Julien Gravier

Influence of size, surface coating and fine chemical composition on the in vitro reactivity and in vivo biodistribution of lipid nanocapsules versus lipid nanoemulsions in cancer models

UNLABELLED Lipid nanocapsules (LNCs) and lipid nanoemulsions (LNEs) are biomimetic synthetic nanocarriers. Their in vitro and in vivo performance was evaluated as a function of their size (25, 50 and 100 nm) and the surface PEG chain length. Analysis methods included complement activation test, particle uptake in macrophage and HEK293(β3) cells and biodistribution studies with tumor-grafted mice by fluorescence imaging. A particular attention was paid to keep the concentration of each nanocarrier and to the amount of fluorescent dye in comparable conditions between the in vitro and in vivo studies. Under these conditions, no significant differences were found among the three tested particle sizes and the two nanocarrier types. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers. Passive accumulation of LNCs and LNEs in different tumor types depended on the degree of tumor vascularization. FROM THE CLINICAL EDITOR This study of lipid nanocapsules and lipid nanoemulsions compares their vitro and in vivo performance as a function of size and surface PEG chain length, demonstrating no significant difference among the tested particle sizes. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers.

Phosphonated Near-Infrared Fluorophores for Biomedical Imaging of Bone†

The conventional method for creating targeted contrast agents is to conjugate separate targeting and fluorophore domains. A new strategy is based on the incorporation of targeting moieties into the non-delocalized structure of pentamethine and heptamethine indocyanines. Using the known affinity of phosphonates for bone minerals in a model system, two families of bifunctional molecules that target bone without requiring a traditional bisphosphonate are synthesized. With peak fluorescence emissions at approximately 700 or 800 nm, these molecules can be used for fluorescence-assisted resection and exploration (FLARE) dual-channel imaging. Longitudinal FLARE studies in mice demonstrate that phosphonated near-infrared fluorophores remain stable in bone for over five weeks, and histological analysis confirms their incorporation into the bone matrix. Taken together, a new strategy for creating ultra-compact, targeted near-infrared fluorophores for various bioimaging applications is described.

Fate of paclitaxel lipid nanocapsules in intestinal mucus in view of their oral delivery.

The bioavailability of paclitaxel (Ptx) has previously been improved via its encapsulation in lipid nanocapsules (LNCs). In this work, the interactions between LNCs and intestinal mucus are studied because they are viewed as an important barrier to successful oral delivery. The rheological properties of different batches of pig intestinal mucus were studied under different conditions (the effect of hydration and the presence of LNCs). Fluorescence resonance energy transfer (FRET) was used to study the stability of LNCs in mucus at 37°C for at least 3 hours. Diffusion through 223, 446, and 893 μm mucus layers of 8.4, 16.8, and 42 μg/mL Ptx formulated as Taxol® (Bristol-Myers Squibb, Rueil-Malmaison, France) or encapsulated in LNCs (Ptx-LNCs) were investigated. The effect of the size of the LNCs on their diffusion was also investigated (range, 25–110 nm in diameter). Mucus behaves as a non-Newtonian gel with rheofluidifying properties and a flow threshold. The viscous (G″) and elastic (G′) moduli and flow threshold of the two mucus batches varied with water content, but G′ remained below G″. LNCs had no effect on mucus viscosity and flow threshold. The FRET efficiency remained at 78% after 3 hours. Because the destruction of the LNCs would lead to a FRET efficiency below 25%, these results suggest only a slight modification of LNCs after their contact with mucus. The diffusion of Taxol® and Ptx-LNCs in mucus decreases if the mucus layer is thicker. Interestingly, the apparent permeability across mucus is higher for Ptx-LNCs than for Taxol® for drug concentrations of 16.8 and 42 μg/mL Ptx (P<0.05). The diffusion of Ptx-LNCs through mucus is not size-dependent. This study shows that LNCs are stable in mucus, do not change mucus rheological properties, and improve Ptx diffusion at low concentrations, thus making these systems good candidates for Ptx oral delivery. The study of the physicochemical interaction between the LNC surface and its diffusion in mucus is now envisioned.

FRET Imaging Approaches for in Vitro and in Vivo Characterization of Synthetic Lipid Nanoparticles

DiI and DiD, two fluorophores able to interact by FRET (Förster resonance energy transfer), were coencapsulated in the core of lipid nanocapsules (LNCs) and nanoemulsions (LNEs), lipophilic reservoirs for the delivery of drugs. The ability of FRET imaging to provide information on the kinetics of dissociation of the nanoparticles in the presence of bovine serum albumin (BSA) or whole serum, or after incubation with cancer cells, and after systemic administration in tumor-bearing mice, was studied. Both microscopic and macroscopic imaging was performed to determine the behavior of the nanostructures in a biological environment. When 2 mg/mL FRET LNEs or LNCs were dispersed in buffer, in the presence of unloaded nanoparticles, BSA, or in whole serum, the presence of serum was the most active in destroying the particles. This occurred immediately with a diminution of 20% of FRET, then slowly, ending up with still 30% intact nanoparticles at 24 h. LNCs were internalized rapidly in cultured cells with the FRET signal decreasing within the first minutes of incubation, and then a plateau was reached and LNCs remained intact during 3 h. In contrast, LNEs were poorly internalized and were rapidly dissociated after internalization. Following their iv injection, LNCs appeared very stable in subcutaneous tumors implanted in mice. Intact particles were found using microscopic FRET determination on tumor sections 24 h after injection, that correlated well with the 8% calculated noninvasively on live animals. FRET investigations showed the potential to determine valid and reliable information about in vitro and in vivo behavior of nanoparticles.

Pancreas-Targeted NIR Fluorophores for Dual-Channel Image-Guided Abdominal Surgery

Objective: Pancreas-related complications are some of the most serious ones in abdominal surgery. The goal of this study was to develop and validate novel near-infrared (NIR) fluorophores that would enable real-time pancreas imaging to avoid the intraoperative pancreatic injury. Design: After initial screening of a large NIR fluorophore library, the performance of 3 selected pancreas-targeted 700 nm NIR fluorophores, T700-H, T700-F, and MB, were quantified in mice, rats, and pigs. Dose ranging using 25 and 100 nmol, and 2.5 µmol of T700-F, and its imaging kinetics over a 4 h period were tested in each species. Three different 800 nm NIR fluorophores were employed for dual-channel FLARE™ imaging in pigs: 2 μmol of ZW800-1 for vessels and kidney, 1 μmol of ZW800-3C for lymph nodes, and 2 μmol of ESNF31 for adrenal glands. Results: T700-F demonstrated the highest signal to background ratio (SBR), with peak SBR at 4 h postinjection in mice. In pigs, T700-F produced an SBR ≥ 2 against muscle, spleen, and lymph nodes for up to 8 h after a single intravenous injection. The combination of T700-F with each 800 nm NIR fluorophore provided simultaneous dual-channel intraoperative imaging of pancreas with surrounding organs in real time. Conclusion: Pancreas-targeted NIR fluorophores combined with the FLARE dual-channel imaging system enable the real-time intraoperative pancreas imaging which helps surgeons perform safer and more curative abdominal surgeries.

Renal Clearable Organic Nanocarriers for Bioimaging and Drug Delivery.

Renally cleared zwitterionic nanocarriers (H-Dots) are composed of ε-polylysine backbone for charge variations, near-infrared fluorophores for bioimaging, and β-cyclodextrins for potential drug delivery. H-Dots show ideal systemic circulation and rapid distribution and excrete from normal tissue/organ via renal excretion after complete targeting to the tumor site without nonspecific uptake by the immune system.

An MRI-based classification scheme to predict passive access of 5 to 50-nm large nanoparticles to tumors

Nanoparticles are useful tools in oncology because of their capacity to passively accumulate in tumors in particular via the enhanced permeability and retention (EPR) effect. However, the importance and reliability of this effect remains controversial and quite often unpredictable. In this preclinical study, we used optical imaging to detect the accumulation of three types of fluorescent nanoparticles in eight different subcutaneous and orthotopic tumor models, and dynamic contrast-enhanced and vessel size index Magnetic Resonance Imaging (MRI) to measure the functional parameters of these tumors. The results demonstrate that the permeability and blood volume fraction determined by MRI are useful parameters for predicting the capacity of a tumor to accumulate nanoparticles. Translated to a clinical situation, this strategy could help anticipate the EPR effect of a particular tumor and thus its accessibility to nanomedicines.

Charge and Hydrophobicity Effects of NIR Fluorophores on Bone-Specific Imaging

Recent advances in near-infrared (NIR) fluorescence imaging enabled real-time intraoperative detection of bone metastases, bone growth, and tissue microcalcification. Pamidronate (PAM) has been widely used for this purpose because of its high binding affinity toward bone and remarkable therapeutic effects. Herein we describe the development of a series of PAM-conjugated NIR fluorophores that varied in net charges and hydrophobicity, and compared their bone targeting efficiency, biodistribution, and blood clearance. Since the targeting moiety, PAM, is highly negatively charged but small, the overall in vivo bone targeting and biodistribution were mediated by the physicochemical properties of conjugated fluorophores.

Central C-C Bonding Increases Optical and Chemical Stability of NIR Fluorophores.

Functional near-infrared (NIR) fluorophores have played a major role in the recent advances in bioimaging. However, the optical and physicochemical stabilities of NIR fluorophores in the biological and physiological environment are still a challenge. Especially, the ether linkage on the meso carbon of heptamethine core is fragile when exposed to serum proteins or other amine-rich biomolecules. To solve such a structural limitation, a rigid carbon-carbon bond was installed onto the framework of ether-linked NIR fluorophores through the Suzuki coupling. The robust fluorophores replaced as ZW800-1C and ZW800-3C displayed enhanced optical and chemical stability in various solvents and a 100% warm serum environment (> 99%, 24 h). The biodistribution and clearance of C-C coupled ZW800 compounds were almost identical to the previously developed oxygen-substituted ZW800 compounds. When conjugated with a small molecule ligand, ZW800-1C maintained the identical stable form in warm serum (>98%, 24 h), while ZW800-1A hydrolyzed quickly after 4 h incubation (34%, 24 h).

Sentinel Lymph Node Mapping of Liver

AbstractBackgroundAlthough the sentinel lymph node (SLN) hypothesis has been applied to many tissues and organs, liver has remained unstudied. Currently, it is unclear whether hepatic SLNs even exist. If so, they could alter the management of intrahepatic cholangiocarcinoma and other hepatic malignancies by minimizing the extent of surgery while still providing precise nodal staging. This study investigated whether invisible yet tissue-penetrating near-infrared (NIR) fluorescent light can provide simultaneous identification of both the SLN and all other regional lymph nodes (RLNs) in the liver.MethodsIn 25 Yorkshire pigs, this study determined whether SLNs exist in liver and compared the effectiveness of two clinically available NIR fluorophores [methylene blue and indocyanine green (ICG)], and two novel NIR fluorophores previously described by our group (ESNF14 and ZW800-3C) for SLN and RLN mapping.ResultsIn this study, ESNF14 showed the highest signal-to-background ratio and the longest retention time in SLNs without leakage to second-tier lymph nodes. The findings showed that ICG had apparent leakage to second-tier nodes, and ZW800-3C had poor migration after intraparenchymal injection. However, when injected intravenously, ZW800-3C was able to highlight all RLNs in liver during a 4- to 6-h period. Simultaneous dual-channel imaging of SLN (ESNF14) and RLN (ZW800-3C) permitted unambiguous identification and image-guided resection of SLNs and RLNs in liver.ConclusionThe NIR imaging technology enables real-time intraoperative identification of SLNs and RLNs in the liver of swine. If these results are confirmed in patients, new strategies for the surgical management of intrahepatic malignancies should be possible.