Designing of a pH-activatable carbon dots as a luminescent nanoprobe for recognizing folate receptor-positive cancer cells.

During recent years, cancer has been recognized as a well-known disorder all over the world. One of the important factors to tackle this problem better than past decades is early diagnosis that takes into practice by state-of-the-art visual equipment for detection cancer cells. Herein, in this research, we synthesized carbon dots with pH-dependent behavior from a green source by hydrothermal method with high quantum yield and blue fluorescence. Folic acid-conjugated carbon dots by an efficient and optimal conjugation method were set upped which determined cancer cells visually. These synthesized and conjugated nanoparticles entered into the cancer cells more comprehensive than normal cells by receptor-mediated endocytosis and could distinguish cancer cells from normal ones by fluorescence imaging. Ultimately, synthesized nanoparticles in this research can be considered as an efficient fluorescent nanoprobe for cancer pre-diagnosis.

Methotrexate-based amphiphilic prodrug nanoaggregates for co-administration of multiple therapeutics and synergistic cancer therapy.

The goal of nanomedicine is to seek strategies that are more efficient to address various limitations and challenges faced by conventional medicines, including lack of target specificity, poor bioavailability, premature degradability, and undesired side effects. Self-assembling drug amphiphiles represent a prospective nanomedicine for cancer therapy owing to their favorable route of administration and therapeutic efficiency compared with pristine drug counterparts. In this work, we report a class of self-deliverable prodrug amphiphiles consisting of the hydrophilic drug methotrexate (MTX) and the hydrophobic anticancer drugs camptothecin (CPT) and doxorubicin (DOX) for targeted and combinational chemotherapy. The disulfide bond and hydrazone bond, which are subject to stimuli-triggered bond cleavage, were introduced to link these therapeutic agents and form two prodrug amphiphiles, named as MTX-CPT and MTX-DOX, respectively, which could self-assemble into stable prodrug nanoaggregates (NAs) in aqueous media. MTX molecules in the prodrug NAs facilitated NA uptake into tumor cells with high expression of folic acid receptors (FRs). This systemic study provided clear evidence of the synergistic therapeutic effect by co-administrating dual prodrug NAs on various tumor cells in vitro and a xenograft tumor model in vivo. The obtained prodrug amphiphiles provide an efficient strategy for the design of multifunctional drug delivery systems and elaborate therapeutic nanoplatforms for cancer chemotherapy. STATEMENT OF SIGNIFICANCE: This work presents two kinds of prodrug amphiphiles that are carrier free and integrate targeted drug delivery, stimuli-triggered drug release, synergistic therapy, and theranostic function into a single system. Reduction/acid active prodrug amphiphiles can self-assemble into micellar nanoaggregates (NAs) at a very low critical aggregation concentration. These NAs exhibit superior stability in physiological environment and disassemble in the presence of tumor cells expressing folic acid receptors or the high glutathione or in low pH tumoral endosomal environment. The induced disassembly of prodrug NAs can "switch on" the inherent fluorescence of the internalized camptothecin or doxorubicin for the detection of tumor cells. Compared to a single type of prodrug NA, co-administration of dual prodrug combination can produce an evident synergistic therapeutic effect against various tumor cells in vitro and inhibit xenograft tumor growth in vivo. The methotrexate-based prodrug amphiphiles may provide a potential strategy for developing multifunctional nanoplatforms and delivery of multiple therapeutics in chemotherapy.

Therapeutic efficacy of folate receptor-targeted amphiphilic cyclodextrin nanoparticles as a novel vehicle for paclitaxel delivery in breast cancer.

PURPOSE: The aim of this study is to test folate-conjugated cyclodextrin nanoparticles (FCD-1 and FCD-2) as a vehicle for reducing toxicity and increasing the antitumor efficacy of paclitaxel especially for metastatic breast cancer. METHODS: For the evaluation of PCX-loaded FCD nanoparticles, animal studies were realised in terms of survival rate, tumour size, weight change, metastazis and histopathological examination. RESULTS: FCD-1 displayed significant advantages such as efficient targeting of folate receptor positive breast cancer cells and having considerably lower toxicity compared to that of Cremophor®. When loaded with paclitaxel, FCD-1 nanoparticles, which have smaller particle size, neutral zeta potential, high encapsulation efficiency and better loading capacity for controlled release, emerged as an effective formulation in terms of cytotoxicity and high cellular uptake. In an experimental breast cancer model, anticancer activity of these nanoparticles were compatible with that of paclitaxel in Cremophor® however repeated administrations of FCD-1 nanoparticles were better tolerated by the animals. These nanoparticles were able to localise in tumour site. Both paclitaxel-loaded FCD-1 and FCD-2 significantly reduced tumour burden while FCD-1 significantly improved the survival. CONCLUSIONS: Folate-conjugated amphiphilic cyclodextrin nanoparticles can be considered as promising Cremophor®-free, low-toxicity and efficient active drug delivery systems for paclitaxel.

Functionalized gold nanoparticle-enhanced competitive assay for sensitive small-molecule metabolite detection using surface plasmon resonance.

The development of sensing systems for the measurement of small molecules is an active area of research. A sensor based approach for the measurement of metabolites can potentially provide the simplicity and portability required for widespread use. Rapid detection and quantitation of small-molecule metabolites can potentially emerge as an effective way to link the metabolite profile to the disease state. Surface plasmon resonance (SPR) combined with molecular recognition elements to deliver high specificity is a sensing platform that has been widely applied for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. The work described here combines a periplasmic binding protein for recognition with target modified gold nanoparticles (AuNPs) in a competitive assay format for folic acid (FA) detection. Specifically, a SPR imaging substrate containing immobilized folate binding protein (FBP) is used to measure the adsorption of FA conjugated AuNPs. The immobilization of the FBP and the binding of the FA conjugated AuNPs are characterized and optimized. It is shown that free FA in solution can be quantitatively measured by competition for the surface binding sites with the functionalized AuNPs. We demonstrate that the dynamic range can be lowered from micromolar to nanomolar by simply decreasing the concentration of FA conjugated AuNPs, thus lowering the limit of detection to 2.9 nM. This type of competitive assay can be applied to a range of small molecules, which paves the way for future multiplexed analysis of metabolites using SPR.

An unprecedented mechanism of nucleotide methylation in organisms containing thyX.

In several human pathogens, thyX-encoded flavin-dependent thymidylate synthase (FDTS) catalyzes the last step in the biosynthesis of thymidylate, one of the four DNA nucleotides. ThyX is absent in humans, rendering FDTS an attractive antibiotic target; however, the lack of mechanistic understanding prohibits mechanism-based drug design. Here, we report trapping and characterization of two consecutive intermediates, which together with previous crystal structures indicate that the enzyme's reduced flavin relays a methylene from the folate carrier to the nucleotide acceptor. Furthermore, these results corroborate an unprecedented activation of the nucleotide that involves no covalent modification but only electrostatic polarization by the enzyme's active site. These findings indicate a mechanism that is very different from thymidylate biosynthesis in humans, underscoring the promise of FDTS as an antibiotic target.

From invisible structures of SWCNTs toward fluorescent and targeting architectures for cell imaging.

Single-walled carbon nanotubes (SWNTs) are unique nanostructures used as cargo systems for variety of diagnostic and therapeutic agents. For taking advantage of these structures in biological processes, they should be visible. Therefore, fluorescence labeling of SWCNTs with various probes is a significant issue. Herein, we demonstrate a simple approach for cell specific imaging and diagnosis by combining SWCNTs with a copolymer poly(para-phenylene) (PPP) containing polystyrene (PSt) and poly(ε-caprolactone) (PCL) side chains (PPP-g-PSt-PCL). In this approach PPP-g-PSt-PCL is noncovalently attached on carboxyl functional SWCNTs. The obtained fluorescent probe is bound to folic acid (FA) for targeted imaging of folate receptor (FR) positive HeLa cells. In vitro studies demonstrate that this conjugate can specifically bind to HeLa cells and indicate great potential for targeting and imaging studies.

Synthesis and biological evaluation of novel folic acid receptor-targeted, ß-cyclodextrin-based drug complexes for cancer treatment.

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5-2.5 nm. The host-guest association constant K a was 1,639 M(-1) as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated ß-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Crystal structure of a folate energy-coupling factor transporter from Lactobacillus brevis.

ATP-binding cassette (ABC) transporters, composed of importers and exporters, form one of the biggest protein superfamilies that transport a variety of substrates across the membrane, powered by ATP hydrolysis. Most ABC transporters are composed of two transmembrane domains and two cytoplasmic nucleotide-binding domains. Also, importers from prokaryotes usually have extra solute-binding proteins in the periplasm that are responsible for the binding of substrates. Structures of importers have been reported that suggested a two-state model for the transport mechanism. Energy-coupling factor (ECF) transporters belong to a new class of ATP-binding cassette importers. Each ECF transporter comprises an energy-coupling module consisting of a transmembrane T protein (EcfT), two nucleotide-binding proteins (EcfA and EcfA'), and another transmembrane substrate-specific binding S protein (EcfS). Despite the similarities with ABC transporters, ECF transporters have different organizational and functional properties. The lack of solute-binding proteins in ECF transporters differentiates them clearly from the canonical ABC importers. Previously reported structures of the EcfS proteins RibU and ThiT clearly demonstrated the binding site of substrate riboflavin and thiamine, respectively. However, the organization of the four different components and the transport mechanism of ECF transporters remain unknown. Here we present the structure of an intact folate ECF transporter from Lactobacillus brevis at a resolution of 3 Å. This structure was captured in an inward-facing, nucleotide-free conformation with no bound substrate. The folate-binding protein FolT is nearly parallel to the membrane and is bound almost entirely by EcfT, which adopts an L shape and connects to EcfA and EcfA' through two coupling helices. Two conserved XRX motifs from the coupling helices of EcfT have a vital role in energy coupling by docking into EcfA-EcfA'. We propose a transport model that involves a substantial conformational change of FolT.

A simple and rapid electrochemical strategy for non-invasive, sensitive and specific detection of cancerous cell.

Developing non-invasive, sensitive and specific sensing strategies for cancerous cell detection with simple and low cost instrumentations provide great advantages in cancer research and early diagnosis of diseases. In the present work, gold nanoparticles (Au NPs) functionalized with recognition components (folic acid) and signal indicator (ferrocene) was designed to fabricate electrochemical cytosensor. The Au NPs can not only accelerate electron transfer between signal indicator and the underlying electrode but also accumulate more ferrocene on the cytosensor surface to magnify signal for improving detection sensitivity. The surface-tethered folic acid plays a key role in specific binding folate receptor-riched HeLa cells on the cytosensor surface, resulting in corresponding current signal change measured by differential pulse voltammetry method. A wide detection range from 10 to 10(6) cells/mL with a detection limit as low as 10 cells/mL for cancerous cells was reached in the presence of a large amount of normal ones with fast differential pulse voltammetry measurement. Detection of the captured cells can be finished within 1 min. The developed strategy provides a new way for operationally simple, rapid, sensitive and specific detection of cancerous cells.

Folate-linked drugs for the treatment of cancer and inflammatory diseases.

Folic acid, also known as vitamin B9 (Fig. 9.1), is an essential co-enzyme in one-carbon metabolism pathways, including the biosynthesis of nucleotides (i.e. purines, thymidine) and several amino acids. In general, two functionally different systems mediate the cellular uptake of folate: (1) the reduced folate carrier (RFC, Kd ∼ 10-6 M), an anion transporter that delivers folates across the plasma membrane in a bidirectional fashion, and (2) the folate receptor (FR, Kd ∼ 10-10 M), which internalizes folate through active receptor-mediated endocytosis. The RFC, a membrane-spanning anion transporter, is present in virtually all tissues and is responsible for the majority of folate transport in and out of cells. In contrast, FR expression is largely restricted to malignant cells, activated macrophages, and the proximal tubule cells of the kidneys. Because a variety of important diseases are caused by the former two cell types, interest in exploiting FR for drug targeting applications has rapidly increased. And achievement of this targeting objective, primarily through conjugation of drugs to folic acid is believed to enable (1) enhanced net drug uptake by pathologic cells, and more importantly (2) reduction in drug deposition into non-pathologic cells, thereby mitigating collateral toxicity to normal tissues.

Pharmacokinetics and tissue distribution of docetaxel by liquid chromatography-mass spectrometry: evaluation of folate receptor-targeting amphiphilic copolymer modified nanostructured lipid carrier.

A novel amphiphilic copolymer, folate-poly(PEG-cyanoacrylate-co-cholesteryl cyanoacrylate) (FA-PEG-PCHL) was synthesized to modify docetaxel-loaded nanostructured lipid carrier to lead to a long blood circulating effect and targeting ability for the delivery of antitumor drug in cancer. To investigate the characteristics of modified docetaxel-loaded nanostructured lipid carrier in vivo, a liquid chromatography-mass spectrometry method was developed and validated for the determination of docetaxel in rat plasma and tumor-bearing mouse tissue samples. The biosamples were extracted by liquid-liquid extraction method with ether and separated on a C(18) column (150 mm×4.6 mm, 5 µm) using a mobile phase consisting of methanol-0.01% formic acid water (82:18, v/v). The standard curves were linear over the ranges of 0.01-4.0 µg/mL for plasma and 0.02-8.0 µg/g for tissue samples, respectively. The validated method was successfully applied to the pharmacokinetic study in rat plasma and tissue distribution study in mouse tissues of docetaxel after an intravenous administration of docetaxel injection (DTX injection), docetaxel-loaded nanostructured lipid carrier (DTX-NLC) and FA-PEG-PCHL-modified docetaxel-loaded nanostructured lipid carrier (FA-DTX-NLC), respectively. The results indicated that the FA-DTX-NLC led to significant differences in pharmacokinetic profile and tissue distribution. Nanostructured lipid carrier modified by FA-PEG-PCHL could be one of the promising suspensions for the delivery of docetaxel in cancer.