Machine learning to determine optimal conditions for controlling the size of elastin-based particles

Abstract

This paper evaluates the aggregation behavior of a potential drug and gene delivery system that combines branched polyethyleneimine (PEI), a positively-charged polyelectrolyte, and elastin-like polypeptide (ELP), a recombinant polymer that exhibits lower critical solution temperature (LCST). The LCST behavior of ELP has been extensively studied, but there are no quantitative ways to control the size of aggregates formed after the phase transition. The aggregate size cannot be maintained when the temperature is lowered below the LCST, unless the system exhibits hysteresis and forms irreversible aggregates. This study shows that conjugation of ELP with PEI preserves the aggregation behavior that occurs above the LCST and achieves precise aggregate radii when the solution conditions of pH (3, 7, 10), polymer concentration (0.1, 0.15, 0.3 mg/mL), and salt concentration (none, 0.2, 1 M) are carefully controlled. K-means cluster analyses showed that salt concentration was the most critical factor controlling the hydrodynamic radius and LCST. Conjugating ELP to PEI allowed crosslinking the aggregates and achieved stable particles that maintained their size below LCST, even after removal of the harsh (high salt or pH) conditions used to create them. Taken together, the ability to control aggregate sizes and use of crosslinking to maintain stability holds excellent potential for use in biological delivery systems.

Figure 1

DLS results demonstrate single and bimodal curves can be obtained by varying the salt concentration. The second transition for the bimodal curve for the ELP-PEI10K (grey) occurs at the same point (black box) as neat ELP (blue) and ELP/ELP-PEI800 (orange), indicating that either the ELP-PEI10K transitions earlier than neat ELP or it has more than one ELP molecule conjugated to the PEI10K. The bimodality of the ELP/ELP-PEI800 (orange) and the ELP/ELP-PEI10K (grey) curves disappears with an increase in NaCl concentration to 1 M (blue box). All DLS curves were obtained at pH = 7.

Figure 2

LCST of aqueous solution samples sorted from highest to lowest with respect to the concentration of (a) ELP, (b) ELP/ELP-PEI800, and (c) ELP/ELP-PEI10K as determined by dynamic light scattering. Statistical significance between samples can be found in Supplementary Figure S1.

Figure 3

Dynamic light scattering results of aqueous solution samples for the R_h, sorted from largest to smallest with respect to polymer concentration of (a) ELP, (b) ELP/ELP-PEI800, and (c) ELP/ELP-PEI10K. Statistical significance between samples can be found in Supplementary Figure S2.

Figure 4

When heated above the LCST, the ELP/ELP-PEI polymers form aggregates that remain present in solution so long as the temperature is maintained (light grey bars). Upon crosslinking, the aggregates remain stable below their LCST at 20 °C (black bars), while the aggregates of the non-crosslinked polymers dissolve in the solution below their LCST at 20 °C (dark grey bars).

Figure 5

(a) A weak relationship was seen between R_h and T_t with an R² of 0.3. (b) K-means cluster analysis was used to elucidate the primary driver of the relationship between R_h and T_t. The three groups indicated by K-means cluster analysis to be important for determining R_h versus T_t are based on the concentration of salt. (c) Separating the R_h versus T_t graph by polymer type shows that increasing the molecular weight of PEI attached to ELP decreased the R_h and T_t simultaneously. Graphs for (d) ELP, (e) ELP/ELP-PEI800, and (f) ELP/ELP-PEI10K demonstrate that increasing polymer concentration decreases T_t, but has a small influence on R_h. (g) ELP exhibits minimal effects from a change in pH. Coupling ELP to PEI imposes a pH sensitivity that allows for a control over R_h for (h) ELP/ELP-PEI800 and (i) ELP/ELP-PEI10K.

Figure 6

ELP/ELP-PEI800 and ELP/ELP-PEI10K systematically form nano-and micro-aggregates above LCST when the solution conditions of salt concentration, polymer concentration, and pH are carefully controlled. The PEI block provides the ability to crosslink the copolymers and to achieve particles that remain stable below LCST even after removal of the harsh (high salt or pH) conditions used to create them.