Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems

Abstract

The counterions neutralizing the charges on polyelectrolytes such as DNA or heparin may dissociate in water and greatly influence the interaction of such polyelectrolytes with biomolecules, particularly proteins. In this Review we give an overview of studies on the interaction of proteins with polyelectrolytes and how this knowledge can be used for medical applications. Counterion release was identified as the main driving force for the binding of proteins to polyelectrolytes: Patches of positive charge become multivalent counterions of the polyelectrolyte and lead to the release of counterions from the polyelectrolyte and a concomitant increase in entropy. This is shown from investigations on the interaction of proteins with natural and synthetic polyelectrolytes. Special emphasis is paid to sulfated dendritic polyglycerols (dPGS). The Review demonstrates that we are moving to a better understanding of charge-charge interactions in systems of biological relevance. Research along these lines will aid and promote the design of synthetic polyelectrolytes for medical applications.

Keywords: complementary binding; counterion release; heparin; inflammation; polyelectrolytes.

Figure 1

Interaction of proteins with highly charged polyelectrolytes, for example, DNA, by counterion release: Proteins bear negative (red) and positive charges (blue) on their surface. Above the isoelectric point, the overall surface charge is negative, but the positive patches remain. The polyelectrolyte bears a large number of charges that will lead to counterion condensation, that is, a certain fraction of the counterions are highly correlated with the polyelectrolyte, as shown here. Upon binding of the protein to the polyelectrolyte, a positive patch on the surface of the protein becomes a trivalent counterion of the polyelectrolyte. Thus, three counterions condensed on the polyelectrolyte are released upon binding. The free energy of binding will, therefore, be dominated by the entropic gain through the release of the counterions.[ ¹² , ⁴⁴ , ⁴⁵ ] For the sake of clarity, only the condensed counterions are shown here. However, all the charges on the protein and the polyelectrolyte are balanced by an equal number of counterions.

Figure 2

Interaction of polyelectrolytes with biosystems at different levels of complexity: Linear polyelectrolytes may be assembled into networks[ ⁵⁵ , ⁵⁶ ] and branched systems. Ultimately, they may become building blocks for systems with higher complexity, for example, micelles with core–shell structures. Complexity on the biological side starts with single protein molecules that can interact with polyelectrolyte systems with various architectures. On this level, the therapeutic activity of polyelectrolytes can often be traced back to a blocking of proteins by a suitable polyelectrolyte system.[ ³¹ , ³⁶ , ³⁹ , ⁴⁰ , ⁴² , ⁴⁵ ] Cells present the next level of complexity and their interaction with charged polymeric systems must be understood when considering these systems for, for example, drug delivery or gene transfection.[ ²⁰ , ²² , ⁵⁷ , ⁵⁸ , ⁵⁹ ] Organs present the highest level of complexity and the understanding of their interaction with synthetic polyelectrolyte systems is in its infancy. However, cationic polyelectrolytes with suitable architectures have recently been introduced as agents with anticoagulant reversal activity in blood.[ ⁴¹ , ⁴² , ⁴³ ] The entire matrix of systems and problems gives a good overview of the possible medical problems to which synthetic polyelectrolytes may provide solutions.

Figure 3

Chemical structure of the monomer unit of heparin.

Figure 4

Interaction of linear synthetic polyelectrolytes with proteins. a) The binding constant of poly(acrylic acid) to human serum albumin (HSA) is plotted against the log of the salt concentration. At high salt concentrations, there is a linear relationship, the slope of which gives the number of released counterions according to Equation (3). Deviations at low salt concentrations point to a residual Debye–Hückel repulsion between the protein and the polyelectrolyte. b) MD simulation of the interaction of the interaction of HSA with poly(acrylic acid). The polyelectrolyte is bound to the Sudlow II site of HSA. ^[50]

Figure 5

Sulfated dendritic polyglycerol (dPGS) and its interaction with proteins. a) Chemical structure of dPGS. The scaffold consists of a highly hydrophilic dendritic or hyperbranched structure, with each end group carrying a sulfate group. ^[185] b) Snapshot of the coarse‐grained structure of a second‐generation dPGS. Red beads mark the terminal sulfate groups of the dendritic structure, and yellow beads mark its scaffold. The counterions are displayed as green beads. ^[51] c) Interaction of a second‐generation dPGS with lysozyme measured by ITC at different ionic strengths. The incremental heat per injection is plotted against the molar ratio of lysozyme to dPGS in aqueous solution. The inset displays the log of the resulting binding constant as a function of the log of the salt concentration according to Equation (3). ^[45] d) Coarse‐grained MD simulations of the interaction of a third‐generation dPGS with lysozyme. The snapshot shows a complex of the central dPGS molecule with four lysozymes. ^[45]

Figure 6

Thermodynamic analysis of the binding of lysozyme to a second‐generation dPGS. ^[54] Top: A plot of log K_b versus log c _s as suggested by Equations (4) and (6). There is a perfectly linear relation in this double‐logarithmic plot, in which the slope gives the number of released counterions, as discussed in conjunction with Equation (3a). The linear relationship is used to extrapolate the binding constant ΔG_b at a salt concentration of 1 m. K_b(1 m) is related to ΔG_res, the residual of the Gibbs free energy of binding according to Equation (5,) and reflects all contributions to ΔG _b not related to counterion release. Bottom: Enthalpy–entropy compensation for the data obtained on the system dPGS‐G2/lysozyme. The enthalpy ΔH_b is plotted against TΔS_res=TΔS_b−TΔS_ci according to Equation (7). The solid line denotes the fit by Equation (8). The dashed line shows the master curve derived by Dragan et al. ^[11] for a wide variety of systems in which DNA interacts with proteins.

Figure 7

The anti‐inflammatory effect of dPGS. As shown by Dernedde et al., ^[36] dPGS inhibits an overwhelming inflammatory response and reduces the extravasation of leukocytes. dPGS targets the adhesion molecules L‐ and P‐selectin, while no binding to E‐selectin is observed. The same finding was made in our recent study by MD simulations. ^[45] Thus, dPGS acts by preventing leukocyte extravasation through the binding of the selectins. Moreover, binding to complement factors C3 and C5 inhibits the formation of the proinflammatory anaphylatoxins. Here, the reduction of the C5a level decreases further leukocyte activation and recruitment. As a result, the adhesion cascade is balanced and contributes to initiate the healing process. ^[196]

Figure 8

Complement pathway: dPGS interferes with the three pathways of complement activation and reduces formation of the membrane attack complex (MAC), which is a pore that is inserted into the cytoplasmic membrane and thereby leads to cell death. Reduced activity of the C3 and C5 convertase results in IC₅₀ values of 60 nm (lectin pathway), 300 nm (classical pathway), and 900 nm (alternative pathway). ^[200]

Figure 9

Modulatory effects of dPGS in neuroinflammation caused by Aß oligomers. a) Exposure of microglia to Aβ oligomers causes the activation of microglia and loss of dendritic spines in the hippocampal excitatory neurons. Hyperactive microglia activate astrocytes and these glial cells (reactive astrocytes) produce excessive amounts of lipocalin 2 (LCN2). LCN2 in combination with cytokines released from hyperactive microglia contribute to the impairment of synaptic functions. b) dPGS attenuates microglia hyperactivity, binds to Aβ₄₂ and normalizes the number and function of dendritic spines. ^[40]

Figure 10

Uptake of proteins by a spherical polyelectrolyte brush (SPB). ^[208] Top: The polyelectrolyte brushes consist of a solid polystyrene core (gray sphere) with a radius R _h,core between 50 and 100 nm. Onto its surface are grafted long chains of polyelectrolytes, for example, poly(acrylic acid). Red spheres on the PAA chains represent the negative charge of the acidic residues, while blue spheres represent the positive counterions. Nearly all of the counterions of the brushes are confined within the brush layer (osmotic brush). The protein molecules are represented by green spheres. Their uptake will lead to the release of a concomitant number of counterions. Adsorption of proteins by polyelectrolyte brushes is hence mainly entropy‐driven.[ ¹⁰⁹ , ²⁰⁸ ] Bottom: a) The Gibbs free energy of binding ΔG_b of HSA to a spherical polyelectrolyte brush carrying long chains of poly(acrylic acid) (black squares) compared to the results for HSA binding to dPGS and of HSA interacting with linear chains of poly(acrylic acid). In all cases, the ITC‐determined ΔG_b exhibits only a weak dependence on temperature, which is followed by a strong enthalpy–entropy compensation (EEC) shown in (b) for HSA interacting with a SPB: Both ΔH_b as well as TΔS_b vary strongly with temperature, whereas ΔG_c stays nearly constant because of the EEC. ^[208]

Figure 11

Modeling the competitive adsorption of proteins onto charged networks as exemplified by charged core–shell microgels.[ ⁷³ , ⁷⁴ ] The shell consists of a charged network built up of hydrophilic chains. The network contains negatively charged monomer units, which lead to a charge density c_g. The concentration of the counterions and the co‐ions within the network are regulated by the Donnan potential. The proteins are modeled by charged spheres with charge numbers z ₁ and z ₂, respectively, whereas the overall radii are given by R ₁ and R ₂, respectively. The uptake of proteins is governed by the interaction of the charged proteins with the Donnan potential of the network. ^[73] The model can consider the competitive adsorption of several proteins onto the network. ^[74] Here, two different proteins with effective charges z ₁ and z ₂ undergo competitive adsorption to the charged core–shell particle. The model leads to a fully quantitative understanding of the experimental results with four different proteins. ^[74]

Figure 12

Micelles formed by the block copolymer from dPGS and poly(ϵ‐caprolactone) for active targeting of inflammation‐related tumor tissues. ^[38] The micelles are assembled from a block copolymer consisting of a highly charged dPGS block and a hydrophobic poly(caprolactam) block. Both parts are interlinked by a sulfur bridge, which will be cleaved in the reductive environment of the cell. The drug doxorubicin (DOX) can be encapsulated in the hydrophobic core and, thus, brought to the infected tissue by intravenous injection. After uptake in the cells, the S‐S bridges are cleaved and the drug is released.

Figure 13

Design of UHRA and its interaction with heparin. a) Structure of UHRA and heparin binding groups (R). The chemical structure consists of an HPG‐based core and mPEG₃₅₀ brush layer with hexamethylated tris(2‐aminoethylamine) as HBGs arranged in a multivalent fashion. b) The mechanism of the antidote action of UHRA. The antithrombin (AT) bound heparin complex responsible for the anticoagulant activity of polyanionic heparin is dissociated by interaction with the UHRA because of its high binding affinity. This process restores the generation of thrombin.[ ⁴¹ , ²³⁹ ]