Noninvasive Fluorescence Resonance Energy Transfer Imaging of <i>in Vivo</i> Premature Drug Release from Polymeric Nanoparticles

Understanding <i>in vivo</i> drug release kinetics is critical for the development of nanoparticle-based delivery systems. In this study, we developed a fluorescence resonance energy transfer (FRET) imaging approach to noninvasively monitor <i>in vitro</i> and <i>in vivo</i> cargo release from polymeric nanoparticles. The FRET donor dye (DiO or DiD) and acceptor dye (DiI or DiR) were individually encapsulated into poly­(ethylene oxide)-<i>b</i>-polystyrene (PEO-PS) nanoparticles. When DiO (donor) nanoparticles and DiI (acceptor) nanoparticles were coincubated with cancer cells for 2 h, increased FRET signals were observed from cell membranes, suggesting rapid release of DiO and DiI to cell membranes. Similarly, increased FRET ratios were detected in nude mice after intravenous coadministration of DiD (donor) nanoparticles and DiR (acceptor) nanoparticles. In contrast, another group of nude mice i.v. administrated with DiD/DiR coloaded nanoparticles showed decreased FRET ratios. Based on the difference in FRET ratios between the two groups, <i>in vivo</i> DiD/DiR release half-life from PEO-PS nanoparticles was determined to be 9.2 min. In addition, it was observed that the presence of cell membranes facilitated burst release of lipophilic cargos while incorporation of oleic acid-coated iron oxide into PEO-PS nanoparticles slowed the release of DiD/DiR to cell membranes. The developed <i>in vitro</i> and <i>in vivo</i> FRET imaging techniques can be used to screening stable nanoformulations for lipophilic drug delivery

Application of a new volumetric microsampling device for quantitative bioanalysis of immunosuppression: Supplementary material

Background: Volumetric absorptive microsampling may reduce the blood collection burden associated with therapeutic drug monitoring of immunosuppression to prevent organ transplant rejection. This work describes the development of a laboratory and analytical technique for quantifying tacrolimus and mycophenolic acid (MPA) from the Tasso-M20™ in human whole blood using bead-based impact-assisted extraction. Results: The sampled blood volume was accurate with estimated volumes within expected 20 μl. Recovery using impact-assisted extraction was 73–87% for MPA and 100% for tacrolimus and was hematocrit-independent for both analytes. The LC-MS/MS assay is precise and accurate within the acceptance criteria of 15%. Conclusion: The sampling and extraction procedures allowed for accurate quantification of tacrolimus and MPA. Exploration of abuse scenarios identified important education points for patients conducting home-based sample collections in the future.</p

Facile Fabrication of Near-Infrared-Resonant and Magnetic Resonance Imaging-Capable Nanomediators for Photothermal Therapy

Although many techniques exist for fabricating near-infrared (NIR)-resonant and magnetic resonance imaging (MRI)-capable nanomediators for photothermal cancer therapy, preparing them in an efficient and scalable process remains a significant challenge. In this report, we exploit one-step siloxane chemistry to facilely conjugate NIR-absorbing satellites onto a well-developed polysiloxane-containing polymer-coated iron oxide nanoparticle (IONP) core to generate dual functional core–satellite nanomediators for photothermal therapy. An advantage of this nanocomposite design is the variety of potential satellites that can be simply attached to impart NIR resonance, which we demonstrate using NIR-resonant gold sulfide nanoparticles (Au₂SNPs) and the NIR dye IR820 as two example satellites. The core–satellite nanomediators are fully characterized by using absorption spectra, dynamic light scattering, ζ potential measurements, and transmission electron microscopy. The enhanced photothermal effect under the irradiation of NIR laser light is identified through in vitro solutions and in vivo mice studies. The MRI capabilities as contrast agents are demonstrated in mice. Our data suggest that polysiloxane-containing polymer-coated IONPs can be used as a versatile platform to build such dual functional nanomediators for translatable, MRI-guided photothermal cancer therapy

Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in a Tumor Microenvironment

Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid–polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-l-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pH_e: 7.0–6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pH_endo: 6.5–4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use

C1QBP Negatively Regulates the Activation of Oncoprotein YBX1 in the Renal Cell Carcinoma As Revealed by Interactomics Analysis

The Y-box-binding protein 1 (YBX1) plays a critical role in tumorigenesis by promoting cell proliferation, overriding cell-cycle check points, and enhancing genomic instability. In this study, the interactome of YBX1 in renal cell carcinoma (RCC) was analyzed by coimmunoprecipitation and mass spectrometry to better understand its function and regulatory mechanism. A total of 129 proteins were identified as potential YBX1 binding partners. The interaction between the complement component 1, q subcomponent binding protein (C1QBP), and YBX1 was further confirmed by immunoprecipitation and Western blotting. Knockdown of C1QBP enhanced the phosphorylation of YBX1and its nuclear translocation, indicating that C1QBP negatively regulated YBX1 activation. The clinical significance of these two proteins was analyzed in the tissues from 52 RCC patients by immunohistochemistry. Expression of YBX1 was markedly elevated in the carcinoma tissues, and its nuclear expression was associated with histological T stage and metastasis. Meanwhile, the level of C1QBP in the carcinoma tissues was significantly lower than that in the adjacent healthy tissues, which was negatively correlated with the nuclear localization of YBX1 in the RCC tissues (<i>P</i> = 0.011). These data suggest that C1QBP is a novel regulator of YBX1, and the expression of C1QBP and the nuclear expression of YBX1 could both be used as independent prognostic makers for cancer progression in the RCC patients. The proteomics data have been deposited to the ProteomeXchange with identifier PXD001493

Core–Shell Nanoparticles Based on Pullulan and Poly(β-amino) Ester for Hepatoma-Targeted Codelivery of Gene and Chemotherapy Agent

This study designs a novel nanoparticle system with core–shell structure based on pullulan and poly­(β-amino) ester (PBAE) for the hepatoma-targeted codelivery of gene and chemotherapy agent. Plasmid DNA expressing green fluorescent protein (pEGFP), as a model gene, was fully condensed with cationic PBAE to form the inner core of PBAE/pEGFP polycomplex. Methotrexate (MTX), as a model chemotherapy agent, was conjugated to pullulan by ester bond to synthesize polymeric prodrug of MTX-PL. MTX-PL was then adsorbed on the surface of PBAE/pEGFP polycomplex to form MTX-PL/PBAE/pEGFP nanoparticles with a classic core–shell structure. MTX-PL was also used as a hepatoma targeting moiety, because of its specific binding affinity for asialoglycoprotein receptor (ASGPR) overexpressed by human hepatoma HepG2 cells. MTX-PL/PBAE/pEGFP nanoparticles realized the efficient transfection of pEGFP in HepG2 cells and exhibited significant inhibitory effect on the cell proliferation. In HepG2 tumor-bearing nude mice, MTX-PL/PBAE/pEGFP nanoparticles were mainly distributed in the tumor after 24 h postintravenous injection. Altogether, this novel codelivery system with a strong hepatoma-targeting property achieved simultaneous delivery of gene and chemotherapy agent into tumor at both cellular and animal levels

Analogues of the Allosteric Heat Shock Protein 70 (Hsp70) Inhibitor, MKT-077, As Anti-Cancer Agents

The rhodacyanine, MKT-077, has antiproliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogues designed for greater stability. The most potent molecules, such as <b>30</b> (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC₅₀ values of 0.4 ± 0.03 and 0.7 ± 0.2 μM, respectively). The analogues modestly destabilized the chaperone clients, Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (<i>t</i>_1/2 = 37 min) compared to MKT-077 (<i>t</i>_1/2 < 5 min). Finally, NMR titration experiments suggested that these analogues bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogues as chemical probes for studying Hsp70s roles in cancer

Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein–Protein Interaction between Menin and Mixed Lineage Leukemia (MLL)

Development of potent small molecule inhibitors of protein–protein interactions with optimized druglike properties represents a challenging task in lead optimization process. Here, we report synthesis and structure-based optimization of new thienopyrimidine class of compounds, which block the protein–protein interaction between menin and MLL fusion proteins that plays an important role in acute leukemias with <i>MLL</i> translocations. We performed simultaneous optimization of both activity and druglike properties through systematic exploration of substituents introduced to the indole ring of lead compound <b>1</b> (MI-136) to identify compounds suitable for in vivo studies in mice. This work resulted in the identification of compound <b>27</b> (MI-538), which showed significantly increased activity, selectivity, polarity, and pharmacokinetic profile over <b>1</b> and demonstrated a pronounced effect in a mouse model of MLL leukemia. This study, which reports detailed structure–activity and structure–property relationships for the menin–MLL inhibitors, demonstrates challenges in optimizing inhibitors of protein–protein interactions for potential therapeutic applications

Design, Synthesis, and Biological Evaluation of 4‑Quinoline Carboxylic Acids as Inhibitors of Dihydroorotate Dehydrogenase

We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues <b>41</b> (DHODH IC₅₀ = 9.71 ± 1.4 nM) and <b>43</b> (DHODH IC₅₀ = 26.2 ± 1.8 nM). A cocrystal structure between <b>43</b> and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine <b>46</b> (DHODH IC₅₀ = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound <b>41</b> possesses significant oral bioavailability (<i>F</i> = 56%) and an elimination <i>t</i>_1/2 = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development

Design, Synthesis, and Biological Evaluation of 4‑Quinoline Carboxylic Acids as Inhibitors of Dihydroorotate Dehydrogenase

We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues <b>41</b> (DHODH IC₅₀ = 9.71 ± 1.4 nM) and <b>43</b> (DHODH IC₅₀ = 26.2 ± 1.8 nM). A cocrystal structure between <b>43</b> and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine <b>46</b> (DHODH IC₅₀ = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound <b>41</b> possesses significant oral bioavailability (<i>F</i> = 56%) and an elimination <i>t</i>_1/2 = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development