amide groups more likely. The extent of thisinteraction is also probably affected by the polymer topol-ogy, as noted in sec. 4.2, and favored in network systemssuch as microgels.We then explored the features of PNIPAM hydrationin the two solvents, in particular to evaluate if the pres-ence of trehalose leads to local differences. The radialdistribution functions between water oxygens and PNI-PAM atoms (Fig. S10) have a very similar shape in waterand water-trehalose, with three distinct hydration shells.The coordination number of PNIPAM atoms by water oxy-gens, obtained by integration of the radial distribution asa function of the distance from PNIPAM (Fig. S11), isequal in the two solvents up to distances greater than thethickness of the first hydration shell, both before and af-ter the coil-to-globule transition. Therefore trehalose, atthe investigated concentration, does not inhibit hydrationof PNIPAM. This finding is confirmed by the analysis ofPNIPAM-water HBs, whose results are summarized in Fig.6c. We calculated the different components of this inter-action, namely the HBs formed between water moleculesand polymer’s N-H or C=O groups, and also identified theHBs where a water molecule is bridged between two dif-ferent amide groups of the polymer chain. The numberof PNIPAM-water HBs decreases moving from the coilstate at 283 K to the globule state at 318 K, althoughthe chain maintains a large hydration also after the tran-sition, as pointed out in previous studies [88, 89, 91, 92]and confirmed by the Raman spectra of PNIPAM micro-gels reported in this work. Notably, it clearly emerges thatPNIPAM hydrogen bonding with water in the two solventmedia is very similar. Only a small difference is detected inthe number of HBs involving the carbonyl group, slightlylower in the presence of trehalose for the competition oftrehalose-PNIPAM HBs. As a consequence of these find-ings, the decrease of transition enthalpy per mole of poly-mer residues that we measured by calorimetry cannot beascribed to a less dehydration of amide groups but is likelydue to a lower molar partial enthalpy of water in the bulkof the water-trehalose solution [93], making thermodynam-ically more favoured the transition to the collapsed state[21]. By considering overall the results on the chain sizeand hydration, the simulations suggest that, to maintainthe same hydration level as in water, the chain adopts moreextended conformations. This allows for higher WASAvalues compensating for the trehalose presence in the sur-rounding of the polymer. The similarity of the hydrationfeatures of PNIPAM in water and in water-trehalose mix-ture, observed in the simulations, is consistent with the re-sults of Raman measurements discussed in sec. 4.2, whichindicate that the presence of trehalose does not sensiblychange the hydration of PNIPAM amide groups, eitherbelow or above the transition (see Fig. 3b). We note,12
83 3180.00.51.01.52.02.5283 3180.000.040.080.12283 3180.000.050.100.150.20 allCO NH bridge w w/t (c) P N I PA M - w a t e r H B s T (K) (b)
PN/w ; PN/w/t P N I PA M - P N I PA M H B s T (K) all ; CO ; NH P N I PA M - t r eha l o s e H B s T (K) (a) (a) Average number of PNIPAM-trehalose HBs per PNIPAM repeating unit at 283 and 318 K. The total number of HBs and its contributionsinvolving carbonyl (C=O) and amine (N-H) groups are shown, as indicated in the legend. (b) Average number of PNIPAM-PNIPAM HBsper PNIPAM repeating unit at 283 and 318 K, calculated in water and water-trehalose solution. (c) Average number of PNIPAM-water HBsper PNIPAM repeating unit at 283 and 318 K, calculated in water (open symbols) and water-trehalose solution (closed symbols). The totalnumber of HBs and its contributions involving carbonyl (C=O) and amine (N-H) groups are shown, as indicated in the legend. The numberof water molecules H-bonding two different amide groups of the polymer chain is also reported (stars). Error bars are within the symbol size.Snapshots showing some representative configurations from the sim-ulations of a PNIPAM chain in water-trehalose at a) 283 K and b)318 K and from the simulations in water at c) 283 K and d) 318 K.PNIPAM backbone carbon atoms are shown in red, while hydrogenand side-chain atoms are represented in gray. Water and trehalosemolecules in the first solvation shell are also displayed in blue andyellow, respectively. instead, that the hydration pattern of PNIPAM in water-trehalose is different from that detected in water-ethanolin a corresponding simulation study [22]. Contrary to tre-halose, ethanol preferentially adsorbs on PNIPAM becauseof the interaction between ethyl and isopropyl groups, sig-nificantly competing with water in hydrogen bonding topolymer amide groups.The effect of carbohydrates on PNIPAM LCST has beenattributed to the presence of hydrated-sugar complexes,acting as effective cosolutes in the ternary mixture. Thehigher is the sugar size and hydration number, the largeris the decrease of the LCST as compared to the aqueoussolution mainly for entropic effects [46]. We analysed thehydration features of trehalose in our model, by detectinga hydration number of 18.0 ( ± ∼ ± ± τ T W − HB (Table3), confirm the strong and preferential interaction of tre-halose with water.Another relevant aspect that should be taken into ac-count is the trehalose-induced effect on the solution dy-namics. In biological contexts, the mechanism of cryop-reservation of biomacromolecules by trehalose is thoughtto be related to a strong slowdown effect on the dynamicsof water, especially for those molecules that are locatedclose to the protein surface [35, 43]. To verify this con-dition in our system and to explore the dynamics of thedifferent constituents, we analyzed the time behavior of theMSD of hydrogen atoms belonging to PNIPAM and tre-halose, and of bulk and hydration water molecules at theinvestigated temperatures (Fig. S12). The data allowedus to estimate the average diffusion coefficient of PNIPAM( D P ), trehalose ( D T ) and bulk water ( D w ), as reported inTable 3. It is found that the addition of trehalose reducesthe water diffusivity in the bulk of the solution to ∼ ∼
30% of the value in water. Notably, the diffu-sion coefficient ratio of PNIPAM across the coil-to-globuletransition significantly differs in the two solvent media,and has a different relationship with the solvent mobil-13 olvation properties from MD simulations of PNIPAM in water (w) and in water-trehalose mixture (m). HB TW is the average number oftrehalose-water HBs and τ TW − HB their characteristic lifetime; D P is the diffusion coefficient of PNIPAM; D T and D W are the diffusioncoefficient, respectively, of trehalose and water; τ PW − HB is the lifetime of PNIPAM-water HBs and τ F HS is the exchange time of water inthe first hydration shell. Errors on D values are within 5%. T HB TW τ TW − HB D P (w) D P (m) D T D W (w) D W (m) τ PW − HB (w) τ PW − HB (m) τ FHS (w) τ FHS (m)(K) (ps) ( cm s − ) ( cm s − ) ( cm s − ) ( cm s − ) ( cm s − ) (ps) (ps) (ps) (ps)283 15 . ± .
1) 93( ±
3) 4 . · − . · − . · − . · − . · − ±
32) 363( ±
12) 490( ±
10) 760( ± . ± .
1) 23( ±
3) 1 . · − . · − . · − . · − . · − ±
3) 88( ±
3) 135( ±
10) 230( ± ity. Indeed, in the binary system D P roughly scales withthe diffusivity of the solvent, while it is not the case inthe trehalose-containing system, where the increase of D P across the transition is higher than that suffered both bytrehalose and water. Concerning the hydration water oftrehalose and PNIPAM, the behavior of the MSD, whichdoes not reach a long-time diffusive regime within the sam-pling interval, prevents us from estimating the value of thediffusion coefficient. However, the comparison between theMSDs shown in Fig. S12, leads us to conclude that the wa-ter molecules in proximity to the polymer in the absenceof trehalose are slower than those surrounding trehalosein the ternary mixture, in which the mobility of PNIPAMhydration water is further reduced. These characteristics,observed at 283 and 318 K, highlight the strong slowingdown effect of trehalose on the dynamics of PNIPAM andof its hydration shell. To better characterize the hydra-tion water dynamics, we also calculated two characteristictimes, namely, the PNIPAM-water HB lifetime ( τ P W − HB )and the exchange time of water in the first hydration shell( τ F HS ), both given in Table 3. All things being equal, wefind that τ F HS is higher than τ P W − HB . This is a conse-quence of the more restraining geometric conditions im-posed on water molecules by being H-bonded rather thanresiding in the polymer first hydration shell. Besides, it isalso due to the possibility for water molecules of breakingHBs with amide groups without escaping the surround-ing of hydrophilic or hydrophobic domains of PNIPAM.The increased stability of the PNIPAM hydration shell inthe presence of trehalose is confirmed by these character-istic times, which are significantly higher in the ternarysystem, both below and above the transition: the HB life-time is ∼ . ∼ .
5. Conclusions
PNIPAM is the premier example of polymer with re-sponsive properties. The sensitivity to temperature, chem-ical, and/or environmental changes of the polymer chainconformation is reflected by a volume phase transition incrosslinked polymer architectures, such as microgels, valu-able for many practical applications. Due to this respon-sivity, which is capable to reproduce in a simple homopoly-meric structure many aspects of the folding/unfoldingtransition typical of proteins, PNIPAM microgels also con-stitute a model synthetic biomimetic material, useful tostudy the effect of specific cosolvents or cosolutes on thethermal stability of biomolecules. Among additives, tre-halose is one of the most effective bioprotectants, althoughthe molecular mechanisms involved in this action still needclarification. By using a multi-technique approach com-bining experimental and numerical simulation studies, thiswork provides new insights into the effect of trehalose onthe temperature-induced volume phase transition of PNI-PAM microgels. We monitored the single-particle behav-ior in highly dilute suspension by means of dynamic lightscattering, and explored the local changes of solvation anddynamics of the system’s constituents at the molecularscale by means of Raman spectroscopy and atomistic sim-ulations. Analysis of the temperature-dependent hydro-dynamic volume provided thermodynamic parameters as-sociated to the transition, and allowed us to study the14welling ability of the microgel. At the investigated con-centration, trehalose decreases the phase transition tem-perature by ≈
10 K, the decrease being justified by a lowertransition enthalpy of PNIPAM, not compensated by acorresponding decrease of transition entropy. The parti-cles in their thermodynamically stable state, either swollenor collapsed, remain ≈
20% more expanded than in purewater, without changing their thermal contraction coeffi-cient and capacity to swell. Our experimental findings,supported by the numerical evidence, highlight that tre-halose in the presence of PNIPAM remains preferentiallyhydrated and mainly develops water-mediated interactionswith the polymer, preserving the polymer hydration stateboth before and after the transition. The molecular dy-namics simulations, performed on a linear segment of themicrogel network (PNIPAM 30-mer), allowed us to studythe properties of the system with atomistic detail, provid-ing novel information on the trehalose-induced effects onthe dynamics of both polymer and water. Our investiga-tion reveals that, differently from the structural charac-teristics of the PNIPAM hydration shell, the dynamics ofPNIPAM and water are strongly affected by the presenceof trehalose: in the system before and after the transitionthe diffusivity of PNIPAM is drastically reduced, as well asthe mobility of water molecules in proximity to the poly-mer and in the bulk of the solution. Moreover, althoughtrehalose is preferentially excluded from the PNIPAM sur-face and no transient clustering of trehalose at the inves-tigated concentration is detected around the polymer, thelifetime of PNIPAM-water HBs is around 40% longer inthe trehalose-containing system, and the exchange of wa-ter molecules from the PNIPAM hydration shell to thebulk needs about 60% more time; besides, the exchange oftrehalose molecules in proximity to the PNIPAM surfaceis much slower than water, contributing to confine a smallfraction of slowed down water molecules close to the poly-mer surface. Altogether these results highlight the strongslowdown effect of trehalose on the dynamics of PNIPAMand of its hydration shell, the latter being stabilized bothfrom a structural point of view, by trehalose preferentialexclusion, and from a dynamical point of view, by an in-creased time persistence.These results found in PNIPAM are quite remarkablesince, in biological contexts, the effect of preferential ex-clusion from the protein’s surface layer and the slowdownof its hydration water and internal motions, are invokedfor explaining the outstanding effectiveness of trehalose instabilizing proteins against denaturation. A reduced co-operativity of the PNIPAM microgel transition here ob-served in the presence of trehalose, moreover, appears asan analogue of the reduced cooperativity of the unfold-ing transition associated to an increased protein stability,further concurring to strengthen the analogy of PNIPAMmicrogel with a protein system.In conclusion, the present combination of experimen-tal and computational study reveals a coherent pictureof the mechanism by which trehalose at moderate con- centration perturbs the thermal stability of PNIPAM mi-crogels, fixing some points in explaining the sugar effec-tiveness in protein stabilization, and providing clues asto how a cryoprotectant action is possible. Future workwill further exploit the biomimetic behavior of PNIPAMmicrogels to understand the mechanisms of interaction be-tween water, cosolutes and biomolecules, by extending thepresent study to higher trehalose concentrations and toother water-cosolvent mixtures, in order to rationalize theeffect of added molecules in terms of their hydrophilicityand size.
Acknowledgments
B.R., L.C, S.C. and C.P. acknowledge support fromUniversit`a di Perugia (“CarESS” project, D.R. n. 597);L.T., M.B., E.C. and E.Z. from European Research Coun-cil - ERC (ERC-CoG-2015, Grant No. 681597 MIMIC);L.T., E.B., E.C. and E.Z. from Ministero dell’Istruzione,dell’Universit`a e della Ricerca - MIUR (FARE projectR16XLE2X3L, SOFTART). Support for computationaltime by CINECA-ISCRA grants (Nos. HP10C1IX5O andHP10C9V0IP) is also acknowledged.
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Experiments • Figure S1 reports a characterization of the solvent media in which the microgels were suspended. • Figure S2 shows the intensity autocorrelation function, G (2) ( t ), obtained by dynamic light scattering (DLS) atdifferent temperatures for microgels in water-trehalose solution. • Figure S3 illustrates the cumulants analysis performed on the DLS spectra of the microgel suspension in water-trehalose solution. • Figure S4 compares the temperature dependence of the hydrodynamic radius R h of microgels in water and water-trehalose solution. • Figure S5 illustrates the normalization procedure of the Raman spectra of microgel suspensions in water and water-trehalose solution, needed to the solvent subtraction procedure. • Figure S6 illustrates the solvent subtraction procedure performed on the Raman spectra of microgel suspensions inwater and water-trehalose solution.
280 290 300 310 3200.981.001.021.041.061.081.10 280 290 300 310 3200.51.01.52.02.53.03.54.0 280 290 300 310 3201.331.341.351.361.37 H O from ref. [1]H O/trehaloseH O (x 0.9125) r ( g / c m ) T (K) b) H O this workH O/trehaloseH O from ref. [2] h ( c P ) T (K)a) c) H O from ref. [1]H O/trehalose n D T (K)
Fig. S1: (a) Temperature dependence of mass density ρ , (b) dynamic viscosity η , and (c) refractive index n D , of water and water-trehalosesolution at trehalose mole fraction x tr = 0 . G ( t ) ( a . u . ) t (ms) Fig. S2: Intensity autocorrelation function, G (2) ( t ) = A (cid:2) β | G (1) ( t ) | (cid:3) , measured at different temperatures in a highly diluted sampleof PNIPAM microgels suspended in water-trehalose solution. Symbols indicate experimental data, solid lines represent the fitting curvesobtained as explained in Fig. S3. The correlation functions are normalized to their total amplitude as obtained from the fit. The decay atsmall times reveals the presence in the autocorrelation function of the scattered field, G (1) ( t ), of a relaxation term due to the dynamics oftrehalose molecules in aqueous solution. exp. fit d) G ( ) ( t ) ( a . u . ) t (ms)A exp. fit [ G ( ) ( t )- A ] / t (ms) exp. fit l n [ C ( t ) ] t (ms) exp. fit b)c) C ( t ) t (ms) a) Fig. S3: As an illustration, we show the steps followed to analyze the DLS signal of the microgel suspension in water-trehalose at 280.5 K.
First step:
The quantity G (2) ( t ) = A (cid:2) β | G (1) ( t ) | (cid:3) directly measured by DLS is shown in panel (a) with solid symbols. Second step: G (2) ( t ) is used to calculate the quantity [ G (2) ( t ) − A ] / , proportional to G (1) ( t ). This quantity can be modeled as the superposition oftwo decay contributions, the one al smaller times due to the relaxation of trehalose in water, and the other due to the Brownian motion ofmicrogel particles in suspension. Both contributions are formally reproduced by stretched exponential functions. Accordingly, the data ofpanel (a) are fitted with G (2) ( t ) = A + (cid:2) A exp( − ( t/τ ) β + A exp( − ( t/τ ) β ) (cid:3) , with β , < β = 1 within the uncertainty, to minimize the number of free fit-parameters the fitting procedure is performed atall temperatures by setting β = 1, i.e., by reproducing the trehalose relaxation function with a simple exponential. The red solid line inpanel (a) demonstrates a perfect fit to the experimental data. This line translates into the green line of panel (b), also reported in panel (c)for comparison. Third step:
By subtracting from the data of panel (b) the quantity A exp( − t/τ ) obtained from the fit, the contribution C ( t ) due to the Brownian motion of microgel particles is finally obtained (panel (c)). Fourth step:
The deviation of C ( t ) from a singleexponential decay is analyzed by the method of cumulants (panel (d)). The logarithm of C ( t ) is fitted by a fourth-order polynomial, whosefirst and second-order terms are respectively related to the z-average value ( < D > z ) and variance ( < ( δD ) > z ) of the diffusion coefficientdistribution of microgel particles.
280 290 300 310 3201520253035404550
PN/w PN/w/t R h ( n m ) T (K)
Fig. S4: Hydrodynamic radius R h as a function of temperature of PNIPAM microgels in water and water-trehalose solution. Error is withinthe symbol size. H O/trehalose PN/H O PN/H O/trehalose
T=283 K -C-C- stretching
Fig. S5: At each temperature, the spectra of PNIPAM microgels in the two solvents, water and water-trehalose, were normalized to theC-C stretching vibration peak at about 950 cm − where both trehalose and water negligibly contribute to the Raman signal, i.e., they werenormalized to the sample’s content of PNIPAM. Analogously, the two solvent spectra were normalized to the H-O-H bending mode of water inthe 1500-1700 cm − frequency range, i.e., to the water content of the sample. The red arrow indicates the peak at about 800 cm − , the onlytrehalose signal with no superposition with those of PNIPAM. Notice that, for graphical reasons, the solvent and PNIPAM/solvent spectraare represented on different arbitrary scales. H O PN/H O PN/H O (solvent-free)
T=283 K
Fig. S6: Starting from the normalized PNIPAM/solvent spectra and the normalized solvent spectra as described in the previous figure, thesolvent-free spectra were obtained by subtracting from each normalized PNIPAM/solvent spectrum the corresponding normalized solventsignal, as much as to avoid negative residues in the difference spectrum. It has to be noted that, in the trehalose-containing system thisoperation resulted in the complete removal of the peak at about 800 cm − (red arrow), indicating that the subtraction was carried out ateach temperature with spectra put on the same relative scale, as shown in each panel of the present figure. . Molecular dynamics simulations We report the following additional characterization of the solution behavior of PNIPAM in water and water-trehaloseas obtained from the MD simulations: • Figure S7 compares the distribution of values of PNIPAM radius of gyration ( R g ) in water and water-trehalose. • Figure S8 compares the distribution of values of water accessible surface area (WASA) of PNIPAM in water andwater-trehalose. • Figure S9 shows the radial distribution functions, g ( r ), calculated between the anomeric oxygen of trehalose andspecific atoms of PNIPAM belonging to hydrophilic and hydrophobic groups. • Figure S10 reports the radial distribution functions characterizing PNIPAM hydration. • Figure S11 shows the water coordination number, CN ( r ), of PNIPAM specific groups, as obtained by integrationof the corresponding radial distribution functions reported in Fig. S10. • Figure S12 shows a comparison between the mean square displacements (MSDs) of water oxygen atoms, PNIPAMhydrogen atoms, and trehalose hydrogen atoms. • Figure S13 reports the time behavior of the number fraction of trehalose and water molecules in the first solvationshell. G ( R G ) R G [nm] Fig. S7: Distribution of values of PNIPAM R g in water (blue and magenta line at 283 K and 318 K, respectively) and in water-trehalose(light blue and pink line at 283 K and 318 K, respectively).
30 35 40 450.000.020.040.06 G ( W ASA ) WASA [nm ] Fig. S8: Distribution of values of WASA for PNIPAM in water (blue and magenta line at 283 K and 318 K, respectively) and in water-trehalose(light blue and pink line at 283 K and 318 K, respectively). (c) g O t r eh - C pn (r) r [¯] (b) g O t r eh - N pn (r) r [¯] g O t r eh - O pn (r) r [¯] (a) Fig. S9: Radial distribution functions between the anomeric oxygen atom of trehalose and PNIPAM (a) oxygen, (b) nitrogen and (c) methylcarbon atoms at the two investigated temperatures (283 K: blue line, 318 K: pink line). (c) g O w - C pn (r) r [¯] water/trehalose 283 K water/trehalose 318 K water 283 K water 318 K g O w - O pn (r) r [¯] (a) water/trehalose 283 K water/trehalose 318 K water 283 K water 318 K water/trehalose 283 K water/trehalose 318 K water 283 K water 318 K (b) g O w - N pn (r) r [¯] Fig. S10: Radial distribution function between water oxygen and PNIPAM (a) oxygen, (b) nitrogen, and (c) methyl carbon atoms. g(r)calculated in water are shown with a blue/magenta line, while those in water-trehalose are displayed with a light blue/pink line at the twoinvestigated temperatures of 283K/318K. The higher values of radial distribution functions of the water-trehalose-PNIPAM system are dueto a normalization effect. CN O w - O pn (r) r (¯) w/t 283 K w/t 318 K w 283 K w 318 K CN O w - N pn (r) r (¯) w/t 283 K w/t 318 K w 283 K w 318 K CN O w - C pn (r) r (¯) w/t 283 K w/t 318 K w 283 K w 318 K a) b) c) Fig. S11: Coordination number of water oxygen atoms around PNIPAM (a) oxygen, (b) nitrogen, and (c) methyl carbon atoms as a functionof the distance. CN(r) calculated in water are shown with a blue/magenta line, while in water-trehalose are displayed with a light blue/pinkline at the two investigated temperatures of 283K/318K. Vertical dashed lines indicate the distance of the first solvation shell.
283 K M S D ( n m ) Time (ps)
Bulk water (w) Bulk water (w/t) PNIPAM (w) PNIPAM (w/t)
283 K M S D ( n m ) Time (ps)
Hydration water PNIPAM (w) Hydration water PNIPAM (w/t) Hydration water trehalose Trehalose (a)(c) (d)
318 K
Bulk water (w) Bulk water (w/t) PNIPAM (w) PNIPAM (w/t) M S D ( n m ) Time (ps) (b)
318 K
Hydration water PNIPAM (w) Hydration water PNIPAM (w/t) Hydration water trehalose Trehalose M S D ( n m ) Time (ps)
Fig. S12: Comparison between the mean square displacements (MSDs) of water oxygen atoms, PNIPAM hydrogen atoms and trehalosehydrogen atoms at 283 K (upper panels) and 318 K (lower panels). Data calculated for bulk water are shown with black empty and closedcircles in water and water-trehalose solution, respectively; for PNIPAM hydrogen atoms in water with green full diamonds; for hydrationwater of PNIPAM in water with green empty diamonds; for PNIPAM hydrogen atoms in water-trehalose mixture with blue full triangles; forhydration water of PNIPAM in water-trehalose mixture with blue empty triangles; for trehalose hydrogen atoms with magenta full triangles,and for trehalose hydration water with magenta empty triangles. In all panels, a dotted black line with slope 1 is displayed. A c t r eha l o s e ( t ) Time (ns)
Trehalose 283 K 318 K a) b) A c w a t e r ( t ) Time (ns)
Water water 283 K water 318 K water/trehalose 283 K water/trehalose 318 K
Fig. S13: Time behavior of the number fraction of trehalose (a) and water (b) molecules residing in the first solvation shell. Data calculatedfor trehalose at 283 and 318 K are shown in yellow and blue, respectively. Data calculated for water in the simulations in pure water arerepresented in brown/light green at 283/318 K. Data calculated for water in the simulations in water-trehalose mixture are displayed in darkgreen/pink at 283/318 K.
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