Molecular Imaging and Preclinical Studies of Radiolabeled Long-Term RGD Peptides in U-87 MG Tumor-Bearing Mice.

The Arg-Gly-Asp (RGD) peptide shows a high affinity for αvß3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvß3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.

Synthesis and Evaluation of 18F-INER-1577-3 as a Central Nervous System (CNS) Histone Deacetylase Imaging Agent.

BACKGROUND: Epigenetic dysfunction is implicated in many neurologic, psychiatric and oncologic diseases. Consequently, histone deacetylases (HDACs) inhibitors have been developed as therapeutic and imaging agents for these diseases. However, only a few radiotracers have been developed as HDACs imaging agents for the central nervous system (CNS). We report herein the synthesis and evaluation of [18F]INER-1577-3 ([18F]5) as an HDACs imaging agent for CNS. METHODS: [18F]INER-1577-3 ([18F]5) was synthesized by two methods: one-step (A) and two-step (B) methods. Briefly, radiofluorination of the corresponding precursors (11, 12) with K[18F]/K2.2.2 followed by purifications with HPLC gave ([18F]5). The quality of [18F]INER- 1577-3 synthesized by these methods was verified by HPLC and TLC as compared to an authentic sample. The inhibitions of [18F]INER-1577-3 and related HDACs inhibitors on tumor cells growth were carried out with breast cancer cell line 4T1 and MCF-7. The whole-body and brain uptake of [18F]INER-1577-3 in rats and AD mice were determined using a micro-PET scanner and the data was analyzed using PMOD. RESULTS: The radiochemical yield of [18F]INER-1577-3 synthesized by these two methods was 1.4 % (Method A) and 8.8% (Method B) (EOB), respectively. The synthesis time was 115 min and 100 min, respectively, from EOB. The inhibition studies showed that INER-1577-3 has a significant inhibitory effect in HDAC6 and HDAC8 but not HDAC2. PET studies in rats and AD mice showed a maximum at about 15 min postinjection for the whole brain of a rat (0.47 ± 0.03 %ID/g), SAMP8 mice (5.63 ± 1.09 %ID/g) and SAMR1 mice (7.23 ± 1.21 %ID/g). CONCLUSION: This study showed that INER-1577-3 can inhibit tumor cell growth and is one of a few HDACs inhibitors that can penetrate the blood-brain barrier (BBB) and monitor HDAC activities in AD mice. Thus, [18F]INER-1577-3 may be a potent HDACs imaging agent, especially for CNS.

Apoptosis induction and AKT/NF-κB inactivation are associated with regroafenib-inhibited tumor progression in non-small cell lung cancer in vitro and in vivo.

Non-small cell lung cancer (NSCLC) is a malignant lung cancer type with poor prognosis. NF-κB, the oncogenic transcription factor, has been recognized as an important mediator in progression of NSCLC. Regorafenib, a multikinase inhibitor, was demonstrated to inhibit tumor progression through suppression of ERK/NF-κB signaling in hepatocellular carcinoma cells in vitro and in vivo. However, whether regorafenib inhibit progression of NSCLC is ambiguous. Thus, the major purpose of present study was to evaluate anticancer efficacy and underlying mechanism of regorafenib on tumor progression in NSCLC in vitro and in vivo. CL-1-5-F4 cells were treated with regorafenib, NF-κB (QNZ) or AKT (LY294002) inhibitor for 24 or 48 h. Then, we performed cell viability assay, NF-κB reporter gene assay, transwell invasion assay and apoptosis related flow cytometry assay on cellular level to verify anti-cancer effect and mechanism of regorafenib. CL-1-5-F4 bearing animal model was treated with vehicle or regorafenib for 28 days. The therapeutic efficacy and mechanism of regorafenib in CL-1-5-F4 bearing animal model were investigated by tumor size evaluation, whole body computer tomography (CT) scan, Haemotoxylin and Eosin (H&E) stain and immunohistochemistry (IHC) stain. Our results demonstrated regorafenib significantly inhibited tumor growth and induced apoptosis through extrinsic/intrinsic pathways in NSCLC in vitro and in vivo. Furthermore, we also found the suppression of AKT/NF-κB signaling was required for regorafenib inhibited expression of progression-related and invasion-related proteins. Our finding indicated apoptosis induction and suppression of AKT/NF-κB signaling were associated with regorafenib-inhibited progression of NSCLC in vitro and in vivo.

Ligand Characteristics Important to Avidity Interactions of Multivalent Nanoparticles.

Multivalent interactions involve the engagement of multiple ligand-receptor pairs and are important in synthetic biology as design paradigms for targeted nanoparticles (NPs). However, little is known about the specific ligand parameters important to multivalent interactions. We employed a series of oligonucleotides as ligands conjugated to dendrimers as nanoparticles, and used complementary oligonucleotides on a functionalized SPR surface to measure binding. We compared the effect of ligand affinity to ligand number on the avidity characteristics of functionalized NPs. Changing the ligand affinity, either by changing the temperature of the system or by substitution noncomplementary base pairs into the oligonucleotides, had little effect on multivalent interaction; the overall avidity, number of ligands required for avidity per particle, and the number of particles showing avidity did not significantly change. We then made NP conjugates with the same oligonucleotide using an efficient copper-free click chemistry that resulted in essentially all of the NPs in the population exceeding the threshold ligand value. The particles exceeding the threshold ligand number again demonstrated high avidity interactions. This work validates the concept of a threshold ligand valence and suggests that the number of ligands per nanoparticle is the defining factor in achieving high avidity interactions.

Evaluating binding avidities of populations of heterogeneous multivalent ligand-functionalized nanoparticles.

Ligand-functionalized, multivalent nanoparticles have been extensively studied as targeted carriers in biomedical applications for drug delivery and imaging. The chemical synthesis method used, however, generates nanoparticles that are heterogeneous with respect to the number of ligands on each nanoparticle. This article examines the role this heterogeneity in ligand number plays in multivalent interactions between nanoparticle ligands and targeted receptors. We designed and synthesized a model heterogeneous multivalent nanoparticle system and developed a unique kinetic analysis to quantify the avidity interactions. This system used mono-dispersed poly(amidoamine) (PAMAM) dendrimers that were then chemically functionalized with ssDNA oligonucleotides as to yield the heterogeneous nanoparticle platform (ligand valencies n = 1.7, 3.1, 6), and employed complementary oligonucleotides as targeted receptors on a surface plasmon resonance (SPR) biosensor to evaluate the multivalent binding of the nanoparticle population. Kinetic analysis of both parallel initial rate and dual-Langmuir analyses of SPR binding curves was performed to assess avidity distributions. We found that batches of multivalent nanoparticles contain both fast- and slow-dissociation subpopulations, which can be characterized as having "weak" and "strong" surface interactions ("binding"), respectively. Furthermore, we found that the proportion of "strong" binders increased as a function of the mean oligonucleotide valence of the nanoparticle population. These analyses allowed an assessment of how avidity distributions are modulated by the number of functionalized ligands and suggested that there are threshold valences that differentiated fast- and slow-dissociation nanoparticles.

DNA condensation by partially acetylated poly(amido amine) dendrimers: effects of dendrimer charge density on complex formation.

The ability of poly(amido amine) (or PAMAM) dendrimers to condense semiflexible dsDNA and penetrate cell membranes gives them great potential in gene therapy and drug delivery but their high positive surface charge makes them cytotoxic. Here, we describe the effects of partial neutralization by acetylation on DNA condensation using light scattering, circular dichroism, and single molecule imaging of dendrimer-DNA complexes combed onto surfaces and tethered to those surfaces under flow. We find that DNA can be condensed by generation-five (G5) dendrimers even when the surface charges are more than 65% neutralized, but that such dendrimers bind negligibly when an end-tethered DNA is stretched in flow. We also find that when fully charged dendrimers are introduced by flow to end-tethered DNA, all DNA molecules become equally highly coated with dendrimers at a rate that becomes very fast at high dendrimer concentration, and that dendrimers remain bound during subsequent flow of dendrimer-free buffer. These results suggest that the presence of dendrimer-free DNA coexisting with dendrimer-bound DNA after bulk mixing of the two in solution may result from diffusion-limited irreversible dendrimer-DNA binding, rather than, or in addition to, the previously proposed cooperative binding mechanism of dendrimers to DNA.

Paclitaxel-conjugated PAMAM dendrimers adversely affect microtubule structure through two independent modes of action.

Paclitaxel (Taxol) is an anticancer drug that induces mitotic arrest via microtubule hyperstabilization but causes side effects due to its hydrophobicity and cellular promiscuity. The targeted cytotoxicity of hydrophilic paclitaxel-conjugated polyamidoamine (PAMAM) dendrimers has been demonstrated in cultured cancer cells. Mechanisms of action responsible for this cytotoxicity are unknown, that is, whether the cytotoxicity is due to paclitaxel stabilization of microtubules, as is whether paclitaxel is released intracellularly from the dendrimer. To determine whether the conjugated paclitaxel can bind microtubules, we used a combination of ensemble and single microtubule imaging techniques in vitro. We demonstrate that these conjugates adversely affect microtubules by (1) promoting the polymerization and stabilization of microtubules in a paclitaxel-dependent manner, and (2) bundling preformed microtubules in a paclitaxel-independent manner, potentially due to protonation of tertiary amines in the dendrimer interior. Our results provide mechanistic insights into the cytotoxicity of paclitaxel-conjugated PAMAM dendrimers and uncover unexpected risks of using such conjugates therapeutically.

Bifunctional PAMAM dendrimer conjugates of folic acid and methotrexate with defined ratio.

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.

Photochemical release of methotrexate from folate receptor-targeting PAMAM dendrimer nanoconjugate.

Nanoparticle (NP)-based targeted drug delivery involves cell-specific targeting followed by a subsequent therapeutic action from the therapeutic carried by the NP system. NPs conjugated with methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (DHFR) localized in cytosol, have been under investigation as a delivery system to target cancer cells to enhance the therapeutic index of methotrexate, which is otherwise non-selectively cytotoxic. Despite improved therapeutic activity from MTX-conjugated NPs in vitro and in vivo, the therapeutic action of these conjugates following cellular entry is poorly understood; in particular it is unclear whether the therapeutic activity requires release of the MTX. This study investigates whether MTX must be released from a nanoparticle in order to achieve the therapeutic activity. We report herein light-controlled release of methotrexate from a dendrimer-based conjugate and provide evidence suggesting that MTX still attached to the nanoconjugate system is fully able to inhibit the activity of its enzyme target and the growth of cancer cells.

Dendrimer-based multivalent methotrexates as dual acting nanoconjugates for cancer cell targeting.

Cancer-targeting drug delivery can be based on the rational design of a therapeutic platform. This approach is typically achieved by the functionalization of a nanoparticle with two distinct types of molecules, a targeting ligand specific for a cancer cell, and a cytotoxic molecule to kill the cell. The present study aims to evaluate the validity of an alternative simplified approach in the design of cancer-targeting nanotherapeutics: conjugating a single type of molecule with dual activities to nanoparticles, instead of coupling a pair of orthogonal molecules. Herein we investigate whether this strategy can be validated by its application to methotrexate, a dual-acting small molecule that shows cytotoxicity because of its potent inhibitory activity against dihydrofolate reductase and that binds folic acid receptor, a tumor biomarker frequently upregulated on the cancer cell surface. This article describes a series of dendrimer conjugates derived from a generation 5 polyamidoamine (G5 PAMAM) presenting a multivalent array of methotrexate and also demonstrates their dual biological activities by surface plasmon resonance spectroscopy, a cell-free enzyme assay, and cell-based experiments with KB cancer cells.

Bioanalytical Screening of Riboflavin Antagonists for Targeted Drug Delivery - A Thermodynamic and Kinetic Study.

The present study screened riboflavin mimicking small molecules to determine their binding activity for the riboflavin binding protein. We performed thermodynamic and kinetic binding studies of these molecules using a combination of two analytical approaches; isothermal titration calorimetry and surface plasmon resonance spectroscopy. Screening of a biased set of non-riboflavin based small molecules by microcalorimetry led to the discovery of two known drug molecules, quinacrine and chloroquine, as favorable ligands for the riboflavin receptor with K(D) value of 264, and 2100 nM, respectively. We further demonstrated that quinacrine is a competitive ligand for the receptor as measured by surface plasmon resonance. Thus this study describes the identification of a novel class of dual acting riboflavin antagonists that target riboflavin receptor for cellular uptake and display multifunctional activities upon cellular entry.

Design of riboflavin-presenting PAMAM dendrimers as a new nanoplatform for cancer-targeted delivery.

This communication describes the synthesis and in vitro biological evaluation of novel generation 5 PAMAM dendrimers conjugated with riboflavin as a targeting ligand. Cell-based experiments demonstrated that a dendrimer conjugated with riboflavin is able to undergo cellular binding and uptake in KB cells, and when the dendrimer is also conjugated with methotrexate, the riboflavin dendrimer conjugate can potently inhibit cell growth.

Light-controlled release of caged doxorubicin from folate receptor-targeting PAMAM dendrimer nanoconjugate.

We report the synthesis and in vitro evaluation of folate receptor-targeted nanoconjugate that releases its therapeutic payload via a photochemical mechanism.

Bipolar ion detector based on sequential conversion reactions.

This work demonstrates the feasibility of a novel scintillation detector with greater detection efficiency than that of chevron-type microchannel plate (MCP) detectors. The detection mechanism involves sequential conversion reactions induced by ion-surface impacts. Identical detection conditions can be utilized to monitor both positive and negative ions in mass spectrometers. The proposed detector comprises an ion beam guiding device, a negatively biased washer-shaped conversion dynode, and an aluminum-coated scintillation detector. The beam guide changes the electric field around the washer-shaped conversion dynode, and it allows the primary and secondary ions to propagate toward the scintillation phosphor and the conversion dynode, respectively. The detection is achieved by the detection of electron-induced luminescence on a phosphor. The amplification efficiency of this bipolar ion detector increases as the conversion dynode voltage increases. For ions with a mass-to-charge ratio of up to 90 000, the sensitivity of the BID is 1.4-14.4 times that of the MCP. Further improvement of the sensitivity can be achieved by increasing the conversion dynode voltage or the ion acceleration voltage. Results of this study demonstrate that this detector is a promising alternative for efficient ion detection.