Polyethylene glycol-derived polyelectrolyte-protein nanoclusters for protein drug delivery

Abstract

Polyelectrolyte-protein nanocomplexes prepared under mild and simple conditions which could have biological activity arising from protein have emerged as fascinating protein delivery systems. However, common polyelectrolytes have problems of biocompatibility and metabolism in vivo, which may limit their further applications. Herein, a novel polyethylene glycol polyelectrolyte was synthesized and used for carrying protein drugs. Different from previously reported polyelectrolyte-protein nanoclusters, the polyethylene glycol polyelectrolyte-protein nanoclusters avoid organic solvent and protein modification, and the structure and bioactivity of proteins are well preserved. Moreover, the polyethylene glycol polyelectrolyte-protein nanoclusters have good hemocompatibility and biocompatibility. These novel polyethylene glycol polyelectrolyte-protein nanoclusters would provide a potent tool for fabrication of versatile protein drug carriers.

Scheme 1. Synthesis of PEG-PAGE(R) polyelectrolyte and preparation of PEG polyelectrolyte–protein nanoclusters.

Fig. 1. ¹H NMR spectra (400 MHz, CDCl₃) of (A) PEG-PAGE, (B) PEG-PAGE(COOH), (C) PEG-PAGE(NH₂), and (D) PEG-PAGE(GTAC).

Fig. 2. The potentiometric titration curves (pH–V curves) of PEG_5k-PAGE₁₈(R) copolymers containing different dissociated groups.

Fig. 3. DLS results of protein: (A) bovine serum albumin, (B) insulin, (C) hemoglobin, and (D) lysozyme mixed with polymer in different proportions.

Fig. 4. TEM images of (A) bovine serum albumin nanoclusters, (B) insulin nanoclusters, (C) hemoglobin nanoclusters and (D) lysozyme nanoclusters. Scale bar = 200 nm.

Fig. 5. (A) The particle size and (B) PDI changes of PEG polyelectrolyte–Hb nanoclusters in PBS (10% FBS, pH 7.4, 0.01 M) at 37 °C over 72 h. (C) In vitro protein release of PEG polyelectrolyte–Hb nanoclusters in PBS (pH 7.4, 0.01 M) at 37 °C over 72 h. (D) CD spectra of native Hb solution and PEG polyelectrolyte–Hb nanoclusters (pH 7.4).

Fig. 6. (A) UV-visible spectra of PEG polyelectrolyte–Hb nanoclusters in different gas-binding states. (B) Oxygen dissociation curves and (C) Hill plots of native Hb (P₅₀: 26.6 mmHg; Hill coefficient: 2.129) and PEG polyelectrolyte–Hb nanoclusters (P₅₀: 25.9 mmHg; Hill coefficient: 2.287).

Fig. 7. (A) Cytotoxicity of PEG polyelectrolyte–hemoglobin nanoclusters against L929 cells. Blood cell counting of blood mixed with saline or PEG polyelectrolyte–hemoglobin nanoclusters: (B) white blood cells (WBC) and (C) red blood cells (RBC). Alterations of (D) C3 and (E) PLT levels of mice after i.v. injection of PEG polyelectrolyte–hemoglobin nanoclusters and saline. (F) Blood clearance curve of PEG polyelectrolyte–hemoglobin nanoclusters in mice after intravenous injection. Statistical P-values: no significance, n.s.; *P < 0.05; **P < 0.01.