This article has been accepted for publication in a future issue of this journal, but has not been

fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TBME.2018.2866368, IEEE
Transactions on Biomedical Engineering
TBME-00445-2018.R1 1

Near-Infrared-Fluorescent Erythrocyte-
Mimicking Particles: Physical and Optical
Characteristics
Jack C. Tang, Allen Partono, and Bahman Anvari

 fluorescent materials that emit in this window can enhance the

Abstract—Exogenous fluorescent materials activated by near- imaging contrast as a result of reduced autofluorescence by
infrared (NIR) light can offer deep optical imaging with sub- endogenous fluorophores in this spectral band [1]. Thus, high
cellular resolution, and enhanced image contrast. We have signal to noise, and deep optical penetration, on the order of at
engineered NIR particles by doping hemoglobin-depleted least about one cm, can be achieved by NIR imaging [1, 5].
erythrocyte ghosts (EGs) with indocyanine green (ICG). We refer One NIR-fluorescent contrast agent of immense interest is
to these optical particles as NIR erythrocyte-mimicking
indocyanine green (ICG). It remains as the only FDA-
transducers (NETs). A particular feature of NETs is that their
diameters can be tuned from micrometer to nanometer scale, approved NIR fluorophore for specific applications including
thereby, providing a capability for broad NIR biomedical assessment of cardiac output [6, 7], hepatic function [8-10],
imaging applications. Herein, we investigate the effects of ICG retinal angiography [11-13]. Having been used in thousands of
concentration on key material properties of micrometer-sized patients with rare occurrence of side effects (<0.15%) [14],
NETs, and nanometer-sized NETs fabricated by either sonication ICG is considered to be one of the least toxic agents
or mechanical extrusion of EGs. The zeta potentials of NETs do administered in humans [1]. ICG has also been used for non-
not vary significantly with ICG concentration, suggesting that FDA-approved investigational applications, such as sentinel
ICG is encapsulated within NETs regardless of particle size or lymph node biopsy [15-23], photothermal therapy [24-26], and
ICG concentration. Loading efficiency of ICG into the NETs
photodynamic therapy [26-30]. ICG-based fluorescence
monotonically decreases with increasing values of ICG
concentration. Based on quantitative analyses of the fluorescence angiography for vascular mapping and perfusion assessment is
emission spectra of the NETs, we determine that 20 μM ICG also becoming increasingly attractive [31-35], without the
utilized during fabrication of NETs presents an optimal drawbacks of potential toxicity or radioactivity of some of the
concentration that maximizes the integrated fluorescence contrast agents associated with CT and MR angiography.
emission for micrometer- and nanometer-sized NETs. In recent years, a number of fluorescence imaging systems
Encapsulation of ICG in these constructs also enhanced the have been developed for use with ICG. Several of these
fluorescence stability and quantum yield of ICG. These results systems have reached preclinical or clinical stages, including
guide the engineering of NETs with maximal NIR emission for the Curadel FLARE, Novadaq Spy Elite, Hamamatsu PDE-
imaging applications such as fluorescence-guided tumor
NEO II, Fluoptics Fluobeam, Intuitive Surgical da Vinci
resection, and real-time angiography.
Firefly, and Nawoo Vision FIAT-L imaging systems [36, 37],
Index Terms— biomedical optical imaging, biomembranes, further emphasizing the growing interest in NIR fluorescence
biomimetic materials, biophotonics, molecular imaging, imaging, particularly based on ICG.
nanobiotechnology, nanomedicine, particle production. Nevertheless, despite its established clinical utility, a
disadvantage of ICG is its short circulation half-life (≈2-4
minutes) [38, 39]. Upon intravenous injection, ICG non-
I. INTRODUCTION specifically binds to serum proteins, such as albumin, and is
se of exogenous fluorescent materials activated by near- uptaken by hepatocytes [40-42]. One strategy for extending

U infrared (NIR) excitation provides a capability for deep,

high-resolution biomedical optical imaging at the cellular
the circulation half-life of ICG is to encapsulate the dye into
protective constructs. Encapsulation can also serve to enhance
the stability of ICG, which can degrade due to heat or optical
and molecular levels [1, 2]. These advantages are primarily
due to reduced light absorption and scattering by hemoglobin, exposure [43, 44]. To date, various materials have been used
water, and other biological components in the NIR to encapsulate ICG, including polymers [45, 46], plant-virus
transparency window [3, 4]. Additionally, exogenous capsids [47-49], and liposomes [50, 51].
Encapsulation of ICG into liposomes has been shown to
increase the fluorescence quantum yield of ICG [43, 52].
Submitted for review on March 24, 2018. This work was supported in part However, liposomal formulation lacks the presence of
by grants from the National Science Foundation (CBET-1509218), National immunomodulatory surface proteins that may be required to
Institute of Arthritis and Musculoskeletal and Skin Diseases (R01-AR068067),
and National Cancer Institute (1R43-CA210715).
increase the circulation time of ICG. For example, the reported
J. C. Tang, A. Partono, and B. Anvari are with the Department of half-life of ICG in liposomal nanoparticles following
Bioengineering, University of California, Riverside, Riverside, CA, USA intravenous injection in mice is less than a minute [53].
(correspondence email: [email protected]).

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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TBME.2018.2866368, IEEE
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TBME-00445-2018.R1 2

Recently, we have encapsulated ICG using hemoglobin- To remove intracellular hemoglobin, the isolated RBCs
depleted red blood cells (RBCs), also known as erythrocyte were incubated in increasingly hypotonic conditions.
ghosts (EGs) [54-57]. We refer to these ICG loaded particles Specifically, we incubated the RBCs for 20 minutes in 0.5X
as NIR erythrocyte-mimicking transducers (NETs) since they PBS (1 ml, 4 °C). These RBCs were then collected by
can use NIR light to generate heat, fluorescence, and reactive centrifugation (20,000 × g, 15 minutes, 4 °C), and the red
oxygen species [58]. Several groups, including our own, have supernatant (containing hemoglobin and other intracellular
investigated NETs for imaging, phototherapeutic, and drug- contents) was discarded. The pellet of these erythrocytes with
delivery applications [55-57, 59-62]. EGs have been used to partial hemoglobin depletion was then re-suspended in 1 ml of
encapsulate and deliver other therapeutic and diagnostic 0.5X PBS. This hypotonic treatment in 0.5X PBS was
payloads as well [63-65]. repeated a total of three times. Then, we decreased the tonicity
Native RBCs have long circulation times on the order of of the buffer, and re-suspended the erythrocytes in 0.25X PBS
≈90-120 days, attributed in part to the presence of specific and incubated them for 20 minutes at 4 °C. Then, the particles
“self-marker” membrane proteins such as CD47, that allow were centrifugally washed (20,000 × g, 15 minutes, 4 °C).
them to evade immune cells [66]. We have previously Hypotonic treatment and wash in 0.25X PBS was repeated a
demonstrated that CD47 can remain on the surface of NETs total of three times.
[56]; hence, providing a mechanism for extended circulation Hemoglobin-depleted EGs were then resealed by
of NETs. As constructs that can be fabricated using centrifugally washing the pellet in 1X PBS (20,000 × g, 15
autologous blood or compatible blood types, NETs may prove minutes, 4 °C), and re-suspending the pellet in 1X PBS. The
to be non-toxic materials for biomedical imaging and resulting micrometer-sized EGs (EGs) were diluted to have
therapeutic applications. an absorbance value of two at 280 nm, corresponding to ≈0.04
A useful feature of NETs is that their diameter can be tuned ml of EGs used for each 1 ml of NETs. The 280 nm
to various sizes ranging from nanometer to micrometer scale, wavelength corresponds to the absorption peak of the aromatic
and that their ICG content can be adjusted during the rings found in proteins, which enabled us to control the
fabrication process. Nanometer-sized NETs (nNETs) have amount of EG material in each sample.
relevance to cancer optical theranostics, based on the
enhanced permeability and retention (EPR) effect [67, 68].
Micrometer-sized NETs (NETs) may be useful for
angiography, and photothermal treatment of vascular
cutaneous diseases, such as the port wine stain.
In this study, we fabricated NETs, and nNETs formed by
two methods based on mechanical extrusion, and sonication.
Our objective was to investigate the effects of changing the
ICG concentration utilized during the fabrication process on
the resulting material properties, including zeta potential, ICG
loading efficiency, and optical absorption and fluorescence
characteristics of both NETs and nNETs. We also
demonstrate that NETs can retain their fluorescence
characteristics for at least up to 12 hours in storage (4 °C) and
at physiological (37 °C) temperatures. The fluorescence
characteristics are particularly important for clinical
translation of NETs for biomedical fluorescence imaging, and
provide the basis for selecting an optimized concentration of Fig. 1. Schematic of the processes to fabricate micrometer- and nanometer-
ICG that can produce NETs and nNETs with maximal sized NETs (NETs and nNETs, respectively). After the formation of
micrometer-sized erythrocyte ghosts (EGs), these particles can be loaded
fluorescence emission. with ICG to form NETs. EGs are mechanically extruded, or sonicated to
reduce their size to the nanometer scale before loading with ICG to yield
II. MATERIALS AND METHODS nNETs.

A. Formation of Micrometer-Sized Erythrocyte Ghosts B. Sonication and Mechanical Extrusion of EGs

(EGs) We reduced the size of EGs to the nanometer-scale using
Intact RBCs were isolated from whole bovine blood (1 ml) two methods: sonication and mechanical extrusion (Fig. 1).
(Rockland Immunochemicals, Inc., Limerick, PA, USA) in Under the sonication method, EGs were subject to ultrasonic
citrate by centrifugation (1000 × g, 5 minutes, 4 °C) pulses for eight minutes (30 W, 20 kHz) using a Misonix S-
(Eppendorf 5424 Microcentrifuge, Hamburg, Germany). The 400 probe sonicator (Farmingdale, NY, USA). Each pulse
supernatant containing the plasma and buffy coat was lasted 5 seconds, and pulses were delivered 20 seconds apart.
discarded, and the erythrocytes were re-suspended in 1 ml of To avoid heating of the EGs, the vial containing the solution
≈300 mOsm phosphate-buffered saline (1X PBS) (Fisher was placed in a beaker of ice water during sonication. We
Bioreagents, Fair Lawn, New Jersey, USA). Erythrocytes were replaced the ice after each minute of sonication. After every
isolated by centrifugally washing them three times in 1X PBS two minutes of sonication, the EGs were given five minutes to
(1000 × g, 5 minutes, 4 °C). rest in fresh ice water. After sonication was completed, the
nanometer-sized EGs (nEGs) were centrifugally washed

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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TBME.2018.2866368, IEEE
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TBME-00445-2018.R1 3

(100,000 × g, one hour, 4 °C) in a Type 90ti rotor (Beckman averaged. This was done for three independent samples, and
Coulter, Inc., Palo Alto, CA, USA) and re-suspended in 1X used to estimate the mean  and the corresponding SD.
PBS.
Under the extrusion method, EGs were sequentially E. Fluorescence Microscopy of NETs
extruded through track-etched polyester membranes Fluorescence images of NETs were obtained using a
(Sterlitech Corp., Kent, WA, USA) with defined pore Hamamatsu C9100-13 electron-multiplying charge-coupled
diameters (800, 400, and 200 nm) to form nanometer-sized detector (Hamamatsu, Japan) with a Nikon Eclipse Ti
EGs (nEGs). EGs were passed through each size membrane at microscope (Tokyo, Japan) and a Chroma Li-Cor IR800 filter
least three times. To prevent excessive clogging of the pores, cube (Bellows Falls, Vermont, USA). We used a Nikon oil-
EGs were diluted ten-fold in 1X PBS prior to extrusion and immersion objective lens with 100x magnification (Tokyo,
re-concentrated afterwards. Extrusion was performed using a Japan). The excitation band was 740 ± 20 nm, and the
10 ml LIPEX® extruder (TRANSFERRA Nanosciences Inc., emission was passed through a filter transmitting wavelengths
Burnaby, B.C., Canada). Extruded nEGs were then greater than 780 nm.
centrifugally washed (100,000 × g, one hour, 4 °C), and re- F. UV-Vis-NIR Absorption Spectra of NETs
suspended in their original volume of 1X PBS.
Steady-state absorption spectrum of each formulation of
C. Loading EGs with ICG to form NETs NETs was recorded using a UV-Vis-NIR spectrophotometer
Each type of EG (micrometer-, and nanometer-sized formed (JASCO V-670, Tokyo, Japan) in transmission mode with a
by sonication or extrusion) was loaded with ICG by incubating quartz absorbance microcuvette (1 cm pathlength). The
the EGs in a hypotonic solution containing ICG. The ICG spectral range for all absorbance measurements was from 230-
loading buffer contained a 1:1:1 volume ratio of EGs, 1100 nm to ensure that both the UV and NIR absorption, from
Sørensen’s phosphate buffer (0.1 M, pH 7.4), and a known proteins and ICG respectively, were recorded.
concentration of aqueous ICG. We varied the total The spectral features of ICG provide insight into the
concentration of the ICG loading buffer (0, 1, 5, 10, 20, 50, conformational states of ICG present within NETs. ICG can
75, and 100 M) and incubated the EGs with the indicated be in either a monomeric form, indicated by an absorption
ICG loading buffers for 30 minutes to form NETs. The total peak at 805 nm, or in an H-like aggregated form [73, 74],
tonicity of the ICG loading buffers was ≈175 mOsm. indicated by a secondary absorption peak at 740 nm. We
Following incubation, excess ICG was removed by define the parameter  as the ratio of the monomeric ICG
centrifugation (20,000 × g, 15 minutes for NETs; 100,000 × absorbance to that of the H-like aggregate form of ICG. We
g, one hour for nNETs formed by sonication or extrusion). note that the “absorption” of NETs is effectively a
The supernatant was set aside to quantify the loading combination of optical scattering and photon absorption.
efficiency of ICG into NETs (as described below). The Therefore, the spectra of EGs (formed using 0 M ICG in the
resulting NETs were then washed in 3 ml of 1X PBS to reseal loading buffer) contains both the optical scattering component
the membranes and remove residual ICG. The supernatant of the particles, as well as the absorption component of
from this wash was removed and added to the supernatant for proteins and any residual hemoglobin. To calculate , we first
quantification of the ICG loading efficiency of NETs. NETs removed the contribution of optical scattering and absorption
were then re-suspended in 1 ml of 1X PBS for characterization by other components in NETs by subtracting the baseline
studies. absorption spectrum of EGs (constructs without ICG) from
each absorption spectrum of NETs. Then, we used this
D. Dynamic Light Scattering-Based Measurement of NETs
baseline-subtracted absorbance for the calculation of , as
Diameters and Zeta Potential
follows:
The hydrodynamic diameters of each NETs formulation
was measured by dynamic light scattering (DLS) using a A* (λ = 805 nm)
Malvern Zetasizer NanoZS (Malvern, United Kingdom). ψ= (1)
Samples were suspended in 1 ml 1X PBS within a polystyrene A* (λ = 740 nm)
cuvette (1 cm pathlength). Each DLS measurement was
collected at a scattering angle of 90°. We report the size of where A*( = 805 nm) is the absorbance value of NETs at 805
NETs using the z-average diameter, which is the intensity- nm minus the absorbance value of EGs at 805 nm. Similarly,
based harmonic mean of the light-scattering particles [69, 70]. A*( = 740 nm) is the absorbance value of NETs at 740 nm
Eight measurements were taken for each sample, and minus the absorbance value of EGs at 740 nm. Wavelength is
averaged. The average diameters of three independent samples denoted as .
were then used to calculate the mean z-average diameter and G. Quantification of ICG Loading Efficiency of NETs
the corresponding standard deviation (SD).
The zeta potential () of NETs and EGs in 1X PBS were We define the ICG loading efficiency () of NETs as:
also estimated using the Malvern Zetasizer via the msuper
Smoluchowski approximation, which relates the  to the ε=1- (2)
observed electrophoretic mobility of the particles [71, 72]. minitial
Each measurement was done in a folded capillary cell at 10
where minitial is the amount of ICG introduced into the loading
°C. Similar to the z-average diameter characterizations, the 
buffer, and msuper is the amount of ICG present in the
of each formulation of NETs was measured eight times, and

0018-9294 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TBME.2018.2866368, IEEE
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supernatant upon completing the fabrication of NETs. The FLuorescence-Assisted Resection and Exploration (FLARE)
supernatant was collected immediately after centrifugation and system [17]. We chose this system as an example since its
stored in a dark refrigerator at 4°C to prevent ICG optical specifications needed for our analyses are readily
photobleaching. Supernatant solutions were contained in available in published scientific literature [17]. The system
disposable polystyrene cuvettes (1 cm pathlength), and their consists of two LED sources for excitation in bands of 656-
absorption spectra (400-1100 nm) were obtained using the 678 nm and 745-779 nm. Fluorescence emission is passed
UV-Vis-NIR spectrophotometer. To determine msuper, we through two NIR filters with respective bandpass wavelengths
compared the absorbance value of the supernatant at its of 689-725 nm and 800-848 nm. Mathematically, we define ,
spectral peak (780 nm) to a calibration curve (data not shown) the integrated normalized fluorescence emission, as:
that related peak absorbance values of supernatant ICG to
known concentrations of ICG in the same supernatant buffer. λex = 678 nm λem = 725 nm
Currently, the clinical formulation of ICG is free ICG γ= ∑ [ ∫ F(λex , λem ) dλem ]
dissolved in water. In order to compare the optical absorption
λex = 656 nm λem = 689 nm
of NETs with that of free ICG in water, we determined the
equivalent concentration of free ICG that would have the same λex = 678 nm λem = 848 nm
monomer absorbance value as each of the NETs formulations
fabricated using various ICG concentrations in the loading + ∑ [ ∫ F(λex , λem ) dλem ]
buffer. To do this, we first subtracted the baseline spectrum of λex = 656 nm λem = 800 nm
EGs (i.e., particles not loaded with ICG) from the absorption
spectrum of each formulation of NETs. In this manner, we λex = 779 nm λem = 848 nm

eliminated the contribution of optical scattering and protein + ∑ [ ∫ F(λex ,λem ) dλem ] (4)
absorption from each spectrum. We then compared the λex = 745 nm λem = 800 nm
monomeric absorption peaks of baseline-subtracted NETs to a
calibration curve of the monomeric absorption peaks of free
We also determined the relative fluorescence quantum yield
ICG dissolved in water, in the range of ≈1-26 M, to
(NETs) for each of the NETs formulations in response to 780
determine the equivalent free ICG concentration of NETs.
nm excitation wavelength, which corresponds to the
H. Excitation-Emission Mapping of NETs Fluorescence absorption peak of free ICG, as:
To determine the fluorescence characteristics of each
formulation of NETs, we mapped the excitation-emission ϕNETs (λex = 780 nm) = ϕICG (λex = 780 nm)
(ExEm) spectra using a Fluorolog-3 spectrofluorometer
λem = 850 nm
(HORIBA, Ltd., Edison, New Jersey, USA). Solution of NETs ∫λ 𝑓𝑁𝐸𝑇𝑠 (λex = 780 nm, λem ) dλem
em = 800 nm
were contained within a quartz cuvette (1 cm pathlength). The ×[ -ANETs(λex = 780 nm)
]
excitation wavelengths ranged between 600-830 nm, and were 1 - 10
generated using a 450 W xenon arc lamp. Corresponding to
each excitation wavelength, the bathochromic fluorescence
light was collected by the detector to ensure the collection of 1 - 10-AICG (λex = 780 nm)
×[ λem = 850 nm
] (5)
only the fluorescence, but not the scattered light. ∫λ 𝑓𝐼𝐶𝐺 (λex = 780 nm, λem ) dλem
em = 800 nm
Prior to ExEm mapping, a sample of each NETs
formulation was diluted to have a NIR peak absorbance of 0.2
or lower. To correct for the primary inner-filter effect, which where ICG (ex = 780 nm) is the fluorescence quantum yield (≈
arises from the attenuation of excitation light in strongly 2.58%) [52] of free ICG in water (6.45 M) at 780 nm
absorbing samples [75], the fluorescence intensity values (f) in excitation wavelength, fNETs(ex = 780 nm, em) is the
each ExEm map were normalized with respect to the fluorescence emission intensity of a given NETs formulation
percentage of excitation light absorbed by the sample, as: in response to 780 nm excitation, ANETs (ex = 780 nm) is the
absorbance value of a given NETs formulation at 780 nm, AICG
f(λex , λem ) (ex = 780 nm) is the absorbance value of free ICG in water
F(λex , λem ) = (3) (6.45 M) at 780 nm, and fICG(ex = 780 nm, em) is the
1 - 10-Aex
fluorescence emission intensity of free ICG in water (6.45
where F(ex, em) is the normalized fluorescence emission, ex M) in response to 780 nm excitation wavelength.
is a given excitation wavelength, em is an emission
wavelength in response to excitation at ex, and Aex is the I. Determination of NETs Fluorescence Stability at 4 °C and
absorbance of the diluted NETs formulation at ex. 37 °C
To obtain an estimate of total fluorescence emission for a We investigated the fluorescence stability of the three types
given NETs formulation, we integrated the emission in of NETs fabricated using 20 μM ICG stored at either 4 °C or
response to two bands of excitation wavelengths, 656-678 nm 37 °C for up to 12 hours. Following a given storage time t,
and 745-779 nm. These bands correspond to the excitation each sample of NETs, suspended in 1X PBS, was photo-
light sources utilized in a clinically relevant NIR excited at 780 nm. The normalized fluorescence emission [see
intraoperative fluorescence imaging system, the (3)] at a given t was integrated to yield the quantity I(t) as:

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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TBME.2018.2866368, IEEE
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the increase in diameter of NETs and nNETs formed by

λem = 900 nm
extrusion with increased ICG loading concentration to the
𝐼(𝑡) = ∫ F(λex = 780 nm, λem ) dλem (6) incorporation of ICG molecules into the phospholipid
membrane of NETs. Such localization of the dye may increase
λem = 795 nm
the overall diameter of the constructs. Indeed, fluorescence
We define I* as the value of I normalized to its initial value at microscopy of NETs revealed partial localization of ICG to
t = 0 (immediately after fabrication). the membrane periphery [Fig. 2(c)].
The z-average diameters of nNETs formed by sonication
III. RESULTS AND DISCUSSION are smaller than the diameter of nNETs formed by extrusion
[Fig. 2(b)]. The mean diameters of nNETs formed by
A. Hydrodynamic Diameter and Zeta Potential () of NETs sonication ranged between ≈181-190 nm, and unlike NETs
Based on the dynamic light scattering (DLS) method, the and nNETs formed by extrusion, did not show a statistically
mean intensity-weighted average (z-average) diameter of significant dependence on ICG loading concentration. This
NETs ranged between ≈1.9-2.1 m, depending on the ICG result suggests that ICG may be primarily localized in the
concentration used in the loading buffer [Fig. 2(a)]. When interior of nNETs formed by sonication rather than being
NETs were fabricated using ICG loading concentrations in embedded within the phospholipid membrane. We attribute
the range of 5-100 M, the resulting diameters were this to the disruption and weakening of the membrane and
significantly larger (p < 0.01) than that of micrometer-sized cytoskeleton of EGs during sonication, which may be the
erythrocyte ghosts (EGs). However, diameters of NETs physical basis for localization of ICG to the interior cavity.
approached a plateau level of ≈2.1 m for ICG loading The  values for NETs and nNETs formed by extrusion
concentrations ≥ 20 M. were indistinguishable from the  of untreated bovine RBCs,
which was measured to be -12.78 ± 0.84 mV in 1X PBS (≈300
mOsm) [Fig. 2(d)]. These results suggest that glycophorin A,
and sialic acid, the primary negative charge-bearing
components of the RBC membrane [76], were not removed by
hypotonic treatment or mechanical extrusion.
Maintaining the normal  of RBCs can be a contributing
factor in increasing the circulation time of NETs. Gbadamosi
et al. found a linear relationship between the  of methoxyl
poly(ethylene glycol)-coated polystyrene microspheres and
their uptake by mouse macrophages [77]. Extrapolation of
their data shows that  of approximately -15 mV would result
in none or minimal uptake by phagocytes. This value is
remarkably close to the  value of mouse [78] and human
RBCs [78, 79]. Therefore, NETs and other particle systems
with similar , may be minimally uptaken by macrophages as
compared to neutral or highly charged particles [80, 81].
All formulations of nNETs formed by sonication had 
values that were significantly lower (p < 0.05) than the  of
bovine RBCs, NETs, and nNETs fabricated by extrusion.
Fig. 2. Size and zeta potential of NETs. z-average diameters were estimated
by dynamic light scattering (DLS) for: (a) EGs and NETs, and (b) nEGs
This may be due to degradation of sialic acid residues or
and nNETs. (c) Fluorescence image of NETs (100X magnification) formed membrane surface proteins during sonication. For the three
using 20 M ICG in the loading buffer. (d) Zeta potential () of EGs and types of NETs, there were no statistically significant changes
NETs in 1X (≈300 mOsm) phosphate-buffered saline (PBS). Single, double, in  values in response to changes in the ICG concentration
and triple asterisks indicate statistically significant differences with p < 0.05,
within the loading buffer [Fig. 2(d)]. This result suggests that
0.01, and 0.001 respectively. In panel (d), the single blue asterisk indicates
statistically significant differences between the  values of nEGs formed by the sulfonate portions of ICG, which are responsible for the
sonication, compared to the  values of the other two types of NETs. negative charge of ICG, were completely encapsulated (either
localized within the interior cavity or embedded within the
nNETs formed by extrusion also had significantly larger membrane) [43], and not exposed to the external environment
diameters compared to nEGs formed by extrusion, as shown in of the erythrocyte-derived constructs.
Fig. 2(b). Depending on the ICG concentration in the loading Using DLS, Kuo et al. demonstrated that suspensions of
buffer, z-average diameters of nNETs formed by extrusion nEGs formed by sonication did not aggregate despite freeze-
ranged between ≈203-255 nm. We have previously published thaw cycling up to five cycles [82]. This result was in contrast
SEM [56] and TEM [55] images of extruded nNETs. In these to liposomal formulations that aggregated after the first freeze-
papers, we have also demonstrated that the electron thaw cycle [82]. Our own studies demonstrated that the
microscope- and DLS-based measurements of nNETs diameter and of nNETs formed by extrusion did not change
diameters are in agreement. Estimates of NETs diameter even after eight weeks of storage at -20 °C [83]. Therefore,
based on fluorescence microscopy [Fig. 2(c)] are also in cryopreservation may serve as a suitable method for the long-
agreement with DLS-based estimates in Fig. 2(a). We attribute

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TBME-00445-2018.R1 6

term storage of nNETs since the particles remain colloidally This sandwich-like, - stacking of individual ICG molecules
stable after freeze-thaw cycling. results in interaction of the individual transition dipole
moments that raises the excited-state energy to produce a blue-
B. Absorption characteristics of NETs
shifted absorption peak [49, 73, 74]. In water, even relatively
The absorption spectra of NETs, nNETs formed by low concentrations of ICG (> 10 M) will begin to form H-
extrusion, and nNETs formed by sonication revealed NIR aggregates [74, 85].
peaks that were centered between 801-805 nm, 797-802 nm, As the ICG concentration in the loading buffer increased,
and 799-802 nm, respectively (Fig. 3). These spectral peaks there was a corresponding non-linear increase in ICG
are attributed to the monomer form of ICG, as indicated in absorption in the range of 600-900 nm for the three types of
previous studies [54-56], and were bathochromically shifted NETs. For example, for NETs [Fig. 3(a)], doubling the
with respect to the absorption peak of free ICG (780 nm) by concentration of ICG in the loading buffer, from 50-100 M,
approximately 20 nm. This shift is consistent with the resulted in an increase in peak NIR absorbance of only 9.4%.
behavior of ICG in emulsions and micellar formulations [52, For nNETs formed by extrusion [Fig. 3(b)], the peak NIR
84], and can be attributed to the intercalation of ICG into the absorbance only increased 27.8% in response to the same
lipid bilayer of NETs and binding to the phospholipids and
increase of 50-100 M ICG in the loading buffer. For nNETs
membrane proteins therein [43, 55, 84], leading to changes in
formed by sonication, this same increase in ICG loading
the conformation of ICG and its electronic states.
buffer did not change the absorption spectrum [Fig. 3(c)]. This
The absorption shoulder in the spectral range of 720-760
result suggests that there is a limit to the amount of ICG that
nm corresponds to the H-like aggregate form of ICG [54, 74].
can be loaded into NETs. Nevertheless, the nNETs formed by

Fig. 3. Optical absorption characteristics of NETs. UV-Vis-NIR absorption spectra of: (a) EGs and NETs, (b) nEGs and nNETs formed by extrusion, and (c)
nEGs and nNETs formed by sonication. In panel (d), we present the ratio of the monomer to H-like aggregate absorbance values of ICG ) [see (1)] for NETs
formed using various concentrations of ICG in the loading buffer. The displayed legend (0-100 M) corresponds to the ICG concentration in the loading buffer
to fabricate the three types of NETs and is shared among panels (a), (b), and (c). Each displayed absorption spectrum in panels (a), (b), and (c) is an average of
triplicate samples. Error bars in panel (d) are the SDs associated with the triplicate samples of each formulation.

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sonication had the highest absorption in the range of 600-900

nm, suggestive of higher loading efficiency of ICG into these
nNETs. We present quantitative data regarding ICG loading
efficiency in the following section.
Optical attenuation over the spectral band of 250-500 nm is
in part due to scattering by the erythrocyte-derived particles,
and absorption by proteins at 280 nm. The presence of ICG
also makes small contributions to absorbance values at 280
nm, and 400 nm (data not shown). Despite having nearly the
same amount of phospholipid membrane material as NETs,
nNETs formed by sonication showed less attenuation in the
250-500 nm band, owing to their smaller diameter [Fig. 3(c)].
In a previous study, we showed that the reduced scattering
coefficient of nNETs was less than half (≈45%) of that of
NETs [54], which is consistent with the lower attenuation in
the 250-500 nm spectral band for nNETs formed by
sonication. nNETs formed by extrusion had even lower
attenuation in this spectral band, due to loss of EG material
during the extrusion process, resulting in fewer relative Fig. 4. ICG-loading characteristics of NETs. (a) ICG loading efficiency ()
number of light-scattering particles. The lower amount of EG into NETs [see (2)]. (b) Effective concentration of ICG within NETs. (c) Free
materials in the extruded type is also a contributing factor for ICG equivalent concentration of NETs as functions of ICG concentration of
the loading buffer were determined by quantifying the amount of ICG in the
their relatively low NIR absorbance and low ICG loading supernatant. (d) The free ICG equivalent concentration corresponding to
efficiency [Fig. 4(a)]. various effective concentrations of ICG within NETs. Error bars in each panel
The parameter  provides a quantitative measure of the indicate the SDs associated with triplicate samples.
amount of ICG in its monomeric form relative to its H-like cytoskeletal network and membrane of EGs, which may allow
aggregate form within the NETs, as described by (1). Values ICG to be more readily loaded. The  values for nNETs
of  decreased as the concentration of ICG in the loading formed by extrusion were significantly lower (p < 0.05) than
buffer increased, indicating progressively higher fractions of those of other types of NETs, which may be attributed to the
the H-like aggregate forms of ICG were present in NETs [Fig. lower amount of EG material that remained after the extrusion
3(d)]. The increase in H-like aggregate fractions within NETs process.
is likely due to their presence in the ICG loading buffer prior Multiplying  by the concentration of ICG in the loading
to being loaded into NETs. At ICG loading concentrations of buffer yields the effective concentration of ICG within the
≥ 5 M, values of  were highest for NETs. Thus, the population of NETs [Fig. 4(b)]. Thus, the effective
relative fractions of the monomeric form of ICG in NETs concentration of ICG within NETs is an estimate of the
was higher those in nNETs formed by either sonication or concentration of ICG that would be dispersed in solution if
extrusion. This behavior influences the fluorescence properties NETs were disintegrated to release their ICG content. The
of NETs, as shown later in the text. effective concentration of ICG within NETs increased almost
C. ICG Loading efficiency of NETs and Equivalent Free-ICG sigmoidally toward a maximum limit.
Concentrations The maximum free ICG equivalent concentrations
corresponding to NETs, and nNETs formed by sonication or
We estimated the loading efficiencies of ICG into NETs ( ) extrusion at various ICG loading concentrations were 16.6 ±
using (2). For all three types of NETs, there was a monotonic
0.5, 14.1 ± 1.3, and 5.0 ± 0.6 M, respectively [Fig. 4(c)].
decrease in  as the ICG concentration in the loading buffer Much like the effective concentration of ICG within NETs, the
was increased beyond 1 M [Fig. 4(a)]. The average value of asymptotic trend in free ICG equivalent concentration also
 for NETs, and nNETs formed by extrusion or sonication indicated a maximum upper limit to the NIR absorbance of
methods were 87%, 94%, and 98% respectively when using 1 NETs.
M ICG concentration in the loading buffer [Fig. 4(a)]. The Values of the free ICG equivalent concentration of NETs,
low values of  associated with use of higher concentrations of as a function of the effective concentration ICG within NETs
ICG can be attributed to the fixed amount of EG material are presented in Fig. 4(d). These particular results provide a
available to accept ICG. At high loading concentrations of convenient method to quantitatively determine how the
ICG, the fixed amount of EG material can only uptake a effective ICG concentration within NETs compares with
limited amount of ICG, leaving a large fraction of non- various concentrations of free ICG dissolved in water. As the
encapsulated ICG in the loading buffer. loaded concentration of ICG continued to increase beyond 10
Notably, nNETs formed by sonication had significantly M for nNETs fabricated by extrusion, and 15 M for NETs
higher  values (p < 0.05) for all ICG loading concentrations and nNETs formed by sonication, there were nearly no further
when compared to  for NETs, and nNETs formed by changes in the free ICG equivalent concentrations. This results
extrusion (except nNETs formed by extrusion using 1 M suggests that at higher ICG loading concentrations, the
ICG loading concentration). This is likely the result of the additional ICG loaded into NETs was likely in the H-like
aforementioned sonication-induced disruption of the aggregate form, and is consistent with the trend of lower 

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values when increasing the ICG concentration in loading excitation wavelengths for these formulations were 678, 678,
buffer [see Fig. 3(d)]. and 676 nm, respectively.
Using (4), we determined the formulations of NETs that
D. Fluorescence Characteristics of NETs
generated maximum fluorescence emission for the detection
Normalized excitation emission (ExEm) maps of NETs bands used in the FLARE intraoperative imaging system [17].
were calculated using (3) based on absorption and To do this, we defined parameter  as the integrated
fluorescence spectral data, and are presented as Fig. 5. These normalized emission collected over the excitation and
maps are universal, and can be used by those who may be emission wavelengths used by the system. The two excitation
interested in using a given imaging system. Specifically, by bands were 656-678 nm and 745-779 nm, whereas the two
utilizing these excitation and emission bands, one can select emission collection bands were 689-725 nm and 800-848 nm.
the appropriate NETs formulation suitable for fluorescence NETs with the highest average  were fabricated using 10
detection associated with a system of interest. M ICG in the loading buffer [Fig. 6(a)]. This formulation had
We attribute the emission “hot spots” in the range of ≈770-
a  value that was statistically different from those associated
830 nm to the monomeric forms of ICG within NETs. The
with the 1 and 100 M ICG loading concentrations. nNETs
most intense monomeric emissions were observed from
formed by either extrusion or sonication had a maximum
NETs and nNETs formed by sonication using 5 M ICG
average  when fabricated using 20 M ICG. We also
loading concentration when photo-excited at 778 nm and 780
determined that the maximal values of  for the three types of
nm, respectively. Interestingly, 5 M corresponded to the ICG
NETs fabricated using 20 M ICG were not statistically
concentration at which the maximum value of  for NETs
was determined [Fig. 3(d)]. nNETs formed by extrusion had different from each other. Therefore, the  of NETs and
the highest normalized fluorescence emission intensity value nNETs is effectively maximized when fabricating them using
(F) associated with monomeric ICG when they were 20 M ICG in the loading buffer. The corresponding free ICG
fabricated using 20 M of ICG in the loading buffer and equivalent concentrations for these formulations were 12.3 ±
photo-excited at 780 nm [Fig. 5(b)]. 0.1, 2.8 ± 0.5, and 13 ± 2 M for NETs, nNETs formed by
A general trend associated with all three types of NETs is extrusion, and nNETs formed by sonication [Fig. 4(c)].
that increasing the concentration of ICG in the loading buffer Using (5), we determined the relative fluorescence quantum
beyond their respective values associated with maximum yield, , of NETs in response to excitation at 780 nm, using
monomeric emission as indicated above, produced a second 6.45 M ICG dissolved in water as the reference sample for
emission hot spot associated with the H-like aggregate quantum yield calculations [52]. The maximum values of 
emission. Peak values of F associated with H-like aggregate were 4.38%, 3.27%, and 2.98% for NETs, nNETs formed by
emissions were produced using respective ICG loading extrusion, and nNETs formed by sonication, respectively [Fig.
concentrations of 75, 75, and 100 M for NETs, and nNETs 6(b)]. The ICG concentrations used to fabricate NETs with
formed by extrusion or sonication. The corresponding photo- maximum  were 10, 10, and 1 M for NETs, nNETs

Fig. 5. Normalized excitation-emission (ExEm) maps for: (a) NETs, (b) nNETs formed by extrusion, and (c) nNETs formed by sonication. NETs were
fabricated using ICG concentrations in the range of 0-100 M. Each displayed ExEm map is an average of three ExEm maps generated using triplicate samples.
The scale bar on the right corresponds to the values of F, [see (3)].

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Fig. 6. Integrated normalized fluorescence emission [see (4)], and relative fluorescence quantum yield [see (5)] of NETs. (a) Values of the integrated normalized
fluorescence emission, , and (b) relative fluorescence quantum yield, , as a function of ICG concentration in the loading buffer for the three types of NETs.
Values of  are calculated relative to the  of 6.45 M free ICG in water, in response to excitation at 780 nm. Single, double, and triple asterisks indicate
differences of p < 0.05, 0.01, and 0.001, respectively. Error bars indicate the SDs associated with triplicate samples.
formed by extrusion, and nNETs formed by sonication, summarize some of the key optical and material properties of
respectively. Encapsulation of ICG into NETs resulted in  the three types of fluorescence-optimized NETs in Table I.
values that were up to ≈70, 27, and 16% higher than those for
free ICG for NETs, and nNETs formed by extrusion or
sonication, respectively.
The increase in  upon encapsulation of ICG into NETs can
be attributed to the change in the local environment of ICG.
Free ICG has a low value of , partly due to its flexible
polyene bridge that can rotate and fold, providing multiple
pathways for non-radiative decay and relaxation of its energy
from the excited state [86, 87]. ICG, among other cyanine
dyes, is also prone to aggregation and self-quenching [43, 88,
89], which can also reduce . Physical association of ICG
Fig. 7. Time-dependent fluorescence stability, quantified by the metric I* [see
molecules with NETs constituents (e.g., phospholipids and (6)] for NETs and free ICG at (a) 4 °C, and (b) 37 °C. Error bars indicate
membrane proteins) derived from erythrocytes can stabilize normalized SD (SD divided by I(t = 0)) for triplicate samples. Statistical
the polyene bridges and reduce the rate of vibrational significance is evaluated with respect to the initial values at t = 0. Single,
relaxation from the excited state, which can lead to increased double, and triple asterisks indicate differences of p < 0.05, 0.01, and 0.001,
respectively. The excitation wavelength was 780 nm.
.
E. Fluorescence Stability of NETs and Free ICG
TABLE I
After 12 hours of storage at 4 °C, there were no statistically
MATERIAL PROPERTIES OF NETS FABRICATED USING 20 M ICG
significant changes in the values of I* [see (6)] for the three
Type of NETs z-average  (mV)  (%)  (%)
types of NETs (Fig. 7a). However, free ICG showed ≈ 50% diameter (nm)
reduction in I* after 12 hours of storage at 4 °C. Micron-sized
NETs 2090 ± 30 -12.9 ± 0.4 40 ± 2 3.8 ± 0.1
and nano-sized NETs formed by extrusion did not show
statistically significant changes in I* after 12 hours of storage nNETs formed
225 ± 8 -12.0 ± 1.0 22 ± 3 3.2 ± 0.4
at 4 °C or 37 °C (Fig. 7a and b), indicating their fluorescence by extrusion
stability. NETs formed by sonication showed about 15%
nNETs formed
reduction in I* after 12 hours of storage at 37 °C (Fig. 7b), by sonication
181.1 ± 0.5 -15.1 ± 0.9 72 ± 2 1.8 ± 0.4
suggesting that sonication may have resulted in formation of
NETs with weakened membrane, leading to some ICG leakage For the z-average diameter and , the uncertainty values indicate the SD of
or degradation over this time interval. In comparison, free ICG triplicate samples, where each sample was measured eight times and averaged
after four hours of storage showed 40% reduction in I*, which to obtain a single value. The uncertainty of the estimated  and  values
was further increased to 50% after 12 hours. Finally, we indicate the SD associated with a single measurement for triplicate samples.

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