Binding and Release between Polymeric Carrier and Protein Drug: pH

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Binding and Release between Polymeric Carrier and Protein Drug: pH Mediated Interplay of Coulomb forces, Hydrogen Bonding, van der Waals Interactions and Entropy Sergio De Luca, Fan Chen, Prasenjit Seal, Martina H. Stenzel, and Sean C. Smith Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00657 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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Binding and Release between Polymeric Carrier and Protein Drug: pH Mediated Interplay of Coulomb forces,

Hydrogen

Bonding,

van

der

Waals

Interactions and Entropy Sergio De Luca,† Fan Chen,‡ Prasenjit Seal,† Martina H. Stenzel,*,‡ and Sean C. Smith*,† †

‡

Integrated Material Design Centre (IMDC), School of Chemical Engineering, UNSW Australia, NSW 2052, Sydney, Australia

Centre for Advanced Macromolecular Design, School of Chemical Engineering and School of

Chemistry, The University of New South Wales, Sydney, New South Wales 2052, Australia

ACS Paragon Plus Environment

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ABSTRACT: The accelerating search for new types of drugs and delivery strategies poses the challenge to understand the mechanism of delivery. To this end, a detailed atomistic picture of binding between the drug and the carrier is quintessential. While many studies focus on the electrostatics of drug-vector interactions, it has also been pointed out that entropic factors relating to water and counter ions can play an important role. By carrying out extensive molecular dynamics simulations and subsequently validating with experiment, we shed light herein on the binding in aqueous solution between a protein drug and a polymeric carrier. We examined the complexation between the polymer, poly (ethylene glycol) methyl ether acrylate-bpoly(carboxyethyl acrylate (PEGMEA−b−PCEA) and the protein, egg white lysozyme, a system that acts as a model for polymer-vector / protein-drug delivery systems. The complexation has been visualized and characterized using contact maps and hydrogen bonding analyses for five independent simulations of the complex, each running over 100 ns. Binding at physiological pH is, as expected, mediated by coulombic attraction between the positively charged protein and negatively charged carboxylate groups on the polymer. However, we find that consideration of electrostatics alone is insufficient to explain the complexation behaviour at low pH. Intracomplex hydrogen bonds, van der Waals interactions also water-water interactions dictate that the polymer does not release the protein at pH 4.8 or indeed at pH 3.2, even though the Coulombic attractions are largely removed as carboxylate groups on the polymer become titrated. Experiments in aqueous solution carried out at pH = 7.0, 4.5, and 3.0 confirm the veracity of the computed binding behaviour. Overall, these combined simulation and experimental results illustrate that coulomb interactions need to be complemented with consideration of other entropic forces, mediated by van der Waals interactions and hydrogen bonding, in order to search for adequate descriptors to predict binding and release properties of


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polymer-protein complexes. Advances in computational power over the past decade make atomistic molecular dynamics simulations such as implemented here one of the few avenues currently available to elucidate the complexity of these interactions and provide insights towards finding adequate descriptors. Thus, there remains much room for improvement of design principles for efficient capture and release delivery systems.

Keywords: hydrogen bond, Lysozyme, molecular dynamics, PEGMEA−b−PCEA, pH


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■ INTRODUCTION New types of drug currently in development are often based on large biomolecules such as proteins or DNA as they can target cancer more specifically without causing detrimental side effects. Some of these therapies have however been stalled due to the fragility of the molecule. Issues stemming from the low hydrolytic stability, especially in the presence of enzymes in the body, and the low cellular uptake of these often highly charged drugs can be addressed by the use of a suitable drug carrier. Recent years have witnessed an unprecedented surge of interest in the field of drug delivery.1–11 Drugs such as siRNA and proteins are typically water-soluble and highly charged. However, they display low hydrolytic stability and need to be protected against decomposition using a drug carrier such as polymeric nanoparticles.12–14 Most polymers interact strongly with protein, most often in an unwanted fashion causing problems for implants for example.15 Binding between polymer and proteins, which is often driven by hydrophobic forces and H-bonding,16 is often non-specific, but can also be reversible when stronger binding proteins are present.17 Polymers that reduce protein binding are i) hydrophilic, ii) have hydrogen bond acceptors, iii) do not have hydrogen bond donors and iv) they are neutral,16 which means that polymers that capture proteins for drug delivery should have the opposite properties. A popular way of delivering these types of drugs is by polyion complex formation with a polymer carrying the opposite charge.1-3 Polyion complexes, which are the result of the interaction of biological molecules and synthetic polymers coined polyelectrolytes, have been widely employed as a way not only to deliver drugs,2 but also to design materials for applications such as tissue engineering.4


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The interactions of polyelectrolytes with proteins have intrigued researchers not only because of the possibility to design new materials, but also because of the abundance of proteins in the body that can potentially interact with charged polymers. In particular, the interaction of proteins with charged surfaces, as they may be found on implanted devices, was the focus of many investigations as understanding of these processes may be able to predict biofouling.5,18 Polyion complex micelles are the result of electrostatic interaction between proteins and block copolymers, which carry a neutral hydrophilic block, typically poly (ethylene glycol) or PEG, and a charged block that carry the opposite charges to the protein. These micelles have evolved as one of the most popular ways to deliver protein drugs.3,19-22 The role of PEG is to stabilize the resulting nanoparticle against further aggregation while it is assumed that polyelectrolyte block will bind to the protein by electrostatic forces protecting the protein. Much thought has been given to the nature of this interaction. Experimental methods such as scattering techniques23 (light scattering and small angle neutron scattering (SANS)), can provide information on the dimension of the nanoparticle while isothermal titration calorimetry (ITC)24 can offer information on the thermodynamics of the binding process. These techniques can be complemented by surface plasmon resonance,25 which reveals information on the rate of binding. Many other techniques such as circular dichroism,26 microscopy studies, mass spectrometry can help to analyse these polyion complex micelles.4 The reader is referred to on excellent review on this topic.4 The focal point of polyion complexes in general or polyion complex micelles in particular is the nature of the binding between both parts. In the context of drug delivery approaches that exploit pH variation to achieve the desired binding and release properties, the binding between polymer and protein is usually discussed in terms of electrostatic interactions


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between positive and negative charges. These electrostatic charges are typically affected by pH values, ionic strength, and the nature of the counterion. Many experimental observations support the electrostatic binding model, such as the decline of the binding strength with increasing ionic strength as the counterions will interfere with the electrostatic bond. Moreover, binding is often observed when the protein takes on a net charge that is similar to the polymer, which was assigned to the interaction of the polymer with either hydrophobic pockets in the protein or charged patches.27 This would lead to localized strong attractive forces between polymer and the charged pocket and at the same time long-range repulsive forces. However, the precise origin of the interaction of polyelectrolytes with proteins cannot solely be explained with binding by electrostatic charges as there are, for example, conflicting reports of the influence of the pH value. Although the pH value of the solution will determine the degree of ionization in relation to the pKa value of the polymer and the isoelectric point of the protein, it has in some instances been reported that pH value often has little influence on protein binding28 – but this is disputed in other reports.29 These observations lead to an increased interest in the underpinning mechanism of the binding process on a molecular level. Such discussions point to the question of whether electrostatic interactions based on ion-pair association can provide a suitable design rationale for designing drug vectors that exploit controllable assembly and disassembly of polyion complexes. While many observations such as the dependency on the ionic strength support the traditional picture on electrostatic charges as the driving force, new theories which emerge propose that the binding is predominantly entropic and not electrostatic.30 It should also be acknowledged that the biochemistry literature is replete with studies pointing to the importance of entropic factors beyond simple electrostatics in biomacromolecular complexation.31,32


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The somewhat ambiguous picture summarized above testifies to the fact that the interactions governing complexation between polymers and proteins are indeed highly complex. Despite advances in experimental methods yielding better and more quantitative microscale information about the complexation, simple design rationales derived therefrom are by no means always reliable for developing successful protein drug delivery methods. Advances in computational power and simulation algorithms over the past decade, however, now facilitate atomistic molecular dynamics simulations that have the potential to reveal complexation dynamics and energetics with a level of detail that can facilitate a more sophisticated and better informed approach to developing design strategies. To shed more light on the underpinning mechanism of the interactions between a polymer electrolyte and representative protein drug, we have performed extensive molecular dynamics simulations on a system that is being studied experimentally in our laboratory. The system incorporates a block copolymer polyelectrolyte that facilitates formation of a polyion complex micelle with the positively charge protein lyzosyme (LYZ), a protein that has frequently been used as model protein for testing of this type of drug carrier.26,28,33 The block copolymer used here, poly (ethylene glycol) methyl ether acrylate-bpoly(carboxyethyl acrylate (PEGMEA−b−PCEA) has a measured pKa value of ~ 5.0. Our lab has shown that the polymer is able to condense various cationic proteins of different sizes including LYZ. The PEGMEA block was chosen as an alternative to PEG as the comb-type polymer may form a bushier layer around the protein protecting it against degradation. PEGMEA is furthermore not a hydrogen donor and should therefore display low protein binding in theory. PCEA is in contrast negatively charged at pH 7.4 making it an ideal partner for the condensation of LYZ. In order to probe the complexation interactions between PEGMEA−b−PCEA and LYZ in aqueous solution, the molecular dynamics simulations are performed at three pHs (pH = 7.0,


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4.8, and 3.2). Our simulation results reveal attractive interaction between PEGMEA−b−PCEA and LYZ at pH 7. Although these interactions are somewhat weakened at pH 4.8 as the charge on the polymer decreases, the complex does not dissociate. Neither indeed does it dissociate at a lower pH of 3.2, at which essentially all of the polymer carboxylate groups are titrated leaving no net charge on the polymer. This conclusion is further supported by experimental analyses. The most important finding of our present study is that the binding motif changes upon reduction of pH from physiological (~ 7) to late endosomal (~ 4.5) values, such that complexation is not dominated by coulombic attractions but more by a combination of intra-complex H-bonding and van der Waals interactions.

■ SIMULATIONS The molecular dynamics (MD) simulations, in the present study, are performed using NAMD 2.10 suite of programs.34 The CHARMM force field35 is used to parametrize LYZ and PEGMEA−b−PCEA polymer. This force field has extensively been validated and employed to study proteins.36,37 The amino acids in LYZ are parametrized using the input parameter file parall36-prot as a part of the August 2015 CHARMM update35 collected in the files set top-parc36-aug15. In order to study the interactions between the protein and a synthetic molecule, we used the CHARMM compatible force field, CHARMM General force field or CGENFF.38 Partial charges and parameters were downloaded from the CGENFF webserver39,40 providing atom typing and assignments by chemical analogy. Construction and validation of the PEGMEA−b−PCEA polymer was performed by employing our established strategy,11 which is based upon the CGENFF force field validation in the context of the study of the interactions


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between synthetic polymers and the small anticancer drug 5-Fluorouracil.11 In that work specifically, we constructed several peptide dendritic systems demonstrating that the radius of gyration in function of monomer size are in good agreement with the trend observed for similar sized polymeric systems.41,42 The validation was further supported by the qualitative agreement found between our molecular dynamics results11 and experimental findings about enhancing solubility effects of the 5-Fluorouracil drug in presence of peptide dendrimer.43 The CGENFF force field, recently, has also been validated against prediction of several synthetic PETIM dendrimers44 properties. The PEGMEA−b−PCEA molecular model was built by selecting a small set of appropriate repeating units referring to chemically different parts of the polymer, and edited with the 3D builder in MAESTRO Schrodinger 2.8.013. The file structure was then input into the CGENFF webserver,39,40 providing the atom types, atomic charges, bond, dihedral and improper polymer parameters. The topology and the chemical structure of the polymer is generated with in-house code following the CHARMM force field36 and the NAMD tutorial45 procedures for merging and patching molecular compounds. Figure 1 represents the chemical structure of the polymer PEGMEA−b−PCEA, both schematically and as visualized with VMD.46


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Figure 1. (a) PEGMEA30−b−PCEA chemical structure visualized with VMD. Carbon, hydrogen, oxygen, and sulphur atoms are coloured in cyan, white, red and yellow, respectively. (b) PEGMEA-b-PCEA chemical comb structure, emphasizing two regions differently coloured. The first region is composed of 30 branches composed of 9 C−C−O functional groups, cyan coloured and named residue RS1 or RS1 (PEGMEA) in our nomenclature. The second region ending with carboxyl groups is green coloured and named RS2 (PCEA) with the carboxyl groups belonging to that RS2 is highlighted in pink. These carboxyl groups change their protonation state at different pH, and they can be neutral or negatively charged, as explained in the text. RS1 do not change its protonation state at different pH. The polymer is composed of two major parts, named residue RS1 (RS1) and residue RS2 (RS2) in our nomenclature, and highlighted with different colours in the bottom part of Figure 1. RS1 is neutral at all pHs simulated (7.0, 4.8, and 3.2), hence RS1 keeps the same atomic partial charges in all simulations. RS2, which has a total charge of q = −30e− at pH = 7.0, (e− is the elementary charge: 1.6 × 10−19 C), becomes partially titrated at pH = 4.8 attaining q = −13e− and fully titrated at pH = 3.2 attaining q = 0. To assign the charges in the polyelectrolyte, we used the Henderson-Hasselbalch equation, which gives the percentage of deprotonation of the COOH group on CEA (pKa value for CEA is 4.67) at different pH values to be 100% at pH ~ 7, 68% at pH ~ 5, 43% at pH ~ 4.8, and 1% at pH ~ 3. The charges are positioned in an alternate fashion at pH = 4.8. This is done to avoid the stretching of the polymer due to localized ionization, which is not favoured by entropy. It may be reasonably assumed that this alternate arrangement of the charges is favoured on average in order to minimize energy.


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PROPKA 3.1,47 as a part of the webserver PDB2PQR,48 can be used to predict the pKa values of protein’s ionisable groups. In our set up, PROPKA provided the protonation state of LYZ at pH = 7.0 as q = +8e−, while at pH = 4.8 and pH = 3.2, the protein charge is given as q = +11e- and +12e− respectively. The structure of LYZ (pdb file 1AKI) has been downloaded from the RCSB server,49 from an X−ray diffraction analysis with a resolution of 1.5 Å50 (see Figure 2) for the atomic structure visualized with VMD.46 The CHARMM force field has been successfully applied to study structure and dynamics of unfolded proteins,51 or static and dynamic properties of protein in sugar solutions.52

Figure 2. Egg lysozyme (pdb 1AKI) visualized with VMD. Carbon, nitrogen, hydrogen, oxygen, and sulphur atoms are coloured in cyan, blue, white, red and yellow, respectively. The polymer and LYZ are arranged in a cubic box of side 10 nm using PACKMOL53 and solvated with the water model TIP3P.54 The Na+ ions, added for electroneutrality of the whole system, are randomly distributed in water with VMD.46 Each system contains roughly 93000 atoms with ∼ 29700 water molecules per system. It is worthwhile to mention here that in MD


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simulation, we modelled the interaction of one polymer chain with one protein molecule. One might argue that while this is probably enough to understand the molecular interactions between these two components, it is not a reflection of the real experimental system where the micelles should contain more than one components. However, the MD simulation box, which we set up to capture this essential interaction, is already quite a demanding system in terms of computational efforts. The inclusion of more polymers and more protein, or at the very least, the inclusion of another polymer would have required a larger simulation box and a much larger amount of water that dramatically degrades the performances. In the initial configuration, LYZ and PEGMEA-b-PCEA are positioned in such a way that their minimum distance of separation from the edge of the cubic box is at least 1.5 nm in each direction with the corresponding intermolecular distance large enough to experience very weak interactions at the beginning of the simulation. The minimum allowed distance is no less than roughly 1 nm and reducing at the minimum contact zones. However, the PEGMEA-bPCEA−LYZ minimum distance cannot be too large because of the slow diffusion due to the high molecular weight of the two species (Mlys ∼ 1.4 × 104 gmol−1, Mpegmea-b-pcea ∼ 1.9 × 104 gmol−1). One typical initial configuration, i.e., taken at the time t = 0, is depicted in Figure 3a, where LYZ is coloured in blue, RS1 in red and RS2 in green (water is not shown for clarity). As can be seen, PEGMEA-b-PCEA is sufficiently separated from the protein with RS2 well separated from it having only one small zone at ∼ 0.7−1 nm apart from LYZ. Figure 3b shows the corresponding contact map with the PEGMEA-b-PCEA RS1 and RS2 carbon atoms on the horizontal axis and the nitrogen atoms of LYZ on the vertical axis. The values for the contact maps are obtained using the following expression:


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f (t ) = e d ( t ) d max

……..(1)

where every distance d(t) between two atoms (taken at time t) which is larger than 50 Å has been set to the same 50 (background colour), and where dmax is the largest distance found at time t. Atoms from 1 to 149 belong to RS1 and from 150 to 179 belong to RS2, as indexed on each contact map.


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Figure 3. (a) Initial configuration at the time t = 0 for the system containing LYZ and polymer at pH = 7.0 (named system ‘d’), (b) The contact map corresponding to the snapshot on the left. (c) PEGMEA-b-PCEA–LYZ complex at pH = 7.0 after 100 ns, (d) the contact map plot averaged over the last 50 frames, (e) Complex at pH = 4.8 with the snapshot taken a further 130 ns following the first 100 ns simulation at pH = 7.0, (f) the corresponding contact map averaged over the last 50 frames of this phase, (g) Complex at pH = 3.2 with the snapshot taken a further 130 ns following the simulation at pH = 4.8, and (h) the corresponding contact map on the right averaged over the last 50 frames of this phase. In these figures, LYZ is coloured with blue, PEGMEA-b-PCEA in red and green, with the RS2 (the PCEA part of the


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polymer) in green and RS1 (the PEGMEA part of the polymer) in red. Water is not shown for clarity. Atoms from 1 to 149 belong to RS1 and from 150 to 179 belong to RS2. Only a small atom selection has been chosen to define the contact map, as specified in Figure S1 of the Supporting Information. Each pixel on the grid corresponds to the pair (i, j) representing the distance between the i PEGMEA-b-PCEA atoms and the j LYZ atoms, depicted with different colours as indicated on the vertical bar. The contact map shows a very small region coloured in red corresponding to the RS2 region closest to LYZ visible in the snapshot on its left. Figure 3 will be commented upon in detail below in the “result and discussion” section. The polymer PEGMEA-b-PCEA is first minimised and equilibrated in an aqueous solution without LYZ. Then the full system is assembled and minimised restraining the LYZ coordinates while PEGMEA-b-PCEA and water molecules are allowed to relax. After that the protein is relaxed and the whole system was equilibrated for 20 ns in NPT conditions at T = 298 K and pressure 1 bar. We employed the same conditions for production. The van der Waals interactions are truncated at the cut off distance of 1.2 nm with a smooth switching function.34 All pairs of bonded atoms that match the 1−4 criteria are excluded.34 Periodic boundary conditions are used in each direction. The Particle Mesh Ewald55 method is used to calculate the electrostatic interactions with a grid spacing of 1 Å computed every 4 steps. The temperature is kept constant with the Langevin thermostat and 1 fs time step is used to integrate the equations of motion. The velocity-Verlet integration scheme56 is used to evolve the atom positions. Data analysis has been conducted with the TCL scripts embedded in VMD46 and python. To investigate the complex formation and release, five independent simulations – denoted systems ‘a’ through ‘e’ are performed containing the polymer and protein and two control


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simulations, one with LYZ alone and another one with PEGMEA-b-PCEA alone as reference systems. The polymer – LYZ complex is first simulated for 100 ns at pH = 7.0, then simulated for a further ∼ 130 ns at pH = 4.8 (with protonation states modified according to pH), starting from the final geometry of the previous 100 ns simulation. Finally, the system is simulated for a further 130 ns at pH = 3.2, starting from the final geometry of the previous 130 ns simulation. Since the scope of our investigation is the study of the effects of the pH change on the complex formation and release mechanism, it is essential that while passing from pH = 7.0 to pH = 4.8, the system restarts from the old coordinates to avoid the possibility that a full system reconstruction (resolvation, minimization and equilibration) affects the outcomes more than the pH change itself. Hence, after the first 100 ns simulation time, the protein psf file is corrected according to the PROPKA 3.147 to adjust its protonation state, passing from q = +8e− to q = +11e− keeping the same last coordinate file. The same procedure is applied to the polymer when passing from q = −30e−, where the 30 branches belonging to RS2 are all negatively charged, each with a charge of −1e− (see the bottom part of Figure 1) to q = −13e−, where an appropriate proportion of the RS2 carboxyl functional groups become titrated. The protonation was carried out by rescaling the total charge of the functional group such that it becomes zero when the group becomes protonated, and by distributing the charges in an alternate fashion, i.e., one carboxyl group is left with q = −1e− and the next protonated. The charges of the counterions must be rescaled to guarantee the electroneutrality of the whole system necessary for the convergence of the Ewald sum.55 It is important to note that the counterion rescaling does not influence the interactions between PEGMEA-b-PCEA and LYZ since there are only few ions in solution (≈ 10) in a cubic box of 10 nm side and distant from the zone of intermolecular interaction. A short equilibration time of roughly 5 ns is excluded from the production data to


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guarantee that the small partial charge differences on the relevant subset of atoms are properly accommodated. For similar reasons, the initial configuration of the pH = 3.2 simulation (q = 0) at corresponds to the final frame of the pH = 4.8 simulation. This choice enables us to study the behaviour of PEGMEA-b-PCEA around the protein if or when it might lose its negative charges (from q = −13e− to q = 0). As a necessary preliminary, we examined some properties of the protein, viz. the root mean square deviation (rmsd) and the radius of gyration (Rg), which gives an estimate of the protein stability so as to validate our protocol and to observe if PEGMEA-b-PCEA alters the LYZ structure, compromising the protein integrity. The rmsd is computed according to Eq. 2

 1 rmsd(t ) =   NG

1

∑ (r (t ) − r (0))

2

i

i∈G

i

2  ……………… (2) 

where ri(t) is the position of atom i at time t and ri(0) represents the initial crystal structure coordinates. NG represents the ensemble of the Cα backbone atoms, excluding the most exposed amino acids close to the N−terminus (Lys1–Arg5) and closest to the C−terminus (Arg125– Leu129) which are more flexible, increasing the rmsd and masking the overall stability, however, still remaining around ∼ 1 nm across 100 ns. For the purpose of delivery, it is essential that the protein retains its structural integrity. This is also important for the analysis of the simulations, such that the polymer sees a relatively stable and smooth protein surface and the intermolecular interactions can be studied in function of the pH change. In this sense, small deviations of the amino acids close to the N–terminus and C−terminus do not affect the results. Results are plotted in Figure S2 of the Supporting Information for the first 100 ns where it can be seen that rmsd is very stable after an initial increase, showing a deviation from the crystal structure of roughly ∼ 2


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Å, which is in accord with the literature.57 This result suggests that the CHARMM force field provided protein stability. It can also be seen that the rmsd plot of PEGMEA-b-PCEA−LYZ depicted in black colour in Figure S2 is very similar with that of the lone protein (magenta colour) suggesting that the polymer, even if highly charged (q = −30e−) does not substantially alter the protein morphology, at least on the time scale investigated in our work (∼ 360 ns). This rmsd trend does not substantially change at lower pH, hence we have not shown the corresponding lower-pH traces in Figure S2 of the Supporting Information. To further validate our protocol, we analyze the radius of gyration of the protein, which gives a measure of its structural compactness, and was computed according to Eq. 3

Rg (t ) =

1 M

N

∑ m r (t ) − R (t ) i i

cm

2

…………..(3)

i =1

where ri is the position of atom i computed with respect to the centre of mass of the polymer Rcm, and mi is the mass of atom i. Results for five independent simulations are plotted in Figures S3a– S3e of the Supporting Information. The first 100 ns for the complex at pH = 7.0 ending at the vertical orange dashed line are followed by ∼ 130 ns at pH = 4.8 that ends at the vertical light blue dashed line. Finally, the complex is simulated for further 130 ns at the pH = 3.2 (ending at the time ∼ 360 ns). Time averaged Rg values are taken sampling every 20 ps over the simulation period of 360 ns. It can be seen that the Rg time profile is quite stable for every simulation, indicating that LYZ does not undergo significant conformational transitions when interacting with PEGMEA-b-PCEA. Figure S3a also plots Rg for the protein when the polymer is not present (magenta), showing a similar trend. These Rg results are consistent with the rmsd profiles, confirming that PEGMEA-b-PCEA does not compromise the protein integrity. Only small


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oscillations of amplitude ∼ 1 Å are visible at pH = 7.0, however, those are not the sign of the peptide collapse. One indicator of the effect of the polymer on LYZ can be found by computing the molecular solvent accessible surface area (SASA) of the protein and providing a measure of the contact area between water and LYZ. SASA was computed by rolling a sphere of probe radius 1.4 Å (approximately the size of one water molecule) around the van der Waals sphere of the protein atoms. The result, plotted in Figures S4 of the Supporting Information show that SASA does not increase with time, yielding values between 7000 and 8000 Å2 at pHs of 7.0, 4.8, and 3.2. Moreover, it has also been observed that SASA for the control system (only LYZ without PEGMEA-b-PCEA in the simulation box) appears to agree well with SASA for the complex. These results confirm that the protein surface is not significantly altered by the polymer and also that water does not penetrate inside the LYZ structure. To further check the consistency of the previous results, we analyse the water content of LYZ, counting the number of water molecules penetrating, or being very close to the interior of the protein. The definition of protein interior is arbitrary and corresponds to our amino acids selection plot in Figure 4, where the interior amino acids are coloured in blue, and where a close water is defined as the water molecule within a cutoff of 2.5 Å from the selected amino acid atoms. The result plotted in Figure 5 shows that the water content for every system remains quite stable, around 10 water molecules, hence, water does not penetrate LYZ, for the time scale investigated. Only for the control system, where only lysozyme is present in solution appears to have a slightly larger water content, probably because PEGMEA-b-PCEA–LYZ contacts reduces to some extent, the interactions between the protein and water. Overall, the previous results validate our protocol.


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Figure 4. Interior part of LYZ (blue coloured), defined to measure the water content plot in Figure 5. The remaining part of the protein is coloured in yellow and some water molecules, within a cutoff radius of 2.5 Å from the internal residues are coloured in red.


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Figure 5. Number of water molecules inside LYZ, i.e. within a cutoff distance of 2.5 Å from the internal amino acids of LYZ (as defined and plotted in Figure 4), for five independent simulations (a-e), changing the initial configurations. The vertical, orange dashed line indicates the pH change, from pH = 7.0 (from t = 0 to t = 100 ns, black colour) to pH = 4.8 (from t = 100 ns to t = 230 ns, red colour), for the system. The vertical, light blue dashed line indicates where the simulation for the complex at pH = 4.8 finishes and simulation at pH = 3.2 starts from t = 230 ns to t = 360 ns (green colour). The water content of LYZ without the polymer in solution is plotted in Figure 5a with the magenta colour only for the first ∼ 120 ns for pH = 7.0 and a short run for pH = 4.8. It is interesting to also plot the Rg of PEGMEA-b-PCEA (presented in Figures S5 of the Supporting Information) noting the slow dynamics of the polymer as a consequence of its large mass. We will see, however, that a subset of the polymer atoms, the residue RS2 (Figure 1 bottom part) is characterized by faster dynamics when pH changes. Figure S5a also suggests that when the LYZ is not present (magenta profile) Rg is only slightly larger, since the polymer does not collapse around the protein.

■ EXPERIMENTAL SECTION Polymer synthesis. Synthesis of the block copolymer, poly (ethylene glycol) methyl ether acrylate-b-poly(carboxyethyl acrylate) was reported elsewhere.58 The polydispersity of the resulting copolymer was characterized using size exclusion chromatography (SEC) using DMAc as an eluent and PMMA as a standard.


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Labelling. Synthesis of the block copolymer, poly (ethylene glycol) methyl ether acrylate-b-poly(carboxyethyl acrylate) was reported elsewhere.58 The RAFT polymerized block copolymer, PEGMEA30-b-CEA30, was labelled with cyanine5 amine (cy5, Sigma-Aldrich). The polymer was dissolved in 4 mL DMSO at a concentration of 12.5 mg/mL (0.9 mM, 30 eq), containing N-(3-Dimethylaminopropyl)-N´-ethylcarbodiimide (EDC, Sigma-Adrich) (2 eq) and N-Hydroxysuccinimide (NHS, Sigma-Aldrich) (1 eq) and stirred for 20 min at room temperature. The solution was then added to the cy5 solution (in 1 mL DMSO solution, 20 µg/mL, 1 eq) and continuously stirred overnight in the dark. Unreacted cy5 was removed by dialyzing against methanol for one day and then dialyzed against MilliQ water for another day, followed by freeze-drying. Lysozyme (LYZ, Sigma-Aldrich) was labelled with fluorescein isothiocyanate (FITC, Sigma-Aldrich). 14 mg LYZ was dissolved in 7 mL 0.05 M carbonate buffer, pH 9.5, while 2.34 mg FITC was dissolved in 1 mL DMSO. The reaction was carried out by mixing both the solution at 4 °C overnight in the dark. The crude product was purified by dialyzing against MilliQ water for 2 days at 4 °C, followed by freeze-drying. pKa measurement. The pKa value of the polymer was determined according to a previous report.58 Briefly, PEGMEA30-b-PCEA30 was dissolved in Milli-Q water (0.06 M, 1 mL) in a 4 mL sample tube. Standard NaOH solution (1 M) was used as titrant. The pH value of the solution was measured using Mettler Toledo Seven Compact S220. The polymer solution was firstly titrated by adding the NaOH titrant in 10 µL increments. The volume of titrant was changed to 2 µL increments when the rise in pH was rapid. 10 µL increments of titrant were used again for another 5 times when the rise in pH was gradual until. The pKa of the polymer was determined as the pH value at half equivalence point.59


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Micellization. MilliQ water was used to dissolve labelled PEGMEA-b-PCEA and LYZ separately. 1 mL 0.03 mM LYZ solution was then added into 1 mL 0.06 mM polymer solution dropwise under stirring. The resulting solution was continuously stirred for 2 hours before the next step. To change the pH value of the solution 1M HCl solution was added to the solution while monitoring the pH value with a pH meter. Raster Imaging Cross-Correlation Spectroscopy (RICS). 2 mL of the micelle solution was loaded into a 35 mm glass bottom FluoroDish (World Precision instruments). ccRICS measurements were carried out by laser scanning confocal microscope (Zeiss LSM780) using a 63 × 1.3 NA glycerol immersion objective. The settings for ccRICS measurement was based on a previously reported protocol.60 In brief, a 488 nm argon laser was used to excite FTIC labelled on LYZ and a 633 nm helium neon laser was used to excite cy5 labelled on PEGMEA-b-PCEA. 490-630 nm BP and 650 nm LP filters were used to collect signals from FITC (green channel) and cy5 (red channel) respectively. Frames of 256 × 256 pixel image size were acquired 100 times at a pixel dwell time of 12.61 µsec/pixel with a pixel size of 0.05 µm. Raw data was analyzed in simFCS software (Laboratory for Fluorescence Dynamics) as described in the protocol.60 Confocal imaging for co-localization study. The same micelle preparation method and microscopy as for ccRICS measurement were used for this study. Images with a size of 1024 × 1024 pixel were scanned over 48.11s using the same laser and filters as ccRICS measurement since the same fluorophores were applied. Obtained images were processed using ZEN2011 imaging software (Zeiss).


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Dynamic light scattering (DLS) Measurements. The particle sizes of PEGMEA-bPCEA–LYZ micelles at different pHs were measured using DLS (Malvern Nano-ZS, measurement angle 173°). Two 1 mL of the micelle samples were prepared with 0.05 mM LYZ and 0.1 mM PEGMEA-b-PCEA in MilliQ water. DLS measurements for particle size of each sample were run three times and averaged before pH adjustment. Different pH values were adjusted using HCl solution to pH 3 and 4.5. The particle size of each sample was then measured after 2 h, 4 h and 6 h for three times at 25 °C using DLS.

■ RESULTS AND DISCUSSION To study the interactions between protein and polymer, we measure the centre of mass (cm) distance between the RS2 carbon atoms of PEGMEA-b-PCEA and the nitrogen atoms of LYZ. We do not consider RS1 (PEGMEA part of the polymer) because it is overall neutral and its total charge does not change when the pH changes from 7.0 to 4.8 to 3.2. In contrast, RS2 loses roughly half its charge while passing from q = −30e− (pH 7) to q = −13e− (pH 4.8), and then again from q = −13e− to q = 0 (pH 3.2). Hence, RS2 is expected to play a major role in modulating complexation as pH decreases. The results are plotted in Figure S6 of the Supporting Information. Figure S6a at pH = 7.0 shows that the cm distance between LYZ and RS2 of PEGMEA-b-PCEA slowly decreases in the first ∼ 50 ns, stabilising at ∼ 2 nm. At pH = 4.8, RS2 goes through two quite large oscillations around the same distance of 2 nm indicating that at pH = 4.8, the average cm distance is similar to the pH = 7.0 condition. However, the cm distance between polymer and LYZ slightly increases suggesting that the interactions between the polymer having q = 0 and the protein with q = +11e− are weaker than when the polymer has q =


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−13e−. This result is consistent with a picture of reduced coulomb interactions between the two molecules as the pH drops, however, it also shows that the time scale involved to observe significant variations are of the order of 100 ns. Similar trends are visible in Figures S6c and S6d, even though less clearly. The cm distance profiles of Figure S6b shows a strong oscillating behaviour in the first 100 ns, a decreasing trend on the next phase (pH = 4.8) and a stable signal for pH 3.2. Considering that RS2 is a relatively long chain, it may be possible that while a specific subset of RS2 atom is getting close to LYZ, other RS2 parts may in fact abandon the interaction region, giving an overall stable signal. This indicates that the cm distance, even though a useful parameter in qualitatively probing the interactions should be supported by more measurements. To estimate the effectiveness of the interactions between PEGMEA-b-PCEA and the protein we compute the number of their close contacts, according to Eq. 4. N peg N lys

ri + 3.5 Å

i =1 j =1

ri

N (t ) = ∑∑ ∫

δ (r (t ) − rj (t ))dr …………………(4)

Only the RS2 carbon atoms (Npeg) and lysozyme nitrogen atoms (Nlys) are included in the counting, rj is the distance of the jth lysozyme nitrogen atom from the ith carbon atom of the polymer RS2, with the δ function selecting only the atoms within 3.5 Å from the atom centers of the polymer carbons. Close contacts as a function of time are plotted in Figure 6. The time profiles show several peaks and strong irregularities due to the relative motion of the long, linear RS2 segment of PEGMEA-b-PCEA and the roughly ovoidal LYZ surface. Overall, it is possible to observe a larger number of contacts at pH = 7.0 and pH = 4.8; and somewhat fewer at pH = 3.2. The contact point plotted in Figures 6 for the polymer-protein complex at pH = 3.2 suggests that the uncharged RS2 block (the PCEA part of the polymer) interacts more weakly with LYZ.


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This result should be compared with the charged polymer results, i.e., with at pH = 4.8, which has q = −13e−. The result plotted in Figure 6d, from t = 100 ns to t = 230 ns (red profile), visibly showing a larger number of contacts around t ∼ 230 ns.

Figure 6. Contacts between the LYZ (nitrogen) atoms and RS2 (carbon) atoms of PEGMEA-bPCEA, for five independent simulations (a-e), changing the initial configurations. The vertical, orange dashed line indicates the pH change, from pH = 7.0 (from t = 0 to t = 100 ns, black colour) to pH = 4.8 (from t = 100 ns to t = 230 ns, red colour), for the system including the polymer and protein. The vertical, light blue dashed line indicates where the simulation finishes at pH = 4.8 and starts at pH = 3.2 from t = 230 ns to t = 360 ns (green colour).


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More revealing are the corresponding distance map plots in Figure 3. For this simulation the behaviour of the negatively charged polymer at pH = 4.8 is similar to the behaviour of the polymer at pH = 7.0. This can be seen observing Figure 3d, the distance map for PEGMEA-bPCEA–LYZ at pH = 7.0 at t = 100 ns that is very similar to the distance map for the complex at pH = 4.8 shown in Figure 3f. Consistent with the map result, the corresponding snapshot on its left shows RS2 curving around the lysozyme maximizing the interactions. Comparing Figures 3f and 3h, the distance maps reveal an interesting change in the pattern of attachment between polymer and protein, which is also reflected in the corresponding snapshots to the left. Recalling that backbone atoms 1 to 149 belong to RS1, while 150 to 179 belong to RS2, the distance maps reveal a weakening of RS2 interactions with LYZ (fewer red zones), while at the same time RS1 block has developed a larger number of close contacts. This is indicated by the new purple shading with some red spots in the central region of Figure 3h compared with Figure 3f. Augmenting the results for system ‘d’ shown in Figure 3, Figures S7–S10 of the Supporting Information plot the distance map for the systems ’a’, ’b’, ’c’, and ’e’, respectively, which correspond to the close contact results plotted in Figures S8a, S8b, S8c, and S8e of the Supporting Information. The system ’a’ maps (Figure S7) illustrate again a progressive increase in the extent of RS1 association with LYZ for lower values of pH and the RS2 residues become titrated. This trend is exaggerated in the system ’b’ simulation, which is presented in Figure S8 of the Supporting Information. Clearly at pH = 7.0, the RS2 block with charge q = –30e– dominates the attachment of polymer and LYZ, much like a harpoon mechanism. At lower pHs for both q = –13e– and q = 0 it is apparent that the interaction has broadened out to include a significant degree of association of the RS1 block with LYZ. The snapshots on the left also reveal that at pH 3.2, RS2 is collapsing, whereas at pH 4.8 it has a more open conformation. The


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system ’c’ in Figure S9 of the Supporting Information shows a behaviour similar to system ’a’, with broadened association between RS1 and LYZ evident at lower pH where the coulombic charge of the RS2 residue is reduced or removed. Finally, system ‘e’ in Figure S10 exhibits behaviour similar to that of system ‘b’, with RS2 harpooning the protein at pH = 7.0 followed by a significant broadening of the association across RS1 also at the lower pH. Considered cumulatively, the evidence of the analyses carried out above from the five simulations suggests that (i) at pH = 7.0 the negatively charged RS2 residues drive the complexation of PEGMEA-b-PCEA with LYZ. In essence, RS2 “harpoons” the LYZ and establishes intimate contact between the polymer and protein; (ii) at lower pH when the RS2 residue is partially titrated, the interactions of polymer with protein become more balanced across both blocks of the copolymer, with the RS1 block now associating more closely with the positively charged protein. This trend of balanced RS1−LYZ and RS2−LYZ interactions continues to be pronounced in the complex at pH 3.2, where all the RS2 carboxylate groups are titrated to make the polymer neutral. While the caveat should be borne in mind at all times that the small time scales investigated in our simulations may not be appropriate to distinguish subtle differences between the systems, there is nevertheless a clear suggestion from the snapshots and contact maps that this conclusion is quite robust. Also our simulations can only describe interactions between one isolated polymer and one isolated protein. In the real system, there would certainly be polymer-polymer interactions, which we neglect here. As a consequence, less protein surface would be exposed to interact with the PEGMEA block. However, the fact that there is interaction between the protein and polymer even at lower pH remains valid for both the real and simulated systems.


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To elaborate further on the pattern of interactions between the two moieties summarized above, we examined the respective number of hydrogen bond (HB) interactions between LYZ and blocks RS1 and RS2 of the PEGMEA-b-PCEA copolymer during the total 360 ns simulations. The number of HBs is computed considering as potential donor (D)−acceptor (A) pairs, all the possible combinations including nitrogen and oxygen atoms of LYZ and oxygens atoms of the polymer. The following geometric criterion has been used: the cutoff distance between the donor and the acceptor D···H···A is 3.5 Å, and the corresponding angle is 30°. Figures 7 and 8 show the results of the separate counts for RS1–LYZ and RS2–LYZ HBs over


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Figure 7. Hydrogen bond counting between LYZ nitrogen and oxygen atoms and the polymer RS1 residue oxygens for five independent simulations (a-e), changing the initial configurations. The vertical, orange dashed line indicates the pH change, from pH = 7.0 (from t = 0 to t = 100 ns, black colour) to pH = 4.8 (from t = 100 ns to t = 230 ns, red colour), for the complex. The vertical, light blue dashed line indicates when the pH is switched from 4.8 to 3.2. The final simulation at pH 3.2 runs from t = 230 ns to t = 360 ns, green colour).

Figure 8. Hydrogen bond counting between LYZ nitrogen and oxygen atoms and the polymer RS2 residue oxygens for five independent simulations (a-e), changing the initial configurations. The vertical, orange dashed line indicates the pH change, from pH = 7.0 (from t = 0 to t = 100 ns, black colour) to pH = 4.8 (from t = 100 ns to t = 230 ns, red colour), for the system including


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lysozyme. The vertical, light blue dashed line indicates when the pH is switched from 4.8 to 3.2. The final simulation at pH 3.2 runs from t = 230 ns to t = 360 ns, green colour). the time of the simulations. Figure S11 of the Supporting Information shows the corresponding plot summed over both RS1 and RS2. The results are highly consistent with the corresponding distance maps. For example, for the two simulations ‘b’ and ‘e’ which show exaggerated harpoon association of RS2 with LYZ at pH = 7.0 followed by broadened association of both RS1 and RS2 with LYZ at the lower pHs of 4.8 and 3.2, we see almost no HBs between RS1 and LYZ in the first 100ns of the simulations at pH = 7.0 in Figures 7b and 7d, followed by significant increase in the HB counts for the last 260 ns of the simulations at pHs 4.8 and 3.2. The other frames in Figure 7 show similar but not quite so exaggerated trends in accord with our conclusions derived from the distance plots above. In Figure 8, the numbers of HBs between RS2 and LYZ appear to be relatively similar across the first two segments of the simulations (100 ns at pH = 7.0 and 130 ns at pH = 4.8), which is not too surprising given that while the number of negative charges on RS2 decreases, the number of positive charges on LYZ increases. Further supporting the picture of weakening interactions between RS2 and LYZ as the residues on RS2 become titrated, we see a very pronounced decrease in numbers of HB interactions of RS2 with LYZ in the last 130 ns when RS2 is set to be neutral, corresponding to pH 3.2. Since the PEGMEA-b-PCEA block copolymer (Figure S12 of the Supporting Information for SEC analysis) is designed as a delivery system for LYZ and similar proteins, it should ideally facilitate capture of the protein at physiological pH = 7.0 and also facilitate release of the protein at late endosomal pH’s of ca. 4.5. Our simulations provide the conclusion that capture is indeed working well, but release at low pH 4.8 and 3.2 – at least for an aqueous environment as is represented in the simulations – is not effective. To explore this conclusion we


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have designed experiments to reveal the in vitro capture/release characteristics of the PEGMEALYZ system. In this system, we have chosen molar ratio of polymer to LYZ of 2:1. Although it does not reflect the modelled 1:1 ratio, the resulting nanoparticle was found to have high stability in aqueous solution against aggregation as the zeta potential of the particle is close to zero (Figure S13 of the Supporting Information) allowing long-term monitoring. The different ratio between both blocks is not considered a concern as it will not affect observations on assembly and disassembly. In this experiment, the protein was labelled with FTIC while the polymer was labelled with Cy5 dye. RICS analysis allows monitoring molecular dynamics and concentrations and, likes in this case, co-localization of two fluorescent species. The cross-correlation peak, shown in Figure 9, confirms the interaction between PEGMEA-b-PCEA and LYZ even in an acid environment with pH 4.5 that corroborates well with our molecular dynamics simulations.

14.000 12.000 G(x,y)

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10.000 8.000 6.000 150 135 Pixels 120

4.000 2.000

105 105

120

135 Pixels

150

Figure 9. RICS analysis of PEGMEA-b-PCEA and LYZ in a pH 4.5 aqueous solution. To further strengthen our conclusion, we also performed the co-localisation study of PEGMEAb-PCEA and LYZ at pH = 4.5 using confocal microscopy. Figure 10 depicts the confocal laser scanning microscopy (CLSM) images of the co-localization of PEGMEA and LYZ in pH = 4.5


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aqueous solution. It has been observed that during a measurement of 48.11 s, polymers are colocalized with LYZ at pH = 4.5, which appears as yellow dots in Figure 10. It was not possible to measure at lower pH values as the chosen dyes suffer from photobleaching at lower pH values.

Figure 10. Confocal laser scanning microscopy (CLSM) images of the co-localization of PEGMEA-b-PCEA and LYZ in pH = 4.5 aqueous solution. Red is PEGMEA-b-PCEA; green is LYZ; yellow is the overlap. To further elucidate the high stability of binding between protein and polymer, even at low or almost zero degree of ionization, DLS studies at lower pHs of 4.5 and 3.0 were performed. Figure 11 depicts the particle size of PEGMEA-b-PCEA-LYZ micelles in MilliQ water and after pH adjustment by adding HCl. The particle size in MilliQ water was initially measured at t = 0 and monitored over time at different pH values. The hydrodynamic diameter of the micelles at different time steps, plotted in Figure 11 (see Figure S14 for the size distribution histogram, Figure S15 for the correlation curves, and Table S1 of the Supporting Information for the plotted numbers) reveals that the size remained relatively stable in an acidic environment over a period of 6 h. This in turn illustrates that the protein LYZ cannot be released neither at pH


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4.5 nor even at a lower pH of 3 in aqueous solution, which again supports the present molecular dynamics study.

Figure 11. Particle size of PEGMEA-b-PCEA-LYZ micelles at pH 3, 4.5 and 7 over 6 h as determined by DLS.

■ CONCLUSION The PEGMEA-b-PCEA polymer facilitates in vitro cellular delivery of the protein drug lysozyme. However, in contrast, the simulations carried out in this work reveal that in aqueous solution the polymer carrier is less than optimal, failing to release the drug at a representative late endosomal pH of 4.8. This was unexpected and prompted our experiments in aqueous solution, which then verified the predictions of the simulations. Following the rationale that coulombic pair attractions primarily dictate the binding motif in the polyion complex, one would expect that binding of the positively charged protein with the negatively charged polymer would


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be efficient at pH 7, but should fail at pH 4.8 when the PCEA carboxylate groups become largely titrated, releasing the protein at late-endosomal pH. The simulations suggested convincingly that this does not happen. Our analysis shows that at pHs of 4.8 and 3.2, the mode of interaction within the complex broadens to include more extensive H-bonding interactions of the neutral, hydrophilic PEGMEA block as well as the PCEA block with the protein. Thus, an important finding of the present study is that the binding motif changes at low pH: 4.8 and 3.2, such that it is not dominated by coulombic attractions but more by a combination of intra-complex Hbonding and van der Waals interactions. Returning to the first statement in this conclusion, our study raises the subsequent suggestion that there are features of the intracellular (endosomal or cytoplasmic) environments that facilitate dissociation of the carrier – lysozyme complex in a way that is fundamentally different from the dynamics of the complex in aqueous solution. This highlights directions for future investigation encompassing synergistic application of atomistic simulations and experiment that will yield both a deeper understanding of the delivery process and more sophisticated design principles for polymeric vectors.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The atom subset of the polymer, PEGMEA-b-PCEA that is used to define the contact map, the LYZ rmsd vs time in presence of the PEGMEA-b-PCEA polymer, radius of


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gyration Rg and SASA of LYZ, radius of gyration Rg of PEGMEA-b-PCEA, center of mass (cm) distance between the lysozyme nitrogen atoms and the RS2 carbon atoms of the polymer,

snapshots and corresponding distance map plots for 4 different

configurations of the PEGMEA-b-PCEA–LYZ complex at different pHs, the hydrogen bonding plots between the LYZ nitrogen and oxygen atoms and the polymer RS1 and RS2 residue oxygens for five independent simulations changing the initial configurations, the SEC analysis of PEGMEA30-b-PCEA30, the zeta potential curve, size distribution histogram, and correlation curve of the polymer-protein complex, hydrodynamic diameter of the micelles at different pH and time steps.

■ AUTHOR INFORMATION Corresponding Authors * Phone: +61-293854656. E-mail: [email protected] * Phone: +61-293855132. E-mail: [email protected]

■ ACKNOWLEDGMENTS


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This research was undertaken with the assistance of resources provided by the National Computing Infrastructure (NCI) facility at the Australian National University and the Pawsey Supercomputing Centre; allocated through both the National Computational Merit Allocation Scheme supported by the Australian Government and the Australian Research Council grant LE160100051 (“Maintaining and enhancing merit-based access to the national computational infrastructure facility”, 2016-2018). SDL would like to acknowledge Deva Deeptimahanti of Pawsey Supercomputing Center for giving the support for optimizing performances.

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For Table of Contents Use Only: Binding and Release between Polymeric Carrier and Protein Drug: pH Mediated Interplay of Coulomb forces, Hydrogen Bonding, van der Waals Interactions and Entropy Sergio De Luca,† Fan Chen,‡ Prasenjit Seal,† Martina H. Stenzel,*,‡ and Sean C. Smith*,†


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Binding and Release between Polymeric Carrier and Protein Drug: pH

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