Upconversion nanocrystals (UCNs) display near-infrared (NIR)-responsive photoluminescent properties for NIR imaging and drug delivery. The development of effective strategies for UCN integration with other complementary nanostructures for targeting and drug conjugation is highly desirable. This study reports on a core/shell-based theranostic system designed by UCN integration with a folate (FA)-conjugated dendrimer for tumor targeting and with photocaged doxorubicin as a cytotoxic agent. Two types of UCNs (NaYF4:Yb/Er (or Yb/Tm); diameter = ≈50 to 54 nm) are described, each displaying distinct emission properties upon NIR (980 nm) excitation. The UCNs are surface modified through covalent attachment of photocaged doxorubicin (ONB-Dox) and a multivalent FA-conjugated polyamidoamine (PAMAM) dendrimer G5(FA)6 to prepare UCN@(ONB-Dox)(G5FA). Surface plasmon resonance experiments performed with G5(FA)6 dendrimer alone show nanomolar binding avidity (KD = 5.9 × 10(-9) M) to the folate binding protein. This dendrimer binding corresponds with selective binding and uptake of UCN@(ONB-Dox)(G5FA) by FAR-positive KB carcinoma cells in vitro. Furthermore, UCN@(ONB-Dox)(G5FA) treatment of FAR(+) KB cells inhibits cell growth in a light dependent manner. These results validate the utility of modularly integrated UCN-dendrimer nanocomposites for cell type specific NIR imaging and light-controlled drug release, thus serving as a new theranostic system.

Despite the immense potential, application of existing photocaging technology is limited by the paucity of advanced caging tools. Here we report on the design of a novel thioacetal ortho-nitrobenzaldehyde (TNB) dual arm photocage that enables control of the simultaneous release of two payloads linked to a single TNB unit. Using this cage which was prepared in a single step from commercial 6-nitroverataldehyde, three drug-fluorophore conjugates were synthesized, Taxol-TNB-Fluorescein, Taxol-TNB-Coumarin and Doxorubicin-TNB-Coumarin, and long wavelength UVA light-triggered release experiments demonstrated that dual payload release occurred with rapid decay kinetics for each conjugate. In cell based assays in vitro, dual release could also be controlled by UV exposure, resulting in increased cellular fluorescence and cytotoxicity as potent as unmodified drug towards the KB carcinoma cell line. The extent of such dual release was quantifiable by reporter fluorescence measured in situ, and found to correlate with the extent of cytotoxicity. Thus, this novel dual-arm cage strategy provides a valuable tool which enables both active control and real-time monitoring of drug activation at the delivery site.

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence.MethodsWe characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models.ResultsCARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model.ConclusionTransgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Background: The prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. NK cells can elicit an anti-leukemic response via a repertoire of activating receptors that bind AML surface ligands. NK cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity, and interleukin-15 (IL15) to enhance their persistence. Methods: We characterized in detail NK cell populations expressing a panel of AML (CD123)-specific CARs and/or IL15 in vitro and in AML xenograft models. Results: CARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ CAR-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK cell persistence. Transgenic expression of secretory (s)IL15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK cell subsets. 2B4.ζ/sIL15 CAR and sIL15 NK cells maintained an overall activated NK cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK cell groups. In vivo, 2B4.ζ/sIL15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL15 CAR and sIL15 NK cells induced lethal toxicity in a second model. Conclusion: Transgenic expression of CD123-CARs and sIL15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early phase clinical testing.

The inside cover picture shows the concept of a new binary photocage that enables synchronous controlled release of two payloads linked to a single cage unit. This cage is based on photocleavable thioacetal ortho‐nitrobenzaldehyde, and its utility is illustrated by cross‐tethering both an anticancer agent and a fluorescent reporter to the cage. Caging temporarily retains the drug in an inactive state and quenches reporter fluorescence. The extent of light‐controlled dual release is quantifiable by reporter fluorescence measured in situ. This strategy provides new possibilities for real‐time monitoring of drug or effector activation in cellular systems and at delivery sites. More information can be found in the full paper by P. T. Wong, S. K. Choi et al. on page 126 in Issue 1, 2017 (DOI: 10.1002/cbic.201600494).