Selective filtering of particles by the extracellular matrix: an electrostatic bandpass

Abstract

The transport of microscopic particles such as growth factors, proteins, or drugs through the extracellular matrix (ECM) is based on diffusion, a ubiquitous mechanism in nature. The ECM shapes the local distribution of the transported macromolecules and at the same time constitutes an important barrier toward infectious agents. To fulfill these competing tasks, the hydrogels have to employ highly selective filtering mechanisms. Yet, the underlying microscopic principles are still an enigma in cell biology and drug delivery. Here, we show that the extracellular matrix presents an effective electrostatic bandpass, suppressing the diffusive motion of both positively and negatively charged objects. This mechanism allows uncharged particles to easily diffuse through the matrix, while charged particles are effectively trapped. However, by tuning the strength of this physical interaction of the particles with the biopolymer matrix, the microscopic mobility of formerly trapped particles can be rescued on demand. Moreover, we identify heparan sulfate chains to be one important key factor for the barrier function of the extracellular matrix. We propose that localized charge patches in the ECM are responsible for its highly unspecific but strongly selective filtering effect. Such localized interactions could also account for the observed tunability and selectivity of many other important permeability barriers that are established by biopolymer-based hydrogels, e.g., the mucus layer of endothelial cells or the hydrogel in the nuclear core complex.

Figure 1

Architecture of the extracellular matrix. (A) The extracellular matrix in the basal lamina is composed of three biopolymers: laminin, collagen IV, and heparan sulfate molecules, which are organized in the perlecan complex. These biopolymers are interconnected to each other by nidogen. (B) Trajectory of a PEG-passivated polystyrene bead of 1.1 μm size in an ECM hydrogel for a time range of 60 s. The temporal evolution of the particle position is encoded in the color scheme. The particle travels several meshes of the hydrogel, which appear to have a typical size of ≈2–3 μm.

Figure 2

Particle mobility and particle surface charge are correlated. (A) Trajectories of polystyrene particles (1 μm and 1.1 μm in size) with different surface modifications. The diffusive motion of amine- and carboxyl-terminated particles is strongly suppressed in ECM hydrogels; however, PEG-coated carboxyl-particles can diffuse mostly unhindered. (B) Trajectories of liposomes (∼160–170 nm in size) with different lipid compositions. DOPG/Rh-DPPE and DOTAP/Rh-DPPE liposomes are effectively trapped while DOPC/Rh-DPPE liposomes can diffuse freely. The time range for the data shown in panels A and B is 12 s each. (C and D) Zeta potential of the particles shown in panels A and B as determined in (a) 2 mM Tris buffer containing 100 mM NaCl (pH 7.5) and additional (b) 1 M KCl or (c) 0.5 mM heparin, respectively.

Figure 3

The extracellular matrix constitutes an electrostatic bandpass. (A) Depending on the particular lipid mixture, the microscopic mobility of the liposome particles drastically varies: a bandpass behavior with respect to the surface charge is observed. A mobile and an immobile regime emerge. The dashed red lines represent the two distinct levels of mobility as described in the main text. Liposomes at various sample positions are analyzed; the error bars show the standard deviation characterizing the distribution of the obtained diffusion constant values. The white bar at 60% DOPG represents an immobile subpopulation as described in the main text. (B) The surface potential of liposomes can be gradually varied by changing the lipid content. The error bars represent the standard deviation as determined from triple measurements. (C) Schematic representation of different liposome populations with varying lipid composition and surface charge.

Figure 4

Local heterogeneity and rescue of the microscopic mobility. (Upper panel) Particles at various sample positions are analyzed to characterize the distribution of the local mobility properties in ECM with and without a rescue agent. (A) In standard MEM-α buffer, the distribution width of the diffusion constant is quite narrow for both mobile and immobile polystyrene particles. (B) The mobility of the trapped particles can be rescued by adding 1 M KCl resulting in a very broad distribution of local diffusion coefficients. PEG-passivated particles are unaffected by the addition of 1 M KCl. (C) 0.5 mM heparin can rescue the mobility of the positively charged amine-particles, but not the mobility of the negatively charged carboxyl-particles. (Lower panel) Schematic representation of mobile and immobile particles at different conditions. At high salt concentrations, ions weaken the electrostatic interaction of the charged particles (yellow, blue) with the hydrogel biopolymers by Debye screening. Heparin (blue star) neutralizes the surface charge of amine-beads (yellow) and thus rescues their mobility.

Figure 5

Quantification of the microscopic mobility of the tracer particles in different environments. For each experimental condition, various positions in the hydrogel are analyzed. The bars represent the mean diffusion coefficients, and the error bars denote the standard deviations. In ECM, the diffusion coefficient is reduced > 1000fold to virtually zero for both positively charged amine-terminated and negatively charged carboxyl-terminated particles. PEG-passivated particles, however, exhibit an apparent diffusion constant that is only fourfold smaller than the respective value in pure water. Such a drastic suppression of the particle mobility is observable neither in pure laminin or pure collagen IV hydrogels nor in mixed laminin/collagen IV hydrogels. The bandpass filtering is lost if heparan sulfate is enzymatically digested from the perlecan complex by heparinase I. Conversely, the bandpass filtering is not observable if free heparan sulfate chains are added to the mixed laminin/collagen IV hydrogel.

Figure 6

Biophysical model for the filtering function of the extracellular matrix: The ECM can be represented as a hydrogel with local patches of either positive or negative charge. In this patchworklike hydrogel, charged particles (yellow, blue) are trapped in the respective region of opposite charge (blue, yellow), while neutral particles (gray) can diffuse nearly unhindered.