Jérôme Boutet

Drug development in oncology assisted by noninvasive optical imaging

Early and accurate detection of tumors, like the development of targeted treatments, is a major field of research in oncology. The generation of specific vectors, capable of transporting a drug or a contrast agent to the primary tumor site as well as to the remote (micro-) metastasis would be an asset for early diagnosis and cancer therapy. Our goal was to develop new treatments based on the use of tumor-targeted delivery of large biomolecules (DNA, siRNA, peptides, or nanoparticles), able to induce apoptosis while dodging the specific mechanisms developed by tumor cells to resist this programmed cell death. Nonetheless, the insufficient effectiveness of the vectorization systems is still a crucial issue. In this context, we generated new targeting vectors for drug and biomolecules delivery and developed several optical imaging systems for the follow-up and evaluation of these vectorization systems in live mice. Based on our recent work, we present a brief overview of how noninvasive optical imaging in small animals can accelerate the development of targeted therapeutics in oncology.

LipImage™ 815: novel dye-loaded lipid nanoparticles for long-term and sensitive in vivo near-infrared fluorescence imaging

Abstract. A new contrast agent, LipImage™ 815, has been designed and compared to previously described indocyanine green (ICG)-loaded lipid nanoparticles (ICG-lipidots®). Both contrast agents display similar size (50-nm diameter), zeta potential, high IC50 in cellular studies, near-infrared absorption and emission wavelengths in the “imaging window,” long-term shelf colloidal and optical stabilities with high brightness (>106  L mol−1 cm−1) in ready-to-use storage conditions in aqueous buffer (4°C in dark), therefore being promising fluorescence contrast agents for in vivo imaging. However, while ICG-lipidots® display a relatively short plasma lifetime, LipImage™ 815 circulates in blood for longer times, allowing the efficient uptake of fluorescence signal in human prostate cancer cells implanted in mice. Prolonged tumor labeling is observed for more than 21 days.

Fluorescence time-resolved imaging system embedded in an ultrasound prostate probe

Ultrasound imaging (US) of the prostate has a low specificity to distinguish tumors from the surrounding tissues. This limitation leads to systematic biopsies. Fluorescent diffuse optical imaging may represent an innovative approach to guide biopsies to tumors marked with high specificity contrast agents and therefore enable an early detection of prostate cancer. This article describes a time-resolved optical system embedded in a transrectal US probe, as well as the fluorescence reconstruction method and its performance. Optical measurements were performed using a pulsed laser, optical fibers and a time-resolved detection system. A novel fast reconstruction method was derived and used to locate a 45 µL ICG fluorescent inclusion at a concentration of 10 µM, in a liquid prostate phantom. Very high location accuracy (0.15 cm) was achieved after reconstruction, for different positions of the inclusion, in the three directions of space. The repeatability, tested with ten sequential measurements, was of the same order of magnitude. Influence of the input parameters (optical properties and lifetime) is presented. These results confirm the feasibility of using optical imaging for prostate guided biopsies.

Fluorescence diffuse optical tomography for free-space and multifluorophore studies

We present two major advances in preclinical fluorescence-enhanced diffuse optical tomography (fDOT) system and assess its performance. It is now possible to perform experiments without adaptation liquid or a glass plate over the animal, and our system is equipped with a filter wheel in order to discriminate two injected fluorophores. Evaluation carried out on characterization phantoms and in vivo on mice demonstrates enriched use of the system for biological studies on small animals.

Targeting tumors with cyclic RGD-conjugated lipid nanoparticles loaded with an IR780 NIR dye: In vitro and in vivo evaluation

Like several 50nm-large nanocarriers, lipid nanoparticles (LNPs) can passively accumulate in tumors through the Enhanced Permeability and Retention (EPR) effect. In this study, we developed PEGylated LNPs loaded with IR780 iodide as a contrast agent for NIR fluorescence imaging and modified them with cyclic RGD peptides in order to target integrin avβ3. We demonstrate a specific targeting of the receptor with cRGD-LNPs but not with cRAD-LNP and standard LNP using HEK293(β3), HEK293(β3)-αvRFP, DU145 and PC3 cell lines. We also demonstrate that cRGD-LNPs bind to αvβ3, interfere with cell adhesion to vitronectin and co-internalize with αvβ3 within one hour. We then investigated their biodistribution and tumor targeting in mice bearing DU145 or M21 tumors. We observed no significant differences between cRGD-LNP and the non-targeted ones regarding their biodistribution and accumulation/retention in tumors. This suggested that despite an efficient formulation of the cRGD-LNPs, the cRGD-mediated targeting was not increasing the total amount of LNP that can already accumulate passively in the subcutaneous tumors via the EPR effect.

Enhancement of photoacoustic imaging quality by using CMUT technology: Experimental study

Photoacoustic (PA) signals are known to be wideband thanks to their N-shape. Yet ultrasound (US) transducers commonly used for PA imaging use piezoelectric technology (PZT) and hence present a limited bandwidth in reception. Thus, PA signals can not be fully acquired and are filtered by the receiver. Capacitive micromachined ultrasonic transducer (cMUT) technology has emerged as an alternative to conventional PZT transducers in the field of medical imaging. Among the interesting properties offered by this technology, a theoretically infinite bandwidth in reception may be reached. In this work the interest of cMUT larger bandwidth for PA imaging is studied by comparing a cMUT and a classical PZT linear US probes. PA acquisitions have been made on several homemade bimodal phantoms whose properties are well-known. For each phantom, acquisitions have been realized with both probes in the same conditions. The probes characteristics have been verified for the reception of PA signals and three criteria have been evaluated to compare the two probes: signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and the maximal amplitude of PA signals. The cMUT probe presents indeed a larger fractional bandwidth in reception and the SNR and CNR are enhanced by at least 6dB. Furthermore, the sensitivity of the cMUT probe is higher and it receives 4 to 5 times more signal than the PZT probe. Therefore, this work highlights the potential of cMUT technology for PA imaging through quantitative as well as qualitative parameters.

Localization of fluorescence marked prostate tumor with time-resolved diffuse optical tomography

To increase prostate cancer diagnosis sensibility, we propose to add an optical modality to an US biopsy tool to localize fluorophore marked tumors. Optical signals are acquired on a time-resolved acquisition chain composed by a 770 nm femtosecond laser source and a four channels TCSPC device. The fluorescence concentration is reconstructed by using intensity and mean time of flight acquired from each time-resolved source-detector signal. Validation experiments are performed on a phantom mimicking prostate both on its optical and ultrasound properties with 10 μmol/L ICG 1 cm deep double fluorescent inclusions to simulate marked tumors. An exhaustive search algorithm succeeded in reconstructing the two distinct fluorescence dots with correct locations.

Advances in bi-modal optical and ultrasound detection of prostate cancer diagnosis

Prostate cancer diagnosis is based on PSA dosage and digital rectal examination. In case of positive test, a biopsy is conducted and guided by ultrasound imaging. Today, however, as ultrasound imaging is not able to precisely detect tumors, some biopsies have to be performed in the prostate and the only way to improve detection is to increase the number of those uncomfortable biopsies. In order to decrease this number and to improve the patient wellness, we are studying a way to couple ultrasound and fluorescence optical imaging on an endorectal probe. The ultrasounds are used to get morphological information on the prostate and the optical system to detect and to localize fluorophore marked tumors. To support the development of such a system, we have carried out a new tissue-mimicking phantom which represents the three different kind of tissue concerned during prostate endorectal examination: prostate, rectum, surrounding tissues. It was imaged by ultrasound and by fluorescence diffuse optical imaging. We have proved that the optical system is able to detect and to localize a fluorescing inclusion at different depth inside the phantom which has then been superimposed to the morphological image provided by the ultrasounds.

A quantitative study to design an experimental setup for photoacoustic imaging

During the last decade, a new modality called photoacoustic imaging has emerged. The increasing interest for this new modality is due to the fact that it combines advantages of ultrasound and optical imaging, i.e. the high contrast due to optical absorption and the low acoustic attenuation in biological tissues. It is thus possible to study vascularization because blood has high optical absorption coefficient. Papers in the literature often focus on applications and rarely discuss quantitative parameters. The goal of this paper is to provide quantitative elements to design an acquisition setup. By defining the targeted resolution and penetration depth, it is then possible to evaluate which kind of excitation and reception systems have to be used. First, we recall theoretical background related to photoacoustic effect before to describe the experiments based on a nanosecond laser at 1064 nm and 2.25–5 MHz transducers. Second, we present results about the relation linking fluence laser to signal amplitude and axial and lateral resolutions of our acquisition setup. We verify the linear relation between fluence and amplitude before to estimate axial resolution at 550 μm for a 2.25 MHz ultrasonic transducer. Concerning lateral resolution, we show that a reconstruction technique based on curvilinear acquisition of 30 lines improves it by a factor of 3 compared to a lateral displacement. Future works will include improvement of lateral resolution using probes, like in ultrasound imaging, instead of single-element transducers.

Optical tomograph optimized for tumor detection inside highly absorbent organs

This paper presents a tomograph for small animal fluorescence imaging. The compact and cost-effective system described in this article was designed to address the problem of tumor detection inside highly absorbent heterogeneous organs, such as lungs. To validate the tomographs ability to detect cancerous nodules inside lungs, in vivo tumor growth was studied on seven cancerous mice bearing murine mammary tumors marked with Alexa Fluor 700. They were successively imaged 10, 12, and 14 days after the primary tumor implantation. The fluorescence maps were compared over this time period. As expected, the reconstructed fluorescence increases with the tumor growth stage.