Photosensitizer-conjugated polyacrylamide nanoparticles were prepared for in vivo characterization of the minimally invasive and localized treatment of photodynamic therapy (PDT) on brain tumors. By incorporating a variety of nanoparticle matrixes, choosing methylene blue as a photosensitizer, and targeting the nanoparticle by the use of F3 peptide we have made nanoparticle-based PDT improvements to current PDT efficiency. Quantitative growth patterns were determined through visual observation of the tumorigenic response to various treatments by the use of an animal cranial window model. PDT treatments with methylene blue-polyacrylamide (MB-PAA) nanoparticles produced significant adjournment of tumor growth over control groups, clearly demonstrating the advantages of nanoparticle-based PDT agents for the eradication of local tumors, leading to the potential palliation of the advancing disease.
Hypoxia is a condition where a region of tissue has less than adequate oxygen. It is of particular importance in tumor biology, as the hypoxic core of tumors has been shown to impede the effectiveness of many therapies. We demonstrate a novel method for oxygen sensing in vivo, based on the photoacoustic lifetime measurement of an oxygen sensitive probe. The experimental results derived from the main artery in the rat tail indicated that the lifetime of the probe, quantified by the photoacoustic measurement, shows a good linear relationship with the blood oxygenation level in the targeted artery.
Indocyanine green (ICG) is an FDA approved tricarbocyanine dye. This dye, with a strong absorbance in the near
infrared (NIR) region, has been extensively used for fluorescence and photoacoustic imaging in vivo. ICG in its free
form, however, has a few drawbacks that limit its in vivo applications, such as non-targetability, tendency to form
aggregates which changes its optical properties, fast degradation, short plasma lifetime and reduced fluorescence at
body temperature. In order to bypass these inherent drawbacks, we demonstrate a polyacrylamide based nanocarrier
that was particularly designed to carry the negatively charged ICG molecules. These nanocarriers are biodegradable,
biocompatible and can be specifically targeted to any cell or tissue. Using these nanocarriers we avoid all the
problems associated with free ICG, such as degradation, aggregation and short plasma lifetime, and also enhance
demonstrate its ability towards photoacoustics and fluorescence imaging.
The determination of oxygen levels in blood and other tissues in vivo is critical for ensuring proper body functioning, for monitoring the status of many diseases, such as cancer, and for predicting the efficacy of therapy. Here we demonstrate, for the first time, a lifetime-based photoacoustic technique for the measurement of oxygen in vivo, using an oxygen sensitive dye, enabling real time quantification of blood oxygenation. The results from the main artery in the rat tail indicated that the lifetime of the dye, quantified by the photoacoustic technique, showed a linear relationship with the blood oxygenation levels in the targeted artery.
KEYWORDS: Nanosensors, Tumors, Nanoparticles, Luminescence, Calibration, 3D acquisition, Two photon excitation microscopy, 3D modeling, Two photon imaging, Tissues
Tumors are generally characterized by a pH lower than the surrounding tissues. The mapping of tumor pH is of great
importance as it plays a critical role in drug delivery and its effectiveness. Here we present a pH mapping technique
in tumor spheroids, using targeted, ratiometric, fluorescent, pH nano-sensor that is based on two-photon excitation.
Spheroids are micro-tumors that are widely used as an in-vitro three dimensional tumor model to study the different
properties of the tumor for the purpose of drug delivery, therapy etc. The nanosensor consists of 8-Hydroxypyrene-
1,3,6-trisulfonic acid (HPTS), a pH sensitive dye, encapsulated in polyacrylamide hydrogel nanoparticle matrix and
F3 peptide, conjugated to the nanoparticle's surface. The nanosensor has an average size of 68nm and contains
approximately 0.5% dye by weight. The fluorescence intensity ratio, at the two-photon excitation wavelengths of
900nm and 750nm, increases linearly in the pH range from 6.0 to 8.0 and is used to determine the pH of the local
environment. Our study reveals the pH distribution inside human cervix cancer spheroids (of different sizes) during
the various stages of their formation. This information can be used to develop more efficient drug delivery
mechanisms. The two-photon excitation used for this purpose is especially useful as it drastically minimizes both
photobleaching and autofluorescence, thus leading to an increase in the signal-to-noise ratio. It also enables deep
tissue imaging due to higher photon penetration depth.
Distinguishing the tumor from the background neo-plastic tissue is challenging for cancer surgery such as surgical
resection of glioma. Attempts have been made to use visible or fluorescent markers to delineate the tumors during
surgery. However, the systemic injection of the dyes requires high dose, resulting in negative side effects. A novel
method to delineate rat brain tumors intra-operatively, as well as post-operatively, using a highly sensitive photoacoustic
imaging technique enhanced by tumor targeting blue nanoparticle as contrast agent is demonstrated. The nanoparticles
are made of polyacrylamide (PAA) matrix with covalently linked Coomassie-Blue dye. They contain 7.0% dye and the
average size is 80nm. Their surface was conjugated with F3 peptide for active tumor targeting. These nanoparticles are
nontoxic, chemically inert and have long plasma circulation lifetime, making them suitable as nanodevices for imaging
using photoacoustics. Experiments on phantoms and rat brains tumors ex-vivo demonstrate the high sensitivity of
photoacoustic imaging in delineating the tumor, containing contrast agent at concentrations too low to be visualized by
eye. The control tumors without nanoparticles did not show any enhanced signal. This study shows that photoacoustic
imaging facilitated with the nanoparticle contrast agent could contribute to future surgical procedures for glioma.
A new method is developed to perform local measurements of fluorophore excited state lifetimes in turbid media without collecting the fluorescence emission. The method is based on a pump-probe approach where a first laser pulse excites the dye and then a second laser pulse is used for photoacoustic probing of the transient absorption. The photoacoustic response generated by the probe pulse is recorded by an ultrasound receiver. Repeating the measurement for increasing pump-probe time delays yields a series of photoacoustic signals that are used to extract the lifetime of the excited state. The method is validated by measuring the lifetime of an oxygen sensitive dye solution at different concentrations of dissolved oxygen. The dye is pumped with a 532-nm pulsed laser and the transient absorption at 740 nm is probed using a second pulsed laser system. The photoacoustic-based results are in close agreement with those obtained from time-dependent fluorescent measurements. The method can be extended to photoacoustic lifetime imaging by using a receiver array instead of a single receiver. Potential applications of this method include tissue oxygen imaging for cancer diagnostics and mapping molecular events such as resonant energy transfer and ion collisions in a biological environment.
We have developed a new method to perform local measurements of fluorophore excited state lifetimes in turbid
media without collecting the fluorescence emission. The method is based on a double pulse illumination where a first
laser pulse excites the dye and then a second laser is used for photoacoustic probing of the transient absorption. The
photoacoustic response generated by the probe pulse is recorded by an ultrasound receiver. Varying the time delay
between excitation and probing allows for tracking the relaxation dynamics of the excited state. The method was
validated by measuring the lifetime of an oxygen sensitive dye (Pt(II) octaethylporphine) solution at different
concentrations of dissolved oxygen. The dye was excited with a 532 nm pulsed laser and the transient absorption at 740
nm was probed using a second pulsed laser system. The photoacoustics based results coincide with those obtained from
simultaneous time-resolved fluorescent measurements. The method can be extended to photoacoustic lifetime imaging
by using a receiver array instead of a single receiver. This opens unique possibilities for non-invasive, clinical functional
imaging. For example, combined with oxygen sensitive dye, 3D imaging of tissue oxygenation could be developed for
accurate diagnosis of cancer tumors, better planning of radiation therapy, and monitoring efficacy of treatment. Other
potential applications include: in-vivo mapping of ion (e.g. Ca) concentration and dynamics and imaging of enzymes
activity and metabolic functions, as well as environmental studies in turbid media.
Brian Ross, Alnawaz Rehemtulla, Yong-Eun Koo, Ramachandra Reddy, Gwangseong Kim, Caleb Behrend, Sarah Buck, Randal Schneider, Martin Philbert, Ralph Weissleder, Raoul Kopelman
A paradigm for brain cancer detection, treatment, and monitoring uses synergistic, multifunctional, biomedical nanoparticles for: (1) external delivery to cancer cells of singlet oxygen and reactive oxygen species (ROS), but no drugs, thus avoiding multi-drug resistance, (2) photodynamic generation of singlet oxygen and ROS by a conserved critical mass of photosensitizer, (3) enhancement of magnetic relaxivity providing for MRI contrast, (4) control of plasma residence time, (5) specific cell targeting, (6) minimized toxicity, (7) measurement of tumor kill with diffusion MRI. The 40 nm polyacrylamide nanoparticles contained Photofrin, iron-oxide (or Gd), polyethylene glycol and targeting moieties. In-vivo tumor growth was halted and even reversed.
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