Investigating and Optimizing Insulin Partitioning with Conjugated Au Nanoparticles in Aqueous Two-Phase Systems Using Response Surface Methodology

Abstract

This study investigated the impact of bioconjugation on the partitioning of insulin, a clinically valuable protein, in an aqueous two-phase system. Gold nanoparticles of different sizes were synthesized and conjugated with insulin. Analysis of the conjugated insulin showed that the insulin remains fully active. Conjugated gold nanoparticles (AuNPs/insulin) were used in polyethylene glycol (PEG)-dextran aqueous two-phase systems to investigate the effect of pH, PEG and dextran molecular weights, PEG and dextran concentrations, AuNPs/insulin dosage, and nanoparticle size on the partition coefficient. These systems were chosen for their biocompatibility and low toxicity. Response surface methodology with D-optimal design was used to model and optimize these systems and their affected parameters. At the optimum condition of a pH = 8 system containing 21% PEG 4000, 5% dextran 100,000, and 100 IU AuNPs/insulin, the partition coefficient of AuNPs/insulin was found to be 192.96, which is in agreement with the empirical partition coefficient of 189.2. This is significantly higher than the partition coefficient of free insulin in a similar system. This approach could be used to overcome limitations in the feasibility of aqueous two-phase systems for industrial-scale purification of biomolecules and biopharmaceuticals.

Figure 1

(A) AuNP solution; (B) AuNPs/insulin conjugate solution.

Figure 2

Comparison of actual and predicted partitioning coefficient (K) of AuNPs/insulin.

Figure 3

AuNPs/insulin partition coefficient constant response contours at 21% PEG 4000 and 5% dextran as a function of nanoparticle size and dosage of AuNPs/insulin load for different pH and dextran molecular weights.

Figure 4

3D response surface graph for partitioning coefficient of AuNPs/insulin as a function of pH and dextran molecular weight [the other variables were kept constant at 21% PEG 4000, 5% dextran, AuNPs/insulin 100 (IU/mL), and 30 nm AuNPs].

Figure 5

3D response surface graph for partitioning factors as a function of pH and concentration of dextran [the other variables were kept constant at 21% PEG 4000, dextran 100,000, AuNPs/insulin 100 (IU/mL), and 30 nm AuNPs].

Figure 6

3D response surface graph for partitioning factors as a function of nanoparticle size and dextran molecular weight [the other variables were kept constant at pH 8, 21% PEG 4000, 5% dextran, and AuNPs/insulin 100 (IU/mL)].