Low density lipoproteins (LDL) have long been recognized as a potential delivery system for exogenous
agents. Imaging agents or drugs can be attached to LDL through surface loading, protein loading or core
loading methods. The LDL delivery system has received considerable attention particularly among cancer
biologists as it was observed that numerous cancers over-express the low density lipoprotein receptor
(LDLR). In this paper we investigate the utility of LDL to transport optical imaging contrast agents for
caner detection. The method of loading fluorophores into the core of LDL is attractive as it behaves like an
activatable contrast agent. Surface and protein labeled methods also prove to be effective strategies for
tracing LDL nanoparticle activity. The strengths and limitations of the LDL carrier system are discussed
and novel approaches for imaging cancer with LDL nanoparticles are highlighted.
To improve the labeling efficiency of a low-density lipoprotein (LDL)-based photosensitizer (PS) for achieving high probe to protein payload, a tetra-t-butyl silicon phthalocyanine bearing two oleate moieties at its axial positions, SiPcBOA, is designed and synthesized. Using this novel strategy, SiPcBOA reconstituted LDL (r-SiPcBOA-LDL) with a very high payload (SiPcBOA to LDL molar ratio >3000 to 35001:1) is obtained. Using electron microscopy, we find reconstituted LDL (rLDL) with such a high payload essentially retains the mean particle size of native LDL. Since acetylated LDL binds to scavenger receptors of endothelial and microglial cells instead of LDLR, SiPcBOA reconstituted acetylated LDL (r-SiPcBOA-AcLDL) is also prepared to serve as a negative control to validate the LDL receptor (LDLR) targeting specificity. Confocal microscopy studies demonstrate that the internalization of r-SiPcBOA-LDL by human hepatoblastoma G2 (HepG2) tumor cells is mediated by LDLR pathway. The in vitro photodynamic therapy (PDT) response of HepG2 cells to r-SiPcBOA-LDL is compared to SiPcBOA (free drug control) using a clonogenic assay. The slopes of the linear regression fit to the logarithmic data for these two plots are significantly different from each other (p=0.0007), indicating greatly enhanced efficacy of LDLR-targeted PDT.
Current limitation of both near-infrared (NIR) tumor imaging and photodynamic therapy (PDT) is their lack of sufficient tumor-to-tissue contrast due to the relatively non-specific nature of delivering dye to the tumor, which has led to false negatives for NIR imaging and inadequate therapeutic ratio for PDT. Hence, agents targeting “cancer signatures”, i.e. molecules that accumulate selectively in cancer cells, are particular attractive. One of these signatures is low-density-lipoprotein receptor (LDLR), which is overexpressed in many tumors. We have developed pyropheophorbide cholesterol oleate reconstituted LDL as a LDLR-targeting photosensitizer (PS) and demonstrated its LDLR-mediated uptake in vitro and in vivo. To improve the labeling efficiency for achieving high probe/protein ratio, tetra-t-butyl silicon phthalocyanine bearing two oleate moieties at its axial positions, (tBu)4SiPcBOA, was designed and synthesized. This compound was designed to 1) prevent the PS aggregation; 2) improve the PS solubility in non-polar solvent; and 3) maximize the PS binding to LDL phospholipid monolayer. Using this novel strategy, (tBu)4SiPcBOA was reconstituted into LDL (r-SiPcBOA-LDL) with a very high payload (500:1 molar ratio). In addition, (tBu)4SiPcBOA reconstituted acetylated LDL (r-SiPcBOA)-AcLDL with similar payload was also prepared. Since Ac-LDL cannot bind to LDLR, (r-SiPcBOA)-AcLDL can serve as the negative control to evaluate LDLR targeting specificity. For biological evaluation of these new agents, confocal microscopy and in vitro PDT protocols were performed using LDLR-overexpressing human hepatoblastoma G2 (HepG2) tumor model. These studies suggest that LDL serves as a delivery vehicle to bring large amount of the NIR/PDT agents selectively to tumor cells overexpressing LDLR.
A new concept for photodynamic therapy (PDT) has been developed based on incorporating a photosensitizer (PS) and a singlet oxygen (1O2) quenching/scavenging molecule (Q) onto a disease-targeting carrier, such that the PS becomes activatable by light only when targeting has occurred. This has the potential to give very high disease specificity in PDT treatment. The first model compound designed using this concept was synthesized containing a pyropheophorbide as the PS and a carotenoid as the 1O2 quencher. These were kept in close proximity by the self-folding of a caspase-3 specific peptide sequence. Upon caspase-3-induced cleavage, the 1O2 production increase has been validated by direct 1O2 luminescence and lifetime measurements, providing proof-of-concept of this 'PDT beacon.'
The effect of photodynamic therapy (PDT) treatment on the metabolic state of tumor mitochondria is investigated by imaging of tumor redox status. PDT is performed using the photosensitizer pyropheophorbide-2-deoxyglucosamide (Pyro-2DG), which utilizes the glucose import pathway. It is found that Pyro-2DG-induced PDT resulting in a highly oxidized state of tumor mitochondria. This is determined from the redox ratio changes derived from the intrinsic oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) [i.e., reduced nicotinamide adenine dinucleotide (NADH)] fluorescence signals observed using a cryoimager. Thus, the redox ratio is a sensitive indicator for providing reliable and informative measurements of PDT-induced tissue damage. In the PDT treated region of the tumor, highly oxidized flavoprotein and diminishing NADH fluorescence is detected, suggesting that flavoprotein and NADH are oxidized by singlet oxygen produced in the photosensitization process.
A current limitation of NIR imaging is the lack of sufficient tumor-to-tissue contrast due to the nonspecific nature of delivering the dye to the tumor.
Utilizing one of the most important cancer signatures, the overexpression of GLUTs, we have developed a series of 2-deoxyglucose conjugated NIR dyes (NIR-
2DG) to enhance tumor selectivity. This uptake mechanism is first confirmed in vitro by confocal microscopy and flow cytometry studies with various
cancer cells. Following intravenously administration to animals, NIR-2DGs are selectively accumulated in the tumor compared to the surrounding normal
tissue as observed by ex vivo and in vivo fluorescence imaging techniques.
KEYWORDS: Tumors, Luminescence, Animal model studies, Scattering, Near infrared, Phased arrays, In vivo imaging, Breast, Signal detection, Imaging systems
We have developed a novel imaging system for determining the localization of tumors labeled by fluorescent contrast agents and embedded several centimeters inside the highly scattered medium. This frequency-domain system utilizes the phased cancellation configuration with a goniometric probe. The instrumentation performance on the phantom test can detect 3 mm diameter sphere filled with 1 nM fluorescent dye, Indocyanine Green (ICG), and 3 cm deep inside the scattering medium with similar optical properties as human breast tissue within a 1 mm localization confidence. Mouse tumor model immersed in appropriate scattering/absorbing medium is used for animal test. Intra-tumor injection of ICG demonstrates the localization of the tumor (5 mm in diameter) submerged 3 cm deep inside the highly scattered medium with 2 mm position error. Results with NIR804-D-Glucosamide on the AR42J tumor bearing nude mouse are also presented with 3 mm localization error. The accuracy of the localization suggest that this system would be helpful to guide the clinical fine-needle biopsy for early breast cancer detection.
We utilized the nude mouse model bearing the human hepatoblastoma G2 (HepG2) tumor and B-16 Murine Melanoma tumor to study the delivery and detection of the reconstituted Pyropheophorbide Cholesterol Oleate (r-pyroCE) molecular beacon. The delivery vehicle, low-density lipoprotein (LDL), labeled with the porphyrin derivative, was employed in response of the overexpression of LDL receptors in the HepG2 tumor. The B-16 melanoma tumor was also observed in this study for its overexpression of the LDL receptors. The tumors were imaged using the 3D low temperature scanner to produce images throughout several sliced sections of each tumor. The fluorescence signal of the pyropheophorbide was detected at 720nm when excited at 670nm in the tumor tissue. The uniform distribution of the signal in the HepG2 tumor shows extravasation of the beacon from the blood vessels. The B-16 tumor did not exhibit strong fluorescent signals and successful delivery as the HepG2 tumor outside the blood vessels and into the tumor tissue.
By using composite self-focus rod, LD-pumped frequency doubling Nd:YVO4 laser and 3D fine regulation stand, a new Fizeau-type interferometer is developed. It can apply to measuring the micro-vibration and deformation of micro- surface at the same time. The theoretical analysis and the experimental results for the interferometer of self-focus sensor are given.
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