Pafolacianine for intraoperative molecular imaging of cancer in the lung: The ELUCIDATE trial.

OBJECTIVE: The study objective was to determine the clinical utility of pafolacianine, a folate receptor-targeted fluorescent agent, in revealing by intraoperative molecular imaging folate receptor α positive cancers in the lung and narrow surgical margins that may otherwise be undetected with conventional visualization. METHODS: In this Phase 3, 12-center trial, 112 patients with suspected or biopsy-confirmed cancer in the lung scheduled for sublobar pulmonary resection were administered intravenous pafolacianine within 24 hours before surgery. Participants were randomly assigned to surgery with or without intraoperative molecular imaging (10:1 ratio). The primary end point was the proportion of participants with a clinically significant event, reflecting a meaningful change in the surgical operation. RESULTS: No drug-related serious adverse events occurred. One or more clinically significant event occurred in 53% of evaluated participants compared with a prespecified limit of 10% (P < .0001). In 38 participants, at least 1 event was a margin 10 mm or less from the resected primary nodule (38%, 95% confidence interval, 28.5-48.3), 32 being confirmed by histopathology. In 19 subjects (19%, 95% confidence interval, 11.8-28.1), intraoperative molecular imaging located the primary nodule that the surgeon could not locate with white light and palpation. Intraoperative molecular imaging revealed 10 occult synchronous malignant lesions in 8 subjects (8%, 95% confidence interval, 3.5-15.2) undetected using white light. Most (73%) intraoperative molecular imaging-discovered synchronous malignant lesions were outside the planned resection field. A change in the overall scope of surgical procedure occurred for 29 of the subjects (22 increase, 7 decrease). CONCLUSIONS: Intraoperative molecular imaging with pafolacianine improves surgical outcomes by identifying occult tumors and close surgical margins.

Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC).

The folate receptor alpha (FR), which is overexpressed in solid tumors including NSCLC, can be utilized for active tumor targeting to afford more effective cancer therapies. In this context, cytochrome c (Cyt c) has drawn attention to cancer research because it is non-toxic, yet, when delivered to the cytoplasm of cancer cells, can kill them by inducing apoptosis. Cyt c nanoparticles (NPs, 169 ± 9 nm) were obtained by solvent precipitation with acetonitrile, and stabilized by reversible homo-bifunctional crosslinking to accomplish a Cyt-c-based drug delivery system that combines stimulus-responsive release and active targeting. Cyt c was released under intracellular redox conditions, due to an S-S bond in the NPs linker, while NPs remained intact without any release under extracellular conditions. The NP surface was decorated with a hydrophilic folic acid-polyethylene glycol (FA-PEG) polymer for active targeting. The FA-decorated NPs specifically recognized and killed cancer cells (IC50 = 47.46 µg/mL) that overexpressed FR, but showed no toxicity against FR-negative cells. Confocal microscopy confirmed the preferential uptake and apoptosis induction of our NPs by FR-positive cancer cells. In vivo experiments using a Lewis lung carcinoma (LLC) mouse model showed visible NP accumulation within the tumor and inhibited the growth of LLC tumors.

Optimized PAH/Folic acid layer-by-layer films as an electrochemical biosensor for the detection of folate receptors.

Folate receptor alpha (FR-α) is a glycoprotein overexpressed in tumor cell surfaces, especially in gynecologic cancers, and can be used as a biomarker for diagnostics. Currently, FRα is quantified by positron emission tomography (PET) or fluorescence imaging techniques. However, these methods are costly and time-consuming. We report on the development of an electrochemical biosensor for FRα detection based on the use of nanostructured layer-by-layer (LbL) films as modified electrodes. Multilayer films were deposited on indium tin oxide (ITO) electrodes by the alternately assembling of positively charged polyallylamine hydrochloride (PAH) and negatively charged folic acid (FA), used as the biorecognition element. UV-vis and FTIR spectroscopies revealed the successful PAH and FA adsorption on ITO. Devices performance was evaluated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The [PAH/FA] films presented a good reproducibility (RSD of 1.12%) and stability when stored in the Tris-HCl solution (RSD 6.7%). The biosensor electrochemical response exhibited a linear relationship with FRα concentration in the range from 10 to 40 nM. The limit of detection reached for CV and EIS measurements were 0.7 and 1.5 nM, respectively. As a proof-of-concept, we show that the devices can differenciate tumor cells from healthy cell, showing an excellent selectivity. The biosensor device based on [PAH/FA] films represents a promising strategy for a simple, rapid, and low-cost cancer diagnosis through FRα quantification on the surface of cancer cells.

Functionalized rare earth-doped nanoparticles for breast cancer nanodiagnostic using fluorescence and CT imaging.

BACKGROUND: Breast cancer is the second leading cause of cancer death among women and represents 14% of death in women around the world. The standard diagnosis method for breast tumor is mammography, which is often related with false-negative results leading to therapeutic delays and contributing indirectly to the development of metastasis. Therefore, the development of new tools that can detect breast cancer is an urgent need to reduce mortality in women. Here, we have developed Gd2O3:Eu3+ nanoparticles functionalized with folic acid (FA), for breast cancer detection. RESULTS: Gd2O3:Eu3+ nanoparticles were synthesized by sucrose assisted combustion synthesis and functionalized with FA using EDC-NHS coupling. The FA-conjugated Gd2O3:Eu3+ nanoparticles exhibit strong red emission at 613 nm with a quantum yield of ~ 35%. In vitro cytotoxicity studies demonstrated that the nanoparticles had a negligible cytotoxic effect on normal 293T and T-47D breast cancer cells. Cellular uptake analysis showed significantly higher internalization of FA-conjugated RE nanoparticles into T-47D cells (Folr hi ) compared to MDA-MB-231 breast cancer cells (Folr lo ). In vivo confocal and CT imaging studies indicated that FA-conjugated Gd2O3:Eu3+ nanoparticles accumulated more efficiently in T-47D tumor xenograft compared to the MDA-MB-231 tumor. Moreover, we found that FA-conjugated Gd2O3:Eu3+ nanoparticles were well tolerated at high doses (300 mg/kg) in CD1 mice after an intravenous injection. Thus, FA-conjugated Gd2O3:Eu3+ nanoparticles have great potential to detect breast cancer. CONCLUSIONS: Our findings provide significant evidence that could permit the future clinical application of FA-conjugated Gd2O3:Eu3+ nanoparticles alone or in combination with the current detection methods to increase its sensitivity and precision.

Antiproliferative Activity and VEGF Expression Reduction in MCF7 and PC-3 Cancer Cells by Paclitaxel and Imatinib Co-encapsulation in Folate-Targeted Liposomes.

Co-encapsulation of anticancer drugs paclitaxel and imatinib in nanocarriers is a promising strategy to optimize cancer treatment. Aiming to combine the cytotoxic and antiangiogenic properties of the drugs, a liposome formulation targeted to folate receptor co-encapsulating paclitaxel and imatinib was designed in this work. An efficient method was optimized for the synthesis of the lipid anchor DSPE-PEG(2000)-folic acid (FA). The structure of the obtained product was confirmed by RMN, FT-IR, and ESI-MS techniques. A new analytical method was developed and validated for simultaneous quantification of the drugs by liquid chromatography. Liposomes, composed of phosphatidylcholine, cholesterol, and DSPE-mPEG(2000), were prepared by extrusion. Their surface was modified by post-insertion of DSPE-PEG(2000)-FA. Reaction yield for DSPE-PEG(2000)-FA synthesis was 87%. Liposomes had a mean diameter of 122.85 ± 1.48 nm and polydispersity index of 0.19 ± 0.01. Lyophilized formulations remained stable for 60 days in terms of size and drug loading. FA-targeted liposomes had a higher effect on MCF7 cell viability reduction (p < 0.05) when compared with non-targeted liposomes and free paclitaxel. On PC-3 cells, viability reduction was greater (p < 0.01) when cells were exposed to targeted vesicles co-encapsulating both drugs, compared with the non-targeted formulation. VEGF gene expression was reduced in MCF7 and PC-3 cells (p < 0.0001), with targeted vesicles exhibiting better performance than non-targeted liposomes. Our results demonstrate that multifunctional liposomes associating molecular targeting and multidrug co-encapsulation are an interesting strategy to achieve enhanced internalization and accumulation of drugs in targeted cells, combining multiple antitumor strategies.

Heterologous expression of Homo sapiens alpha-folate receptors in E. coli by fusion with a trigger factor for enhanced solubilization.

The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FRα by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors.

Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies.

Synthesis and Evaluation of Folate-Conjugated Phenanthraquinones for Tumor-Targeted Oxidative Chemotherapy.

Almost all cells are easily killed by exposure to potent oxidants. Indeed, major pathogen defense mechanisms in both animal and plant kingdoms involve production of an oxidative burst, where host defense cells show an invading pathogen with reactive oxygen species (ROS). Although cancer cells can be similarly killed by ROS, development of oxidant-producing chemotherapies has been limited by their inherent nonspecificity and potential toxicity to healthy cells. In this paper, we describe the targeting of an ROS-generating molecule selectively to tumor cells using folate as the tumor-targeting ligand. For this purpose, we exploit the ability of 9,10-phenanthraquinone (PHQ) to enhance the continuous generation of H2O2 in the presence of ascorbic acid to establish a constitutive source of ROS within the tumor mass. We report here that incubation of folate receptor-expressing KB cells in culture with folate-PHQ plus ascorbate results in the death of the cancer cells with an IC50 of ~10 nM (folate-PHQ). We also demonstrate that a cleavable spacer linking folate to PHQ is significantly inferior to a noncleavable spacer, in contrast to most other folate-targeted therapeutic agents. Unfortunately, no evidence for folate-PHQ mediated tumor regression in murine tumor models is obtained, suggesting that unanticipated impediments to generation of cytotoxic quantities of ROS in vivo are encountered. Possible mechanisms and potential solutions to these unanticipated results are offered.

A novel chromatin insulator regulates the chicken folate receptor gene from the influence of nearby constitutive heterochromatin and the ß-globin locus.

The three-dimensional architecture of genomes provides new insights about genome organization and function, but many aspects remain unsolved at the local genomic scale. Here we investigate the regulation of two erythroid-specific loci, a folate receptor gene (FOLR1) and the ß-globin gene cluster, which are separated by 16kb of constitutive heterochromatin. We found that in early erythroid differentiation the FOLR1 gene presents a permissive chromatin configuration that allows its expression. Once the transition to the next differentiation state occurs, the heterochromatin spreads into the FOLR1 domain, concomitant with the dissociation of CTCF from a novel binding site, thereby resulting in irreversible silencing of the FOLR1 gene. We demonstrate that the sequences surrounding the CTCF-binding site possess classical insulator properties in vitro and in vivo. In contrast, the chicken cHS4 ß-globin insulator present on the other side of the heterochromatic segment is in a constitutive open chromatin configuration, with CTCF constantly bound from the early stages of erythroid differentiation. Therefore, this study demonstrates that the 16kb of constitutive heterochromatin contributes to silencing of the FOLR1 gene during erythroid differentiation.