Interaction of Serum and Plasma Proteins with Polyelectrolyte Microparticles with Core/Shell and Shell-Only Structures

Abstract

Polyelectrolyte microparticles (MPs) synthesized on calcium carbonate cores are considered a promising basis for new drug delivery systems. It is known that microparticles entering a physiological environment absorb proteins on their surface, which can change the properties of the microparticles and alter their functional activity. This study aimed to compare the compositions of the adsorbed protein layer formed on microparticles with the core/shell and shell structures obtained by layer-by-layer deposition. The difference in the microparticle structure was associated with changes in their surface topography and ζ-potential. These microparticles were incubated with human serum or plasma at 37°C for 24 h. The adsorbed proteins were eluted and analyzed by means of SDS-PAGE. The protein composition of the eluates was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS); a total of 357 proteins were identified, and 183 of them were detected in all samples. Our results demonstrate that the relative abundance of proteins of different functional groups (immunoglobulins, complement proteins, and apolipoproteins) varied depending on the structure and surface characteristics of the polyelectrolyte microparticles and the incubation medium. Our findings expand the understanding of the influence of the physicochemical properties of the microparticles on their interaction with proteins, which can help to improve the design of microparticles for drug delivery.

Figure 1

Physicochemical and structural characteristics of the microparticles (MPs) with different structures. (A) Scanning electron microscopy images of core/shell polyelectrolyte MPs obtained by layer-by-layer deposition and shell MPs obtained after core removal. (B) Atomic force microscopy images of core/shell and shell MPs in PBS; the size of the zoomed regions is 1.0 × 1.0 μm². (C) Surface charges (ζ-potentials) of core/shell polyelectrolyte MPs obtained by layer-by-layer deposition and shell MPs obtained after core removal, comparison using Studentʼs t test, N = 8 (* p < 0.05). (D) Size distribution of core/shell and shell MPs obtained by means of optical microscopy. (E) Schema of the preparation of core/shell and shell MPs based on CaCO₃ cores. PVA, poly(vinyl alcohol); MC, methylcellulose; PAH, poly(allylamine hydrochloride); PSS, poly(sodium 4-styrenesulfonate); PAA, poly(acrylic acid).

Figure 2

(A) Schematics of the preparation of samples eluted from the surface of core/shell and shell MPs after their incubation with human serum or plasma. Created with BioRender.com. (B) Results of the SDS-PAGE of the samples eluted from the surface of core/shell and shell MPs after incubation at 37 °C for 24 h with human serum (S) or plasma (P). Lane m contains protein molecular weight markers (kDa). (C) Amount of protein per particle adsorbed on the core/shell and shell MPs after their incubation with serum or plasma as estimated by means of densitometry; comparison of samples using one-way ANOVA with Tukey’s test for multiple comparison, N = 3 (* p < 0.05).

Figure 3

(A) Heatmap of label-free quantification intensities of major proteins (>1% of the total protein content) adsorbed on core/shell and shell microparticles after incubation with serum or plasma. Created using the Heatmapper (http://www.heatmapper.ca/). (B) Relative amounts of proteins divided into groups according to their biological functions in samples obtained from core/shell and shell microparticles after their incubation with serum or plasma. The Apolipoproteins group includes 15 proteins; Coagulation, 33 proteins; Complement system, 27 proteins; Immunoglobulins, 34 proteins; Other, 244 proteins. Albumin forms a separate group because of its large amounts in all samples. The data are presented as the mean and standard deviation calculated from two replicates.

Figure 4

Relative label-free quantification intensities of proteins grouped according to their (A) molecular weights (M_w), (B) isoelectric points (pI), and (C) grand average of hydropathicity (GRAVY) scores adsorbed on core/shell and shell MPs upon their incubation with serum or plasma. The M_w, pI, and GRAVY were calculated on the basis of the primary sequences of the proteins using ProtParam software.