Comprehensive Insight into the ProteinâSurface Biomolecular

Article Cite This: J. Phys. Chem. B 2019, 123, 6331−6344

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Comprehensive Insight into the Protein−Surface Biomolecular Interactions on a Smart Material: Complex Formation between Poly(N‑vinyl Caprolactam) and Heme Protein Krishan Kumar, Ritu Yadav, and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi-110 007, India

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S Supporting Information *

ABSTRACT: Proteins are naturally occurring biopolymers that exhibit a wide range of functional applications. Meticulous knowledge about biomolecular interactions between polymeric biomaterials and body fluids or proteins is essential for designing biospecific surfaces and understanding protein−polymer interactions beyond existing limitations. In this regard, we studied the comparative effect of heme proteins such as cytochrome c, myoglobin, and hemoglobin on the phase behavior of poly(N-vinyl caprolactam) (PVCL) aqueous solution and demonstrated various biomolecular interactions in the polymer−protein complex with the aid of various biophysical techniques. Absorption spectroscopy, steady-state fluorescence spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering studies, laser Raman spectroscopy, field emission scanning electron microscopy, and transmission electron microscopy were carried out at room temperature to examine the changes in absorbance, fluorescence intensity, molecular interactions, particle size, agglomeration behavior, and surface morphologies. Furthermore, differential scanning calorimetry studies were also performed to analyze conformational changes, coil to globule transition, and phase behavior in the presence of proteins. With the addition of heme proteins, the lower critical solution temperature of PVCL increases toward higher temperature. The present study may help in designing smart biomaterials and stimulate more novel concepts in polymer−protein interactions. It also helps in the development of a biomimetic polymer for “smart” applications such as pulsatile drug release systems and controlled bioadhesion by temperature-mediated hydrophilic/hydrophobic switching.

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INTRODUCTION Stimuli-responsive polymers (SRPs) have gained interest of the scientific community because of their extensive range of applications in drug delivery systems, hydrogels, bioconjugates, bioseparation, and hybrid particles. Among the various kinds of SRPs, thermoresponsive polymers (TRPs) can be considered as the most promising material in phase-transition systems, with a wide range of applications in sensors, gels, and drug delivery devices such as nanops (nanostructured porous silicon) which are used in biotechnology and pharmacology.1−4 In fact, TRPs are capable of undergoing instant reversible phase transition from hydrophilic to hydrophobic conformation. This phase transition is triggered by small shifts in physical and chemical properties, in the presence of external stimuli, such as temperature, ionic strength, or concentrations of cosolvents. There can be change in conformation, change in solubility, alteration of hydrophilic/hydrophobic balance, and/ or release of bioactive molecules (e.g., drug molecule). © 2019 American Chemical Society

Poly(N-vinyl caprolactam) (PVCL) is an amphiphilic biomedical and preeminent TRP, which is water soluble up to a certain temperature and later becomes turbid or insoluble. PVCL is a chemical analogue of polyvinyl pyrrolidone, a wellknown and widely used pharmaceutical biomaterial, because of its lower toxicity.5 Naturally, PVCL has a repeating unit consisting of a cyclic amide, where the nitrogen of the amide is directly connected to the hydrophobic backbone as shown in Figure 1a. The lower critical solution temperature (LCST) for PVCL depends on the polymerization method and the molar mass range of the polymer but is nonetheless in the range of 30−35 °C, which is close to the physiological temperature.3 Within a temperature range close to the human body temperature, PVCL has good solubility in many organic Received: May 13, 2019 Revised: July 1, 2019 Published: July 2, 2019 6331

DOI: 10.1021/acs.jpcb.9b04521 J. Phys. Chem. B 2019, 123, 6331−6344

Article

The Journal of Physical Chemistry B

The heme proteins are located at specific sites in human body; thus, if we have knowledge about biomolecular interactions between the biomedical polymer and heme proteins, they can be used in target-specific drug designing and protein separation. Cyt c is an electron carrier which contains heme c that is coordinated with a protein scaffold by cysteine residues. Cyt c is in the shape of a prolate spheroid and the heme group is attached to it through two thioester bonds, that is, cysteine (Cys) 14 and cysteine (Cys) 17, and the “closed crevice” where the heme iron is held, with the axial ligands, histidine (His) 18 and the methionine (Met) 80.15 Cyt c contains five α-helical segments which are N-terminal (residues 2−14), C-terminal (residues 87−103), and helical segments 49−55, 60−70, and 70−75.15,16 Mb, which is a cytoplasmic heme protein, belongs to the globin superfamily of proteins containing 153 amino acids. Out of these 153 amino acids, 121 (79%) are present in the helical region while the remaining 32 amino acids are distributed over the nonhelical region.17,18 The histidine group (His-93) is directly attached to iron and a distal histidine group (His-64) is hovered near the opposite face.19 The α-chain of porcine Hb having 141 amino acids consists of seven helical segments and seven nonhelical segments, while the β-chain which has 146 amino acids consists of eight helical segments and six nonhelical segments.18 The dimer (αβ) of Hb contains three tryptophan (Trp) residues. There are totally six Trp residues present in Hb, that is, two α14Trp, two β15Trp, and two β37Trp. Out of the six Trp residues, the one situated at the dimer−dimer interface is important19 and has been widely studied as a substitute for human blood. These heme proteins are located at specific sites in human body and can be used in targeted drug delivery systems. These heme proteins have been the focus of intense attention and certainly influence on phase behavior of TRPs through various biomolecular interactions that will find applications in biomedical and bioseparation, such as the interface between the polymer−protein interactions at molecular level, formulation of protein−polymer composites, protein resistance surfaces, bioconjugates, bioengineering, and protein separation.10,11,20 In this context, to enhance our knowledge in the area of conformational behavior of TRPs in the presence of heme proteins as stimuli, we have chosen to study the effect of heme proteins such as Cyt c, Mb, and Hb on the thermal phase behavior of PVCL. We quantified the comparative study of the effect of these proteins on the phase behavior of PVCL by the aid of multiple techniques such as UV−visible absorption spectroscopy, steady-state fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), Raman spectroscopy, field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC). Each technique shows clear evidence of the influence of biological stimuli on the phase-transition behavior of PVCL. The current investigation results pave the way to prevent the adverse outcomes of protein adsorption such as cell attachment and biofouling. The crystal structures of Cyt c, Mb, and Hb downloaded from the protein data bank and processed with the PyMOL viewer software and also the structure of PVCL are also shown in Figure 1.

Figure 1. Schematic diagram of (a) PVCL and crystal structure of (b) cytochrome c (Cyt c) (1HRC), (c) myoglobin (Mb) (1 MBN), and (d) hemoglobin (Hb) (1 HAC), which was downloaded from the protein data bank and processed with the PyMOL viewer software.

solvents and is nonionic, stable against hydrolysis, and biocompatible. These are some of the features of PVCL that make it useful in biomedical and biotechnological applications.2−8 PVCL can be considered relatively nontoxic during short exposure times, as confirmed by cellular viability [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test] and membrane integrity [lactate dehydrogenase (LDH) test] by Vihola and co-workers.5 The biocompatibility of the polymer can be increased by grafting the polymer with an amphiphilic group as grafting creates steric repulsion against protein adsorption, thus enhancing the biocompatibility.6 PVCL exhibits type 1 “classical” Flory−Huggins miscibility behavior in which the position of critical point can be shifted toward the lower polymer concentration by increasing the chain length of the polymer.8 The balance between the hydrophilic and hydrophobic moieties along the TRPs determines the LCST, and in this context, the LCST of a TRP is easily altered by the addition of chemical or biological stimuli. The influence of the cosolvents on the phase-transition behavior of the “smart” polymer has been explored experimentally, and it has been reported that extended polymeric globules collapse with increasing temperature.9−15 Although a number of studies about a variety of different additives are available in the literature, only a few studies are available to investigate the effect of biomolecules such as proteins on the responsive behavior of polymers. Very recently, our research group delineated the effect of biological stimuli on the phase behavior of polymer14 and found that different proteins affect the coil and globule transition in different ways. From the biomedical application point of view, knowledge of biomolecular interactions between the polymer and protein is essentially required for pharmaceutical industry. In this context, it is very crucial to explore the effect of biological macromolecules as stimuli on the conformational changes of the TRP for obtaining a clear understanding of polymer−protein interactions, which opens a new area of research for scientific community. 6332


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Figure 2. UV−visible absorption spectroscopy of ANS in PVCL aqueous solution with and without heme proteins, 0.0 mg/mL (black line), 0.5 mg/mL of protein (red line), 1.0 mg/mL of protein (blue line), 1.5 mg/mL of protein (cyan line), 2.0 mg/mL of protein (magenta line), and 2.5 mg/mL of protein (green line) at 25 °C (a) Cyt c, (b) Mb, and (c) Hb. Panel (d) represents absorbance maximum at 410 nm for Cyt c (black line), Mb (red line), and Hb (blue line). Inset panel (a−c) represents changes in the absorbance spectrum of ANS in PVCL (black line), PVCL with 0.5 mg/mL of protein (red line), and pure protein in ANS (blue line).

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a 0.22 μm disposal filter (Millipore, Millex-GS) through a syringe before the execution of the measurements. The final PVCL concentration for all measurements was 7 mg/mL. The heme proteins at different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 mg/mL) with aqueous PVCL solution were prepared. The concentration of extrinsic probe used for UV−vis absorption spectroscopy and fluorescence spectroscopy was set at 2 × 10−5 M; therefore, there would be a negligible effect due to the probe on the PVCL aggregation process. Instrumentation. The specifications and comprehensive information on experimental techniques used in the current investigation have been explicitly elucidated in our previous articles2,14,19 and also provided in the Supporting Information

EXPERIMENTAL SECTION Materials. The probe dye 8-anilino-1-naphthalenesulfonic acid (ANS) with the formula C6H5NHC10H6SO3H and a molecular weight of around 299.35 g/mol in powder form with the assay >97% (high-performance liquid chromatography), Hb porcine (molecular weight 66.7 kDa) in powdered form extracted from the porcine gene HBB(407066), HBE1(407067), Mb from the equine skeletal muscle (molecular weight 17 kDa), Cyt c from the horse heart (molecular weight 12.4 kDa) with the assay >95% (sodium dodecyl sulfate−polyacrylamide gel electrophoresis) were all procured from Sigma-Aldrich Chemicals Co. and used without purification. Hexane, azobis(isobutyronitrile), and N-vinyl caprolactam were procured from TCI and used after recrystallization. PVCL was synthesized by solution polymerization and synthetic reaction, and the procedure is shown in Figure 1S. A detailed procedure for the synthesis of PVCL and its spectral characterization are provided in the Supporting Information Sample Preparation of PVCL in the Presence and Absence of Heme Proteins. All samples were prepared using double-distilled deionized water with a resistivity of 18.3 MΩ cm (Ultra 370 series, Rions India, India). A required amount of polymer and protein was weighed by using an analytical balance (Mettler Toledo) with a precision of ±0.0001 g. To attain complete dissolution, the samples were incubated for few hours at room temperature and filtered with

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RESULTS AND DISCUSSION Divulging the Effect of Proteins on the Conformational Behavior of PVCL by Spectroscopic Methods. To understand deeper insights into the protein−polymer interaction and to ascertain the effect of the biological stimuli on the thermal behavior of synthetic PVCL, we used spectroscopic techniques such as UV−visible spectroscopy, steadystate fluorescence spectroscopy, FTIR spectroscopy, DLS, and Raman spectroscopy. PVCL does not contain any group which can respond in the UV−visible region; therefore, we used an external probe that can sense the changes in the absorption spectra of the molecule and reveal small structural behavior changes in the polymer. ANS is an important dye which is used to study the intermediate states in the folding/unfolding 6333


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interaction of aromatic amino acid residues and PVCL monomers. This result is consistent with our previous spectra of PVCL−Cyt c conjugate which also revealed that there is an interaction between the aromatic amino acid residue and the polymer chain which may be due to H-bonding with the oxygen moiety of the polymer. The absorption peak at 380 nm for the polymer is vanished after addition of Mb. The variation of absorbance at 410 nm accompanied by an increase in intensity (Figure 2d) reveals that with an increasing concentration of protein, there are more hydrophobic pockets that are available for binding with the probe. By increasing the concentration of the protein, new absorbance peaks appear at 504 nm and the increase is more pronounced at higher concentrations. At higher concentrations of Mb, new peaks appear at ∼358 nm, which were initially absent for native Mb, pure PVCL, and at lower concentrations of Mb, which might be due to binding of heme iron and reduction of ferrous group.25 Therefore, one can notice that there is a direct interaction of PVCL and the heme group of the polymer, as well as between the PVCL and aromatic amino acid residues with the corresponding bands for these groups being affected by the addition of Mb to PVCL. In Figure 2c, we show the UV−visible absorption spectra of PVCL in ANS in the presence of porcine Hb. Sus scrofa Hb spectra is used to gain information on changes in the environment of amino acid residues that occur because of the changes in structure associated with the polymer interaction. Spectra in this range reflect the changes in environment or interactions with chromophoric groups. The shapes and wavelength location of the extrema depend on chromophores. The characteristic spectra of pure Hb (Figure 2c inset) are consistent with the literature.26 In the polymer complex, the peak at 280 nm due to protein and the peak at 380 nm due to ANS in PVCL are absolutely disappeared. Soret band extrema increases with the increase in the concentration of Hb; however, at higher concentrations, there is no much increase and the maximum shifts toward a higher wavelength. It can be seen that new peaks arise at ∼358 nm, which can be due to polymer−protein interactions at a higher concentration of Hb. As these peaks at higher concentrations also appear for Mb at a similar wavelength, we can say that Mb and Hb have a similar type of arrangement around the heme cleft; therefore, appearance of peaks at higher concentrations may be due to polymer−protein complex interactions between this protein and the polymer. We used three different heme proteins except Cyt c; the peak at 280 nm is completely diminished, and in Cyt c, the peak shifts to a higher wavelength; the absorbance of peak at 410 nm increases with the increase in concentration, and in each case after a particular concentration, there is not much significant enhancement (Figure 2d). This enhancement of absorbance at 410 nm is large in the case of Cyt c, moderate in the case of Mb, and low in the case of Hb. For Cyt c, the band at 529 nm splits into two peaks. Eventually, we can summarize that the change in the spectra of polymer is due to the changes in the environment of amino acid residues because of the associated interaction with the polymer which can be a hydrophobic, hydrophilic, interaction within the subunit, between the oxygen moiety of the polymer with the COOH terminal residue; direct interaction of aromatic amino acid residues and the polymer surface; hydrogen bonding between the polymer−solvent, polymer−protein, protein−protein, and the protein−solvent; and hydrophobic heme pocket with the

pathway of proteins and to check the conformational changes involved in aggregation of proteins or TRPs. ANS is nonfluorescent in aqueous medium and interacts with the solvent exposed to nonpolar (hydrophobic) sites.2 UV−visible spectra of ANS with aqueous PVCL polymer with varying concentrations of heme proteins are shown in Figure 2. The absorption maximum of ANS with polymer is obtained at ∼380 nm and hump around 350 nm, which is the characteristic absorption band of ANS and is evident from the literature.2,21 In the present study, we have recorded the variation in the peak of maximum wavelength (λmax), that is, 380 nm, as it is sensitive to microenvironmental changes. Native conformation of Cyt c (Figure 2a inset) shows bands at 280, ∼410, and 528 nm which are due to n−Π* transition of aromatic amino acids, an intense Soret band due to Π−Π* transition where His 18 and Met 80 stabilize low spin state by coordinating at axial positions,22 and a broad Q (α + β) band due to electronic transition in the heme group, respectively.16,22 The intensity of these bands increases with change in the protein concentration, and the broad Q band splits into α (520 nm) and β (550 nm) bands in the polymer−protein complex, which was initially absent either in the pure protein in ANS (the red line shown in the inset of Figure 2a) or pure polymer in the case of ANS (the black line shown in Figure 2a). This clearly represents that the polymer interacts with the heme moiety of Cyt c, which is responsible for the splitting during absorbance. This indicates that the presence of Cyt c affects the PVCL−ANS interaction to a greater extent as can be seen from the characteristic ANS spectral peak (380 nm) which vanished completely in the presence of protein and hump (350 nm), which is shifted toward a higher wavelength, that is, red shift. The peak for the native Cyt c at 280 nm is also shifted to a higher wavelength, which represents that the PVCL interaction with aromatic amino acid residues by the mode of interaction is still not clear. The heme cleft of Cyt c is surrounded by the hydrophobic pocket of amino acid residues. The increase in absorbance at peak 410 nm indicates that more nonpolar environment is available around the heme cleft as a hyperchromic shift is observed during changes in environment and stability of molecules that is responsible for Π−Π* transition. This results from changing the concentration (from 0.5 to 1.0 mg/mL) where the intensity change diminishes. With the increasing concentration of Cyt c, the absorbance of all the bands (280, ∼410, 520, and 550 nm) increases, but surprisingly, beyond a concentration of 0.5 mg/mL, there is only a marginal increase in the band at ∼410 nm. As clearly shown in Figure 2d, we find a continuous increase in absorbance maximum with the increasing concentration of Cyt c at 410 nm. Thus, we can interpret that the lower concentration of Cyt c favors more affinity for ANS and it may be attributed to the hydrated coiled conformation of PVCL. These changes in spectra are similar to those appeared during conversion of reduced Cyt c into oxidized form.23 Mb absorption spectroscopy can be used to explore the physical properties of protein such as the structural stability of protein and oxidation of heme iron. UV−visible spectra of PVCL in the absence and presence of Mb are presented in Figure 2b. Native horse heart Mb (FeIII) shows peaks at 280, 410, 505, and 632 nm.24,25 Our results are consistent with those in the literature (Figure 2b inset, blue color). In the presence of polymer, the peak at 280 nm is completely diminished (Figure 2b inset, red color), which shows direct 6334


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Figure 3. Steady-state fluorescence spectroscopy of ANS in PVCL aqueous solution with and without heme proteins, 0.0 mg/mL (black line), 0.5 mg/mL of protein (red line), 1.0 mg/mL of protein (blue line), 1.5 mg/mL of protein (cyan line), 2.0 mg/mL of protein (magenta line), and 2.5 mg/mL of protein (green line) at 25 °C (a) Cyt c, (b) Mb, and (c) Hb. Panel (d) represents the fluorescence intensity maximum at 470 nm for Cyt c (black line), Mb (red line), and Hb (blue line).

intermolecular to intramolecular electron transfer which results in quenching of fluorescence intensity at higher concentrations. From Figure 3a−c, it is clear that Hb exhibited the maximum decrease in intensity as further addition of 0.5 mg of protein, intensity quenched by 3-fold (from ∼240 to ∼97 units). Moreover, at higher concentrations of protein, fluorescence is almost diminished, which can be due to interactions between the polymer chain and protein residue, as well as protein residue and water.19 With increasing concentrations of each protein, there is quenching fluorescence for all three heme proteins. One can say that addition of protein influences the hydrophobicity of solution and serves to block the hydrophobic sites available on the polymer that disrupts the binding with ANS resulting in the variation of quantum yield and induces collapse of the hydrated coiled structure of PVCL. To see these effects more clearly, normalization of the fluorescence intensity of the ANS-containing PVCL solution was performed. Figure 3S illustrates the normalized spectra in the presence and absence of heme proteins. There is a trifling change in the ANS wavelength in the presence of proteins, and this effect remains as such irrespective of varying concentrations of proteins. Therefore, we can say that the integrity of the probe in vicinity of PVCL remains unaffected after addition of heme proteins. Moreover, the fluorescence intensity decreases with increasing protein concentration from 0.5 to 2.5 mg/mL. This quenching in the fluorescence intensity follows the order pure PVCL > PVCL + Cyt c > PVCL + Mb > PVCL + Hb.

hydrophobic moiety of polymer and van der Waals interaction between larger polymer chains and protein surface. To observe in more detail the protein−polymer interactions and the effect of protein on the phase behavior of PVCL, we carried out steady-state fluorescence spectroscopy. Figure 3 represents the steady-state fluorescence spectroscopy results of the polymer in the presence and absence of heme proteins at room temperature. ANS is used as an external fluorescent probe. It is very sensitive toward change in mobility, local polarity of the molecule, and microenvironmental changes in polymer structure.2,18,19 Amphiphilic ANS binds with PVCL in aqueous medium and exhibits a fluorescence intensity maximum at ∼470 nm. The fluorescence spectra of ANS depend on the nature of the solvent, and the intensity increases with an increase in the hydrophobicity of medium. As presented in Figure 3, PVCL gives maximum intensity when ANS is surrounded by more nonpolar chain links of the polymer. On the other hand, with the addition of protein, the intensity decreases, and such a decrease in intensity epitomizes an increase in polarity around ANS owing to the presence of protein in PVCL aqueous solution (Figure 3d). With increasing protein concentration, the intensity decreases with a red shift in wavelength maximum, and this decrease is different for all three proteins. This can be explained as each ANS molecule is solvated in different ways in the presence of each protein,21,27 and the presence of protein changes the water environment around ANS in PVCL aqueous solution. The variation in the quantum yield of ANS is due to the change in the excitation of localized electron from 6335


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Figure 4. FTIR spectra of PVCL in D2O with and without heme proteins, 0.0 mg/mL (black line), 0.5 mg/mL of protein (red line), 1.0 mg/mL of protein (blue line), 1.5 mg/mL of protein (cyan line), 2.0 mg/mL of protein (magenta line), and 2.5 mg/mL of protein (green line) at 25 °C (a) Cyt c, (b) Mb, and (c) Hb in the range 1570−1640 cm−1 and (a′) Cyt c, (b′) Mb, and (c′) Hb in the range 2850−3010 cm−1.

amide I (CO) band and the C−H stretching band, respectively. To avoid overlap of δ(O−H) band at ∼1640 cm−1 with ν(CO) of polymer, D2O was used as a solvent and the position of IR bands of polymer in D2O were identical to H2O.19,21 The repeating units in the case of proteins give rise to nine characteristic IR absorption bands (amides A, B, and I−VII). Out of these bands, amide I bands (1700−1600 cm−1) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. Amide I bands are directly related to

FTIR spectroscopy can provide a better insight into proteininduced interactions and structural changes with polymers. Because of the lack of hydrogen donors in the molecular chain of PVCL, it is unable to form inter/intramolecular H-bonding, and carbonyl group is only involved in hydrogen bonding with the surrounding water molecules resulting in a hydration shell.8,11 Figure 4 shows the FTIR spectrum of PVCL in the presence and absence of proteins using D2O as the solvent at room temperature. The spectrum is recorded in range of 1650−1570 and 3000−2850 cm−1, which corresponds to the 6336


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Figure 5. Influence of Heme proteins on hydrodynamic diameter, (dH) of PVCL aqueous solution with and without heme proteins, protein-free (black line), 0.5 mg/mL of protein (red line), 1.0 mg/mL of protein (blue line), 1.5 mg/mL of protein (cyan line), 2.0 mg/mL of protein (magenta line), and 2.5 mg/mL of protein (green line) at 25 °C (a) Cyt c; (b) Mb; and (c) Hb.

hydrated interactions, which can be hydrophobic hydration or hydrogen bonding. With the addition of this protein to PVCL, the C−H stretching band at 2930 cm−1 is observed to shift (Figure 4b) toward a lower wavenumber (around 2930 cm−1) while the 2870 cm−1 band shifts toward a higher wavenumber. The changes in wavenumber observed upon addition of Mb are arbitrary when the protein concentration is increased from 0.5 to 2.5 mg/mL. In the presence of Hb, there is no shift in frequency; however, the intensity increases at peak ∼2930 cm−1 and it follows a random order near peak 2860 cm−1 which represents different modes of interaction with symmetric and asymmetric stretching bands. New peaks (Figure 4c′) at around 1635 cm−1 are observed, and the absorbance increases with varying protein concentrations. Our observation is consistent with a slight change in the carbonyl group of the polymer, all three heme proteins exhibit a band near 1640 cm−1 with an increase in intensity for peak ∼2930 cm−1 with an increasing concentration and a random order near peak 2860 cm−1. A considerable shift is observed in the case of Mb only. One can interpret that the bonding interaction and conformational changes between the polymer and protein residues are different for each protein. The spectra of PVCL carbonyl group slightly change; however, the molecular interaction triggered by the presence of the protein with the C−H bond ultimately depends on the type and nature of the protein used. For better understanding the agglomeration and association behavior of macromolecular assemblies, DLS (or photon correlation spectroscopy) measurements were performed in a sub-micron region.30−32 Figure 5 depicts the particle diameter/

the backbone conformation, and these bands originate from the CO stretching vibration of amide group coupled with inphase bending of the N−H group and stretching of the C−N bond.17,18,26,27 The frequency of amide I band in solvent media is affected due to involvement of lone pair of PVCL ring in hydrogen bonding and transformation of CO···D−O−D to free CO bonds28 followed by the gradual hydrophobic hydration of PVCL with neighboring water molecules that lead to changes in the C−H bands. The hydration/dehydration mechanism of PVCL in the presence of proteins was traced to the frequencies of the amide I and C−H peaks as these bands provide the valuable information regarding the structural changes upon interactions. The characteristic amide I peak for the polymer is at around 1610 cm−1 and the C−H stretching peak appears at around 2930 cm−1, which is consistent with the literature.28,29 Figure 4 clearly represents the changes in the IR spectrum upon addition of three heme proteins, and Figure 4a depicts that upon addition of Cyt c, there are no significant changes in the frequency values of the polymer; however, the peak intensity gradually increases with an increase in the concentration of protein (near 2864 cm−1) in addition to the appearance of peak at 1636 cm−1 (Figure 4a′), which can be due to the stretching vibration of the carbonyl group of amino acid residues or interaction of polymer with Cyt c. With the addition of Mb to the polymer solution, there is a negligible shift in frequency in the carbonyl region; however, there is a considerable change in C−H symmetric and asymmetric stretching. It represents rearrangement of heavy water molecules around the polymer backbone and changes in 6337


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higher concentration of this protein, that is, 2.5 mg/mL. Upon addition of Mb, the change in size is much less (32.42 nm) at a lower concentration (0.5 mg/mL), but beyond this concentration, there is a tremendous increase in size and protein− polymer complex reaching up to 149.96 nm, which indicates change in the polymer diffusion rate because of the increase in size with increasing surface interactions. The increase in size with the increase in Hb concentration is continuous; however, the increase is steeper at lower concentrations. After addition of 0.5 mg/mL of this protein, there is an increase in size from 27.74 to 51.14 nm, which is the largest increase in size at any particular dilution for Hb. Therefore, we can conclude that with the increasing concentrations of Cyt c, there is very less increase in size (dH varies from 27.74 to 34.97 nm); for Mb, the size increases tremendously at higher concentrations of this protein (increase from 27.74 to 149.96 nm), whereas for Hb, the dH values increase monotonically (dH varies from 27.74 to 97.37 nm) with changing concentrations from 0.5 to 2.5 mg/mL (Table 1). The probable reason for increased dH values may be due to the large-size aggregate formation of protein−polymer biomacromolecular assemblies. In practice, a polynomial fit to the logarithm of the function leads to the size (the first cumulant) and its deviation (the second cumulant), where the PDI corresponds to the square of the normalized standard deviation of an underlying Gaussian size distribution. The PDI

hydrodynamic diameter (dH) of PVCL in heme proteins at 25 °C. The average dH value for PVCL in aqueous solution is 27.74 nm, which is consistent with the literature value.2 Table 1 reports the changes in the dH values of PVCL in heme Table 1. Hydrodynamic Diameter (dH)/nm and PDI of PVCL in the Presence and Absence of Heme Proteins Hydrodynamic diameter (dH)/nm

Poly dispersity index (PDI)

Cconcentration of proteins (mg/mL)

Cyt c

Mb

Hb

Cyt c

Mb

Hb

0.0 0.5 1.0 1.5 2.0 2.5

27.74 28.50 30.65 32.95 35.50 34.97

27.74 32.42 77.33 85.51 95.96 149.96

27.74 51.14 70.03 76.59 84.66 97.37

0.269 0.285 0.379 0.398 0.436 0.413

0.301 0.389 0.484 0.512 0.547 0.566

0.263 0.570 0.548 0.537 0.569 0.536

proteins and the change in the polydispersity index (PDI) of the medium as a function of protein concentration. From the table, it is clear that with the addition of protein, the size varies differently depending on the nature of the protein. There is not much increase in the size of PVCL from 28.50 to 35.50 nm with a changing concentration of Cyt c from 0.5 to 2.0 mg/mL. However, the size decreases from 35.50 to 34.97 nm at a

Figure 6. Laser Raman spectra of PVCL (freeze-dried sample) containing (a) Cyt c, (b) Mb, and (c) Hb in varying concentrations, 0.0 mg/mL (black line), 0.5 mg/mL of protein (red line), 1.0 mg/mL of protein (blue line), 1.5 mg/mL of protein (cyan line), 2.0 mg/mL of protein (magenta line), and 2.5 mg/mL of protein (green line). Panel (d) represents the laser Raman spectra of PVCL individually as well as in the presence of 0.5 mg/mL of Cyt c, Mb, and Hb; pure PVCL (black line); PVCL with 0.5 mg/mL of Cyt c (red line); 0.5 mg/mL of Mb (blue line); and 0.5 mg/mL of Hb (cyan line). 6338


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Figure 7. FESEM micrographs of freeze-dried PVCL aqueous solution in the absence and presence of heme proteins at a fixed concentration of (a) pure PVCL, (b) PVCL with 0.5 mg/mL of Cyt c, (c) PVCL with 0.5 mg/mL of Mb, and (d) PVCL with 0.5 mg/mL of Hb. Scale bar = 10 μm.

increasing concentration, which is consistent with the increase in size at higher concentrations. The decay time at higher concentrations is greater than at lower concentrations because of the increase in size with increasing concentration. The size of the polymer increases sharply after the addition of a small amount of porcine Hb; hence, the correlation coefficient is in agreement with our experimental results. Large-size agglomerates are consistent with hydrodynamic radii as shown in Figure 5. Laser Raman Spectroscopy Analysis of PVCL in the Presence of Heme Proteins. Raman spectroscopy is an important technique to determine interaction changes, and it can provide noninvasive information from various samples which are important in protein science and polymer science. In Figure 6, we present the alteration in the scattering intensities of the lyophilized PVCL sample developed upon addition of heme proteins. To avoid degradation of the lyophilized sample, a laser beam of 514 nm was used. For PVCL aqueous solution, three scattering bands are observed between 2850 and 3000 cm−1 (spectra in black), consistent with those reported in the literature.34 The polymer structural unit contains various CH2 groups which are responsible for these characteristic bands. Figure 6a represents changes in symmetric and asymmetric C− H stretching bands observed with the addition of Cyt c with varying concentrations. With the addition of Cyt c, the scattering intensity decreases along with the diminishing C−H stretching bands of PVCL, and at higher concentrations, these peaks completely disappear. In the presence of protein, the water molecules surrounding the PVCL surface polarize which can be the reason for changes in spectra. The scattering intensity displays a pattern very similar to that of Mb or Hb, although Mb persists the stretching bands. The Raman active modes of porphyrin can be enhanced when the excitation is in resonance with the characteristic bands in the electronic absorption spectra of porphyrins, that is, Soret maximum and Q maximum.35 The Raman enhancement during the excitation within Soret maxima is mainly due to the ν4 band.35,36 The three bands observed for PVCL between 2850 and 3000 cm−1 are destroyed to a greater extent in the case of Hb as compared to Mb and Cyt c (Figure 6d). Increasing concentration of proteins leads to decreases in the scattering intensity for Cyt c, whereas for Mb and Hb,

values for our experiment are greater than 0.1, indicating that the simple cumulant fitting is not a complete representation and that more than a single species are present.19,32,33 This was confirmed by the size distribution analysis as shown in Figure 5. For a homogeneous sample, a single peak near the expected size can be anticipated, whereas the peak is broader than that for a single species, indicating the presence of some fragments and oligomeric assemblies. These variations in dH are due to the changes in the preferential interactions between the polymer−proteins and solvophobic interactions among the residues of the protein. It is interesting to note that the dH values increase with increasing concentrations of heme proteins. This signifies that the variation in the hydrogen bonds between the carbonyl group of PVCL and water molecules is substantially provoked by the residues and functional groups of the protein. To study more clearly and obtain deeper knowledge about these macromolecule assemblies, correlation coefficients values as a function of decay time for polymer and polymer−protein complexes at different concentrations are plotted in Figure 4S. Correlation coefficient gives more information on the size of aggregated species. The intensity of signal at time = t is compared to the intensity at a very small time later (t + γt), where γ is very small. If there is correlation between the intensities of two signals, the two signals are strongly or well correlated. If the signal intensity at t is compared with itself, then there is a perfect correlation as the signals are identical. A perfect correlation gives unity (1.00) and no correlation gives zero (0.00). Signals at t + 2γt, t + 3γt, t + 4γt, and so forth were compared with the signal at time, t. For larger particles, the signal changes very slowly and the correlation persists for longer times, whereas if particles are small with a higher diffusion speed, then the correlation will reduce quickly.33 The measured correlogram provides clear evidence of the mean size. Figure 4S(a−c) shows the comparison of the change in the correlation coefficient of the polymer with the changing concentrations of heme proteins. As can be seen from Table 1, it is clear that the addition of Cyt c increases the size of the polymer slightly, an observation which is confirmed by the measured correlogram where the time decay and hence also the mean is nearly the same for all concentrations. By contrast, with the addition of Mb, the decay time increases with 6339


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Figure 8. TEM images of PVCL aqueous solution in the absence and presence of heme proteins at a fixed concentration of (a) pure PVCL, (b) pure Cyt c, (c) PVCL with Cyt c, (d) pure Hb, (e) PVCL with Hb, and (f) PVCL with Mb. Scale bar = 200 nm for (a), (b), (c), (f) and scale bar =500 nm for (d) and (e).

chains vary. However, a considerable morphological change was observed in the presence of heme proteins (Cyt c and Hb). This observation is consistent with our aforesaid results by other techniques also. Morphological modification in the presence of Cyt c and Hb concomitant with variation among polymer chains changes the hydrophobic/hydrophilic balance or hydrogen bonding, which leads to changes in the water structure around the polymer chains. The reason for variation of hydrophobic domain composition can be the presence of different amino acid sequences in heme proteins. With the addition of Cyt c to PVCL, the granular size of the aggregates and the pore size vary. It is also clearly visible from the FESEM micrographs (Figure 7b) that the interconnected polymer chains are linked with the protein surface. PVCL contains interconnected chains with a definite size and diameter. It is clearly seen that the addition of protein causes the size and diameter of these chains to vary. Addition of protein leads to a decrease in the size and diameter of these chains, which is presented in Figure 7a. Moreover, the system becomes denser and agglomerates appear. The size and thickness of these chains decreased to the largest extent in the case of Hb, where thin long chains are visible in the micrograph (Figure 7d). TEM Analysis of the Complex Formation of PVCL with Heme Proteins. Figure 8 portrays the TEM micrographs of different heme proteins and their interaction with TRP PVCL. In Figure 8a, the TEM image of PVCL is shown which shows a typical polymeric assembly with porous nature. In Figure 8b, the TEM images of pure Cyt c is displayed, while Figure 8c shows the complex forms after Cyt c interacting with PVCL, clearly portraying the PVCL molecules surrounding the surface of Cyt c; results indicating this type of interaction are also obtained from our earlier studies. Similarly, the effect of PVCL interaction with Hb is shown in Figure 8d,e, indicating the same as in the case of Cyt c, a complex structure formed by PVCL around Hb molecules. In the case of Mb (Figure 8f), a very contrasting morphology is obtained; when compared to pure Mb (the inset of Figure 8f), a significant morphological change can be seen due to the interaction with PVCL. These TEM results clearly show the complex formation of PVCL with heme proteins. Phase-Transition Behavior of PVCL in the Presence of Heme Proteins by DSC Measurements. Now, we focus on

addition of a lower concentration of protein decreases the scattering intensity maximum; after that, it increases with the increase in the concentration of either Mb or Hb. The aforesaid changes in the Raman spectra can be observed because of different structures of proteins and different modes of interaction with PVCL. Figure 6d presents a shift in the scattering intensity for PVCL studied at a fixed concentration of protein. Addition of Cyt c, Mb, and Hb results in the decrease of scattering intensity of the polymer with diminishing characteristic bands for PVCL and appearance of new bands with enhanced intensity at around ∼1600 and ∼1350 cm−1, which can be due to the interaction between the polymer−protein and formation of conjugates. In our study, we see exceptional behavior of Hb toward interaction with PVCL in Raman spectroscopy. Addition of Hb physiognomies peaks for polymer is destroyed to the largest extent; moreover, no peaks appear in the range 1400−1700 cm−1 and the scattering intensity decreases to the largest extent at lower concentrations. The indirect polarization of proteins brings the abovementioned changes in the Raman spectra of the polymer and diminishing peaks of PVCL as well as appearance of new peaks clearly reflect the interaction induced among the protein and polymer segments. Morphological Changes of Freeze-Dried PVCL with Proteins by FESEM. The changes in the surface morphology of PVCL in the presence of biomolecules were characterized using FESEM and are shown in Figure 7. The changes in the surface properties upon addition of proteins can be seen from the FESEM micrographs. Pure PVCL contains polymer chains which are interconnected with each other as can be seen in Figure 7a. The FESEM images were taken at 10 μm with an extrahigh tension voltage of 5.00 kV, which shows that upon addition of protein, the porosity of the freeze-dried PVCL sample ultimately increased the formation of a polymer− protein conjugate-like geometry. The observed increase in porosity is probably due to the enhanced cross-linking between hydrated PVCL chains because of the presence of heme proteins. With the addition of Mb, the surface morphology of PVCL does not change much. PVCL contains interconnected chains with a definite size and diameter. It is clearly seen from the addition of the protein that the size and diameter of these 6340


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Figure 9. DSC heating curves of PVCL in H2O with and without heme proteins, 0.0 mg/mL (black line), 0.5 mg/mL of protein (red line), 1.0 mg/ mL of protein (blue line), 1.5 mg/mL of protein (cyan line), 2.0 mg/mL of protein (magenta line), and 2.5 mg/mL of protein (green line) (a) Cyt c, (b) Mb, (c) Hb, and (d) Pure PVCL solution. Panel (e) represents the variation of LCST values with an increasing concentration for Cyt c (black line), Mb (red line), and Hb (blue line).

tuning the LCST behavior of the TRP PVCL with varying concentrations of heme proteins at the temperature range 25− 60 °C. The DSC heating curves are shown in Figure 9. Sample and reference cells were heated separately with a temperature scan rate of 1.00 °C/min and monitored using a thermocouple attached to a disk platform. Pure PVCL solution in H2O without any heme proteins shows phase transition at 35.6 °C which is consistent with the literature value.37 The DSC curves for the PVCL aqueous solution are broad and markedly asymmetric with a sharp change below phase transition and a gradual change above phase transition, which is also unswerving with previous findings.9,28,29,37 From the heating curves presented in Figure 9, it is clear that heme proteins have influence on the phase behavior of PVCL and it depends on the type of protein added. Table 2 represents the LCST values of PVCL in the presence of heme proteins. The shape of DSC curve for pure PVCL is similar irrespective of different molecular weight,37 different concentration, and scanning rate,28 and our results are in good agreement with the literature. We varied the scan rate from 1.0 to 0.05 °C and found that the shape of PVCL is similar (Supporting Information 4S). From Figure 9, we can see that there are considerable differences between the PVCL solution and the heme proteins added. Upon addition of Cyt c (Figure 9a), the heat flow

Table 2. LCST Values of PVCL in the Presence of Heme Proteins Obtained from DSC Studies LCST/°C concentration of proteins (mg/mL)

Cyt c

Mb

Hb

0.0 0.5 1.0 1.5 2.0 2.5

35.6 36.8 37.0 37.3 37.5 37.8

35.6 36.4 37.1 37.4 37.3 37.4

35.6 36.9 37.5 37.7 38.1 38.2

diminishes and the process becomes less endothermic. The temperature at which a sharp change in enthalpy on lowtemperature side (onset of transition, i.e., Ton) is also increased37,38 from 31.3 to 31.9 °C with change in LCST from 35.6 to 36.8 °C for 0−0.5 mg/mL of Cyt c. Moreover, addition of Cyt c peak becomes broader, which can be due to the changes in water/polymer/protein interactions taking place when the solution is heated. Below LCST temperature, there are well-solvated PVCL chains which imply that polymer groups are in contact with water molecules. Other weak forces, such as dipole−dipole, dispersion forces,38 and H-bonding are also present in the system. With an increase in temperature, the hydration of the methylene group weakens slightly. The 6341


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The Journal of Physical Chemistry B Scheme 1. Schematic Depiction of the Complex Formation between the Polymer and Proteina

a

The complex formed is due to various types of interactions between the protein and polymer depending on the type of heme protein and concentration of protein.

these proteins. As clearly seen from the FESEM micrographs (Figure 7), the morphology of PVCL changes after addition of proteins and each protein varies morphology differently. Addition of Mb does not lead to much variation in the morphology of PVCL; however, the diameter of PVCL chains decreases with the increase in porosity of the system. Although for Cyt c and Hb there is a considerable change in the surface, for Hb, the thickness of polymer chains reduced to the largest extent. This can be the reason for the far greater change in the LCST for Hb as compared to Mb. Addition of proteins leads to formation of a polymer−protein complex-like geometry which is also visible in TEM images (Figure 8) where the protein is attached on the surface of the polymer with an overall increase in the porosity of the system. The change in PVCL-induced interactions in the presence of proteins is clearly depicted in Scheme 1 through a diagrammatic representation. From the UV−visible spectra of heme proteins, we can interpret that with the addition of protein in the polymer solution, a polymer−protein complex is formed, which shows a complete different property as that of an individual polymer or protein. The complex formed is due to various types of interactions between the protein and polymer depending on the type of protein and the protein concentration. When we compare our results on PVCL with our previous work14,19 on poly-N-isopropyl acrylamide (PNIPAM), we find that Mb and Hb destabilize the cotton ball structure and decrease the LCST. This decrease is mainly attributed due to hydrogen bonding between the polymer and biomolecules and protein-induced conformational changes of PNIPAM chains.

contact between the hydrophobic unit and water molecules becomes thermodynamically less favorable, and as a result, there is a collapse of PVCL chains. With increasing concentrations of Cyt c, there is hardly any change in the onset temperature (Ton); however, at a higher concentration, there is much increase in both LCST (37.7 °C) and onset temperature (32.8 °C). The shape of the thermogram is more or less similar at all concentrations; however, it is broad when compared to the pure PVCL solution. Figure 9b depicts the change in heat flow with the addition of Mb at various concentrations. Endothermic peak becomes much broader after addition of Mb and the broadness increases with the increase in the concentration of protein. Moreover, the onset temperature, which is the temperature at which the sharp increase in heat flow appears, is nearly unaffected by the concentration at lower temperatures, although it decreases at higher concentrations. Water molecules which are bound to the polymer are released into the bulk solvent as the temperature is increased. The number of water molecules released into the bulk could affect the endothermic heat of PVCL during the phase transition. Mb affects endothermic heat to a great extent as it increases the LCST and the peak becomes broader, so we can say that the protein affects the dehydration mechanism of PVCL. With the addition of Hb, the onset temperature rises and the endothermic peak broadens. When we compare all three proteins, Hb has the greatest effect on both the LCST and the onset temperature. By contrast, Mb decreases the onset temperature and broadens the asymmetric peaks, whereas Cyt c has very little influence on either the shape or the LCST value. Figure 9e clearly demonstrates that the addition of heme proteins increases the LCST in a concentration-dependent fashion. The results obtained from the sophisticated biophysical techniques indicate that the biomolecular interactions are dominant and serve as biological stimuli affecting the phase behavior of PVCL. The significant spectral variations illustrate the variety of molecular interactions of PVCL exhibited in the presence of proteins. The rationale behind the variation in LCST values would be the structural variation brought by

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CONCLUSIONS In the present study, the thermally induced molecular conformational behavior of PVCL in the presence of proteins was explored. Various types of biomolecular interactions were also studied with the aid of absorption spectroscopy, steadystate fluorescence spectroscopy, DLS, FTIR spectroscopy, laser Raman spectroscopy, FESEM, TEM, and DSC. With the increase in heme protein concentration, the LCST of PVCL varies in a way depending on the nature and structural 6342


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arrangements of the protein. Changes in the hydration process were put forward based on the asymmetric DSC curves with a sharp increment in heat flow below the LCST and a gradual decrement after the LCST. These observations suggest that there is a decrease in the variety of conformational changes in PVCL because of the topological hampering by the caprolactam ring. When combined, these observations further suggest that the addition of heme proteins increases the LCST of PVCL because of strong protein−water, polymer−water, and polymer−protein interactions. The results from DLS and FESEM micrographs inferred that the size and shape of molecular assembly vary gradually with addition of proteins. Surprisingly, the results obtained with PVCL in the presence of Mb and Hb were completely opposite to those found in the case of PNIPAM. This unexpected alteration can be due to different structural integrities of the polymers. Whereas in the case of PVCL, these two heme proteins act as a bridge between the polymer and water molecules, thereby holding more water molecules around the polymer and preventing from obtaining a sponge-like structure of PVCL mesoglobules. PVCL most likely finds success in the biomedical field and tissue engineering materials because of its good biocompatibility and special three-dimensional structures with a specific distribution gradient of water molecules from hydrophobic core to hydrophilic surface. The smart behavior of PVCL opens the possibility for the development of more complex systems in which the drug release can be triggered by the stimuli associated with the local tumor hyperthermia with certain diseases. This part of the study can help us to design a next-generation smart polymeric biomaterial with improved cytocompatibility and cancer-targeted drug delivery.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.9b04521.

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Experimental details, synthetic reaction, synthesis of PVCL, and 1H NMR spectrum of PVCL (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ac.in. Phone: +91-11-27666646-142. Fax: +91-11-2766 6605. ORCID

Pannuru Venkatesu: 0000-0002-8926-2861 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledged the financial support from the Science and Engineering Research Board (SERB), DST, New Delhi, India (grant no. EMR/2016/001149). The authors also gratefully acknowledged the Sophisticated Analytical Instrument Facility (SAIF)-AIIMS, New Delhi, under the SAIF Program of DST for providing TEM facility.

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REFERENCES

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