Orthogonal Functionalization of Ferritin via Supramolecular Re-Assembly

To investigate if the degree of functionalization of ferritin could be controlled using a supramolecular self-assembly process, two photophysical separable batches of ferritin were created by functionalizing ferritin capsids with either Cy3- or Cy5-dye (loading rate of about 50 %). After dis-assembly, Cy3-, Cy5- as well as non-functionalized ferritin subunits were mixed in variable ratios. Photophysical measurements revealed that the ratio in which the subunits were mixed was indeed indicative for the ratios in which the functionalized subunits were observed in the re-assembled capsids. During re-assembly, however, a slight preference for the inclusion of non-functionalized subunits was observed, indicating the reactivity decreased following functionalization. The iron biomineralization properties of ferritin were retained by the multi-functionalized capsids as FeII diffused rapidly inside making it visible by transmission electron microscopy (TEM). These combined data indicate that it is possible to functionalize ferritin in an orthogonal manner using the supramolecular interaction between ferritin subunits.</p

In vivo stability of supramolecular host–guest complexes monitored by dual-isotope multiplexing in a pre-targeting model of experimental liver radioembolization

Introduction: Cyclodextrin (CD)-based supramolecular interactions have been proposed as nanocarriers for drug delivery. We previously explored the use of these supramolecular interactions to perform targeted hepatic radioembolization. In a two-step procedure the appropriate location of the diagnostic pre-targeting vector can first be confirmed, after which the therapeutic vector will be targeted through multivalent host–guest interactions. Such a procedure would prevent therapeutic errors that come from a mismatch between diagnostic and therapeutic procedures. In the current study we explored the use of dual-isotope imaging to assess the in vivo stability of the formed complex and individual components. Methods: Dual-isotope imaging of the host and guest vectors was performed after labeling of the pre-targeted guest vector, being adamantane (Ad) functionalized macro-aggregated albumin (MAA) particles, with technetium-99 m (99mTc-MAA-Ad). The host vector, Cy50.5CD9PIBMA39, was labeled with indium-111 (111In-Cy50.5CD9PIBMA39). The in situ stability of both the individual vectors and the resulting [MAA-Ad–111In-Cy50.5CD9PIBMA39] complexes was studied over 44 h at 37 °C in a serum protein-containing buffer. In vivo, the host vector 111In-Cy50.5CD9PIBMA39 was administered two hours after local deposition of 99mTc-MAA-Ad in mice. Dual-isotope SPECT imaging and quantitative biodistribution studies were performed between 2 and 44 h post intravenous host vector administration. Results: The individual vectors portrayed <5% dissociation of the radioisotope over the course of 20 h. Dissociation of [MAA-Ad–111In-Cy50.5CD9PIBMA39] complexes remained within a 10–20% range after incubation in serum. In vivo dual-isotope SPECT imaging of host–guest interactions revealed co-localization of the tracer components. Quantitative assessment of the biodistribution revealed that the hepatic accumulation of the host vector nearly doubled between 2 h and 44 h post-injection (from 14.9 ± 6.1%ID/g to 26.2 ± 2.1%ID/g). Conclusions: Assessment of intra-hepatic host–guest complexation was successfully achieved using dual isotope multiplexing, underlining the complex stability that was found in situ (up to 44 h in serum). Overall, the results obtained in this study highlight the potential of supramolecular chemistry as a versatile platform that could advance the field of nanomedicine

In vivo stability of supramolecular host–guest complexes monitored by dual-isotope multiplexing in a pre-targeting model of experimental liver radioembolization

Introduction: Cyclodextrin (CD)-based supramolecular interactions have been proposed as nanocarriers for drug delivery. We previously explored the use of these supramolecular interactions to perform targeted hepatic radioembolization. In a two-step procedure the appropriate location of the diagnostic pre-targeting vector can first be confirmed, after which the therapeutic vector will be targeted through multivalent host–guest interactions. Such a procedure would prevent therapeutic errors that come from a mismatch between diagnostic and therapeutic procedures. In the current study we explored the use of dual-isotope imaging to assess the in vivo stability of the formed complex and individual components. Methods: Dual-isotope imaging of the host and guest vectors was performed after labeling of the pre-targeted guest vector, being adamantane (Ad) functionalized macro-aggregated albumin (MAA) particles, with technetium-99 m (99mTc-MAA-Ad). The host vector, Cy50.5CD9PIBMA39, was labeled with indium-111 (111In-Cy50.5CD9PIBMA39). The in situ stability of both the individual vectors and the resulting [MAA-Ad–111In-Cy50.5CD9PIBMA39] complexes was studied over 44 h at 37 °C in a serum protein-containing buffer. In vivo, the host vector 111In-Cy50.5CD9PIBMA39 was administered two hours after local deposition of 99mTc-MAA-Ad in mice. Dual-isotope SPECT imaging and quantitative biodistribution studies were performed between 2 and 44 h post intravenous host vector administration. Results: The individual vectors portrayed 111In-Cy50.5CD9PIBMA39] complexes remained within a 10–20% range after incubation in serum. In vivo dual-isotope SPECT imaging of host–guest interactions revealed co-localization of the tracer components. Quantitative assessment of the biodistribution revealed that the hepatic accumulation of the host vector nearly doubled between 2 h and 44 h post-injection (from 14.9 ± 6.1%ID/g to 26.2 ± 2.1%ID/g). Conclusions: Assessment of intra-hepatic host–guest complexation was successfully achieved using dual isotope multiplexing, underlining the complex stability that was found in situ (up to 44 h in serum). Overall, the results obtained in this study highlight the potential of supramolecular chemistry as a versatile platform that could advance the field of nanomedicine.</p

A supramolecular approach for liver radioembolization

Hepatic radioembolization therapies can suffer from discrepancies between diagnostic planning (scout-scan) and the therapeutic delivery itself, resulting in unwanted side-effects such as pulmonary shunting. We reasoned that a nanotechnology-based pre-targeting strategy could help overcome this shortcoming by directly linking pre-interventional diagnostics to the local delivery of therapy. Methods: The host-guest interaction between adamantane and cyclodextrin was employed in an in vivo pre-targeting set-up. Adamantane (guest)-functionalized macro albumin aggregates (MAA-Ad; d = 18 μm) and (radiolabeled) Cy5 and β-cyclodextrin (host)-containing PIBMA polymers (99mTc-Cy50.5CD10PIBMA39; MW ~ 18.8 kDa) functioned as the reactive pair. Following liver or lung embolization with (99mTc)-MAA-Ad or (99mTc)-MAA (controls), the utility of the pre-targeting concept was evaluated after intravenous administration of 99mTc-Cy50.5CD10PIBMA39. Results: Interactions between MAA-Ad and Cy50.5CD10PIBMA39 could be monitored in solution using confocal microscopy and were quantified by radioisotope-based binding experiments. In vivo the accumulation of the MAA-Ad particles in the liver or lungs yielded an approximate ten-fold increase in accumulation of 99mTc-Cy50.5CD10PIBMA39 in those organs (16.2 %ID/g and 10.5 %ID/g, respectively) compared to the control. Pre-targeting with MAA alone was shown to be only half as efficient. Uniquely, for the first time, this data demonstrates that the formation of supramolecular interactions between cyclodextrin and adamantane can be used to drive complex formation in the chemically challenging in vivo environment. Conclusion: The in vivo distribution pattern of the cyclodextrin host could be guided by the pre-administration of the adamantane guest, thereby creating a direct link between the scout-scan (MAA-Ad) and delivery of therapy

A supramolecular approach for liver radioembolization

Hepatic radioembolization therapies can suffer from discrepancies between diagnostic planning (scout-scan) and the therapeutic delivery itself, resulting in unwanted side-effects such as pulmonary shunting. We reasoned that a nanotechnology-based pre-targeting strategy could help overcome this shortcoming by directly linking pre-interventional diagnostics to the local delivery of therapy. Methods: The host-guest interaction between adamantane and cyclodextrin was employed in an in vivo pre-targeting set-up. Adamantane (guest)-functionalized macro albumin aggregates (MAA-Ad; d = 18 μm) and (radiolabeled) Cy5 and β-cyclodextrin (host)-containing PIBMA polymers (99mTc-Cy50.5CD10PIBMA39; MW ~ 18.8 kDa) functioned as the reactive pair. Following liver or lung embolization with (99mTc)-MAA-Ad or (99mTc)-MAA (controls), the utility of the pre-targeting concept was evaluated after intravenous administration of 99mTc-Cy50.5CD10PIBMA39. Results: Interactions between MAA-Ad and Cy50.5CD10PIBMA39 could be monitored in solution using confocal microscopy and were quantified by radioisotope-based binding experiments. In vivo the accumulation of the MAA-Ad particles in the liver or lungs yielded an approximate ten-fold increase in accumulation of 99mTc-Cy50.5CD10PIBMA39 in those organs (16.2 %ID/g and 10.5 %ID/g, respectively) compared to the control. Pre-targeting with MAA alone was shown to be only half as efficient. Uniquely, for the first time, this data demonstrates that the formation of supramolecular interactions between cyclodextrin and adamantane can be used to drive complex formation in the chemically challenging in vivo environment. Conclusion: The in vivo distribution pattern of the cyclodextrin host could be guided by the pre-administration of the adamantane guest, thereby creating a direct link between the scout-scan (MAA-Ad) and delivery of therapy

Multimodal Tracking of Controlled Staphylococcus aureus Infections in Mice

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, 99m Tc-UBI 29-41 -Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with 99m Tc-UBI 29-41 -Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99m Tc-UBI 29-41 -Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99m Tc-UBI 29-41 -Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies. </p

Multimodal Tracking of Controlled Staphylococcus aureus Infections in Mice

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, 99m Tc-UBI 29-41 -Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with 99m Tc-UBI 29-41 -Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99m Tc-UBI 29-41 -Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99m Tc-UBI 29-41 -Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies. </p

Tailoring Fluorescent Dyes To Optimize a Hybrid RGD-Tracer

Quantitative assessment of affinity and kinetics is a critical component in the development of (receptor-targeted) radiotracers. For fluorescent tracers, such an assessment is currently not yet applied, while (small) changes in chemical composition of the fluorescent component might have substantial influence on the overall properties of a fluorescent tracer. Hybrid imaging labels that contain both a radiolabel and a fluorescent dye can be used to evaluate both the affinity (fluorescent label) and the in vivo distribution (radiolabel) of a targeted tracer. We present a hybrid label oriented and matrix-based scoring approach that enabled quantitative assessment of the influence of (overall) charge and lipophilicity of the fluorescent label on the (in vivo) characteristics of α_vβ₃-integrin targeted tracers. Systematic chemical alterations in the fluorescent dye were shown to result in a clear difference in the in vivo distribution of the different hybrid tracers. The applied evaluation technique resulted in an optimized targeted tracer for α_vβ₃-integrin, which combined the highest T/M ratio with the lowest uptake in other organs. Obviously this selection concept would also be applicable during the development of other (receptor-targeted) imaging tracers