Tat-Functionalized Near-Infrared Emissive Polymersomes for Dendritic Cell Labeling

Abstract

Dendritic cells (DCs) play a pivotal role in both immune tolerance and the initiation of immunological responses. The ability to track DCs in vivo is imperative for the development of DC-based cellular therapies and to advance our understanding of DC function and pathophysiology. Here, we conjugate a cell permeable peptide, Tat, to near-infrared (NIR) emissive polymersomes in order to enable efficient intracellular delivery for future DC tracking with these optical probes. NIR imaging allows quantitative, repetitive, in vivo detection of fluorophore-laden cells, at centimeter tissue depths without disturbing cellular function. Flow cytometry and confocal microscopy results indicate that Tat-mediated polymersome delivery to DCs is concentration and time dependent, resulting in punctate intracellular localization. Further, loading cells with Tat NIR emissive polymersomes does not interfere with cytokine-induced DC maturation and has modest effects on DC viability, but has a significant effect on mature DC-induced activation of naive T cells. We observe significant uptake of NIR emissive polymersomes when conjugated to the peptide, with a lower detection limit of 5000 labeled DCs. The extent of polymersome delivery is estimated as 70 000 +/- 10 000 vesicles/cell, equivalent to 0.7 +/- 0.1 fmol of NIR fluorophore. Our studies will enable future in vivo tracking of ex vivo labeled DCs by NIR fluorescence based imaging.

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“…As previously mentioned, Christian et al demonstrated that the highly cationic HIV-derived TAT peptide, when coupled to NIR-emissive polymersomes, enhances cellular delivery of polymer vesicles to dendritic cells while moderately affecting cell viability [19]. Intracellular uptake of polymersomes was dependent upon their concentration and incubation time in solution; viability was affected by these factors as well [19].…”

“…Dendritic cells (DCs) play an important role in the immune response and have shown potent anticancer activity leading to DC-based vaccines research [56]. Current progress in DC-based vaccines has been, however, limited by various factors [56], some of which could be overcome by the development of imaging methods for in vivo DC tracking [19]. Christian et al have demonstrated the ability to label DCs ex vivo with polymersomes encapsulating porphyrinbased NIRFs; the TAT peptide, as will be discussed in greater detail below, was conjugated to these NIR-emissive polymersome to facilitate efficient uptake of polymer vesicles by DCs [19].…”

“…Current progress in DC-based vaccines has been, however, limited by various factors [56], some of which could be overcome by the development of imaging methods for in vivo DC tracking [19]. Christian et al have demonstrated the ability to label DCs ex vivo with polymersomes encapsulating porphyrinbased NIRFs; the TAT peptide, as will be discussed in greater detail below, was conjugated to these NIR-emissive polymersome to facilitate efficient uptake of polymer vesicles by DCs [19]. Christian and colleagues determined that DC surfaceassociated polymersomes shed over the first 24-48h; but, polymer vesicles that were fully internalized by the DCs remained stably incorporated over 3 days [19].…”

“…Christian et al have demonstrated the ability to label DCs ex vivo with polymersomes encapsulating porphyrinbased NIRFs; the TAT peptide, as will be discussed in greater detail below, was conjugated to these NIR-emissive polymersome to facilitate efficient uptake of polymer vesicles by DCs [19]. Christian and colleagues determined that DC surfaceassociated polymersomes shed over the first 24-48h; but, polymer vesicles that were fully internalized by the DCs remained stably incorporated over 3 days [19]. They further showed that the NIR-emissive-polymersomelabeled DCs, when administered into the foot pad of mice, traffic to the nearest lymph node (popliteal lymph node) and could be tracked in vivo via optical imaging over 33 days [57].…”

“…In addition, biologically active ligands, such as antibodies, can be readily conjugated to the exterior brush surface to target the vesicles or to provide a therapeutic response [19][20][21][22]. These properties of the vesicle architecture effectively create a multimodal platform, which can be used for therapeutic (drug delivery) and/or diagnostic (imaging) applications (Fig.…”