LETTER pubs.acs.org/JPCL
Effects of PAMAM Dendrimer Salt Solutions on Protein Stability Diwakar Shukla,† Curtiss P. Schneider,† and Bernhardt L. Trout* Department of Chemical Engineering, Massachusetts Institute of Technology, E19-502b, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
bS Supporting Information ABSTRACT: Dendrimers are widely used for biological applications. However, their effect on protein stability has not been studied extensively. Typically, charged cationic dendrimers such as PAMAM dendrimers tend to destabilize proteins due to the cooperative binding of surface groups to the protein surface. We have studied the effect of PAMAM dendrimer salt solutions on protein stability both experimentally and computationally. We show that the effect of dendrimers on protein stability depends on the choice of counterion (e.g., dihydrogen phosphate salts reduce the rate of protein aggregation, while chloride salts increase the rate of protein aggregation). In the presence of dihydrogen phosphate and sulfate counterions, the binding of dendrimers to the protein surface is limited (when compared to chloride and thiocyanate), which enhances the conformational stability of proteins. To the best of the authors’ knowledge, this is the first study that has shown that PAMAM dendrimer salts can significantly suppress the aggregation of proteins. SECTION: Statistical Mechanics, Thermodynamics, Medium Effects
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herapeutic proteins are highly susceptible to degradation via means such as deamidation, isomerization, hydrolysis, oxidation, disulfide scrambling, and so forth.13 However, the most troublesome and probably the least understood form of instability has to be aggregation. Aggregates present in an injected solution can elicit adverse side effects and compromise the efficacy of the product.2 To stabilize proteins against aggregation, solution additives are typically added to the protein solution. A variety of additives have been used for protein stabilization, which include sugars, polyols, amino acids, surfactants and so forth.1,36 In the past decade, dendrimers have been widely used for biological applications, including drug and gene delivery, but there are limited studies on their effect on protein stability and aggregation.7,8 Dendrimers are synthetic, highly branched polymers that have a small volume and a high density of surface functional groups.9 Due to the presence of multiple functional groups, dendrimers provide numerous possibilities for interaction with proteins.10 The binding of multiple surface groups leads to a greatly increased avidity between the dendrimer and a protein when compared to the binding of a single functional group. This avidity has contrasting effects on different proteins. Certain types of dendrimers can act as protein denaturants, which can help in solubilizing protein aggregates. Prion protein aggregates (responsible for spongiform encephalopathies, including mad cow disease and CreutzfeldtJakob’s disease), which are only soluble at high denaturant concentrations, have been shown to be soluble in PPI (polypropylene imine) and PAMAM (polyamido amine) dendrimer solutions.11 r 2011 American Chemical Society
Klanjert et al. have shown that dendrimers are effective amyloid-fibril dissolving agents.12 Strong electrostatic interactions between proteins and dendrimers are responsible for the breaking and dissolution of pre-existing fibrillar aggregates in the above studies. Dendrimers can also bind specifically to proteinprotein interfaces, thereby inhibiting protein oligomerization.13,14 However, the strong avidity observed in proteindendrimer interactions can have a negative effect on the thermostability of proteins. Giehm et al. showed that PPI dendrimers significantly reduce the thermostability of insulin (10 μg/mL of the generation 3 PPI dendrimer reduces the melting temperature of insulin by 30 °C). It was also shown that the stability of four other proteins (lysozyme, cutinase, myoglobin, and TNfn3) was only decreased marginally, with lysozyme showing no change in thermostability, even at 100 μg/mL of the generation 3 PPI dendrimer.11,15 Gabelleri et al. used Trp phosphorescence spectroscopy and found perturbations of the protein native fold in solution by neutral, positively, and negatively charged fifth-generation polyamidoamine (PAMAM) dendrimers.16 Bryszewska and co-workers reported that increasing concentrations of PAMAM dendrimers destabilized bovine serum albumin but slightly increased the stability of human serum albumin.1719 These studies clearly show that dendrimers have destabilizing or neutral
Received: June 6, 2011 Accepted: July 1, 2011 Published: July 01, 2011 1782
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The Journal of Physical Chemistry Letters effects on protein stability, which depends on the nature of both the protein and the dendrimer. The interactions between dendrimers and proteins have been studied experimentally,20,21 but there are limited computational studies regarding such interactions. Computational studies reported in the literature have been limited to simulations of higher-generation dendrimers in water,22,23 simulations of the association or clustering of dendrimer molecules,24 interactions of a single dendrimer with DNA25 or protein,14 and so forth, but there are no reported computational studies on the preferential interaction of dendrimers with proteins. Similarly, there is also a lack of preferential interaction coefficient measurements of proteins in aqueous dendrimer solutions. The reason for the absence of such studies is the large dendrimer size and associated conformational degrees of freedom, which cannot be sampled within a reasonable amount of computational time. However, small, generation zero dendrimers can be studied computationally. The interactions between dendrimers and proteins depend on the nature of surface functional groups on the dendrimer. Molecular insights obtained from the interactions of surface functional groups in generation zero dendrimers can be extrapolated to higher-order dendrimers. Dendrimers contain a large number of surface functional groups, and the number of counterions per dendrimer molecule is proportional to the number of surface groups, which scales as 2(n+2), where n is the dendrimer generation. Due to their large number per dendrimer molecule, counterions are expected to play a critical role in proteindendrimer interactions. We have recently revealed the choice of counterion to be a key factor in how arginine interacts with proteins and inhibits aggregation.26 It was found that counterions that form attractive interactions with arginine tended to limit the binding of arginine to the protein and vice versa. Hydrogen-bond-accepting counterions, like sulfate, phosphate, citrate and so forth, induced the clustering of arginine around the protein surface, which led to the inhibition of protein association. Clustering of dendrimers has been studied extensively, but the self-clustering behavior is only observed for amphiphilic dendrimers with solvophobic cores and a solvophilic shell.24,27 PAMAM dendrimers with a charged core and surface groups can form aggregates only in the presence of an external bridging molecule, like an oppositely charged dendrimer or peptide.28,29 The ammonium surface group in PAMAM dendrimers can form hydrogen bonds with counterions like sulfate, phosphate, and so forth. The effects of counterions on the dendrimer structure and self-interaction have been reported in the literature,30 but there are no studies on the effect of counterions on the interaction of dendrimers with proteins. Therefore, a more thorough understanding of the interaction between dendrimers and protein surfaces in the presence of various counterions is desired. In this study, we performed MD simulations of aqueous generation zero PAMAM dendrimer salt solutions and simulations of R-chymotrypsinogen A (aCgn) in the presence of generation zero PAMAM dendrimer salts. We studied the effect of thiocyanate, chloride, dihydrogen phosphate, and sulfate counterions. The details of the simulation setup and computational methods are included in the Supporting Information. We also performed experimental accelerated aggregation studies to obtain the rate of aggregation in the presence of the dendrimer salts. Experimental data show that the dihydrogen phosphate salt form stabilizes the protein, while the chloride form was found to be a strong denaturant. MD simulations reveal that attractive interactions between the surface groups on the dendrimer and
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Figure 1. Monomer loss profiles for aCgn solutions containing generation 0 PAMAM dendrimer chloride and dihydrogen phosphate salts at varying concentrations. The influence of PAMAM dendrimers on aCgn monomer loss due to aggregation is determined at 52.5 °C. For all experiments, the initial monomer concentration, M0, was 10 mg/mL prepared in a 20 mM sodium citrate pH 5 buffer.
various counterions with hydrogen-bond-accepting characteristics influence how the compounds interact with aCgn and how they influence the rate of aggregation. Figure 1 shows aCgn monomer loss profiles, as determined by size exclusion HPLC, for solutions containing a generation zero PAMAM dendrimer salt. At a low dendrimer concentration (0.075 M), the rate of monomer loss in the presence of the chloride salt is almost the same as that of the reference solution, but the rate is significantly lower in the presence of the dihydrogen phosphate salt. The rate of protein aggregation is reduced to 30% of its original value (buffer-only solution) when in the presence of the dihydrogen phosphate salt. The rate of aggregation is further reduced to 21% of its original value when in the presence of 0.15 M phosphate salt. However, the monomer loss becomes significantly higher for the chloride salt, with the rate of aggregation increasing to 150% of its original value at 0.15 M. The results for the chloride salt are similar to the destabilizing effect of polypropylene imine (PPI) dendrimers.15 The authors in that study were able to show that the destabilizing effect was the result of attractive electrostatic interactions. The pH was adjusted so that insulin had a negative charge while the dendrimers had a positive charge. Other proteins with a positive charge were not destabilized as greatly as the negatively charged insulin. Therefore, the overall charge on the protein significantly affects the proteindendrimer interaction. In this study, aCgn at pH 5 has a strong, positively charged surface, but the PAMAM dendrimer chloride salt still destabilizes the protein. Thus, the electrostatic repulsion between the positively charged protein and the positively charged dendrimer is not strong enough to prevent the dendrimer from forming attractive interactions with the protein in the presence of a chloride counterion. As mentioned, our previous inquiry into various arginine salts showed that the interaction between the additive and a protein is strongly influenced by the counterion. Therefore, it is expected that the interaction between the ammonium surface groups of the dendrimer and the counterions plays a role in protein stabilization when in the presence of dihydrogen phosphate and destabilization when in the presence of chloride. We have also investigated the effect of thiocyanate and sulfate ions as their effect on protein stability is expected to be similar to that of the chloride and dihydrogen phosphate salts, respectively. 1783
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The Journal of Physical Chemistry Letters
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Table 1. Preferential Interaction Coefficient of r-Chymotrypsinogen A in Aqueous 0.2 m PAMAM Dendrimer (Generation Zero) Salt Solutions salt SCN
Γ23
Γ23(dend.)
Γ(anion)
0.25
0
3
Cl
1.75
1
15
SO4
2.33
2
8
H2PO4
2.83
4
10
To gain insight into how the dendrimer salts affect protein protein interactions, the preferential interaction coefficient (commonly referred to as Γ23) for various dendrimer salt solutions was determined computationally via MD simulations.31 Γ23 is a measure of the preference of additives for the protein surface and is defined by the following expression ! ∂m3 ∂μ2 Γ23 ¼ Γμ3 ¼ ¼ ð1Þ ∂m2 T, P, μ3 ∂μ3 T, P, m2
where m, T, P and μ represent the molal concentration, temperature, pressure, and chemical potential, respectively. The subscripts used indicate solution components in Scatchard notation: water (subscript 1), the protein (subscript 2), and the additive (subscript 3).32 Preferential interactions, though weak in nature, significantly influence the solubility and stability of a protein in addition to influencing proteinprotein interactions.33,34 The preferential interaction coefficient is thermodynamically related to the free energy of transfer of the protein from water to the additive solution. Additives with a positive Γ23 are typically described as being preferentially bound to the protein surface due to an increase in the concentration of the additive in the local domain (region around the protein surface), and this favorable interaction, as indicated by eq 1, lowers the chemical potential of the protein. Therefore, additives with Γ23 > 0 tend to destabilize proteins due to their preferential interaction with a protein surface as compared to the bulk solution. The opposite is true for additives with a negative Γ23, which are typically described as being preferentially excluded from the surface of the protein and tend to enhance the conformational stability of the proteins. The procedure used for calculating the preferential interaction coefficient35,36 is included in the Supporting Information. Γ23 values for aCgn in the presence of dendrimer salt solutions are reported in Table 1. It can be seen that both the overall Γ23 and the values for the dendrimer molecule alone decrease in the order thiocyanate > chloride > sulfate > dihydrogen phosphate. The concentration of the cationic dendrimer around the protein surface is influenced by the concentration of anions in the vicinity of the protein and vice versa. The preferential accumulation of thiocyanate ions near the protein surface enhances the concentration of the dendrimer in the vicinity of the protein, which would lead to a reduction in protein conformational stability. The preferential exclusion (Γ23< 0) of chloride, phosphate, and sulfate salts would typically lead to enhanced conformational stability of the protein. However, the effect of the chloride salt on protein aggregation is in contradiction to the predicted influence on conformational stability. Even though there is often a link between preferential interaction coefficient measurements and changes in conformational stability,34 there are some cases in which a compound is excluded from the native state but attracted to the unfolded state
(e.g., 2-methyl-2,4-pentanediol). This seems to be the case for the PAMAM dendrimer chloride salt. The large size of the compound and the electrostatic repulsion between the positively charged compound and the positively charged protein inhibits preferential binding. However, in the unfolded state, when there are more sites for the dendrimer to bind to and when the electrostatic charge on the protein is distributed over a larger area, the dendrimer can bind to the protein with greater affinity. This shifts the folding equilibrium toward the unfolded state, which destabilizes the protein. The decrease in the concentration of the dendrimer around the protein in the presence of chloride, sulfate, and dihydrogen phosphate ions compared to that for thiocyanate can be explained in terms of the local charge balance of the dendrimer solution. The concentration of chloride, sulfate, and dihydrogen phosphate counterions near the protein surface is lower than their bulk concentration (away from the protein surface). Therefore, dendrimer molecules are pulled away from the protein surface in order to maintain the charge neutrality of the bulk solution. However, the trend among chloride, sulfate, and dihydrogen phosphate ions cannot be explained in terms of charge separation alone. Dihydrogen phosphate ions have a Γ23 value of 10, which is less excluded than the value for the chloride ion (15), but the Γ23 value for the dendrimer molecule is larger for the chloride salt (1) as compared to that for the dihydrogen phosphate salt (4). It has been reported that the chloride ion does not have a strong preference for the protein surface as compared to the bulk solution.37 Furthermore, the interaction of chloride ions with the cationic dendrimer is also weak as compared to that of the sulfate and dihydrogen phosphate ions due to its inability to form hydrogen bonds with ammonium ions.26 Therefore, the chloride ion is in the middle of the Hofmeister series, with dihydrogen phosphate and sulfate placed to the left of chloride as more stabilizing anions and thiocyanate placed to the right as a destabilizing anion.38 However, the ranking of the anions based on Γ23 values in this study is not the same as the Hofmeister series for anions. As discussed above, ionion interactions may be contributing to the observed preferential interaction of ions with the protein surface. To quantify this behavior, MD simulations were conducted on aqueous solutions of the generation zero dendrimers. Figure 2a shows the radial distribution functions (RDFs) between the dendrimer and the counterions. It can be seen that sulfate and phosphate ions interact strongly with the ammonium surface groups on the dendrimer as compared to chloride and thiocyanate ions. Furthermore, the RDFs between PAMAM dendrimers shown in Figure 2b indicates a clustering of dendrimers in the presence of dihydrogen phosphate and sulfate ions. MD snapshots of the simulation box of aqueous dendrimer salts confirm that there is significant clustering in the dihydrogen phosphate and sulfate salt solutions as compared to the chloride and thiocyanate salts (see Figure 3). The dihydrogen phosphate and sulfate ions seem to act as a bridge between dendrimer molecules, thus forming clusters in solution. The presence of clusters in solution can be quantified by calculating the number of hydrogen bonds between different species in solution (see Table 2). As shown in Table 2, it can be seen that there is no interaction between dendrimer molecules, as indicated by the small number of hydrogen bonds (
