We have engineered nano-vesicles, derived from erythrocytes, which can be doped with various near infrared (NIR) organic chromophores, including the FDA-approved indocyanine green (ICG). We refer to these vesicles as NIR erythrocyte-mimicking transducers (NETS) since in response to NIR photo-excitation they can generate heat or emit fluorescent light. Using biochemical methods based on reduction amination, we have functionalized the surface of NET with antibodies to target specific biomolecules. We present results that demonstrate the effectiveness of NETs in targeted imaging of cancer cells that over-express the human epidermal growth factor receptor-2 (HER2).
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.
The variety of nanoparticles developed by numerous investigators has presented a diverse platform for various optical imaging applications in biomedicine. We have previously reported that the FDA-approved chromophore Indocyanine Green (ICG) can be successfully encapsulated by cross-linked poly-allylamine hydrochloride (PAH)-Disodium Monophosphate (Na2HPO4) to form a nanoparticle for near-infrared imaging applications. The diameter of the constructs is dependent on the charge ratio between the polymer and salt used to encapsulate the chromophore. Modifications of the synthesis methods can alter the photophysical properties of the capsules, either through the adjustment of the charge ratio between PAH and Na2HPO4 or concentration of ICG successfully impregnated into the capsule. Through understanding the effects of tuning the nanoparticle properties, the photophysical characteristics of the constructs can be optimized. Here we present the results of adjusting the diameter of the nanoparticle and amount of ICG on the hydrodynamic diameters, absorption and fluorescence characteristics, and the relative fluorescence quantum yield. Optimizing the photophysical properties of the constructs can lead to increased imaging sensitivity and contrast for potential translational applications, including tumor imaging, which may utilize these nanoconstructs.
Optical nano-materials present a promising platform for targeted molecular imaging of cancer biomarkers and its
photodestruction. Our group is investigating the use of polymeric nanoparticles, loaded with indocyanine green, an FDA-approved
chromophore, as a theranostic agent for targeted intraoperative optical imaging and laser-mediated destruction
of ovarian cancer. These ICG-loaded nanocapsules (ICG-NCs) can be functionalized by covalent attachment of targeting
moieties onto their surface. Here, we investigate ICG-NCs functionalized with anti-HER2 for targeted fluorescence
imaging and laser-mediated destruction of ovarian cancer cells in vitro. ICG-NCs are formed through ionic cross-linking
between polyallylamine hydrochloride chains and sodium phosphate ions followed by diffusion-mediated loading with
ICG. Before functionalization with antibodies, the surface of ICG-NCs is coated with single and double aldehyde
terminated polyethylene glycol (PEG). The monoclonal anti-HER2 is covalently coupled to the PEGylated ICG-NCs
using reductive amination to target the HER2 receptor, a biomarker whose over-expression is associated with increased
risk of cancer progression. We quantify uptake of anti-HER2 conjugated ICG-NCs by ovarian cancer cells using flow
cytometery. The in-vitro laser-mediated destruction of SKOV3 cells incubated with anti-HER2 functionalized ICG-NCs
is performed using an 808 nm diode laser. Cell viability is characterized using the Calcein and Ethidium homodimer-1
assays following laser irradiation. Our results indicate that anti-HER2 functionalized ICG-NCs can be used as theranostic
agents for optical molecular imaging and photodestruction of ovarian cancers in-vitro.
Our group has constructed a new type of viral nanoparticles (VNPs) from genome-depleted plant infecting
brome mosaic virus (BMV) that encapsulates the FDA-approved near infrared (NIR) indocyanine green (ICG)[1]. We
refer to these VNPs as optical viral ghosts (OVGs) since the constructs lack the genomic content of wild-type BMV. One
of our areas of interest is the application of OVGs for real-time intraoperative NIR fluorescence imaging of small
peritoneal ovarian tumor nodules. We target human epidermal growth factor receptor-2 (HER-2) expression in ovarian
cancer as a biomarker associated with ovarian cancer, since its over-expression is linked to the disease’s progression to
death. We functionalize the OVGs with anti-HER-2 monoclonal antibodies using reductive amination methods. We used
fluorescence imaging to visualize the SKOV-3 cells (high HER-2 expression) after incubation with free ICG, OVGs, and
functionalized OVGs. Our results suggest the possibility of using anti-HER2 conjugated OVGs in conjunction with
cytoreductive surgery to detect small tumor nodules (<5cm) which currently are not excised during surgery.
Targeted delivery of therapeutic and imaging agents using surface modified nanovectors has been explored immensely in
recent years. The growing demand for site-specific and efficient delivery of nanovectors entails stable surface
conjugation of targeting moieties. We have developed a polymeric nanocapsule doped with Indocyanine green (ICG)
with potential for targeted and deep tissue optical imaging and phototherapy. Our ICG-loaded nanocapsules (ICG-NCs)
have potential for covalent coupling of various targeting moieties and materials due to presence of amine groups on the
surface. Here, we covalently bioconjugate polyethylene glycol(PEG)-coated ICG-NCs with monoclonal antibody against
HER2 through reductive amination-mediated procedures. The irreversible and stable bonds are formed between anti-
EGFR and aldehyde termini of PEG chains on the surface of ICG-NCs. We confirm the uptake of conjugated ICG-NCs
by ovarian cancer cells over-expressing HER2 using fluorescent confocal microscopy. The proposed process for
covalent attachment of anti-HER2 to PEGylated ICG-NCs can be used as a methodology for bioconjugation of various
antibodies to such nano-constrcuts, and provides the capability to use these optically active nano-probes for targeted
optical imaging of ovarian and other cancer types.
Near-infrared (NIR) optically active nanoparticles are promising exogenous chromophores for applications in medical imaging and phototherapy. Since nanoparticles can be rapidly eliminated from the body by cells of the reticuloendothelial system, a thriving strategy to increase their blood circulation time is through surface modification with polyethylene glycol (PEG). We constructed polymeric nanocapsules loaded with indocyanine green (ICG), an FDA-approved NIR dye, and coated with aldehyde-terminated PEG. Using optical absorbance spectroscopy and flow cytometry, we investigated the effect of PEG coating and molecular weight (MW) of PEG [5000 and 30,000 Daltons (Da)] on the phagocytic content of human spleen macrophages incubated with ICG-containing nanocapsules (ICG-NCs) between 15 to 360 min. Our results indicate that surface coating with PEG is an effective method to reduce the phagocytic content of ICG-NCs within macrophages for at least up to 360 min of incubation time. Coating the surface of ICG-NCs with the low MW PEG results in lower phagocytic content of ICG-NCs within macrophages for at least up to 60 min of incubation time as compared to ICG-NCs coated with the high MW PEG. Surface coating of ICG-NCs with PEG is a promising approach to prolong vasculature circulation time of ICG for NIR imaging and phototherapeutic applications.
Optically active nanoparticles are widely pursued as exogenous chromophores in diagnostic imaging and phototherapeutic
applications. However, the blood circulation time of nanoparticles remains limited due to the rapid clearance of the
nanoparticles by reticuloendothelial system (RES). Coating with Polyethylene glycol (PEG) is a strategy to extend the
circulation time of nanoparticles. Here, we report synthesis and cellular studies of polymeric-based nanocapsules loaded with
Indocyanine green (ICG), an FDA approved near-infrared dye, and coated with PEG molecules of various molecular weights
through reductive amination. We report the effect of PEG's molecular weight on the uptake of these nanocapsules by human
spleen macrophages and hepatocytes using flow cytometry. Our results indicate that the phagocytic content of PEGylated
nanocapsules in human spleen macrophages was reduced as compared to uncoated nanocapsules. Among PEGylated
nanocapsules, low molecular weight (5000 Da) PEG-coated nanocapsules displayed lower intracellular uptake by spleen
macrophages than high molecular weight (30,000 Da) PEG-coated nanocapsules for up to 90 minutes. Encapsulation within
the polymeric nanocapsules reduced the hepatic content of ICG with normal human hepatocytes for up to two hours, while
the molecular weight of PEG did not have a statistically significant effect on the content of the nanocapsules in liver cells.
Our results suggest that reduced uptake of nanocapsules by RES cells can result in prolonged blood circulation time of these
nanoconstructs.
Indocyanine green (ICG) is an FDA-approved infrared chromophore used for various biomedical applications such as
cardiac and hepatic function evaluation, and ophthalmic angiography. Despite its clinical applications, freely dissolved
ICG binds non-specifically to various plasma proteins resulting in changes in its near infrared (NIR) emission properties
and rapid elimination from the vasculature. To overcome these shortcomings, we have encapsulated ICG within
polymeric nano-constructs composed of poly allylamine hydrochloride (PAH) cross-linked with di-sodium hydrogen
phosphate (Na2HPO4). To optimize the photophysical properties of nano-encapsulated ICG (NE-ICG) for clinical
imaging applications, we report measurements of fluorescent quantum yield (φ) of NE-ICG. Specifically, we constructed
capsules of three different diameters (~130, ~240, and ~450 nm). Our preliminary results indicate that NE-ICG shows
less quantum yield compared to freely-dissolved ICG. We determined that the 240 nm diameter capsule to have the
highest φ and 450 nm diameter capsules to have the least φ at room temperature.
Indocyanine green (ICG) is an FDA approved near infrared dye used in assessment of hepatic function and
ophthalmological vascular imaging. However, given the rapid clearance of ICG from the blood stream, its
imaging and phototherapeutic applications remain very limited. As a potential method to increase
circulation time of ICG, and extend its clinical applications, we have encapsulated ICG within polymeric
based nanoconstructs whose surface can be coated with various materials including polyethylene glycol
(PEG). To gain an understanding of the interaction between ICG-containing nanocapsules (ICG-NCs) and
vascular cells, we are characterizing the uptake of the nanocapsules coated with various materials by
human peripheral blood monocytes and human spleen macrophages using fluorescence microscopy.
Results of these studies will be useful in identifying the appropriate coating material that will result in
increased circulation time of ICG-NCs within the vasculature.
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