Preparation of photothermal palmitic acid/cholesterol liposomes

Abstract

Indocyanine green (ICG) is the only FDA-approved near-infrared dye and it is currently used clinically for diagnostic applications. However, there is significant interest in using ICG for triggered drug delivery applications and heat ablation therapy. Unfortunately, free ICG has a short half-life in vivo and is rapidly cleared from circulation. Liposomes have been frequently used to improve ICG's stability and overall time of effectiveness in vivo, but they have limited stability due to the susceptibility of phospholipids to hydrolysis and oxidation. In this study, nonphospholipid liposomes were used to encapsulate ICG, and the resulting liposomes were characterized for size, encapsulation efficiency, stability, and photothermal response. Using the thin-film hydration method, an ICG encapsulation efficiency of 54% was achieved, and the liposomes were stable for up to 12 weeks, with detectable levels of encapsulated ICG up to week 4. Additionally, ICG-loaded liposomes were capable of rapidly producing a significant photothermal response upon exposure to near-infrared light, and this photothermal response was able to induce changes in the mechanical properties of thermally responsive hydrogels. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1384-1392, 2019.

Keywords: cardiogreen; chromophore; indocyanine green; liposome; nanotechnology; photothermal effect.

Figure 1.

Schematic of thin-film hydration method used to fabricate palmitic acid/cholesterol liposomes loaded with ICG.

Figure 2.

The chemical structure and absorbance spectra of aqueous solutions of free ICG (0.017 μM; dashed lined) and ICG encapsulated in palmitic acid/cholesterol liposomes (solid line).

Figure 3.

Percent encapsulation of ICG within liposomes over four weeks. The encapsulation efficiency decreased from 53.6±3.7% after fabrication to 29.2±0.8% after one week. Data is presented as mean ± standard deviation (n=3).

Figure 4.

A) Change in liposome diameter and PDI over time. The average diameter of ICG-loaded liposomes (■) was greater than blank liposomes (●) over the 12 weeks measured. Temporal changes in liposome diameter within the ICG-liposomes and control blank liposomes was not statistically significantly, and that there was no statistically significant difference between the two groups at any time point as determined by a two-way repeated measures ANOVA with a Greenhouse-Geisser correction (n=5). PDI measurements (dashed lines) were averaged each week, and error bars represent standard deviations. All PDI values were ≤0.30. B) Cryo-TEM image of liposomes in deionized H2O shows mostly spherical unilamellar vesicles although some multilamellar vesicles and rod-like particles were also present. Scale bar = 100 nm. Representative frequency histograms showing size range of C) ICG-loaded liposomes and D) blank liposomes at Day 0.

Figure 5.

A) The presence of ICG significantly increased the measured temperature change over blank liposomes (striped) upon exposure to 750 mW NIR light for two minutes (n=9). Statistical analysis performed using two-tailed t-test with significance at p < 0.001. Asterisks (*) indicate statistical significance between samples. The temperature change of an aqueous solutions of free ICG (13 μM) exposed to 750 mW NIR light for two minutes is shown as a reference (n=3). B) The measured temperature changes of ICG-loaded liposomes after 3× 2 min exposures to 750mW of NIR light show that ICG-loaded liposomes produce > 5°C temperature change and had no statistically significantly difference in photothermal response between exposures as determined by one-way ANOVA (n=9). C) Percent of ICG remaining encapsulated after NIR exposures. Data is presented as a percentage of initial ICG loading (Fig. 2, week 0). There was no statistically significantly difference between exposures in encapsulated ICG as determined by repeated measures ANOVA (n=9).

Figure 6.

Representative storage modulus vs time profiles of gelatin hydrogels with ICG-loaded liposomes (A) and blank liposomes (B). The critical storage modulus of 62 Pa was defined as the sol-gel transition point. Gelatin hydrogels with ICG-loaded liposomes that underwent NIR exposure reached that modulus 4.3 min. after exposure. Hydrogels with blank liposomes did not reach that storage modulus even after 10 min.