Enhanced Therapeutic Effect of Liposomal Doxorubicin via Bio-Orthogonal Chemical Reactions in Tumors.

Liposomes are highly biocompatible drug carriers in drug delivery systems (DDSs). Preferential accumulation of liposomes and acceleration of drug release at target tumor sites are essential for effective cancer therapy using liposomal formulations; however, conventional liposomes are unsuitable for on-demand drug release. We have previously reported that drug release can be accelerated via a bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reaction between amphiphilic tetrazine (Tz)-containing liposomes and norbornene (NB) derivatives in vitro. In this study, we prepared HSTz-liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC) and Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) with particle sizes of 60-80 nm and ζ-potentials of -5 to 0 mV. Similar to our previous report, the addition of 5-norbornene-2-carboxylic acid (NBCOOH) to HSTz-liposomes accelerated drug release from the liposomes in vitro. In the biodistribution study using colon26 tumor-bearing mice, the radiolabeled HSTz-liposomes were accumulated and retained in the tumor at 6-48 h post-injection, whereas the radioactivity in the blood almost disappeared at 48 h. Therefore, the timing of the injection of NBCOOH was selected to be 48 h after the injection of the HSTz-liposome to avoid the IEDDA reaction in the bloodstream. We investigated the in vivo drug release by evaluating the intratumoral localization of doxorubicin (DOX) encapsulated in HSTz-liposomes labeled with fluorescent lipids. In the tumors treated with HSTz-liposomes and NBCOOH, DOX was more widely dispersed in the tumor compared with fluorescent lipid, suggesting that the release of encapsulated drugs (DOX) from HSTz-liposomes was enhanced in the tumor tissue via the bio-orthogonal IEDDA reaction. Furthermore, the combination of DOX-encapsulated HSTz-liposomes with NBCOOH significantly suppressed tumor growth compared to conventional DOX-encapsulated liposomes. In conclusion, the bio-orthogonal IEDDA reactions in the liposomal membrane enabled the acceleration of drug release from HSTz-liposomes in vivo, suggesting a promising strategy for effective cancer therapy.

A Radiolabeled Self-assembled Nanoparticle Probe for Diagnosis of Lung-Metastatic Melanoma.

Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and -33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111In and 125I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.

Inverse Electron Demand Diels-Alder Reactions in the Liposomal Membrane Accelerates Release of the Encapsulated Drugs.

Bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reactions between liposomes containing a tetrazine-based (Tz) compound and 2-norbornene (2-NB) could be a novel trigger for accelerating drug release from the liposomes via temporary membrane destabilization, as shown in our previous report. Herein, we evaluated the in vitro drug release using NB derivatives with carboxyl groups [5-norbornene-2-carboxylic acid (NBCOOH) and 5-norbornene-2,3-dicarboxylic acid (NB(COOH)2)] to investigate the effects of substituents at the NB backbone on the drug release rate. First, POTz-liposome composed of a Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) were prepared. The mass spectrometry analysis revealed the binding of NB derivatives to the Tz compound via the IEDDA reaction after the POTz-liposome reacted with the NB derivatives. Indium-111-labeled diethylenetriaminepentaacetic acid (111In-DTPA) was encapsulated inside the liposomes, and the drug release rate was quantified by measuring radioactivity. At 24 h after incubation with 2-NB, NBCOOH, and NB(COOH)2, the release rates of 111In-DTPA from POTz-liposome were 21.0, 80.8, and 23.3%, respectively, which were significantly higher than those of POTz-liposome that was not treated with NB derivatives (4.2%), indicating the involvement of the IEDDA reaction for prompting drug release. Additionally, a thermodynamic evaluation using Langmuir monolayers was conducted to explore the mechanism of the accelerated drug release. An increase in membrane fluidity and a reduction in intermolecular repulsion between POPC and the Tz compound were observed after the reaction with NB derivatives, especially for NBCOOH. Thus, the IEDDA reaction in the liposomal membrane could be a potent trigger for accelerating the release of encapsulated drugs by regulating membrane fluidity and intermolecular repulsion in the liposomal membrane.

Preparation and evaluation of 111In-labeled liposomes containing phosphatidylglycerol for detection of macrophages in atherosclerotic plaques.

Macrophage infiltration is a characteristic feature of atherosclerotic plaque progression. Since liposomes containing 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) are efficiently phagocytosed by macrophages, we deduced that radiolabeled liposomes containing DSPG could potentially be used for nuclear imaging of vulnerable atherosclerotic plaques. Indium-111 (111In)-labeled liposomes containing different ratios of DSPG were developed with a high labeling efficiency. 111In-labeled liposomes with higher DSPG content showed higher uptake by macrophage-like RAW264 cells. A biodistribution study demonstrated rapid blood clearance and selective accumulation in the liver and spleen, especially in normal mice injected with 111In-labeled liposomes with higher DSPG content. Accumulation in atherosclerotic plaques was evaluated using 111In-labeled DSPG liposomes, which had the highest DSPG content among the studied liposomes. 111In-labeled DSPG liposomes accumulated in the plaques and the radioactive regions were mostly consistent with the distribution of macrophages. The target-to-non-target ratio of 111In-labeled DSPG liposomes was higher than that of 111In-labeled control liposomes without DSPG. These results suggest that 111In-labeled liposomes containing DSPG are useful for nuclear medical diagnosis of atherosclerotic plaques.