Published: July 01, 2011 r2011 American Chemical Society 1782 dx.doi.org/10.1021/jz200758m | J. Phys. Chem. Lett. 2011, 2, 1782–1788 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 b S Supporting Information T herapeutic proteins are highly susceptible to degradation via means such as deamidation, isomerization, hydrolysis, oxi- dation, disulfide scrambling, and so forth. 1À3 However, the most troublesome and probably the least understood form of instabil- ity 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. 1,3À6 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 poly- mers 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 interac- tion 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, in- cluding mad cow disease and CreutzfeldtÀJakob’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 Klanjert et al. have shown that dendrimers are effective amy- loid-fibril dissolving agents. 12 Strong electrostatic interactions between proteins and dendrimers are responsible for the break- ing and dissolution of pre-existing fibrillar aggregates in the above studies. Dendrimers can also bind specifically to proteinÀprotein interfaces, thereby inhibiting protein oligomerization. 13,14 How- ever, the strong avidity observed in proteinÀdendrimer interac- tions 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 poly- amidoamine (PAMAM) dendrimers. 16 Bryszewska and co-work- ers reported that increasing concentrations of PAMAM dendrimers destabilized bovine serum albumin but slightly increased the stability of human serum albumin. 17À19 These studies clearly show that dendrimers have destabilizing or neutral Received: June 6, 2011 Accepted: July 1, 2011 ABSTRACT: Dendrimers are widely used for biological appli- cations. 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