Characterization of Intrinsically Radiolabeled Poly(lactic-co-glycolic acid) Nanoparticles for ex Vivo Autologous Cell Labeling and in Vivo Tracking.

With the advent of novel immunotherapies, interest in ex vivo autologous cell labeling for in vivo cell tracking has revived. However, current clinically available labeling strategies have several drawbacks, such as release of radiolabel over time and cytotoxicity. Poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) are clinically used biodegradable carriers of contrast agents, with high loading capacity for multimodal imaging agents. Here we show the development of PLGA-based NPs for ex vivo cell labeling and in vivo cell tracking with SPECT. We used primary amine-modified PLGA polymers (PLGA-NH2) to construct NPs similar to unmodified PLGA NPs. PLGA-NH2 NPs were efficiently radiolabeled without chelator and retained the radionuclide for 2 weeks. Monocyte-derived dendritic cells labeled with [111In]In-PLGA-NH2 showed higher specific activity than those labeled with [111In]In-oxine, with no negative effect on cell viability. SPECT/CT imaging showed that radiolabeled THP-1 cells accumulated at the Staphylococcus aureus infection site in mice. In conclusion, PLGA-NH2 NPs are able to retain 111In, independent of chelator presence. Furthermore, [111In]In-PLGA-NH2 allows cell labeling with high specific activity and no loss of activity over prolonged time intervals. Finally, in vivo tracking of ex vivo labeled THP-1 cells was demonstrated in an infection model using SPECT/CT imaging.

Multicore Liquid Perfluorocarbon-Loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking.

Ultrasound is the most commonly used clinical imaging modality. However, in applications requiring cell-labeling, the large size and short active lifetime of ultrasound contrast agents limit their longitudinal use. Here, 100 nm radius, clinically applicable, polymeric nanoparticles containing a liquid perfluorocarbon, which enhance ultrasound contrast during repeated ultrasound imaging over the course of at least 48 h, are described. The perfluorocarbon enables monitoring the nanoparticles with quantitative 19F magnetic resonance imaging, making these particles effective multimodal imaging agents. Unlike typical core-shell perfluorocarbon-based ultrasound contrast agents, these nanoparticles have an atypical fractal internal structure. The nonvaporizing highly hydrophobic perfluorocarbon forms multiple cores within the polymeric matrix and is, surprisingly, hydrated with water, as determined from small-angle neutron scattering and nuclear magnetic resonance spectroscopy. Finally, the nanoparticles are used to image therapeutic dendritic cells with ultrasound in vivo, as well as with 19F MRI and fluorescence imaging, demonstrating their potential for long-term in vivo multimodal imaging.

The internal structure of gadolinium and perfluorocarbon-loaded polymer nanoparticles affects 19F MRI relaxation times.

19F magnetic resonance imaging (19F MRI) is an emerging technique for quantitative imaging in novel therapies, such as cellular therapies and theranostic nanocarriers. Nanocarriers loaded with liquid perfluorocarbon (PFC) typically have a (single) core-shell structure with PFC in the core due to the poor miscibility of PFC with organic and inorganic solvents. Paramagnetic relaxation enhancement acts only at a distance of a few angstroms. Thus, efficient modulation of the 19F signal is possible only with fluorophilic PFC-soluble chelates. However, these chelates cannot interact with the surrounding environment and they might result in image artifacts. Conversely, chelates bound to the nanoparticle shell typically have a minimal effect on the 19F signal and a strong impact on the aqueous environment. We show that the confinement of PFC in biodegradable polymeric nanoparticles (NPs) with a multicore structure enables the modulation of longitudinal (T1) and transverse (T2) 19F relaxation, as well as proton (1H) signals, using non-fluorophilic paramagnetic chelates. We compared multicore NPs versus a conventional single core structure, where the PFC is encapsulated in the core(s) and the chelate in the surrounding polymeric matrix. This modulated relaxation also makes multicore NPs sensitive to various acidic pH environments, while preserving their stability. This effect was not observed with single core nanocapsules (NCs). Importantly, paramagnetic chelates affected both T1 and T219F relaxation in multicore NPs, but not in single core NCs. Both relaxation times of the 19F nucleus were enhanced with an increasing concentration of the paramagnetic chelate. Moreover, as the polymeric matrix remained water permeable, proton enhancement additionally was observed in MRI.

The anti-inflammatory peptide Catestatin blocks chemotaxis.

Increased levels of the anti-inflammatory peptide Catestatin (CST), a cleavage product of the pro-hormone chromogranin A, correlate with less severe outcomes in hypertension, colitis, and diabetes. However, it is unknown how CST reduces the infiltration of monocytes and macrophages (Mϕs) in inflamed tissues. Here, it is reported that CST blocks leukocyte migration toward inflammatory chemokines. By in vitro and in vivo migration assays, it is shown that although CST itself is chemotactic, it blocks migration of monocytes and neutrophils to inflammatory attracting factor CC-chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, it directs CX3 CR1+ Mϕs away from pancreatic islets. These findings suggest that the anti-inflammatory actions of CST are partly caused by its regulation of chemotaxis.

PLGA Nanoparticles Co-encapsulating NY-ESO-1 Peptides and IMM60 Induce Robust CD8 and CD4 T Cell and B Cell Responses.

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.

Nanovaccine administration route is critical to obtain pertinent iNKt cell help for robust anti-tumor T and B cell responses.

Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.

Continuous-Flow Production of Perfluorocarbon-Loaded Polymeric Nanoparticles: From the Bench to Clinic.

Perfluorocarbon-loaded nanoparticles are powerful theranostic agents, which are used in the therapy of cancer and stroke and as imaging agents for ultrasound and 19F magnetic resonance imaging (MRI). Scaling up the production of perfluorocarbon-loaded nanoparticles is essential for clinical translation. However, it represents a major challenge as perfluorocarbons are hydrophobic and lipophobic. We developed a method for continuous-flow production of perfluorocarbon-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a modular microfluidic system, with sufficient yields for clinical use. We combined two slit interdigital micromixers with a sonication flow cell to achieve efficient mixing of three phases: liquid perfluorocarbon, PLGA in organic solvent, and aqueous surfactant solution. The production rate was at least 30 times higher than with the conventional formulation. The characteristics of nanoparticles can be adjusted by changing the flow rates and type of solvent, resulting in a high PFC loading of 20-60 wt % and radii below 200 nm. The nanoparticles are nontoxic, suitable for 19F MRI and ultrasound imaging, and can dissolve oxygen. In vivo 19F MRI with perfluoro-15-crown-5 ether-loaded nanoparticles showed similar biodistribution as nanoparticles made with the conventional method and a fast clearance from the organs. Overall, we developed a continuous, modular method for scaled-up production of perfluorocarbon-loaded nanoparticles that can be potentially adapted for the production of other multiphase systems. Thus, it will facilitate the clinical translation of theranostic agents in the future.

Nanoparticles for "two color" 19F magnetic resonance imaging: Towards combined imaging of biodistribution and degradation.

The use of polymeric nanoparticles (NPs) as therapeutics has been steadily increasing over past decades. In vivo imaging of NPs is necessary to advance the therapeutic performance. 19F Magnetic Resonance Imaging (19F MRI) offers multiple advantages for in vivo imaging. However, design of a probe for both biodistribution and degradation has not been realized yet. We developed polymeric NPs loaded with two fluorocarbons as promising imaging tools to monitor NP biodistribution and degradation by 19F MRI. These 200 nm NPs consist of poly(lactic-co-glycolic acid) (PLGA) loaded with perfluoro-15-crown-5 ether (PFCE) and PERFECTA. PERFECTA/PFCE-PLGA NPs have a fractal sphere structure, in which both fluorocarbons are distributed in the polymeric matrix of the fractal building blocks, which differs from PFCE-PLGA NPs and is unique for fluorocarbon-loaded colloids. This structure leads to changes of magnetic resonance properties of both fluorocarbons after hydrolysis of NPs. PERFECTA/PFCE-PLGA NPs are colloidally stable in serum and biocompatible. Both fluorocarbons show a single resonance in 19F MRI that can be imaged separately using different excitation pulses. In the future, these findings may be used for biodistribution and degradation studies of NPs by 19F MRI in vivo using "two color" labeling leading to improvement of drug delivery agents.

Clinically-Applicable Perfluorocarbon-Loaded Nanoparticles For In vivo Photoacoustic, 19F Magnetic Resonance And Fluorescent Imaging.

Photoacoustic imaging (PAI) is an emerging biomedical imaging technique that is now coming to the clinic. It has a penetration depth of a few centimeters and generates useful endogenous contrast, particularly from melanin and oxy-/deoxyhemoglobin. Indocyanine green (ICG) is a Food and Drug Administration-approved contrast agents for human applications, which can be also used in PAI. It is a small molecule dye with limited applications due to its fast clearance, rapid protein binding, and bleaching effect. Methods: Here, we entrap ICG in a poly(lactic-co-glycolic acid) nanoparticles together with a perfluorocarbon (PFC) using single emulsion method. These nanoparticles and nanoparticle-loaded dendritic cells were imaged with PA, 19F MR, and fluorescence imaging in vitro and in vivo. Results: We formulated particles with an average diameter of 200 nm. The encapsulation of ICG within nanoparticles decreased its photobleaching and increased the retention of the signal within cells, making it available for applications such as cell imaging. As little as 0.1x106 cells could be detected in vivo with PAI using automated spectral unmixing. Furthermore, we observed the accumulation of ICG signal in the lymph node after subcutaneous injection of nanoparticles. Conclusion: We show that we can label primary human dendritic cells with the nanoparticles and image them in vitro and in vivo, in a multimodal manner. This work demonstrates the potential of combining PAI and 19F MRI for cell imaging and lymph node detection using nanoparticles that are currently produced at GMP-grade for clinical use.