Erythrocyte-derived photo-theranostic agents: hybrid nano-vesicles containing indocyanine green for near infrared imaging and therapeutic applications

Abstract

Development of theranostic nano-constructs may enable diagnosis and treatment of diseases at high spatial resolution. Some key requirements for clinical translation of such constructs are that they must be non-toxic, non-immunogenic, biodegradable, with extended circulating lifetime. Cell-based structures, particularly those derived from erythrocytes, are promising candidate carrier systems to satisfy these requirements. One particular type of theranostic materials utilize light-sensitive agents that once photo-activated can provide diagnostic imaging capability, and elicit therapeutic effects. Here we demonstrate the first successful engineering of hybrid nano-scale constructs derived from membranes of hemoglobin-depleted erythrocytes that encapsulate the near infrared chromophore, indocyanine green. We show the utility of the constructs as photo-theranostic agents in fluorescence imaging and photothermal destruction of human cells. These erythrocyte-mimicking nano-structures can be derived autologously, and may have broad applications in personal nanomedicine ranging from imaging and photo-destruction of cancerous tissues to vascular abnormalities, and longitudinal evaluations of therapeutic interventions.

Figure 1. Size distribution profiling and TEM image of NETs.

(a), Diameter distributions of EGs and NETs. Using dynamic light scattering, the ranges of the measured diameters are 58–190 nm for the EGs, and 78–190 nm for the NETs formed after five minutes of incubation in hypotonic solution and PBS containing 215 μM ICG. Each measurement was repeated using at least three samples. We present the mean of each measurement, represented as circles for EGs, and squares for NET. The error bars represent standard deviations from the mean values. We fitted Lognormal functions to the measured diameter distributions (solid curves). The estimated mean diameters, as determined by the Lognormal fits, are 95.26 nm (R² = 0.97), and 124.61 nm (R² = 0.98) for EGs and NETs, respectively. (b), Illustrative TEM image of NETs.

Figure 2. Absorption spectra and a physical model of a NET containing ICG.

(a), Absorption spectra corresponding to free (non-encapsulated) ICG (3.22 μM) dissolved in PBS, and EGs and NETs re-suspended in PBS after fabrication. (b), A physical model of a NET showing an ensemble of ICG conformational states comprised of ICG monomers, ICG aggregates, and monomers and aggregates of ICG bound to membrane lipids and/or membrane proteins. For illustration purposes, we present three main membrane integral proteins of erythrocytes: Aquaporin, Band3 and Glycophorin.

Figure 3. Fluorescence spectra and fluorescent images of human dermal microvascular endothelial (HDME) cells incubated with ICG or NETs.

(a), Normalized fluorescence spectra in response to 650 nm photo-excitation of free ICG (3.22 and 43 μM) dissolved in PBS, and EGs and NETs re-suspended in PBS. Emission spectra were smoothed using IGOR Pro software with second order binominal algorithm. (b), Fluorescent images of HDME cells after three hours of incubation in vascular cell basal medium containing 13 μM ICG (control) dissolved in PBS (left panel), or NETs (right panel) at 37°C and 5% CO₂ in dark. A Mercury/Xenon arc lamp was used for photo-excitation at 740 ± 35 nm. Cells nuclei were stained by DAPI, and falsely colored in blue using the ImageJ software. A filter transmitting λ > 780 nm was used to collect the emitted NIR fluorescent, falsely colored in red. Scale bars on both panels correspond to 10 μm. The scale bar, 0–54354, corresponds to the NIR fluorescent emission intensity for both panels. The inset on the left panel represents the same image shown on the panel at the scale of 0–6565.

Figure 4. Photothermal response of NETs, and NETs-mediated photothermal destruction of HDME cells.

(a), Photothermal response of NETs suspended in PBS, 13 μM free ICG dissolved in PBS (positive control), and PBS solution (negative control) in response to laser irradiation at λ = 808 nm with incident intensity (I_o) of 19.7 W·cm⁻². The volume of all samples was 120 μl. The free ICG solution and NETs suspension samples were prepared to have nearly the same absorbance value of 0.6 at 808 nm. Temperatures were measured using a thermistor placed 2 mm outside the irradiated spot. (b), absorption spectra of four different PBS-suspended NETs samples (each 120 μl) following laser irradiation for various durations (60, 90, 140, and 200 s) at 808 nm and I_o = 19.7 W/cm². (c), Fluorescent images of HDME cells after three hours of incubation with PBS (negative control), 13 μM free ICG (positive control) and NETs, followed by laser irradiation (λ = 808 nm, I_o = of 19.7 W·cm⁻²).The radiant exposure time in all three samples was 200 s. The volume of NETs suspension or free ICG added to the cells was 200 μl with nearly the same absorbance value of 0.6 at 808 nm. Live cells were stained using Calcein, and falsely colored in green. Dead cells were distinguished using Ethidium homodimer-1 (EthD-1), and falsely colored in red (Scale bars = 10 μm). (d), Percentage of HDME cells photothermally destroyed by NETs as assessed by a fluorescence microplate reader. Three different spots (each spot diameter = 2.2 mm) were irradiated in each well, resulting in irradiation of ≈80% of cells. Each bar represents the mean fraction of the dead cells for three different wells. Error bars correspond to single standard deviations. There was a statistically significant difference in fraction of cells photothermally destroyed by NETs (identified by the asterisk) as compared to those incubated in PBS or free ICG (p < 10⁻⁴).

Figure 5. Cytotoxicity assessment of NETs.

(a), Percentage of live cells post incubation with NETs for 3 hours (solid green bar) and 24 hours (dashed green bar). Cells incubated with culture medium for 24 hours without any additional reagent were used as the positive control population. Cells incubated with 100 μl methanol for 24 hours were used as negative control. Each bar represents the mean fraction of the live cells for three different wells. Error bars correspond to single standard deviations. Statistical analysis of the results for NETs (3 hours) compared to positive control yielded no significant difference in cell viability. Cells treated with methanol (negative control) and NETs for 24 hours (identified by asterisks) yielded statistically significant viability results as compared to the positive control population (p < 10⁻³). Fraction of the viable cells treated with NETs for 24 hours was significantly higher than those treated with methanol (p < 10⁻⁴). (b), Fluorescent images of HDME cells 24 hours post incubation with NETs (right panel) and culture medium (positive control) (right panel). Live cells were stained using Calcein, and falsely colored in green. Dead cells were distinguished using Ethidium homodimer-1 (EthD-1), and falsely colored in red (Scale bars = 100 μm).