Optimization of Biocompatibility for a Hydrophilic Biological Molecule Encapsulation System

Abstract

Despite considerable advances in recent years, challenges in delivery and storage of biological drugs persist and may delay or prohibit their clinical application. Though nanoparticle-based approaches for small molecule drug encapsulation are mature, encapsulation of proteins remains problematic due to destabilization of the protein. Reverse micelles composed of decylmonoacyl glycerol (10MAG) and lauryldimethylamino-N-oxide (LDAO) in low-viscosity alkanes have been shown to preserve the structure and stability of a wide range of biological macromolecules. Here, we present a first step on developing this system as a future platform for storage and delivery of biological drugs by replacing the non-biocompatible alkane solvent with solvents currently used in small molecule delivery systems. Using a novel screening approach, we performed a comprehensive evaluation of the 10MAG/LDAO system using two preparation methods across seven biocompatible solvents with analysis of toxicity and encapsulation efficiency for each solvent. By using an inexpensive hydrophilic small molecule to test a wide range of conditions, we identify optimal solvent properties for further development. We validate the predictions from this screen with preliminary protein encapsulation tests. The insight provided lays the foundation for further development of this system toward long-term room-temperature storage of biologics or toward water-in-oil-in-water biologic delivery systems.

Keywords: fluorescence spectroscopy; protein encapsulation; reverse micelle; viability.

Figure 1

Example EEMS data are illustrated as contour plots using a heat map coloring scheme as indicated. Here, the numerical value of the contour represents the emitted fluorescence intensity in counts per second (CPS). Spectra are shown for the aqueous (a,c) and organic (b,d) layers of Formulations 2 (a,b) and 60 (c,d) which were prepared in iso-octane and Capmul MCM, respectively. High intensity in the organic layer indicates high encapsulation of PI. High intensity in the aqueous layer corresponds to poor encapsulation efficiency. Details on formulations, listed by numerical index, are provided in Table S2.

Figure 2

The average encapsulation efficiency is shown, by solvent, for all formulations prepared using the solvent displacement method (a) or the PIT method (b). Each bar represents the average of 12 samples, and the error bars represent the uncertainty of the mean. Solvents are listed in order from least lipophilic (Transcutol HP) to most lipophilic (Labrafac PG).

Figure 3

(a) Flow cytometry output is shown for control treatments with PBS (gray, negative control) and CTAB (red, positive control). The CTAB treatment exhibits a large number of cells with strong fluorescence in the red (PE-A channel), indicating permeabilization of these cells. Treatment with PBS yields high cell counts with low fluorescence, indicating minimal permeabilization of the cells to PI. (b) The permeabilization of S. cerevisiae after treatment with solvents containing varying concentrations of PI are shown, as quantitated by flow cytometry. Relative permeabilization was calculated for each treatment using CTAB treatment as representative of 100% permeabilization. Four PI contents were tested for each solvent (presented from lowest to highest logP value). (c) The viability of yeast cells after plating on YPD media is shown for all conditions tested. The controls, spotted in duplicate, are shown in positions A1-A8 as follows: PBS with (A1-2) and without PI (A3-4), CTAB with (A5-6) and without PI (A7-8). Solvent-only treatments were spotted in duplicate for four conditions each, in the following order: without PI, with PI undiluted, with PI diluted 10-fold, with PI diluted 100-fold. Thus, eight culture spots are shown for each solvent in order as follows: Capmul MCM (A9-B4), Capryol 90 (B5-B12), Lauroglycol 90 (C1-C8), Lauroglycol FCC (C9-D4), Transcutol HP (D5-D12), Labrafac PG (E1-E8), iso-octane (E9-F4), Captex 355 (F5-F12). Colonies in row G are additional control treatments of buffer without PI (G1-2) and with PI varying doses: undiluted (G3-4), 10-fold diluted (G5-6), 100-fold diluted (G7-8) also spotted in duplicate. (d) This table summarizes the relative toxicity of solvents of yeast cells from panel C and an average permeabilization value for each solvent from the data in panel B.

Figure 4

(a) HeLa cell viability measurements from the CellTiter Blue assay for PBS and CTAB controls and for treatments of solvent-only without PI, and with PI undiluted, 10-fold dilute, and 100-fold diluted, respectively for each solvent, as labeled. (b) A violin plot is shown for all solvent-only treatments, comparing treatment with PI versus treatment without PI. Thickness of the plot indicates relative percentage of samples with viability as indicated on the x-axis within the sample set that received the treatment indicated on the y-axis. (c) HeLa cell viability is shown for all lethal solvents (solvents with minimal viability in panel a), illustrating slight reduction in toxicity due to dilution of RMs. (d) HeLa cell for all non-lethal solvents (solvents with high viability in panel a) showing that strong viability is seen at the lowest RM concentrations tested.

Figure 5

The Pearson’s correlation is shown for each pairwise set of compositional variables and performance measurements. Circle size scales with strength of the correlation. The correlation coefficient values are color coded, as indicated on the right. Dilution factor cannot be compared with partition coefficient, thus correlation data are not provided for this pair.

Figure 6

EEMS of RFP are shown as contour plots for self-nanoemulsion formulations prepared in (a) Capmul MCM, (b) iso-octane, and (c) Lauroglycol 90. Contours represent emission intensity in counts per second as indicated by the color bar in panel (c). Encapsulation efficiencies were calculated to be 61.0%, 68.5%, and 11.6%, respectively.