Effects of ICG concentration on the optical properties of erythrocyte-derived nano-vectors

Erythrocyte-based nanoparticle platforms can offer long circulation times not offered by traditional drug delivery methods. We have developed a novel erythrocyte-based nanoparticle doped with indocyanine green (ICG), the only FDA-approved near-infrared chromophore. Here, we report on the absorption and fluorescence emission characteristics of these nanoparticles fabricated using ICG concentrations in the range of 161-323 μM. These nanoparticles may serve as biocompatible optical materials for various clinical imaging and phototherapeutic applications.


INTRODUCTION
Conversion of light into other forms of energy has formed the basis for many biomedical applications such as imaging, photothermal therapy, and photodynamic therapy (PDT). The near-infrared (NIR) wavelengths are well-suited for such applications because biological materials absorb minimally, and there is reduced scattering in this spectral region (650-1450 nm) [1]. Thus, deep tissue penetration can be achieved using these wavelengths (λ).
Indocyanine green (ICG), an FDA-approved NIR chromophore has had a long history of usage in clinical applications. It has been used since the 1950s for characterizing valvular and septal defects [2], measuring cardiac output [3], assessing liver function [4], and ophthalmic angiography [5][6][7]. ICG has also been approved for measurement of cerebral blood flow in the European Union. [8] Other applications of ICG under investigation include sentinel lymph node mapping of breast [9], cervical [10,11], prostate [12], and skin [13,14] cancers. It is also reported for investigational studies in PDT of choroidal disease [15], and colon cancers [16].
Despite the widespread utility of ICG in clinical medicine, its drawbacks are its short half-life in the vasculature (≈2-4 minutes), and exclusive uptake by the liver prior to excretion in the bile [17]. Due to its amphipathic nature, ICG can nonspecifically bind to various biomolecules in the vasculature, such as albumin, and high-and low-density lipoproteins. Thus, the potential of free-ICG as a theranostic agent remains limited for medical procedures that require extended vascular circulation time. Encapsulation of ICG within polymer hydrogels [18], viral capsids [19], and erythrocytes [17,20], have been investigated in order to extend the vascular circulation time of ICG. Our group has reported a polymerbased nanoparticle that enhanced the fluorescence image contrast of circulation for up to 90 minutes in mice [18].
In this study, we investigate the effect of ICG concentration, in the range of 161-323 µM, on the absorption and fluorescence characteristics of erythrocyte-derived ICG carriers. We refer to these nanoparticles as near-infrared erythrocyte-mimicking transducers (NETs). Currently, there are no FDA-approved nanocarriers for use in biomedical applications [17]. The approved status of free-ICG for imaging applications, and previous clinical studies using erythrocyte-based drug-delivery, can potentially make NETs a promising technology for clinical translation.

Fabrication of NETs
Whole CD-1 mouse blood was drawn by cardiac puncture and chilled to 4°C. Cold whole blood was centrifuged at 800 relative centrifugal force (rcf) for five minutes at 4°C to separate the plasma from the packed RBCs (the packed RBCs occupied approximately half of the total whole blood volume). Then, the plasma was decanted and the packed RBCs were mixed and separated into 250 µL aliquots. We added 1 mL of cold (4°C) 1X PBS to resuspend the RBCs. The RBCs were then washed by centrifugation at 800 rcf for five minutes at 4°C in cold 1X PBS.
Next, we added 1 mL of 0.25X PBS to the washed RBCs to induce hypotonic swelling. This procedure allowed for depletion of hemoglobin from the RBCs. We then separated the hemoglobin from the suspension of erythrocyte ghosts (EGs) by centrifugation at 3000 rcf for five minutes and immediately suspended the EGs in 1 mL of 1X PBS. The volume of EGs was noticeably larger when suspended in 0.25X PBS compared to the volume of EGs suspended in 1X PBS, indicating that swelling did take place.
The EGs were alternatingly incubated in 0.25X PBS and 1X PBS for five minutes in each tonicity. We repeated this three times to remove most of the hemoglobin from the RBCs. This yielded a pink pellet of EGs. The EGs were then serially extruded through track-etched polycarbonate membranes with 0.4 µm pore size using an Avanti Mini-Extruder. Each sample was passed 20 times through the membrane.
ICG was loaded into the nano-sized EGs by incubating them in Sørenson's phosphate buffer (~300 mOsm) with various concentrations of ICG (161-323 µM) for five minutes. The nano-sized NETs were then separated from any remaining free-ICG by centrifugation for fifteen minutes at 10000 rcf. The resulting NETs were then washed at 10000 rcf for 15 minutes at 4°C in 1 mL of 1× PBS.

Characterization of NETs.
The hydrodynamic diameter of NETs was determined by dynamic light scattering (DLS) in polystyrene cuvettes with path length of 1 cm using a Zetasizer NanoZS90 (Malvern Instruments Ltd.). Prior to extrusion, the EGs were measured to be on the order of one micron in diameter. After 20 passes through a porous polycarbonate membrane with 400 nm pore diameter, the extruded EGs had a peak diameter of ~230 nm (Figure 1). Absorbance spectra of NETs ( Figure 2) were obtained using a Jasco-V670 UV-Vis spectrophotometer in the range of 230-1100 nm using a quartz absorbance microcuvette (Starna Cells, Inc.) with 1 cm path length. We chose this range in order to observe the absorbance of protein in our sample (around 275 nm) and the NIR absorbance of ICG (between 600-1000 nm).
Fluorescence spectra of NETs were obtained using a HORIBA Jobin Yvon Fluorolog-3 spectrofluorimeter in the range of 670-900 nm using a quartz fluorescence microcuvette (Starna Cells, Inc.) with 1 cm path length. Fluorescence wavelength l nm 1000 emission was induced in response to 650 nm excitation from a 450 W xenon arc lamp. We define the relative fluorescence intensity ζ(λ) as: where F(λ) is the wavelength-dependent fluorescence intensity, and A(650) is the absorbance at 650 nm excitation wavelength. All absorbance and fluorescence datasets were processed in Microsoft Excel. The relative fluorescence (Figure 3) was plotted and smoothed using 30 passes of the binomial smoothing algorithm in Igor Pro. The relative fluorescence intensity was then integrated and plotted in Figure 4 to report a measure of total NIR light emitted due to ICG fluorescence.

RESULTS AND DISCUSSION
The monomeric absorption peak of ICG (785 nm) does not change throughout the range of concentrations that was used to fabricate the NETs (Figure 2). The constant value of the protein absorbance at 275 nm suggests that the number of EGs being loaded in each sample was nearly the same.   Fluorescence spectra shown in Figure 3 were integrated to yield the brightness of each sample of NETs ( Figure 4). Three NET samples were fabricated at each concentration (consisting of 161, 215, 269, and 323 µM). We did not find any statistically significant difference among the integrated emission values for NETs fabricated using ICG concentrations in the range of 161-323 µM. NETs fabricated using a range of ICG concentrations between 161 -323 µM show similar absorption and fluorescence characteristics. These results suggest that there may be a limit to the amount of ICG that can be loaded into the NETs fabricated according to the protocol described in this paper. In our future studies, we will investigate the effect of using lower ICG concentrations.