Patent Application
20100028261
There is a need for reliable, non-invasive imaging tools to detect, diagnose, and characterize cancer—one of the leading causes of death in the United States. The early detection of cancer is necessary for effective therapeutic outcome and is a primary indicator for long term survival. Moreover, demarcating tumor boundaries with high specificity is required to direct therapeutic interventions to tumor location and cause less or no damage to the surrounding healthy tissue.
Current imaging modalities suffer from many drawbacks. Optical imaging, for example, suffers from a shallow penetration depth on the order of millimeters. Additionally, ionizing imaging modalities, such as X-ray, CT, and PET, present safety concerns. Furthermore, current technologies employed in cancer treatments cause surrounding healthy tissue damage along with tumor necrosis.
Biological processes that lead to cancer occur at the molecular level. Nanotechnology offers unprecedented access to the machinery of living cells, and therefore provides the opportunity to study and interact with normal and cancerous cells in real time, at the molecular and cellular scales, and during the earliest stages of the cancer process. Studies have shown gold nanoparticles can be functionalized with antibodies to specifically bind to molecular markers that are indicative of highly proliferative cells or are overexpressed in different types of cancer.
Photoacoustic imaging is a technique that can provide functional information based on differences in optical absorption properties of the tissue constituents. The absorption of electromagnetic energy, such as light, and the subsequent emission of an acoustic wave by the tissue is the premise of photoacoustic imaging. Specifically for photoacoustic imaging, the tissue is irradiated with nanosecond pulses of low energy laser light. Broadband ultrasonic acoustic waves are generated within the irradiated volume, as the tissue absorbs the light and then undergoes rapid thermoelastic expansion. An ultrasonic sensor and associated receiver electronics are used to acquire the photoacoustic signal.
Some specific example embodiments of the disclosure may be understood by referring, in part, to the following description and the accompanying drawings.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.
The present disclosure generally relates to methods of imaging. More particularly, the present disclosure relates to photoacoustic microscopy methods for selectively imaging biological tissue.
One of the many advantages of the methods of the present disclosure is that photoacoustic imaging is a non-ionizing imaging method. Another advantage is that little or no additional equipment is needed for therapy. Similarly, the methods of the present disclosure provide for sequential monitoring of biological tissue during therapy.
In one embodiment, the present disclosure relates to a method comprising providing a bioconjugate, providing a biological tissue, contacting the biological tissue with the bioconjugate, irradiating the biological tissue to generate an acoustic wave, and detecting the acoustic wave. As used herein, the term “bioconjugate” is defined to include nanoparticles that have been functionalized with a biologically active moiety.
In certain embodiments, bioconjugates that may be used in conjunction with the methods of the present disclosure may comprise nanoparticles that are functionalized to specifically bind to a molecular marker. For example, highly proliferative or cancerous epithelial cells tend to overexpress epithelial growth factor receptor (“EGFR”). Thus, for embodiments wherein EGFR is the molecular marker of interest, one example of a bioconjugate that may be used in conjunction with the methods of the present disclosure includes gold nanoparticles that have been functionalized with anti-EGFR antibody. By way of explanation, and not of limitation, the anti-EGFR moiety of the bioconjugate may act as a targeting moiety and cause the bioconjugate particles to aggregate on the cellular membranes of cells that overexpress EGFR. This aggregation may lead to plasmon resonance coupling between nanoparticles and a red shift in the plasmon resonance frequency of the gold nanoparticle assembly. As used herein, the term “plasmonic nanoparticle” is defined to include any nanoparticle capable of exhibiting plasmon resonance coupling. The red-shift, among other things, may provide the opportunity to differentiate cancer cells from surrounding benign cells by using a combination of labeling with gold nanoparticles and multi-wavelength illumination.
Other suitable biologically active moieties include, but are not limited to, chlorotoxin, which specifically binds to neural gliomas, and the RGD peptide fragment (arginine-glycine-asparagine), which binds to integrins that are prevalent in tumor vasculature. Antibodies that target telomerase and matrix metalloproteinases may also be suitable moieties. The choice of a particular bioconjugate may depend, among other things, upon the tissue type to imaged, the target cell type, and the nanoparticle composition.
For bioconjugates comprising gold nanoparticles, the gold nanoparticles can be used as contrast agents in photoacoustic imaging, because of their strong optical absorption and scattering properties, and as therapeutic agents in photothermal therapy. As one of ordinary skill in the art is aware, the absorbance spectra of the gold nanoparticles can be modified by varying their shape and size. The gold nanoparticles can be tuned to resonate in the NIR region as light has higher penetration depth in the tissue at these wavelengths.
Biological tissues that are suitable for use with the methods of the present disclosure include any tissue that contains a selective receptor for a biologically active moiety of a bioconjugate. An example of such tissues includes, but is not limited to, epithelial tissue.
The imaging systems of the present disclosure generally comprise a light source and an ultrasonic sensor. Example light sources may include, but are not limited to, tunable pulsed lasers and fixed frequency pulsed lasers. Example ultrasonic sensors may include, but are not limited to, transducers. Examples of suitable transducers may include piezoelectric films, such as polyvinylidene fluoride, optical transducers, and optical interferometers. The imaging systems of the present disclosure may also comprise additional electronic and mechanical components such as a pulser/receiver, a digitizer, a motion controller, a three-dimensional positioning stage, and/or a delay switch. One of ordinary skill in the art, with the benefit of this disclosure, will recognize additional electronic and mechanical components that may be suitable for use in the methods of the present invention.
In certain embodiments, the ultrasonic sensor of an imaging system may also serve as a source of pulsed sound waves utilized to obtain an ultrasound image of the biological tissue. In such embodiments, a delay switch may be coupled to a synchronous trigger of a laser such that, after a photoacoustic image has been acquired, the acoustic detector will itself emit pulsed sound waves. The ultrasonic sensor may then detect echoes of these pulsed sound waves so that the echoes may be utilized to obtain an ultrasound image of the biological tissue. An example of an embodiment of this type imaging system is depicted in
In certain embodiments, the aggregation of bioconjugates, for example gold nanoparticles conjugated with anti-EGFR antibodies, may be exploited to undertake molecule specific phototherapy. In such embodiments, the targeted bioconjugates may be used as guiding templates to create localized necrosis. This creation of localized necrosis, among other things, may result in little or no damage to healthy surrounding tissue. Specifically, localized necrosis may be caused by tissue ablation utilizing laser pulses of an energy higher than that required for photoacoustic imaging. Optionally, the progression of phototherapy may be monitored by ultrasound and/or photoacoustic imaging techniques as described herein.
In certain embodiments, the bioconjugates described herein may be used in other applications, including contrast agents for magnetic resonance imaging (MRI) or vascular imaging. Such embodiments may utilize the imaging systems described herein, with or without modifications for such applications that will be recognizable by one of ordinary skill in the art, with the benefit of this disclosure. In such embodiments, the bioconjugate composition may depend upon, among other things, the composition and location of the tissue and/or cell target to be imaged and the imaging system used.
In certain embodiments, the methods of the present disclosure may be used to monitor functional and morphological changes in tissue growth, including for example, in a variety of tissue engineering applications.
To facilitate a better understanding of the present disclosure, the following examples of certain aspects of some embodiments are given. In no way should the following examples be read to limit, or define, the entire scope of the invention.
Specifically, three epithelial tissue phantoms consisting of human epithelial carcinoma cells (A431 keratinocyte) were used: (1) the control tissue sample with no gold nanoparticles; (2) the targeted tissue sample labeled with EGFR targeted gold nanoparticles; and (3) the non-targeted sample with nanoparticles coated with a polyethylene glycol-thiol (PEG-SH) layer which has no molecular specificity.
The 50 nm gold particles were synthesized via citrate reduction of HAuCl4 under reflux. Anti-EGFR monoclonal antibody (clone 225) was purchased from Sigma and purified using a Centricon 100 kD MWCO filter. Antibodies were conjugated with gold nanoparticles using a protocol described in J. S. Aaron, J. OH, T. A. Larson, S. Kumar, T. E. Milner, and K. V. Sokolov, “Increased Optical Contrast in Imaging of Epidermal Growth fact Receptor Using Magnetically Actuated Hybrid Gold/Iron Oxide Nanoparticles,” Opt Express, (to be published), the relevant portions of which are herein incorporated by reference. Briefly, carbohydrate moieties on the antibodies' Fc region were oxidized to aldehyde groups via exposure to 100 mM NaIO4 for 30 minutes and were allowed to covalently bind to a hydrazide portion of the bifunctional hydrazide-PEG-thiol linker (Sensopath Technologies, Inc.) to facilitate nanoparticle conjugation. This Ab-linker solution was diluted in HEPES pH 8 to 5 μg Ab/mL and mixed 1:1 with the colloid suspension (1012 particles/mL) and allowed to conjugate via a thiol-gold binding reaction on a shaker at room temperature for 30 minutes. Subsequently, a small volume of PEG-SH (M.W. 2 kD, Shearwater) was added and allowed to react for another 30 minutes to passivate any remaining gold surface on the particles. To separate the conjugate from unbound antibody, the suspension was spun down at 1000 g for 30 minutes in the presence of 0.01% PEG polymer (M.W. 15 kD, Sigma) which was added as a surfactant to prevent aggregation during centrifugation. The pellet was resuspended in a 2% PEG (M.W. 15 kD, Sigma) in 1×PBS solution at the original particle concentration of 1012 particles/mL. Particles for the non-targeted sample were conjugated only with PEG-SH and resuspended in a 2% PEG solution.
The A431 cells were purchased from American Type Culture Collection and cultured in DMEM supplemented with 5% fetal bovine serum (FBS) at 37° C. in a 5% CO2 environment. Cells were harvested and resuspended in DMEM at a concentration of 2·106 cells/mL and divided into three 450 μl aliquots. One of the aliquots was mixed with an equal volume of the anti-EGFR gold bioconjugate solution and allowed to interact for 45 minutes at room temperature. This sample was named the targeted sample. The other two aliquots were not exposed to the nanoparticles. The three cell suspensions were then spun down at 200 g and resuspended separately using 250 μL aliquots of a buffered collagen solution (2.1 mg/mL, pH 7.4). To determine the amount of nanoparticles attached to cells in the targeted sample, the optical density of the solution of gold bioconjugates at concentration used for labeling was compared to the optical density of the supernatant obtained after the labeled cells were spun down. The UV-Vis measurements showed ca. 260,000 particles per cell corresponding to approximately 25% of the total number of receptors per cell. The targeted sample contained approximately 4·1011 gold nanoparticles/mL. Approximately 4·1012 PEGylated Au particles/mL were added to the buffered collagen solution of the non-targeted sample. The control tissue sample had no gold nanoparticles. The cell/collagen solutions (200 μL) were pipetted into separate stacked spacers (0.5 mm silicone isolators, Molecular Probes) in Petri dishes for optical characterization and photoacoustic imaging. The cell/collagen solution in the Petri dish was allowed to gel in a 37° C. incubator for 1 hour. This procedure resulted in phantoms with randomly distributed cells in a three-dimensional collagen matrix. The 3-D arrangement of cells gives an opportunity to study imaging approaches having depth resolution. The phantoms were then covered with 50 μL of media and stored in an incubator for several hours prior to imaging.
The tissue phantoms were characterized using a Leica DM 6000 upright microscope in epi-illuminated darkfield mode. A 75 W Xenon light source was used for illumination. Images were collected through a 20×, 0.5 NA darkfield objective and detected using a Q-Imaging Retiga EXi ultra-sensitive 12-bit CCD camera.
The extinction spectra were collected with a PARISS hyperspectral imaging device (Lightform, Inc.) in transmitted brightfield mode and a halogen light source. The hyperspectral device was coupled to the Leica microscope and was used to measure the extinction spectra at each pixel in the image. A single vertical section of the sample image was projected onto a prism through a 25 μm slit. The prism spectrally dispersed the one-dimensional image onto a two-dimensional Q-imaging Retiga EXi CCD detector. The sample was translocated laterally via a piezoelectric stage and the imaging process was repeated to construct the three-dimensional hyperspectral data cube. The spatial resolution of hyperspectral image was 1.25 μm and the spectral resolution was 1 nm. Transmitted brightfield spectral data cubes were acquired from 20×300 μm areas and normalized to the illumination lamp spectra, which was acquired through a blank slide containing only 1×PBS.
The extinction spectra of the control sample, the targeted sample, and the non-targeted sample are shown in
The darkfield images of the control, targeted and non-targeted phantoms are presented in
A block diagram of the experimental setup for ultrasound and photoacoustic imaging is shown in
A Q-switched Nd:YAG laser operating at wavelength of 532 nm (5 ns pulses, 20 Hz pulse repetition frequency) was used to obtain photoacoustic images of the samples. The tissue phantoms were also imaged with a tunable OPO laser operating at a wavelength of 680 nm and capable of producing 7 ns pulses at 10 Hz pulse repetition frequency. The ultrasound and photoacoustic images were obtained by mechanically scanning the tissue samples over the desired region. The sampling interval of the mechanical scan (12 μm) was set to be smaller than half the beamwidth of the ultrasonic transducer (42 μm) to satisfy the Nyquist criterion. The photoacoustic response of the sample being imaged was captured using the same receiver electronics as ultrasonic imaging. Specifically the master trigger from the laser source, delayed by several microseconds, was sent to the pulser/receiver to initiate the pulse-echo ultrasound regime. The synchronous trigger from the laser also commenced the data acquisition with 8-bit, 500 MHz digitizer and the data was stored for offline processing. In addition, digital bandpass (20-70 MHz) filtering was employed to reduce noise in the signals. An acquired A-line, therefore, contained the spatially co-registered photoacoustic signal followed by the conventional ultrasound signal.
The ultrasonic images of the three tissue phantoms are presented in of
At 532 nm laser irradiation, the photoacoustic image of the non-targeted sample (
Bioconjugation of gold nanoparticles and preparation of gelatin solution with MDA-MB-468 cells
50 nm gold particles were synthesized via citrate reduction of HAuCl4 under reflux. Anti-EGFR monoclonal antibody (clone 225) was purchased from Sigma (Sigma-Aldrich Inc., Saint Louis, Mo.) and purified using a Centricon 100 kD MWCO filter. The carbohydrate moieties on the antibodies' Fc region were oxidized to aldehyde groups via exposure to 100 mM NaIO4 for 30 minutes and were allowed to covalently bind to a hydrazide portion of the bifunctional hydrazide-PEG-thiol linker (Sensopath Technologies, Inc.) to facilitate nanoparticle conjugation. Diluent of Ab-linker was exchanged to HEPES pH 8 to the final concentration of antibody 50 μg Ab/mL. This solution was mixed 1:10 with the gold colloid suspension (4×1010 particles/mL) and allowed to conjugate via a thiol-gold binding reaction on a shaker at room temperature for 30 minutes. Subsequently, a small volume of PEG-SH (M.W. 2 kD, Shearwater) was added and allowed to react for another 30 minutes to passivate any remaining gold surface on the particles. To separate the conjugate from unbound antibody, the suspension was centrifuged at 1000×g for 30 minutes in the presence of 0.01% PEG polymer (M.W. 15 kD, Sigma) which was added as a surfactant to prevent aggregation during centrifugation. The pellet was resuspended in a phenol red free DMEM at the concentration of 4·1011 particles/mL. Particles for the non-targeted injection were conjugated only with PEG-SH and centrifuged.
The MDA-MB-468 cells were cultured in MEM supplemented with 10% fetal bovine serum at 37° C. in a 5% CO2 environment. To specifically label the cells with gold nanoparticles, the cells were harvested and resuspended in anti-EGFR gold conjugate solution at a concentration of 2·107 cells/mL and incubated for one hour at 37° C. Brief estimation of number of gold conjugates bound to cells gave ˜200,000 conjugates/cell. The cells harvested and resuspended in phenol red free DMEM were used for the second gelatin solution. Both cells aliquots were centrifuged and the supernatant was removed. Cells labeled with targeted gold nanoparticles were resuspended in warm (˜37° C.) gelatin solution (10% by weight) at a concentration of 9·106 cells/mL. The second aliquot of was resuspended in gelatin solution (10% by weight) and PEGylated gold nanoparticles to obtain the final concentration of approximately 1012 gold nanoparticles/mL. Both the gelatin suspensions were maintained at approximately 37° C. and were injected into the mouse abdomen using 30-gauge needle syringe.
Intraperitoneal Injection of Gelatin Solution in Mouse
An euthanized BL6 mouse was obtained from the Animal Resource Center at The University of Texas at Austin. A commercially available depilatory solution was used to remove hair from the abdominal region of the mouse. To mimic a tumor specifically targeted with gold nanoparticles, 500 μL gelatin solution with MDA-MB-468 (breast adenocarcinoma) cells labeled with EGFR targeted gold nanoparticles was injected into the abdominal cavity of the mouse (
Experimental Setup for Combined Photoacoustic and Ultrasound Imaging
A block diagram of the experimental setup for the combined photoacoustic and ultrasound imaging is shown in
The mouse was placed in a water tank attached to a 3-D positioning stage. The 2-D photoacoustic and ultrasound images were obtained by mechanically scanning over the desired region with 100 μm lateral steps to satisfy Nyquist criterion. At each step, the pulsed laser light irradiated the sample and the trigger signal from the laser source initiated the data acquisition by an 8-bit, 500 MHz digitizer. The same trigger signal, delayed by several microseconds, was sent to the pulser/receiver to initiate the pulse-echo ultrasound imaging. Therefore, a captured A-line contained the photoacoustic signal and the conventional ultrasound radio-frequency (RF) data separated by the user defined delay. The A-line records obtained at each lateral step of the mechanical scan were processed offline to obtain spatially co-registered 2-D photoacoustic and ultrasound images. During the offline processing, the photoacoustic and ultrasound signals are extracted from the A-line records and a digital bandpass (5-45 MHz) filter was applied to these raw RF signals to reduce noise. The analytic signals obtained from the photoacoustic and ultrasound RF data were spatially interpolated. The photoacoustic image was overlaid on the corresponding ultrasound image in the region of interest and displayed over a 40 dB dynamic range.
The combined ultrasound and photoacoustic images of the mouse abdomen before (
In congruence with the absorbance spectra shown in
While the photoacoustic images shown in
To ensure molecular specificity, A431 cells were exposed to excess anti-EGFR antibody (C225) in PBS to block available receptors. A separate aliquot of A431 cells was exposed to non-specific (anti-goat) antibody to verify that the blocking was molecular specific. A431 cells not exposed to antibody were used as the positive control. Finally, MDA-MB-435 cells, which do not express EGFR, were used as the negative control. Targeted anti-EGFR nanoparticles were added to the two blocked samples and the positive and negative controls and allowed to interact for 20 minutes. The suspensions were then centrifuged, the O.D. of the supernatants were collected and compared with the original nanoparticle solution (diluted appropriately in PBS) to determine labeling efficiency, and the cells were imaged in dark-field reflectance mode to verify the results (
Ten nanometer diameter iron oxide nanoparticles were synthesized via reduction of FeCl2 and FeCl3 in a 2:1 molar ratio. Gold ions were reduced onto the surface of the iron via an iterative hydroxylamine seeding technique resulting in ca. 50 nm diameter particles. Following synthesis the particles were functionalized with anti-EGFR Ab (Neomarker c225). The nanoparticles were injected into a mouse to demonstrate in vivo MR contrast. T1-, T2-, and T2*-weighted images were collected before and after injection of 100 uL, 1010 particles/ml into the abdominal fat pad of a normal mouse. Imaging was done using a 4.7 T Biospec experimental MR system (Bruker Biospin MRI, Billerica, Mass., USA). The functionalized nanoparticles were clearly distinguished in a mouse in vivo, providing negative T2 and T2* contrast. Representative T2-weighted images are shown in
Vascular imaging experiments were performed using tissue-mimicking phantoms simulating a vessel wall with occlusions (
First, 50 nm diameter spherical gold nanoparticles were synthesized via citrate reduction of HAuCl4 under reflux. Then, they were coated with polyethylene glycol-thiol (PEG-SH) to passivate the surface of the nanoparticles. A small volume of 10−4M mPEG-SH solution (MW 5000 kD, Shearwater) was added to the particle suspension and allowed to react for 30 minutes. After incubation, small volume of 2% PEG polymer (MW 15 kD, Sigma) was added to the mixture to serve as surfactant and prevent aggregation of nanoparticles during centrifugation. The mixture was then centrifuged at 2500×g for 30 minutes, resulting in the pellet of PEGylated gold nanoparticles. Finally, the pellet was resuspended in either warm 10% gelatin (35-40° C., temperature of gelatinization 24° C.) with approximate concentration of 2×1011 particles/ml or phenol red free DMEM.
The mouse monocytes—macrophages (J774A.1 cell line) are characterized by a high rate of non-specific uptake, similar to most cells of macrophage phenotype. Cells were cultured in DMEM supplemented with 5% FBS at 37° C. in 5% CO2. To load cells with gold nanoparticles (
The optical absorbance spectrum of pure gold nanoparticles has a peak at 530 nm, whereas the spectrum of macrophages loaded with gold particles has a peak in the region of 540 nm, i.e. there is a slight red shift of the spectrum compared to pure nanoparticles (
Prior to the imaging experiments, the intact and loaded cells were harvested, mixed with warm (35° C.) 10% gelatin and loaded into the corresponding compartments of the phantom (
During the imaging experiment, the phantom was placed in a water tank filled with a physiological solution (
Finally, the spectroscopic IVPA imaging was performed by 10 nm incremental change of optical wavelength from 680 nm to 750 nm. At each wavelength, the photoacoustic transients were detected and the energy of the photoacoustic response (integral or area under the curve) within small region of interest was computed. The wavelength-dependent behavior of the IVPA signal was then analyzed to reveal spectral properties of optical absorption within specific regions of the phantom.
The results of the IVUS and IVPA imaging studies at two discrete optical wavelengths—532 nm and 680 nm, are presented in
In contrast with IVUS images, two compartments with gold nanoparticles are visualized in the IVPA image obtained at 532 nm wavelength (
Therefore, macrophages loaded with gold nanoparticles can be easily identified in IVPA or combined IVUS/IVPA images (
To avoid the effect of strong optical absorption of blood, the imaging wavelength in IVPA imaging can be changed. Indeed, luminal blood has minimum optical absorption at 680 nm wavelength. In addition, there is a sharp decrease of optical absorption in non-aggregated (i.e., single) nanoparticles after 530 nm and specifically at 680 nm while the absorption spectra of aggregated nanoparticle is relatively broad (
The IVPA imaging studies performed at 680 nm wavelength (
To further confirm the presence of aggregated particles and potentially to differentiate the gold nanoparticles from other tissue constituents such as blood or lipid, spectroscopic IVPA imaging can be performed.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims.
This application is a continuation of International Application No. PCT/US08/053862 filed Feb. 13, 2008, which claims the benefit of U.S. Provisional Application Ser. No. 60/889,603 filed Feb. 13, 2007, both of which are incorporated herein by reference.
This disclosure was made with support under Grant Numbers EB008101, EB004963, CA110079 and CA103830, awarded by National Institutes of Health. The U.S. government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60889603 | Feb 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US08/53862 | Feb 2008 | US |
| Child | 12539936 | US |
