Engineering of Biocompatible Coacervate-Based Synthetic Cells

Abstract

Polymer-stabilized complex coacervate microdroplets have emerged as a robust platform for synthetic cell research. Their unique core-shell properties enable the sequestration of high concentrations of biologically relevant macromolecules and their subsequent release through the semipermeable membrane. These unique properties render the synthetic cell platform highly suitable for a range of biomedical applications, as long as its biocompatibility upon interaction with biological cells is ensured. The purpose of this study is to investigate how the structure and formulation of these coacervate-based synthetic cells impact the viability of several different cell lines. Through careful examination of the individual synthetic cell components, it became evident that the presence of free polycation and membrane-forming polymer had to be prevented to ensure cell viability. After closely examining the structure-toxicity relationship, a set of conditions could be found whereby no detrimental effects were observed, when the artificial cells were cocultured with RAW264.7 cells. This opens up a range of possibilities to use this modular system for biomedical applications and creates design rules for the next generation of coacervate-based, biomedically relevant particles.

Keywords: biocompatibility; block copolymers; complex coacervates; polycations; protocells; self-assembly; synthetic cells.

Figure 1

Self-assembly of stabilized coacervate-based synthetic cells. (A) Schematic illustrating the coacervation of the oppositely charged polycation (12–16 kDa) quaternary amylose (Q-am) and polyanion (12–16 kDa) carboxymethyl amylose (CM-am) followed by self-assembly of terpolymer poly(ethylene glycol)–poly(caprolactone-gradient-trimethylene carbonate)–poly(glutamic acid) (PEG–PCL-g-TMC–PGlu) on the droplet interface. (B) Confocal microscopy image of synthetic cells formed in cell culture medium. The terpolymer is visualized with Nile Red. Scale bar represents 50 μm.

Figure 2

Toxicity of coacervates and their components. Cell viability of HeLa cells, RAW264.7 macrophages, and human umbilical vein endothelial cells (HUVECs) after 24 h of incubation with (A) coacervates, (B) terpolymer, (C) CM-amylose with a degree of substitution (DS) of 0.4, (D) Q-amylose_DS=1.0, and (E) Q-amylose_DS=0.5. Error bars represent the standard deviation of 2 independent experiments.

Figure 3

Toxicity of terpolymer-stabilized, coacervate-based synthetic cells. (A, B) Cell viability after 24 h of incubation with entire synthetic cell mixtures prepared with either Q-amylose_DS=1.0 or Q-amylose_DS=0.5 for (A) HeLa and (B) RAW264.7 cells. (C) Turbidity measurements of coacervates with different molar charge ratios prepared in cell culture medium. (D) Cell viability after 24 h of incubation with entire synthetic cell mixtures prepared with Q-amylose_DS=1.0 and molar charge ratios ([Q]⁺/[CM]⁻) of 1, 2, and 3 for (D) HeLa cells and (E) RAW264.7 cells. Concentrations on the x-axis are given in μg mL^–1. Error bars represent the standard deviation of 2 independent experiments.

Figure 4

Terpolymer-dependent, coacervate-based synthetic cell toxicity. (A) Confocal microscopy image of coacervates prepared using 1.2 mg mL^–1 terpolymer (final concentration) or (B) 0.5 mg mL^–1 terpolymer (final concentration). The terpolymer is visualized with Nile Red. Scale bar represents 25 μm. (C, D) Cell viability after 24 h of incubation with synthetic cells for (C) HeLa cells or (D) RAW 264.7 cells. [Q]⁺/[CM]⁻ = 3, Q-amylose_DS=1.0. The final terpolymer concentration is 1.2 or 0.5 mg mL^–1. Error bars represent the standard deviation of 2 independent experiments. Significance was assessed using the Student’s t test. Significance level is indicated by **p < 0.05.

Figure 5

Purification of coacervate-based synthetic cells. (A) Schematic illustrating the purification process of synthetic cells. Coacervates are centrifuged, after which the supernatant is removed, and the purified pellet is redispersed in an equal amount of cell culture medium. (B, C) Confocal microscopy image of purified coacervates prepared using 1.2 mg mL^–1 terpolymer (final concentration) (B) pellet and (C) supernatant. The terpolymer is visualized with Nile Red. Scale bar represents 25 μm. (D, E) Fraction of compounds present in the pellet and supernatant for (D) terpolymer and (E) Q-amylose. Fractions were calculated from ¹H NMR spectra, as the ratio of peak integrals normalized to the control. Error bars represent the standard deviation of 4 different resonances for (D) δ, 2.32–2.22 (t, 2H; CH₂); δ, 2.00–1.85 (m, 2H; CH₂); δ, 1.65–1.45 (m, 4H; CH₂); and δ, 1.35–1.25 (m, 2H; CH₂) (Figure S10A); and 2 different resonances for (E) δ, 4.50–4.35 (m, 1H; CH); and δ, 3.30–3.15 (m, 9H; CH₃) (Figure S10B). (F, G) Cell viability after 24 h of incubation with DMEM, coacervates, purified coacervates, or supernatant for (F) HeLa cells and (G) RAW264.7 cells. [Q]⁺/[CM]⁻ = 3, Q-amylose_DS=1.0. The Q-amylose and terpolymer concentrations are 0.5 and 1.2 mg mL^–1, respectively. Error bars represent the standard deviation of 2 independent experiments. Significance was assessed using the Student’s t test. Significance levels are indicated by *p < 0.1, ***p < 0.025, or ****p < 0.01.