An Investigation into the Acidity-Induced Insulin Agglomeration: Implications for Drug Delivery and Translation

Abstract

Insulin undergoes agglomeration with (subtle) changes in its biochemical environment, including acidity, application of heat, ionic imbalance, and exposure to hydrophobic surfaces. The therapeutic impact of such unwarranted insulin agglomeration is unclear and needs further evaluation. A systematic investigation was conducted on recombinant human insulin-with or without labeling with fluorescein isothiocyanate-while preparing insulin suspensions (0.125, 0.25, and 0.5 mg/mL) at pH 3. The suspensions were incubated (37 °C) and analyzed at different time points (t = 2, 4, 24, 48, and 72 h). Transmission electron microscopy and nanoparticle tracking analysis identified colloidally stable (zeta potential 15 ± 5 mV) spherical agglomerates of unlabeled insulin (100-500 nm). Circular dichroism established the preservation of insulin's secondary structure rich in α-helices despite exposure to an acidic environment (pH 3) for 72 h. Furthermore, fluorescence lifetime imaging microscopy illustrated an acidic core inside these spherical agglomerates, while the acidity gradually lessened toward the periphery. Some of these smaller agglomerates fused to form larger chunks with discrete zones of acidity. The data indicated a primary nucleation-driven mechanism of acid-induced insulin agglomeration under physiologically relevant conditions.

Figure 1

TEM image (16,500×) of unlabeled human insulin suspension (0.125 mg/mL) after 2 h of incubation at 37 °C showing spherical agglomerates (400–500 nm). A 1 μm scale bar is shown.

Figure 2

TEM image (105,000×) of human insulin suspension (0.125 mg/mL) after 24 h incubation at 37 °C showing a detailed morphology of an individual spherical agglomerate. The core of the agglomerate seemed dense, while the density kept decreasing toward the periphery. A 100 nm scale bar is shown.

Figure 3

ZP measurements on insulin suspensions (0.125, 0.25, and 0.5 mg/mL) at t = 2, 4, 24, 48, and 72 h. Results are shown as mean ± standard deviation (n = 2).

Figure 4

Pooled CD data are shown based on various time points (t = 2, 4, 24, 48, and 72 h). The absorbance overall varied according to the dilution factor with characteristic negative stretches around 208 and 222 nm wavelengths due to α-helices. The overall pattern of CD spectra showed resemblance across the different concentrations and time points.

Figure 5

FLIM imaging was conducted at room temperature (25 °C) of a FITC-labeled human insulin suspension (0.125 mg/mL) after 24 h incubation at 37 °C (pixel size 57 nm). The color scale of the phase lifetime (τ_p) of FITC is shown on the left. Some formations showed a merger of smaller agglomerates. Three larger agglomerates with fused foci are marked within red circles as regions R1, R2, and R3. These regions R1–R3 were then zoomed and shown in a column on the right. The cores of these regions were acidic (blue), while the acidity decreased along a gradient toward the periphery, with transitioning zones marked in green, yellow, and red, indicative of the periphery being the least acidic. A scale bar of 20 μm is embedded within the figure.

Figure 6

FLIM image (37 °C) of a FITC-labeled human insulin suspension (0.125 mg/mL) after 24 h incubation at 37 °C is shown (pixel size 152 nm). The color scale of the phase lifetime (τ_p) of FITC is shown on the left. Multiple larger agglomerates with fused smaller foci were noticed. There were distinctive areas of acidity ranging from highly acidic (blue) regions transitioning gradually toward more basic domains (green, yellow, and red) surrounding these isolated pockets of acidity. A scale bar of 20 μm is embedded within the figure.

Figure 7

FLIM imaging (37 °C) of an individual insulin agglomerate formed within a FITC-labeled human insulin suspension (0.125 mg/mL) after 24 h incubation at 37 °C (pixel size 38 nm). The color scale of the phase lifetime (τ_p) of FITC is shown on the left. The core of the agglomerate was highly acidic (blue) that gradually transitioned toward more basic domains (green, yellow, and red) at the fringe zones. A scale bar of 100 nm is embedded within the figure.

Figure 8

(A) Fluorescence lifetime decay of a FITC-labeled insulin agglomerate imaged by FLIM elicited a three-component fit. (B) Phasor plot showing the distribution of photons detected from the sample. Two distinct clouds of photons were noticed from the background and sample. Both areas lay within the universal circle, indicating a multiexponential decay. The color scale of phasor fluorescence lifetime was created on the photons received from the agglomerates.